RESUMO
Cten is an oncogene promoting EMT in many signaling pathways, namely through Snail. We investigated whether Cten function could be mediated through Src. Cten levels were modulated by forced expression in HCT116 and gene knockdown in SW620 CRC (colorectal cancer) cell lines. In all cell lines, Cten was a positive regulator of Src expression. The functional importance of Src was tested by simultaneous Cten overexpression and Src knockdown. This resulted in abrogation of Cten motility-inducing activity and reduction of colony formation ability together with failure to induce Cten targets. In SW620ΔCten reduced Src expression increased following restoration of Cten, also leading to increased cell motility and colony formation, which were lost if Src was concomitantly knocked down. By qRT-PCR we showed modulation of Cten had no effect on Src mRNA. However, a CHX pulse chase assay demonstrated stabilization of Src protein by Cten. Finally, expression of Cten and Src was tested in a series of 84 primary CRCs and there was a significant correlation between them (P = 0.001). We conclude that Src is a novel and functionally important target of the Cten signaling pathway and that Cten protein causes post-transcriptional stabilization of Src in promoting EMT and possibly metastasis in CRC.
Assuntos
Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Genes src , Tensinas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/fisiologia , Neoplasias Colorretais/genética , Técnicas de Silenciamento de Genes , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismoRESUMO
Cten promotes cell migration however the knowledge of underlying signalling pathways is sparse. We have shown that Cten downregulates E-cadherin, a feature of epithelial to mesenchymal transition (EMT). This prompted us to investigate whether Cten further contributed to EMT processes to regulate cell motility. The regulation of Snail by Cten was investigated following overexpression, knockdown (by RNA-interference) or knockout of Cten in HCT116, Caco-2 and SW620 colorectal cancer (CRC) cell lines. Subsequently, the cycloheximide (CHX) pulse chase assay was used to investigate changes in Snail protein stability and the functional relevance of Cten-Snail signalling was investigated. Snail was identified as a downstream target of Cten signalling using multiple approaches of Cten expression manipulation. Furthermore, this activity was mediated through the SH2 domain of Cten. The CHX assay confirmed that Cten was regulating Snail at a post transcriptional level and this was through the prevention of Snail degradation. Cell migration, invasion and colony formation efficiency were increased following forced expression of GFP-Cten but subsequently lost when Snail was knocked down, demonstrating a functional Cten-Snail signalling axis. In conclusion, we have described a novel Cten-Snail signaling pathway that contributes to cell motility in CRC, mediated by the stabilization of Snail protein. This finding potentially furthers the understanding of EMT regulatory networks in cancer metastasis.
Assuntos
Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição da Família Snail/genética , Tensinas/genética , Western Blotting , Células CACO-2 , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Inativação Gênica , Células HCT116 , Humanos , Estabilidade Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Fatores de Transcrição da Família Snail/metabolismo , Tensinas/metabolismoRESUMO
CTEN/TNS4 is a member of the Tensin gene family. It localizes to focal adhesions and induces cell motility. The mechanisms regulating Cten expression are unclear although we have shown regulation by Kras in the colon and pancreas. In normal mammary cell lines, it is reportedly upregulated by epidermal growth factor receptor (EGFR) and STAT3 signalling and upregulation is accompanied by downregulation of Tensin 3 (Tensin switch). In this study, we investigated the roles of EGFR and STAT3 signalling in the regulation of Cten in colorectal cancer (CRC). In addition, we investigated calpain--a regulator of focal adhesion-associated proteins whose relevance to Cten has not been investigated. CRC cell lines were stimulated with epidermal growth factor (EGF). This resulted in an increase in Cten and Tensin 3 protein. Kras was knocked down and this resulted in downregulation of Cten and Tensin 3. We next investigated the role of STAT3 signalling. Activation and knockdown of STAT3 resulted in downregulation and upregulation, respectively, of Cten. Inhibition of calpain resulted in upregulation of both Cten and Tensin 3. As the regulators of Cten also seemed to regulate Tensin 3, we tested the interaction between Cten and Tensin 3. Cten was forcibly expressed or knocked down resulting, respectively, in upregulation and downregulation of Tensin 3. We conclude that in CRC, Cten is upregulated by EGFR and Kras but downregulated by STAT3. We show that calpain may be a negative regulator of Cten and that a Tensin switch does not occur and, if anything, Cten stabilizes Tensin 3.
Assuntos
Neoplasias Colorretais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/agonistas , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Proteínas dos Microfilamentos/genética , Estabilidade Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Tensinas , TransfecçãoRESUMO
BACKGROUND: Placenta has long been a neglected organ as far as its pathology is concerned. This study is an attempt to observe the morphological features of placentae both gross and microscopic in normal pregnancy, preeclampsia, and eclampsia. MATERIALS AND METHODS: A total of 150 placentae were collected; of these, 50 belonged to normal pregnancy, 50 belonged to pre-eclamptic women, and 50 were from patients of eclampsia. RESULTS: Placental trimmed weight was seen to be decreased in patients of preeclampsia and eclampsia. Placental infarcts were more commonly seen in the diseased group and they were more centrally located. Hypertrophy of the spiral arterioles was observed in the decidual portion found in placental disc and membranes. Distal villous hypoplasia was also frequently seen in the diseased group. Fetal membranes were thickened and showed infarcts in preeclampsia and eclampsia. CONCLUSIONS: Morphological features seen in eclamptic placentae were similar but exaggerated compared to preeclampsia. In conclusion, the pathological changes were found to be more severe and frequent in preeclampsia and eclampsia, but more so in eclamptic placentae as compared with placenta of normal pregnancy. CD56 immunomarker was also used to identify NK cells. They were found to be present only in the diseased group and were located in the decidual portion of the basal plate, implicating their role in the development of the disease.