Cisplatin (CDDP; cis-diamminedichloroplatinum) resistance presents a major impediment in the treatment of several gynecologic solid tumors, including ovarian and cervical tumors. p53, a critical regulator of cellular apoptosis, is a determinant of CDDP sensitivity. In our study, we have observed that the dysregulation of p53 regulators, checkpoint kinase 1 (Chk1) and protein phosphatase magnesium-dependent 1 (PPM1D), significantly reduced CDDP responsiveness in human cancer cells. Isogenic wt-p53 CDDP-sensitive (OV2008) and -resistant (C13*) cervical cancer cells, and isogenic wt-p53 CDDP-sensitive (A2780s) and p53 mutant resistant (A2780cp) ovarian cancer cells, along with CDDP-resistant ovarian cancer cell lines (OCC-1 and OVCAR-3, mutant p53; SKOV-3, p53 null) were used to elucidate the mechanisms of p53 regulation in human gynecologic cancer cells. We have complemented our study with a xenograft model (A2780s) and a tissue microarray of human ovarian tumors to validate our in vitro observations. We have demonstrated that CDDP differentially regulated the p53 activator Chk1 in sensitive and resistant cancer cells; it enhances Chk1 activation in sensitive but not resistant cells. This differential regulation also extended to PPM1D, whereby CDDP enhanced PPM1D content in resistant but not sensitive cells. PPM1D knockdown sensitized resistant cells to CDDP, which was associated with up-regulation of Chk1 and p53 activations, while PPM1D over-expression had the opposite effect. We have also shown that CDDP sensitivity in response to PPM1D down-regulation was p53-dependent. Moreover, CDDP promotes PPM1D nuclear localization in resistant cells and nuclear exclusion in sensitive cells and xenograft tumors. Enhanced PPM1D expression in human ovarian tumors is significantly associated with tumor aggression. Dysregulation of the oncogene Akt has been implicated in a variety of human malignancies, including ovarian cancer. We have demonstrated that Akt regulates PPM1D stability, since activated Akt over-expression in sensitive cells rescued PPM1D from CDDP-induced proteasomal degradation and Akt down-regulation in resistant cells lead to PPM1D de-stabilization and down-regulation. We have shown for the first time that PPM1D is downstream of Akt through which it can modulate CDDP sensitivity in human cancer cells. These findings extend the current knowledge on the molecular basis of CDDP resistance in gynecological cancers and may help in developing effective therapeutic strategies.

Cancer, despite significant advances in diagnosis and treatment, is the second most common cause of death in economically advanced countries. The main reason for the failure of anticancer therapy is the development of chemoresistance, which can be either internal or acquired, and is primarily mediated by the activation of various key regulators (eg MDR, PI3K/Akt, etc.). Genetic and epigenetic mechanisms are involved in activating these pathwa- ys. Significant epigenetic mechanisms that can participate in chemoresistance include regula- tion of gene expression by microRNA (miRNA) and long noncoding RNA (lncRNA). Dere- gulated expression of these non-coding RNAs has been observed in many diseases and their involvement in the initiation and progression of malignant tumors has been demonstrated. In this study, we investigated the expression of long non-coding RNA MIAT in hypoxia (1% O2) in chemosensitive and chemoresistant neuroblastoma cell lines (NBL), as hypoxia is a significant negative prognostic factor of many tumors and is involved in chemoresistance. Relative expression of MIAT was influenced by the number of cultured cells, where expression was increased by culturing more cells. MIAT expression was also significantly increased after 6 hours of NBL culture UKF-NB-4 in hypoxic conditions, and...

No description available.

High Mobility Group AT-hook protein 2 (HMGA2) is a non-histone chromatin binding protein expressed in stem cells, cancer cells but not in normal human somatic cells. The presence of HMGA2 in cancer correlates with advanced neoplastic disease and poor prognosis. HMGA2 plays important roles in Base Excision Repair (BER) and at replication forks. HMGA2 is present at mammalian metaphase telomeres and its loss induces chromosomal aberrations. However, the functional role of HMGA2 at telomeres remains elusive. We hypothesized a protective role of HMGA2 that guards telomeres and modulates DNA damage repair signaling pathways. Employing different HMGA2+ human tumor cell models, we investigated the HMGA2-mediated functions that contribute to chemoresistance in glioblastoma (GB). This study presents a novel interaction of HMGA2 with telomeric protein TRF2 (Telomere Repeat-Binding Factor 2). This interaction retains TRF2 at telomeres, thus capping the telomeres and reducing telomere-dysfunction induced foci despite induced telomere stress. Loss of HMGA2 coincides with increased phosphorylation of TRF2, decreased TRF2 retention at telomeres and increased formation of telomeric aggregates, anaphase bridges and micronuclei. These findings provide new evidence for a unique role of HMGA2 at telomeres as a novel contributor of telomeric integrity. We show that upon DNA damage, HMGA2 causes increased and sustained phosphorylation of Ataxia Telangiectasia and Rad3-related kinase (ATR) and checkpoint kinase 1 (CHK1). Prolonged presence of pCHK1Ser296 coincides with prolonged G2/M block and increased tumor cell survival. The relationship between (ATR)-CHK1 DNA damage response pathway and HMGA2 identifies a novel mechanism by which HMGA2 can alter DNA repair function in cancer cells. We identified HMGA2 as a novel factor contributing to temozolomide (TMZ) resistance in GB. HMGA2 knockdown sensitizes the GB cells to TMZ. We propose a specific combination of FDA-approved drugs, TMZ and Dovitinib (DOV), to increase GB cell death. We show that DOV downregulates key BER proteins, attenuates pSTAT3-coordinated Lin28A and HMGA2 expression. Our results suggest that a sequential therapeutic strategy of pretreating GB cells with DOV followed by a sequence of TMZ and DOV diminishes TMZ resistance and enhances the ability of TMZ to induce GB cell death. Overall, we identified HMGA2 as a multifunctional survival factor in human cancer cells and showed that targeting HMGA2 is a valid strategy to combat HMGA2+ cancer cells. / February 2016

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.

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.

Le cancer du sein est une cause importante de mortalité féminine en France et l’émergence de chimiorésistance, en particulier avec les taxanes, dont le paclitaxel (Taxol®), est une limite importante à l’emploi de ces molécules. Comme de nombreux anticancéreux, les taxanes ciblent les microtubules (MTs), des polymères de tubuline intervenant dans de nombreuses fonctions cellulaires. Ces derniers alternent entre des phases de croissance et de désassemblage, leur conférant ainsi un caractère dynamique. En se liant à la beta-tubuline, le Taxol stabilise les MTs et bloque la mitose, conduisant la cellule vers l’apoptose. La résistance au Taxol est un processus multifactoriel impliquant des mécanismes tels que la surexpression de pompes d’efflux (P-glycoprotéine), des mutations des gènes d’alpha- et de beta-tubuline ou une expression altérée de protéines associées aux MTs (MAPs) telles que MAP4 (stabilisatrice des MTs) ou la stathmine (dépolymérisante), contribuant ainsi à la restauration de la dynamique microtubulaire.Le projet repose sur l’emploi de la lignée tumorale mammaire MDA-MB 231 rendue résistante par paliers à 25nM de Taxol dans des conditions de blocage des pompes d’efflux. L’objectif étant d’explorer les variations protéiques de l’environnement des MTs associées au phénotype chimiorésistant, nous avons comparé le protéome d’extraits microtubulaires de cellules sensibles (Tv) et résistantes au Taxol (T8). Parmi les 112 protéines statistiquement enrichies dans une des deux populations de MTs, on retrouve notamment une augmentation des tubulines beta III et IV, une augmentation et une diminution respectives des moteurs moléculaires kinésine-1 et dynéine ainsi que l’enrichissement de plusieurs septines (SEPT2, 8, 9 et 11) dans les fractions de MTs T8. Les septines sont des GTPases associées en complexes hétéro-oligomériques impliquées dans la cytocinèse et l’organisation du cytosquelette de MTs et d’actine. Le recrutement de ces protéines candidates sur les MTs des cellules T8 a été validé par Western blot (Froidevaux-Klipfel et al., 2011) et immunofluorescence. Dans la littérature, les septines sont décrites comme étant localisées soit au niveau de l’actine soit au niveau des MTs dans les cellules de mammifères. De façon intéressante, dans nos cellules MDA-MB 231, nous observons un recrutement partiel de SEPT2 sur les fibres d’actine dans les cellules Tv alors qu’elle colocalise avec les MTs des cellules T8. Bien que la dépolymérisation de l’actine dans les cellules Tv n’entraine aucun déplacement des septines vers les MTs, cette localisation différentielle des septines sur les MTs des cellules résistantes pourrait néanmoins participer au phénotype chimiorésistant.Par ailleurs, il a été décrit dans la littérature que SEPT2 se lie aux MTs polyglutamylés afin de faciliter le transport vésiculaire à la membrane de protéines impliquées dans la polarisation cellulaire. La polyglutamylation est une modification post-traductionnelle permettant la formation de chaines latérales d’un à plusieurs résidus glutamate sur les tubulines alpha ou beta, régulant ainsi les interactions entre MTs et MAPs. Nos résultats montrent que l’accumulation des septines sur le réseau de MTs des cellules T8 s’accompagne d’une augmentation de la polyglutamylation mais aussi de la tyrosination de la tubuline. De plus, des tests de viabilité cellulaire ont mis en évidence que l’inhibition partielle par RNAi des septines ainsi que des polyglutamylases et de la tubuline tyrosine ligase, comme la surexpression d’enzymes responsables de la déglutamylation de la tubuline, permettent de restaurer une certaine sensibilité au Taxol des cellules T8. Inversement, la surexpression de certaines enzymes responsables de la polyglutamylation et de la tyrosination de la tubuline dans les cellules Tv permet d’instaurer une résistance au Taxol des cellules sensibles.La compilation de nos résultats permet de proposer un nouveau mécanisme de résistance au Taxol des cellules cancéreuses mammaires MDA-MB 231 : une augmentation du niveau de tyrosination de l’alpha-tubuline serait à l’origine de l’allongement des chaînes polyglutamylées sur le MT entraînant une diminution de la liaison de la protéine stabilisatrice MAP4 ainsi que le recrutement des septines sur les MTs. Ces modifications favoriseraient alors le recrutement du facteur de sauvetage CLIP-170 et de kinésines dépolymérisantes telles que MCAK à l’extrémité en croissance du MT des cellules résistantes T8, permettant une certaine restauration de la dynamique microtubulaire et contribuant ainsi à l’apparition du phénotype chimiorésistant.Ces études sont indispensables pour établir les bases d’un nouveau mécanisme de résistance au Taxol impliquant les septines, ainsi qu’un lien de causalité avec la tyrosination et la polyglutamylation. Au-delà, la recherche de ces modulations clés pourra être réalisée sur des cancers mammaires sensibles et résistants au Taxol, issus de biopsies de patients. Ainsi, il sera possible à terme, de déterminer si les septines, la tyrosination et la polyglutamylation de la tubuline ont une véritable importance fonctionnelle dans la résistance de cancers du sein au Taxol. / Breast cancer remains the leading cause of women mortality in France, and chemoresistance emergence, in particular to taxanes, including paclitaxel (Taxol®), is an important limitation to the use of these molecules. As do many anticancer drugs, taxanes target microtubules (MTs), tubulin polymers involved in many cellular functions. These alternate between growing and shrinking stages, thus providing dynamic instability. By binding to beta-tubulin, Taxol stabilizes MTs and prevents successful mitosis, leading to apoptosis. Taxol resistance is a multifactorial process including mechanisms such as overexpression of drug efflux pumps (P-glycoprotein), mutations in the genes for alpha- and beta-tubulin or altered expression of MT-associated proteins (MAPs) like the MT-stabilizing MAP4 or the depolymerizing stathmin, therefore contributing to MT dynamics restoration.The current project relies on the use of the breast carcinoma cell line MDA-MB 231 made gradually resistant to 25nM of Taxol under blocking conditions of efflux pumps. To get a broader insight into the protein modifications from the MT environment associated with the chemoresistant phenotype, we compared the proteomic profiles of total MT fractions from Taxol-sensitive (Tv) and Taxol-resistant (T8) cells. Among the 112 differentially enriched proteins found in one of the two populations, we evidenced increased levels of betaIII and betaIV-tubulins, a slight increase and a decrease of molecular motors kinesin-1 and dynein, respectively, and the enrichment of several septins (SEPT2, 8, 9 and 11) in MT fractions of T8 cells. Septins are GTPases that associate into hetero-oligomeric complexes involved in cytokinesis and in MT and actin cytoskeleton organization. Recruitment of these candidate proteins on MTs of T8 cells has been validated by Western blot analysis (Froidevaux-Klipfel et al., 2011) and immunofluorescence experiments.In the literature, septins localize either on actin or MTs in mammalian cells. Interestingly, in our MDA-MB 231 cells, SEPT2 is recruited partially on actin stress fibers in sensitive Tv cells, whereas it colocalizes with MTs in T8 ones. Although actin depolymerization in Tv cells does not induce any shift towards MTs, this differential localization of septins on MTs of resistant cells could nevertheless participate in the chemoresistant phenotype.Furthermore, it has been described in the literature that SEPT2 binds to polyglutamylated MTs to facilitate vesicular transport to the plasma membrane of proteins implicated in cell polarization. Polyglutamylation is a post-translational modification allowing the formation of side-chains of several glutamate residues on alpha- or beta-tubulins, thus regulating interactions between MTs and MAPs. Our results show that septin accumulation on the MT network of T8 cells is associated with an increase in polyglutamylation but also tyrosination of tubulin. In addition, cell viability assays showed that partial inhibition by RNAi of septins as well as polyglutamylases and tubulin tyrosine-ligase, but also overexpression of enzymes responsible for tubulin deglutamylation, could restore Taxol sensitivity of T8 cells. By contrast, overexpression of enzymes responsible for tubulin polyglutamylation and tyrosination in Tv cells could induce Taxol resistance of sensitive Tv cells.The compilation of our results enables us to provide a new mechanism of Taxol resistance of the breast cancer cells MDA-MB 231: an increased level of alpha-tubulin tyrosination would induce the lengthening of polyglutamylated chains on the MT, resulting in a reduced binding of the stabilizing protein MAP4 as well as the recruitment of septins on MTs. These modifications would promote the recruitment of the rescue factor CLIP-170 and that of depolymerizing kinesins such as MCAK to the growing end of the MT of resistant T8 cells, leading to a restoration of MT dynamics, thus contributing to the emergence of the chemoresistant phenotype.These studies are essential to lay the basis for a new mechanism of Taxol resistance involving septins, and a causal relationship to tyrosination and polyglutamylation. Beyond, the search for these key modulations will be performed on Taxol sensitive and resistant breast cancers, from patients’ biopsies. Thus, it will be eventually possible to determine whether septins, tubulin tyrosination and polyglutamylation have a real functional importance in breast cancer resistance to Taxol.

The tumor suppressor gene TP53 sits at the crux of response to cellular stresses. This is the most frequently inactivated gene in human tumors, being the target of somatic mutations. The protein product of TP53 is p53, and plays a crucial role in anti-proliferative signals through the induction of apoptosis, senescence, and cell-cycle arrest when activated by stresses such as genotoxic chemotherapeutic drugs. Therefore, the status of TP53 mutation in a tumor has profound implications for the tumorigenic potential as well as the response to anti-cancer therapies. Indeed, numerous studies have shown a predictive and prognostic value of TP53 mutations to the response to chemotherapy, but just as many studies show no significant contribution of TP53 mutations to chemotherapy response. This controversy is partly due to the lack of standard methods of TP53 mutation detection, but more importantly, it is due to the categorization of all TP53 mutations into one group. Certain mutations in TP53 can confer a mutant p53 with new, gained activities, not normally present in the WT p53 protein. These have been commonly called "gain of function" (GOF) p53 proteins, and some GOF p53 proteins can even confer oncogenic properties. However, not all gained functions are necessarily implicated in oncogenicity. Using stringent criteria, we have defined a select group of GOF TP53 mutations that do function as oncogenic proteins as oncomorphic TP53 mutations. In this work, we utilize data available from a large patient population through The Cancer Genome Atlas (TCGA) as well as data available from the University of Iowa Gynecologic Oncology Tumor Bank to examine the association of oncomorphic TP53 mutations with patient outcome using advanced serous ovarian cancer as a model. We demonstrate that oncomorphic TP53 mutations are associated with worse progression-free survival, chemoresistance, and higher rates of recurrence than other mutations in TP53 that have no evidence of oncomorphic abilities. We identify molecular alterations in patients with oncomorphic TP53 mutations, particularly the increased expression of β-catenin. We also observe that oncomorphic p53 proteins lose the normal protein:protein interactions with the microRNA microprocessing complex, implicating the role of dysregulated miRNAs in pathways associated with chemoresistance. The cumulative results from our studies provide human evidence for the consideration of different classes of TP53 mutations. Patients with oncomorphic TP53 mutations deserve careful follow-up therapy and may require novel treatment regimens to improve outcomes. We propose that stratification of patients should be considered based upon the individual TP53 mutation identified from their tumors.

No description available.

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