Acyl-CoA synthetase-4 is implicated in drug resistance in breast cancer cell lines involving the regulation of energy-dependent transporter expression

RIOS MEDRANO, MAYRA AGUSTINA; PODESTA, ERNESTO JORGE; ORLANDO, ULISES DANIEL; SOLANO, ANGELA ROSARIA; CASTILLO, ANA FERNANDA; MALOBERTI, PAULA MARIANA

Sao Paulo

Conferencia; Second AACR International Conference: Translational Cancer Medicine, Cancer Discoveries for Clinical Application; 2018

American Association for Cancer Research. Latin American Cooperative, Oncology Group.

Chemotherapeutic treatment of disseminated breast cancer is usually faced with obstacles associated to therapy resistance. The most common reason for the acquisition of drug resistance is the expression of ATP-binding cassette (ABC) transporters which detect and eject anticancer drugs from cells. In addition, we have previously shown that acyl-CoA synthetase 4 (ACSL4) is implicated in hormone therapy resistance. In this context, the aim of the present work was to study the role of ACSL4 in cell resistance to cisplatin, doxorubicin and paclitaxel treatment and the involvement of ABC transporters in the underlying mechanisms. To this end, we used MCF-7 Tet-Off/ACSL4 and MDA-MB-231 mock cells, which overexpress ACSL4, and control line MCF7 Tet-Off empty vector, MDA-MB-231 shRNA ACSL4 and MDA-MB-231 wild type cells. Assays were conducted on cell viability (MTT), cell proliferation (BrdU), drug efflux (flow cytometry), ACSL4-responsive drug resistance ABC transporters (RNA-Seq), transporter mRNA expression, protein levels and signaling pathway participation (real-time PCR and Western blot). MTT assays rendered higher survival rates upon chemotherapeutic treatment in MCF7 Tet-Off/ACSL4 and MDA-MB-231 mock cells as compared to MCF-7 Tet-Off empty vector and MDA-MB-231 shRNA ACSL4, respectively. Doxycycline-induced ACSL4 inhibition in MCF7 Tet-Off/ACSL4 cells counteracted cell survival, rendering results comparable to MCF7 Tet-Off empty vector. BrdU incorporation experiments showed a synergic effect on the inhibition of MDA-MB-231 wild type cell proliferation upon treatment with ACSL4 inhibitor triacsin C and chemotherapeutic drugs. RNA-Seq studies identified ACSL4-responsive drug resistance genes which were significantly and differentially expressed in MCF-7 Tet-Off/ACSL4 as compared to MCF-7 Tet-Off empty vector. Real time PCR and Western blot results validated the changes observed in the expression of transporter genes ABCG2, ABCC4, and ABCC8. MCF-7 Tet-Off/ACSL4 cells showed an increase in protein expression of ABC transporters, while MDA-MB-231 shRNA ACSL4 cells showed inhibition of both protein expression and chemotherapeutic drug efflux. Also, ABCG2 and ABCC4 exhibited higher activity in cells overexpressing ACSL4. Cell survival was further analyzed in the presence of Ko 143 and Ceefourin 1, inhibitors of ABCG2 and ABCC4, respectively, upon chemotherapeutic treatment, with results showing greater participation of ABCG2 in drug anticancer drug resistance in cells overexpressing ACSL4. In addition, ACSL4 inhibition and chemotherapeutic treatment combined with rapamycin-induced mTOR inhibition synergically inhibited proliferation and reduced ABCG2 expression in cells overexpressing ACSL4. In sum, ACSL4 may be regarded as a novel therapeutic target regulating the expression of transporters involved in anticancer drug resistance through the mTOR pathway to restore drug sensitivity in tumors with poor prognosis for disease-free and overall survival.