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. 2016 Jul 12;115(2):203-11.
doi: 10.1038/bjc.2016.190. Epub 2016 Jun 23.

Efficacy of focal adhesion kinase inhibition in non-small cell lung cancer with oncogenically activated MAPK pathways

Hao Zhang  1 , Huanjie Shao  2 , Vita M Golubovskaya  3 , Hongbin Chen  1 , William Cance  3 , Alex A Adjei  1 , Grace K Dy  1
Affiliations

Efficacy of focal adhesion kinase inhibition in non-small cell lung cancer with oncogenically activated MAPK pathways

Hao Zhang et al. Br J Cancer. .

Abstract

Background: Focal adhesion kinase (FAK) is overexpressed in many types of tumours, including lung cancer. Y15, a small molecule which inhibits Y397 FAK autophosphorylation, decreases growth of human neuroblastoma, breast and pancreatic cancers. In this study, we investigated the in vitro and in vivo effects of Y15, and the underlying mechanism on non-small cell lung cancer cells.

Methods: The cytotoxic effects of Y15 targeting FAK signalling were evaluated. Gene-knockdown experiments were performed to determine the anti-cancer mechanism. Xenografts with RAS or EGFR mutations were selected for in vivo Y15 treatment.

Results: Y15 blocked autophosphorylation of FAK in a time- and dose-dependent manner. It caused dose-dependent decrease of lung cancer cell viability and clonogenicity. Y15 inhibited tumour growth of RAS-mutant (A549 with KRAS mutation and H1299 with NRAS mutation), as well as epidermal growth factor receptor (EGFR) mutant (H1650 and H1975) lung cancer xenografts. JNK activation is a mechanism underlying Y15-induced Bcl-2 and Mcl-1 downregulation. Moreover, knockdown of Bcl-2 or Bcl-xL potentiated the effects of Y15. The combination of various inhibitors of the Bcl-2 family of proteins with FAK inhibitors demonstrated synergy in multiple lung cancer cell lines in vitro.

Conclusions: FAK inhibition demonstrated efficacy both in vitro and in vivo in lung cancers with either oncogenic RAS or EGFR mutations. In addition, FAK inhibition in combination with inhibitors of Bcl-2 family of anti-apoptotic proteins has synergistic activity in these MAPK-activated non-small cell lung cancer cell line models.

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Figures

Figure 1
Figure 1
Y15 decreased viability and clonogenicity of lung cancer cell lines in a dose-dependent manner.(A) Expression of Y397-pFAK and FAK in lung cancer cell lines. Western blotting with Y397-pFAK and FAK was performed on different lung cancer cell lines. β-Actin was used as a loading control. Y397-pFAK and FAK levels are variable across different cell lines. (B) MTS assay. RAS-mutant (H1299, H727, H358, A549) or EGFR-mutant (H1650) cell lines were treated with Y15 or control FAK inhibitor C4 (not targeting Y397-pFAK) for 72 h. Y15 increased cell death in all cell lines, whereas control C4 did not. (C) Different lung cancer cells were treated with increasing doses of Y15 for 2 weeks and clonogenicity assay was performed. Y15 significantly decreased clonogenicity in a dose-dependent manner.
Figure 2
Figure 2
Y15 decreased Ras-mutant and EGFR-mutant lung cancer tumour growth in vivo.(A) KRAS-mutant cell A549 were injected subcutaneously into the flank of mice (n=5), and Y15 was administrated at 100 mg kg−1 orally by gavage 5 days a week. 1 × PBS was administered orally by gavage to untreated group. Tumour volume was measured as described in the ‘Materials and Methods' section. *P<0.05, Student's t-test. (B) The same experiment was performed with NRAS-mutant cell H1299 xenograft tumour growth. Y15 was administrated at 180 mg kg−1 by gavage (based on dose–response across multiple cell lines showing that 180 mg kg−1 can consistently show difference in tumour growth inhibition compared with 100 mg kg−1 dose). (C) Y15 decreased Y397-pFAK in tumour xenograft tissues. Tumours from A549 xenograft were collected and used for immunohistochemical staining with Y397-pFAK, FAK and caspase-3. Y15-treated tumours expressed less Y397-pFAK than untreated tumours. Activation of caspase-3 was observed in Y15-treated tumours. (D, E) Same treatments performed with EGFR-mutant H1650 (D) with intrinsic resistance to EGFR TKI and H1975 cell line (E) with known EGFR T790M resistance mutation.
Figure 3
Figure 3
Y15 inhibits Y397 FAK autophosphorylation and downregulates Bcl-2 family members.(A) Y15 decreased Y397-pFAK and downstream p-Src, p-Akt targets in a time-dependent manner. H1299 lung cancer cell line was treated with 5 μM of Y15 for 0–72 h and western blotting was performed with Y397-pFAK, FAK, p-Src, Src, p-Akt, Akt and other targets. Y15 increased cleaved PARP and caspase-3 starting from 24 h. (B) Y15 performed similar effects in H727 and H1650 in a time-dependent manner. (C) Downregulation of Bcl-2 family members was independent of caspase activation. Twenty μM of broad-spectrum caspase inhibitor, z-VAD-fmk, was treated with or without 5 μM Y15 at different time points after 8–48 h in the H1299 cells. Western blotting was performed to evaluate the effect on Bcl-2 family members.
Figure 4
Figure 4
Knockdown of JNK by siRNA significantly attenuates Y15-induced cytotoxicity, and subsequently abrogates Mcl-1 and Bcl-2 downregulation induced by Y15.H1299, H1650 or H358 cells were transfected with siRNA targeting JNK and were treated with Y15 for 48 h, after which whole-cell lysates were prepared and subjected to western blot analysis (A). The extent of apoptosis was determined as described in the ‘Materials and Methods' section (B). Twenty-four hours after siRNA transfection, cells were treated with Y15 (2.5 μM for H1299, and 5 μM for H358 and H1650) for 24 h. Then cells were collected and live cells were quantified with trypan blue exclusion methods. Results of Y15-treated cells were presented as % relative to values of vehicle-treated cells (defined as 100%). Data represents means±s.d. of three independent experiments (*P<0.05, compared with scrambled siRNA control).
Figure 5
Figure 5
Abrogation of anti-apoptotic Bcl-2 family members synergizes the antitumour efficacy of Y15 against lung cancer cells in vitro.(A) H1299 cells with empty vector and their respective shBcl-2, shBcl-xL or shAkt1 clones were treated with Y15 for 72 h, trypan blue assay was performed to evaluate cell viability. (B) Western blots showing differences in the expression of Bcl-2, Bcl-xL and Akt1 in control H1299 and respective shRNA clone cells, with or without Y15. (C) Western blot showing the effect on downstream signalling proteins with the combination of Y15 at 5 μM and the Bcl-2/Bcl-xL inhibitor ABT263 at 2.5 μM in H1299 cells after 24 h treatment. The combination of Y15 and ABT263 decreased Y397-pFAK, p-Src and p-Akt more than each agent alone. (D) ABT263 potentiates Y15-mediated apoptosis. The cell growth inhibition was determined as described in the ‘Materials and Methods' section. Data represent means±s.d. of three independent experiments. (E) Median dose–effect analysis was performed to calculate CI using CalcuSyn software (Biosoft) to determine interactions of Y15 combined with ABT263.
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