5584

PRKCI

epithelial-to-mesenchymal transition (EMT) has an important role in invasion and metastasis of hepatocellular carcinoma (HCC). To explore the regulatory mechanism of atypical protein kinase C iota (aPKCiota) signaling pathways to HCC development, and find an agent for targeted therapy for HCC, immortalized murine hepatocytes were employed to establish an EMT cell model of HCC, MMH-RT cells. Our study showed that EMT took place in MMH-R cells under the effect of transforming growth factor-beta1 (TGF-beta1) overexpressing aPKCiota. Furthermore, we showed that the aPKCiota blocking agent aurothiomalate (ATM) inhibited EMT and decreased invasion of hepatocytes. Moreover, ATM selectively inhibited proliferation of mesenchymal cells and HepG2 cells and induced apoptosis. However, ATM increased proliferation of epithelial cells and had little effect on apoptosis and invasion of epithelial cells. In conclusion, our result suggested that aPKCiota could be an important bio-marker of tumor EMT, and used as an indicator of invasion and malignancy. ATM might be a promising agent for targeted treatment of HCC.

Cholangiocarcinoma (CCA) invasion and metastasis are the primary causes of poor survival rates in patients. The epithelial-mesenchymal transition (EMT) is a crucial step in cancer invasion and metastasis. However, it is still unclear of the molecular mechanism. In this study, the expression of 14-3-3zeta and atypical protein kinase C-iota (aPKC-iota) was further detected in CCA tissues and cell lines. Meanwhile, we established the EMT model of CCA cells and investigated 14-3-3zeta and aPKC-iota co-regulatory effect on the EMT in vitro and in vivo. Further, we identified the downstream molecular glycogen synthase kinase 3 beta (GSK-3beta)/Snail signalling pathway that contribute to regulating the EMT. Our data showed that the expression of 14-3-3zeta and aPKC-iota was synergistically increased in CCA tissues compared with adjacent noncancerous tissues and was intimately associated with differentiation and the tumour-node-metastasis (TNM) stage. Multivariate Cox regression analysis indicated that high 14-3-3zeta and aPKC-iota expression separately predicted a poor prognosis and were independent prognostic indicators in patients with CCA. The CO-IP experiment confirmed that the mutual binding relationship between 14-3-3zeta and aPKC-iota. Small interfering RNAs and siRNA rescue experiment demonstrated that 14-3-3zeta and aPKC-iota regulated each other. In addition, 14-3-3zeta and aPKC-iota pretreatment by si-RNA inhibit the phosphorylated GSK-3beta and Snail expression during EMT. Meanwhile, silence of 14-3-3zeta or aPKC-iota suppressed CCA cells migration, metastasis and proliferation in vitro and in vivo. Our study demonstrates that 14-3-3zeta and aPKC-iota synergistically facilitate EMT of CCA via GSK-3beta/Snail signalling pathway, and may be potential therapeutic target for CCA.

Cholangiocarcinoma (CCA) is a highly malignant bile duct cancer that tends to invade and metastasize early. The epithelial-mesenchymal transition (EMT) has been implicated in cancer cell invasion and metastasis, as well as in cancer cell evasion of host immunity. In this study, we investigated the interaction between atypical protein kinase C-iota (aPKC-iota) and Snail in the regulation of EMT and its relationship to CCA immunosuppression. Our results demonstrated that aPKC-iota, Snail, and infiltrated immunosuppressive cells were significantly up-regulated in CCA tumor tissues and linked to poor prognosis. aPKC-iota induced EMT and immunosuppression by regulating Snail in vitro and in vivo, although aPKC-iota did not directly interact with Snail in coimmunoprecipitation experiments. To further clarify the molecular interaction between aPKC-iota and Snail in relation to EMT, quantitative iTRAQ-based phosphoproteomic analysis and liquid chromatography-tandem mass spectrometry were conducted to identify the substrates of aPKC-iota-dependent phosphorylation. Combined with coimmunoprecipitation, we showed that specificity protein 1 (Sp1) was directly phosphorylated by aPKC-iota on Ser59 (P-Sp1). Both Sp1 and P-Sp1 were up-regulated in CCA tumor tissues and associated with clinicopathological features and poor prognosis in CCA patients. Moreover, using chromatin immunoprecipitation assays, we found that P-Sp1 regulated Snail expression by increasing Sp1 binding to the Snail promoter. P-Sp1 also regulated aPKC-iota/Snail-induced EMT-like changes and immunosuppression in CCA cells. Our findings further indicated that CCA cells with EMT-like features appear to generate immunosuppressive natural T regulatory-like cluster of differentiation 4-positive (CD4(+) )CD25(-) cells rather than to increase CD4(+) CD25(+) natural T regulatory cells, in part by mediating T regulatory-inducible cytokines such as transforming growth factor beta1 and interleukin 2. CONCLUSION: These results demonstrate that aPKC-iota promotes EMT and induces immunosuppression through the aPKC-iota/P-Sp1/Snail signaling pathway and may be a potential therapeutic target for CCA. (Hepatology 2017;66:1165-1182).CI - (c) 2017 by the American Association for the Study of Liver diseases.

Atypical protein kinase Cs (aPKC) are involved in cell cycle progression, tumorigenesis, cell survival and migration in many cancers. We believe that aPKCs play an important role in cell motility of melanoma by regulating cell signaling pathways and inducing epithelial to mesenchymal transition (EMT). We have investigated the effects of two novel aPKC inhibitors; 2-acetyl-1,3-cyclopentanedione (ACPD) and 3,4-diaminonaphthalene-2,7-disulfonic acid (DNDA) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocytes. Molecular docking data suggested that both inhibitors specifically bind to protein kinase C-zeta (PKC-zeta) and PKC-iota (PKC-iota) and kinase activity assays were carried out to confirm these observations. Both inhibitors decreased the levels of total and phosphorylated PKC-zeta and PKC-iota. Increased levels of E-cadherin, RhoA, PTEN and decreased levels of phosphorylated vimentin, total vimentin, CD44, beta-catenin and phosphorylated AKT in inhibitor treated cells. This suggests that inhibition of both PKC-zeta and PKC-iota using ACPD and DNDA downregulates EMT and induces apoptosis in melanoma cells. We also carried out PKC-iota and PKC-zeta directed siRNA treatments to prove the above observations. Immunoprecipitation data suggested an association between PKC-iota and vimentin and PKC-iota siRNA treatments confirmed that PKC-iota activates vimentin by phosphorylation. These results further suggested that PKC-iota is involved in signaling pathways which upregulate EMT and which can be effectively suppressed using ACPD and DNDA. Our results summarize that melanoma cells proliferate via aPKC/AKT/NF-kappaB mediated pathway while inducing the EMT via PKC-iota/Par6/RhoA pathway. Overall, results show that aPKCs are essential for melanoma progression and metastasis, suggesting that ACPD and DNDA can be effectively used as potential therapeutic drugs for melanoma by inhibiting aPKCs.