The Wnt signaling pathway is highly conserved and participates in many important cellular functions including differentiation, embryonic development and tissue generations. £]-catenin, the central mediator of the Wnt signaling, interacts with the TCF/LEF family of transcription factors in the nucleus and initiates downstream gene transcription. In addition, £]-catenin is known as a proto-oncogene implicated in numerous cancers including colorectal, cervical, endometrial and skin cancer. Chibby (Cby) is evolutionarily conserved in many species and acts as a repressor of Wnt/£]-catenin signaling. In our previous study, we have established that Cby over-expression attenuated £]-catenin translocation to nucleus and its transcriptional activity. Thus, it was hypothesized that Cby may possess potential tumor suppressing capabilities. In the present study, we first explored endogenous Cby expression status in human cervical cancer cells: HeLa and SiHa cell lines. It was observed that Cby mRNA and protein levels were significantly down-regulated in both cancer lines compared with primary cervical cells. We then conducted functional assays of tumorigenicity on both cells using adenoviral-encoded Cby and its NLS (nuclear localization signaling) deleted variant (Cby∆NLS). It was found that gene delivery of Cby or Cby∆NLS inhibited the proliferation, invasiveness, and colony forming in HeLa and SiHa cells. Immunofluorescent analysis revealed that Cby or Cby∆NLS gene transfer reduced the expression of Ki-67, a cell proliferative marker. Furthermore, Cby or Cby∆NLS restoration induced apoptosis and perturbed cell cycle progression in both cervical cancer cells. Finally, Cby over-expression decreases the expression of £]-catenin/TCF4 regulated genes such as c-myc and PCNA, which might contributed to the anti-neoplastic mechanism for Cby in cervical cancer cell lines. Our results strongly suggest that Cby may serve as a tumor suppressor gene during cervical carcinogenesis, and may facilitate in creation of new therapeutic methods.

ABSTRACT Chibby (or PIGEA-14) is a novel antagonist of the Beta-catenin pathway in nucleus. However, the tumor-suppressing function of Chibby and the importance of nuclear targeting to the cellular functions of Chibby have not been validated. By fusion of Chibby cDNA with green fluorescent protein (GFP) or Flag-tag, it was found that exogenous Chibby expression was detected in the nucleus as well as cytoplasm of transfected HeLa cells, but with a preferential nuclear localization (more than 50% cells with nuclear Chibby expression). Chibby overexpression significantly abrogated the cellular Beta¡Vcatenin activities and induced apoptosis in HeLa cells. Moreover, Chibby gene delivery attenuated the proliferation, migration, and anchorage-independent growth of HeLa cells, supporting the tumor suppressor function of Chibby. Mutation or deletion of the predicted nuclear localization sequence (NLS), at residues 123-126, significantly promoted the cytoplasmic localization of Chibby, indicating residues 123-126 is the NLS domain of Chibby. Interestingly, ecotopic expression of Chibby NLS mutants remained capable of inducing apoptosis and inhibiting Beta¡Vcatenin activities in HeLa cells. Besides, overexpression Chibby NLS mutants effectively attenuated the viability, motility and colonies formation of HeLa cells. Expression analysis revealed that Chibby NLS mutants retained Beta-catenin in the cytoplasm and prevented its nuclear entry, thereby inhibiting the Beta-catenin transcriptional activities. In summary, Chibby shuttles between nucleus and cytoplasm, and possesses the functions of tumor suppressor and Beta-catenin antagonist.

Les cils et flagelles sont des organites cellulaires retrouvés des protozoaires aux mammifères. Une dérégulation de l’assemblage (ciliogenèse) ou de la fonction des cils, entraîne diverses maladies chez l’homme. Parmi les acteurs de la ciliogenèse, se trouvent les facteurs de transcription RFX. La recherche de gènes cibles de RFX chez la drosophile a permis d’identifier le gène Chibby (Cby), précédemment décrit comme un antagoniste de la voie de signalisation Wnt/wingless. Contrairement aux vertébrés, chez les invertébrés aucun lien n’est encore établi entre cil et voie wg. L’identification de cby comme cible de dRFX chez la drosophile suggère une fonction ciliaire de cby et permettrait l’établissement du lien cil/voie wg. CBY se localise à la zone de transition des cils des neurones sensoriels du système nerveux périphérique et aux centrioles des spermatides. Les drosophiles invalidées pour cby présentent un phénotype de non coordination, mais aucun phénotype de type wg. Ces mutants ont des défauts des cils sensoriels, ainsi que des défauts d’organisation des spermatides. De plus, chez les embryons, les protéines actrices du transport intra-flagellaire, NompB et CG11356, sont mal distribuées lorsque CBY est absente. Enfin, chez les mutants, la localisation de la protéine UNC est affectée dans les cellules germinales en fin de spermatogenèse. En conclusion, chez la drosophile, CBY est impliquée dans le tri protéique organisé à la base du cil de neurones sensoriels, de concert avec les autres protéines localisées à la zone de transition. Dans le testicule, CBY est nécessaire à la maturation des spermatides. En revanche, CBY n‟intervient pas dans la régulation de la voie wg / Cilia and flagella are organelles found from protozoa to mammals. Deregulation of the assembly (ciliogenesis) or function of cilia, causes various diseases in humans. Among those involved in ciliogenesis are the RFX transcription factors. The search for RFX target genes in Drosophila identified the Chibby (CBY) gene, previously described as an antagonist of the Wnt / wingless pathway. Unlike in vertebrates, in invertebrates is still no link established between cilia and the wg pathway. The identification of CBY as a target of dRFX in Drosophila suggests a ciliary function of CBY and would allow the establishment of the link cilia / wg pathway. CBY is localized at the transition zone of cilia of sensory neurons of the peripheral nervous system and at centrioles in spermatids. Drosophila invalidated for CBY present a phenotype of uncoordination, but no wg phenotype. These mutants have defects in sensory cilia and defects in organization of spermatids. In addition, in embryos, the distribution of proteins involved in intra-flagellar transport, NompB and CG11356, is affected when CBY is absent. Finally, in the mutants, the localization of the UNC protein is affected in germ cells at the end of spermatogenesis. In conclusion, in Drosophila, CBY is involved in the protein sorting organized at the base of cilia of sensory neurons, with the other proteins located at the transition zone. In the testes, CBY is necessary for the maturation of spermatids. However, CBY is not involved in the regulation of the wg pathway