RESUMEN
Asymmetric cell division is a key tumor suppressor mechanism that prevents the uncontrolled expansion of the stem cell (SC) compartment by generating daughter cells with alternative fates: one retains SC identity and enters quiescence and the other becomes a rapidly proliferating and differentiating progenitor. A critical player in this process is Numb, which partitions asymmetrically at SC mitosis and inflicts different proliferative and differentiative fates in the two daughters. Here, we show that asymmetric Numb partitioning per se is insufficient for the proper control of mammary SC dynamics, with differential phosphorylation and functional inactivation of Numb in the two progeny also required. The asymmetric phosphorylation/inactivation of Numb in the progenitor is mediated by the atypical PKCζ isoform. This mechanism is subverted in breast cancer via aberrant activation of PKCs that phosphorylate Numb in both progenies, leading to symmetric division and expansion of the cancer SC compartment, associated with aggressive disease. Thus, Numb phosphorylation represents a target for breast cancer therapy.
Asunto(s)
Neoplasias de la Mama , Proteínas de la Membrana , Células Madre Neoplásicas , Proteínas del Tejido Nervioso , División Celular Asimétrica , Neoplasias de la Mama/genética , Femenino , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitosis , Células Madre Neoplásicas/citología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , FosforilaciónRESUMEN
BACKGROUND: The transcription factor Pax8 is expressed during thyroid development and is involved in the morphogenesis of the thyroid gland and maintenance of the differentiated phenotype. In particular, Pax8 has been shown to regulate genes that are considered markers of thyroid differentiation. Recently, the analysis of the gene expression profile of FRTL-5 differentiated thyroid cells after the silencing of Pax8 identified Wnt4 as a novel target. Like the other members of the Wnt family, Wnt4 has been implicated in several developmental processes including regulation of cell fate and patterning during embryogenesis. To date, the only evidence on Wnt4 in thyroid concerns its down-regulation necessary for the progression of thyroid epithelial tumors. RESULTS: Here we demonstrate that Pax8 is involved in the transcriptional modulation of Wnt4 gene expression directly binding to its 5'-flanking region, and that Wnt4 expression in FRTL-5 cells is TSH-dependent. Interestingly, we also show that in thyroid cells a reduced expression of Wnt4 correlates with the alteration of the epithelial phenotype and that the overexpression of Wnt4 in thyroid cancer cells is able to inhibit cellular migration. CONCLUSIONS: We have identified and characterized a functional Pax8 binding site in the 5'-flanking region of the Wnt4 gene and we show that Pax8 modulates the expression of Wnt4 in thyroid cells. Taken together, our results suggest that in thyroid cells Wnt4 expression correlates with the integrity of the epithelial phenotype and is reduced when this integrity is perturbed. In the end, we would like to suggest that the overexpression of Wnt4 in thyroid cancer cells is able to revert the mesenchymal phenotype.
Asunto(s)
Factores de Transcripción Paired Box/metabolismo , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/genética , Proteína Wnt4/genética , Animales , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Humanos , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box/genética , Fenotipo , Regiones Promotoras Genéticas , Ratas , Glándula Tiroides/citología , Glándula Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Tirotropina/metabolismo , Proteína Wnt4/metabolismoRESUMEN
BACKGROUND: PAX8 is a member of the paired box (Pax) multigene family of transcription factors, which are involved in the developmental and tissue-specific control of the expression of several genes in both vertebrates and invertebrates. Previously, several studies reported that PAX8 is expressed at high levels in specific types of tumors. In particular, PAX8 has been recently reported to be conspicuously expressed in human ovarian cancer, but the functional role of PAX8 in the carcinogenesis of this type of tumor has not been addressed. In this study, we investigated the contribution of PAX8 in ovarian cancer progression. METHODS: Stable PAX8 depleted ovarian cancer cells were generated using short hairpin RNA (shRNA) constructs. PAX8 mRNA and protein were detected by RT-PCR, immunoblot and immunofluorescence. Cell proliferation, motility and invasion potential of PAX8 silenced cells were analyzed by means of growth curves, wound healing and Matrigel assays. In addition, PAX8 knockdown and control cells were injected into nude mice for xenograft tumorigenicity assays. Finally, qPCR was used to detect the expression levels of EMT markers in PAX8-overexpressing and control cells. RESULTS: Here, we show that PAX8 plays a critical role in the migration, invasion and tumorigenic ability of ovarian cancer cells. Our results show that RNA interference-mediated knockdown of PAX8 expression in SKOV-3 ovarian cancer cells produces a significant reduction of cell proliferation, migration ability and invasion activity compared with control parental SKOV-3 cells. Moreover, PAX8 silencing strongly suppresses anchorage-independent growth in vitro. Notably, tumorigenesis in vivo in a nude mouse xenograft model is also significantly inhibited. CONCLUSIONS: Overall, our results indicate that PAX8 plays an important role in the tumorigenic phenotype of ovarian cancer cells and identifies PAX8 as a potential new target for the treatment of ovarian cancer.
Asunto(s)
Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Ováricas/genética , Factores de Transcripción Paired Box/biosíntesis , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Neoplasias Ováricas/patología , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Congenital hypothyroidism (CH) is a common endocrine disease that occurs in about 1:3000 newborns. In 80-85% of the cases, CH is presumably secondary to thyroid dysgenesis (TD), a defect in the organogenesis of the gland leading to an ectopic (30-45%), absent (agenesis, 35-40%) or hypoplastic (5%) thyroid gland. The pathogenesis of TD is still largely unknown. Most cases of TD are sporadic, although familial occurrences have occasionally been described. Recently, mutations in the PAX8 transcription factor have been identified in patients with TD. OBJECTIVE: Our aim was to identify and functionally characterize novel PAX8 mutations with autosomal dominant transmission responsible for TD. DESIGN: The PAX8 gene was sequenced in a mother and child both suffering from congenital hypothyroidism (CH) because of thyroid hypoplasia. Subsequently, expression vectors encoding the mutated PAX8 were generated, and the effects of the mutation on both the DNA-binding capability and the transcriptional activity were evaluated. RESULTS: PAX8 gene sequencing revealed a heterozygous mutation that consists of the substitution of a histidine residue with a glutamine at position 55 of the PAX8 protein (H55Q). When tested in cotransfection experiments with a thyroglobulin promoter reporter construct, the mutant protein turned out to be still able to bind DNA in Electrophoretic Mobility Shift Assay assays but transcriptionally inactive. CONCLUSIONS: Our findings confirm the important role of PAX8 in normal thyroid development and support the evidence that in humans haploinsufficiency of PAX8 is associated with TD.