Department of Protein Phosphorylation, Cancer Research UK London Research Institute, London WC2A 3LY, UK.
Carcinogenesis. 2014 Feb;35(2):396-406. doi: 10.1093/carcin/bgt313. Epub 2013 Sep 26.
Protein kinase C iota (PKCι), a serine/threonine kinase required for cell polarity, proliferation and migration, is commonly up- or downregulated in cancer. PKCι is a human oncogene but whether this is related to its role in cell polarity and what repertoire of oncogenes acts in concert with PKCι is not known. We developed a panel of candidate oncogene expressing Madin-Darby canine kidney (MDCK) cells and demonstrated that H-Ras, ErbB2 and phosphatidylinositol 3-kinase transformation led to non-polar spheroid morphogenesis (dysplasia), whereas MDCK spheroids expressing c-Raf or v-Src were largely polarized. We show that small interfering RNA (siRNA)-targeting PKCι decreased the size of all spheroids tested and partially reversed the aberrant polarity phenotype in H-Ras and ErbB2 spheroids only. This indicates distinct requirements for PKCι and moreover that different thresholds of PKCι activity are required for these phenotypes. By manipulating PKCι function using mutant constructs, siRNA depletion or chemical inhibition, we have demonstrated that PKCι is required for polarization of parental MDCK epithelial cysts in a 3D matrix and that there is a threshold of PKCι activity above and below which, disorganized epithelial morphogenesis results. Furthermore, treatment with a novel PKCι inhibitor, CRT0066854, was able to restore polarized morphogenesis in the dysplastic H-Ras spheroids. These results show that tightly regulated PKCι is required for normal-polarized morphogenesis in mammalian cells and that H-Ras and ErbB2 cooperate with PKCι for loss of polarization and dysplasia. The identification of a PKCι inhibitor that can restore polarized morphogenesis has implications for the treatment of Ras and ErbB2 driven malignancies.
蛋白激酶 C iota（PKCι）是一种丝氨酸/苏氨酸激酶，对于细胞极性、增殖和迁移是必需的，在癌症中通常上调或下调。PKCι 是人类癌基因，但这是否与其在细胞极性中的作用有关，以及哪些癌基因与 PKCι 协同作用尚不清楚。我们开发了一组表达候选癌基因的 Madin-Darby 犬肾（MDCK）细胞系，并证明 H-Ras、ErbB2 和磷酸肌醇 3-激酶转化导致非极性球体形态发生（发育不良），而表达 c-Raf 或 v-Src 的 MDCK 球体则主要是极化的。我们表明，靶向 PKCι 的小干扰 RNA（siRNA）减少了所有测试球体的大小，并仅部分逆转了 H-Ras 和 ErbB2 球体中异常极性表型。这表明 PKCι 的需求不同，而且这些表型需要不同的 PKCι 活性阈值。通过使用突变体构建、siRNA 耗竭或化学抑制来操纵 PKCι 功能，我们已经证明 PKCι 是基质中亲本 MDCK 上皮小囊极化所必需的，并且 PKCι 活性存在一个阈值，高于或低于该阈值，会导致组织形态发生紊乱。此外，用一种新型 PKCι 抑制剂 CRT0066854 处理能够恢复发育不良的 H-Ras 球体中的极化形态发生。这些结果表明，紧密调节的 PKCι 是哺乳动物细胞正常极化形态发生所必需的，H-Ras 和 ErbB2 与 PKCι 合作导致极化丧失和发育不良。鉴定出一种能够恢复极化形态发生的 PKCι 抑制剂，可能对治疗 Ras 和 ErbB2 驱动的恶性肿瘤具有重要意义。
