Academic literature 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.<br>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.<br>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.<br>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.