Epigenetic Input Dictates the Threshold of Targeting of the Integrin-Dependent Pathway in Non-small Cell Lung Cancer
Yang Zhang, Kai Cheng, Bingwei Xu, Junfeng Shi, Jun Qiang, Shujin Shi, Yuanqin Yi, Hongxia Li, Tengchuan Jin, Ruihua Guo, Yadi Wu, Zeyi Liu, Xiaowei Wei, Jian‐An Huang, Xiuwei H. Yang
Abstract
With increasing link of integrin/FAK-dependent signaling to NSCLC malignancy, we investigated therapeutic potential of targeting this adhesion receptor-mediated pathway. Our analysis of the TCGA cohort showed that a subset of pro-tumorigenic integrins, including α1β1, α2β1, α3β1, α5β1and α6β4, were frequently amplified or upregulated at genomic or mRNA level in KRAS or EGFR mutation/overexpression-enriched adenocarcinomas. These alterations appeared complimentary and correlated with poor patient survival, regardless of KRAS mutation-coupled αv integrins (p <0.0072). Since the integrin/FAK-dependent signaling is tightly coupled with normal human physiology, we sought for a synthetic lethal-type targeting with VS-6063, a chemical inhibitor of integrin-mediated FAK activity, and A549 cells, which carry KRAS mutation and EGFR overexpression. Our screening analysis revealed that JQ1 and IBET-762 -inhibitors of epigenetic reader BRD4, and LBH589 -a pan inhibitor of histone deacetylases (HDACs), exhibited a synergy with VS-6063 in terms of mitigating tumor cell viability. This epigenetic link was corroborated by strong effects of additional inhibitors and RNAi-mediated knockdown of FAK and BRD4 or its downstream effector c-Myc. Intriguingly, low doses of JQ1 (≤ 0.5µM) markedly escalated efficacy of VS-6063 across a panel of 10 NSCLC cell lines. This catalyst-like effect was in line with our pathway analysis of the TCGA cohort, where c-Myc falls downstream of KRAS and EGFR oncogenes. Mechanistically, co-inhibiting the integrin-FAK and BRD4/c-Myc axes synergistically induced apoptotic cell death and DNA damage response, and impaired stemness-associated tumorsphere formation. These effects were accompanied by a marked inhibition of Akt-and p130Cas/Src-dependent signaling, but not Erk1/2 activity. Meanwhile, JQ1 alone or in combination with VS-6063 attenuated cellcell adhesion and extracellular matrix (ECM)-dependent cell spreading, reminiscent of phenotypic changes caused by malfunctional E-cadherin or integrins. Paradoxically, this phenotypic impact also coincided with downregulation of EMT-inducting transcription factor ZEB1 and Snail. Moreover, effect of the VS-6063/JQ1 combination was nearly equivalent to that of VS-6063 plus Carboplatin or Osimertinib in terms of cell apoptosis, DNA damage response, and suppression of c-Myc. Taken together, our results suggest that the integrin/FAK and BRD4/c-Myc axes cooperatively drive NSCLC virulence, and a cotargeting may serve as a new line of therapy capable of overcoming EGFR-or RAS/MEK/Erk1/2-driven malignancy.