title: 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.
authors.affiliation: Departments of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Laurence
authors.initials: L
authors.lastname: Booth
authors.affiliation: Departments of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Jane L
authors.initials: JL
authors.lastname: Roberts
authors.affiliation: Departments of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Mehrad
authors.initials: M
authors.lastname: Tavallai
authors.affiliation: Departments of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Timothy
authors.initials: T
authors.lastname: Webb
authors.affiliation: Departments of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Leon
authors.affiliation: Departments of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Jesse
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: William P
authors.initials: WP
authors.lastname: McGuire
authors.affiliation: Department of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Poklepovic
authors.affiliation: Departments of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Dent
mesh_terms: D000077716:Afatinib; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D000069439:Dasatinib / Q000008:administration & dosage / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D008819:Mice, Nude; D009154:Mutation; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism*; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics / Q000378:metabolism*; D011799:Quinazolines / Q000008:administration & dosage / Q000494:pharmacology*; D034622:RNA Interference; D020893:Receptor, ErbB-3 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics; D023041:Xenograft Model Antitumor Assays; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D011799:Quinazolines; D000077716:Afatinib; C581292:ERBB3 protein, human; D019009:Proto-Oncogene Proteins c-kit; D019859:Proto-Oncogene Proteins c-met; D020893:Receptor, ErbB-3; D019061:src-Family Kinases; D000069439:Dasatinib
keywords: ERBB1 T790M L858R; ERBB3+c-MET+c-KIT; H1975; afatinib resistance; dasatinib