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Targeting CDK4 overcomes EMT-mediated tumor heterogeneity and therapeutic resistance in KRAS-mutant lung cancer
Aparna Padhye, … , Christopher A. Bristow, Don L. Gibbons
Aparna Padhye, … , Christopher A. Bristow, Don L. Gibbons
Published July 26, 2021
Citation Information: JCI Insight. 2021;6(17):e148392. https://doi.org/10.1172/jci.insight.148392.
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Research Article Oncology

Targeting CDK4 overcomes EMT-mediated tumor heterogeneity and therapeutic resistance in KRAS-mutant lung cancer

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Abstract

Lack of sustained response to therapeutic agents in patients with KRAS-mutant lung cancer poses a major challenge and arises partly due to intratumor heterogeneity that defines phenotypically distinct tumor subpopulations. To attain better therapeutic outcomes, it is important to understand the differential therapeutic sensitivities of tumor cell subsets. Epithelial-mesenchymal transition is a biological phenomenon that can alter the state of cells along a phenotypic spectrum and cause transcriptional rewiring to produce distinct tumor cell subpopulations. We utilized functional shRNA screens, in in vitro and in vivo models, to identify and validate an increased dependence of mesenchymal tumor cells on cyclin-dependent kinase 4 (CDK4) for survival, as well as a mechanism of resistance to MEK inhibitors. High zinc finger E-box binding homeobox 1 levels in mesenchymal tumor cells repressed p21, leading to perturbed CDK4 pathway activity. Increased dependence on CDK4 rendered mesenchymal cancer cells particularly vulnerable to selective CDK4 inhibitors. Coadministration of CDK4 and MEK inhibitors in heterogeneous tumors effectively targeted different tumor subpopulations, subverting the resistance to either single-agent treatment.

Authors

Aparna Padhye, Jessica M. Konen, B. Leticia Rodriguez, Jared J. Fradette, Joshua K. Ochieng, Lixia Diao, Jing Wang, Wei Lu, Luisa S. Solis, Harsh Batra, Maria G. Raso, Michael D. Peoples, Rosalba Minelli, Alessandro Carugo, Christopher A. Bristow, Don L. Gibbons

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Figure 3

ZEB1 regulates p21 expression and causes differential CDK4 pathway activation.

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ZEB1 regulates p21 expression and causes differential CDK4 pathway activ...
(A) Relative expression of Cdkn1a mRNA in a panel of murine lung cancer cells and 393P vehicle and AZDR cell lines. (B) Cluster plot analysis of correlation between CDKN1A mRNA and EMT score in 29 KRAS-mutant human lung adenocarcinoma cell lines. (C) IHC on tumors derived from 393P, 344SQ, and 393P-AZDR cells. Scale bar: 50 μm. (D) Cluster plot analysis of correlation between ZEB1 and p21 H-scores in NSCLC specimens. (E and F) Relative expression of Cdkn1a mRNA and p21 protein levels upon induction of EMT or MET. ZEB1 induction for 48 hours with 2 μg/mL of doxycycline in murine and human epithelial lung cancer cell lines. miR-200 induction for 48 hours with 2 μg/mL of doxycycline in murine and human mesenchymal lung cancer cell lines. (G) Luciferase reporter assay to determine relative luciferase activity of CDKN1A promoter reporter construct transfected into epithelial H358 and H441 cells with induced ZEB1 expression or mesenchymal H1299 with induced miR-200 expression. Relative luciferin signal was normalized to promoter-less vector control signal. (H) Fold enrichment by qPCR analysis of CDKN1A promoter containing ZEB1 binding site after endogenous ZEB1 ChIP in H441 cells with inducible ZEB1 expression or H1299 cells with inducible miR-200 expression, using ZEB1 antibody or IgG control antibody. Data are presented as mean ± SD, and 1-way ANOVA was used for statistical analysis in all the panels. ****P < 0.0001; ***P < 0.005; **P < 0.001; *P < 0.05.

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