The afatinib resistance of in vivo generated H1975 lung cancer cell clones is mediated by SRC/ERBB3/c-KIT/c-MET compensatory survival signaling

Abstract

We generated afatinib resistant clones of H1975 lung cancer cells by transient exposure of established tumors to the drug and collected the re-grown tumors. Afatinib resistant H1975 clones did not exhibit any additional mutations in proto-oncogenes when compared to control clones. Afatinib resistant H1975 tumor clones expressed less PTEN than control clones and in afatinib resistant clones this correlated with increased basal SRC Y416, ERBB3 Y1289, AKT T308 and mTOR S2448 phosphorylation, decreased expression of ERBB1, ERBB2 and ERBB3 and increased total expression of c-MET, c-KIT and PDGFRβ. Afatinib resistant clones were selectively killed by knock down of [ERBB3 + c-MET + c-KIT] but not by the individual or doublet knock down combinations. The combination of the ERBB1/2/4 inhibitor afatinib with the SRC family inhibitor dasatinib killed afatinib resistant H1975 cells in a greater than additive fashion; other drugs used in combination with dasatinib such as sunitinib, crizotinib and amufatinib were less effective. [Afatinib + dasatinib] treatment profoundly inactivated ERBB3, AKT and mTOR in the H1975 afatinib resistant clones and increased ATG13 S318 phosphorylation. Knock down of ATG13, Beclin1 or eIF2α strong suppressed killing by [ERBB3 + c-MET + c-KIT] knock down, but were only modestly protective against [afatinib + dasatinib] lethality. Thus afatinib resistant H1975 NSCLC cells rely on ERBB1- and SRC-dependent hyper-activation of residual ERBB3 and elevated signaling, due to elevated protein expression, from wild type c-MET and c-KIT to remain alive. Inhibition of ERBB3 signaling via both blockade of SRC and ERBB1 results in tumor cell death.

RETRACTED: Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamine‐induced hepatocellular carcinoma

“…The UPR is an ER stress‐activated signaling pathway that modifies cell proliferation and survival and plays an important role in HCC development . During ER stress different transcription factors regulate the expression of ER chaperones which are often overexpressed in tumor tissues . Different studies have shown that the UPR is regulated by the circadian clock .…”

EPHA2 blockade reverses acquired resistance to afatinib induced by EPHA2‐mediated MAPK pathway activation in gastric cancer cells and avatar mice

“…Finally, we explored the potential mechanism of acquired resistance to afatinib. Previous studies revealed distinct mechanisms of acquired resistance to afatinib in lung cancer including MET amplification, IGF1R and SRC upregulation, and AKT phosphorylation . In the present study, we observed not only the elevated phosphorylation of AKT/S6 and MEK/ERK/JNK2/p38, but also the enrichment of genes involved in cell cycle pathway in the afatinib‐resistant PDX model.…”

Multi-kinase inhibitors interact with sildenafil and ERBB1/2/4 inhibitors to kill tumor cellsin vitroandin vivo

“…One of the key observations made in this manuscript and in the recent Booth et al manuscript that examines the biology of the afatinib resistant H1975 cells in greater detail is that afatinib resistant H1975 clones do not exhibit any additional “hotspot” mutations in proto-oncogenes compared to controls that would have been a priori predicted [10]. In the ADOR PDX isolate, again to our surprise, no well described proto-oncogene driving mutation was identified.…”

“…Control treated tumors were also isolated when they had a volume of ~500 mm 3 . Of significant note for clonal characterization, the isolated afatinib treated tumor clones’ cells were only growth inhibited by afatinib when cultured in vitro with daily supplementation at concentrations >> 2 μM, and as such these afatinib resistant cells were routinely passaged in a pulsatile fashion between experiments in growth media containing only 1 μM afatinib to maintain the afatinib resistant phenotype but not to promote further selective pressure on drug resistance and thus also did not cause selection of surviving clones due to non-ERBB1/2/4 off-target effects [10]. …”