Wang HongBin, Gutierrez-Uzquiza Alvaro, Garg Rachana, Barrio-Real Laura, Abera Mahlet B, Lopez-Haber Cynthia, Rosemblit Cinthia, Lu Huaisheng, Abba Martin, Kazanietz Marcelo G
From the Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and.
the Centro de Investigaciones Inmunológicas Básicas y Aplicadas, Universidad Nacional de La Plata, CP1900 La Plata, Argentina.
J Biol Chem. 2014 Jul 11;289(28):19823-38. doi: 10.1074/jbc.M114.548446. Epub 2014 May 13.
Overexpression of PKCϵ, a kinase associated with tumor aggressiveness and widely implicated in malignant transformation and metastasis, is a hallmark of multiple cancers, including mammary, prostate, and lung cancer. To characterize the mechanisms that control PKCϵ expression and its up-regulation in cancer, we cloned an ∼ 1.6-kb promoter segment of the human PKCϵ gene (PRKCE) that displays elevated transcriptional activity in cancer cells. A comprehensive deletional analysis established two regions rich in Sp1 and STAT1 sites located between -777 and -105 bp (region A) and -921 and -796 bp (region B), respectively, as responsible for the high transcriptional activity observed in cancer cells. A more detailed mutagenesis analysis followed by EMSA and ChIP identified Sp1 sites in positions -668/-659 and -269/-247 as well as STAT1 sites in positions -880/-869 and -793/-782 as the elements responsible for elevated promoter activity in breast cancer cells relative to normal mammary epithelial cells. RNAi silencing of Sp1 and STAT1 in breast cancer cells reduced PKCϵ mRNA and protein expression, as well as PRKCE promoter activity. Moreover, a strong correlation was found between PKCϵ and phospho-Ser-727 (active) STAT1 levels in breast cancer cells. Our results may have significant implications for the development of approaches to target PKCϵ and its effectors in cancer therapeutics.
蛋白激酶Cε（PKCε）是一种与肿瘤侵袭性相关的激酶，广泛参与恶性转化和转移过程，在包括乳腺癌、前列腺癌和肺癌在内的多种癌症中均有高表达。为了阐明调控PKCε表达及其在癌症中上调的机制，我们克隆了人PKCε基因（PRKCE）约1.6 kb的启动子片段，该片段在癌细胞中表现出较高的转录活性。全面的缺失分析确定了两个富含Sp1和STAT1位点的区域，分别位于-777至-105 bp（区域A）和-921至-796 bp（区域B），它们是癌细胞中观察到的高转录活性的原因。随后进行的更详细的诱变分析、电泳迁移率变动分析（EMSA）和染色质免疫沉淀分析（ChIP）确定，相对于正常乳腺上皮细胞，乳腺癌细胞中-668/-659和-269/-247位置的Sp1位点以及-880/-869和-793/-782位置的STAT1位点是导致启动子活性升高的元件。在乳腺癌细胞中对Sp1和STAT1进行RNA干扰沉默可降低PKCε的mRNA和蛋白表达以及PRKCE启动子活性。此外，在乳腺癌细胞中发现PKCε与磷酸化丝氨酸727（活性）STAT1水平之间存在强相关性。我们的结果可能对开发针对PKCε及其效应器的癌症治疗方法具有重要意义。
