Abstract LB-B02: Acquisition of stem cell-like properties accompanying with Src family kinase/Focal adhesion kinase activation contributes to acquired resistance to afatinib in lung cancer cells

Abstract

[Background] Most NSCLC patients harboring activating EGFR mutations benefit from treatment with EGFR-TKIs, but the final clinical efficacy of EGFR-TKIs varies because of development of tumor resistant to EGFR-TKIs. Multiple kinase-targeted 2nd generation TKIs such as afatinib have been developed to overcome drug resistance to the 1st generation TKIs. Afatinib is an irreversible multitargeted TKI targeting EGFR including T790M, HER2 and HER4. To develop further personalized therapeutics and drug resistance modifiers, we should understand the molecular based mechanism of drug resistance to 2nd as well as 3rd generation TKIs. In our present study, we present a novel finding that acquisition of cancer stem-like cells properties accompanying with activation of residual Src family kinase (SFK)/focal adhesion kinase (FAK) is responsible for the survival of afatinib-resistant lung cancer cells when expression of targeted EGFR, HER2 and HER4 is abraded.

[Materials and methods] We have established afatinib-resistant subclones from human lung cancer cell lines, HCC827, harboring activating EGFR mutation after stepwise exposure to afatinib. The afatinib-resistant clones do not harbor T790M, K-ras mutation or PTEN loss. We have characterized biochemical properties of drug-resistant subclones as compared with their drug-sensitive counterparts by Western blot and RT-PCR. ALDEFLUOR kit was used to isolate cell populations with the stem cell specific ALDH enzymatic activity.

[Results] We characterized two independent afatinib-resistant subclones, BR1-8 and BR2-3. [1] Afatinib-resistant subclones showed markedly decreased expression of EGFR, HER2, HER3, c-Met and PDGFRβ as compared with their parental cell lines, HCC827; [2] Akt phosphorylation in afatinib-resistant subclones was not suppressed by afatinib; [3] SFK and FAK were activated in resistant subclones, and combination of afatinib with c-Src siRNA or with dasatinib moderately suppressed cell growth and AKT phosphorylation; [4] Combination of afatinib with siFAK moderately blocked cell growth of resistant subclones. [5] Cancer stem like cell marker, ALDH1, positive cell population increased in afatinib-resistant sublines compared with HCC827 cell lines.

[Conclusions] Together, our present study presents that increase of cancer stem like cells and SFK/FAK activation could be a mechanism responsible for acquired resistance to afatinib. We would further discuss the association of ALDH1 expression and SFK/FAK activation with afatinib resistance in lung cancer.

Citation Format: Yuichi Murakami, Kahori Sonoda, Koichi Azuma, Kosuke Watari, Michihiko Kuwano, Mayumi Ono. Acquisition of stem cell-like properties accompanying with Src family kinase/Focal adhesion kinase activation contributes to acquired resistance to afatinib in lung cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B02.