aPKC-ι/P-Sp1/Snail signaling induces epithelial-mesenchymal transition and immunosuppression in cholangiocarcinoma

Abstract

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-ι) and Snail in the regulation of EMT and its relationship to CCA immunosuppression. Our results demonstrated that aPKC-ι, Snail, and infiltrated immunosuppressive cells were significantly up-regulated in CCA tumor tissues and were linked to poor prognosis. aPKC-ι induced EMT and immunosuppression by regulating Snail in vitro and in vivo, although aPKC-ι did not directly interact with Snail in co-immunoprecipitation (co-IP) experiments. To further clarify the molecular interaction between aPKC-ι and Snail in relation to EMT, quantitative iTRAQ-based phosphoproteomic analysis and LC-MS/MS were conducted to identify the substrates of aPKC-ι-dependent phosphorylation. Combined with co-IP, we showed that Sp1 was directly phosphorylated by aPKC-ι on Ser59 (P-Sp1). Both Sp1 and P-Sp1 were up-regulated in CCA tumor tissues and were also associated with clinicopathological features and poor prognosis in CCA patients. Moreover, using chromatin immunoprecipitation assays, we found that P-Sp1 regulated the Snail expression by increasing Sp1 binding to the Snail promoter. P-Sp1 also regulated aPKC-ι/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 (Treg)-like CD4⁺CD25^- cells rather than to increase CD4⁺CD25⁺ nTreg cells, in part by mediating Treg-inducible cytokines such as TGF-β1 and IL-2.

Conclusion: These results demonstrated that aPKC-ι promotes EMT and induces immunosuppression via the aPKC-ι/P-Sp1/Snail signaling pathway and may be a potential therapeutic target for CCA. This article is protected by copyright. All rights reserved.

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