Cyclin D3/CDK11p58 complex is involved in the repression of androgen receptor.

Androgen receptor (AR) is essential for the maintenance of the male reproductive systems and is critical for the carcinogenesis of human prostate cancers (PCas). D-type cyclins are closely related to the repression of AR function. It has been well documented that cyclin D1 inhibits AR function through multiple mechanisms, but the mechanism of how cyclin D3 exerts its repressive role in the AR signaling pathway remains to be identified. In the present investigation, we demonstrate that cyclin D3 and the 58-kDa isoform of cyclin-dependent kinase 11 (CDK11p58) repressed AR transcriptional activity as measured by reporter assays of transformed cells and prostate-specific antigen expression in PCa cells. AR, cyclin D3, and CDK11p58 formed a ternary complex in cells and were colocalized in the luminal epithelial layer of the prostate. AR activity is controlled by phosphorylation at specific sites. We found that AR was phosphorylated at Ser-308 by cyclin D3/CDK11p58 in vitro and in vivo, leading to the repressed activity of AR transcriptional activation unit 1 (TAU1). Furthermore, androgen-dependent proliferation of PCa cells was inhibited by cyclin D3/CDK11p58 through AR repression. These data suggest that cyclin D3/CDK11p58 signaling is involved in the negative regulation of AR function.

Identification of the p28 subunit of eukaryotic initiation factor 3(eIF3k) as a new interaction partner of cyclin D3.

Cyclin D3 is found to play a crucial role not only in progression through the G1 phase as a regulatory subunit of cyclin-dependent kinase 4 (CDK 4) and CDK 6, but also in many other aspects such as cell cycle, cell differentiation, transcriptional regulation and apoptosis. In this work, we screened a human fetal liver cDNA library using human cyclin D3 as bait and identified human eukaryotic initiation factor 3 p28 protein (eIF3k) as a partner of cyclin D3. The association of cyclin D3 with eIF3k was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscopic analysis. We found that cyclin D3 specifically interacted with eIF3k through its C-terminal domain. Immunofluorescence experiments showed that eIF3k distributed both in nucleus and cytoplasm and colocalized with cyclin D3. In addition, the cellular translation activity in HeLa cells was upregulated by cyclin D3 overexpression and the mRNA levels are constant. These data provide a new clue to our understanding of the cellular function of cyclin D3.

Interleukin-8 signaling promotes androgen-independent proliferation of prostate cancer cells via induction of androgen receptor expression and activation

The aim of our study was to assess the importance of the CXC chemokine and interleukin (IL)-8 in promoting the transition of prostate cancer (CaP) to the androgen-independent state. Stimulation of the androgen-dependent cell lines, LNCaP and 22Rv1, with exogenous recombinant human interleukin-8 (rh-IL-8) increased androgen receptor (AR) gene expression at the messenger RNA (mRNA) and protein level, assessed by quantitative polymerase chain reaction and immunoblotting, respectively. Using an androgen response element-luciferase construct, we demonstrated that rh-IL-8 treatment also resulted in increased AR transcriptional activity in both these cell lines, and a subsequent upregulation of prostate-specific antigen and cyclin-dependent kinase 2 mRNA transcript levels in LNCaP cells. Blockade of CXC chemokine receptor-2 signaling using a small molecule antagonist (AZ10397767) attenuated the IL-8-induced increases in AR expression and transcriptional activity. Furthermore, in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, coadministration of AZ10397767 reduced the viability of LNCaP and 22Rv1 cells exposed to bicalutamide. Our data show that IL-8 signaling increases AR expression and promotes ligand-independent activation of this receptor in two androgen-dependent cell lines, describing two mechanisms by which this chemokine may assist in promoting the transition of CaP to the androgen-independent state. In addition, our data show that IL-8-promoted regulation of the AR attenuates the effectiveness of the AR antagonist bicalutamide in reducing CaP cell viability.

miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes.

Overexpression of the microRNA miR-433 promotes resistance to paclitaxel through the induction of cellular senescence in ovarian cancer cells

Annually, ovarian cancer (OC) affects 240,000 women worldwide and is the most lethal gynecological malignancy. High-grade serous OC (HGSOC) is the most common and aggressive OC subtype, characterized by widespread genome changes and chromosomal instability and is consequently poorly responsive to chemotherapy treatment. The objective of this study was to investigate the role of the microRNA miR-433 in the cellular response of OC cells to paclitaxel treatment. We show that stable miR-433 expression in A2780 OC cells results in the induction of cellular senescence demonstrated by morphological changes, downregulation of phosphorylated retinoblastoma (p-Rb), and an increase in β-galactosidase activity. Furthermore, in silico analysis identified four possible miR-433 target genes associated with cellular senescence: cyclin-dependent kinase 6 (CDK6), MAPK14, E2F3, and CDKN2A. Mechanistically, we demonstrate that downregulation of p-Rb is attributable to a miR-433-dependent downregulation of CDK6, establishing it as a novel miR-433 associated gene. Interestingly, we show that high miR-433 expressing cells release miR-433 into the growth media via exosomes which in turn can induce a senescence bystander effect. Furthermore, in relation to a chemotherapeutic response, quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that only PEO1 and PEO4 OC cells with the highest miR-433 expression survive paclitaxel treatment. Our data highlight how the aberrant expression of miR-433 can adversely affect intracellular signaling to mediate chemoresistance in OC cells by driving cellular senescence.

Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma:analysis of a clinic-based population

Five to ten percent of individuals with melanoma have another affected family member, suggesting familial predisposition. Germ-line mutations in the cyclin-dependent kinase (CDK) inhibitor p16 have been reported in a subset of melanoma pedigrees, but their prevalence is unknown in more common cases of familial melanoma that do not involve large families with multiple affected members. We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families). Five independent, definitive p16 mutations were detected (18%, 95% confidence interval: 6%, 37%), including one nonsense, one disease-associated missense, and three small deletions. No mutations were detected in CDK4. Disease-associated mutations in p19ARF, whose transcript is derived in part from an alternative codon reading frame of p16, were only detected in patients who also had mutations inactivating p16. We conclude that germ-line p16 mutations are present in a significant fraction of individuals who have melanoma and a positive family history.

PIAS1 is increased in human prostate cancer and enhances proliferation through inhibition of p21

Prostate cancer development and progression are associated with alterations in expression and function of elements of cytokine networks, some of which can activate multiple signaling pathways. Protein inhibitor of activated signal transducers and activators of transcription (PIAS)1, a regulator of cytokine signaling, may be implicated in the modulation of cellular events during carcinogenesis. This study was designed to investigate the functional significance of PIAS1 in models of human prostate cancer. We demonstrate for the first time that PIAS1 protein expression is significantly higher in malignant areas of clinical prostate cancer specimens than in normal tissues, thus suggesting a growth-promoting role for PIAS1. Expression of PIAS1 was observed in the majority of tested prostate cancer cell lines. In addition, we investigated the mechanism by which PIAS1 might promote prostate cancer and found that down-regulation of PIAS1 leads to decreased proliferation and colony formation ability of prostate cancer cell lines. This decrease correlates with cell cycle arrest in the G0/G1 phase, which is mediated by increased expression of p21(CIP1/WAF1). Furthermore, PIAS1 overexpression positively influences cell cycle progression and thereby stimulates proliferation, which can be mechanistically explained by a decrease in the levels of cellular p21. Taken together, our data reveal an important new role for PIAS1 in the regulation of cell proliferation in prostate cancer.

Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA is lethal. Importantly, uracil misincorporation is a mechanism of cytotoxicity induced by fluoropyrimidine chemotherapeutic agents including 5-fluorouracil (5-FU) and elevated expression of dUTPase is negatively correlated with clinical response to 5-FU-therapy. In this study we performed the first functional characterization of the dUTPase promoter and demonstrate a role for E2F-1 and Sp1 in driving dUTPase expression. We establish a direct role for both mutant and wild-type forms of p53 in modulating dUTPase promoter activity. Treatment of HCT116 p53(+/+) cells with the DNA-damaging agent oxaliplatin induced a p53-dependent transcriptional downregulation of dUTPase not observed in the isogenic null cell line. Oxaliplatin treatment induced enrichment of p53 at the dUTPase promoter with a concomitant reduction in Sp1. The suppression of dUTPase by oxaliplatin promoted increased levels of dUTP that was enhanced by subsequent addition of fluoropyrimidines. The novel observation that oxaliplatin downregulates dUTPase expression may provide a mechanistic basis contributing to the synergy observed between 5-FU and oxaliplatin in the clinic. Furthermore, these studies provide the first evidence of a direct transcriptional link between the essential enzyme dUTPase and the tumor suppressor p53.

Dissecting cell cycle protein complexes using the pptimized yeast cytosine deaminase protein-fragment complementation assay “You too can play with an edge”

Les protéines sont les produits finaux de la machinerie génétique. Elles jouent des rôles essentiels dans la définition de la structure, de l'intégrité et de la dynamique de la cellule afin de promouvoir les diverses transformations chimiques requises dans le métabolisme et dans la transmission des signaux biochimique. Nous savons que la doctrine centrale de la biologie moléculaire: un gène = un ARN messager = une protéine, est une simplification grossière du système biologique. En effet, plusieurs ARN messagers peuvent provenir d’un seul gène grâce à l’épissage alternatif. De plus, une protéine peut adopter plusieurs fonctions au courant de sa vie selon son état de modification post-traductionelle, sa conformation et son interaction avec d’autres protéines. La formation de complexes protéiques peut, en elle-même, être déterminée par l’état de modifications des protéines influencées par le contexte génétique, les compartiments subcellulaires, les conditions environmentales ou être intrinsèque à la croissance et la division cellulaire. Les complexes protéiques impliqués dans la régulation du cycle cellulaire sont particulièrement difficiles à disséquer car ils ne se forment qu’au cours de phases spécifiques du cycle cellulaire, ils sont fortement régulés par les modifications post-traductionnelles et peuvent se produire dans tous les compartiments subcellulaires. À ce jour, aucune méthode générale n’a été développée pour permettre une dissection fine de ces complexes macromoléculaires. L'objectif de cette thèse est d'établir et de démontrer une nouvelle stratégie pour disséquer les complexes protéines formés lors du cycle cellulaire de la levure Saccharomyces cerevisiae (S. cerevisiae). Dans cette thèse, je décris le développement et l'optimisation d'une stratégie simple de sélection basée sur un essai de complémentation de fragments protéiques en utilisant la cytosine déaminase de la levure comme sonde (PCA OyCD). En outre, je décris une série d'études de validation du PCA OyCD afin de l’utiliser pour disséquer les mécanismes d'activation des facteurs de transcription et des interactions protéine-protéines (IPPs) entre les régulateurs du cycle cellulaire. Une caractéristique clé du PCA OyCD est qu'il peut être utilisé pour détecter à la fois la formation et la dissociation des IPPs et émettre un signal détectable (la croissance des cellules) pour les deux types de sélections. J'ai appliqué le PCA OyCD pour disséquer les interactions entre SBF et MBF, deux facteurs de transcription clés régulant la transition de la phase G1 à la phase S. SBF et MBF sont deux facteurs de transcription hétérodimériques composés de deux sous-unités : une protéine qui peut lier directement l’ADN (Swi4 ou Mbp1, respectivement) et une protéine commune contenant un domain d’activation de la transcription appelée Swi6. J'ai appliqué le PCA OyCD afin de générer un mutant de Swi6 qui restreint ses activités transcriptionnelles à SBF, abolissant l’activité MBF. Nous avons isolé des souches portant des mutations dans le domaine C-terminal de Swi6, préalablement identifié comme responsable dans la formation de l’interaction avec Swi4 et Mbp1, et également important pour les activités de SBF et MBF. Nos résultats appuient un modèle où Swi6 subit un changement conformationnel lors de la liaison à Swi4 ou Mbp1. De plus, ce mutant de Swi6 a été utilisé pour disséquer le mécanisme de régulation de l’entrée de la cellule dans un nouveau cycle de division cellulaire appelé « START ». Nous avons constaté que le répresseur de SBF et MBF nommé Whi5 se lie directement au domaine C-terminal de Swi6. Finalement, j'ai appliqué le PCA OyCD afin de disséquer les complexes protéiques de la kinase cycline-dépendante de la levure nommé Cdk1. Cdk1 est la kinase essentielle qui régule la progression du cycle cellulaire et peut phosphoryler un grand nombre de substrats différents en s'associant à l'une des neuf protéines cycline régulatrice (Cln1-3, Clb1-6). Je décris une stratégie à haut débit, voir à une échelle génomique, visant à identifier les partenaires d'interaction de Cdk1 et d’y associer la cycline appropriée(s) requise(s) à l’observation d’une interaction en utilisant le PCA OyCD et des souches délétées pour chacune des cyclines. Mes résultats nous permettent d’identifier la phase(s) du cycle cellulaire où Cdk1 peut phosphoryler un substrat particulier et la fonction potentielle ou connue de Cdk1 pendant cette phase. Par exemple, nous avons identifié que l’interaction entre Cdk1 et la γ-tubuline (Tub4) est dépendante de Clb3. Ce résultat est conforme au rôle de Tub4 dans la nucléation et la croissance des faisceaux mitotiques émanant des centromères. Cette stratégie peut également être appliquée à l’étude d'autres IPPs qui sont contrôlées par des sous-unités régulatrices.

The orphan nuclear receptor, liver receptor homolog-1 (LRH-1, NR5A2) regulates decidualization

La période de réceptivité endométriale chez l’humain coïncide avec la différentiation des cellules stromales de l’endomètre en cellules hautement spécifiques, les cellules déciduales, durant le processus dit de décidualisation. Or, on sait qu’une transformation anormale des cellules endométriales peut être à l’origine de pertes récurrentes de grossesses. LRH-1 est un récepteur nucléaire orphelin et un facteur de transcription régulant de nombreux évènements relatif à la reproduction et comme tout récepteur, son activation promouvoit l’activité transcriptionnelle de ses gènes cibles. Nous avons déjà montré que LRH-1 et son activité sont essentiels pour la décidualisation au niveau de l’utérus chez la souris et nous savons qu’il est présent dans l’utérus chez l’humain au moment de la phase de prolifération mais aussi de sécrétion du cycle menstruel, et que son expression augmente dans des conditions de décidualisation in vitro. Notre hypothèse est alors la suivante : LRH-1 est indispensable à la décidualisation du stroma endométrial, agissant par le biais de la régulation transcriptionnelle de gènes requis pour la transformation de cellules stromales en cellules déciduales. Afin d’explorer le mécanisme moléculaire impliqué dans la régulation transcriptionnelle effectuée par l’intermédiaire de ce récepteur, nous avons mis en place un modèle de décidualisation in vitro utilisant une lignée de cellules stromales de l’endomètre, cellules humaines et immortelles (hESC). Notre modèle de surexpression développé en transfectant les dites cellules avec un plasmide exprimant LRH-1, résulte en l’augmentation, d’un facteur 5, de l’abondance du transcriptome de gènes marqueurs de la décidualisation que sont la prolactine (PRL) et l’insulin-like growth factor binding protein-1 (IGFBP-1). En outre, la sous-régulation de ce récepteur par l’intermédiaire de petits ARN interférents (shRNA) abolit la réaction déciduale, d’un point de vue morphologique mais aussi en terme d’expression des deux gènes marqueurs cités ci-dessus. Une analyse par Chromatin ImmunoPrécipitation (ou ChIP) a démontré que LRH-1 se lie à des régions génomiques se trouvant en aval de certains gènes importants pour la décidualisation comme PRL, WNT 4, WNT 5, CDKN1A ou encore IL-24, et dans chacun de ces cas cités, cette capacité de liaison augmente dans le cadre de la décidualisation in vitro. Par ailleurs, des études structurelles ont identifié les phospholipides comme des ligands potentiels pour LRH-1. Nous avons donc choisi d’orienter notre travail de façon à explorer les effets sur les ligands liés à LRH-1 de traitements impliquant des agonistes et antagonistes à notre récepteur nucléaire. Les analyses par q-PCR et Western blot ont montré que la modulation de l’activité de LRH-1 par ses ligands influait aussi sur la réaction déciduale. Enfin, des études récentes de Salker et al (Salker, Teklenburg et al. 2010) ont mis en évidence que les cellules stromales humaines décidualisées sont de véritables biocapteurs de la qualité embryonnaire et qu’elles ont la capacité de migrer en direction de l’embryon. La série d’expériences que nous avons réalisée à l’aide de cellules hESC placées en co-culture avec des embryons de souris confirme que la migration cellulaire est bien dirigée vers les embryons. Cette propriété quant à l’orientation de la migration cellulaire est notoirement diminuée dans le cas où l’expression de LRH-1 est déplétée par shRNA dans les hESC. Nos données prouvent donc que LRH-1 régule non seulement la transcription d’un ensemble de gènes impliqués dans le processus de décidualisation mais agit aussi sur la motilité directionnelle de ces cellules hESC décidualisées in vitro.

Oocyte activation and preimplantation development of bovine embryos obtained by specific inhibition of cyclin-dependent kinases

Realizaram-se dois experimentos para avaliar a eficiência da bohemina e roscovitina associadas à ionomicina para ativação partenogenética e desenvolvimento embrionário inicial de bovinos. No primeiro, foram testadas diferentes concentrações (0, 50, 75 ou 100µM) e diferentes tempos de exposição (2, 4 ou 6 horas) à bohemina ou à roscovitina na ativação de oócitos bovinos maturados in vitro (MIV) pré-expostos à ionomicina. Os melhores tratamentos, bohemina 75µM e roscovitina 50µM, ambos por seis horas, foram utilizados no segundo experimento, no qual oócitos bovinos MIV foram expostos à ionomicina seguido ou não pelo tratamento com inibidores específicos das quinases dependentes de ciclina (CDKI), e avaliados quanto à configuração nuclear, taxa de ativação e desenvolvimento até blastocisto. Os tratamentos combinados (ionomicina+CDKI) apresentaram melhor taxa de ativação (77,3%) e desenvolvimento embrionário inicial (35,2%) do que a ionomicina sozinha (69,4% e 21,9%, respectivamente), e também promoveram ativação mais uniforme (aproximadamente 90% de formação de um pronúcleo). Estes resultados demonstram que os CDKIs potencializam o efeito da ionomicina na ativação e desenvolvimento embrionário inicial e podem auxiliar na obtenção de protocolos de ativação mais eficientes, aumentando a capacidade de desenvolvimento de embriões produzidos por meio de biotécnicas reprodutivas.

The genetics of hypertension modifies the renal cell replication response induced by experimental diabetes

To investigate whether the genetics of hypertension modifies renal cell responses in experimental diabetes, we studied the renal cell replication and its regulation by two cyclin-dependent kinase (Cdk) inhibitors, p27(Kip1) and p21(Cip1), in prehypertensive spontaneously hypertensive rats (SUR) and their genetically normotensive counterparts, Wistar Kyoto (WKY) rats, with and without streptozotocin-induced diabetes. In diabetic SIIR, the number of proliferating glomerular (0.6 +/- 0.3 positive cells/50 glomeruli) and tubulointerstitial (2.8 +/- 0.6 positive tubulointerstitial cells/50 grid fields) cells assessed by the bromodeoxyuridine technique was significantly (P = 0.0002) lower than in control SIIR (13.2 +/- 1.7 and 48.6 +/- 9.7, respectively) and control (14.0 +/- 1.8 and 63.9 +/- 10.6) and diabetic (14.3 +/- 3.5 and 66.4 +/- 11.5) WKY rats. Proliferating cell nuclear antigen, another marker of cell proliferation, was significantly reduced in replicating glomerular (P = 0.0002) and tubulointerstitial (P < 0.0001) cells in diabetic SHR. In freshly isolated glomeruli, the level of p27(Kip1) detected by Western blotting was significantly higher In diabetic SIIR than in nondiabetic SHR (1.52 +/- 0.14 vs. 1.00 +/- 0.10% of control, P = 0.014). The expression of p21(Cip1) in isolated glomeruli did not differ among the groups of rats. In conclusion, the response of renal cell replication to diabetes differs markedly between prehypertensive SIIR and their WKY control rats. The decreased glomerular cell proliferation in prehypertensive diabetic SIIR is at least partly mediated by a higher expression of the Cdk inhibitor p27(Kip1).

Structural bioinformatics study of cyclin-dependent kinases complexed with inhibitors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

p16INK4A immunohistochemical overexpression in premalignant and malignant oral lesions infected with human papillomavirus

Human papillomavirus (HPV) is believed to promote the oncogenic process, and the correlation between viral oncoproteins and dysfunction of p16 INK4A tumor suppressor protein in oral lesions is controversial. To test the hypothesis that anogenital HPV types participate in disruption of the regulation of p16INK4A suppressor protein in oral lesions, we analyzed 46 oral biopsy specimens for the presence of HPV 6/11 and 16/18 by in situ hybridization (ISH) and for p16INK4A expression by immunohistochemistry (IHC). Eighteen (39%) of the 46 oral lesions were HPV-positive and 28 (61%) were HPV-negative. HPV 6/11 DNA was found in 5 (11%) and HPV 16/18 in 13 (28%) of 46 biopsies. Nine of the 18 HPV-positive oral lesions (50%), assessed by catalyzed signal amplification coupled to ISH (CSA-ISH), gave high-intensity p16INK4A immunostaining. Focal and diffuse patterns were observed in 11/13 (77%) lesions with HPV 16/18, focal immunopositivity in 3/5 (80%) with HPV 6/11, and negative or sporadic p16-labeling in 18/28 (64%) without the presence of HPV DNA. These results showed a strong association between overexpression of p16 protein and malignant oral lesions, mainly those infected by HPV 16/18. We can conclude that high-risk HPV types are associated with p16 overexpression, and p16 may serve as a biomarker in oral cancer related to high-risk HPV infection.