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CDK6-PI3K signaling axis is an efficient target for attenuating ABCB1/P-gp mediated multi-drug resistance (MDR) in
Lei Zhang1,2,3, Yidong Li4, Chaohua Hu5
1State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, 350002, China. zhangl@fjirsm.ac.cn.
Background:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Insights
The CDK6-PI3K signaling axis reverses multidrug resistance (MDR) in cancer by downregulating ABCB1 expression. Targeting this axis enhances chemotherapy sensitivity and inhibits tumor growth and metastasis.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- Multidrug resistance (MDR) mediated by ATP binding cassette subfamily B member 1 (ABCB1/P-gp) is a significant challenge in cancer chemotherapy.
- The precise regulatory mechanisms underlying ABCB1-mediated MDR remain largely unelucidated.
Purpose of the Study:
- To investigate the regulatory role of the CDK6-PI3K signaling axis in ABCB1-mediated MDR in human cancer cells.
- To explore the potential of targeting this axis for overcoming chemotherapy resistance.
Main Methods:
- Utilized CRISPR/Cas9 gene editing to knockout CDK6 or CDK4 in KB-C2 cells.
- Employed Western blot, RT-PCR, and transcriptome analysis to assess gene expression and signaling pathways.
- Conducted flow cytometry for cell apoptosis and cell cycle analysis.
- Performed in vivo studies to evaluate tumor sensitivity to doxorubicin, growth, and metastasis.
Main Results:
- CDK6 deficiency led to significant downregulation of ABCB1 expression and reversal of MDR.
- Transcriptomic analysis revealed alterations in PI3K, KRAS, and MAPK signaling pathways upon CDK6 knockout.
- The CDK6-PI3K axis demonstrated synergistic regulation of ABCB1 expression, with reciprocal regulation observed between PI3K and CDK6.
- Alternative splicing of ABCB1 mRNA was identified as a mechanism influenced by CDK6, CDK4, or PI3K levels.
- In vivo, loss of CDK6 enhanced doxorubicin sensitivity, inhibited tumor growth and metastasis, and improved survival in mice.
Conclusions:
- The CDK6-PI3K axis represents a novel therapeutic target for reversing ABCB1-mediated MDR in cancers.
- Inhibition of cancer cell proliferation pathways is associated with CDK6 deficiency.
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