Les cancers sont caractérisés par une prolifération anarchique des cellules causée par une dérégulation des mécanismes de contrôle du cycle cellulaire, comme la protéine p27Kip1 (p27). Dans le noyau, p27 inhibe les complexes cycline-CDK, bloquant ainsi la progression du cycle de division cellulaire, et par conséquent, la prolifération cellulaire. Cette propriété confère à p27 un rôle de suppresseur de tumeurs. Toutefois, dans certains cancers, p27 est relocalisé dans le cytoplasme où il exerce un rôle oncogénique, cependant les mécanismes moléculaires par lesquels p27 agit comme un oncogène restent largement inconnus. Des études récentes montrent que sa localisation cytoplasmique permet à p27 d'activer l'autophagie, un processus de recyclage des constituants intracellulaires dans les cellules carencées en nutriments. Dans les cellules cancéreuses, l'autophagie est un des mécanismes d'adaptation au microenvironnement tumoral et leur permet de survivre malgré des conditions défavorables. En outre, l'autophagie peut être induite par le stress causé par les traitements anti-cancéreux, ce qui retarde l'apoptose en permettant aux cellules d'échapper aux traitements. L'autophagie induite par la localisation cytoplasmique de p27 pourrait ainsi compromettre l'efficacité des thérapeutiques anti-tumorales. Le but de mon projet de thèse était de déterminer par quels mécanismes p27 contrôle l'autophagie et participe ainsi à la survie des cellules dans des conditions de stress métabolique. Durant ma thèse, j'ai pu mettre en évidence un rôle essentiel de p27 dans la régulation de l'autophagie et de la mort cellulaire. Mes travaux indiquent que le statut de p27 cytoplasmique ou nucléaire détermine à la fois le degré d'autophagie et la susceptibilité des cellules à l'apoptose induite par la privation nutritionnelle. En utilisant des méthodes de biologie cellulaire et moléculaire, j'ai disséqué les voies de signalisation et le mécanisme moléculaire expliquant le rôle pro-autophagique de p27. De manière surprenante, il apparait que p27 régule l'autophagie par différents mécanismes en fonction de la carence infligée aux cellules. [...] / P27 controls cell cycle progression via its ability to block cyclin-CDK activity. Thus, it acts as a tumor suppressor in the nucleus. However, in certain cancers, p27 relocalizes in the cytoplasm where it may promote tumorigenesis by still largely unknown mechanisms. Recent studies have shown that the cytoplasmic localization of p27 induces autophagy, a catabolic process whereby intracellular constituents are recycled in response to nutrient depletion. In cancer cells, autophagy acts as as an adaptive response to metabolic stress in tumor tissues. Furthermore, autophagy may be induced by various cancer therapies, leading to chemotherapeutic resistance and promoting cancer cell survival. The aim of my PhD project was to determine by which mechanisms p27 controls autophagy and cell survival upon metabolic stress conditions. My results indicate that p27 plays a prominent role in the regulation of autophagy and cell death during nutrient deprivation. The status of p27 determines the rate of autophagy and the susceptibility of cells to apoptosis. Importantly, the mechanisms underlying the role of p27 in autophagy appears to be different in function of the nature of the metabolic stress. Amino acid deprivation leads to translocation of p27 to lysosomes where it participates in the inhibition of mTOR, a kinase that acts as a master regulator of cellular metabolism and autophagy. In contrast, the effect of p27 in glucose starved cells depends mostly on its role in the regulation of microtubule dynamics, which controls intracellular vesicle trafficking. Thus, in glucose starved cells, p27 promotes the fusion of autophagic vesicles and degradation of autophagy cargo. To conclude, my results show that p27 is a critical modulator of starvation-induced autophagy and its status determines the response of cells to metabolic stress. Therefore, p27 may serve as a predictive marker for treatment response targeting specific metabolic pathways and may constitute a promising target for anticancer treatment affecting these pathways.

Identifying the mechanisms that regulate neural precursor cell (NPC) proliferation and differentiation is important for understanding CNS development among different vertebrates. My work has focused specifically on understanding how mitogenic factors, like basic fibroblast growth factor (FGF2), regulate the NPC cell cycle. Mitogenic factors and serum are thought to drive cell cycle and therefore proliferation mainly by activating G1-type cyclin-dependent kinases (CDKs). The general hypothesis being addressed here is that FGF2 also promotes cell cycle progression of NPCs through the degradation of the cell cycle inhibitor p27Kip1. I show that, in the presence of FGF2 in vitro, embryonic rat cortical NPCs express high protein levels of the CDC28 protein kinase regulatory subunit 1b (Cks1b), a component of the SCFSkp2 E3 ubiquitin ligase complex that targets p27Kip1 for proteasomal degradation. I also show that NPCs maintained in FGF2 express undetectable levels of p27KIP1, while removal of FGF2 results in increased p27Kip1 protein expression and decreased protein expression of Cks1b. RNA expression data shows that Cks1b mRNA is reduced in non-dividing NPCs but is present in dividing NPCs, suggesting that Cks1b is being regulated at the transcriptional level. Analysis of the putative promoter of Cks1b reveals numerous conserved transcription factor consensus sites that could potentially play a role in regulation of Cks1b transcription, including consensus sites for E2F and the cell cycle-dependent element (CDE) cell cycle genes homology region (CHR) tandem repressor element. I use chromatin immunoprecipitation and luciferase assays to identify which E2Fs occupy and regulate the transcription of Cks1b under different conditions of mitogen stimulation. The data show that E2F4 occupies the promoter of Cks1b in non-dividing NPCs while E2F1 binds exclusively in proliferating NPCs. Mutation of either the E2F or CDE/CHR consensus sites independently de-represses the activity of a Cks1b promoter reporter in NPCs in G0/G1, while mutation of both sites delays induction of promoter activity. Finally, I use in ovo electroporation to determine if p27Kip1 has an additional role in neuronal differentiation during early spinal cord development. I show that ectopic expression of p27Kip1 is insufficient to induce neuronal differentiation in spinal cord progenitors.

The complex PI3K/mTOR pathway regulates tumor progression via effects on cellular proliferation, apoptosis, autophagy, and motility. New drugs that inhibit the catalytic site of both PI3K and mTOR have shown promise in clinical trials. Here, we report the first use of a novel, dual PI3K/mTOR catalytic site inhibitor (PF-04691502, PF1502) in a xenograft model of breast cancer metastasis to bone. Metastatic MDA-MB-1833 cells showed PI3K/mTOR activation relative to parental MDA-MB-231. Low-dose PF1502 significantly impaired tumor cell motility and invasion in vitro without causing cell cycle arrest, apoptosis, or reduced proliferation. Pre-treatment of tumor cells at this dose reduced bone metastatic outgrowth in vivo. The atypical tumor suppressor, p27KIP1, is phosphorylated in its C-terminal region by multiple AGC kinases downstream of PI3K/mTOR. These phosphorylation events promote cytoplasmic mislocalzation of p27 which, in turn, facilitates inhibition of the RhoA cytoskeletal regulatory protein. The resulting turnover of the actin cytoskeleton is thought to underlie the increased cellular motility attributed to cytoplasmic p27. In MDA-MB-1833 cells, PI3K/mTOR inhibition reduced p27 C-terminal phosphorylation at T157 and T198 and reduced cytoplasmic p27 levels. Overexpression of a p27T157D/T198D phospho-mimetic mutant conferred resistance to the anti-motility effects of PF1502 in vitro. MDA-MB-1833 cells demonstrate p27-dependent inhibition of RhoA-ROCK signaling, as well as p27-dependent motility and invasion in vitro, however, RhoA knockdown did not confer resistance to the anti-motility effects of PF1502. p27shRNA dramatically impaired the bone metastatic outgrowth of MDA-MB-1833 in vivo. In an effort to explore potentially novel RhoA-independent mechanisms whereby cytoplasmic p27 might drive tumor cell motility and metastasis, we turned to the process known as epithelial-to-mesenchymal transition (EMT). The EMT program has been implicated as a critical driver of tumor metastasis in a variety of cancer models. PI3K/mTOR inhibition and shRNA p27 treatment both reversed expression of EMT markers in MDA-MB-1833. Thus, PI3K/mTOR appears to drive p27-dependent motility and metastasis at least in part by induction of an EMT-like phenotype, a novel mechanism through which p27 might act to promote tumor progression. These results provide an important new clinical rationale supporting the use of PI3K/mTOR inhibitors as anticancer agents via their inhibition of tumor invasion and metastasis.

No description available.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecological malignancy in Western countries. A better understanding of the etiology and risk factors associated with this disease is crucial for the development of early detection protocols as well as more effective therapies. Epidemiological data has shown that the risks of EOC are highest among peri- or post- menopause women, while increased parity or the use of oral contraceptives is preventive. These data suggest that alterations in reproductive factors are associated with ovarian cancer risks; however, the molecular mechanisms underlying such a link remain to be understood. For decades, EOC was believed to arise from the epithelium that surrounds the ovarian surface, yet this concept fails to explain the morphological resemblance of ovarian epithelial neoplasms with the epithelial cells of the Müllerian-derived female reproductive tract. Alternative ideas have argued that EOC may originate from extra- or para-ovarian tissues such as the fallopian tube and ovarii rete. Studies of the origin of EOC will provide a better understanding of the disease and advance the protocols for early diagnosis. The aims for this thesis are to establish in vivo ovarian tumor models based on the germ cell deficient Wv/Wv mice that mimicking menopausal physiology. The Wv mice harbor a point mutation in c-Kit, which reduces its tyrosine kinase activity to about 1%, resulting in a premature loss of ovarian germ cells and follicles that recapitulates the initiation of menopause in human. We have developed ovarian tumor models by deleting the tumor suppressor genes p53 or p27kip1 in Wv/Wv mice. We found that both Wv/Wv:p27+/- and Wv/Wv :p27 -/- mice developed ovarian epithelial tumors, which consist of papillary structures lined by hyperchromatic neoplastic cells. Positive Cytokeratin 8 (CK8) staining indicated the epithelial origin of these tumors. In vitro primary cultures of mouse ovarian surface epithelial (MOSE) cells from wildtype, p27+/- and p27 -/- mice further confirmed the growth advantage caused by p27 deficiency. However, neither p27 +/- nor p27 -/- MOSE cells were transformed in vitro, probably due to the compensatory increase of cyclin dependent kinase inhibitor (CKI) proteins including p21, p16, p19. When p53 was deleted unilaterally in the ovarian surface epithelial cells of Wv/Wv:p53 loxP/loxP mice by single administration of Adenovirus containing Cre activity (Ad-Cre), ovarian tumors developed after long latency. The ovarian tumors were significantly enlarged when compared with the uninfected ovary from the same mouse. However, most of the lesions in Wv:p53 conditional knockout tumors was negative for epithelial and follicular markers. In vitro deletion of p53 in MOSE cells significantly increased the proliferation and passage numbers of these cells. A compensatory increase of the CKI protein p16, as well as the cellular senescence level was also observed in p53 deleted MOSE cells, suggesting that p53 deletion alone was not sufficient to bypass p16- mediated tumor defense mechanisms in MOSE cells. Taken together, single deletion of p27 and p53 significantly amplified the phenotype of benign tubular adenomas in Wv/Wv mouse. However, neither p27 nor p53 deletion was sufficient to induce the development of malignant ovarian carcinomas in Wv/Wv mice, probably due to the up-regulation of CKI family proteins such as p21, p16 or p19.

Gliomas are the most common human brain tumors and are divided into four stages by WHO classification scheme. Benign gliomas are defined as grades I (Pilocytic astrocytomas) and II (Astrocytomas), whereas grade III (Anaplastic Astrocytomas, AA) and grade IV (Glioblastoma Multiforme, GBM) are malignant. Although both grades III and IV are malignant, the prognoses for these tumors are quite different. The 2-year survival rate for grade III gliomas is 50%, and grade IV is < 20 %. Mechanisms of tumorigenesis are not exactly elucidated in brain tumor cells. The thesis is to study the role of p27 kip1 in human malignant brain tumors. The experimental methods include ribonuclease protection assay (RPA), western blotting, immunohistochemical staining and immunocytochemical staining. mRNAs of p130, p107, Rb, p53 and p27 kip1 in normal brain tissues and brain tumors were overexpressed in most case. The p27kip1 mRNA were expressed in all astrocytomas and GBM, and mRNA quantity of p27kip1 were more in brain tumors than in normal brain tissues. PI3K/Akt pathway regulates several cellular functions such as cell survival and cell proliferation. Active Akt can phosphorylate p27kip1 that may contribute cell cycle from G1 phase to S phase. Skp2 identifies phospho-p27kip1 and promotes p27kip1 degradation. We found p27kip1 overexpression and Akt activation in astrocytomas and GBM. The expression of p-Akt were found in 20 %, 87 % and 71 % in normal brain tissues, astrocytomas and GBM, respectively. Expression of p27kip1 and p-Akt has shown significant correlation in GBM (P = 0.0236). Overexpression of p27kip1 mRNA in brain tumors may be consequence of p-Akt and Skp2.

The F-box protein Skp2 (S-phase kinase associated protein 2) positively regulates the G1-S transition by controlling the cell cycle inhibitor p27Kip1. The p42/p44 mitogen-activated protein (MAP) kinase activation is also necessary for the cell cycle progression. p27Kip1 acts as a negative regulator of the cell cycle by inhibiting the activity of cyclin/cdk complexes during G0 and G1. RT-PCR, Western blotting and immunohistochemical staining were used to assay their relationship with cervical lesion development. In RT-PCR and western blotting, Skp2 mRNA and protein were expressed mostly in carcinoma tissues. At Fisher¡¦s exact test showed that Skp2, p27Kip1 and p42/p44 MAP Kinase are strongly associated with disease progession respectively (P < 0.0001, P < 0.0001, P = 0.0043). We also found a postive correlation between the expression of Skp2 and p42/p44 MAP Kinase (P = 0.0097).

The extracellular signal-regulated protein kinase 5 (ERK5), also known as big mitogen-activated protein (MAP) kinase 1 (BMK1), is a non-redundant mitogen-activated protein kinase (MAPK) implicated in mediating the response of cells to mitogens, oxidative and osmotic stresses. The molecular complexity of the ERK5 cascade has been mostly delineated by over-expression studies. For example, like other MAPKs, ERK5 activity increases upon phosphorylation by a MAPK/ERK kinase, namely MEK5. However, the physiological role of ERK5 is not rigorously established by these data. Furthermore, in comparison to the other members of the family, little is known about the downstream targets of ERK5. This constitutes an obstacle for the molecular understanding of the signalling mechanisms that account for the effect of ERK5 activation in vivo. To clarify these issues, I have tested the effect of the conditional loss of ERK5 in primary mouse embryonic fibroblasts (MEFs). My results indicate that ERK5 is required for the proliferation of MEFs, at least in part, by promoting the entry into S phase of the cell cycle. ERK5 suppressed the expression of the cyclin-dependent protein kinase (CDK) inhibitors, p21 and p27. As a result, low-level CDK2 activity detected in ERK5-deficient MEFs correlated with hypo-phosphorylation of the retinoblastoma (Rb) protein and with a defect in G1 to S phase transition of the cell cycle. ERK5 blocks p21 expression by decreasing the stability of the p21 transcript. This process might, at least partially, involve a mechanism implicating c-Myc-induced transcriptional up-regulation of the miR-17-92 cluster. Concerning p27, ERK5 decreases p27 protein stability. The stabilisation of p27 in the absence of ERK5 resulted in the accumulation of the protein in the nucleus. To examine the relevance of my findings in cancer, I tested the effect of pharmacological inhibition of ERK5 in two human breast cancer cell lines, MCF7 and MDA- MB-231, using XMD8-92, a novel potent and selective inhibitor of ERK5. My results show that these cells are dependent on ERK5 to proliferate. Furthermore, I found that incubation of MDA- MB-231 cells with XMD8-92 compromised their ability to invade. In both breast cancer cell lines, ERK5 down-regulates p21 and p27 expression. Together with evidence that cancer patients with poor prognosis display a high-level of expression of components of the ERK5 signalling pathway, these findings support the hypothesis that ERK5 can be a potential target for cancer therapy.

O mecanismo de oncogênese na laringe pode ser controlado por vários fatores, entre eles fatores envolvidos na regulação do ciclo celular e outros de risco, tais como exposição prolongada ao fumo e álcool. O desenvolvimento do câncer de laringe também pode estar associado à infecção pelo HPV. Este estudo, análise imunoistoquímica quantitativa de p53, p27 e Mdm2, foi realizado em 54 pacientes com carcinoma invasivo de laringe subdivididos em: carcinoma sem metástase (laryngeal squamous cell carcinoma without metastasis - LSCCWT), com metástase (laryngeal squamous cell carcinoma with metastasis - LSCCW) e linfonodos cervicais (limph nodes biopsies - LB). A detecção e tipificação do HPV foram realizadas pela reação em cadeia da polimerase (PCR) e os tipos de HPV avaliados foram HPV 6, 11, 16, 18, 31 e 33. Na análise quantitativa, alta expressão de p53, p27 e Mdm2 foi observada nos grupos LSCCW e LSCCWT assim como nas biópsias dos linfonodos cervicais, indicando que a avaliação dessas proteínas poderia não discriminar carcinomas de laringe metastáticos e não-metastáticos. Detecção do HPV foi verificada em apenas 7.4% dos casos. Dentre os pacientes HPV positivos, verificou-se expressão negativa de p53. Por outro lado, alta expressão de p27 e Mdm2 foi observada. Em conclusão, a avaliação quantitativa de p53, p27 e Mdm2 não permite traçar um perfil complementar em lesões metastáticas de laringe. / The mechanism of larynx oncogenesis could be controlled by various factors, most of them involved in cell cycle regulation and other risk factors such as smoking and alcohol abuse. The development of laryngeal carcinoma is associated with human papillomavirus (HPV) infection. In this study, quantitative immunohistochemistry was perfomed for p53, p27 and Mdm2 in 54 patients with invasive laryngeal squamous cell carcinoma without metastasis (LSCCWT), with metastasis (LSCCW) and cervical lymph nodes (LB). HPV detection and typing was performed by PCR and the HPV types evaluated were HPV 6, 11, 16, 18, 31 and 33. In the quantitative analysis higher p53, p27 and Mdm2 expression was observed in both LSCCW and LSCCWT, as well in cervical lymph node biopsies with metastasis, may indicating that evaluation of these proteins may not discriminate between metastatic and non-metastatic laryngeal carcinoma. HPV was found in 7.4% of the cases. Among HPV- positive patients, p53 expression was negative. On the other hand, high p27 and Mdm2 expression was observed. In conclusion, these data suggest that quantitative evaluation of p53, p27 and Mdm2 does not permit to determine a complementary profile in metastatic laryngeal lesions.