Thèses sur le sujet « Tumor chemoresistance »

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

Regulation of cancer cell phenotype by the tumor microenvironment has motivated further investigation into how microenvironmental factors could contribute to tumor initiation, development, and therapeutic resistance. Analyzing how the microenvironment drives tumor development and cancer cell heterogeneity is particularly important in cancers such as glioblastoma multiforme (GBM) that have no known risk factors and are characterized by a high degree of heterogeneity. GBM patients have a median survival of 15 months and therefore are in great need of more effective therapeutic options. The goal of this research is to generate in vitro models of the heterogeneous brain tumor microenvironment, with a focus on vascular dynamics, to probe the impact of microenvironmental cues on tumor progression and to integrate the tumor models with highly sensitive analytical tools to characterize the epigenome of discrete phenotypic subpopulations that contribute to intratumoral cellular heterogeneity. As GBM tumors are characterized by a dense vasculature, we delved into microenvironmental factors that may be promoting angiogenesis. The correlations emerging between inflammation and cancer led to analysis of the inflammatory molecule lipopolysaccharide (LPS). We utilized 3D micro-tissue models to simulate vascular exposure to ultra-low chronic inflammatory levels of LPS and observed an increase in vascular formation when brain endothelial cells were exposed to ultra-low doses of LPS. We also utilized our micro-tissue models to analyze histone methylation changes across the epigenome in response to microenvironmental cues, namely culture dimensionality and oxygen status. The H3K4me3 modification we analyzed is associated with increased gene transcription, therefore the alterations we observed in H3K4me3 binding across the genome could be a mechanism by which the tumor microenvironment is regulating cancer cell phenotype. Lastly, we developed a microfluidic platform in which vascular dynamics along with microenvironmental heterogeneities can be modeled in a more physiologically relevant context. We believe the studies presented in this dissertation provide insight into how vasculature primed by chronic inflammation and epigenetic alterations in tumor cells could both contribute to enhanced tumor development. Modeling these biological processes in our advanced microfluidic platform further enables us to better understand microenvironmental regulation of tumor progression, uncovering new potential therapeutic targets.
PHD

Il colangiocarcinoma (CCA) è una neoplasia epiteliale che origina dai dotti biliari, ed è caratterizzata da una prognosi infausta, imputabile alla spiccata invasività e alla resistenza alla chemioterapia. L’aggressività delle cellule di CCA è esacerbata dallo stroma desmoplastico che si sviluppa contestualmente alla crescita tumorale, contenente fibroblasti cancro-associati (CAF), macrofagi tumore-associati e cellule endoteliali linfatiche (LEC). Durante il mio dottorato, ho analizzato la natura e la rilevanza biologica dei segnali paracrini intercorrenti tra cellule stromali e tumorali nel CCA, nel tentativo di chiarire i meccanismi molecolari che guidano la progressione tumorale. In un primo studio, abbiamo esaminato la citochina pleiotropica Leukemia Inhibitory Factor (LIF), che abbiamo scoperto essere secreta non solo dalle cellule di CCA, ma anche dalle cellule infiammatorie e dai CAF all’interno del microambiente tumorale. Abbiamo dimostrato che il LIF ostacolava l’induzione di apoptosi in cellule di CCA trattate con gemcitabina e cisplatino, tramite attivazione del pathway PI3K/Akt, e conseguente up-regolazione della proteina anti-apoptotica Myeloid Cell Leukemia (Mcl)-1. In un secondo studio, abbiamo considerato un tipico readout delle interazioni stroma-tumore, ovvero la transizione epitelio-mesenchimale (EMT) delle cellule carcinomatose, un fenomeno alla base dell’invasione tumorale. In precedenza, avevamo dimostrato che S100A4, un biomarcatore della EMT, promuove attivamente l’invasività del CCA quando espresso nel nucleo delle cellule tumorali. Abbiamo quindi osservato che la nuclearizzazione di S100A4 era dipendente dalla sua SUMOilazione, che poteva essere inibita trattando le cellule di CCA con dosi nanomolari di paclitaxel. La riduzione dei livelli nucleari di S100A4 si associava ad una diminuzione dell’attività di RhoA e Cdc42, della secrezione della metalloproteinasi della matrice (MMP)-9, e dell’espressione della MMP di membrana di tipo 1. Inoltre, il paclitaxel a basse dosi attenuava l’invasività delle cellule di CCA, sia in vitro sia in un modello xenograft. In un terzo studio, abbiamo ipotizzato che la comunicazione tra cellule di CCA e CAF potesse promuovere la linfoangiogenesi tumorale, un processo di fondamentale importanza per la metastatizzazione del CCA. Abbiamo dimostrato che, dopo stimolazione con il Platelet-Derived Growth Factor (PDGF)-D, un mediatore cruciale del reclutamento dei CAF da parte delle cellule di CCA, i fibroblasti incrementavano la secrezione del Vascular Endothelial Growth Factor (VEGF)-A e del VEGF-C, in virtù dell’attivazione delle chinasi ERK1/2 e JNK. Coerentemente, il medium condizionato derivato da fibroblasti trattati con il PDGF-D promuoveva la migrazione delle LEC e il loro assemblaggio in strutture vascolari 3D, ed entrambi gli effetti erano prevenuti bloccando il recettore β del PDGF a livello dei fibroblasti, o i recettori 2 e 3 dei VEGF a livello delle LEC. Anche la permeabilità di LEC in monostrato era aumentata dai fibroblasti stimolati con il PDGF-D, così da favorire la migrazione trans-endoteliale delle cellule di CCA. In conclusione, i nostri risultati confermano che lo stroma tumorale è in grado di alimentare la progressione del CCA, sia modulando direttamente il comportamento delle cellule tumorali, sia allestendo un microambiente favorevole alla diffusione metastatica. Una piena comprensione delle interazioni tra cellule neoplastiche e stromali nel CCA potrebbe portare in futuro allo sviluppo di terapie innovative e “multi-target” in grado di eradicare più efficacemente il tumore.
Cholangiocarcinoma (CCA) is an epithelial cancer arising from the biliary tree. CCA carries a poor prognosis, owing to early and pronounced invasiveness, and resistance to chemotherapy. The aggressiveness of CCA cells is exacerbated by the desmoplastic stroma developing in conjunction with tumor outgrowth, which mainly consists of cancer-associated fibroblasts (CAFs), tumor-associated macrophages, and lymphatic endothelial cells (LECs). During my PhD studies, I sought to dissect the nature and the biological relevance of the dense paracrine communications between stromal and cancer cells in CCA, in an effort to unveil the molecular mechanisms driving tumor progression. In a first study, we focused on a pleiotropic cytokine named leukemia inhibitory factor (LIF), which we found to be released not only by CCA cells, but also by inflammatory cells and CAFs within the tumor microenvironment. We showed that LIF hindered the induction of apoptosis in CCA cells treated with gemcitabine plus cisplatin, an effect dependent on the up-regulation of the anti-apoptotic protein myeloid cell leukemia (Mcl)-1, occurring downstream of PI3K activation. Therefore, targeting the LIF/PI3K/Mcl-1 axis may represent a feasible strategy to increase CCA responsiveness to chemotherapy. In a second study, we considered a classic readout of tumor-stroma interactions, i.e., the epithelial-to-mesenchymal transition (EMT) of cancer cells, a process underlying carcinoma invasion and metastasis. Previously, we had shown that S100A4, an EMT biomarker, acts as a mechanistic determinant of CCA invasiveness when expressed in the nucleus of cancer cells. We then demonstrated that the nuclearization of S100A4 was dependent on its SUMOylation, which could be inhibited by treating CCA cells with paclitaxel at nanomolar doses. Down-modulation of nuclear S100A4 hampered the activity of RhoA and Cdc42, the secretion of matrix metalloproteinase (MMP)-9, and the expression of membrane-type (MT)1-MMP. Moreover, low-dose paclitaxel significantly impaired CCA cell invasiveness, both in vitro and in a SCID mouse xenograft model, implying that a selective reduction in S100A4 nuclear expression may prevent tumor dissemination in CCA patients. In a third study, we aimed at clarifying whether the interplay between CCA cells and CAFs could drive tumor lymphangiogenesis, a process of utmost importance for CCA metastatization. We showed that, upon stimulation with platelet-derived growth factor (PDGF)-D, a major mediator of CAF recruitment by CCA cells, fibroblasts increased the secretion of vascular endothelial growth factor (VEGF)-A and VEGF-C, due to the activation of ERK1/2 and JNK. Consistently, conditioned medium from PDGF-D-treated fibroblasts promoted the recruitment of LECs, along with their assembly in 3-D vascular structures, and both effects could be prevented by antagonizing either PDGF receptor β on fibroblasts, or VEGF receptors 2 and 3 on LECs. The permeability of LEC monolayers was also increased by PDGF-D-treated fibroblasts, supporting the trans-endothelial migration of CCA cells. Overall, we unveiled the presence of a sequential cross-talk among CCA cells, CAFs and LECs, whose disruption may interfere with CCA metastatic spread. In conclusion, our results validate the notion that the tumor stroma strongly promotes the progression of CCA, both by directly shaping the behavior of cancer cells, and by setting up a microenvironment conducive to metastasis. Hopefully, a comprehensive understanding of the mutual interactions between cancer and stromal cells will lead to the development of innovative, multitargeted therapies that may more effectively eradicate the tumor.

Tumors are similar to organs, with unique physiology giving rise to an unusual set of transport barriers to drug delivery. Cancer therapy is limited by non-uniform drug delivery via blood vessels, inhomogeneous drug transport into tumor interstitium from the vascular compartment, and hindered transport through tumor interstitium to the target cells. Four major abnormal physical and physiological properties contribute to these transport barriers. Accumulated solid stress compresses blood vessels to diminish the drug supply to many tumor regions. Immature vasculature with high viscous and geometric resistances and reduced pressure gradients leads to sluggish and heterogeneous blood flow in tumors to further limit drug supply. Nonfunctional lymphatics coupled with highly permeable blood vessels result in elevated hydrostatic pressure in tumors to abrogate convective drug transport from blood vessels into and throughout most of the tumor tissue. Finally, a dense structure of interstitial matrix and cells serves as a tortuous, viscous, and steric barrier to diffusion of therapeutic agents. In this dissertation, I discuss the origins and implications of these barriers. I then highlight strategies I have developed for overcoming these barriers by modulating either drug properties or the tumor microenvironment itself to enhance the delivery and effectiveness of drugs in tumors.
Engineering and Applied Sciences

OVCA is the most lethal gynecological cancer, due primarily to late diagnosis and chemoresistance (Canada, 2014; Society, 2014b). CDDP resistance is a major hurdle to successful therapy (MayoClinic, 2014). The mechanism of chemoresistance is multi-factorial including defects in apoptotic pathway and key tumor suppressor as well as dysregulation of metabolism (Borst et al., 2000; Galluzzi et al., 2012a; Siddik, 2003). Elevated aerobic glycolysis is a major source for fulfilling high energy demand of cancer, but the role of metabolic reprogramming and its regulatory mechanism in OVCA cells remain unknown. p53 is a key tumor suppressor involved in apoptosis and frequent defect of p53 (> 80%) exist in epithelial OVCA. HKII is a key metabolic enzyme involved in the first step of glycolysis and its frequent presence in the mitochondria (80% >) has been reported in multiple cancers. We demonstrate here that CDDP-induced, p53-mediated HKII down-regulation and mitochondrial p53-HKII interaction are determinants of chemosensitivity in OVCA. CDDP decreased HKII (mRNA abundance, protein level), altered its cellular localization and glycolysis in p53-wt chemosensitive OVCA cells, a response loss or attenuated in p53 deficient cells. HKII depletion sensitized chemoresistant cells to CDDP -induced apoptosis in a p53- dependent manner. In addition, p53 binds to HKII and facilitates its nuclear localization. Mechanistically, our data suggest that CDDP-activated p53 (phosphorylated p53; P-p53 Ser15) interacts with HKII in the nucleus for its regulation. Upon entry to the nucleus, P-p53(Ser15) transcriptionally regulates HKII by promoter binding, contributing to the regulation of HKII and aerobic glycolysis, eliciting apoptosis in chemosensitive OVCA cells. Conversely, this response is compromised in p53 defect chemoresistant cells. Using proximity ligation assay (PLA) in human OVCA cell lines and primary tumor cells and tumor sections from OVCA patients, we have demonstrated that nuclear HKII-P-p53(Ser15) intracellular trafficking is associated with chemosensitivity in vitro and in vivo. Furthermore, the nuclear HKII-P-p53(Ser15) interaction may be useful as a biomarker for chemosensitivity in multiple epithelial subtypes of OVCA.

Le cancer du sein est le cancer le plus fréquemment observé chez les femmes en France et dans le monde. Bien que le nombre de cas observés chaque année a tendance à diminuer depuis 2005, notamment grâce au dépistage organisé, cette maladie reste la première cause de décès par cancer chez les femmes. De nombreux travaux ont montré que la progression tumorale est dépendante des cellules tumorales mais également des cellules " saines " du microenvironnement (ou stroma) qui entourent la tumeur. Dans le cas du cancer du sein, les adipocytes, type cellulaire majeur du stroma mammaire, représentent des acteurs émergents dans la progression tumorale. Mon équipe est l'une des premières à avoir montré que les adipocytes péritumoraux étaient impliqués dans l'agressivité des cancers du sein. Du dialogue bidirectionnel qui s'instaure entre les adipocytes et les cellules cancéreuses mammaires résulte des modifications des deux types cellulaires : (i) les cellules tumorales " éduquées " par les adipocytes présentent des capacités invasives augmentées et une plus grande résistance aux traitements et (ii) les adipocytes co-cultivés avec les cellules tumorales acquièrent un phénotype activé avec des modifications spécifiques telles que délipidation, perte de marqueurs adipocytaires, surexpression de cytokines pro-inflammatoires et sécrétion de protéines de la matrice extracellulaire, qui nous ont amené à les nommer CAA pour " Cancer-Associated Adipocytes ". De façon intéressante, le dialogue paracrine entre les tumeurs et les adipocytes pourrait être amplifié dans l'obésité, où l'équilibre normal des protéines sécrétées par le tissu adipeux est perturbé. Dans le cancer du sein, l'obésité est associée à une augmentation des risques de survenue après la ménopause et une aggravation du pronostic indépendamment du statut ménopausique s'expliquant par une augmentation de la dissémination locale et à distance et par une réponse diminuée aux traitements, notamment par une résistance plus importante. L'objectif de ma thèse a été d'évaluer le rôle des adipocytes dans la chimiorésistance des cellules tumorales mammaires. En effet, la résistance est une limite majeure à l'efficacité des traitements et contribue à l'apparition de rechutes, lesquelles sont augmentée chez les patientes obèses. Au moyen d'un modèle de co-culture en 2D, nous avons montré que les adipocytes sont capables de promouvoir une résistance pléïotropique (doxorubicine, paclitaxel, 5-fluorouracile et cyclophosphamide) dans diverses lignées tumorales mammaires, indépendamment du type de tumeur. Grâce aux propriétés de fluorescence des anthracyclines, nous avons montré que cette résistance implique une augmentation de l'efflux de doxorubicine, l'empêchant d'agir au niveau de son site d'action nucléaire. Ce mécanisme d'efflux implique un processus original, qui fait intervenir la protéine de voûte majeure MVP / LRP (Major vault protein / Lung resistance protein), un transporteur nucléocytoplasmique dont la fonction reste à ce jour mal comprise. Suite à l'efflux nucléaire de drogue, celle-ci s'accumule dans des vésicules cytoplasmiques avant d'être effluée hors de la cellule via des vésicules extra cellulaires. Nous avons également pu montrer que cette résistance médiée par MVP s'explique par la sécrétion de facteurs solubles adipocytaires et est amplifiée en conditions d'obésité. En conclusion, nos résultats montrent que les adipocytes péritumoraux sont capables d'influencer la progression tumorale en favorisant la chimiorésistance via un mécanisme original impliquant la protéine MVP, qui pourrait potentiellement devenir un marqueur de résistance aux traitements. Ces travaux pourraient expliquer, au moins en partie, le mauvais pronostic des cancers du sein chez les patientes obèses et ouvrent donc, à plus long terme, des perspectives thérapeutiques intéressantes, destinées à interrompre le dialogue délétère entre adipocytes et cellules tumorales, en particulier chez les patients obèses
Breast cancer is the most common cancer among women in France, as well as in the European Union and the United States. Although the number of cases observed each year has tended to decrease since 2005, notably due to organized screening, this disease remains the leading cause of cancer death in women. Many studies have shown that tumor progression is dependent on tumor cells but also on the "healthy" cells of the microenvironment (or stroma) that surround the tumor. In the case of breast cancer, adipocytes, the major cell type of the mammary stroma, represent emerging actors in tumor progression. My team is one of the first to have shown that peritumoral adipocytes were involved in the aggressiveness of breast cancers. From the bi-directional dialogue that takes place between adipocytes and mammary cancer cells results some changes in both cell types : (i) the tumor cells "educated" by the adipocytes have increased invasive capacities and greater resistance to treatments and (ii) the adipocytes co-cultured with the tumor cells acquire an activated phenotype with specific modifications such as delipidation, loss of adipocyte markers, overexpression of pro-inflammatory cytokines and secretion of proteins of the extracellular matrix, which led us to name them CAA for "Cancer-Associated Adipocytes". Interestingly, the paracrine dialogue between tumors and adipocytes could be amplified in obesity, where the normal balance of proteins secreted by adipose tissue is disrupted. In breast cancer, obesity is associated with an increased risk of occurrence after menopause and a worsening prognosis independent of menopausal status due to increased dissemination (local and remote) and decreased response to treatments, in particular by a greater resistance. The objective of my thesis was to evaluate the role of adipocytes in the chemoresistance of mammary tumor cells. Indeed, resistance is a major limit to the effectiveness of treatments and contributes to the onset of relapses, which are increased in obese patients. Using a 2D co-culture model, we have shown that adipocytes are able to promote pleiotropic resistance (doxorubicin, paclitaxel, 5-fluorouracil and cyclophosphamide) in various mammary tumor lines, irrespective of tumor type. By taking advantage of the fluorescence properties of anthracyclines, we have shown that this resistance implies an increase in the doxorubicin efflux, preventing it from acting at its site of nuclear action. This efflux mechanism implies an original process involving the major vault protein MVP / LRP (Major Vault Protein / Lung Resistance Protein), a nucleocytoplasmic transporter whose function remains poorly understood to date. Following nuclear drug efflux, it accumulates in cytoplasmic vesicles before before being expelled from the cell via extracellular vesicles. We also showed that this resistance mediated by MVP could be explained by the soluble factors secreted from adipocytes and is amplified in obesity conditions. In conclusion, our findings highlight that peritumoral adipocytes are able to influence tumor progression by promoting chemoresistance via an original mechanism involving the MVP protein, which could potentially become a marker of resistance to treatments. This work may explain, at least in part, the poor prognosis of breast cancers in obese patients and thus could provide interesting therapeutic perspectives, in order to interrupt the deleterious dialogue between adipocytes and tumor cells, particularly in obese patients

L’impatto della dieta (in particolare quella associate a cicli di restrizione calorica) sui benefici della salute sono stati già dimostrati. Questi includono il miglioramento delle malattie cardiovascolari, insulino resistenza, diabete, disordini immuni, rallentamento dei processi di invecchiamento ed in particolare il ridotto rischio per il cancro. Recenti studi in modelli animali ed in modelli in vitro hanno scoperto un nesso tra i cicli di restrizione calorica ed il ridotto rischio di cancro ed un migliorata efficacia della chemioterapia che è stata già descritta per alcuni tipi di cancro. L’obiettivo di questo progetto di ricerca è stato quello di elucidare il ruolo dei cicli di restrizione calorica nelle vie di segnale intracellulari coinvolti nei meccanismi di chemioresistenza del cancro del pancreas che è tra i cancri più aggressivi ed è classificato come la quarta causa di morte cancro-correlata al fine di implementare una formulazione nutrizionale che mimasse la restrizione calorica in grado di reversare la chemioresistenza o di inibire la crescita tumorale. Avvantaggiandoci di modelli animali xenograft e di linee cellulari del cancro del pancreas, mediante l’utilizzo di approcci biochimici e biomolecolari abbiamo inizialmente cercato di comprendere a fondo il ruolo dei cicli di restrizione calorica nella progressione del cancro del pancreas in un modello murino predisposto e poi elucidato i meccanismi molecolari coinvolti nella chemioresistenza. A doggi è di fondamentale importanza identificare i targets potenziali che posso essere utilizzati come predittori di malattia utili per la prevenzione, la prognosi ed il trattamento. I risultati di questo progetto saranno di aiuto per gli scienziati impegnati nell’identificazione di nuovi target terapeutici.
The impact of nutrition (particularly associated with short term starvation (STS)) on major health benefits have been already demonstrated. These include amelioration of cardiovascular diseases, diabetes, insulin resistance, immune disorders, slowing of the aging process and in particular reduced risks of cancer. Recent studies in rodent and in in vitro models uncovered a potential link between STS and improved efficacy of chemotherapy which has already been demonstrated for some types of cancer. The broader objective of the research project developed during the PhD program was to elucidate the role of fasting (or short term starvation, STS) on the intracellular signaling events involved in the chemo-resistance of pancreatic cancer (PC) amidst the most aggressive types of cancer ranked as the fourth leading cause of cancer-related deaths worldwide, in order to implement a new diet formulation, mimicking calories restriction, in order to reverse chemoresistance or inhibit tumor growth. Taking advantage of in vivo xenograft mouse model for pancreatic cancer and in vitro PC cell lines, using biochemical and biomolecular approaches we first aimed to understand in depth the role of STS during the onset of pancreatic cancer in an ad hoc murine model and we then elucidate the molecular mechanisms involved in PC chemoresistance. It is important to systematically identify potential targets, which could serve as biomarkers for cancer prevention, prognosis and treatment. By elucidating the mechanisms involved in PC chemoresistance the results of this study will help scientists to identify new therapeutic targets.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.
Approximately 25% of patients with locoregional esophageal adenocarcinoma (EC) are resistant (marked by minimal tumor regression; TRG 3) to preoperative chemoradiation, including 5FU‐based and CROSS regimens. Previously, an immunohistochemistry (IHC) test that accurately identifies patients as responders (TRG 0‐2) or non‐responders (TRG 3) to neoadjuvant CTRT was developed and validated. The current study was designed to identify gene expression profile (GEP) signatures able to predict response to preoperative treatment. Methods: Formalin‐fixed, paraffin‐embedded (FFPE) tumor tissue from 24 diagnostic biopsies (14 responders, 10 non‐responders) was collected. RNA was isolated, and RT‐PCR performed to assess the expression of 96 candidate genes chosen from in silicoanalysis. Genetic signatures incorporating genes with significant expression differences in pathologically determined responders versus non‐responders were identified, and linear and non‐linear predictive modeling methods were used to assess the accuracy of the signatures for predicting treatment response. Cross validation was performed to attain corrected accuracy values. Ten‐, 18‐, and 24‐gene signatures were identified with significantly different gene expression levels in responders compared to non‐responders (p < 0.05). Functional groups represented by the signatures included DNA damage repair, extracellular matrix remodeling, and 5FU metabolism. Partial Least Squares (PLS) prediction of treatment response was compared to pathologic TRG determined by blinded pathologic reading, and resulted in an area under the curve (AUC) of 0.99 and overall accuracy of 100% for the 24‐gene signature. Corrected AUC of 0.99 and accuracy of 95% resulted from five‐fold cross validation with 20 iterations. Heatmap analysis of the 24‐gene signature separated the EC cases into two distinct clusters, the first with 93% responders and the second with 90% non‐responders. The current study identifies novel gene signatures able to accurately predict EC patient response to preoperative treatment. The GEP may allow non‐responders to avoid unnecessary toxicities associated with chemoradiation therapy.

Prostate cancer is one of the most common cancers affecting one of every six men in United States. It is increasingly appreciated that tumor or cancer stem cells are the cells responsible for initiating tumor formation and therefore should be targeted for eradication in cancer treatment. But the mechanism involved in the acquisition of unlimited self-renewal and tumor initiation by cancer stem cells is unknown. Nanog, along with Oct3/4 and Sox-2, constitute the core transcriptional circuitry for the maintenance of stemness in embryonic stem cells. Herein we report that Nanog expression was detected at mRNA and protein levels in prostate cancer cells. The Nanog-expressing LNCaP-T and DU145 cells were enriched by infection with lentiviruses expressing GFP under the control of Nanog promoter. The Nanog-enriched prostate cancer cells had stronger expressions of stem and progenitor cell surface markers, including CD44 and CD133, when compared with those in the control group. Colony formation assay found that the Nanog-enriched LNCaP-T and DU145 cells formed more holoclones and prosta-spheres, which contained more self-renewing cells, than the control cells did. On the other hand, knockdown of Nanog in DU145 or LNCaP-T cells, via shRNAs, reduced their ability to form holoclones. Instead, most clones derived were meroclone and paraclones as result of increased differentiation and senescence due to knockdown of Nanog. When injected into mice, Nanog-enriched DU145 cells were found to possess increased tumorigenic potentials when compared to the vector controls. On the other hand, LNCaP-T cells with Nanog knocked down did not form tumors, while the vector controls readily formed tumors. Taken together, our data suggest an essential role for Nanog in the self-renewal and tumor initiation of prostate cancer cells. Chemotherapy is the major salvage therapeutic modality available for the patients with advanced cancers. However, drug resistance by some prostate cancer cells is a major barrier to efficacious chemotherapy. It has been increasingly appreciated that cancer stem cells are responsible for resistance to chemo- or radio-therapy, in addition to tumor initiation. However, the mechanisms involved remain unknown. In this study, we examined whether Nanog plays an essential role of Nanog in resistance to chemotherapy. In the surviving fractions of prostate cancer cells, we found increased levels of Nanog protein when compared to the cells treated with solvent control. To determine the role of Nanog in resistance of prostate cancer cells, we marked and enriched Nanog-expressing prostate cancer DU145 and LNCaP-T cells using a reporter gene under control of 2.5 kb hNanog1 promoter. When compared to the control, the prospectively enriched Nanog-expressing cells presented increased resistance to Taxol, vinblastine, and doxorubicin. Profiling of genes in drug resistance and metabolism revealed a marked increase in the mRNA level of ATP-binding cassette (ABC) efflux transporters B1 and G2 in tumor cells enriched with endogenous Nanog expression. The increased expression of ABCB1 and ABCG2 at protein levels in Nanog expressing cells was confirmed by Western blot and immunocytochemistry. Inhibition of ABCB1 activities sensitized Nanog expressing cells toward Taxol and vinblastine, and to less extent, doxorubicin. Blocking of ABCG2 activity sensitized Nanog expressing cells toward doxorubicin, but not Taxol and vinblastine. In addition, the tumor cells enriched with Nanog expression showed reduced apoptosis in response to Taxol treatment. Interestingly, Nanog-enriched prostate carcinoma cells displayed aberrantly activated â-catenin signaling, which is potentially associated with their increased chemo-resistant ability as well as the increased acquisition of epithelial to mesenchymal transition. In summary, Nanog is expressed in prostate cancer cells, especially in those positive for stem/progenitor markers. Enrichment of Nanog expressing cells led to enrichment of tumor cells with increased tumor initiating ability and increased resistance toward chemotherapy. Knockdown of Nanog reduces tumor initiating ability of prostate cancer cells and further sensitizes them toward chemotherapy. The gain-of-function and loss-of-function studies suggest an essential role of Nanog for prostate cancer cells to initiate tumor formation and resist chemotherapy.

El cáncer de próstata (CaP) metastásico es actualmente incurable. Prostate tumor overexpressed 1 (PTOV1) se expresa a niveles altos en CaP y otras neoplasias asociado a estadíos más agresivos y mal pronóstico clínico. Un objetivo principal de esta tesis es estudiar la función de PTOV1 en la progresión del CaP y su papel en el desarrollo de resistencia a la quimioterapia, principalmente con taxanos. Hemos demostrado que PTOV1 induce una transición epitelio-mesénquima parcial promoviendo la sobreexpresión de c-Jun, Snail1 y vimentina. Como soporte de su relevancia en la progresión del cáncer de próstata, su downregulation significativamente inhibe el potencial tumorigénico y metastásico de células de cáncer de próstata PC3. La sobreexpresión de PTOV1 en células de cáncer de próstata sensibles al tratamiento con docetaxel incrementa la supervivencia de estas células tras la exposición a dicho fármaco e induce la sobreexpresión de genes relacionados con el fenotipo resistente al docetaxel (ABCB1, CCNG2 y TUBB2B). Además, PTOV1 favorece “in vitro” la formación de prostatoesferas y la expresión de genes de autorenovación (ALDH1A1, LIN28A, MYC y NANOG). Por el contrario, la disminución de expresión de PTOV1 disminuye la supervivencia celular provocando un arresto del ciclo celular en la fase G2/M seguido de muerte celular por apoptosis. Estos efectos son mayores en células resistentes al docetaxel. Análisis de bases de datos públicas de tumores de próstata sin tratar muestra que la expresión de PTOV1 correlaciona positivamente con mayor grado de los tumores, y con la expresión de CCNG2 y ALDH1A1. También observamos que PTOV1, CCNG2 y ALDH1A1 están significativamente sobreexpresados en el grupo de pacientes que posteriormente a la prostatectomía radical desarrolló metastatasis y que el análisis de las metástasis muestra gran expresión y amplificación de estos 3 genes, sugiriendo que estos genes podrían ser utilizados como predictores de metástasis y mal pronóstico. En otros tumores como máma y ovario, la mala respuesta a la quimioterapia está asociada a niveles más altos de PTOV1. En conjunto, nuestros datos confirman el papel pro-oncogénico de PTOV1 e indican que podría ser un gene candidato para determinar el pronóstico de pacientes con cáncer de próstata. Un segundo objetivo era estudiar el papel de la desregulación de la traducción de proteínas en cáncer de próstata resistente a la supresión androgénica y a la quimioterapia. La mayoría de intentos para determinar el perfil genético de células de próstata resistente a las terapias han sido llevados a cabo mediante el análisis de la expresión génica usando el RNA total. Sin embargo, datos de otros y de nuestro grupo sugieren que el control a nivel de la traducción de proteínas es un importante regulador de la expresión génica en cáncer. En modelos desarrollados in vitro de células de cáncer de próstata resistentes a la supresión androgénica y la quimioterapia con docetaxel, hemos observado una disminución significativa de la proliferación y la vía de señalización PI3K/ AKT/mTOR, principal regulador de la síntesis de proteínas. Mediante secuenciación masiva (NGS) hemos analizado el conjunto de RNAm asociados a polisomas de estos modelos resistentes, como un reflejo más directo de aquellos transcritos que activamente están siendo traducidos. Mediante estos análisis, pretendemos discernir los mecanismos que operan la regulación de la traducción del RNA en la progresión del cáncer de próstata especialmente en el desarrollo de resistencia a las terapias.
Metastatic prostate cancer is presently incurable. Prostate tumor overexpressed 1 (PTOV1) is expressed at high levels in prostate cancer and other neoplasias in association with a more aggressive disease and poor clinical outcomes. One main objective of this thesis is to study PTOV1 function in prostate cancer progression and its role in the development and maintenance of resistance to current therapies, mainly chemotherapy with taxanes. We showed here that PTOV1 increased cell motility and invasion of prostate cancer cells inducing a partial epithelial-to-mesenchymal transition promoting the translation of c-Jun protein, Snail1 and Vimentin. In support of the relevance of PTOV1 in prostate cancer progression, its knockdown significantly inhibited the tumorigenic and metastatic potentials of PC-3 prostate cancer cells. Transduction of PTOV1 in docetaxel-sensitive Du145 and PC3 cells significantly increased cell survival after docetaxel exposure and induced docetaxel-resistance genes expression (ABCB1, CCNG2 and TUBB2B). In addition, PTOV1 induced prostatospheres formation and self-renewal genes expression (ALDH1A1, LIN28A, MYC and NANOG). In contrast, Du145 and PC3 cells knockdown for PTOV1 significantly accumulated in the G2/M phase, presented a concomitant increased subG1 peak, and cell death by apoptosis. These effects were enhanced in docetaxel-resistant cells. Analyses of tumor datasets show that PTOV1 expression significantly correlated with prostate tumor grade, drug resistance (CCNG2) and self-renewal (ALDH1A1, MYC) markers. These genes are concurrently overexpressed in most metastatic lesions. Metastases also show PTOV1 genomic amplification in significant co-occurrence with docetaxel-resistance and self-renewal genes. Our findings identify PTOV1 as a promoter of docetaxel-resistance and self-renewal characteristics for castration resistant prostate cancer. The concomitant increased expression of PTOV1, ALDH1A1 and CCNG2 in primary tumors, may predict metastasis and bad prognosis. In addition high expression of PTOV1 in breast and ovarian tumors are associated with bad response to chemotherapy and lower overall survivals.​ Altogether, data point to a pro-oncogenic role of PTOV1 in different tumors and indicate that it is a candidate gene for patient prognosis. A second main objective is to study the role of translation deregulation in aggressive prostate cancer, in particular in the cells resistance to most conventional therapies used in metastatic in prostate cancer. Most attempts to determine the mRNA profile of androgen independent cells and chemoresistant cells have made use of total RNA for gene expression analysis. However, several lines of evidence suggest that translational control is a central regulator of gene expression in cancer. By using polysome profiling coupled to RNA-seq approach we aim to discern selective regulation of a subset of mRNAs among resistant cells that might help us to elucidate the mechanisms operating in the control of RNA translational in prostate cancer progression, especially in the development of resistance to current therapies.

Les cancers de l’ovaire représentent la première cause de mortalité due aux cancers gynécologiques dans les pays développés. La plupart de ces cancers sont diagnostiqués à des stades tardifs quand les cellules cancéreuses ovariennes ont disséminé et colonisé les parois de la cavité abdominale. En conséquence, ces cancers sont associés à un sombre pronostic.Plus d’un tiers des patientes présentent une accumulation de liquide d’ascite au moment du diagnostic. L’ascite est un fluide exsudatif ayant une composante cellulaire et également une composante liquide acellulaire, constituant un réservoir dynamique de molécules bioactives. Malgré une réponse efficace aux traitements standards, les taux de récidive de cette pathologie restent élevés. L’acquisition d’une chimiorésistance et le mode de propagation atypique des cellules cancéreuses sont deux éléments importants impliqués dans la récidive des cancers ovariens.Le rôle de l’ascite et plus particulièrement de la composante matricielle ascitique, dans le processus de dissémination et dans la chimiorésistance des cellules cancéreuses ovariennes reste peu étudié et constitue le cœur de ce travail de thèse.L’objectif des travaux a été d’étudier l’influence de l’ascite et de deux glycoprotéines matricielles purifiées à partir d’échantillons d’ascites sur les comportements cellulaires propices à la dissémination et à la récidive des cancers ovariens. De plus, l’influence de l’ascite sur la réponse des cellules aux traitements à base de platine a également été recherchée et estimée.L’étude démontre que l’ascite est un microenvironnement propice à la dissémination des cellules cancéreuses ovariennes et que la fibronectine et la vitronectine ascitiques ainsi que leurs récepteurs d’adhérence spécifiques sont des éléments qui participent à la régulation de cette dissémination. L’ascite exerce également un effet protecteur contre l’effet cytotoxique des agents chimiothérapeutiques.Ces résultats mettent en perspectives l’intérêt potentiel de la fibronectine et de la vitronectine ascitiques comme outils diagnostiques et/ou cibles thérapeutiques des cancers ovariens et nous engagent à approfondir l’étude de ces deux molécules et de leurs conséquences sur la progression de la maladie
Ovarian cancers are the leading cause of death among gynaecological cancers in western countries. Most of these cancers are diagnosed at a late stage, when ovarian cancer cells have spread and colonized the walls of the abdominal cavity. Therefore, these cancers are associated to a poor prognosis.More than one third of the patients show an accumulation of ascites at the time of the diagnosis. Ascites are exudative fluids composed of a cellular and also an acellular fraction. In fact, ascites constitute a dynamic reservoir of bioactive molecules. Even if the response of ovarian cancers to the current first-line therapy, that consit in debulking surgery followed by chemotherapy, is satisfactory, the rate of recurrence remains important. The gain of a chemoresistance and the atypical widespread of cancer cells are two important factors involved in the recurence of the ovarian cancers.The role of ascites and more particularly, of the ascitic matrix componants on the dissemination process and on the chemoresitance of the ovarian cancer cells remain poorly studied and is the aim of this work.The objective of this study was to investiguate the influence of ascites and of two matrix glycoproteins purified from samples of ascites i) on the cells behavior convenient to the dissemination and the recurrence of the ovarian cancers and ii) on the response of cells to therapeutics treatments with platinum.This study suggests that ascites are a permissive microenvironment to the dissemination of ovarian cancer cells and that ascitic fibronectin and vitronectin as well as their specific receptors are actors which participate to the regulation of this dissemination. Ascites also protect ovarian cancer cells against the cytotoxicity of chemotherapeutic drugs.These results illustrate the potential interest of ascites derived fibronectin and vitronectin as diagnosis tools and/or therapeutic targets for ovarian cancers and encourage us to deepen the study of these two molecules and their consequences in the progress of the disease

Parmi les cellules stromales présentes dans le microenvironnement du cancer du sein, les adipocytes représentent des acteurs émergents dans la progression tumorale. Etudier le dialogue croisé entre adipocytes et cellules tumorales est d'importance en médecine, car l'obésité est un facteur reconnu de mauvais pronostic dans de nombreux cancers, notamment le cancer du sein. Nos résultats montrent que lorsque les adipocytes sont maintenus en présence des cellules tumorales pendant un temps prolongé, ils se réorientent progressivement en cellules fibroblastiques, présentant des caractéristiques de fibroblastes-Associés au Cancer. Nous avons nommé ces cellules, ADFs pour " Adipocytes Derived Fibroblasts " et montré que ces derniers, présents dans des tumeurs mammaires humaines, favorisent de manière importante l'invasion des cellules tumorales. D'autre part, nous avons démontré que les adipocytes favorisent une résistance pléiotropique aux cellules tumorales via un mécanisme d'efflux original et que cette chimiorésistance médiée par les adipocytes est amplifiée en conditions d'obésité. Ces travaux pourraient expliquer, en moins en partie, le mauvais pronostic des cancers du sein chez les patientes obèses
Among stromal cells present in breast cancer, adipocytes represent emerging players in tumor progression. Studying the role of adipocytes in cancer is a major interest since epidemiological studies have convincingly established that obesity is associated with a poor outcome for several cancers, especially breast cancer. We demonstrated that some CAFs (Cancer Associated Fibroblasts) present in the breast tumor stroma arise from the "dedifferenciation" of adipocytes upon prolonged stimulation by tumor cells. This population was named ADFs (Adipocytes-Derived Fibroblasts) and was found in clinical samples of breast cancer. We further demonstrated that ADFs stimulate the invasive capacities of tumor cells. Moreover, our results suggest that adipocytes promote multidrug resistance in breast cancer cell lines mediated by an original efflux mechanism. Finally, we demonstrated that adipocyte-induced chemoresistance is amplified by obesity. This work may explain, at least in part, the poor prognosis of breast cancer in obese patients

Le cancer ovarien est la 1ère cause de décès par cancer gynécologique. A ce jour, le traitement le plus efficace consiste en une exérèse la plus complète possible de la tumeur associée à une chimiothérapie à base de sel de platine et de taxanes. Cependant, la survie globale des patientes à 5 ans reste faible (environ 40%), due à un fort taux de récidive et au développement d'une résistance aux traitements. Les interactions entre les cellules tumorales ovariennes (CTO) et le microenvironnement semblent être impliquées dans la progression de la maladie et l'acquisition de cette chimiorésistance. Parmi les cellules du microenvironnement tumoral, nous avons pu isoler des cellules stromales mésenchymateuses (MSC) à partir de biopsies de carcinome ovarien. Ces MSC associées au cancer (CA-MSC) ont la capacité d'induire une résistance des CTO au traitement par carboplatine. Afin de mieux comprendre les mécanismes conduisant des MSC à sécréter des facteurs pro-tumoraux, dans un contexte de cancer ovarien, nous avons mis en place un modèle à partir de MSC issues de donneurs ne présentant pas de cancer. Nous avons observé que la présence d'un environnement tumoral ovarien modifie le phénotype de MSC de moelle osseuse (BM-MSC) physiologiques, en entrainant notamment la sécrétion par ces CA-MSC " induites " de facteurs chimioprotecteurs pour les CTO. Parmi les facteurs sécrétés, nous avons identifié des chimiokines telles que CXCL1, CXCL2 et IL-8 comme des cibles thérapeutiques intéressantes pour lutter contre la résistance à la chimiothérapie. En effet, les CA-MSC et les CA-MSC " induites " sécrètent plus de CXCL1, CXCL2 et d'IL-8 que les BM-MSC. En outre, l'inhibition des récepteurs de ces chimiokines (CXCR1 et CXCR2) sensibilise les CTO au carboplatine, même en présence des sécrétions des CA-MSC et des CA-MSC " induites ". Ces expériences in vitro ont été confirmées in vivo dans un modèle expérimental murin. En effet, la co-injection à des souris immunodéprimées de MSC avec des CTO humaines entraine une protection des CTO vis-à-vis du carboplatine comparée à l'injection de CTO seules. Le co-traitement avec un inhibiteur de CXCR1 et de CXCR2 a permis de sensibiliser les CTO au carboplatine et d'empêcher la chimiorésistance induite par les MSC. En effectuant une étude rétrospective évaluant la concentration de ces chimiokines au moment du diagnostic, nous avons confirmé que les patientes qui seront a posteriori " résistantes " au carboplatine ont une concentration sérique de ces chimiokines qui est plus élevées que les patientes du groupe " sensibles " au carboplatine. Outre leur rôle direct dans l'acquisition de la chimiorésistance, les chimiokines comme CXCL1, CXCL2 et IL-8 peuvent être impliquées dans la régulation du système immunitaire. Nous avons montré que les CA-MSC étaient capables de modifier le phénotype des macrophages vers un phénotype M2 décrit comme pro-tumoral. En effet, ces macrophages ainsi polarisés ont un pouvoir cytotoxique dirigé contre les CTO inférieur aux macrophages non stimulés. L'ajout d'un inhibiteur de CXCR1 et de CXCR2 restaure le pouvoir cytotoxique initial des macrophages, même en présence des sécrétions des CA-MSC. Ainsi nos travaux suggèrent que les CA-MSC pourraient provenir de MSC physiologiques qui, au contact d'un environnement tumoral ovarien, vont acquérir un phénotype capable d'induire la sécrétion de facteurs chimioprotecteurs pour les CTO et de polariser les macrophages vers un phénotype moins cytotoxique pour les CTO. Ces deux phénomènes pro-tumoraux peuvent être inhibés par l'utilisation d'un inhibiteur des récepteurs de CXCR1 et de CXCR2. Ainsi ces récepteurs des chimiokines, semblent être des cibles thérapeutiques intéressantes afin de sensibiliser les CTO au carboplatine et traiter plus efficacement la tumeur. Ceci pourrait permettre d'éviter les récidives des cancers ovariens qui sont, à l'heure actuelle, observées chez plus de 70% des patientes
Ovarian cancer is the leading cause of gynecological cancer death. To date, the most effective treatment consists of the complete excision of the tumor associated with chemotherapy based on platinum salts and taxanes. However, the 5-year overall survival remains low (close to 40%) due to a high rate of recurrence and development of resistance to treatments. Disease progression and the acquisition of this chemoresistance seem to be due to interactions between ovarian tumor cells (OTC) and the microenvironment. Amidst the cells of the tumor microenvironment, we were able to isolate mesenchymal stromal cells (MSC) from tumor biopsies of patients with ovarian adenocarcinoma. These cancer-associated MSC (CA-MSC) have the ability to induce resistance to carboplatin in OTC. In order to understand the mechanisms leading to the secretion of pro-tumoral factors by the CA-MSC in the context of ovarian cancer, we have developed a model based on the in vitro MSC culture of from healthy donors in tumor conditioning media. We have observed that an ovarian tumor environment modifies the physiological phenotype of bone marrow MSC (BM-MSC), leading in particular to the secretion by these "induced" CA-MSC of chemoprotective factors for OTC. Among these secreted factors, we have identified chemokines such as CXCL1, CXCL2 and IL-8 as therapeutic targets in order to control drug resistance. In fact, CA-MSC and "induced" CA-MSC secrete more CXCL1, CXCL2 and IL-8 than BM-MSC and the use of an inhibitor of their receptors (CXCR1 and CXCR2) sensitized OTC to carboplatin even in the presence of CA-MSC and " induced " CA-MSC secretions. These in vitro experiments have been confirmed in an experimental mouse model in vivo. Indeed, the co-injection of MSC with OTC yielded a greater protection of OTC to carboplatin compared with the OTC injection alone. Co-treatment with a CXCR1 and CXCR2 inhibitor resulted in sensitization of OTC to carboplatin and prevention of MSC-induced chemoresistance. We conducted a retrospective study evaluating the concentration of these chemokines at the time of diagnosis. We thus showed that patients who are a posteriori "resistant" to carboplatin have a higher concentration of chemokines than patients belong to the "sensitive" group to carboplatin. In addition to their direct role concerning the acquisition of chemoresistance, chemokines such as CXCL1, CXCL2 and IL-8 may be involved in the immune system regulation. In this context, we showed that CA-MSC were able to modify the phenotype of macrophages into a M2 phenotype described in literature to have a pro-tumoral activity. Indeed, these polarized macrophages present a lower cytotoxic capacity against OTC than unstimulated macrophages. CXCR1 and CXCR2 inhibitor restores the initial cytotoxic activity of macrophages even in the presence of CA-MSC secretions. Thus, our work suggests that CA-MSC could originate from physiological MSC which, in contact with an ovarian tumor environment, acquire a phenotype capable of inducing the secretion of chemoprotective factors for CTO and of polarizing macrophages into a less cytotoxic phenotype for OTC. These two pro-tumoral mechanisms can be inhibited by the use of CXCR1 and CXCR2 receptor inhibitors emphasizing the role of these chemokines in the development of a chemoresistance and showing how important is to go further is this study. Finally, these chemokines receptors seem to be therapeutic targets in order to sensitize OTC to carboplatin and to potentialize actual treatments. This could prevent the recurrence of ovarian cancers that are presently observed in more than 70% of patients

This study evaluated comparative genomic hybridisation (CGH) as a tool to detect candidate regions of the genome associated with chemoresistance. Using a variation on conventional CGH, DNA from three cell lines that were resistant to thymidylate synthase (TS) inhibitors (tomudex [TDX] or 5-fluorouracil [5-FU]) and their sensitive parent cells were evaluated. In MCF-7 and H630, cells that were resistant to TDX, a specific TS inhibitor with no other known cytotoxic potential, only a single region of change (18p gain) was apparent. The third cell line H630R10, which was resistant to 5-FU, had changes in several genomic regions following the acquisition of resistance, including 18p. Gain in the chromosomal region containing the TS gene (18pll.32) was detected by CGH in all three resistant cell lines. However, additional novel regions of interest were identified in the cells that were resistant to 5-FU, a cytotoxic agent known to have several other modes of cytotoxicity besides TS inhibition. These results suggested that CGH is of potential use in the detection of regions of the genome involved in chemoresistance. Having shown the potential of CGH as a tool for assessing chemoresistance at the genomic level, steps toward clinical application of this technique were evaluated. A prerequisite for study in archival pathology samples was successful DNA extraction and universal amplification of tumour DNA from paraffin-embedded tumour sections for CGH analysis. Degenerate oligonucleotide primed - polymerase chain reaction (DOP-PCR) was performed on minute quantities (50ngs) of fresh cell line DNA (H630R10) and tumour DNA (osteosarcoma), as well as paraffin-embedded DNA from the same case. The results of these DOP-PCR CGH reactions were compared with conventional CGH using l|0.g quantities of fresh DNA from both H630R10 cell line and osteosarcoma. The CGH profiles of the conventional CGH and DOP-PCR CGH did not show a high level of concordance, only 55% of the gains and 83.3% of losses detected by conventional CGH were detected by DOP-PCR CGH The use of universal amplification by DOP-PCR in paraffin-embedded sections was not taken forward into clinical evaluation. A study of colorectal cancer (CRC) was initiated which involved the microdissection of 29 Dukes' C CRC tumours from fresh frozen material for CGH analysis. This conventional CGH analysis of CRC tumours involved assessing each tumour twice by reversal of fluorochromes. Only genomic regions that were detected as changed in both forward and reverse profiles were accepted. This approach detected several regions of genome as changed across the 29 tumours. In all, 108 gains (a mean number of 3.7 aberrations per tumour, range 1-12) and 85 losses (a mean number of 2.9 aberrations per tumour, range 0-11) were detected in the 29 tumours. CGH analyses identified certain chromosomal regions as more likely to be changed than others. The most frequent aberrations detected across the 29 tumours was a loss of chromosomal arm 18q, seen in 31% of the tumours assessed. Gain was also common at some sites throughout the genome, for example, gain of chromosomal arms, 13q and 20q was seen in 27.6% of cases. Mann-Whitney U tests investigating the association between specific chromosomal aberrations such as gain of 20q or loss of 18q and known markers of CRC tumourigenesis (p53, p27, p21, Rb, cyclin Dl, PCNA, P-catenin, e-cadherin, c-erbB-2, bcl2, EGFR and c-erbB-2) assessed by immunohistochemistry (IHC) in 29 tumours found no association. Testing of the total number of genomic aberrations detected (loss + gain = genetic grade) rather than the frequency of aberration at specific chromosomal loci also found no association with the CRC tumour markers. Finally, the association between the chromosomal aberrations detected by CGH was investigated in relation to patient survival. This thesis has demonstrated the value of a global approach to the study of chemoresistance and tumourigenesis through the application of powerful technology such as CGH.

Trop souvent diagnostiqués à des stades tardifs du fait de leur quasi asymptomatie, les adénocarcinomes séreux ovariens posent un véritable problème de santé publique. Malgré les progrès récents de prise en charge chirurgicale, l’émergence d’une maladie résiduelle microscopique chimiorésistante impacte grandement le pronostic des patientes.Le microenvironnement tumoral est un acteur clé de la progression tumorale et de l’émergence de résistances aux traitements anticancéreux. Durant ces travaux de thèse, nous nous sommes intéressés à deux composants majeurs du stroma tumoral, d’une part les cellules souches mésenchymateuses, d’autre part les cellules endothéliales.Nous avons pu démontrer que les cellules souches mésenchymateuses participent à la progression tumorale et l’émergence de résistances. Enfin nous avons démontré que les cellules endothéliales, via la production de facteurs angiocrines, participent à la chimiorésistance des cellules tumorales ovariennes.Dans ce travail, nous avons pu définir de nouvelles cibles thérapeutiques mettant en jeu la relation entre les cellules tumorales ovariennes et l’hôte
Ovarian cancers constitute a poor prognosis disease. Due to their absence of symptoms, ovarian cancers are generally diagnosed at late stages. Despite major breakthrough regarding ovarian cancer surgery, minimal residual disease-induced relapse is still a hurdle for clinicians.Tumor microenvironment is a key actor on disease progression and resistance to therapy. In this study, we have focused on two major components of the tumor stroma, on one hand, the mesenchymal stem cells, and the endothelial cells on the other hand.We were able to demonstrate that mesenchymal stem cells are critically involved in ovarian cancer progression and resistance to therapy, while the endothelium, through production of angiocrine factors, is deeply involved in resistance of ovarian cancer cells to platinum and taxane based therapy.Here, we set forth the idea that disrupting the relationship between ovarian cancer cells and their host stroma constitute a new therapeutic window

Le cancer du pancréas est l’un des cancers les plus mortels avec un pronostic extrêmement sombre. En 2020, le taux de survie à 5 ans reste très faible (de 3 à 9 % seulement) et la médiane de survie est de moins de 6 mois. Malgré des progrès dans la prise en charge des patients, les thérapies actuelles n’ont pas l’efficacité espérée. Ceci est dû à la forte chimiorésistance observée dans ce cancer. Le facteur clé de cette résistance est le microenvironnement tumoral complexe composé majoritairement de stroma et d’une matrice extracellulaire dense limitant l’accès des thérapies à la tumeur. Les limitations des modèles d’études actuels, notamment en termes de pertinence physiologique, sont un frein important dans la compréhension de cette chimiorésistance. En réponse à cette problématique, les chercheurs se tournent vers de nouvelles approches en développant de nouveaux modèles d’études alternatifs à ceux déjà disponibles (in vitro et in vivo).Les objectifs de ce travail ont été : (i) de développer un dispositif de culture in vitro 3D microfluidique permettant de reproduire le microenvironnement tumoral sur les plans biologique et mécanique, ainsi que de modéliser les écoulements et transports de masse présents dans une tumeur pancréatique, et (ii) d’aborder les changements morphologiques de la co-culture par l’étude de marqueurs épithélio-mésenchymateux et d’étudier l’impact de la chimiothérapie FOLFIRINOX dans ce modèle.Dans un premier temps nous avons montré numériquement et expérimentalement la faisabilité d’un tel modèle in vitro. La matrice extracellulaire choisie est une association de collagène I et d’acide hyaluronique créant une structure rigide proche des conditions in vivo. Elle permet le maintien de la culture à long terme en ne se dégradant pas ou peu sous l’effet de la perfusion ainsi que l’activation des cellules stellaires pancréatiques. La vitesse de perfusion choisie permet quant à elle d’appliquer un flux interstitiel équivalent à celui observé dans le microenvironnement in vivo, induisant une pression hydrostatique et une contrainte de cisaillement sur les cellules.Nous avons ensuite mis en évidence l’apport biologique de cette modélisation en montrant une chimiorésistance accrue au protocole FOLFIRINOX des cellules tumorales à la fois en mono- et en co-culture dans le dispositif microfluidique. Nous montrons également la mise en place d’un processus ayant des caractéristiques de la transition épithélio-mésenchymateuse et d’une possible promotion d’un phénotype dédifférencié des cellules tumorales par les cellules stellaires pancréatiques activées.En conclusion, nous présentons dans cette thèse un modèle d’étude microfluidique original permettant de modéliser une tumeur (co-culture de cellules épithéliales et mésenchymateuses) et d’étudier la cinétique d’une chimiothérapie multidrogues complexe. Ce dispositif devrait à l’avenir nous permettre d’étudier plus en profondeur les mécanismes de chimiorésistance et les interactions tumeur-stroma dans le cancer du pancréas
Pancreatic cancer is one of the deadliest cancers with an extremely poor prognosis. In 2020, the 5-year survival rate remains very low (only 3 to 9%) and the median is less than 6 months. Despite significant progress in the patient care, current therapies do not have the expected effectiveness. This is due to the strong chemoresistance observed in this cancer. The key factor of this resistance is the complex tumor microenvironment mainly composed of stroma and a dense extracellular matrix limiting the access of therapies to the tumor. The current models’ limitations, particularly in terms of physiological relevance, are a major obstacle in understanding this chemoresistance. In response to this issue, researchers are turning to different approaches by developing brand new models that are alternatives to those already available (in vitro and in vivo).The objectives of this work were: (i) to develop an in vitro 3D microfluidic culture device allowing to reproduce the tumor microenvironment both biologically and mechanically, as well as to model the flows and mass transport present in a pancreatic tumor, and (ii) to approach the morphological changes of the co-culture by studying epithelio-mesenchymal markers and to study the impact of FOLFIRINOX chemotherapy in this model.First, we have shown numerically and experimentally the feasibility of such an in vitro model. The chosen extracellular matrix is a combination of collagen I and hyaluronic acid creating a rigid structure close to in vivo conditions. It allows long-term culture maintenance under the effect of the perfusion as well as the activation of pancreatic stellate cells. The chosen perfusion rate allows to apply an interstitial flow in the model equivalent to the one observed in the in vivo microenvironment, inducing hydrostatic pressure and shear stress on the cancerous cells.Then, we demonstrated the biological contribution of this model by showing an increased chemoresistance to the FOLFIRINOX protocol of tumor cells both in mono- and in co-culture in the microfluidic device. We also show the establishment of a process presenting characteristics of epithelial-mesenchymal transition and a possible promotion of a dedifferentiated phenotype of tumor cells by activated pancreatic stellate cells.In conclusion, we present in this thesis an original microfluidic model allowing to mimic a tumor (co-culture of epithelial and mesenchymal cells) and to study the kinetics of a complex multidrug chemotherapy. In the future, the device should allow us to further study the mechanisms of drug resistance and tumor-stroma interactions in pancreatic cancer

Multidrug resistance (MDR) consists in the ability of cancer cells to become resistant towards different drugs and is frequently mediated by ABC-transporters efflux pumps, such as P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), which are also infamous for conferring cancer cell stemness and aggressiveness, thereby imparting a poor prognosis. MDR has been extensively studied in human oncology, but less is known in veterinary medicine. The aims of the past three years of investigation on canine mammary tumors have been to determine the distribution of P-gp and BCRP in the different cellular components of hyperplasia and neoplasia, to compare P-gp and BCRP expression in the histological stages and grades of canine mammary carcinomas (CMSs), to describe P-gp and BCRP expression in the stroma associated with neoplasia, and to examine P-gp and BCRP expression in two aggressive types of CMSs, namely canine inflammatory mammary cancer and histological grade 3 non-inflammatory carcinomas. P-gp and BCRP immunohistochemical expression was significantly higher in malignant vs benign epithelial cells and hyperplastic epithelium of the mammary gland, in aggressive histotypes (simple vs complex carcinomas; inflammatory carcinoma vs non-inflammatory carcinoma, only for P-gp), and in histological grade 2 and 3 carcinomas vs grade 1. Neoplasia-associated fibroblasts showed an increased expression in stage II and grade 2 and 3 carcinomas compared with stage I and grade 1. An increased expression of P-gp and BCRP was found in a canine relapsing and chemoresistant cutaneous mast cell tumor after chemotherapy with Vinblastine e Prednisolone. Chemoresistance in this case could be related to an increased efflux of the drugs mediated by these transmembrane pumps. Evaluation of P-gp and BCRP could help in the identification of aggressive, invasive and chemoresistant canine tumors, and the dog could provide a useful spontaneous model for chemoresistant human tumors.
La resistenza multifarmaco (MDR) conferisce alle cellule neoplastiche resistenza verso diversi composti chemioterapici ed è frequentemente dovuta all’azione di pompe di efflusso transmembrana (ABC-transporters), tra le quali la glicoproteina-P (P-gp) e la Breast Cancer Resistance Protein (BCRP), conosciute inoltre, per conferire caratteristiche di malignità e “staminalità” associate ad una prognosi infausta. La MDR è oggetto di molteplici studi in oncologia umana, mentre poco è noto in veterinaria. Gli obbiettivi di questi tre anni di ricerca sui tumori mammari della cagna sono stati: determinare l’espressione di P-gp e BCRP nelle componenti cellulari della mammella iperplastica e neoplastica, confrontarne l’espressione tra i diversi gradi e stadi istologici dei carcinomi, descriverne l’espressione nello stroma associato alla neoplasia, ed esaminarne e confrontarne l’espressione in due gruppi di neoplasie mammarie aggressive quali il carcinoma infiammatorio e il carcinoma di grado istologico 3. Mediante l’immunoistochimica è emerso che l’espressione di P-gp e BCRP era significativamente più elevata nei tumori mammari maligni (nelle cellule epiteliali maligne rispetto all’epitelio iperplastico), negli istotipi più aggressivi (nei carcinomi semplici rispetto ai complessi e nei carcinomi infiammatori rispetto ai carcinomi non-infiammatori, per P-gp), e nei carcinomi di grado istologico 2 e 3 rispetto al grado 1. I fibroblasti esprimevano maggiormente P-gp e BCRP nello stroma associato ai carcinomi di stadio II e di grado 2 e 3, rispetto a quelli di stadio I e grado 1. Un aumento dell’espressione di P-gp e BCRP è stato riscontrato in un cane con mastocitoma cutaneo recidivante dopo chemioterapia con Vinblastina e Prednisolone. La chemioresistenza sviluppata potrebbe essere dovuta all’aumento dell’efflusso dei farmaci dal comparto intracellulare mediato da P-gp e BCRP. Determinare l’espressione di P-gp e BCRP potrebbe essere utile ad identificazione le neoplasie aggressive e chemioresistenti, ed il cane potrebbe fornire un valido modello spontaneo per lo studio della chemioresistenza nei tumori dell’uomo.

Tumour necrosis has long been associated with poor prognosis and reduced survival in cancer. Hypotheses to explain this include the idea that as aggressive tumours tend to grow rapidly, they outgrow their blood supply leading to areas of hypoxia and subsequently necrosis. However whilst this and similar hypotheses have been put forward to explain the association, the biological significance of the cells which make up necrotic tissue has been largely ignored. This stems from the belief that because a tumour is more aggressive and fast growing it develops areas of necrosis, rather than, the tumour is more aggressive because it contains areas of necrosis. Which came first like the egg and chicken is yet to be determined, however to date most research has only considered the possibility of the former. Viable cells were found in the necrotic core of Multicellular Tumour Spheroids. When examined these cells were found to be different to the original cell line in terms of proliferation, migration, and chemosensitivity. A proteomic analysis showed that these phenotypical changes were accompanied by changes in a large number of proteins within the cells, some of which could be potential therapeutic targets. Furthermore this has led to a new hypothesis for tumour necrosis and its association with poor prognosis. Necrotic tissue provides a microenvironemental niche for cells with increased survival capabilities. Protected from many chemotherapeutics by their non-proliferative status once conditions improve these cells can return to proliferation and repopulate the tumour with an increasingly aggressive population of cells.
Yorkshire Cancer Research

Tumour necrosis has long been associated with poor prognosis and reduced survival in cancer. Hypotheses to explain this include the idea that as aggressive tumours tend to grow rapidly, they outgrow their blood supply leading to areas of hypoxia and subsequently necrosis. However whilst this and similar hypotheses have been put forward to explain the association, the biological significance of the cells which make up necrotic tissue has been largely ignored. This stems from the belief that because a tumour is more aggressive and fast growing it develops areas of necrosis, rather than, the tumour is more aggressive because it contains areas of necrosis. Which came first like the egg and chicken is yet to be determined, however to date most research has only considered the possibility of the former. Viable cells were found in the necrotic core of Multicellular Tumour Spheroids. When examined these cells were found to be different to the original cell line in terms of proliferation, migration, and chemosensitivity. A proteomic analysis showed that these phenotypical changes were accompanied by changes in a large number of proteins within the cells, some of which could be potential therapeutic targets. Furthermore this has led to a new hypothesis for tumour necrosis and its association with poor prognosis. Necrotic tissue provides a microenvironemental niche for cells with increased survival capabilities. Protected from many chemotherapeutics by their non-proliferative status once conditions improve these cells can return to proliferation and repopulate the tumour with an increasingly aggressive population of cells.

RUOLO DELLA PARAOXONASI-2 NEL TUMORE DELLA VESCICA: EFFETTO DEL SILENZIAMENTO E DELLA OVERESPRESSIONE DELLA PON2 SULLA PROLIFERAZIONE, MIGRAZIONE E CHEMORESISTENZA SCOPO: Nella popolazione mondiale, il tumore della vescica occupa il 5° posto tra i tumori più frequenti. Lo scopo di questo studio è quello di comprendere il coinvolgimento della Paraoxonasi-2 (PON2) nella carcinogenesi dello stesso. Le cellule T24 (linea cellulare di carcinoma della vescica umana) sono state trasfettate utilizzando i plasmidi pcDNA3 e plKO.1 per indurre rispettivamente l’overespressione o il silenziamento del gene PON2. MATERIALI E METODI: L’influenza della PON2 sulla migrazione cellulare è stata analizzata mediante il test di rimarginazione delle ferite in vitro. Le cellule T24 overespresse e silenziate per il gene della PON2, sono state trattate per 24 ore con Cisplatino e Gemcitabina e mediante il saggio colorimetrico MTT e l’utilizzo della 2,7-diclorofluoresceina diacetato. (DCFH-DA) sono state valutate, rispettivamente, la proliferazione cellulare e la suscettibilità allo stress ossidativo. Per comprendere l’implicazione della PON2 nell’apoptosi sono state valutate l’attività della Caspasi-3 e Caspasi-8. RISULTATI: l’overespressione della PON2 ha determinato, nelle cellule T24 trattate con chemioterapici, un aumento della proliferazione cellulare, dei livelli intracellulari dei ROS e della Caspasi-3 e Caspasi-8. Al contrario, il silenziamento della PON2 ha portato ad una riduzione della vitalità cellulare delle cellule T24 dopo trattamento chemioterapico. Infine si è osservato un aumento della produzione di ROS ed un'attivazione di entrambe le Caspasi 3 ed 8. CONCLUSIONI: sebbene questi risultati richiedano ulteriori conferme per comprendere appieno le potenziali implicazioni fisiopatologiche della PON2 nel cancro della vescica, i nostri dati propongono l'enzima PON2 quale biomarker utile per il monitoraggio della crescita tumorale e dei fenomeni di chemioresistenza.
ROLE OF PARAOXONASE-2 IN BLADDER CANCER: EFFECT OF GENE SILENCING AND OVEREXPRESSION ON CELL PROLIFERATION, MIGRATION AND CHEMORESISTANCE. Purpose: Bladder cancer (BC) represents the 7th most common neoplasm in men and the 17th most common in women, worldwide. The identification of reliable biomarkers becomes essential for both early diagnosis and effective therapy of BC. Paraoxonase-2 (PON2) exhibits anti-oxidant and anti-apoptotic activity. Therefore, the aim of this study was to explore the involvement of PON2 in BC cell metabolism. To analyze the potential role of PON2 enzyme in the physiopathology of bladder cancer, we transfected T24 cells (human bladder cancer cell lines) by using pcDNA3 and plKO.1 plasmid to induce the overexpression or the silencing of PON2 gene, respectively. Materials and methods: we tested the PON2 biological influence on cell migration by monolayer wound healing assay. Subsequently, T24 transfected cells were treated for 24 h with cisplatin and gemcitabine and then we measured the cell proliferation by MTT colorimetric assay and susceptibility to oxidative stress. In the harvested cells, we also investigated the activity levels of both Caspase-3 and Caspase-8 as key regulators of the apoptotic response. Results: the PON2 overexpression was responsible for an increased T24 cell viability against chemotherapy agents that resulted also in a ROS production and Caspases activation lower than the control. Conversely, the PON2 silenced gene reduced the strength of T24 cells to the chemotherapy treatment. The decreased cell viability was correlated to an increased production of ROS and to an activation of Caspases higher compared to the control. Conclusions: although these results require further confirmations to fully understand the potential implications of PON2 modulation in BC our data define the PON2 enzyme as a strong and novel molecular approach to control the tumor growth and its susceptibility to chemotherapeutics.

Dans les leucémies aiguës myéloïdes (LAM), un taux élevé d’espèces réactives de l’oxygène (ROS) est connu pour favoriser la prolifération de blastes, alors qu’un niveau faible promeut la quiescence des cellules souches leucémiques. La faible oxygénation, ou hypoxie, de la niche médullaire pourrait contribuer à la chimiorésistance des LAM en diminuant le stress oxydatif. Les facteurs induits par l’hypoxie (HIF) sont impliqués dans le contrôle du métabolisme et des enzymes antioxydantes. Leur inhibition conduit à un stress et à la mort des cellules de LAM. Mon objectif était d’étudier un lien entre l’hypoxie, le métabolisme oxydatif et la chimiorésistance dans un modèle in vitro de culture de cellules de LAM. L’acquisition d’un profil hypoxique par les cellules souches hématopoïétiques (CSH), cultivées avec des cellules stromales mésenchymateuses (CSM) médullaires, a été montré. Nous avons postulé que les cellules leucémiques pouvaient également l’acquérir en coculture avec des CSM humaines. Pour le démontrer, nous avons cultivé des cellules de LAM primaires ou de la lignée MV4-11 sur des CSM humaines primaires ou de la lignée HS-27a. A l’instar des CSH, nous avons identifié trois populations leucémiques en fonction de leur capacité d’adhésion sur les CSM : en suspension, adhérentes aux CSM et nichées dans les CSM. Les cellules nichées, les plus adhérentes, ont une plus forte expression du CXCR4 que les autres. Elles sont aussi plus résistantes de 2 à 7 fois à la cytarabine. Cependant, aucune modification du phénotype souche et des capacités clonogéniques, de repopulation ou de xénogreffe, n’a pu être associée aux cellules nichées comparées aux deux autres populations. En revanche, les cellules nichées présentent un profil hypoxique, une plus faible prolifération avec une augmentation de la phase G0, et de plus faibles niveaux de ROS en lien avec une masse mitochondriale diminuée. Ceci suggère donc un lien entre la chimiorésistance et l’hypoxie ou le métabolisme, plutôt qu’avec une capacité souche. Nous avons aussi montré que l’acriflavine, un inhibiteur non spécifique des HIF, pouvait avoir un effet synergique avec la cytarabine sur les cellules nichées chimiorésistantes. Nos résultats montrent que le surnageant ou le simple contact avec les CSM ne suffisent pas à induire le changement métabolique et la résistance à la cytarabine. Nous pensons que l’hypoxie dans la niche peut moduler le métabolisme oxydatif et donc la chimiorésistance par des mécanismes directs et/ou indirects via l’expression de CXCR4, montré récemment comme impliqué dans la régulation du stress oxydatif des CSH
In acute myeloid leukemia, a high level of ROS is known to favor blasts proliferation, whereas a low level promotes stem cells quiescence. The low oxygenation, or hypoxia, of the bone marrow niche could contribute to chemoresistance of AML cells by reducing the oxidative stress. Hypoxia-inducible factors (HIF) are involved in the control of the cell metabolism and antioxidant enzymes. HIFs inhibition leads to AML cells stress and death. The purpose of this work was to study a link between hypoxia, oxidative metabolism and chemoresistance in an in vitro model of leukemic cell culture. The acquisition of a hypoxic profile by hematopoietic stem cells (HSC) cultured with medullary mesenchymal stromal cells (MSC), has been shown. We hypothesized that AML cells may also acquire such profile in a coculture with human MSCs. To demonstrate that, we cultivated primary AML cells or the MV4-11 cell line on primary human MSCs or the HS-27a cell line. Like HSCs, we identified three leukemic populations according to their adhesion capacity to MSCs: in suspension, adherent to MSCs and embedded in MSCs. Embedded cells, the most adherent, have stronger CXCR4 expression compared to the others. They are also 2- to 7-fold more resistance to cytarabine. However, no change in the stem cell phenotype profile and in the clonogenic, repopulation or xenograft capacities, could be associated with the embedded cells compared to other populations. In contrast, embedded cells present a hypoxic profile, a weak proliferation with increased G0 phase, and lower ROS level that may rely on lower mitochondrial mass. This suggests that chemoresistance mainly relies on hypoxia or cell metabolism rather than a higher stem cell capacity. Furthermore, we have shown that acriflavine, a non-specific HIF inhibitor, could synergize with the cytarabine to eliminate embedded chemoresistant cells. Our results show that the MSC supernatant or a simple contact are not sufficient to induce metabolic change and resistance to cytarabine. We assume that hypoxia in the niche may modulate the oxidative metabolism and the chemoresistance by direct mechanisms and/or indirect ones through CXCR4 expression, a chemokine receptor shown to be involved in the regulation of the oxidative stress in HSC

The primary focus of this research is the mechanisms of cell death in head and neck squamous cell carcinoma (HNSCC) treatment. These cancers typically originate in squamous cells that line the moist mucosal surfaces of head and neck. HNSCC is commonly treated with a platinum based agent, cisplatin. While the drug does offer strong antitumor effects, its prolonged use often results in tumor-acquired resistance, which limits treatment effectiveness. We have shown that cisplatin treatment induces the expression of a pro-apoptotic BCL-2 family member Noxa, which then initiates caspase- dependent apoptosis through its binding and sequestration of pro-survival protein MCL-1 for its inactivation. Without Noxa induction, cell death is significantly reduced when treating HNSCCs with cisplatin. The objectives of this study are (1) to determine the molecular mechanisms by which Noxa induces cell death in HNSCC cells; (2) to determine the molecular mechanisms of cisplatin-resistance in isogenic HNSCC cell lines. We observed an increase of apoptosis by ectopic expression of Noxa in all HNSCC cell lines tested, but not in immortalized human normal oral keratinocytes (NOK), suggesting that Noxa overexpression is sufficient to induce tumor-specific cell death. Noxa-induced cell death was mediated by BAX and BAK activation. BAK activation was mediated through Noxa binding to MCL-1, but not BCL-XL. Cisplatin- resistant cells induced less Noxa and apoptosis, supporting that Noxa induction is prerequisite for apoptosis induced by cisplatin. Taken together, Noxa induces tumor- specific cell death in HNSCC cells primarily through BAX and BAK activation, which suggests the therapeutic potential of this protein.

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
Glioblastoma (GBM) is the most aggressive and common form of primary brain tumour characterized by fast proliferation, high invasion, and resistance to current standard treatment. The average survival rate post-diagnosis is only of 14.6 months, despite the aggressive standard post-surgery treatment approaches of radiotherapy concomitant with chemotherapy with temozolomide (TMZ). Currently, efforts are being endowed to develop a new and more efficient therapeutic approach capable to overcome chemoresistance, and to inhibit tumour progression and improve overall patient survival rate. Abnormal microRNA (miRNA) expression levels have been correlated with chemoresistance, proliferation and resistance to apoptosis, which result from their master regulatory role of molecular pathways important for these tumoral features. Additionally, altered cell metabolism, favouring glycolysis, has been identified as an emerging cancer hallmark and has been intensively described in GBM, thus offering a new target for new GBM therapies. In this work, we hypothesized that gene therapy based on modulation of miRNAs with aberrant expression in GBM and predicted to target crucial metabolic enzymes might promote a shift of GBM cell metabolism, decreasing the glycolytic dependence of tumor cells and contributing to their sensitization to chemotherapeutic agents. We have found that the transient increase of hsa-miR-200-3p and hsa-miR-144-3p levels, shown to be underexpressed in U87 and DBTRG human GBM cell lines, promoted the downregulation of mRNA of enzymes involved in bioenergetic pathways, with consequent alterations in cell metabolism. In this context, both miRNAs showed to be effective in inhibiting glycolysis in U87 cells, whereas in DBTRG cells hsa-miR-144 inhibited mitochondrial respiration and hsa-miR-200c-3p inhibited both mitochondrial respiration and glycolysis. Furthermore, modulation of both miRNAs impaired the migratory capacity of DBTRG cells, which, per se, indicates the potential therapeutic value of these miRNAs. Additionally, the ability of miRNA modulation to sensitize U87 and DBTRG cells to the chemotherapeutic drugs dichloroacetate (DCA) and TMZ was evaluated. Although no differences in U87 cell viability were found between cells treated with either drug and those submitted to the drug treatment combined with miRNA mimics, DBTRG cells became more sensitive to DCA and TMZ after hsa-miR-200c-3p modulation. Overall, our results show that the bioenergetic pathways constitute a promising therapeutic target of miRNA modulation to overcome chemoresistance mechanisms in GBM cells.
O glioblastoma (GBM) é o tipo mais agressivo e comum de tumor cerebral primário, caracterizado por uma rápida proliferação, elevada capacidade invasiva e resistência à terapia convencional. A taxa média de sobrevivência após diagnóstico é de apenas 14.6 meses, após uma terapia agressiva pós-cirúrgica, consistindo em radioterapia e quimioterapia com temozolamide (TMZ). Atualmente, têm sido desenvolvidos esforços no sentido de encontrar novas terapias, mais eficientes e capazes de obviar a quimioresistência, inibir a progressão do tumor e aumentar a taxa de sobrevivência dos doentes. Em virtude do papel dos microRNAs (miRNAs) como reguladores de mecanismos moleculares envolvidos no desenvolvimento de GBM, a expressão anómala destas moléculas tem-se revelado responsável pelas características de extrema agressividade deste tumor. Alterações metabólicas que favorecem a dependência energética da glicólise, amplamente descritas em GBM, foram recentemente identificadas como características típicas das células cancerígenas, constituindo desse modo novos alvos terapêuticos para GBM. Neste trabalho, concebeu-se uma estratégia de terapia génica para GBM, baseada na regulação de miRNAs, cujos níveis se encontram alterados neste tumor e cujos alvos previstos incluem enzimas metabólicas. Esta abordagem resultaria numa alteração do metabolismo das células de GBM, diminuindo a sua dependência da via glicolítica e contribuindo para a sua sensibilização a agentes quimioterapêuticos. Demonstrou-se que o aumento transiente dos níveis de expressão dos miRNAs hsa-miR-200c-3p e hsa-miR-1443p, em linhas celulares humanas de GBM (U87 e DBTRG), promoveu uma diminuição da expressão dos seus alvos envolvidos nas vias do metabolismo bioenergético, com consequentes alterações no metabolismo celular. Neste contexto, ambos os miRNAs inibiram a via glicolítica em células U87, ao passo que em células DBTRG o hsa-miR-144-3p demonstrou ser efetivo na inibição da glicólise e o hsa-miR-200c-3p inibiu a respiração mitocondrial e a glicólise. Adicionalmente, o aumento dos níveis de ambos os miRNAs resultou na diminuição da capacidade migratória das células DBTRG, o que aponta para um potencial terapêutico destes miRNAs. Além disso, avaliou-se o efeito da terapia combinada, consistindo no aumento dos níveis de miRNAs, seguido de incubação com os fármacos dicloroacetato (DCA) e TMZ, na viabilidade de células U87 e DBTRG. Apesar de não terem sido encontradas diferenças entre células U87 incubadas com cada um dos fármacos e com o tratamento combinado, em células DBTRG o aumento dos níveis do hsa—miR-200c-3p resultou num maior efeito tanto do DCA como do TMZ. O conjunto dos nossos resultados demonstra que as vias do metabolismo bioenergético são alvos promissores da ação de miRNAs, que, em combinação com quimioterapia, poderá constituir uma abordagem promissora no tratamento de GBM.

碩士
國立中山大學
生物醫學研究所
98
Pancreatic ductal adenocarcinoma (PDAC) is one of the most insidious forms of cancer whose incidence nearly equals its death rate. Despite extensive research studies, no effective therapeutic approaches for diminishing the morbidity associated with this disease are available. PDAC is characterized by activating Kras mutations and inactivation of Ink4a and the p53-Arf pathway in virtually all cases, while SMAD4—a central regulator of Transforming growth factor-beta (TGF-β) signaling—is inactivated in 55% of PDAC. Our overall goal is to understand how perturbations in the inactivation of SMAD4 pathway contribute to the late stages of PDAC pathogenesis, and to elucidate the role of SMAD4 inactivation on the conversion of a benign form of the cancer to a more aggressive metastatic form. To address this important topic in cancer biology, we have devised a strategy to develop model cell lines to dissect the role of SMAD4 defect in PDAC cell lines and the potential synergistic effects of hypoxia and/or TGF-β1 upon SMAD4 inactivation in their metastatic properties. Experiment results showed SMAD4 restored in PDAC model cell lines were down regulate HIF-1α, VEGF, FGF10 and FGFR2 genes expression level, and also inhibited migration, chemoresistance and angiogenesis of cancer cells. We hypothesize that these effects are due to SMAD4 suppresses some cancer genes in PDAC. Further detailed investigations are also needed to fully elucidate the detail mechanisms for our findings here therefore, the future works of this study will go step on looking for those important downstream effect genes regulated by Smad4 protein in PDAC cells and try to find out the connection of all the dependence proteins.

博士
中山醫學大學
醫學研究所
101
Background: Tumor cycling hypoxia is now a well-recognized phenomenon in animal and human solid tumors. However, how tumor cycling hypoxia impacts chemotherapy is unclear. In the present study, we explored the impact and the mechanism of cycling hypoxia on tumor microenvironment-mediated chemoresistance. Methods: Hoechst 33342 staining and hypoxia-inducible factor-1 (HIF-1) activation labeling together with immunofluorescence imaging and fluorescence-activated cell sorting were utilized to isolate hypoxic tumor subpopulations from human glioblastoma xenografts. ABCB1 expression, P-glycoprotein function, and chemosensitivity in tumor cells derived from human glioblastoma xenografts or in vitro cycling hypoxic stress-treated glioblastoma cells were determined by western blot analysis, drug accumulation and efflux assays, and MTT assay, respectively. Results: ABCB1 expression and P-glycoprotein function were upregulated under cycling hypoxia in glioblastoma cells concomitant with decreased responses to doxorubicin and BCNU. However, ABCB1 knockdown inhibited these effects. Moreover, immunofluorescence imaging and flow cytometric analysis for ABCB1, HIF-1 activation, and Hoechst 3342 in glioblastoma revealed highly localized ABCB1 expression predominantly in potentially cycling hypoxic areas with HIF-1 activation and blood perfusion within the solid tumor microenvironment. The cycling hypoxic tumor cells derived from glioblastoma xenografts exhibited higher ABCB1 expression, P-glycoprotein function, and chemoresistance than chronic hypoxic and normoxic cells. Tumor-bearing mice that received YC-1, an HIF-1α inhibitor, exhibited suppressed tumor microenvironment-induced ABCB1 expression and enhanced survival rate in BCNU chemotherapy. Conclusions: Cycling hypoxia plays a vital role in tumor microenvironment-mediated chemoresistance via the HIF-1-dependent induction of ABCB1. HIF-1 blockade before and concurrent with chemotherapy could suppress cycling hypoxia-induced chemoresistance.

Questa tesi si inserisce in un filone di ricerca in cui viene studiato il coinvolgimento di hERG, canale del potassio voltaggio dipendente nella patogenesi tumorale. In particolare è noto il suo ruolo nella chemioresistenza delle leucemie dipendente dal microambiente midollare e questo ha suggerito hERG come possibile bersaglio per la terapia antileucemica. In questo lavoro di tesi sono stati sviluppati due possibili approcci uno farmacologico e l’altro molecolare al fine di inibire l’attività di questo canale. Nel primo caso abbiamo scelto la claritromicina, un antibiotico macrolide già in commercio la cui attività inibitoria nei confronti di hERG è già stata documentata studiandone l’ effetto in combinazione o in assenza di citarabina (chemoterapico) su una linea cellulare di leucemia promielocitica acuta (HL60). Questi esperimenti sono stati effettuati su cellule in sospensione oppure in co-coltura con cellule stromali mesenchimali. Dagli esperimenti in vitro è stato dimostrato che la combinazione dei due farmaci riduce l’attività chemoprotettiva indotta dal microambiente midollare (CI=0.657). I risultati incoraggianti ci hanno permesso di passare a studiare l’effetto dei due farmaci su topi SCID (Severe Combined Immunodeficency Disease) inoculati con HL60 marcate con la luciferasi. Inizialmente è stata dimostrata la capacità della claritromicina di ridurre lo sviluppo tumorale e la sua invasività. Successivamente l’effetto combinato con citarabina garantisce una maggior sopravvivenza rispetto ai controlli, con almeno 20 giorni di differenza. Per quanto riguarda l’approccio molecolare abbiamo valutato la possibilità di silenziare hERG mettendo a punto nuovi sistemi per veicolare il siRNA corrispondente. Per questo scopo sono stati scelti quattro tensioattivi cationici: 12-3-12, 12-6-12, 12-12-12 e SH14 le cui micelle possono essere impiegate come vettori per la terapia genica. E’ stata studiata la cinetica di formazione dei complessi tra micelle e siRNA per conoscerne la struttura e quindi la stabilità, parametri chiave per l’efficienza di transfezione. La caratterizzazione chimico-fisica è stata seguita mediante SAXS presso European Synchrotron Radiation Facility (ESRF) di Grenoble. Questa tecnica sfrutta l’utilizzo di un fascio di raggi X che, una volta diffuso dalla nuvola elettronica degli atomi specialmente da quelli con numero atomico più elevato che compongono i complessi, fornisce un segnale dipendente dalla struttura. E’ stato possibile misurare la variazione di forma e di dimensioni durante il processo di formazione dei complessi. Le due soluzioni di siRNA e di micelle sono state caricate separatamente mediante un dispositivo di miscelamento controllato via computer (stopped-flow) e accoppiato alla tecnica SAXS. Si è osservato che il processo di formazione dei complessi è estremamente rapido e avviene subito dopo il mixing, in tempi inferiori a 0.05s. All’interno dei nanoaggregati le due componenti (siRNA e micelle) si alternano con distanze ripetitive di 3-4nm e con l’aumentare del tempo il numero di unità che li compongono aumenta (fino a 4-8 unità a seconda del tipo di tensioattivo). Gli aggregati contenenti SH14 sono meno ordinati rispetto agli altri. Alla fine della registrazione (circa 15 minuti dopo il mixing) il raggio di girazione dei complessi è compreso tra 7 e 27nm; queste dimensioni sono compatibili con un’elevata efficienza di transfezione.

Cancer is characterised as a set of diseases that is involved in uncontrolled cell growth with the ability to invade or spread to the other part of the body. Carcinogenesis is recognized as a process through aggregation of genetic and epigenetic changes in normal cell that ultimately leading to unlimited growth proliferation and invasion. Pancreatic neuroendocrine tumour (PNET) is a rare tumour that arise from neuroendocrine gland, occurs in various part of the body. The prevalence rate of PNETs is near about 25–30 per 100,000 population in the United States and according to Surveillance Epidemiology and End Results (SEER), the incidence rate of PNETs increased five-fold from 1973 to 2011. PNETs comprises approximately 7% of all types of cancer in the pancreas. Normally, 5 years survival rate of PNETs near about 42%. PNETs is a heterogenous group of disorder with less 5 years survival rate due to lack of effective therapeutic options for patients with advanced stages, absence of symptomatology specially in case of non-functional PNETs and also to the phenomenon of chemoresistance, dependent on multiple mechanisms. Recent data shows that incidence rate of this tumour increases as a result of germline genetic mutations. Concerning to genetic change, it is very important to explain the differences that occurs at the level of chemoresistance. The treatment plan of the PNETs varies on type, location and aggressiveness of the tumour. Surgery is the only curative treatment in early stage but in advanced stages chemotherapy and radiotherapy are the most palliative treatment option of PNETs. Chemotherapy which is mainly based cisplatin combined with capecitabine and the response rate of treatment is near about 30%. Cisplatin is responsible for the formation of DNA adducts, leading to DNA damage, and induces generation of ROS, that consequently leads to oxidative stress, cell damage and death. Glutathione (GSH) plays an important role in the maintenance of intercellular redox balance and detoxification. Chemoresistance can be based on the alteration of the detoxification mechanisms and GSH system has been pointed as one of the most important. Cysteine is a rate limitant substrate for GSH synthesis, and xCT cyst(e)ine transporter is implicated in cancer severity and chemoresistance. The Hypothesis of the project is: the disruption of xCT and uptake of cysteine leads to the reversal of resistance to alkylating agents in pancreatic neuroendocrine tumours (PNETs). To accomplish the hypothesis we defined 3 aims: 1st aim will be to address the expression of xCT in PNETs cell lines, and the modulation of xCT expression by cysteine and cisplatin; 2nd aim will be focused on the effect of xCT inhibition in PNETs cell death, using erastin and sulfasalazine, and 3rd aim will be focused on the effect of new nanoformulations in order to disturb cysteine uptake (Sechry and Sechry@PUREG4-FA) and glutathione synthesis (BSO@PUREG4-FA). Our work allowed to reveal the role of xCT transporter and the role of cysteine in PNETs cell line resistance. This cell line showing different response patterns in cysteine transporters activity helped to reveal the differences of the transporter in chemoresistance mechanism. It also showed that besides xCT transporter other cysteine transporter such as EAAT3 also appeared to be involved in the dynamics of chemoresistance mechanism. This work was also important to undercover the effect of new nanoformulations in order to disturb cysteine uptake by using SeChry and GSH synthesis by using BSO in PNETs cell lines. SeChry, but not SeChry@PUREG4-FA, induced cell death in BON-1 cell lines. SeChry cytotoxicity can be selective for cancer cells and this was taken in consideration in our new strategy by using SeChry@PUREG4-FA, however the assay was not successful and new markers for targeted delivery must be investigated in PNETs. BSO@PUREG4-FA induced cell death in combination with platinum salts in PNETs cell lines. Possibly, the use of folate functionalised particles will help to bypass the critical step in the non-specific delivery of BSO to non-cancer cell. The targeted BSO delivery to cancer cells can be explored as a novel strategy in cancer therapeutics. Moreover, more assays with cancer and non-cancer cells must be done in order to determine if folate receptor is in fact a suitable target to delivery drugs to PNETS cells, and find new and more specific targets.