Here we showed that the c-Myc oncogene is responsible for overexpression of pyruvate carboxylase (PC) in highly invasive MDA-MB-231 cells. Pharmacological inhibition of c-Myc activity with 10074-G5 compound, resulted in a marked reduction of PC mRNA and protein, concomitant with reduced cell growth, migration and invasion. This growth inhibition but not migration and invasion can be partly restored by overexpression of PC, indicating that PC is a c-Myc-regulated pro-proliferating enzyme. Analysis of chromatin immunoprecipitation sequencing of c-Myc bound promoters revealed that c-Myc binds to two canonical c-Myc binding sites, locating at nucleotides -417 to -407 and -301 to -291 in the P2 promoter of human PC gene. Mutation of either c-Myc binding site in the P2 promoter-luciferase construct resulted in 50-60% decrease in luciferase activity while double mutation of c-Myc binding sites further decreased the luciferase activity in MDA-MB-231 cells. Overexpression of c-Myc in HEK293T cells that have no endogenous c-Myc resulted in 250-fold increase in luciferase activity. Mutation of either E-boxes lowered luciferase activity by 50% and 25%, respectively while double mutation of both sites abolished the c-Myc transactivation response. An electrophoretic mobility shift assay using nuclear proteins from MDA-MB-231 confirmed binding of c-Myc to both c-Myc binding sites in the P2 promoter. Bioinformatic analysis of publicly available transcriptomes from the cancer genome atlas (TCGA) dataset revealed an association between expression of c-Myc and PC in primary breast, as well as in lung and colon cancer tissues, suggesting that overexpression of PC is deregulated by c-Myc in these cancers.

中文翻译：

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c-Myc直接针对高度浸润性乳腺癌中丙酮酸羧化酶的过度表达。

在这里，我们证明了c-Myc癌基因负责丙酮酸羧化酶（PC）在高侵袭性MDA-MB-231细胞中的过表达。用10074-G5化合物对c-Myc活性进行药理学抑制，导致PC mRNA和蛋白质显着减少，同时细胞生长，迁移和侵袭减少。PC的过表达可以部分恢复这种生长抑制，但不能迁移和入侵，这表明PC是c-Myc调节的促增殖酶。c-Myc结合的启动子的染色质免疫沉淀测序分析表明，c-Myc与两个典型的c-Myc结合位点结合，位于人PC基因P2启动子的核苷酸-417至-407和-301至-291。P2启动子-荧光素酶构建体中任一c-Myc结合位点的突变导致荧光素酶活性降低50-60％，而c-Myc结合位点的双重突变进一步降低了MDA-MB-231细胞中的荧光素酶活性。没有内源性c-Myc的HEK293T细胞中c-Myc的过度表达导致萤光素酶活性增加了250倍。两个E-box的突变分别将荧光素酶活性降低50％和25％，而两个位点的双重突变均消除了c-Myc反式激活反应。使用来自MDA-MB-231的核蛋白进行的电泳迁移率变动分析证实了c-Myc与P2启动子中的两个c-Myc结合位点结合。