Multidrug resistance (MDR) mediated by ATP binding cassette subfamily B member 1 (ABCB1/P-gp) is a major cause of cancer chemotherapy failure, but the regulation mechanisms are largely unknown. Based on single gene knockout, we studied the regulation of CDK6-PI3K axis on ABCB1-mediated MDR in human cancer cells. CRISPR/Cas9 technique was performed in KB-C2 cells to knockout cdk6 or cdk4 gene. Western blot, RT-PCR and transcriptome analysis were performed to investigate target gene deletion and expression of critical signaling factors. The effect of cdk4 or cdk6 deficiency on cell apoptosis and the cell cycle was analyzed using flow cytometry. In vivo studies were performed to study the sensitivity of KB-C2 tumors to doxorubicin, tumor growth and metastasis. Deficiency of cdk6 led to remarkable downregulation of ABCB1 expression and reversal of ABCB1-mediated MDR. Transcriptomic analysis revealed that CDK6 knockout regulated a series of signaling factors, among them, PI3K 110α and 110β, KRAS and MAPK10 were downregulated, and FOS-promoting cell autophagy and CXCL1-regulating multiple factors were upregulated. Notably, PI3K 110α/110β deficiency in-return downregulated CDK6 and the CDK6-PI3K axis synergizes in regulating ABCB1 expression, which strengthened the regulation of ABCB1 over single regulation by either CDK6 or PI3K 110α/110β. High frequency of alternative splicing (AS) of premature ABCB1 mRNA induced by CDK6, CDK4 or PI3K 110α/110β level change was confirmed to alter the ABCB1 level, among them 10 common skipped exon (SE) events were found. In vivo experiments demonstrated that loss of cdk6 remarkably increased the sensitivity of KB-C2 tumors to doxorubicin by increasing drug accumulation of the tumors, resulting in remarkable inhibition of tumor growth and metastasis, as well as KB-C2 survival in the nude mice. CDK6-PI3K as a new target signaling axis to reverse ABCB1-mediated MDR is reported for the first time in cancers. Pathways leading to inhibition of cancer cell proliferation were revealed to be accompanied by CDK6 deficiency.

中文翻译：

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CDK6-PI3K 信号轴是减弱癌细胞中 ABCB1/P-gp 介导的多药耐药 (MDR) 的有效靶点

由 ATP 结合盒亚家族 B 成员 1 (ABCB1/P-gp) 介导的多药耐药 (MDR) 是癌症化疗失败的主要原因，但其调节机制在很大程度上是未知的。基于单基因敲除，我们研究了CDK6-PI3K轴对ABCB1介导的人癌细胞MDR的调控。在 KB-C2 细胞中进行 CRISPR/Cas9 技术以敲除 cdk6 或 cdk4 基因。进行蛋白质印迹、RT-PCR 和转录组分析以研究靶基因缺失和关键信号因子的表达。使用流式细胞仪分析cdk4或cdk6缺乏对细胞凋亡和细胞周期的影响。进行体内研究以研究 KB-C2 肿瘤对多柔比星、肿瘤生长和转移的敏感性。cdk6 的缺乏导致 ABCB1 表达的显着下调和 ABCB1 介导的 MDR 的逆转。转录组学分析显示，CDK6敲除调节了一系列信号因子，其中PI3K 110α和110β、KRAS和MAPK10下调，FOS促进细胞自噬和CXCL1调节多因子上调。值得注意的是，PI3K 110α/110β 缺乏反过来下调 CDK6 和 CDK6-PI3K 轴协同调节 ABCB1 表达，这加强了 ABCB1 对 CDK6 或 PI3K 110α/110β 单一调节的调节。CDK6、CDK4或PI3K 110α/110β水平变化诱导的早产ABCB1 mRNA的高频选择性剪接（AS）被证实改变了ABCB1水平，其中发现了10个常见的跳跃外显子（SE）事件。体内实验表明，cdk6 的缺失通过增加肿瘤的药物积累显着增加了 KB-C2 肿瘤对阿霉素的敏感性，从而显着抑制了肿瘤的生长和转移，以及裸鼠中 KB-C2 的存活。CDK6-PI3K 作为逆转 ABCB1 介导的 MDR 的新靶信号轴在癌症中首次被报道。导致抑制癌细胞增殖的途径被发现伴随着CDK6缺乏。