Dissertations / Theses on the topic 'Cancer cells metabolism'

The TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) gene was described in 2006 by Dr. Karen Vousden's group in response to the induction of the tumour suppressor gene p53. Since then, numerous studies have focused on clarifying the role of this gene in the metabolism of tumour cells. TIGAR was initially described as an enzyme with bisphosphatase activity on fructose-2,6-bisphosphate, a key metabolite in the positive allosteric regulation of phosphofructokinase-1, which catalyses a key reaction in glycolysis. Through this bisphosphatase activity, TIGAR reduces the levels of fructose-2,6-bisphosphate and, consequently, decreases the glycolytic flux and redirects the metabolites to the pentose phosphate pathway, which is determinant for the antioxidant capacity of cells. Overexpression of TIGAR has been described in multiple tumours, as well as in different cell lines, indicating that this gene confers a growth advantage to these cells. The present doctoral thesis has focused on studying the metabolic function of TIGAR in tumours, as well as the mechanisms that regulate its transcription. The study was carried out on cell lines of cervical cancer and lung cancer in which we have been able to confirm that TIGAR exerts a bisphosphatase function on fructose-2,6-bisphosphate. TIGAR has proven to be key in the response of the cervical cancer cell line HeLa to the blockage of glycolysis, either by inhibiting the expression of the PFKFB3 gene by interfering RNA technology, or by blocking the PFK-2 protein by the drug 3PO. The blockage of glycolysis increases oxidative stress and the phosphorylation of the kinase Akt, which is required for the induction of TIGAR. Furthermore, through metabolomic studies we have been able to describe for the first time the involvement of TIGAR in the entrance of pyruvate to the tricarboxylic acid cycle, in the mitochondria. Finally, and in relation to the mechanisms that regulate the transcription of TIGAR, we have proved that the transcription factor Nrf2, key in the regulation of the antioxidant activity of tumour cells, controls the expression of TIGAR in HeLa cells. In lung cancer cells, where the over activation of Nrf2 is related to chemo resistance and radiotherapy, the relationship between Nrf2 and TIGAR seems to be indirect. With the results presented in this doctoral thesis we have contributed to a better understanding of the role of TIGAR in tumour metabolism and have laid the foundations for future studies aimed at blocking this protein in tumours.
El gen TP53-Induced Glycolysis and Apoptosi Regulator (TIGAR) va ser descrit l'any 2006 pel grup de la Dra. Karen Vousden en resposta a l’activació del supressor tumoral p53. Des de llavors, nombrosos estudis s'han centrat en aclarir el paper d'aquest gen en el metabolisme de les cèl·lules tumorals. Inicialment, la funció atribuïda a TIGAR va ser la de bisfosfatasa de la fructosa-2,6-bisfosfat, metabòlit clau en la regulació al·lostèrica positiva de l’enzim fosfofructoquinasa-1, que catalitza la una reacció clau en la glucòlisi. Mitjançant aquesta activitat bisfosfatasa, TIGAR redueix els nivells de fructosa-2,6-bisfosfat i, en conseqüència, frena en flux glicolític i redirigeix els metabòlits a la via de les pentoses fosfat. És per aquest motiu que TIGAR es va descriure com un gen amb capacitat antioxidant. La present tesi doctoral s'ha centrat en estudiar la funció metabòlica de TIGAR en línies tumorals, així com els mecanismes que regulen la seva transcripció. Amb aquests estudis hem pogut demostrar que TIGAR és clar en la resposta de les cèl·lules al bloqueig de la glucòlisi, ja sigui per la inhibició de l'expressió del gen PFKFB3 mitjançant la tecnologia de RNA d'interferència, com pel bloqueig de la proteïna PFK-2 mitjançant el fàrmac 3PO. El bloqueig de la glucòlisi provoca un augment de l'estrès oxidatiu i de la fosforil·lació de la quinasa Akt, necessària per a la inducció de TIGAR.que al seu torn condueix a una inducció de TIGAR. D’altra banda, estudis metabolòmics ens han permès descriure per primera vegada l’acció de TIGAR en nivells inferiors de la glicòlisi, afectant l’entrada del piruvat al cicle de Krebs. Finalment, hem pogut comprovar que el factor de transcripció Nrf2, clau en la regulació de l'activitat antioxidant de les cèl·lules, controla l'expressió de TIGAR en una línia cel·lular de càncer de cèrvix. En cèl·lules de càncer de pulmó, en canvi, la relació entre Nrf2 i TIGAR sembla ser indirecta. Amb els resultats presentats en aquesta tesi doctoral hem contribuït a entendre millor el paper de TIGAR en el metabolisme tumoral i hem establert les bases per a futurs estudis dirigits al bloqueig d'aquesta proteïna als tumors.

The purpose of this thesis was to characterize cancer metabolism in vitro using epithelial ovarian cancer as a model on an untargeted, systems-level, basis with particular attention paid to the difference between cancer stem cell metabolism and cancer cell metabolism. Two-dimensional gas chromatography coupled to mass spectrometry was used to measure the metabolite profiles of the ovarian cancer and cancer stem cell lines under normal baseline conditions and also under chemotherapeutic and environmental perturbations. These two cell lines exhibited significant metabolic differences under normal baseline conditions and results demonstrated that metabolism in the ovarian cancer stem cell line was distinct from that of more differentiated isogenic cancer cells, showing similarities to stem cell metabolism that suggest the potential importance of metabolism for the cancer stem cell phenotype. Glucose deprivation, hypoxia, and ischemia all perturbed ovarian cancer and cancer stem cell metabolism, but not in the same ways between the cell types. Chemotherapeutic treatment with docetaxel caused metabolic changes mostly in amino acid and carbohydrate metabolism in ovarian cancer cells, while ovarian cancer stem cell metabolism was not affected by docetaxel. Overall, these metabolic differences between the two cell types will deepen our understanding of the metabolic changes occurring within the in vivo tumor and will help drive development of cancer stem cell targeted therapeutics.

Academic: Ovarian cancer is the leading cause of death among female gynecologic cancers. Current treatments include surgical debulking, and chemotherapy. However, better interventions are needed to reduce the mortality rate of metastatic disease. Ovarian cancer cells have displayed the ability to aggregate and form 3D homogeneous and heterogeneous spheroids, which can function as micrometastases. Ovarian cancer spheroids survive independently prior to adhering to an endothelial tissue. Since aggregation has been shown to provide a survival advantage to the spheroids and increased their aggressive phenotype, this study aimed to investigate how the metabolism of ovarian cancer cells change in 3-dimensional (3D) culture. Examining metabolic pathways and identifying markers of metabolic change could provide the scientific base for new, targeted interventions for this disease. Spheroids of both homogeneous and heterogeneous composition demonstrated overall lower metabolic capacity than their adherent counterparts. Spheroids had a lower basal energetic demand than adherent cells, paralleled by lower maximal respiration capacity, glycolytic capacity, and spare respiratory capacity. We conclude that the lower energetic demand of spheroids may be a mechanism to prolong death by reserving energy and metabolic cellular processes; this may render anti-metabolic drug treatment with AICAR or metformin ineffective against disseminating ovarian cancer aggregates. General: Ovarian cancer is currently the leading cause of death among female gynecologic cancers. While treatments exist, better interventions are needed to reduce the mortality rate in this form of cancer. Ovarian cancer cells have displayed the ability to aggregate and form 3D homogeneous and heterogeneous spheroids, which can function as micrometastases. Ovarian cancer spheroids survive independently prior to adhering to an endothelial tissue. Since aggregation has been shown to provide a survival advantage to the spheroids and increased their aggressive phenotype, this study aims to investigate how the metabolism of ovarian cancer cells change in 3-dimensional (3D) culture. Examining metabolic pathways and identifying markers of metabolic change could provide the scientific base for new, targeted interventions for this disease.
Master of Science

Metabolic rearrangements are essential to satisfy the different needs of cancer cells during tumorigenesis. Recent studies highlighted a role for such metabolic reprogramming in adaptation to therapies and chemo-resistance development. 5-fluorouracil (5-FU) is an antimetabolite drug widely used as a first-line treatment for colorectal cancer. Despite several advantages of 5-FU, its clinical application is still greatly limited, due to the acquisition of drug resistance. In the first part of this thesis, we illustrate the role of micro RNAs (miRNAs) in reprogramming colon cancer cells toward a resistant phenotype as well as their involvement in the response of resistant cells to acute treatment with 5-FU. We performed a global gene expression profile for the entire miRNA genome, and we found a change in the expression of four miRNAs following acute treatment with 5-FU in cells resistant to this drug. Among them, we focused on miR-210-3p, previously described as a key regulator of DNA damage repair mechanisms and mitochondrial metabolism. Here we show that miR-210-3p downregulation enables resistant cells to counteract the toxic effect of the drug increasing the expression of RAD-52 protein, involved in DNA damage repair. Moreover, miR-210-3p downregulation enhances mitochondrial oxidative metabolism, increasing the expression levels of succinate dehydrogenase subunits D, decreasing intracellular succinate levels and inhibiting HIF-1α expression. These results suggest that miR-210-3p downregulation following 5-FU treatment sustains DNA damage repair and metabolic adaptation to counteract drug treatment, thus supporting the resistant phenotype. In the second part of this thesis, we reveal important adaptations in serine and one-carbon metabolism associated with the acquisition of 5-FU resistance in colorectal cancer cells. 5-FU resistant cells showed an increase in both serine up-take from extracellular medium and de novo serine synthesis pathway. Together with increased serine availability, dynamic labeling experiment after 13C-serine incubation underlined a different utilization of serine-derived carbons in resistant cells with a sustained flux into the mitochondrial compartment supporting increased purine nucleotides synthesis. Accordingly, we found a strong decrease in the expression of the cytosolic isoform of the enzyme serine hydroxy-methyltransferase (SHMT1) and a concomitant increase in the expression of the mitochondrial isoform (SHMT2) in 5-FU resistant cells compared to parental cells, confirming the shift toward mitochondrial one-carbon branch activity. Accordingly, higher expression levels of the mitochondrial serine transporter SFXN1 have been observed in resistant cells with respect to the sensitive ones. Silencing SHMT2 in 5-FU resistant cells increases the efficacy of the treatment with 5-FU against resistant cells confirming the importance of the reported adaptation in the acquisition of resistance to 5-FU. In conclusion, the data shown in this thesis underline different adaptations related to both miRNAs expression and nutrient metabolism carried out by 5-FU resistant cells. This reprogramming supports the response of 5-FU resistant cells to overcome the toxic effect of the drug. The identification of such alterations opens the possibility of new therapeutic approaches to tackle resistant cells and overcome colon cancer relapse.

Ovarian cancer is the fourth leading cause of cancer-related death in women and the leading cause of gynecologic cancer death. Moreover, it is regarded as a therapy resistant tumor, because it shows the formation of more aggressive recurrence of the primary tumor as a result of chemotherapy. This chemo-resistance is thought to be related to the presence of the Cancer Stem Cells (CSC). Tumor cells are characterized by a high glycolytic metabolism even in the presence of oxygen, the so-called Warburg effect; however, it is unclear whether this condition is also shared by CSC. We identified ovarian CSC, according to their co-expression of CD44 and CD117 markers, in 40 samples of ascitic effusions from ovarian cancer-bearing patients. We have analyzed phenotipic characteristics by investigating stemness marker expression by flow-cytometry, spheroid formation assay, tumorigenicity in vivo and gene expression in RT-PCR. For the analysis of metabolic characteristics, ovarian cancer cells were FACS-sorted in to CD44+CD117+ and CD44+CD117- cell populations and analyzed through specific metabolic gene-cards. Results were confirmed also through Western Blot for specific metabolic enzymes and functional assay of mitochondrial activity. We have demonstrated that CD44+CD117+ EOC cells presented high tumorigenicity and expressed stemness-associated markers and multidrug resistance pumps. Moreover, the CD44+CD117+ cell population overexpressed genes associated with glucose uptake, oxidative phosphorylation (OXPHOS), and fatty acid -oxidation, indicating higher ability to direct pyruvate towards the Krebs cycle. Consistent with a metabolic profile dominated by OXPHOS, the CD44+CD117+ cells showed higher mitochondrial reactive oxygen species (ROS) production and elevated membrane potential, and underwent apoptosis upon inhibition of the mitochondrial respiratory chain. The CSC also had a high rate of pentose phosphate pathway (PPP) activity, which is not typical of cells privileging OXPHOS over glycolysis, and may rather reflect the PPP role in recharging scavenging enzymes. Furthermore, CSC resisted in vitro and in vivo glucose deprivation, while maintaining their CSC phenotype and OXPHOS profile. In this study, we show that a subpopulation of CD44+CD117+ EOC cells fulfilling the canonical properties of CSC does not preferentially exploit a glycolytic metabolism, privileging instead the mitochondrial respiratory pathway. These observations could explain the CSC resistance to anti-angiogenic therapies, and indicate this peculiar metabolic profile as a possible target of novel treatment strategies.
Il cancro all’ovaio viene considerato un tumore resistente alla terapia e questa farmaco-resistenza si pensa sia correlata alla presenza delle cellule staminali tumorali (CSC). Le cellule staminali tumorali sono una rara e piccola popolazione cellulare responsabile dell’insorgenza del tumore, del mantenimento della sua crescita, dei casi di recidive e metastasi, in seguito alla loro proprietà di farmaco-resistenza. Considerando queste premesse, è indispensabile caratterizzare queste cellule in modo da trovare un possibile bersaglio terapeutico e migliorare i risultati delle terapie attuali. Le cellule tumorali sono caratterizzate da un metabolismo altamente glicolitico anche in presenza di ossigeno, denominato “Effetto Warburg”. Poco si conosce riguardo al metabolismo delle cellule staminali tumorali, e soprattutto non è noto se l’effetto Warburg è una condizione condivisa. Questo progetto di ricerca si prefigge di: - caratterizzare le CSC nel campioni primari di liquido ascitico di cancro all’ovaio; - studiare il profilo metabolico delle CSC isolate, per identificare eventuali differenze con la controparte differenziata. RISULTATI: Inizialmente abbiamo identificato le CSC, secondo la co-espressione dei marcatori CD44 (il recettore dell’acido ialuronico), e CD117 [c-kit, recettore della citochina SCF (Stem Cell Factor)] in 40 campioni di liquido ascitico di cancro all’ovaio di pazienti in cura all’ospedale di Padova. Questa rara popolazione cellulare CD44+CD117+ è in grado di formare strutture sferoidali; è altamente tumorigenica in topi immunodeficienti; presenta farmaco-resistenza, dimostrata con trattamenti in vitro con farmaci solitamente utilizzati in clinica; ed è caratterizzata da un’alta espressione di geni codificanti: pathway di staminalità (Nanog, Oct4, Sox2), pompe o enzimi detossificanti, coinvolti nei fenomeni di farmaco-resistenza (ABCG2, MRP1, MRP2 e ALDH1A) e enzimi coinvolti nel fenomeno della transizione epitelio-mesenchimale, importante nei processi di metastasi (SNAIL1, SNAIL2, ZEB1, ZEB2, TWIST1). Complessivamente, questi risultati dimostrano che le cellule CD44+CD117+ rappresentano una popolazione con caratteristiche di staminalità. A seguito di questa caratterizzazione fenotipica, abbiamo studiato il profilo metabolico delle cellule CD44+CD117+, confrontandolo con quello della controparte non-staminale (CD44+CD117-). In primo luogo, abbiamo esaminato l’espressione di geni coinvolti in diverse importanti vie metaboliche, tra cui: il metabolismo del glucosio, il ciclo dell'acido tricarbossilico (TCA), la catena di trasporto degli elettroni (ETC) nel processo della respirazione mitocondriale, la via dei pentoso fosfati (PPP), e la β-ossidazione degli acidi grassi. Le cellule CD44+CD117+ mostrano alti livelli di espressione dei geni associati alla glicolisi, e sono caratterizzate da una forte dipendenza dalla via dei pentoso fosfati e della β-ossidazione degli acidi grassi, dimostrata da una significativa diminuzione della loro vitalità in seguito a trattamento in vitro con due inibitori specifici delle due vie metaboliche (DHEA e Etomoxir rispettivamente). Inoltre le cellule CD44+CD117+ sono caratterizzate da un'alta espressione dei geni codificati enzimi coinvolti nel ciclo di Krebs e nella fosforilazione ossidativa (OXPHOS). Questo risultato ci ha permesso di analizzare l'espressione di un enzima chiave del ciclo di Krebs, la piruvato deidrogenasi (PDH), fondamentale nel trasporto del piruvato dalla glicolisi alla respirazione cellulare. Abbiamo verificato livelli di espressione comparabili dell’enzima PDH nelle due popolazioni cellulari CD44+CD117+ e CD44+CD117-, mentre l’enzima PDHK1, che inattiva la piruvato deidrogenasi tramite fosforilazione, risulta meno espressa nella popolazione CD44+CD117+. Questi dati suggeriscono che nelle cellule staminali tumorali venga privilegiato il trasporto del piruvato verso i mitocondri, per catalizzare il metabolismo della respirazione mitocondriale. Alla luce di questi risultati, abbiamo studiato l’attività mitocondriale nella popolazione staminale e nella controparte non staminale. In particolare le cellule CD44+CD117+ sono caratterizzate da bassi livelli di ROS (specie reattive dell’ossigeno) totali, da alti livelli di ROS mitocondriali, da una iper-polarizzazione del potenziale di membrana mitocondriale in seguito a trattamento con oligomicina (inibitore dell’ATP-sintasi) e da una drammatica diminuzione della vitalità cellulare in seguito a trattamento con inibitori specifici della catena di trasporto degli elettroni (ETC) (oligomicina inibitore dell’ATP-sintasi; rotenone inibitore del complesso I e antimicina inibitore del complesso III). Complessivamente, questi risultati ci hanno suggerito un modello sperimentale del profilo metabolico delle cellule staminali tumorali CD44+CD117+, le quali privilegiano la via della respirazione mitocondriale, a discapito della via glicolitica. Inoltre, abbiamo dimostrato che un trattamento in vitro e in vivo (2DG) di deprivazione di glucosio o blocco della via glicolitica seleziona una popolazione di cellule con caratteristiche di staminalità: incremento dell’espressione dei marcatori CD44 e CD117, farmaco-resistenza, tumorigenicità in vivo, formazion dii sferoidi in vitro ed espressione di geni convolti in pathway tipici delle cellule staminali. Questa popolazione cellulare ha mostrato una down-regolazione della maggior parte delle vie metaboliche, entrando in uno stato di quiescenza pur mantenendo livelli di espressione significativi dei geni codificanti enzimi del metabolismo ossidativo e iper-polarizzazione del potenziale di membrana mitocondriale, nonché dell’attività dei mitocondri. A conclusione del progetto e come ulteriore dimostrazione del profilo metabolico ossidativo delle cellule staminali tumorali, contrario all’effetto Warburg sfruttato dalle cellule tumorali, abbiamo eseguito degli esperimenti in vitro con due farmaci che colpiscono le vie metaboliche della respirazione cellulare: Metformina e CPI-613. Metformina inibisce il complesso I della catena di trasporto degli elettroni ed è attualmente in uso in studi clinici come farmaco antitumorale promettente; CPI-613 è un farmaco innovativo che inibisce due enzimi chiave del ciclo degli acidi tricarbossilici, PDH e α-KGH. Trattamenti in vitro con questi farmaci hanno dimostrato una significativa diminuzione della vitalità delle cellule CD44+CD117+, fondamentale verifica della loro dipendenza da questo profilo metabolico. CONCLUSIONI: In questo studio abbiamo investigato il profilo metabolico delle cellule staminali tumorali, isolate ex-vivo da campioni di liquidi ascitici di pazienti con carcinoma ovarico, dimostrando che le CSC ovariche, a differenza delle cellule differenziate neoplastiche, sfuggono all’effetto Warburg, utilizzando preferibilmente una respirazione ossidativa. Questa osservazione può indicare nuove strade e nuove strategie per approcci di terapie mirate nei confronti delle CSC, alla luce delle peculiari caratteristiche del loro metabolismo.

Background There are complex metabolic processes occurring in normal, benign and malignant breast cells. To date, there have been relatively few NMR and metabonomic studies investigating these processes in either cultured breast cells or in human biofluids. Aims To develop a combination method of harvesting and lysing cultured breast cells without using any chemicals, which may affect their metabolism, and to determine and compare the metabolic profile of MCF-7 and MCF_7MMU-2 human breast cancer cells by 1 H-NMR spectroscopy and metabonomics. A second aim was to collect and compare by 1 H-NMR-based metabonomics, plasma and urine from women with breast cancer and from healthy controls, in order to determine whether there are any metabolites in these biofluids which differ between the two groups. Methods A series of experiments was performed to optimise a combination method of harvesting and lysing cultured malignant and normal breast cells for 1H-NMR analysis, without using any chemicals. This method was subsequently used to determine the metabolic profile of four different breast cell lines (MCF-7, MCF_7MMU- 2, MDA-MB-231, and HB2), and compare the metabolic profile of MCF-7 and MCF- 7MMU-2 cells. Following ethical approval by the local' Research Ethics Committee (REC), plasma and urine samples were obtained from 41 post-menopausal women with newlydiagnosed breast cancer, and 30 healthy post-menopausal controls. The plasma and urine samples were analysed by 1 H-NMR spectroscopy, and their spectral profiles subjected to Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) for multivariate statistics.

Aquest és el primer informe que demostra que l'autofàgia està mecànicament vinculat al manteniment de les cèl•lules tumorals que expressen alts nivells de CD44 i baixos nivells de CD24, que són típics de les cèl•lules mare del càncer de mama. Els nostres resultats actuals proporcionen una nova visió de com la divisió mitocondrial s'integra a la xarxa de la transcripció impulsada per factors de reprogramació, especifica de la pluripotència única de les cèl•lules mare. L'autofàgia pot controlar la refractarietat de novo de carcinomes de mama amb el gen HER2 amplificat per l'anticòs monoclonal trastuzumab (Herceptin). Per tant, el tractament de combinació amb trastuzumab i cloroquina, com a fàrmac anti-malàric i inhibidor de l’autofàgia, suprimeix radicalment el creixement del tumor en un xenoempelt de tumor completament refractari a trastuzumab en un model murí. L’addició de cloroquina amb els règims amb trastuzumab pot, per tant, millorar els resultats en les dones amb càncer de mama HER2. Aquesta és una àrea molt emocionant i molt prometedora de la investigació del càncer, com la modulació farmacològica de l'autofàgia sembla augmentar l'eficàcia dels règims contra el càncer disponibles en l'actualitat i s'obre el camí per al desenvolupament de noves estratègies terapèutiques combinatòries que s'espera que contribueixin a l'eradicació del càncer.
This is the first report demonstrating that autophagy is mechanistically linked to the maintenance of tumor cells expressing high levels of CD44 and low levels of CD24, which are typical of breast cancer stem cells. Our current findings provide new insight into how mitochondrial division is integrated into the reprogramming of the factors-driven transcriptional network that specifies the unique pluripotency of stem cells. Autophagy may control the de novo refractoriness of HER2 gene-amplified breast carcinomas to the monoclonal antibody trastuzumab (Herceptin). Accordingly, treatment with trastuzumab and chloroquine, as antimalarial drug and inhibitor of autophagy, radically suppresses tumor growth in a tumor xenograft completely refractory to trastuzumab in a mouse model. Adding chloroquine to trastuzumab-based regimens may therefore improve outcomes among women with autophagy-addicted HER2-positive breast cancer. This is a very exciting and highly promising area of cancer research, as pharmacologic modulation of autophagy appears to augment the efficacy of currently available anticancer regimens and opens the way to the development of new combinatorial therapeutic strategies that will hopefully contribute to cancer eradication.

Many kind of cancer cells exploit glycolysis rather than oxidative phosphorylation for energy production even in the presence of oxygen. This kind of metabolism, although less efficient in terms of ATP production, generates high levels of glycolytic intermediates necessary to support the high biosynthetic flux of rapidly proliferating cells. . This mechanism is further enhanced in cancer cells by the expression of a particular form of pyruvate kinase -M2 (PKM2) which promote a low efficiency glycolysis (in terms or ATP production) and consequently an increase in the formation of biosynthetic metabolites. In this work we investigate the role of Bisphosphogliceratemutase (BPGM) an enzyme involved in the metabolic reprogramming of highly proliferating cancer cells. BPGM acts both as a mutase, converting the glycolytic intermediate 1,3-bisphosphoglycerate to 2,3-bisphosphoglycerate and as a phosphatase, converting the 1,3-bisphosphoglycerate to 3-phosphoglycerate. BPGM is an erythrocyte-specific enzyme but our real time PCR and western blotting experiments show its expression in many cancer cell lines and in proliferating primary human fibroblasts. BPGM silencing lead to a strong decrease of cell proliferation rate BPGM activity in cancer cell lead to the skipping of the first ATP production in the glycolytic pathway of glycolysis, causing an increase of glycolytic flux necessary to sustain the high rate of intermediates production needed for support cancer cells growth.

Doctor of Philosophy(PhD)
Iron (Fe) is essential for cell growth and replication as many Fe-containing proteins catalyse key reactions involved in energy metabolism (cytochromes, mitochondrial aconitase and Fe-S proteins of the electron transport chain), respiration (hemoglobin and myoglobin) and DNA synthesis (ribonucleotide reductase). If not appropriately shielded, Fe could participate in one-electron transfer reactions that lead to the production of extremely toxic free radicals. The Fe storage protein, ferritin, is essential to protect cells against Fe-mediated oxidative stress by accommodating excess Fe into its protein shell (Xu et al., 2005). However, despite intensive research over the last few decades, many questions relating to intracellular Fe metabolism, e.g. Fe release from ferritin remain unanswered. Therefore, it is important to elucidate the molecular mechanisms of Fe trafficking in cells. At the beginning of my candidature, little was understood regarding the effect of anti-cancer agents, anthracyclines on the Fe-regulated genes, including transferrin receptor-1 (TfR1), N-myc downstream-regulated gene-1 (Ndrg1) and ferritin. Furthermore, the mechanisms of ferritin-Fe release and anthracycline-mediated ferritin-Fe accumulation are unclear. The work presented in Chapters 3 and 4 has addressed these issues. Apart from the studies examining the molecular interactions of anthracyclines with Fe, a mouse model with perturbed Fe metabolism was used and the marked alterations of protein expression in the heart of this knockout mouse model was discussed in Chapter 5. Chapter 3 Anthracyclines are effective anti-cancer agents. However, their use is limited by cardiotoxicity, an effect linked to their ability to chelate iron (Fe) and perturb Fe metabolism (Xu et al., 2005). These effects on Fe-trafficking remain poorly understood, but are important to decipher as treatment for anthracycline cardiotoxicity utilises the chelator, dexrazoxane. Incubation of cells with doxorubicin (DOX) up-regulated mRNA levels of the Fe-regulated genes, transferrin receptor-1 (TfR1) and N-myc downstream-regulated gene-1 (Ndrg1). This effect was mediated by Fe-depletion, as it was reversed by adding Fe and was prevented by saturating the anthracycline metal-binding site with Fe. However, DOX did not act like a typical chelator, as it did not induce cellular Fe mobilisation. In the presence of DOX and 59Fe-transferrin, Fe-trafficking studies demonstrated ferritin-59Fe accumulation and decreased cytosolic-59Fe incorporation. This could induce cytosolic Fe-deficiency and increase TfR1 and Ndrg1 mRNA. Up-regulation of TfR1 and Ndrg1 by DOX was independent of anthracycline-mediated radical generation and occurred via HIF-1α-independent mechanisms. Despite increased TfR1 and Ndrg1 mRNA after DOX treatment, this agent decreased TfR1 and Ndrg1 protein expression. Hence, the effects of DOX on Fe metabolism were complex due to its multiple effector mechanisms. Chapter 4 The Fe storage protein, ferritin, can accommodate up to 4500 atoms of Fe in its protein shell (Harrison and Arosio, 1996). However, the underlying mechanism of ferritin-Fe release remains unknown. Previous studies demonstrated that anti-cancer agents, anthracyclines, led to ferritin-59Fe accumulation (Kwok and Richardson, 2003). The increase in ferritin-59Fe was shown to be due to a decrease in the release of Fe from this protein. It could be speculated that DOX may impair the Fe release pathway by preventing the synthesis of essential ferritin partner proteins that induce Fe release. In this study, a native protein purification technique has been utilised to isolate ferritin-associated partners by combining ultra-centrifugation, anion-exchange chromatography, size exclusion chromatography and native gel electrophoresis. In addition to cells in culture (namely, SK-Mel-28 melanoma cells), liver taken from the mouse was used as a physiological in vivo model, as this organ is a major source of ferritin. Four potential partner proteins were identified along with ferritin, e.g. aldehyde dehydrogenase 1 family, member L1 (ALDH1L1). Future studies are required to clarify the relationship of these proteins with cellular Fe metabolism and ferritin-Fe release. Chapter 5 A frequent cause of death in Friedreich’s ataxia patients is cardiomyopathy, but the molecular alterations underlying this condition are unknown. We performed two dimensional electrophoresis to characterise the changes in protein expression of hearts using the muscle creatine kinase frataxin conditional knockout (KO) mouse. Pronounced changes in the protein expression profile were observed in 9-week-old KO mice with severe cardiomyopathy. In contrast, only a few proteins showed altered expression in asymptomatic 4-week-old KO mice. In hearts from frataxin KO mice, components of the iron-dependent complex-I and -II of the mitochondrial electron transport chain and enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) displayed decreased expression. Interestingly, the KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilisation and pyruvate decarboxylation. This constitutes evidence of metabolic compensation due to decreased expression of electron transport proteins. There was also pronounced up-regulation of proteins involved in stress protection, such as a variety of chaperones, as well as altered expression of proteins involved in cellular structure, motility and general metabolism. This is the first report of the molecular changes at the protein level which could be involved in the cardiomyopathy of the frataxin KO mouse.

Ovarian cancer is the fourth most common cancer among women worldwide, leading to high mortality rates. Platinum anti-cancer drugs have been widely used in the treatment of ovarian cancers, but the development of resistance to these drugs is a rapidly growing impediment for their clinical use. Studies of the cell morphology using IncuCyte Zoom and HoloMonitor M4 have shown that A2780.CisR cells contained heterogeneous cell populations that partially underwent epithelial-to-mesenchymal transition (EMT). Mesenchymal A2780.CisR cells were likely to be more resistant to a cytotoxic natural killer (NKL) cell line, compared with the epithelial A2780 cells. Mesenchymal phenotype was associated with lower total Cu content and higher total Fe content in A2780.CisR compared with A2780 cells under physiologically relevant conditions (2.0 M Cu(II) or 10 M Fe(III) for 24 h). Conversely, live cell confocal microscopy studies with a novel ratiometric Cu(I)-sensitive fluorescent dye, InCCu1, revealed higher cellular content of labile Cu in A2780.CisR cells compared with A2780 cells. Moreover, the InCCu1 dye showed promise for differential staining of multiple cellular organelles, including mitochondria, endosomes/lysosomes, fat droplets and budding extracellular vesicles. Fat droplets were more abundant in A2780.CisR compared with A2780 cells and concentrated at the advancing edges of cellular protrusions (shown by InCCu1 and Nile Red staining). Comparison of the biochemical content of cell membranes using ATR-FTIR (attenuated total reflection Fourier transform infrared) spectroscopy technique showed higher relative content of fatty acids and cholesterol in the membranes and surrounding environments of A2780.CisR compared with A2780 cells.

Iron (Fe) is essential for cell growth and replication as many Fe-containing proteins catalyse key reactions involved in energy metabolism (cytochromes, mitochondrial aconitase and Fe-S proteins of the electron transport chain), respiration (hemoglobin and myoglobin) and DNA synthesis (ribonucleotide reductase). If not appropriately shielded, Fe could participate in one-electron transfer reactions that lead to the production of extremely toxic free radicals. The Fe storage protein, ferritin, is essential to protect cells against Fe-mediated oxidative stress by accommodating excess Fe into its protein shell (Xu et al., 2005). However, despite intensive research over the last few decades, many questions relating to intracellular Fe metabolism, e.g. Fe release from ferritin remain unanswered. Therefore, it is important to elucidate the molecular mechanisms of Fe trafficking in cells. At the beginning of my candidature, little was understood regarding the effect of anti-cancer agents, anthracyclines on the Fe-regulated genes, including transferrin receptor-1 (TfR1), N-myc downstream-regulated gene-1 (Ndrg1) and ferritin. Furthermore, the mechanisms of ferritin-Fe release and anthracycline-mediated ferritin-Fe accumulation are unclear. The work presented in Chapters 3 and 4 has addressed these issues. Apart from the studies examining the molecular interactions of anthracyclines with Fe, a mouse model with perturbed Fe metabolism was used and the marked alterations of protein expression in the heart of this knockout mouse model was discussed in Chapter 5. Chapter 3 Anthracyclines are effective anti-cancer agents. However, their use is limited by cardiotoxicity, an effect linked to their ability to chelate iron (Fe) and perturb Fe metabolism (Xu et al., 2005). These effects on Fe-trafficking remain poorly understood, but are important to decipher as treatment for anthracycline cardiotoxicity utilises the chelator, dexrazoxane. Incubation of cells with doxorubicin (DOX) up-regulated mRNA levels of the Fe-regulated genes, transferrin receptor-1 (TfR1) and N-myc downstream-regulated gene-1 (Ndrg1). This effect was mediated by Fe-depletion, as it was reversed by adding Fe and was prevented by saturating the anthracycline metal-binding site with Fe. However, DOX did not act like a typical chelator, as it did not induce cellular Fe mobilisation. In the presence of DOX and 59Fe-transferrin, Fe-trafficking studies demonstrated ferritin-59Fe accumulation and decreased cytosolic-59Fe incorporation. This could induce cytosolic Fe-deficiency and increase TfR1 and Ndrg1 mRNA. Up-regulation of TfR1 and Ndrg1 by DOX was independent of anthracycline-mediated radical generation and occurred via HIF-1α-independent mechanisms. Despite increased TfR1 and Ndrg1 mRNA after DOX treatment, this agent decreased TfR1 and Ndrg1 protein expression. Hence, the effects of DOX on Fe metabolism were complex due to its multiple effector mechanisms. Chapter 4 The Fe storage protein, ferritin, can accommodate up to 4500 atoms of Fe in its protein shell (Harrison and Arosio, 1996). However, the underlying mechanism of ferritin-Fe release remains unknown. Previous studies demonstrated that anti-cancer agents, anthracyclines, led to ferritin-59Fe accumulation (Kwok and Richardson, 2003). The increase in ferritin-59Fe was shown to be due to a decrease in the release of Fe from this protein. It could be speculated that DOX may impair the Fe release pathway by preventing the synthesis of essential ferritin partner proteins that induce Fe release. In this study, a native protein purification technique has been utilised to isolate ferritin-associated partners by combining ultra-centrifugation, anion-exchange chromatography, size exclusion chromatography and native gel electrophoresis. In addition to cells in culture (namely, SK-Mel-28 melanoma cells), liver taken from the mouse was used as a physiological in vivo model, as this organ is a major source of ferritin. Four potential partner proteins were identified along with ferritin, e.g. aldehyde dehydrogenase 1 family, member L1 (ALDH1L1). Future studies are required to clarify the relationship of these proteins with cellular Fe metabolism and ferritin-Fe release. Chapter 5 A frequent cause of death in Friedreich’s ataxia patients is cardiomyopathy, but the molecular alterations underlying this condition are unknown. We performed two dimensional electrophoresis to characterise the changes in protein expression of hearts using the muscle creatine kinase frataxin conditional knockout (KO) mouse. Pronounced changes in the protein expression profile were observed in 9-week-old KO mice with severe cardiomyopathy. In contrast, only a few proteins showed altered expression in asymptomatic 4-week-old KO mice. In hearts from frataxin KO mice, components of the iron-dependent complex-I and -II of the mitochondrial electron transport chain and enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) displayed decreased expression. Interestingly, the KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilisation and pyruvate decarboxylation. This constitutes evidence of metabolic compensation due to decreased expression of electron transport proteins. There was also pronounced up-regulation of proteins involved in stress protection, such as a variety of chaperones, as well as altered expression of proteins involved in cellular structure, motility and general metabolism. This is the first report of the molecular changes at the protein level which could be involved in the cardiomyopathy of the frataxin KO mouse.

Epithelial Ovarian Cancer (EOC) is a very malignant neoplasm, accounting for 5% of cancer mortality in women. Although progress has been made in EOC treatments by improved debulking surgery and platinum-taxane regimens, the 5-year survival rate of advanced-stage EOC remains below 30%. This poor prognosis relies on the one hand on the late diagnosis and on the other on the chemo-resistance occurring after only a few months from the completion of treatment. The reasons for recurrence and cancer drug resistance remain uncertain. Recent evidence suggests that EOC, akin most tumors, contains a tiny population of cells, named cancer stem cells (CSC), probably responsible for chemotherapy resistance and tumor recurrence. Ovarian CSC are characterized by the co-expression of two surface markers: CD44 (hyaluronic acid receptor) and CD117 (stem cell factor receptor or c-kit). Recently, our research group demonstrated that ovarian CD44/CD117 co-expressing cells, which represent 1-2% of cancer cells from ascitic effusions of EOC-bearing patients, are endowed with canonical stemness properties and are able to resist in vitro and in vivo glucose starvation. We reported that this glucose deprivation resistance is mostly due to the ability of ovarian CSC to privilege oxidative phosphorylation, rather than the aerobic glycolysis (Warburg effect) exploited by the non-stem tumor bulk. However, independently of CSC fraction, our experiments also highlighted that not all the analyzed EOC samples presented a similar glucose addiction. Thus, investigating this issue and its related metabolic aspects is the first aim of the current project. Concerning this aim, we showed that tumor cells from EOC patients can be categorized, according to their in vitro viability under glucose starvation, into glucose deprivation-sensitive (glucose-addicted, GA) and glucose deprivation-resistant (glucose non-addicted, GNA). Although deregulated glucose metabolism is usually observed in cancer, whether this metabolic trait influences response to or is modulated by cytotoxic drugs is unknown; therefore, we addressed the possible correlation of these glucose addiction profiles with the patient response to platinum (PLT) regimens. In this regard, when EOC cells were cultured in the absence of glucose, all samples from PLT-sensitive patients felt into the GA group; compared to GNA samples, they disclosed higher expression of glucose metabolic enzymes, higher proliferation rates and in vitro sensitivity to PLT, as well as reduced multi-drug resistance pump expression. On the other hand, the samples derived from PLT-resistant patients felt into the GNA category. The close association between PLT sensitivity and glucose metabolic profile was confirmed in a xenograft model, where a stringent parallelism between PLT sensitivity/resistance and glucose metabolism was identified. Finally, in a cohort of naïve EOC patients categorized as GA or GNA at diagnosis, Kaplan Meier curves showed that the GA phenotype was associated with significantly better progression-free survival, compared to GNA patients. Overall, these results suggest that in vitro glucose addiction of EOC cells could be regarded as a reliable marker to predict the patient response to platinum regimens. Investigating the molecular traits leading to the distinctive glucose metabolism of EOC samples is the second aim of this project. In this regard, microRNA (miRNA), that are small non-coding RNA molecules, represent a promising field, in view of their ability to modulate many genes and pathways. Moreover, their involvement in cancer development and progression has already been reported in ovarian cancer, and recently miRNA also emerged to regulate cell metabolism in several normal and cancer tissues. Thus, we performed a miRNA profile on EOC patient-derived samples, comparing both GA versus GNA samples and CSC versus non-CSC, in order to establish whether the miRNA signature behind the metabolic differences of EOC samples is associated with the total tumor bulk or rather with a specific cell fraction. The data did not reveal any miRNA differentially expressed in GA compared to GNA cells, but several miRNA resulted significantly deregulated in CSC versus non-CSC. We focused our attention on mir-602, which was found up-regulated in CSC; indeed, despite little information about this miRNA, its target Casein Kinase 1 Delta (CSNK1D), which displays a key role in cell proliferation and asymmetric division, seemed very interesting in view of its involvement in breast cancer progression. We demonstrated that CSNK1D is down-regulated in CSC, according to mir-602 over-expression. Moreover, the in vitro inhibition of mir-602 reduced the expression of the major stemness-associated genes in CSC, suggesting that mir-602 could regulate some cell stemness pathways. Since none of the analyzed stemness genes is directly targeted by mir-602, it is reasonable to advance that mir-602 could indirectly activate the expression of such genes through its inhibitory function on CSNK1D; thus, we propose that cell stemness signaling is inhibited by casein kinase, whose translation is in turn repressed by mir-602. Although a few further experiments are needed to validate our idea, this project highlights a possible miRNA-mediated regulatory mechanism of cell stemness features in EOC.
Il tumore ovarico di tipo epiteliale (EOC – Epithelial Ovarian Cancer) rappresenta la prima causa di morte per neoplasia ginecologica. Sebbene la chirurgia e la chemioterapia a base di carbo/cis-platino abbiano migliorato la prognosi delle pazienti affette da tale carcinoma, il tasso di sopravvivenza a 5 anni dalla diagnosi di EOC in stadio avanzato rimane inferiore al 30%. Tale elevata mortalità è riconducibile alla diagnosi in fase tardiva ed all’insorgenza di resistenza alla chemioterapia di prima linea, che porta a frequenti ricadute entro pochi mesi dalla conclusione dei trattamenti. Le cause della comparsa di fenomeni di resistenza alla terapia, e della conseguente ricorrenza del tumore, è ancora incerta. Recenti studi hanno indicato che la crescita del carcinoma epiteliale dell’ovaio è sostenuta da una minima popolazione di cellule, dette cellule tumorali staminali (CSC - Cancer Stem Cells), probabilmente responsabili della ricomparsa del tumore al termine delle sedute farmacologiche. Un consenso generale supporta l’idea che l’eliminazione di questa popolazione rappresenti uno dei più importanti obiettivi della terapia anti-tumorale. Tuttavia, si ha ancora una scarsa conoscenza dei meccanismi che conferiscono un vantaggio di sopravvivenza alle CSC sulle cellule tumorali non staminali, rendendo così arduo lo sviluppo di terapie mirate anti-CSC. Le CSC del carcinoma epiteliale ovarico sono caratterizzate dall’espressione di due marcatori di superficie cellulare: CD44 (recettore dell’acido ialuronico) e CD117 (c-kit o recettore del fattore cellulare staminale SCF). Recentemente, il nostro gruppo di ricerca ha dimostrato che le cellule ovariche co-esprimenti CD44 e CD117, che rappresentano l’1-2% delle cellule tumorali provenienti dall’ascite delle pazienti affette da EOC, possiedono le canoniche proprietà staminali e sono in grado di sopravvivere in vitro ed in vivo alla deprivazione di glucosio. Abbiamo inoltre osservato che tale resistenza all’assenza di glucosio è principalmente dovuta alla capacità delle CSC, contrariamente alle cellule tumorali non staminali, di privilegiare la fosforilazione ossidativa anziché la glicolisi aerobica (Warburg Effect). Tuttavia, indipendentemente dalla frazione delle cellule tumorali staminali, l’analisi comparativa tra i diversi campioni di EOC ha evidenziato che non tutti presentano la stessa dipendenza dai glucidi; per alcuni, infatti, pochi giorni in vitro senza glucosio sono sufficienti a ridurre significativamente la vitalità cellulare, mentre per altri lo stesso effetto è ottenibile solo dopo molte settimane di coltura nelle stesse condizioni. Dunque, approfondire tale questione e gli aspetti metabolici ad essa correlati è il primo scopo di questo progetto. A tal proposito, sulla base della vitalità cellulare in condizioni di coltura senza glucosio, abbiamo potuto suddividere le cellule derivanti da asciti di pazienti con EOC in due categorie: sensibili alla deprivazione di glucosio (GA – Glucose-Addicted) e resistenti a tale deprivazione (GNA – Glucose Non-Addicted). Sebbene variazioni nella regolazione del metabolismo del glucosio siano state frequentemente osservate nei casi di neoplasia, non è ancora noto se questo diverso tratto metabolico influenzi la risposta dei pazienti alle terapie, o se sia da queste modulato. Pertanto, abbiamo deciso di ricercare una eventuale correlazione tra i diversi profili di dipendenza dal glucosio da noi riscontrati e la risposta dei pazienti al trattamento a base di carbo/cis-platino. Infatti, da un punto di vista clinico, i pazienti vengono categorizzati come platino-resistenti o platino-sensibili, a seconda che la neoplasia recidivi entro od oltre i 6 mesi, rispettivamente, dalla fine della chemioterapia di prima linea. I nostri esperimenti hanno rivelato che, quando le cellule di EOC vengono coltivate in assenza di glucosio, tutti i campioni provenienti da pazienti platino-sensibili ricadono all’interno del gruppo GA; confrontati con i campioni GNA, i GA mostrano una maggiore produzione degli enzimi del metabolismo glucidico, un maggior tasso di proliferazione, e una minore espressione delle pompe cellulari per l’espulsione dei farmaci. Parallelamente, i campioni derivanti dai pazienti platino-resistenti rientrano nella categoria GNA. La stretta associazione tra la sensibilità ai chemioterapici e il profilo cellulare di utilizzo del glucosio è stata confermata in un modello murino di xenotrapianti, nel quale è stato identificato uno stringente parallelismo tra risposta al platino e metabolismo glucidico. Infine, in una coorte di pazienti non chemio-trattate affette da EOC, le quali erano state categorizzate come GA o GNA alla diagnosi, le curve di Kaplan Meier hanno messo in luce che il fenotipo GA, rispetto a quello GNA, è associato con un maggior periodo di sopravvivenza senza recidive, in modo statisticamente significativo. Nel complesso, questi dati suggeriscono che il grado di dipendenza dai glucidi delle cellule di EOC, osservabile in vitro, può rappresentare un valido marcatore per predire la risposta dei pazienti alla chemioterapia a base di platino. Analizzare il tratto molecolare che determina il peculiare metabolismo glucidico dei campioni di EOC costituisce il secondo obiettivo del nostro progetto di ricerca. A tal riguardo, i microRNA (miRNA), ossia piccole molecole di RNA non codificante, rappresentano un promettente settore di studio, in qualità della proprietà di queste strutture molecolari di regolare molti geni e vie di segnale. Inoltre, il loro coinvolgimento nello sviluppo e nella progressione tumorale è già stato dimostrato per il carcinoma ovarico, e recentemente i miRNA sono risultati essere importanti modulatori del metabolismo cellulare in molti tessuti normali e neoplastici. Pertanto, il nostro gruppo di ricerca ha prodotto un profilo di espressione di miRNA su cellule derivanti da pazienti affette da EOC, confrontando sia campioni GA contro GNA, sia CSC contro non-CSC; il nostro fine è stabilire se il pattern di miRNA alla base delle differenze metaboliche tra i campioni di EOC sia associato alla totale massa tumorale o piuttosto ad una specifica frazione cellulare neoplastica. Questi dati non hanno rivelato alcun miRNA differentemente espresso tra cellule GA e GNA; tuttavia, molti miRNA sono risultati deregolati nelle CSC rispetto alle non-CSC. Noi ci siamo focalizzati sul mir-602, up-regolato delle CSC; infatti, nonostante la scarsa conoscenza su questo miRNA, il suo target chinasi Caseina 1 Delta (CSNK1D), che detiene un ruolo chiave nella proliferazione cellulare e nella divisione asimmetrica, ci è parso molto interessante in virtù del suo già dimostrato coinvolgimento nella progressione del carcinoma mammario. In questo contesto, i nostri esperimenti hanno dimostrato che CSNK1D è down-espressa nelle CSC, in accordo alla up-modulazione del mir-602. Inoltre, l’inibizione in vitro del mir-602 riduce nelle CSC l’espressione della maggior parte dei geni associati alle proprietà staminali, suggerendo che il mir-602 potrebbe controllare alcune delle vie di segnale correlate alla staminalità. Dato che nessuno dei geni di staminalità analizzati si lega direttamente al mir-602, appare ragionevole supporre che suddetto miRNA possa indirettamente attivare l’espressione di tali geni tramite la sua funzione inibitoria su CSNK1D. Dunque, secondo la nostra ipotesi, le caratteristiche staminali sarebbero inibite dalla chinasi Caseina, la quale sarebbe a sua volta repressa dal mir-602. Nonostante molti altri esperimenti siano necessari per confermare questa nostra teoria, il progetto qui presentato mette in rilievo la possibile esistenza di un meccanismo di regolazione, mediato dal mir-602, responsabile delle proprietà di staminalità delle cellule del carcinoma epiteliale ovarico.

Phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme that catalyses decarboxylation of oxaloacetate to phosphoenolpyruvate and it is part of gluconeogenic/glyceroneogenic pathway. There are two known isoforms of PEPCK, the mitochondrial and the cytosolic isozyme that are catalysing chemically identical reactions, but they differ in regulation and expression pattern. Selective presence of mitochondrial isoform of this enzyme (PEPCK-M, PCK2) in all types of cancer examined and in cycling neuroprogenitors, suggests a functional relationship with the metabolic adaptations of these cells. This thesis has had as its main objectives the characterization of the role of PEPCK-M in tumour cells and in neuronal progenitor cells. Metabolism of cell in CNS is not completely elucidated yet. Here we demonstrate that Tbr2 positive neuronal progenitors are metabolically dependent on lactate, which is favouring maintenance of their undifferentiated state. Lactate as metabolite can feed anabolic pathways and sustain ATP production by its oxidation in the TCA cycle. However, essential pathways like PPP, glycerol synthesis or one carbon metabolism pathways require carbons to feed the glycolytic intermediate pool. PEPCK-M in this setting, with lactate as sole carbon source is the only known pathway to fulfil the above-mentioned anabolic requirements. By using inhibitor of PEPCK-M we were able to prove that Tbr2 positive neuronal progenitors are metabolically dependent on PEPCK-M activity and their number significantly decrease after inhibiting PEPCK-M in vitro and in vivo. PEPCK-M activity in tumour cells is necessary for survival and growth in 2D and in cultures on semi-solid agar (anchorage-independent growth), which suggests that this enzyme has a fundamental role in the survival program to cell stress. A Kaplan-Meier analysis from datasets available in the GEO database (> 5000 patients) shows that elevated PCK2 expression is significantly associated with a worse prognosis in patients with breast cancer. Despite its potential relevance for metabolic adaptations in cancer, the mechanisms responsible for its pro- survival activity are not known. Therefore, we have proposed to study these mechanisms through metabolomic analysis where we wanted to examine whether PEPCK-M feeds an alternative pathway to glucose using carbons from glutamine in an experimental model with reduced and overexpressed levels of PEPCK-M activity. We demonstrated the functionality of PEPCK-M driven cataplerosis in MCF7 cells grown under glucose deprivation by showing synthesis of serine and glycine from glutamine by observing contribution of 13C-labeled carbons from [U-13C] glutamine into these metabolites. In the absence of nutritional stress (high abundance of glucose and amino acids), the silencing of PEPCK-M induces oxidative stress and the accumulation of succinate, with the consequent induction of p21 and deficiencies in cell growth. Glutamine cataplerosis is not affected by alterations in PEPCK-M activity. However, a higher enrichment of all carbons with 13C in intermediates of the Krebs cycle (TCA cycle) suggests a reduction in flux through this pathway. Together, these data increase our understanding of metabolic adaptations in tumours and the role of PEPCK in providing alternative carbon fluxes to deal with nutritional stress. Finally, these studies allow us to propose PEPCK-M as a new target for the treatment of tumorigenic processes that will need to be validated in the future.
El fosfoenolpiruvato carboxiquinasa mitocondrial (PEPCK-M; PCK2) se regula transcripcionalmente por limitación de aminoácidos y por ER-estrés, de una manera dependiente de ATF4, aumentando así la supervivencia de la célula. La presencia selectiva de esta isoenzima en todos los tipos de cáncer examinado y en células neuroprogenitoras, sugiere una relación funcional con las adaptaciones metabólicas de estas células. Esta tesis ha tenido como objetivos fundamentales la caracterización del rol de la PEPCK-M en célula tumoral y en célula neuroprogenitora En cultivos neuronales, los neuroprogenitores Tbr2 positivos requieren lactato como sustrato metabólico para el mantenimiento de su fenotipo y su metabolismo. La PEPCK-M se expresa a niveles altos en este tipo celular y su actividad es necesaria para mantener la viabilidad de estos progenitores y cumplir con los requerimientos anabólicos a partir de carbonos provenientes del lactato. La actividad PEPCK-M en célula tumoral es necesaria para la supervivencia y crecimiento. A pesar de su potencial relevancia para las adaptaciones metabólicas en cáncer, no se conocen los mecanismos responsables de su actividad pro-supervivencia. Por ello, nos hemos propuesto estudiar estos mecanismos mediante análisis de metabolómica con los que hemos querido examinar si la PEPCK-M alimenta una vía alternativa a la glucosa utilizando carbonos provenientes de glutamina en un modelo experimental con niveles de actividad PEPCK-M reducidos y sobreexpresados. La contribución de carbonos marcados con 13C a partir de [U- 13C] glutamina en los productos de ramificación de glicolisis como serina y glicina, esta correlacionando directamente con los niveles de actividad PEPCK-M en condiciones de estrés nutricional (baja glucosa). La cataplerosis de glutamina no se ve afectada por alteraciones en la actividad de PEPCK-M. Sin embargo, un mayor enriquecimiento de 13C en intermediarios del ciclo de Krebs sugieren una reducción del flujo a través de esta vía. En conjunto, estos datos aumentan nuestra comprensión de las adaptaciones metabólicas en los tumores y el papel de la PEPCK en la provisión de flujos de carbono alternativas para lidiar con el estrés nutricional. Finalmente, estos estudios nos permiten proponer a la PEPCK-M como una nueva diana para el tratamiento de procesos tumorogénicos que necesitará ser validada en el futuro.

Vitamin D is a precursor to a steroid hormone, 1,25 dihydroxyvitamin D (1,25(OH)2D). After its discovery and the characterization of its receptor, the vitamin D receptor (VDR), it was initially thought only to be involved in calcium homeostasis, but further research revealed an important role for vitamin D in the regulation of cell growth and differentiation of such cells as osteoblasts and bone marrow adipocytes. 1,25(OH)2D has also been shown to be a strong inhibitor and pro-differentiator of keratinocytes. The anti-proliferative and pro-differentiative properties of this hormone have led to studies where 1,25(OH)2D anticancer properties were assessed and initial findings that showed a requirement of other factors beyond VDR to induce 1,25(OH)2D signaling led to the identification of the retinoid X receptor, a common heterodimeric partner for several hormone receptors. The focus of thesis was to further elucidate the structure-function relationship of both the vitamin D receptor and the retinoid X receptor. Additionally, contributions to work directed towards further identifying the effects of vitamin D on osteoblast differentiation and survival. Interactions of 1,25(OH) 2D3 with its cognate receptor, identifying a key amino acid (Tryptophan 286) required for ligand contact and transcriptional activation, are described in Chapter 2. Mechanisms of vitamin D action on mesenchymal stem cell differentiation, promotion of osteoblast induction and maturation, and inhibition of adipocyte differentiation, are eluicidated in Chapter 3. Chapter 4 illustrates the effects of RAS/RAF/Mitogen-activated protein kinase mediated RXRalpha phosphorylation on the three-dimensional structure of the RXR/nuclear receptor partner heterodimers. Furthermore, this chapter reveals the inhibitory effect of the phosphorylation of a critical amino acid (serine 260) on the interaction of the AF-2 domain of the RXR with several coactivators, resulting in a decrease in the signaling potential of multiple steroid hormone receptors. The findings of this thesis further the knowledge of several areas of vitamin D biology, including both the canonical areas of bone formation, and the non-canonical area of vitamin D and cancer.

Die Entstehung von Tumoren und damit einhergehenden Veränderungen wurden insbesondere im letzten Jahrzehnt kontrovers diskutiert. Bisher standen nur wenige Datensätze mit ausreichender Datendichte zur Verfügung um eine umfassende Untersuchung der Regulation des Stoffwechsels durchzuführen. Die in dieser Arbeit zusammengefassten Projekte adressieren verschiedene Aspekte der Stoffwechselregulation und beschreiben die Verknüpfung von Zellkulturexperimenten mit innovativen Hochdurchsatz-Technologien, komplexer Datenanalyse und Computer-basierter Modellierung zur Bestimmung der Stoffwechselflüsse in eukaryotischen Zellen. Die Kombination von GC-MS und LC-MS basierten Technologien ermöglicht die quantitative Analyse des zentralen Kohlenstoffwechsels. Markierungsexperimente mit stabilen Isotopen (pSIRM) erlauben die dynamische Analyse der Stoffwechselaktivität. In verschiedenen Projekten wurden das Proteom und Metabolom von Krebszellen, humanen Stammzellen (hESCs), induzierten pluripotenten Stammzellen (iPS) und deren dazugehörigen differenzierten Vorläufer- oder Nachfolgerzellen bestimmt. Die multivariate, statistische Analyse der Daten ermöglichte die Differenzierung verschiedener Zelltypen basierend auf der Kombination aller quantitativ bestimmten Daten. Quantitative Bestimmungen der Poolgrössen, Isotopeninkorporationen, sowie der extrazellulären Raten in neuronalen, pluripotenten Vorläuferzellen (Luhmes d0) und Neuronen (Luhmes d6) ermöglichte die Bestimmung der Stoffwechselflusskarte beider Zelltypen unter Verwendung der instationären metabolischenen Flussanalyse (INST-MFA). Die Etablierung einer Qualitätskontrolle für GC-MS basierte Daten (MTXQC), sowie die Zuordnung der GC-MS Fragmente zur Molekülstruktur, ermöglichten den Ausbau des Netzwerkes des zentralen Kohlenstoffwechsels und die Implementierung der Daten für die metabolische Flussanalyse.
Metabolic reprogramming of the central carbon metabolism (CCM) is highly debated during the last decade. It describes the rearrangement of nutrient consumption for providing energy and building blocks for cellular proliferation and maintenance. So far, only sparse data are available for an in-depth analysis of metabolic reprogramming events. The herein summarised projects address metabolic programming from different perspectives and show the implementation of cell culture experiments, cutting-edge high-throughput technologies, bioinformatics, and computational modelling into one workflow providing the determination of metabolic flux maps of mammalian cells. The combination of GC-MS and LC-MS-based methodologies enable the quantitative analysis of proteins and metabolites of the CCM. Pulsed stable isotope-resolved metabolomics (pSIRM) experiments allow monitoring the fate of nutrients within the network of the CCM. The time-dependent and position-specific incorporation of carbon-13 leads to an indirect measurement of the metabolic flux, the only one functional readout of a cell. High-throughput technologies were applied in four projects to gain insights in metabolic reprogramming in cancer cell lines, human embryonic stem cells (hESCs), induced pluripotent stem (iPS) cells and their derived fibroblasts. A global principal component analysis demonstrated the discrimination of phenotypes by different classes of quantitative data. The comparison of metabolic and protein levels confirms the presence of the Warburg effect in both cell types. Though, the executing enzymes vary regarding their isoenzyme identity and expression levels. Methodological improvements provided the implementation of GC-MS derived data for INST-MFA. The mapping of GC-MS derived fragments to the molecule structure enables an extension of the CCM network. Robustness of the input data has been improved by the development of a R-scripting based quality control tool (MTXQC).

Ovarian cancer is known as the "silent killer," due to its late diagnosis and frequent recurrence after initial treatment.  Finding a new way to diagnose and treat ovarian cancer in conjunction with current therapies is paramount.  By capitalizing on metabolic changes that occur during cancer progression, interventions can be developed.  The Nobel laureate Otto Warburg is credited with discovering an altered metabolic state within cancer cells known as the Warburg effect.  In the Warburg effect, cancer cells participate in an increased rate of aerobic glycolysis with an excess secretion of lactate, allowing for carbon flux into biosynthetic pathways.  Exactly which metabolic pathways are altered in ovarian cancer and at which stage in the progression of ovarian cancer they are occurring was unknown.  Therefore using the recently established mouse ovarian surface epithelial (MOSE) progression model, we were able to measure metabolic changes in varying states of disease and levels of aggressiveness.  As cells progressed from a benign early stage (MOSE-E), through a transitional intermediate stage (MOSE-I), to an aggressive late stage (MOSE-L), the MOSE cells became more glycolytic and lipogenic, establishing the MOSE model as a valuable model for studying ovarian cancer metabolism.  Treating the MOSE cells with the naturally occurring chemotherapeutic agent sphingosine decreased p-AKT  protein levels in the cell, decreased the glycolytic rate and decreased de novo cholesterol synthesis.  Cancer stem cells are known to be resistant to chemotherapy treatments and targeting their metabolism may be promising for combinatorial treatments.  Therefore, the metabolism of highly aggressive tumor-initiating cells (TIC), harvested from ascites of C57Bl/6 mice injected with MOSE-L cells were characterized.  Although the basal metabolism of the TICs was similar to the MOSE-L cells, TICs were more resistant to cell death as a consequence of external stresses and substrate depletion.  The TICs could also up-regulate oxygen consumption rate (OCR) when uncoupled and increase glycolysis when ATP Synthase was inhibited, highlighting their resiliency.  Taken together, we have identified targets for treatment strategies that could suppress the growth of primary tumors and may be effective against TICs, thereby suppressing tumor recurrence and possibly prolonging the life of women with ovarian cancer.
Ph. D.

In this thesis, tumor metabolic reprogramming has been exploited in order to propose new targets in cancer treatment. On one hand, we explored the pentose phosphate pathway (PPP) enzymes as putative therapeutic targets against breast and colon cancer. We observed that inhibition of ox-PPP enzymes G6PD in colon cancer cells and 6PGD in breast cancer cells halted cell prolifertion, resulted in cell cycle arrest and apoptosis. We also demosntrated that in colon cancer cells G6PD is strongly regulated by the glutamine availability mediated by NRF2 transcription factor. Moreover, 6PGD inhibition decreased mammosphere formation capacity of breast cancer cells implying that stem cell characteristics of breast cancer cells were altered by 6PGD inhibition. Besides that, 6PGD inhibition also altered the central carbon metabolism of breast cancer cells leading to decreased glucose consumption and increased glutamine consumption. Observing that both pathways are deeply related to glutamine metabolism, we decided to investigate the metabolic network adaptations that breast cancer cells undergo when the glutamine is scarce. Knowing that hypoxic conditions are common features of tumor microenvironments, we also investigated the characterization of a hypoxia mimicking condition which leads to defective mitochondria. In fact, in these two conditions, we produced huge amount of transcriptomics, metabolomics and fluxomics data in order to produce a genome scale metabolic model (GSMM) combining multi-omics data in the frame of a European project which helps us to understand the regulation of metabolic alterations in breast cancer cells. While produced data is to be used in production of GSMM, we also took advantage of the data to study the metabolic adaptations that breast cancer cells undergo in the deprivation of glutamine or when mitochondria are defected. We propose that increased pyruvate cycle with glutamine deprivation and increased reductive carboxylation with not fully functional mitochondria could be targeted in combination therapies to fight against cancer. All in all, besides showing the importance of metabolism in cancer cell proliferation and survival, the results presented in this study also highlights the importance of Systems Biology approaches to understand the molecular mechanisms underlying complex multifactorial diseases in order to develop new potential therapeutic targets.

2013/2014
Lo scopo del presente progetto di Dottorato riguarda la possibilità di riconoscere ed identificare le cellule mesenchimali del cancro sfruttando il metabolismo iper-attivato, nonché de-regolato, delle stesse. Una delle differenze principali tra le cellule epiteliali e quelle mesenchimali, del cancro, è il metabolismo ed in particolare il così detto Effetto Warburg (da Otto Einrich Warburg il suo scopritore e sostenitore, nel 1926). Nello specifico, questo effetto riguarda la caratteristica delle cellule tumorali di preferire, come via metabolica, la glicolisi alla classica fosforilazione ossidativa, anche in presenza di ossigeno. Questo processo, infatti, porta a produzione ed accumulo di acido lattico e ad una riduzione della quantità disponibile di glucosio nel mezzo esterno. Negli ultimi anni si è scoperto quanti e quali vantaggi competitivi, dal punto di vista proliferativo, comporti questa trasformazione, consentendo così anche lo sviluppo di numerose tecniche terapeutiche e diagnostiche basate proprio sulla trasformazione della fisiologica normalità. Questo progetto di dottorato si è proposto di sviluppare una nuova metodica diagnostica in grado di distinguere le cellule mesenchimali del cancro da quelle epiteliali tramite lo studio del loro metabolismo e senza l’ausilio di anticorpi. Essendosi prefisso, infatti, come scopo finale quello di fornire un contributo nel campo della diagnosi preventiva e della prognosi a basso costo; una degli intenzioni principali del presente progetto era quello di ridurre al minimo l’utilizzo di anticorpi nel processo di identificazione dei due sottotipi tumorali. Questo progetto ha così sviluppato un sistema basato su nanoparticelle magnetiche (MNPs), in particolare di cobalto ferrite, con lo scopo di favorire l’ avanzamento nel campo delle attuali tecniche di isolamento magnetico. Nello specifico le MNPs sono state sintetizzate e funzionalizzate con un analogo fluorescente del glucosio (il 2-2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose, 2-NBDG) e caratterizzate con spettroscopia infrarossa e microscopia elettronica. Al fine di poterne monitorare in vitro la captazione da parte delle cellule tumorali, sono state utilizzate due ben note linee cellulari di tumore alla mammella, le MCF7 e le MDA-MB-231, definite rispettivamente: epithelial-like e mesenchymal-like. Ne è stata valutata la captazione e quindi l’internalizzazione da parte di queste due linee di cancro al seno, dimostrandola con 3 differenti metodiche (microscopia confocale, saggio di immuno-cito-chimica e analisi con microscopia elettronica accoppiata a milling ionico micro-guidato). In particolare è stato dimostrato come aumentando la concentrazione di glucosio nel mezzo (da 5.5 a 25 mM), le cellule epiteliali riducano drasticamente l’uptake di MNPs mentre esso si conservi nel sottotipo mesenchimale. Tale differente comportamento è infatti basato sul differente metabolismo dei due sottotipi tumorali, il quale consente, senza l’utilizzo di anticorpi, di individuare il sottotipo mesenchimale, più metabolicamente attivo (e tipicamente più aggressivo), da quello epiteliale. Successivamente è stato ricercato il meccanismo molecolare responsabile di tale internalizzazione tramite l’inibizione del più espresso trasportatore di membrana del glucosio, Glut1, con un inibitore selettivo (STF-31). La specifica captazione da parte delle cellule mesenchymal-like è stata infine studiata come potenziale caratteristica da sfruttare per eventuali trattamenti di ipertermia, o termoterapia. Nello specifico è stato utilizzato un laser infrarosso che focalizzato in maniera accurata e precisa su uno degli aggregati di MNPs (dimensionalmente compatibile con la risoluzione della microscopia ottica), presenti all’interno delle cellule, ne ha permesso l’induzione selettiva di morte (necrosi o apoptosi in base tipicamente al tempo e all’intensità dell’esposizione). La possibilità di indurre una morte selettiva nelle cellule tramite la somministrazione di nanoparticelle magnetiche, è attualmente ben nota sia in ricerca che in terapia, ma esclusivamente tramite l’ausilio di campi magnetici oscillanti, onde radio o raggi infrarossi non focalizzati. Infine per le applicazioni diagnostiche, che questo progetto si era prefissato, è stato studiato e sviluppato un dispositivo microfluidico utile all’isolamento di cellule tumorali tramite il principio di displacement magnetico. Tale dispositivo è stato studiato tramite simulazioni a computer ed in seguito prodotto per i successivi test. Ne sono stati valutati i parametri fondamentali ed infine studiato sperimentalmente con biglie magnetiche di dimensioni comparabili alle cellule, riportando infine i calcoli teorici per l’applicazione su campioni contenenti cellule tumorali. Le dimostrazioni applicative che questa tesi di Dottorato ha fornito potranno portare, nel prossimo futuro, ad un avanzamento nel campo della diagnostica del cancro, consentendo di sviluppare sistemi a basso costo e quindi check-up più frequenti e mirati. Infine le prove di ipertermia eseguite, sfruttando le nanoparticelle magnetiche, forniscono interessanti spunti per possibili nuove terapie mirate.
In cancer diagnosis the recognition of epithelial and mesenchymal cancer cells is one of the most difficult challenges. The former subtype have a well recognized method for their identification that uses an anti Epithelial Cell Adhesion Molecule (Ep-CAM) antibody. In contrast, the mesenchymal subtype lacks of a well spread and well expressed membrane marker lowering, consequently, the possibilities to develop a widely-usable assay. Currently, the proposed methods and techniques use several specific antibodies to recognize and isolate the mesenchymal cancer cells from whole blood. In this Doctorate thesis we exploit the possibility to use metabolism instead of membrane markers to recognize and isolate mesenchymal cancer cell from a complex environment like the one in the presence of cells with other subtype characteristics. The metabolism of cancer cells is characterized by a higher rate of glycolysis respect to non-neoplastic cells. Mesenchymal cells, in particular, exhibit a hyper activated ATP production and an enhanced glucose uptake. Based on these fundamentals, we developed a new approach for mesenchymal cancer cells detection by means of magnetic CoFe2O4 nanoparticles (MNPs) coated by 2-2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG), a fluorescent glucose analogue and by D-glucose molecule, as control. MNPs were synthesized, functionalized and finally characterized by Fourier Transform Infra Red (FTIR) spectroscopy and Scanning Electron Microscopy (SEM). MNPs mean size was around 27 nm for every sample. Considering two types of BC cells: epithelial-like (MCF-7) and mesenchymal-like (MDA-MB-231), it has been demonstrated that increasing the glucose concentration in the medium from 1 g/L (low glucose) to 4.5 g/L (high glucose), results in a selectively MNPs internalization by the mesenchymal subtype. In particular we used a breast cancer (BC) cell lines co-culture and thus we demonstrated the mesenchymal predisposition to uptake glucose coated MNPs both in normal and in high glucose conditions. We observed that the mesenchymal-like cells (MDA-MB-231), respect to the luminal-epithelial ones (MCF7), internalized a statistically significant higher amount of glucose coated CoFe2O4 NPs in both glucose concentrations. Internalization was investigated using advanced techniques as immunofluorescence, immuno-cyto-chemical assay, confocal microscopy and Focused Ion Beam (FIB) - SEM. The internalization MNPs mechanism has been further investigated by the selective blockage of glucose transporter channels, via a specific inhibitor (STF-31), resulting in a proportional uptake decrease as a consequence of the treatment. From a therapeutic point of view, the presence of MNPs inside the mesenchymal-like cells has been exploited for hyperthermia studies by exposing the cells to a localized Infra Red (IR) laser beam. Cells without MNPs were not affected by the IR laser while cells positive to MNPs have been warmed due to the IR absorbance characteristics of cobalt ferrite core, leading to cell damages and rearrangements. We, moreover, investigated the ability of White Blood Cells (WBCs), obtained by healthy donor, to internalize the CoFe2O4 – 2-NBDG NPs at both glucose medium concentrations, leading to a possible diagnostics applications in whole blood cells analysis. For this purpose, a microfluidics device has been developed for a possible isolation of MNPs-positive cells by applying an external magnetic field. The microfluidics chip was fabricated by the soft optical lithography method and the so obtained PDMS mold was bound to a glass slide by oxygen plasma treatment. Finite element computer simulation has been carried out for better understanding the magnetic displacement principles in microfluidics devices. Finally, the set up has been fabricated and tested with micron-sized magnetic beads for the separation yield evaluation. Concluding, thinking in terms of diagnostics we could infer that this method may lead to an alternative technique for mesenchymal cancer cells detection and isolation thanks to the magnetic properties of the MNPs used. Indeed, by applying an external magnetic field to a mixed sample of mesenchymal/epithelial cancer cells and WBCs it would be possible to isolate the mesenchymal-MNPs-positive ones. Moreover, our demonstration of localized heating could lead in the future to a possible therapeutics application in thermotherapy field.
XXVII Ciclo
1984

Metastasis represents the most life-threatening aspect of tumorigenesis and is the leading cause of death by cancer. Intensive research in this field has shed light on some of the molecular strategies employed by the heterogeneous cancer cell populations to leave the primary tumor, disseminate and grow new colonies in distant organs. In any given tumor, one important functional category of cancer cells is represented by cancer stem cells (CSCs), endowed with self-renewal and tumor-initiating potentials. Moreover, the epithelial-mesenchymal transition (EMT) program represents a process of fundamental importance conducive to tumor dissemination and metastatic spread of cancer cells. Some studies have pointed out that the EMT is responsible for the acquisition of the CSC-like state whereas others have shown that both cell entities can exist separately and cooperate to accelerate the process of metastasis. Here, we propose the combined use of metabolomics and fluxomics strategies to shed light on the metabolic reprogramming and vulnerabilities accompanying specific cancer cell phenotypes that differs in their metastatic and invasive capacities. The main objective of this thesis is focused on the characterization of the metabolic reprogramming and vulnerabilities of uncoupled CSC and EMT phenotypes present in a dual-cell prostate cancer cell model and represented by the highly related cell subpopulations PC-3M and PC-3S cells, respectively. Our results indicated that epithelial PC-3M cells, displaying CSC features and a high metastatic potential, preferentially rely on aerobic glycolysis (Warburg effect) for bioenergetics. Although these cells show low coupling between glycolysis and oxidative phosphorylation (OXPHOS) because of low pyruvate dehydrogenase activity, they display an increased metabolic flexibility to utilize different carbon sources, such as fatty acids, glutamine and other amino acids, that offset the decreased diversion of glucose-derived carbons into the tricarboxylic acid cycle and OXPHOS. The characterization of the non-CSC mesenchymal PC-3S cells expressing the EMT program and endowed with a high invasive capacity, showed a strong coupling between aerobic glycolysis and OXPHOS and a strong dependence on the mitochondrial metabolism for bioenergetics, which leads to higher levels of ROS that require increased levels of glutathione to provide an adequate antioxidant defense system. PC-3M and PC-3S cells differentially reprogram the use of the oxidative and non-oxidative branches of the pentose phosphate pathway to sustain their distinct metabolic needs. Glycolytic intermediates are preferentially directed to ribose synthesis in PC-3M cells to build up nucleotides whereas the generation of NADPH is more crucial for PC-3S cells to counteract their higher oxidative stress and sustain their increased fatty acid synthesis. Glutamine metabolism substantially contributes to TCA reactions in PC-3. For PC-3S cells, both glucose and glutamine are necessary to display a proper mitochondrial function. PC-3M cells are more dependent than PC-3S cells on the glutaminase reaction for proliferation and survival and this reliance lies mainly on the increased need for PC-3M cells to neutralize the excessive levels of protons (lactic acid) that result from their marked Warburg effect, which is achieved by the ammonia molecules released from glutamine metabolism. The high metabolic flexibility displayed by the CSC subpopulation including the participation of serine, glycine and one-carbon metabolism, the uptake of ketogenic amino acids, proline metabolism, among others, provide PC-3M cells with an extensive metabolic dynamics to obtain not only precursors but also to balance their redox status (NAD+/NADH and NADP+/NADPH) for metabolic processes to continue (e.g. glycolysis) and protect them from excessive acidity derived from a high glycolytic rate. Collectively, these results strengthen the notion that specific metabolic signatures are associated to CSC and EMT programs and highlight the importance of studying uncoupled cell phenotypes in order to univocally associate their characteristic metabolic reprogramming.
El proceso de la metástasis es la principal causa de mortalidad en pacientes de cáncer. En los últimos años se ha desvelado la importancia de la cooperación entre distintas subpoblaciones celulares que coexisten en el tumor. Entre estas subpoblaciones, encontramos las células denominadas cancer stem cells (CSCs), con un elevado potencial de autorenovación, pluripotencia y capacidad de iniciar tumores. Por otro lado, ciertas subpoblaciones celulares del tumor son capaces de incrementar sus capacidades migratorias e invasivas, mediante el proceso de epithelial-mesenchymal transition (EMT). Diversos estudios han demostrado que la cooperación entre CSCs y células que han activado el programa EMT facilita la colonización metastásica. Dado que la reprogramación metabólica es responsable de proveer a las células tumorales aquellos recursos bioenergéticos y de biosíntesis necesarios para el mantenimiento de su fenotipo tumoral, en este trabajo se ha caracterizado el metabolismo y las vulnerabilidades metabólicas de dos subpoblaciones celulares derivadas de la línea celular PC-3, con características diferenciadas de CSCs por un lado (PC-3M) y de EMT por otro (PC-3S). El estudio metabólico de estas subpoblaciones celulares desveló que las células PC-3M presentan una mayor preferencia para el uso de la glucólisis (efecto Warburg más marcado), mientras que las PC-3S son más dependientes del metabolismo energético mitocondrial. Estas subpoblaciones también difieren en el uso de las ramas oxidativa y no oxidativa de la vía de las pentosas fosfato y en las reacciones de biosíntesis y degradación de ácidos grasos, con el fin de satisfacer las distintas necesidades metabólicas que caracterizan estos fenotipos. Por otro lado, las PC-3M muestran una elevada flexibilidad y adaptación metabólica, siendo capaces de metabolizar numerosos substratos, entre ellos diferentes tipos de amino ácidos. Particularmente, el metabolismo de la glutamina en las PC-3M es más esencial que en las PC-3S, no sólo por su papel anaplerótico, si no por su función de tamponamiento de los excesos de ácido. El conjunto de estos resultados han desvelado las particularidades metabólicas y vulnerabilidades asociadas a los fenotipos, no solapados, de CSCs y EMT. El conocimiento adquirido podrá contribuir en el diseño de nuevas estrategias terapéuticas para el tratamiento de la metástasis.

The development of acquired resistance to chemotherapeutic drugs is often a limiting factor in the treatment of cancer. In this study, I investigated alterations in MAP kinase signalling and metabolism in a doxorubicin/epirubicin-resistant breast cancer cell line. Doxorubicin, also known as adriamycin, is one of the most important drugs in the treatment of breast cancer. The MAP kinases ERK, JNK, and p38 have all been linked to drug response and tumorigenesis, as well as apoptosis, in different cell types. I found that ERK activity was downregulated in the resistant cell line, while transcripts of the ERK phosphatases DUSP5 and DUSP6 were enriched. The resistant cells also contain less FOXO3a, a broadly pro-apoptotic transcription factor that regulates many aspects of cellular activity. I also used NMR-based metabonomics to generate a metabolic profile of the parental and doxorubicin-resistant cell lines. Many metabolic changes are seen during tumorigenesis, with further changes seen after the development of drug resistance. An increase in glycolysis is the best known, but alterations in choline metabolism and glutamine usage are also commonly seen in cancer. My results confirmed an increase in glycolysis in the resistant cells, as well as altered glutamine metabolism, and also provided novel findings for future work. The reduction in intracellular glutamine in the resistant cells was correlated with a loss of expression of the metabolic enzyme glutamine synthetase. My results show that the doxorubicin-sensitive parental cell line expresses glutamine synthetase, which was required for maximal proliferation rate. Conversely, the lack of glutamine synthetase in the doxorubicin-resistant cells caused them to be dependent on the provision of extracellular glutamine for growth. This may have implications for the treatment of drug resistant breast cancers.

Las células tumorales presentan alteraciones metabólicas. Una de las diferencias con las células no transformadas es que los tumores usan más glucosa incluso en condiciones de normoxia, lo que se utiliza actualmente para visualizar tumores mediante la técnica de PET. Ultimamente se están desarrollando terapias basadas en estas particularidades metabólicas de las células tumorales, que las hacen más sensibles a inhibición del metabolismo glicolítico. Aquí estudiamos la muerte producida por la falta de glucosa en sarcomas y otros tipos tumorales. La privación de glucosa induce apoptosis o necrosis dependiendo de la línea celular. Mostramos cómo la privación de glucosa induce actividad caspasa en células deficientes de Bax/Bak, mientras que en las líneas de rabdomiosarcoma induce necrosis. Además la caspasa-8 está implicada en la muerte por ausencia de glucosa en estas células deficientes en Bax/Bak y también en células humanas HeLa. Investigamos el uso de 2-deoxiglucosa como inductor de muerte celular frente a líneas de rabdomiosarcoma alveolar y embrional. La 2-deoxiglucosa promueve muerte celular en líneas de rabdomiosarcoma alveolar. También induce diferenciación acompañada por una bajada de PAX3/FOXO1a, una proteína surgida de la translocación cromosómica en las células de rabdomiosarcoma alveolar y crítica en el desarrollo oncogénico de las mismas. Caracterizamos la muerte celular apoptótica inducida por 2-deoxiglucosa en líneas de sarcoma, in vitro. Estudiamos las moléculas de las rutas de apoptosis que determinan la sensibilidad de estas células a la 2-deoxiglucosa, con especial interés en las proteínas de la familia del oncogén Bcl-2. La muerte celular inducida por el tratamiento está asociada a la activación de Bax y Bak. Observamos que la sobre-expresión de proteínas anti-apoptóticas de la familia Bcl-2 como Bcl-xL y de Mcl-1 previene la apoptosis, indicando que la muerte sucede a través de la ruta mitocondrial. Enseñamos que la bajada de Mcl-1 y la subida de Noxa son críticos en la muerte por 2-DG. Además, la 2-DG promueve estrés reticular acompañado de la inducción de ATF4 y chaperonas. Al bajar los niveles de ATF4, protegemos a las células de la muerte inducida por 2-DG y prevenimos la pérdida de Mcl-1. Incubamos células en presencia de manosa, que revierte el estrés inducido por la 2-DG y previnimos la muerte sin subir los niveles de ATP. Así, el estrés energético causado por la 2-DG no es la principal causa de muerte celular. También la 2-DG promueve la fosforilación de eIF2α, un inductor de ATF4, y la inactivación de mTOR. Nuestros resultados sugieren que el uso de inhibidores glicolíticos como la 2-DG pueden ser efectivos en el tratamiento de los rabdomiosarcoma alveolares y que Noxa podría ser un marcador pronóstico de la eficiencia de estas drogas. Por otro lado, caracterizamos las respuestas a la falta de glucosa y 2-DG, en particular la respuesta autofágica que puede determinar que las células mueran o no en respuesta a la falta de nutrientes. Observamos que las células están sensibilizadas al tratamiento con 2-DG tras el uso de cloroquina. Sin embargo, la presencia de cloroquina no afecta a la privación de glucosa. Intentamos clarificar si la falta de glucosa está induciendo macroautofagia. El flujo de LC3 por western blot nos indica que no hay más macroautofagia ni en células DKO ni en las Rh4 en ausencia de glucosa. Por microscopía confocal y analizando células HeLa y Rh4, tampoco vemos mayores acúmulos de GFP-LC3 tras la retirada de glucosa comparando con tratamientos clásicos de inducción de autofagía como la retirada de aminoácidos o con rapamicina. Estos datos sugieren que la combinación de 2-DG con inhibidores de la autofagia podría ser útil en el tratamiento contra rabdomiosarcomas y que la falta de glucosa no induce autofagia.
Characterization of cell death induced by inhibition of glucose metabolism Rhabdomyosarcoma (RMS) is the most common soft-tissue tumor of childhood characterized by high malignancy, local invasiveness and a marked predisposition to metastasize. Recently, a number of therapies targeting tumor cell metabolism are being developed, since oncogenic changes make tumors more sensitive to inhibition of glycolytic metabolism. We observed that the glycolytic inhibitor 2-deoxyglucose (2-DG) can efficiently promote cell death in p53-deficient alveolar rhabdomyosarcoma (the rhabdomyosarcoma subtype with poorer prognosis), but not in embryonal rhabdomyosarcoma. Differential expression of HIF-1 could not explain the different sensitivity of tumor subtypes. 2-deoxyglucose induced nuclear chromatin condensation, cleavage of caspase substrates and DNA degradation which was inhibited by caspase inhibitors. Cell death triggered by 2-DG was associated with its ability to activate Bax and Bak and was prevented by overexpression of the anti-apoptotic Bcl-2 homologs Bcl-xL and Mcl-1. Conversely, siRNA against Bcl-xL or Mcl-1 accelerated death. 2-DG promoted downregulation of the anti-apoptotic protein Mcl-1 and accumulation of two BH3-only proteins, Noxa and Bim. siRNA against these proteins indicated that Noxa mediates 2-DG-induced cell death. Addition of different carbon/energy sources indicated that apoptosis is not associated with loss of ATP but rather with endoplasmic reticulum stress and the eIF2-alpha-ATF4 pathway. 2-DG promoted Mcl-1 loss, probably due to general inhibition of translation, since both eIF2-alpha phosphorylation and inactivation of the mTOR pathway were observed. All these events (ER stress, energetic stress and inhibition of the mTOR pathway) are known to induce autophagy. Indeed, 2-DG induces autophagy, and inhibition of autophagy at different levels sensitizes cells to 2-DG. Together, our findings suggest that glycolysis inhibitors such as 2-DG may be effective in treating alveolar rhabdomyosarcoma.

Molte cellule tumorali, al fine di generare ATP e sostenere i processi anabolici, si servono principalmente della glicolisi piuttosto che della respirazione mitocondriale. Di conseguenza, il glucosio assume un ruolo critico per la sopravvivenza e la proliferazione delle cellule tumorali. Inoltre, attraverso la via dei pentosi fosfati, il glucosio porta alla formazione di NADPH, contribuendo al mantenimento nelle cellule dell’equilibrio ossidativo. Nondimeno, il glucosio può entrare anche nel pathway biosintetico delle esosammine (HBP), sostenendo la N- e O-glicosilazione di lipidi e proteine, importante per lo sviluppo tumorale. Considerando l’essenziale ruolo del glucosio, un possibile approccio per la terapia antitumorale è l’utilizzo del metabolismo del glucosio come target, non solo attraverso la glicolisi ma sfruttando anche gli altri processi glucosio-dipendenti. A tal proposito, la deprivazione di glucosio e la seguente analisi del destino cellulare a livello fenotipico e molecolare possono costituire una strategia utile per smascherare tutti i meccanismi mediati dal glucosio che partecipano alla crescita e alla sopravvivenza delle cellule tumorali. Tale strategia potrebbe essere poi sfruttata per offrire nuovi target e progettare nuove terapie antitumorali. Sebbene alcuni dati indichino che i tumori originino da cellule con persistenti difetti alla catena respiratoria mitocondriale, l’inibizione della fosforilazione ossidativa (OXPHOS) sembra una condizione di adattamento più che una causa della riprogrammazione metabolica delle cellule tumorali. In questo scenario, i meccanismi di regolazione post-traduzionali, di natura essenzialmente reversibile, a carico di proteine mitocondriali potrebbero assumere un importante ruolo regolatorio. Una delle principali modificazioni post-traduzionali è la fosforilazione dei residui Ser/Thr e, a tal proposito, la chinasi PKA presenta numerosi target a livello mitocondriale ed è coinvolta nella regolazione di biogenesi, trasporto e attività dei Complessi I e IV e della morfologia mitocondriale. Poiché è stato osservato che K-ras può causare la diminuzione dell’espressione di geni codificanti per componenti della via cAMP/PKA, nelle cellule K-ras-trasformate la deregolazione di tale via potrebbe portare alla disfunzione mitocondriale ed allo switch metabolico caratteristico delle cellule tumorali. A conferma di questa ipotesi, le cellule K-ras-trasformate mostrano minori livelli di attività dell’enzima PKA rispetto alle cellule normali. Inoltre, la stimolazione esogena della attività della PKA, ottenuta mediante trattamento con forskolina (FSK), protegge le cellule K-ras-trasformate, sia murine sia umane, dalla morte indotta dalla deplezione di glucosio. Tale protezione è dovuta alla stimolazione dell’attività del Complesso I, all’aumento dell’ATP intracellulare e della fusione mitocondriale e alla riduzione dei livelli di ROS. L’inibizione specifica di PKA previene quasi completamente molti di questi effetti. Inoltre, il breve trattamento con Mdivi-1, molecola in grado di favorire la fusione mitocondriale, riduce fortemente i livelli di ROS specialmente nelle cellule trasformate, indicando una stretta relazione tra morfologia e attività mitocondriale. Queste osservazioni supportano l’idea che l’apoptosi indotta dalla deprivazione di glucosio nelle cellule K-ras-trasformate è associata alla deregolazione della via cAMP/PKA che a sua volta causa la diminuzione dell’attività del Complesso I, la riduzione della produzione di ATP e la prevalenza della fissione mitocondriale rispetto alla fusione. Tale scoperta può aprire nuovi scenari per lo sviluppo di farmaci antitumorali. Poiché la carenza di glucosio si può riscontrare nell’ambiente in cui cresce e si sviluppa il tumore, tale condizione può essere sfruttata per potenziare l’azione di specifici agenti, come alcuni modulatori dell’OXPHOS. Infatti, l’inibizione delle funzioni mitocondriali in condizioni di deprivazione di glucosio potrebbe risultare letale per le cellule tumorali. In accordo, in questo lavoro viene mostrato che la deprivazione di glucosio e gli inibitori del Complesso I, come rotenone, piericidina A e capsaicina, hanno un effetto sinergico nell’indurre la morte delle cellule tumorali. Nello specifico, basse dosi d’inibitori del Complesso I, inefficaci sulle cellule normali e su cellule cresciute in alto glucosio, diventano citotossiche per le cellule tumorali cresciute in basso glucosio. L’effetto citotossico degli inibitori del Complesso I sulle cellule tumorali è ulteriormente e fortemente aumentato quando l’attività OXPHOS viene stimolata tramite il trattamento con FSK. Queste osservazioni dimostrano che la riattivazione della funzione mitocondriale associata alla deplezione di glucosio e al trattamento con basse dosi di inibitori del Complesso I riduce fortemente la sopravvivenza delle cellule tumorali e potrebbe quindi essere valutato come approccio terapeutico. Come indicato in precedenza, nelle cellule tumorali il glucosio è implicato in numerosi processi. L’analisi trascrittomica e proteomica di cellule murine K-ras-trasformate e della loro controparte normale mostra che la deprivazione di glucosio modula l’espressione di molti geni legati allo stress del reticolo endoplasmatico e all’Unfolded Protein Response (UPR). L’attivazione di tale risposta si osserva in entrambe le linee cellulari ma più fortemente nelle cellule trasformate, dove è associata anche alla morte cellulare. Infatti, la sua attenuazione tramite l’inibitore della traduzione proteica, cicloesimide, o lo chaperone chimico, 4-fenil-butirrato, protegge specificatamente le cellule trasformate dalla morte cellulare in basso glucosio. Anche l’inibizione della chinasi proapoptotica JNK, attivata a valle dell’UPR, previene specificatamente la morte delle cellule trasformate. Questa osservazione è in accordo col fatto che in basso glucosio le cellule trasformate mostrano una maggiore attivazione di JNK rispetto alle cellule normali. Inoltre, l’attivazione dell’UPR e la morte glucosio-dipendente delle cellule trasformate è completamente prevenuta dall’aggiunta nel terreno di coltura di un substrato dell’HBP, N-Acetyl-D-glucosammina, cosa che suggerisce una stretta relazione tra i due processi. È interessante notare che anche cellule umane esprimenti l’oncogene K-ras e caratterizzate da un fenotipo iperglicolitico mostrano simili effetti in seguito alla modulazione dell’UPR o dell’HBP. Quindi, la deprivazione di glucosio nelle cellule K-ras-trasformate può indurre un meccanismo di morte cellulare UPR-dipendente, attivato dall’eccessivo accumulo di proteine mal foldate, probabilmente come conseguenza della riduzione della N-glicosilazione delle proteine. La piena delucidazione di questa risposta potrebbe essere importante per progettare nuove strategie terapeutiche antitumorali. Oggi la nuova sfida della ricerca e della terapia antitumorale è il totale sradicamento del tumore, uccidendo anche le cellule staminali tumorali (cancer stem cells, CSCs). Considerando l’importante ruolo del metabolismo e della sua riprogrammazione nello sviluppo tumorale, la caratterizzazione del metabolismo delle CSCs può essere considerata un importante mezzo per lo sviluppo di nuove strategie antitumorali. Recentemente, è stata ottenuta la linea cellulare staminale di osteosarcoma umano, 3AB-OS. In questo lavoro di tesi ho svolto una prima caratterizzazione del suo profilo metabolico, paragonato a quello delle cellule tumorali MG63, da cui le cellule 3AB-OS sono state selezionate. Si è osservato che le cellule 3AB-OS dipendono più fortemente dalla glicolisi rispetto alle cellule MG63. Infatti, quando cresciute in presenza di galattosio e piruvato (substrati mitocondriali) le cellule 3AB-OS riducono maggiormente la propria capacità proliferativa rispetto alle cellule MG63. Esse risultano anche essere fortemente sensibili alla deprivazione di glucosio e al trattamento con inibitori della glicolisi mentre sono insensibili all’inibizione della catena respiratoria. Inoltre, diversamente dalle cellule MG63, le cellule 3AB-OS presentano principalmente mitocondri frammentati, in particolare in basso glucosio. Tutte queste osservazioni suggeriscono che il metabolismo energetico delle cellule 3AB-OS presenti caratteristiche paragonabili a quello delle cellule staminali normali e delle cellule tumorali caratterizzate da un metabolismo glicolitico. Può essere interessante notare che il profilo trascrizionale delle cellule 3AB-OS è simile a quello delle cellule K-ras-trasformate, confermando la similitudine tra le CSCs e le cellule tumorali glicolitiche. Quindi, alcune strategie sviluppate per il trattamento delle cellule tumorali glucosio-dipendenti potrebbero essere usate anche per trattare specifiche CSCs.
Several cancer cells, in order to generate ATP and sustain different anabolic processes, rely mainly on glycolysis instead of Oxidative Phosphorylation (OXPHOS). Thus, glucose assumes a critical role for cancer cell survival and proliferation. Moreover, through the penthose phospate pathway glucose leads to production of NADPH contributing to maintenance of cellular oxidative equilibrium. Besides, glucose can also enter Hexosamine Biosynthesis Pathway (HBP), sustaining lipid and protein N- and O-glycosylation that cover an important role in cancer development. Taking in consideration the essential role of glucose in cancer, one important anticancer therapeutic approach is to target its metabolism namely glycolysis and the other processes in which it is involved. On this regard, glucose deprivation and consequent analysis of cancer cell fate both at phenotypical and molecular level can be a useful strategy to unmask all mechanisms that participate to glucose-mediated cancer cell growth and survival. Such a strategy could be subsequently exploited to provide new targets and to set new anticancer therapies. Although there is evidence that tumors originate from cells with persistent defects in the mitochondrial respiratory system, inhibition of OXPHOS activity seems to be an adaptation to cancer metabolism reprogramming rather than a cause. In this scenario, reversible post-translational modifications of mitochondrial components could assume an important regulatory role. Among the most important post-translational modifications there is Ser/Thr phosphorylation and, on this regard, the protein kinase PKA has numerous mitochondrial targets being involved in the regulation of the biogenesis, the import and the activity of mitochondrial Complex I or IV as well as of mitochondrial morphology. Since it has been observed that oncogenic K-ras may lead to a depression of genes encoding for components of the cAMP/PKA signaling pathway, in K-ras-transformed cells the deregulation of cAMP/PKA pathway could cause OXPHOS depression and “glucose addiction” of cancer cells. In agreement with such a hypothesis, K-ras-transformed cells show lower PKA activity as compared to normal cells. Moreover, exogenous stimulation of PKA activity, achieved by Forskolin (FSK) treatment, protects mouse and human K-ras-transformed cells from apoptosis induced by glucose deprivation, by enhancing Complex I activity, intracellular ATP levels and mitochondrial fusion and by decreasing intracellular ROS levels. Worth noting, several of these effects are almost completely prevented by inhibition of PKA activity. Moreover, short time treatment with Mdivi-1, a molecule that favors mitochondrial fusion, strongly decreases the cellular ROS levels especially in transformed cells, indicating a close relationship between mitochondrial morphology and activity. These findings support the notion that glucose shortage-induced apoptosis, specific of K-ras-transformed cells, is associated to a derangement of PKA signaling that leads to mitochondrial Complex I decrease, reduction of ATP formation and prevalence of mitochondrial fission over fusion. Such a discovery can thereby open new approaches for the development of anticancer drugs. Given that glucose shortage is often encountered in the tumor microenvironment, it can be exploited to potentiate the action of specific agents, such as the mitochondrial OXPHOS activity modulators, that in condition of glucose deprivation could be lethal for cancer cells. Accordingly, it is shown that glucose deprivation and Complex I inhibitors, i.e., rotenone, piericidin A and capsaicin, synergize in inducing cancer cell death. In particular, low doses of Complex I inhibitors, ineffective on normal cells and on cells grown in high glucose, become specifically cytotoxic on cancer cells cultured in low glucose. Importantly, the cytotoxic effect of Complex I inhibitors is strongly enhanced when mitochondrial OXPHOS activity is stimulated by FSK. These findings demonstrate that the reactivation of the mitochondrial function associated with glucose depletion and low doses of mitochondrial Complex I inhibitors strongly affect cancer cell survival. This therapeutic approach might be valuable to eradicate cancer cells. As above indicated, glucose is implicated in numerous processes in cancer cells. Transcriptomic and proteomic analyses applied to mouse K-ras-transformed cells as compared to normal cells show that glucose deprivation modulates the expression of several genes linked to endoplasmic reticulum stress and the Unfolded Protein Response (UPR). The activation of such a response, as confirmed by mRNA and protein expression, is observed in both cell lines, but only in transformed cells is strictly associated to their death. In fact, its attenuation by protein translation inhibitor cycloheximide or chemical chaperone 4-Phenyl-butyrate specifically rescues transformed cells from death. Moreover, glucose deprivation-induced transformed cell death is also prevented by inhibition of an UPR downstream pro-apoptotic kinase, JNK, whose activation is observed specifically in transformed cells as compared to normal cells. Interestingly, UPR activation and death of transformed cells is completely prevented by addition of a specific HBP substrate, namely N-Acetyl-D-glucosamine, suggesting a strict relation between the two processes. Notably, also oncogenic K-ras expressing human glycolytic cells show similar effects after UPR modulating treatments. Thus, we show that glucose deprivation can induce an UPR-dependent transformed cell death mechanism, which is activated by harmful accumulation of unfolded proteins, probably as consequence of N-glycosylation protein reduction. The full elucidation of this response could be relevant to design new therapeutic strategies. Today the new challenge of anticancer research and therapy is the total eradication of the cancer, targeting cancer stem cells (CSCs). Considering the important role of metabolism and metabolic reprogramming in cancer development, also the definition of CSCs metabolism can be considered an important tool for future strategies targeting these cells. Recently, a human osteosarcoma 3AB-OS CSC-like line has been developed. Therefore we have decided to characterize its metabolic features as compared to the parental osteosarcoma MG63 cells, from which 3AB-OS cells were previously selected. 3AB-OS cells depend on glycolytic metabolism more strongly than MG63 cells. Indeed, addition to the growth medium of galactose and pyruvate -mitochondrial specific substrates- instead of glucose markedly reduces 3AB-OS growth, as compared to MG63 cells. In line with these findings 3AB-OS cells, compared to MG63 cells, are strongly sensitive to glucose depletion, glycolysis inhibition and less sensitive to respiratory inhibitors. Additionally, in contrast to MG63 cells, 3AB-OS display mainly fragmented mitochondria, particularly in low glucose. Overall, these findings suggest that 3AB-OS energy metabolism is more similar either to normal stem cells or to cancer cells characterized by a glycolytic metabolism. Interestingly, the transcriptional profile of CSCs is similar to that of K-ras-transformed cells, confirming a possible similarity to glycolytic cancer cells. Therefore, some strategies developed for glucose addicted cancer cells could be used also to treat specific CSCs.

Abstract 17β-Hydroxysteroid dehydrogenases (17HSDs) catalyze the reactions between 17-hydroxy and 17-keto steroids. In the present study, the enzyme activities and tissue distribution of 17HSD type 1, type 2 and type 4 were characterized. Furthermore, the role of 17HSD type 1 in estrogen-dependent growth was studied in MCF-7 breast cancer cells which were stably transfected with type 1 cDNA. Endogenous oxidative 17HSD activity found in COS-m6 monkey kidney cells was first compared with that of human placental 17HSD. Cultured COS-m6 cells exclusively possessed oxidative 17HSD activity, converting estradiol (E2) to less active estrone (E1). When placental 17HSD was transfected into these cells, highly reductive activity appeared. The 17HSD enzyme in COS-m6 cells also catalyzed the conversion of testosterone to androstenedione, whereas the placental enzyme was estrogen-specific. These results further proved the existence of different 17HSD isoenzymes. The enzymatic properties and cell- and tissue-specific expression of 17HSD type 1, type 2 and oxidative type 4 were further characterized. The data confirmed that in cultured cells the direction of 17HSD activity is determined by the expression of different isoenzymes and not by the intracellular environment. In addition, the 17HSD type 1 gene expresses two mRNA signals, 1.3 kb and 2.3 kb in size. The expression of 1.3 kb mRNA, but not 2.3 kb mRNA was related to enzyme concentration in all the cell types studied. The type 1 enzyme was expressed in the placenta, ovary and in some breast cancer specimens and in the cell lines originated from these tissues. 17HSD type 2 was more widely expressed in both steroidogenic and in target tissues of steroid action. 17HSD type 4 was expressed in almost all cell lines and in all tissues studied, but no correlation with 17HSD activity was detected. These results suggest that 17HSD type 1 is involved in E2 production in females and 17HSD type 2 is responsible for inactivation of sex steroids. However, the oxidation of 17β-hydroxysteroids seems not to be the primary activity of 17HSD type 4. The mRNAs for 17HSD type 1, type 2 and type 4 were found to be expressed in human mammary epithelial cells. In breast tissue samples both 17HSD type 1 and type 2 were detected by in situ hybridization. Despite the presence of 17HSD type 1 mRNA in human mammary epithelial cells, only oxidative 17HSD activity was detected. The reason for the lack of reductive activity is not yet known. Finally, MCF-7 breast cancer cells were stably transfected with 17HSD type 1 cDNA in order to study the effect of 17HSD type 1 on estrogen-dependent growth. In wild type MCF-7 cells, very low 17HSD activity was detected and E1 did not have any effect on cell growth. In the cells expressing 17HSD type 1, E1 was rapidly converted to E2. Hence in these cells E1 had a similar growth-promoting effect as E2 as a result of the action of 17HSD type 1. The presence of 17HSD type 1 in breast cancer cells may thus be an important factor regulating estrogen exposure and the estrogen-responsive growth of breast cancer tissue.

La mitochondrie occupe un rôle essentiel au sein des cellules cancéreuses. Etant la source principale de synthèse d’ATP mais est également le lieu de réactions anaboliques et cataboliques, la mitochondrie supporte le développement tumoral. De plus, la mitochondrie est également impliquée dans la réponse aux stress cellulaire en régulant notamment l’autophagie ou la mort des cellules cancéreuses.Dans ce contexte, nous avons démontré que la fonction mitochondriale peut altérer la réponse au stress réticulaire permettant la survie des cellules tumorales. En effet, la surexpression de la protéine GILZ (Glucocorticoid-Induced Leucine Zipper) atténue la mort cellulaire induite par le stress réticulaire. Ceci est permis grâce au maintien du réseau mitochondrial et à l’augmentation de la fonction mitochondriale. Dans cette étude, nous avons démontré que le maintien de la fonction mitochondriale est important pour l’effet protecteur de GILZ puisque l’utilisation de lignées cellulaires de mélanome dépourvues d’activité mitochondriale (lignées ρ0) et surexprimant GILZ sont sensibles à la mort induite par les inducteurs de stress réticulaire. Nos études ont également démontré que l’augmentation de la fonction mitochondriale induite par GILZ peut être utilisée pour resensibiliser les cellules cancéreuses à la mort notamment en utilisant des molécules prooxydantes comme l’elesclomol.Dans un autre contexte tumoral, nous avons également démontré qu’une sous population de cellules de mélanome BRAFV600E peuvent augmenter leur métabolisme mitochondrial dans le but de survivre à la mort cellulaire induite par les inhibiteurs de MAPK. La résistance aux MAPKi implique une augmentation significative de l’OxPHOS mitochondriale associée un remodelage du réseau mitochondrial autour du réticulum endoplasmique facilitant la recapture du calcium mitochondrial. Nos résultats ont permis de démontrer que la fonction mitochondriale est cruciale pour la survie des cellules cancéreuses. De part son rôle cellulaire multiple, il apparaît clairement que la mitochondrie constitue une cible thérapeutique de choix dans le traitement des cancers
Mitochondria occupies a key role in cancer cells. As the main source of ATP synthesis and the site of anabolic and catabolic reactions, mitochondria support tumor development. Besides, mitochondria are also involved in the response to cellular stress regulating autophagy or cancer cell death.In this context, we have demonstrated that mitochondrial function may alter the ER stress response thus promoting tumor cell survival. Indeed, overexpression of the Glucocorticoid-Induced Leucine Zipper protein (GILZ) protein attenuates endoplasmic reticulum stress mediated cell death. This is achieved by maintaining the mitochondrial network and the increase of mitochondrial function. In this study, we demonstrated that maintaining mitochondrial function is important for the protective effect of GILZ since using melanoma cell lines lacking mitochondrial activity (ρ0 cell lines) and overexpressing GILZ are susceptible to death induced by reticular stress inducers. Our studies have also shown that the increase of mitochondrial function induced by GILZ can be used to re-sensitize the cancer cells to death induced by prooxidant molecules as elesclomol.In another tumoral context, we have also demonstrated that a sub-population of BRAF mutated melanoma cells can increase mitochondrial metabolism to survive to ER stress-mediated cell death induced by several MAPK inhibitors. Resistance to MPAki involves a significant increase in mitochondrial OXPHOS associated with mitochondrial network remodeling around the ER, which facilitates mitochondrial calcium uptake. Our results have shown that mitochondrial function is crucial for the survival of cancer cells. Altogether our data indicate that given their multiple cellular roles, cancer cell mitochondria constitute attractive therapeutic targets

IF1, the endogenous inhibitor protein of mitochondrial F1Fo-ATPase, has raised interest in cancer research due to its overexpression in solid tumours compared to normal tissues. Physiologically, IF1 protects cells from energy depletion by limiting the ATP hydrolytic activity of ATP synthase triggered by mitochondrial depolarization caused by oxygen deficiency as it occurs during ischemic episodes. Considering both the physiological function of IF1 and that cancer cells in solid tumour are frequently exposed to oxygen deprivation, we hypothesized that IF1 overexpression represents a strategy that cancer cells develop to protect themselves from energy depletion under conditions of low oxygen availability. To assess this, we assayed the bioenergetic changes in 143B and HCT116 cancer cells with different metabolic features following stable silencing of IF1. Interestingly, we found that in both cell lines exposed to oxygen deprivation conditions the presence of IF1 limits the energy dissipation due to the activation of the ATP hydrolytic activity of ATP synthase. Furthermore, the analyses of cellular growth and viability revealed that the IF1 silencing inhibited proliferation in the highly glycolytic 143B cells, while it induced more than 50% of cellular death in HCT116 OXPHOS-dependent cells, indicating that the energetic advantage conferred by IF1 is essential for cancer cell proliferation or survival depending on the energy metabolism of each cell line. Moreover, under mitochondrial depolarization conditions, both mitophagy and mitochondrial biogenesis markers were found up-regulated in IF1-expressing cells only, thus indicating a continuous renewal and preservation of the mitochondrial mass. Taken together, our results sustain the idea that IF1 overexpression supports cancer cell adaptation to hypoxic or anoxic conditions also favouring the proliferation of re-oxygenated cells by promptly providing functional mitochondria.

Die zirkadiane Uhr ist ein endogenes Zeitmesssystem, das die Anpassung physiologischer Prozesse an die geophysikalische Zeit ermöglicht. Die zirkadiane Uhr besteht aus einem zentralen Schrittmacher und peripheren Uhren in jeder Zelle. Bei Säugern ist eine bestimmte Anzahl von Uhr-Genen in regulatorischen Schleifen miteinander verbunden, wodurch Oszillationen in der Expression der Uhr-Gene sowie in zahlreichen Zielgenen erzeugt werden. Zielgene der zirkadianen Uhr sind unter anderem an zellulären Prozessen beteiligt, die eine Rolle bei der Tumorentstehung und -progression spielen. Funktionsstörungen der zirkadianen Uhr stehen im Zusammenhang mit verschiedenen Krankheitsbildern, unter anderem Krebs. Ziel dieses Projekts war es, die Rolle der zirkadianen Uhr bei der Tumorentstehung und entwicklung zu untersuchen. Der Fokus lag dabei auf tumorspezifischen Stoffwechselwegen. Die Rolle einer deregulierten zirkadianen Uhr wurde in einem in vitro Zellmodel untersucht. Die SW480 Zelllinie wurde aus einem Primärtumor isoliert und die SW620 Zelllinie aus einer Lymphknotenmetastase desselben Patienten. Die untersuchten Zelllinien zeigten deutliche Unterschiede in Bezug auf ihre Uhr Phänotypen mit globalen Konsequenzen auf oszillierende Gene und Stoffwechselwege. Die Runterregulation des Uhrgens Bmal1 führte in SW480 Zellen zu einem metastatischen Phänotyp, der stark dem von SW620 Wildtypzellen ähnelte. Darüber hinaus führte die Runterregulation von Bmal1 zu einer Veränderung des metabolischen Phänotyps und zu einer modifizierten Antwort auf die Behandlung mit einem Glykolyseinhibitor. Die in diesem Projekt erzielten Ergebnisse unterstützen die postulierte Rolle von Bmal1 als Tumorsuppressor und verdeutlichen das reziproke Wechselspiel zwischen der zirkadianen Uhr und dem Stoffwechsel von Krebszellen und zeigen mögliche Auswirkungen einer deregulierten Uhr auf den Zellmetabolismus während der Tumorentwicklung.
The circadian clock is an internal timing system that allows the entrainment of physiological and behavioural processes to the geophysical time with a periodicity of about 24 hours. It consists of a central pacemaker and peripheral clocks in every cell. In mammals, a distinct set of genes is interconnected in regulatory feedback loops, thereby generating oscillations in gene expression in the core-clock itself as well as in many target genes. Clock target genes are, among others, involved in cellular processes connected to tumour development and progression, including metabolic pathways, drug response pathways and the cell cycle. Malfunctions of the circadian clock are associated with different pathologies including cancer. The aim of this project was to study the role of the circadian clock in tumour development and progression with a focus on cancer metabolism and treatment response. The role of a deregulated clock was investigated in SW480 cells derived from a primary tumour and SW620 cells derived from a lymph node metastasis of the same patient. The investigated cell lines showed clear differences with respect to their clock phenotypes with consequences on global oscillating gene expression and alterations in metabolic pathways. A knockdown of the core-clock gene Bmal1 in SW480 cells induced a metastatic phenotype similar to SW620 wild type cells, as indicated by faster proliferation, lower apoptosis rate and a highly energetic metabolic phenotype. Furthermore, Bmal1-KD induced metabolic phenotype rewiring as seen by altered glycolytic activity and mitochondrial respiration and modified treatment response to metabolism-targeting anticancer treatment. The results obtained in this project reinforce the postulated role of Bmal1 as a tumour suppressor and elucidate a reciprocal interplay between the circadian clock and cancer metabolism with implications in metabolic phenotype rewiring during tumour progression.

The development of therapy resistance is a major obstacle in the successful treatment of advanced prostate cancer. This thesis investigated mechanisms that help drive therapy resistance and discovered that prostate cancer cells can utilise different metabolic pathways in order to become resistant to current therapies. This project also explores new therapeutic strategies to use in combination with current treatments to help fight disease progression and improve outcomes for men with prostate cancer.

Thesis (MSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: In this study, the influence of rooibos on the catalytic activity of enzymes 17β -hydroxysteroid dehydrogenase type 3 (17βHSD3), 17β-hydroxysteroid dehydrogenase type 5 (AKR1C3), 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2), 5α-reductase type 1 (SRD5A1) and 5α-reductase type 2 (SRD5A2), which catalyse prostate androgen metabolism, was investigated. The activities of both 17βHSD3 and AKR1C3 heterologously expressed in CHO-K1 and HEK293 cells were inhibited significantly by rooibos, with rooibos reducing the conversion of androstenedione (A4) and 11keto-androstenedione (11KA4) to testosterone (T) and 11ketotestosterone (11KT), respectively. The catalytic activity of 17βHSD2 towards T, 11hydroxytestosterone (11OHT) and 11KT was also significantly inhibited by rooibos in transiently transfected HEK293 cells. In transiently transfected HEK293 cells rooibos did not inhibit SRD5A1 while the rate of T conversion to dihydrotestosterone (DHT) by SRD5A2 was decreased. Analysis of steroid metabolism in PNT2 cells also suggests that rooibos does not modulate the catalytic activity of endogenously expressed SRD5A towards A4, however, the conversion of T to DHT was reduced. In addition, reductive 17βHSD activity towards A4 was inhibited in the presence of rooibos in both PNT2 and BPH-1 cells. In contrast, the conversion of 11KA4 to 11KT was inhibited in BPH-1, PC-3 and LNCaP cells, with negligible conversion of 11KA4 in PNT2 cells. Interestingly, data suggests inhibition of 3α-hydroxysteroid dehydrogenase type 3 (AKR1C2) activity in the production of androsterone (AST) from 5α–androstenedione (5α-dione), as well as the dehydrogenase reaction of T to A4 in PNT2 cells by rooibos. Androgen metabolism pathways were subsequently investigated in LNCaP cells to determine androgen metabolism by endogenous enzymes. Rooibos resulted in the reduced conversion of A4 in LNCaP cells to the same extent as indomethacin, a known AKR1C3 inhibitor. Rooibos also modulated T, DHT and AST metabolism in LNCaP cells. Furthermore, uridine diphosphate glucuronosyltransferase (UGT) activity in LNCaP cells was inhibited by rooibos, decreasing T-, DHT– and AST-glucuronide formation. These data prompted subsequent investigations into the influence of rooibos at cellular level, and prostatespecific antigen (PSA) levels were assayed in the presence of rooibos. PSA was significantly inhibited by rooibos in the absence and presence of DHT, suggesting possible interaction of rooibos with the mutated androgen receptor (AR) or estrogen receptor-β (ERβ) expressed in LNCaP cells. Taken together, rooibos inhibited the catalytic activity of key enzymes that catalyse the activation of androgens in the prostate, as well as inhibiting enzymes involved in the conjugation of androgens. At cellular level, PSA levels were also decreased by rooibos, possibly through AR or ERβ interactions – clearly indicating a modulatory role for rooibos in active androgen production.
AFRIKAANSE OPSOMMING: In hierdie studie was die invloed van rooibos ten opsigte van die katalitiese aktiwiteite van die ensieme 17β-hidroksi-steroïed-dehidrogenase tipe 2, tipe 3 en tipe 5 (17βHSD2, 17βHSD3, AKR1C3), asook 5α-reduktase tipe 1 en tipe 2 (SRD5A1, SRD5A2) ondersoek. Hierdie ensieme is betrokke in die produksie van androgene in die prostaat. Rooibos het die katalitiese aktiwiteit van 17βHSD3 en AKR1C3 in CHO-K1 en HEK293 selle beïnvloed en het vermindere omskakeling van androstenedioon (A4) en 11keto-androstenedioon (11KA4) na testosteroon (T) en 11-ketotestosteroon (11KT), afsonderlik, veroorsaak. Die katalitiese aktiwiteit van 17βHSD2 teenoor T, 11-hidroksie-testosteroon (11OHT) en 11KT was ook beïnvloed in die teenwoordigheid van rooibos in HEK293 selle. Die katalitiese aktiwiteit van SRD5A1 teenoor A4 en T is nie beïnvloed deur rooibos nie, alhoewel dit voorkom asof rooibos die omsettingstempo van T na dihidrotestosteroon (DHT) deur SRD5A2, getransfekteer in HEK293 selle, verminder het. Verdere ondersoeke is in normale prostaat epiteel selle, in die teenwoordigheid van rooibos uitgevoer. Rooibos het geen invloed op die katalitiese aktiwiteit van SRD5A teenoor A4 gehad nie, alhoewel vermindere omskakeling van T na DHT aangetoon kon word. Rooibos het ook die omskakeling van A4 na T in beide PNT2 en BPH-1 selle tot „n mate geïnhibeer. Die omskakeling van 11KA4 na 11KT was ook verminder in BPH-1, PC-3 en LNCaP selle. Die omskakeling van 11KA4 na 11KT was beduidend laer in PNT2 selle en kon die invloed van rooibos nie aangetoon word nie. Bykomende data toon dat rooibos ook die omskakeling van 5α-androstenedioon (5α-dione) na androsteroon (AST), gekataliseer deur 3α-hidroksi-dehidrogenase tipe 3 (AKR1C2), verminder, gesamentlik met die vermindere omskakeling van T na A4, deur 17βHSD2, in PNT2 selle. Hierdie studie het ook ondersoek ingestel, na die metabolisme van androgene in LNCaP selle. Vermindere A4 metabolisme is in die teenwoordigheid van rooibos asook in die teenwoordigheid van indometasien, „n bekende AKR1C3 inhibitor, gevind. Rooibos verminder dus die aktiwiteit van reduktiewe 17βHSD in LNCaP selle. Verandering in die metabolisme van T, DHT en AST in LNCaP selle, in die teenwoordigheid van rooibos, is ook gevind. Verdere ondersoek in LNCaP selle het gewys dat rooibos „n vermindering in die produksie van gekonjugeerde T, DHT en AST veroorsaak. Die studie het die invloed van rooibos op prostaat-spesifieke antigeen (PSA) ook ondersoek. Daar is vasgestel dat rooibos die vlakke van PSA verminder in die afwesigheid en teenwoordigheid van DHT in LNCaP selle. Hierdie resultaat dui op moontlike interaksie van rooibos met die androgeen (AR) of estrogeen-reseptor-β (ERβ), teenwoordig in LNCaP selle. Rooibos het die katalitiese aktiwiteit van ensieme, wat bydra tot androgeen produksie, geïnhibeer, asook die konjugasie van androgene. Op „n sellulêre vlak, het rooibos die vlakke van PSA-sekresie verminder, wat moontlike interaksie met die AR en ERβ aandui. Hierdie bevindings dui daarop dat rooibos wel n rol het om te speel in die modulasie van aktiewe androgene in die prostaat.

L’objectiu d’aquesta tesi doctoral és profunditzar en els mecanismes que fan de PFKFB3 un gen clau en la reprogramació metabòlica. En concret, s’estudia els mecanismes de connexió de la insulina i el TGF-β1 amb el metabolisme de la glucosa i PFKFB3 en les línies cel·lulars de carcinoma de colon (HT29) i glioblastoma multiforme (T98G), respectivament. PFKFB3 és un enzim bifuncional homodimèric que pertany a la família de la 6-fosfofructo-2-quinasa/fructosa-2,6- bisfosfatasa (PFK-2/FBPasa-2), que controla la conversió entre la fructosa-6-fosfat (Fru-6-P) a fructosa-2,6-bisfosfat (Fru-2,6-P2). Aquest metabòlit és important per a la regulació del flux glicolític ja que és un potent activador al·lostèric de l’enzim limitant de la glicòlisi fosfofructoquinasa-1 (PFK-1). S’ha demostrat que PFKFB3 regula la glicòlisi durant la progressió del cicle cel·lular i el creixement. La seva activitat està regulada per HIF-1α, Akt, p38 i PTEN i es necessari per a la supervivència i creixement de molts tipus de càncer. S’ha descrit que la insulina té un paper important en el creixement cel·lular i la supervivència del càncer. Als anys 70 es va descriure que concentracions fisiològiques d’insulina estimulen la proliferació de les cèl·lules de càncer de mama. També s’ha demostrat que l’expressió de TGF-β en gliomes malignes proporciona avantatges de supervivència de les cèl·lules tumorals potenciant el creixement cel·lular, la migració, la invasió, la angiogènesi, la immunosupressió i les propietats de les cèl·lules mare. A més, la presencia d’insulina i la sobrexpressió de TGF-β s’ha relacionat amb la inducció de l’efecte Warburg. En canvi, no es coneix amb detall els mecanismes moleculars que relacionen tant la insulina com el TGF-β amb una glicòlisi millorada en els sistemes tumorals i, especialment, amb la PFKFB3. Tenint en conte això, la tesi es va organitzar en dos capítols de resultats. En el primer capítol, s’estudia la regulació de PFKFB3 en resposta a insulina en les cèl·lules HT29. Els resultats obtinguts demostren que la insulina provoca un augment significatiu de la concentració de Fru-2,6-P2 i de lacat intracel·lular ja als 30 minuts de tractament, que correlacionen amb l’activació de PFKFB3 i de PFKFB2 per fosforilació a través de la via de PI3K/Akt. En canvi, a temps més llargs, la insulina indueix l’expressió gènica de PFKFB3 i també participa la via de PI3K/Akt. La seqüència localitzada entre els nucleòtids -1269 i -1297 del promotor humà de PFKFB3 és la responsable d’aquesta inducció possiblement a través del factor SREB-1c. Al segon capítol, es presenta la regulació de diversos gens glicolítics, entre ells PFKFB3, en resposta a TGF-β1 en les cèl·lules de glioblastoma T98G. Els resultats obtinguts demostren que PFKFB3 és regulat a nivell transcripcional en resposta a aquest factor a través de l’activació conjunta de la via de Smad, p38 MAPK i PI3K/Akt. També es demostra que PFKFB3 és indispensable per al augment de la capacitat de les cèl·lules de formar colònies en resposta a TGF-β1. En resum, els resultats presentats confirmen que PFKFB3 té un paper important en el metabolisme glicolític en les cèl·lules tumorals. Per tant, conèixer en profunditat els factors que dirigeixen l’expressió de PFKFB3 com els mecanismes moleculars que participen en la seva complexa regulació mitjançant diferents vies de transducció de senyals permet pensar en nous mètodes terapèutics basats en la seva inhibició, i així en la inhibició de l’elevada taxa glicolítica que suposa tenir-la activada.
PFKFB3 is a homodymeric bifunctional enzyme, belonging to the family of 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatases, that controls the conversion of fructose-6-phosphate (Fru- 6-P) to fructose-2,6-bisphosphate (Fru-2,6-P2). This metabolite is important for the dynamic regulation of glycolytic flux by allosterically activating phosphofructokinase-1, a rate-limiting enzyme in glycolysis. Cancer cell lines produce markedly elevated levels of Fru-2,6-P2 when compared to non-malignant cells and it has been shown that PFKFB3 is highly expressed and regulated by HIF-1α, Akt, p38 and PTEN, and required for the survival and growth of multiple cancer types. In the present thesis we investigate the mechanism by which the expression of PFKFB3 gene is regulated by insulin and TGF-β1 in human colon adenocarcinoma (HT29) and glioblastoma (T98G) cell lines, respectively. Although insulin and TGF-β1 contribute to the metabolic reprogramming of cancer cells and tumor-associated stromal cells, little is known of the molecular mechanisms connecting these factors with enhanced glycolysis. We demonstrate that the effect of insulin on Fru-2,6-P2 concentration correlates with changes in PFKFB3 phosphorylation state. Moreover, siRNA experiments confirm that PFKFB3 expression also contributes to the increase in Fru-2,6-P2 concentration induced by insulin. Then, we focus on the signalling cascades activated by insulin in HT29 cells using specific inhibitors of MAPK to analyze, at molecular level, the kinase pathway responsible of PFKFB3 regulation. We report here that insulin produce a significant induction of PFKFB3 mRNA in HT29 cell line and this induction is dependent on the PI3K/Akt pathway. Furthermore, our study reveals a 29nt sequence located at-1279 and -1288 in the PFKFB3 promoter that is implicated in the induction of PFKFB3 in response to insulin. Moreover, we demonstrate that TGF-β1 upregulates PFKFB3 mRNA and protein expression resulting in an increase in Fru-2,6-P2 concentration, glucose uptake, glycolytic flux and lactate production. Moreover, these increases in PFKFB3 mRNA and protein expression and Fru-2,6-P2 concentration were reduced when the Smad3, p38 mitogen- activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways were inhibited. We demonstrate that inhibition of PFKFB3 activity with 3PO or siRNA-mediated knockdown of PFKFB3 significantly eliminated the capacity of the T98G cells to form colonies by TGF-β1, one of the hallmarks of transformation. Taken together, these results suggest a multimodal mechanism of insulin affecting PFKFB3 transcriptional regulation and kinase activation by protein phosphorylation, resulting in an increase in Fru-2,6-P2 concentration and stimulation of glycolysis in cancer cells.

Cisplatin is one of the most potent anticancer agents used in the treatment of various solid tumors. Unfortunately the onset of resistance is the main limit of this therapy and severely compromises the treatment effectiveness. Although several studies regarding cisplatin resistance have been performed, the molecular mechanisms are not completely understood. Classically, cisplatin is studied as a DNA-damaging chemotherapy agent, but more recent investigations showed that only 5-10% of intracellular platinum is bound to nuclear DNA, while the great majority of the intracellular drug can interact with a variety of cellular component including proteins, RNA and mitochondrial DNA. MtDNA, unlike nDNA, does not possess efficient repair systems; therefore it is more susceptible to the onset of mutations often associated to cancer development, loss of tumor suppressor, activation of oncogenes and mitochondrial dysfunctions related with an increase of glycolytic activity. The Warburg effect indicates the alteration of energetic metabolism used by tumor cells as a strategy to adapt and grow independently from the substrate availability. This evidence suggested us to verify the hypothesis that a similar metabolic strategy might be of relevance in cisplatin resistance. Therefore, our aim was to investigate the energetic metabolism and the mitochondrial dynamic of cisplatin-resistant and sensitive cancer cells with different experimental approaches, in order to reveal targets useful to overcome the resistance. In our laboratory we have already revealed that cisplatin resistant ovarian cancer cell line C13, as compared to sensitive line 2008, exhibits metabolic changes. Indeed, resistant clone showed a different mitochondrial and metabolic profile characterized by an increase of glucose and glutamine uptake, a decrease of the mitochondrial membrane potential and mitochondrial mass. In this scenario, we proceeded to phenotype other cancer cells that present acquired or intrinsic resistance in order to identify new targets to sensitize to cisplatin treatment. Our results pointed out alterations in mitochondrial fusion and fission in chemoresistant cancer cells. Moreover, data obtained showed that resistant clones, with an imbalance toward fission process, present a faster mitochondrial turn-over using mitophagy as a mitochondrial quality control mechanism. Furthermore, the data showed a mitochondrial network differently organized in resistant variants underlining a probable implication of dynamic process in resistance mechanisms. Having regard to the data about metabolic reprogramming, breast cancer cells that have an innate resistance to cisplatin were evaluated. The expression of c-Myc nuclear transcription factor, involved in the metabolic reprogramming of tumor cells, has been evaluated highlighting a different expression of some of its target genes involved in the glycolylisis and glutaminolysis, besides an increased dependency of glucose in cisplatin resistant cells.
Il cisplatino è uno dei più potenti agenti antitumorali utilizzati nel trattamento di vari tumori solidi. Purtroppo l'insorgenza della resistenza è il limite principale di questa terapia e compromette gravemente l'efficacia del trattamento. Anche se sono stati eseguiti numerosi studi per quanto riguarda la resistenza al cisplatino, i meccanismi molecolari non sono del tutto chiari. Classicamente, il cisplatino è studiato come agente chemioterapico che crea danno a livello del DNA, ma studi più recenti hanno dimostrato che solo il 5-10% del platino è legato al DNA nucleare, mentre la maggior parte del farmaco intracellulare può interagire con diverse componenti cellulari tra cui proteine, RNA e DNA mitocondriale. Il DNA mitocondriale, a differenza del DNA nucleare, non possiede sistemi di riparazione efficienti ed è quindi più suscettibile alla comparsa di mutazioni spesso associate allo sviluppo del cancro, alla perdita di oncosoppressori, attivazione di oncogeni e ad alterazioni della funzionalità mitocondriale correlata ad aumento dell'attività glicolitica. L'aumento della glicolisi anaerobica, anche in presenza di alte concentrazioni di ossigeno (effetto Warburg), è l'alterazione del metabolismo energetico utilizzata dalle cellule tumorali come strategia per adattarsi e crescere in modo indipendente dalla disponibilità del substrato. Queste evidenze scientifiche ci hanno suggerito di verificare l'ipotesi che una simile strategia possa essere rilevante nell'insorgenza della resistenza al cisplatino. Pertanto, lo scopo di questo studio è stato quello di indagare il metabolismo energetico e la dinamica mitocondriale delle cellule tumorali sensibili e resistenti al cisplatino con diversi approcci sperimentali, al fine di rivelare utili targets per superare questa importante forma di resistenza. Nel nostro laboratorio abbiamo già dimostrato che la linea di carcinoma ovarico resistente al cisplatino C13, rispetto alla linea sensibile del 2008, presenta cambiamenti metabolici. Infatti, il clone resistente ha mostrato un profilo mitocondriale e metabolico differente, caratterizzato da un aumento della dipendenza da glucosio e glutammina, una diminuzione del potenziale di membrana e della massa mitocondriale. In questo scenario, lo studio ha proseguito con la valutazione del meccanismo di resistenza cisplatino fenotipizzando altre cellule tumorali che presentano resistenza acquisita o intrinseca. I nostri risultati indicano una alterazione dei meccanismi di fusione e fissione mitocondriale nelle cellule tumorali chemioresistenti. I dati ottenuti hanno dimostrato che i cloni resistenti, che presentano uno squilibrio verso processo di fissione, attivano un turn-over mitocondriale più veloce, utilizzando la mitofagia come meccanismo di controllo della qualità mitocondriale. Inoltre, i dati ottenuti hanno mostrato un network mitocondriale diversamente organizzato nelle resistenti sottolineando una probabile implicazione della dinamica mitocondriale nei meccanismi di resistenza. Per quanto riguarda i dati relativi alla riprogrammazione metabolica, sono state prese in esame cellule del cancro al seno che hanno una resistenza innata al cisplatino. È stata valutata l'espressione del fattore di trascrizione c-Myc che è coinvolto nella riprogrammazione metabolica delle cellule tumorali, per di più si è evidenziata una diversa espressione di alcuni geni bersaglio di c-Myc coinvolti nella glicolisi e glutamminolisi, oltre che una maggior dipendenza dal glucosio nelle linee resistenti di carcinoma al seno.

The endothelium plays a pivotal role in the development of cardiovascular disease (CVD) and emerging evidence indicates that pathological blood vessel responses and endothelial dysfunction are associated with metabolic alterations in endothelial cells (ECs). This project aims at performing a complete characterization of the metabolic profiles of an endothelial pathological Acute Myocardial Infarction (AMI) model of 8 patients. The results discussed throughout this thesis are part of this attempt, and brought to the identification of the insights and causes of the AMI pathology, as a consequence of the metabolic alterations related to the endothelium dysfunction which occurs in patients. Due to patients variability, finding a single and clear mechanism among all is quite hard to grasp. However, we have been able to find some metabolic feature to be exploited as possible biomarker for the identification of this CVD. Patients cells presented a low proliferation rate and unveiled a dependence to mitochondrial metabolism, which results in an increased ROS-oxidative stress. Consequently, these cells express increased level of glutathione that supplies the antioxidant defense and prevent ROS (Reactive oxygen species) accumulation. Glutamine seems to play a key role in this AMI model; first of all it is necessary for these cells to display a proper mitochondrial function and in addition, it is required for the synthesis of glutathione as antioxidant against the high level of ROS detected. Additionally, finding a higher content of glutaminase C (GAC) in patients, has opened the possibility that these cells rely more on glutaminase reaction for their survival, and this dependence gathered with the augmented need to neutralize the acidic pH , which results from the increased lactate production, by the ammonia molecules released from glutamine metabolism. This findings point that in AMI model is occurring a metabolic adaptation similar to the Warburg effect, usually described in cancer cells. In the frame of finding the same origin among different pathologies ,in the second part of this work we focused on the crosstalk between dysfunctional endothelium and tumor microenvironment. Moreover, nowadays there is an increasing interest in supporting the existence of a link between cardiovascular pathologies and cancer. One of the wide possibilities which lies these two lethal morbidities is a an alteration of the DNA repair system, crucial for the recovery of the healthy cells against the diseased ones, when a pathological event takes place. Through this study we found that: alternative splicing governs cell‐type regulated expression of variant forms of mRNAs and their encoded proteins that exert differential function. So, employing cancer cell model in which distinct tumor cell subpopulations display differentiated epithelial or mesenchymal phenotype, we have identified alternatively spliced mRNAs with potential impact on the self‐renewal capacities of these cell subpopulations. More in details, among all the genetic characters which can be involved in this process, we provide evidences that RAP80 (UIMC1), an adaptor protein with critical functions in homology-dependent DNA repair (HDR), is expressed as alternatively spliced isoforms in epithelial and mesenchymal cells, as a function of ESRP1/2 expression. More specifically, we have found that the ratio of expression of a full-length isoform to a short isoform of RAP80 is significantly higher in epithelial cells than mesenchymal cells in a prostate cancer cell model for EMT. RAP80 contains a region required for interaction with Abraxas , a core component of the BRCA1-A complex involved in DNA-damage repair. We propose that the ratio of full-length RAP80 to the short isoform lacking AIR is a new mechanism for the regulation of HDR mediated by BRCA1. A higher long/short RAP80 isoform ratio will favor, and lower ratios will counter, the recruitment of BRCA1-A complexes to DSBs.

This study investigated molecular signals involved in sustaining CSCs and ways to target their activity using sFRP4 alone or in combination with chemotherapeutic drugs, possible involvement of methylation-mediated silencing of the sFRP gene family, and metabolic reprogramming by relocating metabolic flux to glycolysis or oxidative phosphorylation. sFRPs have an ability to sensitize tumour cells to chemotherapeutic drugs, thereby enhancing cell death. Altogether, sFRP4 treatment compromises the cell proliferation, and critically affects the cell survival mechanisms of CSCs.

Thesis advisor: Thomas N. Seyfried
Two major metabolic phenomena observed in cancer cells include the Warburg effect and Crabtree effect. The Crabtree effect is the in vitro inhibition of respiration by glucose. The influence of glucose on the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of tumorigenic RAW264.7 and VM-M3 macrophage cells, as well as non-tumorigenic BV-2 microglia cells, was studied using the Seahorse XF96 extracellular flux analyzer. RAW264.7, VM-M3, and BV-2 cells incubated in glucose medium displayed a significantly lower OCR and higher ECAR compared to cells incubated in no glucose medium. Furthermore, when glucose medium was added to the RAW264.7 and BV-2 cells in real-time using the Seahorse XF96 injection ports, a rapid decrease in OCR and increase and ECAR was observed. Therefore, RAW264.7, VM-M3, and BV-2 cells display a robust Crabtree effect in vitro, as assessed by OCR and ECAR. Additionally, it is important to consider the Crabtree effect when studying in vitro energy metabolism of all cell and tissue types. It was also found that the elimination of the Crabtree effect through glucose deprivation resulted in dynamic cardiolipin (CL) fatty acid changes in VM-M3 cells. VM-M3 cells incubated in 10 mM glucose medium for four hours displayed a short-chain, saturated (immature) CL fatty acid composition, while VM-M3 cells incubated in no glucose media for four hours displayed long-chain, unsaturated (mature) CL fatty acid composition. Cardiolipin (CL) is a phospholipid highly enriched in the inner mitochondrial membrane. Mature, long-chain, unsaturated CL molecular species are involved in maintaining mitochondrial function and membrane integrity. Overall, these data suggest that CL fatty acid composition may function as a structural component of the Crabtree effect in vitro. The Warburg effect, or aerobic glycolysis, is the observation that tumor cells consume less oxygen and more glucose than normal, untransformed cells in the presence of oxygen. It has been shown that immune cells display a Warburg effect upon activation by changing their core metabolism from oxidative phosphorylation to glycolysis. In this study, it was observed that both RAW264.7 macrophage cells and BV-2 microglia cells display a significantly lower OCR and higher ECAR following LPS-activation. However, this observation is dependent on the concentration of LPS. Therefore, these data suggest that both RAW264.7 and BV-2 cells display a LPS concentration-dependent change in metabolism from oxidative phosphorylation to glycolysis upon LPS-activation in vitro. The in vitro lipid profiles that resulted from the Crabtree effect and the LPS-activated Warburg effect were also studied in the RAW264.7 cell line. The lipids phosphatidylserine (PS) and cardiolipin (CL) displayed the most robust changes in the RAW264.7 cells. Both PS and CL have been shown to be associated with cellular respiration
Thesis (MS) — Boston College, 2016
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

Abstract Malignant tissue growth induces marked biochemical and structural changes in the extracellular matrix of the tumour and its surrounding tissues. In the present study, we evaluated the prognostic value of the serum concentration of the markers of synthesis of type I collagen (PICP, PINP) and type III collagen (PIIINP) as well as the marker of type I collagen degradation (ICTP) and compared them with the conventional indicators of prognosis (clinical stage, grade of differentiation, histological subtype, residual tumour load and the age of the patient). The prognostic value of peritoneal cytological findings at operation was an additional object in our studies. High preoperative serum ICTP (>5.6μg/L) and PIIINP (>3.2μg/L) concentrations and a low PICP:PINP ratio (>2) correlated with poor prognosis in ovarian carcinoma in univariate analysis and in multivariate analysis when each variable was analyzed separately with the conventional factors. However, ICTP concentration was the only prognostic variable in multivariate analysis including PIIINP, PINP, ICTP and CA125. When analyzed with conventional prognostic factors (clinical stage, grade, residual tumour, presence of ascites, histology), clinical stage and ICTP were independent indicators of prognosis. In addition, malignant cells in the peritoneal fluid aspirate at primary operation, grade and the age of the patient predicted poor prognosis in multivariate analysis. Postoperative serum ICTP concentration 9-months after the operation was the strongest prognostic factor as compared to the preoperative ICTP and CA125 values and clinical variables. These results indicate that serum collagen metabolites, especially ICTP, are indicators of prognosis in epithelial ovarian cancer. The present ICTP-test does not detect the degradation products of immature type I collagen, the dominating form in ovarian cancer tissue. Therefore, the excess ICTP in invasive ovarian cancer might originate through the degradation of trivalently matured collagens in non-malignant tissues surrounding the malignancy. ICTP may thus be an indicator of invasive properties of the tumor and its determination opens up new perspective to predict the clinical outcome of ovarian cancer.

Cytotoxic chemotherapeutic agents have a major role in the treatment of cancers. However, many cytotoxic agents have a narrow therapeutic window with best treatment response achieved only within a small range of drug concentrations.

Darmkrebszellen und T-Zellen regulieren ihren zentralen Kohlenstoffmetabolismus um ihren anabolen Bedarf zu erfüllen. Tumorzellen mit einer KRAS- oder BRAF-Mutation zeigen ein schnelles Wachstum, welches eine Umprogrammierung des Metabolismus vor aussetzt. Der mitochondriale T-Zellen-Aktivierungsinhibitor (TCAIM) ist bekannt dafür die mitochondriale Zellstruktur zu beeinflussen. Der Einfluss auf den Metabolismus nicht klar. In dieser Arbeit präsentiere ich erstmalig ein mathematische Model des zentralen Kohlen stoffmetabolismus in Darmkrebszellen und T-Zellen. Mithilfe dieses Modells analysiere ich, wie sich die Regulation in ähnlichen Zelllinien unterscheidet. In Bezug auf die Darm krebszellen vergleiche ich BRAF-(CaCO2-BRAFV600E), KRAS-(CaCO2-KRASG12V) mu tierte Zelllinien mit einer Basiszelllinie (CaCO2-control) und zeige, dass der Kohlenstoff metabolismus in BRAF-mutierten Zellen im Vergleich zu den beiden übrigen Zelllinien herabreguliert ist. Das Modell bestätigt außerdem, dass der Monocarboxylattransporter (MCT) in den Darmkrebszellen eine wichtige Rolle, insbesondere in den KRAS mu tierten Zellen, spielt. In T-Zellen zeigt der Vergleich von Wildtypzellen (CD8 T-Zellen) mit TCAIM homozygoten Zellen (TCAIM homozygote CD8 T-Zellen), dass der Kohlen stoffmetabolismus in zweiteren überwiegend herabreguliert und weniger aktiv ist. Diesen Effekt konnte ich durch die Analyse von RNASeq-Daten der jeweiligen Zelltypen bestä- tigen. Des Weiteren stelle ich fest, dass sich der Tricarbonsäurezyklus umkehrt, wenn durch die Glykolyse nicht ausreichend Laktat exportiert und die Biomasseproduktion unterstützt werden kann. Meine Arbeit stellt damit insgesamt einen neuartigen Ansatz zur Integration von Meta bolomik und RNAseq Daten dar, um die Regulation des zentralen Kohlenstoffmetabo lismus zu verstehen.
Colon cancer cells and T cells regulate central carbon metabolism to meet their anabolic needs. In KRAS and BRAF tumors, metabolic reprogramming is a premise to support rapid proliferation. In T cells, the mitochondrial T cell activation inhibitor (TCAIM) is known to affect mitochondrial morphology but its effect on cellular metabolism is not well understood. Via mathematical modelling, I investigate the differential regulation of closely related cell lines. I present the first mathematical model for colon cancer and T cell metabolism, unraveling differential regulation between related cell lines. The model shows that CaCO2-BRAFV600Ecells are mostly downregulated compared to CaCO2-KRASG12Vand CaCO2-control. Additionally, it demonstrates the critical role of monocarboxylate transporter (MCT), especially for CaCO2-KRASG12V. Concerning T cells, I compare wild-type T cells to homozygous TCAIM T cells. This unveils that TCAIM homozygous cells have a mostly downregulated TCA cycle, validated by RNASeq data, and are less metabolically active than wild-type T cells. Furthermore, if the glycolytic flux is not sufficient to support lactate export and biomass production, the model reveals that the TCA cycle is reversed as it requires less regulation. Taken together, this work presents a novel approach to integrate data referring to metabolic and genetic regulation of metabolism. On this basis, we can now better discriminate the metabolic capacity of CaCO2-control, CaCO2-BRAFV600E, CaCO2-KRASG12V, wildtype CD8 T cells, and homozygous TCAIM CD8 T cells.