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    Biochemical Pharmacology
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    Acyl-CoA synthetase-4 is implicated in drug resistance in breast cancer cell T lines involving the regulation of energy-dependent transporter expression
    Ulises Daniel Orlando1, Ana Fernanda Castillo1, Mayra Agustina Ríos Medrano, Angela Rosaria Solano, Paula Mariana Maloberti, Ernesto Jorge Podesta
    Biomedical Research Institute, INBIOMED (UBA, CONICET), Department of Biochemistry, School of Medicine, University of Buenos Aires, CABA C1121ABG, Argentina
    Breast cancer
    Chemotherapeutic drug resistance ACSL4
    mTOR pathway
    ABC transporter 
    Acyl-CoA synthetase-4 (ACSL4) is an enzyme implicated in estrogen receptor α (ERα) negative regulation and hormone therapy resistance in breast cancer. In addition, ACSL4 has been associated to certain types of hormone resistance in prostate cancer.
    Chemotherapeutic treatment of disseminated breast cancer is usually faced with therapy resistance associated to ATP-binding cassette (ABC) transporter expression, which detect and eject anti-cancer drugs from cells. In this context, the aim of the present work was to study the role of ACSL4 in anti-cancer drug resistance 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 MCF-7 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-re-sponsive drug resistance ABC transporter genes (RNA-Seq), transporter mRNA expression, protein levels and signaling pathway participation (real-time PCR and Western blot).
    Higher survival rates upon chemotherapeutic treatment were obtained in MCF-7 Tet-Off/ACSL4 and MDA-MB-231 mock cells, an effect counteracted by doxycycline- or shRNA-induced ACSL4 inhibition, respectively. A synergic effect of ACSL4 inhibitor triacsin C and chemotherapeutic drugs was observed on the inhibition of MDA-MB-231 wild type cell proliferation. MCF-7 Tet-Off/ACSL4 Methoctramine showed greater doxorubicin, Hoechst 33342 and calcein AM efflux. In contrast, MDA-MB-231 shRNA ACSL4 cells evidenced inhibition of chemotherapeutic drug efflux. ABCG2, ABCC4, and ABCC8 were identified as ACSL4-responsive drug resistance genes whose expression was increased in MCF-7 Tet-Off/ACSL4 cells but inhibited in MDA-MB-231 shRNA ACSL4 cells. Further cell survival assays in the presence of Ko 143 and Ceefourin 1, inhibitors of ABCG2 and ABCC4, respectively, upon chemotherapeutic treatment showed greater participation of ABCG2 in anti-cancer drug resistance in cells overexpressing ACSL4. In addition, ACSL4 inhibition and chemotherapeutic treatment combined with rapa-mycin-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 in-volved in anticancer drug resistance through the mTOR pathway to restore drug sensitivity in tumors with poor prognosis for disease-free and overall survival.
    1. Introduction arachidonic acid metabolism [1–5]. Regarding physiological processes,
    ACSL4 participates in the regulation of steroidogenesis in adrenal and Acyl-CoA synthetase-4 (ACSL4) is an enzyme taking part in Leydig cells [6–8], the modulation of prostaglandin E2 (PGE2) release
    Abbreviations: ACSL4, acyl-CoA synthetase 4; MBC, metastatic breast cancer; ER, estrogen receptor; Tet, tetracycline; ABC, ATP-binding cassette; shRNA, short hairpin RNA; BrdU, 5-bromo-2′-deoxyuridine; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide; qRT-PCR, quantitative reverse transcription-PCR; ERBB2, Erb-B2 receptor tyrosine kinase 2; PGE2, prostaglandin E2; mTORC1/2, mammalian target of rapamycin (mTOR) complexes 1/2; p70S6K, ribosomal protein S6 kinase 70 kDa polypeptide 1; Rictor, rapamycin-insensitive companion of mTOR; GSK3 α/β, glycogen synthase kinase α/β Corresponding author at: Department of Biochemistry, School of Medicine, University of Buenos Aires, Paraguay 2155 5th floor, CABA C1121ABG, Argentina.
    E-mail address: (E.J. Podesta). 1 These authors have contributed equally to this work.
    by human arterial smooth muscle cells [9], the regulation of axonal transport of synaptic vesicles in Drosophila melanogaster [10], ferrop-tosis-mediated production of 5-hydroxyeicosatetraenoic acid [11] and zebrafish embryogenesis [12].
    In pathological processes, the ACSL4 gene has been found to be deleted in a family with Alport syndrome, elliptocytosis, and mental retardation [13]. ACSL4 has been linked to ferroptosis-associated dis-ease [14] and adipocyte dysfunction and obesity [15]. ACSL4 associa-tion with cancer has been inferred from abnormal expression in colon, breast, prostate and hepatocellular carcinoma., and its high expression has been found to correlate with triple-negative breast cancers [16–19] and tumor cell aggressiveness in different types of cancer [19–22], particularly metastatic breast cancer (MBC) [18–21,23].