Дисертації з теми "Cancer drug resistance, tumor metabolism"

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.

Chemoresistance remains a major clinical obstacle to effective management of ovarian cancer. Cancer stem cells (or tumor-initiating cells, TICs) have been discovered recently, and have played a pivotal role in changing the view of cancer development; however, the molecular mechanisms by which these cells escape conventional therapies remain elusive. In this study, TICs were isolated from ovarian cancer cells as tumor spheres with specific stem properties under TIC-selective conditions. Unlike non-TICs, TICs strongly express stem cell factor (SCF) and c-Kit. Blocking SCF-c-Kit by SCF neutralizing antibodies, c-Kit small interfering RNA (siRNA) or imatinib (Gleevec), a clinical drug that inhibits c-Kit signaling, significantly inhibited TIC proliferation. Although cisplatin and paclitaxel killed the non-TICs, they did not eliminate TICs. Importantly, the combination of cisplatin/paclitaxel with c-Kit siRNA or imatinib inhibited the growth of both non-TICs and TICs. Similar results were obtained when patient-derived TICs were used. The findings also indicate that tumor-predisposing microenvironment, such as hypoxia, may promote ovarian TICs through upregulating c-Kit expression. Furthermore, I have showed that c-Kit expression induced activation of Phosphatidylinositol 3-kinases (PI3K)/Akt, -catenin, and ATP-binding cassette G2, which could be reversed by treatment with the PI3K/Akt inhibitor or -catenin siRNA. I further studied potential gene expression in TICs using cDNA and microRNA (miRNA) microarrays. The result from these microarrays provided a general profile in gene expression of TICs compared with the bulk tumor cells. In particular, let-7a, b, and c were shown to be downregulated in TICs compared to bulk tumor cells, suggesting that their loss may contribute to ovarian cancer development. Together, this study reveals a previously undescribed therapeutic effect of SCF-c-Kit signaling blockade to prevent ovarian cancer progression by eliminating TICs and the altered genes or miRNAs may represent possible molecular targets.
published_or_final_version
Biological Sciences
Master
Master of Philosophy

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2007.
Committee Chair: Nie, Shuming; Committee Member: Lyon, L. Andrew; Committee Member: McIntire, Larry V.; Committee Member: Murthy, Niren; Committee Member: Prausnitz, Mark R.

La récidive tumorale est l'un des principaux obstacles à surmonter à l'avenir pour améliorer la survie globale des patients atteints de cancer du côlon (CCR). Les échecs thérapeutiques observés chez les patients sont compatibles avec une accumulation de cellules souches cancéreuses (CSCs) résistantes aux médicaments. Dans cette étude, nous démontrons que le récepteur nucléaire PXR (NR1I2) agit comme un régulateur important de la chimiorésistance des CSCs coliques et de leur capacité à initier la rechute tumorale après traitement. Nous avons d'abord montré que l'expression de PXR augmente avec celle de certains marqueurs des CSCs dans des cellules cancéreuses de patients CCR traitées par chimiothérapies. Nous avons constaté que PXR est préférentiellement exprimé dans les CSCs coliques et qu'il contribue à l'enrichissement des CSCs après chimiothérapies in vitro et in vivo. Par des approches de transcriptomiques, nous avons observé qu'au sein des CSCs coliques, PXR contrôle l'expression d'un large réseau de gènes marqueurs des CSCs coliques, ainsi que des gènes impliqués dans la résistance aux médicaments ou à l'apoptose, ou impliqués dans la dissémination métastatique. Enfin, l'inhibition de PXR par interférence à ARN diminue la survie et auto-renouvèlement des cellules souches cancéreuses du côlon in vitro, ainsi que leur capacité à résister à la chimiothérapie après xénogreffes, conduisant à des retards importants de rechute tumorale après traitements par chimiothérapies in vivo. Cette étude suggère fortement que l'inhibition ciblée de PXR peut représenter une stratégie de traitement néo-adjuvant afin de diminuer la résistance aux médicaments et la récidive des patients CCR via la sensibilisation des cellules souches cancéreuses aux chimiothérapies classiques
Tumor recurrence is one of the major obstacles to overcome in the future to improve overall survival of patients with colon cancer. High rates and patterns of therapeutic failure seen in patients are consistent with a steady accumulation of drug-resistant cancer stem cells (CSCs). Here, we demonstrate that the nuclear receptor PXR (NR1I2) acts as a key regulator of colon CSC chemoresistance and of their ability to generate post-treatment tumor relapse. We first determined that the enrichment of PXR paralleled that of CSC markers upon treatment of colon cancer cells with standard of care chemotherapy. We found that PXR was highly expressed in colorectal cancer cells displaying CSC markers and function and that it was instrumental for the emergence of CSCs following chemotherapy in vitro and in vivo. mRNA profiling experiments in colon CSCs indicated that PXR transcriptionally controls a large network of genes including markers of stemness, genes involved in resistance to drug/apoptosis or migration/invasion. Finally, PXR down-regulation altered the survival and self-renewal of colon CSCs in vitro and hampered their capacity to resist chemotherapy in vivo, leading to significant delays of post-chemotherapy tumor relapse. This study strongly suggests that targeting PXR may represent a novel treatment strategy to prevent drug resistance and recurrence through the sensitization of CSCs to standard chemotherapy. Taken together, our data strongly suggest that PXR plays an instrumental role in the so-called "intrinsic" pan-resistance of CSCs against therapy

Tumor cells exhibit metabolic reprogramming according to microenvironmental scenarios (i.e. stroma composition and/or anticancer drugs burden) to meet their demands for energy, rapid proliferation, metastasis and progression. In our experimental model, a vicious metabolic synergy between CAFs and prostate cancer (PCa) cells has been described as a pivotal engine allowing cancer cells to achieve aggressive features and evolve their malignancy. Such metabolic crosstalk is mainly based on the OXPHOS rewiring of PCa cells induced by highly glycolytic CAFs through the establishment of tumor:stroma lactate shuttle. In the first part of this study, we highlighted a peculiar CAFs conditioning of PCa cells in terms of OXPHOS upgrading and enhancement. Indeed, we observed that CAFs induce a SIRT1/PGC-1α axis activation in PCa cells, leading to the accumulation of mitochondrial ROS and TCA cycle oncometabolites (succinate/fumarate) that are both closely related to EMT engagement and PCa invasiveness. Indeed, we found that CAFs-exacerbated mitochondrial ROS are crucial for the oxidation of critical targets (Src, PKM2) needed for the metabolic reprogramming toward OXPHOS established in CAFs-exposed PCa cells. On the other hand, succinate is able to maintain CAFs-induced HIF-1α activation and the HIF-1- dependent malignant phenotype of PCa cells. Furthermore, we intriguingly observed a mechanism of mitochondrial transfer elicited by CAFs in order to further boost OXPHOS exploitation, mitochondrial ROS generation and invasiveness of PCa cells. Among microenvironmental cues, chemotherapy resistance has been increasingly and finely associated to the metabolic reprogramming of resistant cancer cells. In the second part of this study, we metabolically characterized docetaxel-resistant PCa cells and we clearly outlined a metabolic adaptation of resistant cancer cells compared to the sensitive counterpart. Docetaxel-resistant PCa cells undergo a Warburg escape towards OXPHOS addiction in order to ensure metabolic advantages during acquisition of resistant phenotype. Together with lactate and glucose, we also found an higher glutamine mitochondrial exploitation by docetaxel-resistant cells. Furthermore, we appreciated a role of CAFs in modulating the response to drug exposure by protecting sensitive and resistant PCa cells. We found that such metabolic/resistant phenotype can be counteracted either by metformin (or other mitocans) treatment or by overexpressing miR-205, a downregulated miRNA orchestrating prostate tumor:stroma crosstalk. Taken together, all the data obtained in our study highlight the role of OXPHOS as an important shared metabolic state between chemotherapy resistance and symbiosis with microenvironment PCa cells.

Metastasis and drug resistance represent the two main causes of therapeutic failure in oncology. In the present dissertation, the interplay between them has been interrogated using metabolomics, systems biology and biophysical approaches, in an attempt to find common phenotypic adaptations and metabolic vulnerabilities of metastatic and resistant cancer cells, potentially exploitable in novel combination therapies. The obtained results unveil that highly metastatic e-CSC phenotypes of CRPC present particular metabolic vulnerabilities that can potentially lead to establishing putative biomarkers and metabolic targets that are specific for PCa subsets with high tumorigenic potential. Moreover, by generating isogenic cell models of multiplatinum resistance in CRPC and CRC we also identified that metastatic solid tumors with originally opposed metabolic profiles can lead to different metabolic adaptations as they acquire platinum resistance, but that a common metabolic signature of acquired platinum resistance arises, which also includes alterations in proline and one carbon metabolism, glutathione synthesis and ROS production. In addition to characterizing in deep the metabolic reprogramming associated to resistance to platinum compounds already used in the clinics, we also explored the possibility to design of novel platinum drugs able to counter platinum-resistant tumors. In this regard, we identified novel families of cyclometallated platinum (II) and platinum (IV) compounds exhibit strong antiproliferative effects in the low micromolar range against a wide variety of solid tumors. The leading compounds of each series also exhibit remarkable selectivity for cancer cells and the capacity to arrest the cell cycle at S and G2/M phases, induce apoptosis and increase intracellular ROS levels. The multiple combinations of equatorial and axial ligands explored in this work, allowed us to conclude that octahedral Pt (IV) compounds containing tridentate [C,N,N’] ligands are the optimal design to improve efficacy and selectivity against cancer cell lines. Remarkably, we have also identified that these novel cyclometallated Pt (IV) exhibit a complete absence of cross-resistance with the platinum-resistant CRC and CRPC models generated in this work. Indeed, platinum-based chemotherapy can severely affect internal cell architecture, causing fluctuations in the levels of macromolecular crowding inside cells and having an impact on the supramolecular organization of cell metabolism. In turn, this has been proved to have a profound impact on the kinetic behavior of metabolic enzymes that govern the rate of metabolic pathways that we have identified as important throughout this work. Thus, we have explored the kinetic behavior of lactate dehydrogenase (LDH), as a representative of aerobic glycolysis, under the presence of globular obstacles that do not introduce specific interactions with either LDH or its substrates, dextran polymers, obtaining that LDH kinetics is impaired in an obstacle size- and concentration-dependent manner. Additionally, we unveiled that LDH kinetic behavior shifts from activation control to diffusion control as crowding increases, implying that the behavior of LDH inside cells could be significantly different than previous dilute solution kinetic studies of this enzyme had predicted. On the other hand, the effect of macromolecular crowding on glutaminolysis had not been explored prior to this work. By studying the kinetic behavior of glutamate dehydrogenase (GLDH) in crowded media and characterizing its negative cooperativity, we have concluded that its kinetics is impaired by crowding in an obstacle size- and concentration-dependent manner, but that negative cooperativity is not significantly altered by macromolecular crowding. The actual impact of macromolecular crowding on cell metabolism has been scarcely explored and we are just scratching the surface of the understanding of the multiple implications that this phenomenon may entail for cell physiology and, in particular, for the metabolic alterations of cancer cells. Our observations throughout this work will hopefully have contributed to set grounds onto this enthralling enterprise, as long as meaningfully contributed to encounter valuable therapeutic tools against metastatic CRPC and CRC that can circumvent platinum resistance, both with new generations of platinum compounds and novel metabolic targets that selectively target metastatic solid tumors.

Breast cancer is a highly heterogeneous disease, having not only several intrinsic sub-types but also significant sub-clonal heterogeneity within tumors. Intra-tumor heterogeneity can have profound impact on tumor evolution, disease progression and response to therapy. Furthermore, these phenomena can also be influenced by interactions of cancer cells with those of the microenvironment, thereby adding an extra layer of complexity to the study of tumor biology. To investigate the impact of sub-clonal heterogeneity on tumor phenotypes, we developed a heterogeneous mouse xenograft model of breast cancer. Our model revealed that tumor growth can be driven by a minor clone, expressing IL11, in a non-cell autonomous fashion mediated through the microenvironment. We also found that non-cell autonomous driving and clonal interference stabilizes sub-clonal heterogeneity, thereby enabling inter-clonal interactions leading to new phenotypic traits. Utilizing the same model, we identified cooperative interactions between IL11- and FIGF- expressing sub-clones that enhance the metastatic behavior of the tumor as a whole. We found that metastatic cooperation between these two populations result in larger and heterogeneous lung metastasis. Using expression profiles from primary tumors and corresponding metastatic lesions, we identified several key immune-regulatory and extracellular matrix (ECM) remodeling pathways that promote metastasis in our model system. Lastly, we examined heterotypic interactions between tumor cells and cancer associated fibroblasts (CAFs) to understand the mechanism of resistance to lapatinib. Using a 3D co-culture model, we identified significant sub-type-specific changes in gene expression, metabolic, and therapeutic sensitivity profiles of breast cancer cells induced by CAFs. We identified JAK2/STAT3 pathway and CAF-secreted hyaluronan as major factors contributing to CAF-mediated protection. We also found that close spatial proximity to CAFs impacts therapeutic responses by affecting proliferation rates of cancer cells. In summary, we have used in vitro and in vivo models systems to identify key interactions within populations of tumors cells, as well as between tumor microenvironmental components and cancer cells, to identify mechanisms that influence tumorigenesis, metastasis and drug response. We believe that these findings will increase our understanding of breast cancer heterogeneity and enable us to design better therapeutic regimens to eradicate the disease.
Medical Sciences

Cancer cells have been known for some time to have very different metabolismas compared to that of normal non proliferating cells. As metabolism is involvedin almost every aspect of cell function, there has been a recent resurgence ofinterest in inhibiting cancer metabolism as a therapeutic strategy. Inhibitors thatspecifically target altered metabolic components in cancer cells are being developedas antiproliferative agents. However, many such inhibitors have not progressedinto the clinic due to limited efficacy either in vitro or in vivo. In this study weexplore the hypothesis that this is often due to the robustness of the metabolicnetwork and the differences between individual cancer cell lines in their metaboliccharacteristics. We take a systems biology approach. We investigate the cellular bioenergetic profiles of a panel of five non-small celllung cancer cell lines before and after treatment with a novel inhibitor of theglutaminase-1 (GLS1) enzyme. Additionally, we explore the effects of this inhibitoron intracellular metabolism of these cell lines as well as on the uptake and secretionof glucose, lactate and amino acids. To be able to do the latter robustly, wehad to modify the experimental assay considerably from procedures that seemto be standard in the literature; using these earlier procedures the metabolicenvironment of the cells was highly variable, leading to misleading results onthe metabolic effects of the inhibitor. We reduced cell density, altered mediumvolume and changed the time-window of the assay. This led to the cells growingexponentially, appearing indifferent to the few remaining changes. In this newassay, the metabolic effects of the glutaminase inhibitor became robust. One of the most significant results of this study is the metabolic heterogeneitydisplayed across the cell line panel under basal conditions. Differences in themetabolic functioning of the cell lines were observed in terms of both theirbioenergetic and metabolic profile. The amount of respiration attributed tooxidative phosphorylation differed between cell lines and respiratory capacity wasattenuated in most cells. However, the rate of glycolysis was similar betweencell lines in this assay. These results suggest that the Warburg effect arisesthrough a greater diversity of mechanisms than traditionally assumed, involvingvarious combinations of changes in the expression of glycolytic and mitochondrialmetabolic enzymes. The effects of GLS1 inhibition on cellular bioenergetics and metabolism alsodiffered between cell lines, even between resistant cell lines, indicating that theremay also be a diversity of resistance mechanisms. The metabolomic response ofcell lines to treatment suggests potential resistance mechanisms through metabolicadaptation or through the prior differences in the metabolic function of resistantcell lines. Part of the metabolome response to GLS1 inhibition was quite specificfor sensitive cells, with high concentrations of IMP as the strongest marker. Our results suggest that the metabolome is a significant player in what determinesthe response of cells to metabolic inhibitors, that its responses differ between cancercells, that responses are not beyond systems understanding, and that thereforethe metabolome should be taken into account in the design of and therapy withanti-cancer drugs.

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.

Protein tyrosine phosphorylation in eukaryotes is a key mechanism for cellular control, since it is involved in several processes, such as cellular metabolism, proliferation, differentiation and oncogenic transformation. A fine balancing of cellular protein tyrosine phosphorylation levels is determined by regulating the activities of protein-tyrosine kinases and/or protein-tyrosine phosphatases (PTPs) (Alonso et al., 2004). The PTP superfamily comprises almost 70 enzymes that, despite very limited sequence similarity, share a common CX5R active-site motif and an identical catalytic mechanism. LMW-PTPs are a group of cytosolic enzymes of 18 kDa that are widely expressed in different tissues. They are represented by two most abundant isoforms (arised from mutually exclusive alternative splicing), named fast and slow, according to their electrophoretic mobility (Tabernero et al., 2008; Wo et al., 1992; Dissing et al., 1991; Xing et al., 2007). LMW-PTP interacts with several receptor tyrosine kinases and docking proteins that may be involved in cancer progression, but the identification of functionally relevant interactors is not yet conclusive. Several works have shown how LMW-PTP overexpression is associated with human tumorigenesis, especially in breast, colon and kidney. In fact, results obtained with a wide array of human carcinomas indicate a significant increase in the expression of LMW-PTP in tumor tissue and a correlation between higher expression of LMW-PTP on one hand and worse prognosis and reduced survival on the other (Malentacchi et al., 2005). LMW-PTP acts also as a positive regulator of tumor onset and growth in in vivo animal models. (Chiarugi et al., 2004). Moreover there are many findings that LMW-PTP plays a key role in chemoresistance: Ferreira et al., demonstrated how the phosphatase overexpression induce resistance towards vincristine and imatinib, in a leukemia cell lines (Ferreira et al., 2012). Our studies were conducted on a Melanoma cell line, A375, to study in depth the role of LMW-PTP in skin tumor onset, in order to elucidate the molecular mechanism and pathways in which this enzyme is involved and also in the view of identifying new possible therapeutic targets. Using transient silencing technique, we tested the apoptotic response of A375 cells: citofluorimetric analysis showed that upon phosphatase silencing, treatment with 5FU increase cell mortality up to 50%. In agreement with these results, western blot analysis of apoptotic markers (Caspase3, Bcl2 and Bim) confirmed that LMW-PTP silencing sensitize cancer cells towards chemotherapic drugs. Looking for a link between LMW-PTP and apoptosis, we speculate that the phosphatase exerts its action through Cav1-Bcl2 pathways. In fact the phosphorylation on Tyr14 of Cav-1 leads to Bcl2 degradation, increasing apoptosis. Our data demonstrate how LMW-PTP knocking-down lead to Cav-1 Tyr phosphorylation, and Bcl-2 down-regulation. Several studies have demonstrated how tumor cells can develop chemoresistance increasing the activity or the expression level of membrane transporters that actively expel chemotherapy drugs from inside. Our experiments suggest that LMW-PTP may confer resistance against anti-tumoral drugs, increasing the activity of some of these membrane transporters, thus limiting the toxic effect of chemotherapy. One of the most important treatment for Melanoma is Radiotherapy. Using therapeutic doses of radiation (2Gy), we demonstrated that silenced cells were more responsive to this treatment, with respect to control samples, confirming that LMW-PTP plays a key-role not only in chemio- but also in radio-resistance. Furthermore resistant cancer cells, usually, show common phenotypes: one of this features is self-renewal capability. Colony formation assay demonstrates that melanoma cells are able to reform new colonies, even when cells are exposed to a damage, such as 5FU treatment or 2Gy irradiation. When LMW-PTP is knocked-down and cells treated with 5FU or radiated, we observed no colony formation: we can assume that LMW-PTP silencing leads to the loss of some characteristics of self- renewal, fundamental for metastasis. LMW-PTP exerts its action also through some molecules implicated in cell migration and adhesion. Adhesion and Detachment assay showed that LMW-PTP silencing lead cells to be more adherent to a substrate. Accordingly Wound Healing Assay demonstrated that melanoma cells are able to migrate and refill the wound very quickly. We tested also the Invasiveness of A375 cells: silenced cells showed a decreased ability to invade, respect to untreated cells: in fact LMW-PTP down-regulation lead to a MMP-9 reduction. Considering the importance of LMW-PTP in tumor onset we investigate the possibility to inhibit this enzyme as a new therapeutic approach.. Previous work demonstrated that a natural compound, Morin, is an enzymatic inhibitor of LMW-PTP. Morin is not toxic for Melanoma cells, but its combination with 5FU, causes a strong increase of apoptotic cells. Interestingly, this sensitization is not reproducible in non tumoral cell line, such as C2C12 myoblast: this co-treatment could be specific for cancer cells. Furthermore our studies demonstrate that Morin doesn’t act only as an inhibitor of LMW-PTP: western blot analysis showed that the flavonol lead to phosphatase degradation, in a dose and time-dependent manner. This down-regulation may be due to different mechanism, but since this effect start 2h after Morin incubation, we hypothesized that a proteasome activation may be involved. Incubation with Morin together with a proteasome inhibitor (MG132), confirmed our hypothesis. The enhancement of chemotherapic action, obtained with Morin is reproducible even with Radiation therapy. A375 cells pre-treated with Morin and then radiated with 2Gy, decrease dramatically their viability. Moreover irradiation exposition didn’t influence self-renewal capability of Melanoma cells: on the contrary, Morin treatment before irradiation is able to affect the formation of new colonies after the treatment. LMW-PTP is involved in cell-adhesion, migration and invasion: in fact Morin can affect this markers. After Morin treatment Melanoma cells are less able to migrate and invade; furthermore cells increase the number of focal adhesions. To better understand the role of LMW-PTP in tumor onset, we looked for new substrates. To analyze this aspect we studied the phospho-proteomic profile of silenced Melanoma cells. Through these analysis we identified different proteins showing an higher levels of Tyr phosphorylation, upon LMW-PTP silencing. One of the new substrates identified with this experimental approach is Annexin-A1, a protein involved in apoptosis: this finding suggest a further possible link between LMW-PTP and apoptosis resistance, a link that will be further investigated. More interestingly, four of the proteins identified with the proteomic analysisbelong to glycolysis pathway, PKM2, α-Enolase, GAPDH and TIM. To confirm this results we performed immune and co-immunoprecipitations: these analyses confirmed that the mentioned enzymes get in contact with LMW-PTP and, presumably, are direct substrates of the phosphatase. It is well known that the “Warburg effect” causes alteration in cancer cell energetic metabolism, leading cells to consume large quantity of glucose, metabolizing it predominantly through glycolysis, and producing high level of lactate. Considering that four of these substrates are involved in glycolysis pathway, we tested some metabolic parameters. When LMW-PTP is silenced A375 cells consume less O2, and consequently their glucose up-take is higher, producing more lactate respect to controls. Pyruvate kinase controls the final and rate-limiting reaction of glycolysis: PKM2 undergoes conformational conversion between a tetrameric/full active and a dimeric/less active state. The conversion to a less active state, induced by Tyr phosphorylation, confers to PKM2 “non-metabolic” abilities. Indeed, PKM2 translocates into the nucleus and acts as a transcriptional co-activator of β-catenin and hypoxia-inducible factor 1α (HIF-1α), cooperating to control cell proliferation and glucose catabolism, respectively. Western Blot Analysis of the Glucose transporter GLUT-1 and Hesokinase II, confirmed our previous data. When LMW-PTP is down-regulated both proteins had an increased expression level, explaining, at least in part, the glycolytic metabolism showed by silenced cells. Moreover LMW-PTP influences not only the Tyr phosphorylation state of PKM2, but also its expression level: in fact when the phosphatase is down-regulated PKM2 protein level is higher. In conclusion, our results demonstrated that LMW-PTP plays a key role in chemo and radio-resistance acquisition of Melanoma cells: in fact when the phosphatase is knocked-down cells are more responsive to therapy. Considering that gene silencing cannot be used in patients, the discovery that Morin is able to reproduce the same phenotype, open new possibilities for therapies. Moreover LMW-PTP seems to influence metabolism of Melanoma cells, a parameter often deregulated in cancer cells. Further studies will be conducted to better characterize the role of this enzyme, and its role in the regulation of tumor metabolism.

Metabolic reprogramming in cancer cells has recently been connected to IF1, a key regulator of ATP synthase activity, which is found to be overexpressed in many human carcinomas. Considering the pivotal role of mitochondria inside the cells, mitochondrial alteration is crucial for tumors to grow and IF1 may help tumor progression by conserving cellular ATP in hypoxic conditions. Although the inhibitor has being deeply investigated, its role in tumorigenesis and/or cancer progression is still debated. Therefore, we prepared stable IF1-silenced clones from the human osteosarcoma 143B cell line and assayed the main bioenergetic parameters, to examine both the role played by IF1 and the mechanism the inhibitor adopts in tumor cells to control mitochondrial mass, structure and function, besides regulate energy homeostasis. In our model, overall data indicate that the inhibitor protein can enhance the rate of ATP synthesis via OXPHOS, thus representing a successful strategy used by cancer cells to produce more energy and proliferate under oxygen availability. In addition, recent literature has clearly evidenced that epigenetic alterations play a crucial role in modifying genes expression and modulating cancer cell metabolism, sustaining tumor growth and dissemination. Moreover, these molecules have been recently addressed as responsible for chemoresistance in several common therapies, prompting further investigations over miRNAs-driven metabolic alterations. However, compelling data emerging from a microRNA expression profiling, revealed an up-regulation of four miRNAs in BRAF(V600E) mutation-carrying melanomas, when developing resistance to BRAF-I, the major breakthrough in the treatment of these poor-prognosis malignancies. Intriguingly, three of these miRNAs target SUFU, a protein inhibiting the Shh pathway, which is altered in various forms of cancer. Since therapeutic failure still accounts for death in over 90% of patients with metastatic cancers, we also focused on the role exerted by these miRNAs in the molecular mechanisms of drug-resistance.

Cancer remains the second leading cause of death in the United States and the world, despite years of research and the development of different treatments. One reason for this is cancer cells are able to survive through adaptation to their environment and aberrantly activated growth signaling. As such, developing new therapies that overcome these hurdles are necessary to combat cancer. Previous work in our laboratory using RNA interference screening identified genes that regulate the survival of glioblastoma (GBM) or autophagy in chronic myelogenous leukemia (CML) cancer cells. One screen identified Phosphatidylinositol-4,5-bisophosphate 3-kinase catalytic subunit beta (PIK3CB) in the family of Phosphatidylinositol 3-kinases (PI3K) as a survival kinase gene in GBM. Work contained in this dissertation set out to study PIK3CB mediated GBM cell survival. We report that only PIK3CB, in its family of other PI3K genes, is a biomarker for GBM recurrence and is selectively important for GBM cell survival. Another screen identified the long non-coding RNA, Linc00467, as a gene that regulates autophagy in CML. Autophagy is a dynamic survival process used by all cells, benign and cancerous, where cellular components are broken down and re-assimilated to sustain survival. Work contained in this dissertation set out to characterize the role that Linc00467 serves in regulating autophagy in a myriad of cancers. Collectively our data have showed Linc00467 to actively repress levels of autophagy in cancer cells. Further, our data revealed an important role for Linc00467 in regulating the stability of the autophagy regulating protein serine-threonine kinase 11 (STK11). Because of the unique role that Linc00467 serves in regulating autophagy we renamed it as, autophagy regulating long intergenic noncoding RNA or ARLINC. Taken together the work in this dissertation unveils the inner-workings of two important cancer cell survival pathways and shows their potential for development into therapeutic targets to treat cancer.
Ph. D.

Cancer remains the second leading cause of death in the United States and the world, despite years of research and the development of different treatments. One reason for this is cancer cells are able to survive through adaptation to their environment and aberrantly activated growth signaling. As such, developing new therapies that overcome these hurdles are necessary to combat cancer. Previous work in our laboratory using RNA interference screening identified genes that regulate the survival of glioblastoma (GBM) or autophagy in chronic myelogenous leukemia (CML) cancer cells. One screen identified Phosphatidylinositol-4,5-bisophosphate 3-kinase catalytic subunit beta (PIK3CB) in the family of Phosphatidylinositol 3-kinases (PI3K) as a survival kinase gene in GBM. Work contained in this dissertation set out to study PIK3CB mediated GBM cell survival. We report that only PIK3CB, in its family of other PI3K genes, is a biomarker for GBM recurrence and is selectively important for GBM cell survival. Another screen identified the long non-coding RNA, Linc00467, as a gene that regulates autophagy in CML. Autophagy is a dynamic survival process used by all cells, benign and cancerous, where cellular components are broken down and re-assimilated to sustain survival. Work contained in this dissertation set out to characterize the role that Linc00467 serves in regulating autophagy in a myriad of cancers. Collectively our data have showed Linc00467 to actively repress levels of autophagy in cancer cells. Further, our data revealed an important role for Linc00467 in regulating the stability of the autophagy regulating protein serine-threonine kinase 11 (STK11). Because of the unique role that Linc00467 serves in regulating autophagy we renamed it as, autophagy regulating long intergenic noncoding RNA or ARLINC. Taken together the work in this dissertation unveils the inner-workings of two important cancer cell survival pathways and shows their potential for development into therapeutic targets to treat cancer.
Ph. D.

The eradication of glioblastoma multiforme (GBM) (WHO grade 4) remains a tremendous clinical challenge in human oncology. Indeed, this tumor accounts for the most common, aggressive and lethal primary brain cancer in adults, exhibiting a dismal prognosis, despite extensive surgical resection and adjuvant radio- and chemo-therapy. The finding that GBM contains functional subsets of cells with stem-like properties named glioblastoma stem cells (GSCs) has opened up novel opportunities and promises for the development of new therapies for this devastating cancer. GSCs are self-renewing, multipotent cells, with the capacity to establish and maintain glioma tumors at the clonal level, leading to the hypothesis that they are tumor-initiating cells. Moreover, these rare subpopulations of cells possess an elevated proliferative potential, and intrinsic resistance to therapy, being thus considered a key determinant driving tumor growth, and relapse after resection and therapy. In serum free culture conditions, GSCs form neurospheres. free-floating cell aggregates with a spheroid morphology. These neurospheres preserve many of the important characteristics of the parent tumor, as cell heterogeneity and the ability to drive the parent tumor’s cell invasiveness, when transplanted in murine brain. In addition, GSCs possess the capacities to give rise to a heterogeneous population of cells such as endothelial cells (glioblastoma endothelial cells, GECs) which may directly participate in the vascularization, a critical step in tumors, particularly in malignant GBM, one of the most vascularized/angiogenic tumor described. Moreover, it has been observed that microvasculature structures are the regions responsible for the localization and the maintenance of GSCs. Different molecules, including lipid mediators appear to play a key role in the GBM microenvironment. Among lipid mediators involved in GSC properties, ceramide (Cer) and sphingosine-1-phosphate (S1P) has recently emerged as key signals, able to control growth, invasion, and therapy resistance in various human cancers, including GBMs. Of relevance, the presence of S1P in both glioma cell lines and human gliomas is critical for tumor cell proliferation and survival, a down-regulation of sphingosine kinase 1 (SK1) suppressed growth of human GBM cells and xenografts, and a higher expression of S1P receptor 1 (S1PR1) in GBM has been correlated with poor prognosis. However, little is known on the role of Cer and S1P in GSCs. Recent studies reported that S1P acts as an invasive signal in GSCs, and that the inhibition of SKs, or the administration of a S1PR antagonist, results in GSC death. Very recently it was reported that GSCs derived from U87GBM cells, and those isolated from a human GBM specimen can release S1P extracellularly, and that S1P acts as a first messenger to enhance GSC chemoresistance to Temozolomide. In GSC invasiveness and chemoresistance very little is known about autocrine machinery controlling GSC proliferation and particularly on the significance of S1P in these events. In order to expand previous investigation on S1P and to better understand its role on GSC activity and chemoresistance in this project I focused on the pivotal role of S1P on GSC stemness properties.

Solid tumors are complex biological structures which are composed of cellular and matrix components, everything being perfused by blood vessels. During tumor development, modifications of both biochemical and mechanical parameters are observed and can feedback on one another. Cancer cells constantly interact with their mechanical environment and the whole tissue is mostly confined by its surrounding. Compressive mechanical stress develops in part from cell proliferation and could eventually result in the clamping of blood vessels leading to increased interstitial fluid pressure (hydrostatic pressure). The consequent hypoperfusion poses important obstacles to drug delivery and nanomedicines. In fact, the tortuous tumor microvasculature has blood velocities up to one order of magnitude lower compared to healthy capillary networks. Moreover, the fast angiogenesis during tumor progression leads to high vascular density in solid tumors, large gaps exist between endothelial cells in tumor blood vessels, and tumor tissues show selective extravasation and retention of macromolecular drugs (Enhanced Permeation Retention – EPR – effect). These effects have served as a basis for the development of drug delivery systems which are aimed at enhancing tumor tissue targeting and drug therapeutic effectiveness. Over the last 15 years, a plethora of materials and different formulations have been proposed for the realization of nanomedicines. Yet, drug-loading efficiency, sequestration by phagocytic cells, and tumor accumulation of nanoparticle-loaded agents - nanomedicines - are sub-optimal. Starting from these considerations, during my PhD, I studied two complementary approaches: in the first two years my work was focused on implementing the characteristics of Discoidal Polymeric Nanoconstructs designed with controlled geometries and mechanical properties. In the last year, I investigated the role of mechanical stress on chemotherapeutic efficacy. More precisely, this work first reviews the use of deformable discoidal nanoconstructs (DPNs) as a novel delivery strategy for therapeutic and imaging agents. Inspired by blood cell behavior, these nanoconstructs are designed to efficiently navigate the circulatory system, minimize sequestration by phagocytic cells, and recognize the tortuous angiogenic microvasculature of neoplastic masses. In this work, the synthesis, drug loading and release, and physico-chemical characterization of DPNs were enhanced with particular emphasis on the ability to independently control size, shape, surface properties, and mechanical stiffness. Two different loading strategies were tested, namely the straightforward “direct loading” and the “absorbance loading”. In the former case, the agent was directly mixed with the polymeric paste to realize DPNs whereas, in the latter case, DPNs were first lyophilized and then rehydrated upon exposure to a concentrated aqueous solution of the agent. Under these two loading conditions, the encapsulation efficiencies and release profiles of three different molecules and their corresponding prodrugs were systematically assessed (1,2-Distearoyl-sn-glycero-3-phosphorylethanolamine lipid chains or 1 kDa PEG chains were directly conjugated with Cy5.5 or methotrexate and Doxorubicin). Moderately hydrophobic compounds with low molecular weight showed encapsulation efficiencies of 80%, with absorption loading (direct loading has efficiencies around 1%). The DOX-DPN showed on triple negative breast cancer cells a toxicity comparable to free DOX. Preliminary in vivo preliminary studies conducted with directly loaded Cy5-DPN demonstrated a fairly solid integration of the imaging compound with the polymer matrix of the particles. The second part of the work dissect what happens to free drugs or to drugs carried by nanovectors once they reach the tumor site. As we mention above, the elevated mechanical stress derived from tumor progression could result in blood vessels clamping with consequent reduction of drug efficacy. It is quite obvious to imagine that if the drug fails to reach the tumor it cannot act on it. Indeed, mechanical stress within the tumor site is present from the early stages of the disease. Our goal was to understand what happens when mechanical stress is not yet so large enough to fully collapse the blood vessels. Are there mechanical alterations that can affect the efficacy of a chemotherapeutic? We studied how mechanical perturbations of the tumor microenvironment could contribute to the mechanical-form of Gemcitabine drug resistance. Specifically, we developed a new in vitro strategy to mimic the mechanical compression stress induced by the stroma during tumor progression. We embedded pancreatic tumor spheroids into agarose polymeric matrix in order to demonstrate the effect of mechanical compressive stress on tumor proliferation. Then, we validated our results with other types of mechanical stresses. Finally, we investigated the therapeutic efficacy of a proliferation-based chemotherapy: Gemcitabine. Collectively, having the physical cues of cancer in mind, it can be important to cross-fertilize the fields of physical oncology and nanomedicine.

Einer der Gründe für das Scheitern derzeitiger Behandlungsmethoden beim Brustkrebs ist die Resistenz gegenüber der angewandten Chemotherapie. Eine große Rolle bei der Entstehung der Multiplen Medikamentenresistenz spielt das MDR1-Gen und sein Genprodukt, das P-Glykoprotein. Das Y-Box-Protein YB-1 reguliert die Expression des MDR1-Gens; eine Überexpression und nukleäre Lokalisation von YB-1 geht im Brustkrebs mit einer gesteigerten P-Glykoprotein Expression einher. In dieser Arbeit wurden Gewebeproben von 83 Brustkrebspatientinnen auf eine YB-1 Überexpression im Tumor und im peritumoralen Epithel untersucht. YB-1 wurde mittels der immunhistochemischen APAAP-Methode an Formalin-fixierten, in Paraffin eingebetteten Brustkrebsgewebeproben nachgewiesen. Die klinische Relevanz der YB-1 Expression wurde untersucht, indem sie mit dem klinischen Verlauf in einem mittleren Beobachtungszeitraum von 61 Monaten und etablierten biologischen Tumorfaktoren wie Lymphknotenstatus, histologisches Grading, Tumorgröße, Hormonrezeptorstatus, uPA und PAI-1 verglichen wurde. In der Kohorte der Patientinnen mit einer postoperativen adjuvanten Chemotherapie zeigte sich eine 5-Jahres-Rezidivrate von 68 % bei einer hohen YB-1 Expression im Tumor und eine Rückfallrate von 39 % bei einer niedrigen YB-1 Expression. Unter Beachtung auch der YB-1 Expression im peritumoralen Epithel konnte ein noch größerer Unterschied hinsichtlich der 5-Jahres-Rezidivrate festgestellt werden. Diese betrug bei Patientinnen mit einer hohen YB-1 Expression 66 %, während bei Patientinnen mit einer niedrigen YB-1 Expression im Nachbeobachtungszeitraum kein Rezidiv festgestellt wurde. Bei der Gegenüberstellung der 5-Jahres-Rezidivraten in der Kohorte der Patientinnen ohne Zytostatikatherapie zeigte sich eine Rückfallrate von 30 % bei einer hohen YB-1 Expression und eine Rückfallrate von 0 % bei einer niedrigen YB-1 Expression. Eine hohe YB-1 Expression war demnach in beiden Kohorten mit einer schlechteren klinischen Prognose assoziiert. Das Ergebnis in der Gruppe der Patientinnen ohne postoperative Chemotherapie zeigt, dass YB-1 mit der Tumoraggressivität beim Brustkrebs korreliert. Eine Korrelation zwischen der YB-1 Expression und den etablierten prognostischen Faktoren Lymphknotenstatus, Tumorgröße und histologisches Grading konnte nicht festgestellt werden. Es wurde jedoch eine signifikante negative Korrelation zwischen der YB-1 Expression und dem Hormonrezeptorstatus und eine positive Korrelation zwischen YB-1 und den Faktoren uPA und PAI-1 gefunden. In dieser Arbeit wurde gezeigt, dass YB-1 eine klinische Relevanz besitzt mit Hinblick sowohl auf eine prognostische als auch eine prädiktive Bedeutung bei der Identifikation von Hoch-Risiko-Patientinnen im Brustkrebs in Ab- und Anwesenheit einer postoperativen Chemotherapie.
Intrinsic or acquired resistance to chemotherapy is one of the reasons for failure of current treatment regimens in breast cancer patients. P-glycoprotein and its gene mdr1 plays a major role in the development of a multi-drug resistant tumor phenotype. The Y-box protein YB-1 regulates the expression of mdr1. In human breast cancer, overexpression and nuclear localization is associated with upregulation of P-glycoprotein. In this study, tissues of 83 breast cancer patients have been analyzed with regard to YB-1 overexpression in tumor tissue and in surrounding benign breast epithelial cells. YB-1 has been detached by the immunohistochemical APAAP-method using formalin-fixed, paraffin-embedded breast cancer tissues. Clinical relevance of YB-1 expression was analyzed by comparing it with clinical outcome after a median follow-up of 61 months and with tumor biological factors lymph-node status, tumor size, histological grading, hormone-receptor status and the factors uPA and PAI-1. In patients who received postoperative chemotherapy, the 5-year-relapse rate was 68% in patients with high YB-1 expression in tumor cells and 39% in patients with low expression. With regard to YB-1 expression in surrounding benign breast epithelial cells, the 5-year-relapse rate was 66% in patients with high YB-1 expression whereas in patients with low expression no relapse has been observed so far. YB-1 thus indicates clinical drug resistance in breast cancer. In patients who received no chemotherapy, the 5-year-relapse rate was 30% in patients with high YB-1 expression whereas in patients with low YB-1 expression no relapse occurred. YB-1 thus correlates with breast cancer aggressiveness. In both groups high YB-1 expression was associated with poor clinical outcome. A correlation between YB-1 and tumor biological factors lymph-node status, tumor size and histological grading has not been found. But a significant negative correlation has been observed between YB-1 and hormone-receptor status and a positive correlation between YB-1 and uPA and PAI-1. This dissertation could show the clinical relevance of YB-1 with regard to a prognostic and predictive significance by identifying a high-risk group of breast cancer patients both in presence and absence of postoperative chemotherapy.

Tese de Doutoramento em Ciências da Saúde
In the last years a new revival interest has been demonstrated in the reprogrammed metabolism of cancer cells. Described by Otto Warburg, the altered metabolism characterized mainly by a high dependence on lactic acid fermentation, even in the presence of oxygen, is an emergent hallmark of cancer cells. The increase of the glycolytic flux induces a high acidity of the extracellular space, maintained by overexpression of different pH regulators at the plasma membrane, and enhances the more aggressive characteristics of tumour cells, such as increased migration and invasion abilities and resistance to therapy. Therefore, the altered metabolism can be an excellent target for the development of new therapies in cancer field. The reverse pH gradient establish an interplay between cancer metabolism and the surrounding environment. Furthermore, the ablation of pH regulation on cancer cells can be a second way to overcome the major obstacle in antitumour therapy, the multidrug resistance. This phenotype is identified in other cell population that reside inside a tumour mass, beside the tumour parenchymal cells, the cancer stem cells (CSCs), the main responsible for tumour relapse. Recently, it has been described that the reprogrammed metabolism is an emergent target to eliminate this tumour population and to increase the survival rates in many different types of cancer. However, new efforts are needed to improve the knowledge obtained until now on this cancer hallmark. For that reason, this work aims to study and characterize the role of reprogrammed metabolism in different types of cancer, gliomas and pancreatic ductal adenocarcinoma (PDAC) cells and the derived CSCs. Additionally, we aimed to study how this altered phenotype can be modulated, using bioenergetic modulators (BMs), combined or not with conventional drugs. The metabolic profile of the different cell lines was analysed through quantification of lactate production, glucose consumption and intracellular ATP. Concerning the inhibition of the main energetic pathways, it was performed using the glycolytic inhibitors dichloroacetate (DCA), 2-deoxy-D-glucose (2-DG) and the OXPHOS inhibitor and antidiabetic drug, phenformin. All BMs induced a decrease in tumour cell proliferation, and when combined with the conventional antitumour drugs, temozolomide (TMZ) in case of glioma cells, and paclitaxel albumin nanoparticles (NAB-PTX) for pancreatic cancer cells, an increase of drug cytotoxicity was found. Furthermore, when using an in vivo glioma model, the chicken chorioallantoic membrane, all BMs showed an elevated specificity targeting only tumour cells. Additionally, all BMs, namely the glycolytic inhibitors, induced an altered metabolic profile and decrease in migration and invasion abilities in glioma cells. Regarding pancreatic cancer cells, we observed a higher dependence on glycolysis for both cell lines in 2D cell culture. In CSCs, this metabolic profile was more evident in 3D conditions, when an extracellular matrix with higher percentage of collagen was used. Additionally, we verified that the BMs affected the metabolic behaviour of both cell lines. For the parenchymal cells, glycolysis and OXPHOS were important in PANC-1 cell proliferation, but the effect was dependent of the growth substrate. CSCs presented a very complex pattern, showing metabolic plasticity, where inhibition of one pathway can be compensated by others. For instance, it was verified that the CSCs redirected their metabolism to glycolysis as the main energy source, when OXPHOS was inhibited by phenformin. The combination of NAB-PTX with BMs decreased cell proliferation and increased cell death, namely for phenformin in CSCs. The second objective was to unravel the role of the pH regulators, in cancer characteristics. The hyper-glycolytic acid-resistant phenotype has been described in many type of cancers, namely in breast cancer, the other model used in this work. For that, the expression of pH regulators was evaluated both in breast cancer clinical samples and breast cancer cell lines. We observed an overexpression of these proteins, indicating that they can be used as predictive biomarkers in breast cancer diagnosis. Specific inhibitors for these proteins were used and the main characteristics of tumour cells were evaluated. All the compounds decreased cell viability as well as, the migration and invasion abilities of cancer cells. Furthermore, when combined with the conventional drug, doxorubicin, one of the first line drugs used in breast cancer chemotherapeutic regimens, a synergistic effect we observed. To conclude, this study suggests that tumour metabolism behaves as a mediator between tumour cells and the tumour microenvironment, being an important player in tumourigenesis and in the aggressive phenotype of cancer cells. Thus, blockage of the main players involved in this relationship, can disrupt the mechanism responsible for treatment failure in these three types of cancers and improve the existent therapeutic options used in clinical practice.
Nos últimos anos tem sido registado um aumento do interesse na reprogramação metabólica das células tumorais. Descrito pelo cientista Otto Warburg, a reprogramação metabólica caracterizada nomeadamente pela elevada dependência da fermentação láctica, mesmo na presença de oxigénio, é uma característica emergente das células tumorais. O aumento do fluxo glicolítico leva a uma maior acidez do espaço extracelular, mantida pela sobreexpressão de reguladores de pH na membrana plasmática celular, o que induz caraterísticas mais agressivas por parte das células tumorais, como a capacidade de migração e invasão e resistência à terapia. Portanto, o metabolismo alterado pode ser um excelente alvo no desenvolvimento de novas terapias no ramo da oncobiologia. O gradiente de pH reverso, mantido pela sobre-regulação de reguladores de pH nas células tumorais, estabelece uma relação entre o metabolismo das células tumorais e o microambiente circundante. Para além disso, o bloqueio da regulação de pH nas células tumorais pode ser uma segunda alternativa para ultrapassar o maior problema na terapia anti-tumoral, a resistência a múltiplos fármacos. Este fenótipo foi igualmente identificado numa outra população de células que reside dentro da massa tumoral, para além das células tumorais parenquimatosas, as células estaminais tumorais, principais responsáveis pela recorrência do tumor. Recentemente, tem sido descrito que o metabolismo reprogramado é um alvo terapêutico emergente para eliminar a população tumoral e aumentar as taxas de sobrevivência em diferentes tipos de cancro. No entanto, são necessários mais estudos para aumentar o conhecimento obtido até então acerca desta característica tumoral. Por esta razão, este trabalho tem como objetivo estudar e caracterizar o papel do metabolismo alterado em diferentes tipos de cancro, gliomas e adenocarcinoma ductal do pâncreas (ACDP) e as células estaminais tumorais derivadas. Adicionalmente, pretendeu-se estudar como se poderia modificar este fenótipo, usando moduladores bioenergéticos (MB), combinados ou não com fármacos convencionais. O perfil metabólico das diferentes linhas celulares foi analisado através da quantificação da produção de lactato, consumo de glucose e ATP intracelular. Relativamente à inibição das principais vias metabólicas, esta foi realizada usando os inibidores glicolíticos dicloroacetato e 2-desoxiglucose e o inibidor da fosforilação oxidativa e fármaco antidiabético, a fenformina. Todos os MBs induziram uma diminuição da proliferação das células tumorais, e quando combinados com fármacos antitumorais convencionais, a temozolamida no caso das células de glioma, e paclitaxel associado à albumina no caso das células tumorais pancreáticas, foi observado um aumento da citotoxicidade do fármaco. Para além disso, quando utilizado um modelo in vivo de glioma, a membrana corioalantóide de galinha, todos os MBs mostraram uma elevada especificidade, atuando apenas nas células tumorais. Relativamente ao ACDP, observamos uma elevada dependência pela glicólise nas duas linhas celulares em culturas em 2D. Nas células estaminais tumorais, este perfil metabólico foi mais evidente em condições de crescimento 3D, quando se utilizou uma matriz extracelular com elevada percentagem de colagénio. Adicionalmente, verificou-se que os MBs afetaram o comportamento metabólico de ambas as linhas celulares. Nas células parenquimatosas a glicólise e a fosforilação oxidativa foram importantes na proliferação das células PANC-1, mas este efeito foi dependente do substrato de crescimento. As células estaminais tumorais apresentaram um padrão muito complexo, mostrando uma plasticidade metabólica, onde a inibição de uma via metabólica pode ser compensada por outras. Por exemplo, foi verificado que as células estaminais tumorais redirecionaram o seu metabolismo para a glicólise como principal fonte de energia, quando a fosforilação oxidativa foi inibida pela fenformina. A combinação do paclitaxel associado à albumina com os MBs diminuiu a proliferação celular e aumentou a morte celular, nomeadamente para a fenformina nas células estaminais tumorais. O segundo objetivo deste trabalho foi o estudo do papel dos reguladores de pH nas características tumorais. O fenótipo hiper-glicolítico ácido-resistente tem sido descrito em vários tipos de cancro, nomeadamente no cancro de mama, outro dos modelos utilizados neste trabalho. Assim, a expressão dos reguladores de pH foi avaliada em amostras humanas de tecidos e linhas celulares de cancro de mama. Observamos uma sobre-expressão dessas proteínas, indicando que podem ser usadas como biomarcadores preditivos no diagnóstico deste tipo de cancro. Foram utilizados inibidores específicos destes reguladores foram utilizados e as principais características tumorais foram avaliadas nestas condições. Todos os inibidores diminuíram a viabilidade celular, assim como, a capacidade de migração e invasão por parte das células tumorais. Adicionalmente, quando utilizados simultaneamente com o fármaco convencional doxorrubicina, um dos fármacos de primeira linha utilizada nos regimes de quimioterapia no cancro de mama, foi observado um efeito sinérgico. Em conclusão, este estudo sugere que o metabolismo das células tumorais funciona como mediador entre estas células e o microambiente tumoral, sendo um fator importante na carcinogénese e no fenótipo mais agressivo das células tumorais. Portanto, a inibição dos principais intervenientes nesta correlação pode bloquear o mecanismo responsável pela falta de eficácia do tratamento nestes três tipos de cancro e potenciar as opções terapêuticas utilizadas na prática clinica.
The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), University of Minho. Financial support was provided by grants from the Fundação para Ciência e Tecnologia (FCT) through individual fellowship (SFRH/BD/103025/2014), by NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal Partnership Agreement, through the European Regional Development Fund (FEDER), and through the Competitiveness Factors Operational Programme (COMPETE) and by National funds, through the Fundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038. This work was also supported by an internal CESPU project 02-GBMC-CICS-2011 MetabRes_CESPU_2017.

PURPOSE: Cytochrome P450 2W1 (CYP2W1) is a monooxygenase detected in 30% of colon cancers, whereas its expression in nontransformed adult tissues is absent, rendering it a tumor-specific drug target for development of novel colon cancer chemotherapy. Previously, we have identified duocarmycin synthetic derivatives as CYP2W1 substrates. In this study, we investigated whether two of these compounds, ICT2705 and ICT2706, could be activated by CYP2W1 into potent antitumor agents. EXPERIMENTAL DESIGN: The cytotoxic activity of ICT2705 and ICT2706 in vitro was tested in colon cancer cell lines expressing CYP2W1, and in vivo studies with ICT2706 were conducted on severe combined immunodeficient mice bearing CYP2W1-positive colon cancer xenografts. RESULTS: Cells expressing CYP2W1 suffer rapid loss of viability following treatment with ICT2705 and ICT2706, whereas the CYP2W1-positive human colon cancer xenografts display arrested growth in the mice treated with ICT2706. The specific cytotoxic metabolite generated by CYP2W1 metabolism of ICT2706 was identified in vitro. The cytotoxic events were accompanied by an accumulation of phosphorylated H2A.X histone, indicating DNA damage as a mechanism for cancer cell toxicity. This cytotoxic effect is most likely propagated by a bystander killing mechanism shown in colon cancer cells. Pharmacokinetic analysis of ICT2706 in mice identified higher concentration of the compound in tumor than in plasma, indicating preferential accumulation of drug in the target tissue. CONCLUSION: Our findings suggest a novel approach for treatment of colon cancer that uses a locoregional activation of systemically inactive prodrug by the tumor-specific activator enzyme CYP2W1.

The statin family of hydroxymethylglutaryl coenzyme A reductase (HMGCR) inhibitors, used to control hypercholesterolemia, triggers apoptosis of various human tumour cells. HMGCR is the rate-limiting enzyme of the mevalonate (MVA) pathway, a fundamental metabolic pathway required for the generation of a number of biochemical end-products including cholesterol and isoprenoids, but the contribution of the MVA pathway to human cancer remains largely unexplored. Furthermore, as only a subset of tumour cells has been shown to be highly responsive to statins, the identification of appropriate subsets of patients will be required to successfully advance these agents as anticancer therapeutics. To this end, there were two major aims to this work: 1) Elucidate a molecular rationale for the observed therapeutic index of statin-induced apoptosis in normal and tumour cells; 2) Identify molecular determinants of sensitivity for statin-induced apoptosis in human tumour cells. To address the first aim we demonstrated that dysregulation of the MVA pathway, achieved by ectopic expression of either full length HMGCR (HMGCR-FL) or its novel splice variant lacking exon 13 (HMGCR-D13), increases transformation. Ectopic HMGCR promotes growth of transformed and non-transformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice. We also show that high mRNA levels of HMGCR and four out of five other MVA pathway genes correlate with poor prognosis in primary breast cancer, suggesting the MVA pathway may play a role in the etiology of human cancers. To address the second aim, we show that dysregulation of the MVA pathway is a key determinant of sensitivity to statin-induced apoptosis in multiple myeloma. In a panel of 17 distinct myeloma cell lines, half were sensitive to statin-induced apoptosis and the remainder were insensitive. Interestingly, in sensitive cells, the classic feedback response to statin exposure is lost, a feature we demonstrated could distinguish a subset of statin-sensitive primary myeloma cells. We further illustrated that statins are highly effective and well tolerated in an orthotopic model of myeloma using cells harboring a dysregulated MVA pathway. Taken together, this work provides a molecular rationale and determinants of sensitivity for statin-induced apoptosis of human tumour cells.

碩士
國立中山大學
生物醫學研究所
98
VCAM-1 (CD106) is a transmembrane glycoprotein and involved in many pathological inflammatory processes. VCAM-1 plays an important role in leukocyte adhesion, leukocyte transendothelial migration and cell activation by binding to integrin VLA-1 (α4β1). In our preliminary data, we observed 2-3 fold increase in the expression of VCAM-1 in the side population of ovarian cancer, which exhibits stem cell-like properties in ovarian cancer. In addition, we have also found VCAM-1 is upregulated in many breast cancer epithelial cells and directly correlated with breast tumor progression; however, its mechanism of action in tumor biology remains unknown. Here, we describe the establishment and use of breast cancer cell line model systems to dissect the functional roles of VCAM-1 activation in the manifestation of malignant phenotype of human breast cancer. We show that VCAM-1 overexpression in the NMuMG breast epithelial cells increase cell motility rates and chemoresistance to doxorubicin and cisplatin in vitro, conversely, in an established metastatic breast cancer cell line, MDAMB231, we find that knockdown endogenous VCAM-1 expression by small interfering RNA reduced the migration rate . Furthermore, we also demonstrated that knockdown endogenous VCAM-1 expression in MDAMB231 cells reduced the tumor formation in SCID xenograft mouse model. In conclusion, our findings are consistent with the hypothesis that overexpression of VCAM-1 facilitates breast cancer progression by enhancing the malignant properties of breast cancer cells and suggests that targeting of VCAM-1 induced pathways are attractive strategies for therapeutic intervention.

博士
國立成功大學
基礎醫學研究所
97
Spontaneous IL-6 production has been observed in various tumors and has been implicated in cancer pathogenesis, progression, and drug resistance. Most studies emphasized the importance of NF-κB and AP-1 on IL-6 regulation. Others pointed out the potential roles of HIF-1α and p53. However, the molecular mechanism of IL-6 autocrine in tumor cells and the impact of IL-6 on drug resistance are not well defined yet. The previous study in our laboratory has demonstrated high IL-6 levels in malignant pleural effusions of patients with lung adenocarcinoma than those of non-malignant patients. In addition, in Stat3 downstream of IL-6 also plays an important role in the metastatic property and drug sensitivity of lung cancer cells. In this study, we planed to uncover the mechanism of IL-6 autocrine in cancer cells and the impact of IL-6 on cancer drug resistance. First of all, we demonstrated that autocrine IL-6 was able to activate downstream signals and result in drug resistance in cancer cells. We showed that Jak2/Stat3 pathway as well as previously reported MEK/Erk, PI3-K/Akt and NF-κB pathways contribute to IL-6 autocrine secretion in an aggressive lung cancer cell line, various drug resistant cancer cell lines, and patients' primary lung cancer cells. We showed that Stat3 not only contributed to the regulation of endogenous IL-6 autocrine but also to the exogenous-stimulation induced IL-6 expression. In addition, Stat3 may regulate IL-6 autocrine by modulating endosome associated secretion pathway in parallel with controlling IL-6 expression. In spite of the importance of autocrine IL-6 in cancer drug resistance, the Stat1 activation responding to IFN-γ stimulation instead of the Stat3 activation responding to IL-6 stimulation was shown to be an ideal indicator of the development of drug resistance in human lung cancers using single cell phospho-specific flow cytometry analysis.

La chemioterapia è uno degli standard di cura per diversi tipi di tumori solidi. Sebbene i benefici dell’impiego clinico dei chemioterapici siano stati ampiamente riconosciuti, la loro efficacia terapeutica è limitata dall'insorgenza della resistenza farmacologica, che causa la recidiva del tumore, il fallimento dei trattamenti successivi e l'eventuale morte dei pazienti. La farmacoresistenza è un fenomeno multifattoriale, i cui meccanismi molecolari non sono stati ancora completamente compresi. Negli ultimi decenni, l’alterazione del metabolismo energetico cellulare è stata introdotta come segno distintivo della chemio-resistenza in aggiunta ai meccanismi più comunemente descritti, come ad esempio l'aumento dell'ingresso o la diminuzione dell’efflusso del farmaco, il potenziamento dei sistemi di riparazione del DNA e l'aumento dei sistemi antiossidanti. Numerosi studi hanno dimostrato una correlazione tra le alterazioni del metabolismo mitocondriale e il cancro, e successivamente anche con la resistenza ai farmaci, evidenziando così il ruolo fondamentale dei mitocondri nella progressione e nell'aggressività tumorale. Nonostante le ricerche recenti, il ruolo dei mitocondri nell'insorgenza della resistenza al cisplatino e alla doxorubicina non è ancora del tutto chiaro. Lo scopo di questo progetto è stato quello di caratterizzare il profilo mitocondriale di cellule tumorali sensibili e resistenti sia al cisplatino che alla doxorubicina. Inizialmente, abbiamo valutato alcuni aspetti relativi allo stato e alla funzione del mitocondrio, come la morfologia mitocondriale, il potenziale di membrana e la massa. I nostri risultati hanno evidenziato diversi profili mitocondriali tra le cellule tumorali sensibili e resistenti. In particolare, le cellule tumorali resistenti al cisplatino hanno mostrato alterazioni nei livelli proteici del complesso OXPHOS, mentre i cloni resistenti alla doxorubicina hanno evidenziato dei cambiamenti nei processi di dinamica mitocondriale, ed in particolare una ridotta biogenesi mitocondriale. Le alterazioni identificate attraverso la fenotipizzazione mitocondriale rappresentano il punto di partenza per colpire le cellule tumorali resistenti con approcci farmacologici selettivi per superare la resistenza alla chemioterapia. Successivamente, l’obiettivo della mia tesi si è focalizzato sulla valutazione di strategie di terapie combinate tra modulatori mitocondriali ed agenti chemioterapici per ripristinare l'effetto dei farmaci nei cloni resistenti. Gli approcci hanno coinvolto l’utilizzo di plumbagina, che è un modulatore mitocondriale, e di quercetina, che è un induttore della biogenesi mitocondriale, in associazione rispettivamente con il cisplatino e la doxorubicina, dimostrando così che la terapia combinata è in grado di sensibilizzare nuovamente i cloni resistenti al chemioterapico. La seconda parte del mio progetto è stata orientata sull’identificazione di altri approcci farmacologici per ripristinare l'efficacia delle terapie endocrine nelle cellule umane di tumore al seno. I nostri risultati hanno evidenziato che la combinazione di Olaparib con Fulvestrant e Tamoxifene ha sensibilizzato le cellule di tumore al seno al trattamento endocrino. Inoltre, l'associazione di Olaparib, anti-PDL1 e anti-CXCR2 ha contrastato l'effetto pro-tumorigenico indotto dalle cellule mieloidi immature umane. I risultati emersi in questo lavoro pongono nuove basi per l'identificazione di approcci farmacologici innovativi per colpire selettivamente le alterazioni delle cellule tumorali resistenti, con l'obiettivo finale di superare la farmacoresistenza.

According to the prediction of the Global Cancer Observatory (GCO), an interactive web platform that presents global cancer statistics, pancreatic ductal adenocarcinoma (PDAC) will be second cause of death in Western societies within the next decade. To date, the overall survival of patients at 5 years after diagnosis is only 6%, and 25% in patients undergoing surgical resection, but these represent only 15% of total cases. These data emphasize the need to find new effective drug treatments. Indeed, current therapeutic approaches have great limitations such as chemoresistance, both innate and acquired after the treatment, lack of biomarkers for prediction of therapy response, and intrinsic toxicity of chemotherapeutic agents that causes several side effects. In this thesis highlights two novel anticancer agents that could be used for the treatment of particularly resistant PDAC tumors: gemcitabine molecules capable of releasing nitric oxide (NO-GEMs) and selective and reversible benzylpiperidine-based MAGL inhibitors. The first ones act on mechanisms of direct resistance to GEM by increasing its concentration inside the cells through the use of NO; the second ones, act at the level of lipid metabolism by preventing the formation of signal molecules. The use of MAGL inhibitors has been further studied and are proposed to may be use as target therapy to treat PDAC patients with GOF mutant p53 R273H.

博士
國立成功大學
基礎醫學研究所
104
Acquisition of drug resistance to conventional chemotherapy is a challenge in treating recurrent cancer. Bladder cancer has the highest recurrence rate of any malignancy. Although primary tumors can be eliminated by surgery, chemotherapy, and radiotherapy, the tumors recur frequently and progress to muscle-invasive disease. Here we investigated whether anticancer drugs induced Oct4 expression and ultimately results in tumor recurrence in bladder cancer. We identified a positive correlation of the expression levels of Oct4 with the rate of tumor recurrence in 122 clinical specimens of superficial high-grade (stages T1-2) bladder transitional cell carcinoma. Chemotherapy induced Oct4 expression in bladder cancer cells. Notably, treatment with cisplatin increased CD44-positive bladder cancer cells expressing Oct4, representing cancer stem-like cell subpopulation. Overexpression of Oct4 reduced, whereas knockdown of Oct4 enhanced, drug sensitivity in bladder cancer cells. Furthermore, there was a poor response to cisplatin treatment in vivo when tumor cells overexpressed Oct4. In terms of clinical relevance, inhibition of Oct4 by all-trans retinoic acid synergistically increased sensitivity to cisplatin in bladder cancer cells. Furthermore, the combination of cisplatin and all-trans retinoic acid was superior to cisplatin alone in suppressing tumor growth. At last, Luciferase reporter and chromatin immunoprecipitation (ChIP) assays demonstrated that Oct4 could transactivate the MDR1 promoter by binding to the Oct4 response element. These results indicated Oct4 involved in chemodrugs efflux. Patients with Oct4 and/or MDR1 high-expressing tumors were associated with shorter recurrence-free intervals compared with patients with Oct4 and/or MDR1 low-expressing tumors. Therefore, our findings provide evidence that Oct4 can mediate acquired drug resistance to anticancer drugs, and implicate that targeting Oct4 may be a therapeutic strategy to circumvent drug resistance.

OBJECTIVE: Development of treatment resistance and adverse toxicity associated with classical chemotherapeutic agents highlights the need for safer and effective therapeutic approaches. Herein, we examined the effectiveness of a combination treatment regimen of 5-fluorouracil (5-FU) and curcumin in colorectal cancer (CRC) cells. METHODS: Wild type HCT116 cells and HCT116+ch3 cells (complemented with chromosome 3) were treated with curcumin and 5-FU in a time- and dose-dependent manner and evaluated by cell proliferation assays, DAPI staining, transmission electron microscopy, cell cycle analysis and immunoblotting for key signaling proteins. RESULTS: The individual IC50 of curcumin and 5-FU were approximately 20 microM and 5 microM in HCT116 cells and 5 microM and 1 microM in HCT116+ch3 cells, respectively (p<0.05). Pretreatment with curcumin significantly reduced survival in both cells; HCT116+ch3 cells were considerably more sensitive to treatment with curcumin and/or 5-FU than wild-type HCT116 cells. The IC50 values for combination treatment were approximately 5 microM and 1 microM in HCT116 and 5 microM and 0.1 microM in HCT116+ch3, respectively (p<0.05). Curcumin induced apoptosis in both cells by inducing mitochondrial degeneration and cytochrome c release. Cell cycle analysis revealed that the anti-proliferative effect of curcumin and/or 5-FU was preceded by accumulation of CRC cells in the S cell cycle phase and induction of apoptosis. Curcumin potentiated 5-FU-induced expression or cleavage of pro-apoptotic proteins (caspase-8, -9, -3, PARP and Bax), and down-regulated anti-apoptotic (Bcl-xL) and proliferative (cyclin D1) proteins. Although 5-FU activated NF-kappaB/PI-3K/Src pathway in CRC cells, this was down-regulated by curcumin treatment through inhibition of IkappaBalpha kinase activation and IkappaBalpha phosphorylation. CONCLUSIONS: Combining curcumin with conventional chemotherapeutic agents such as 5-FU could provide more effective treatment strategies against chemoresistant colon cancer cells. The mechanisms involved may be mediated via NF-kappaB/PI-3K/Src pathways and NF-kappaB regulated gene products.

Une résistance aux agents anticancéreux utilisés dans le traitement du cancer du sein est souvent associée à un échec de traitement. Des variations dans le devenir des agents anticancéreux dans l’organisme, sont des facteurs pouvant expliquer des phénomènes de résistance. Notre but était d’évaluer l’impact des isoenzymes du CYP450s, dans le métabolisme local des agents anticancéreux. Notre premier objectif était de valider un gène rapporteur pour nos analyses de PCR en temps réel. Pour ce faire, nous avons criblé l’expression de 6 gènes rapporteurs dans 23 lignées cellulaires. NUP-214 a été démontré comme étant le gène rapporteur le plus stable avec un écart-type de seulement 0.55 Ct. Notre deuxième objectif était de déterminer le niveau d’expression des ARNm de 19 isoformes du CYP450 dans plusieurs lignées cellulaires du cancer du sein. Les ARNm des CYP450s ont démontré une très grande variabilité entre les lignées cellulaires. Les isoformes CYP1B1 et CYP2J2 démontrent l’expression la plus importante pour la majorité des lignées. Notre troisième objectif était d’évaluer la corrélation entre l’expression des isoformes des CYP450s et leur activité métabolique en utilisant les substrats spécifiques du CYP1B1 et 2J2, 7-éthoxyrésorufine et ébastine, respectivement. Une forte corrélation (r2=0.99) fut observée entre l’activité métabolique vis-à-vis l’ébastine et l’expression du CYP2J2. De même, le métabolisme du 7-éthoxyrésorufine était fortement corrélé (r2=0.98) avec l’expression du CYP1B1. En résumé, ces résultats suggèrent que le métabolisme local des agents anticancéreux pourrait significativement moduler le devenir des agents anticancéreux dans l’organisme, et pourrait être ainsi, une source de résistance.
Several types of cancer cells have shown an innate or accute resistance to anti-cancer agents which in turn causes a failure in treatment. This resistance has been suggested to be caused by the expression of membrane transporters in cancer cells, as well as inter-individual variability in metabolism. Our interest was to evaluate the implication of CYP450 enzymes in the local metabolism of cancer cells. Our first objective was to screen the expression level of six housekeeping genes (HKG) using 23 different cell lines to determine which gene was the most stable. We found that NUP-214 was the most stable HKG across the panel of cell lines tested, with a standard deviation of only 0.55 Ct. Our second objective was to determine the expression level of 19 CYP450 mRNA isoforms in various breast cancer cell lines by RT-PCR. The CYP450 mRNAs showed a large variability between the different cell lines analyzed, where CYP1B1 and 2J2 were strongly expressed in most cell lines. Our third objective was to determine if measurable metabolic activity was present and correlates with mRNA expression in these same breast cancer cell lines using the specific substrates 7-ethoxyresorufin and ebastine for CYP1B1 and 2J2 activities, respectively. The metabolism of 7-ethoxyresorufin showed an excellent correlation of 0.98 with CYP1B1 expression while ebastine demonstrates a strong correlation (r2=0.99) with 2J2 expression. Overall, these results suggest that local metabolism of anti-cancer agents could significantly affect drug disposition and be a source of chemoresistance.

Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a significant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumors are defective in nonhomologous end-joining (NHEJ), and this phenotype may be used clinically by combining radiotherapy with a radiosensitizing drug that targets homologous recombination, thereby sparing normal tissues with intact NHEJ. The response of the homologous recombination protein RAD51 to radiation is inhibited by the small-molecule tyrosine kinase inhibitor imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using short hairpin RNA technology to mimic the invasive tumor phenotype and also RAD51 knockdown (RAD51KD) cells to show imatinib's pathway selectivity. Ku80KD, RAD51KD, nonsilencing vector control, and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ and cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (nontoxic) levels. Imatinib radiosensitized Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitized RAD51KD and RT112 cells but not Ku80KD cells. Taken together, our findings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cancer. Cancer Res; 73(5); 1611-20. (c)2012 AACR.