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  • br In terms of the mechanisms underlying ACSL


    In terms of the mechanisms underlying ACSL4-regulated cell re-sistance to chemotherapeutic drugs, the evidence reported here sug-gests that ACSL4-dependent resistance could involve the activation of the mTOR pathway. Our current results show that mTOR inhibition and ACSL4 inhibition work in combination to increase cell sensitivity (Fig. 7). ACSL4 is known to regulate components of both mTORC1 and 2, along with its upstream regulators and substrates [20]. In addition, our previous findings revealed a significant increase in the phosphor-ylation of p70S6K on Thr389 and its substrate, ribosomal protein S6. An increase has also been observed in the phosphorylation of Rictor (ra-pamycin-insensitive companion of mTOR) on Thr1135, substrate of p70S6K and component of mTORC2 complex [20].
    Moreover, the current report shows that p70S6K inhibition pro-duced a marked decrease in ABCG2 protein expression in Fulvestrant (ICI 182,780) over-expressing ACSL4 (Fig. 7), which is in line with results previously ob-tained in prostate cancer studies showing increased sensitivity to
    chemotherapy upon p70S6K inhibition [72,73]. Furthermore, mTOR has been shown to act on ABCG2 cellular localization [47]. Another key ACSL4-regulated component of mTOR signaling, glycogen synthase kinase α/β (GSK3 α/β) activity inhibition increases mTOR activity [20], whereas GSK3 β inhibition increases hormonal and chemother-apeutic resistance in breast cancer cells, an effect counteracted by ra-pamycin [74].
    Altogether, these results indicate that ACSL4 participation in in-creasing multiple drug resistance may involve the activation of the 
    mTOR pathway increasing ABCG2-expression and activity, and that the role of ACSL4 activity inhibition in reducing chemoresistance and in-hibiting cell proliferation and survival may involve synergistic actions. Therefore, combination treatment with submaximal concentrations of the chemotherapeutic agents and the inhibition of ACSL4 activity ap-pears to be effective in inhibiting cell proliferation and survival in cells expressing high levels of energy-dependent transporters.
    For these reasons, ACSL4 inhibition in combination with che-motherapy may be thought to provide an effective treatment option for
    Fig. 7. Signaling pathway mediating ACSL4 effects on cell resistance to chemotherapeutic agents in MCF-7 Tet-Off cells. A) MCF-7 Tet-Off empty vector and MCF-7 Tet-Off/ACSL4 cells were treated with cisplatin (1 μM), doxorubicin (0.025 μM), paclitaxel (0.1 μM), rapamycin (5 nM) and triacsin C (0.5 µM) or in the combinations indicated. After 48 h, cell survival was evaluated through MTT assay. ***p < 0.001 MCF-7 Tet-Off/ACSL4 triple-treated cells vs. MCF-7 Tet-Off/ACSL4 cells treated with corresponding single drug or with corresponding two-agent combination. B) MCF-7 Tet-Off empty vector and MCF-7 Tet-Off/ACSL4 cells were treated with or without p70S6K inhibitor PF4708671 (10 µM, 48 h). Whole cell extracts protein levels were analyzed by Western blot using the indicated antibodies. Protein level integrated optical density was quantified and normalized to β-tubulin signal. Data represent the means ± SD of three independent experiments. ***p < 0.001 vs. untreated MCF-7 Tet-Off/ACSL4 cells.
    highly metastatic triple-negative breast cancer, which may limit the toxicity and side effects of existing therapies. In other words, blocking ACSL4 activity together with chemotherapeutic treatment may leave the tumor with extremely few options, preventing the development of cell resistance or restoring cell sensitivity to drugs in patients with poor prognosis and for whom no specific treatment guidelines are available. As a future perspective, there is hope that further elucidation of the cellular and molecular processes that allow tumor cells to develop re-sistance and the use of new agents to prevent these processes will im-prove outcome for patients with triple-negative breast cancer.
    We thank Cristina Paz for her critical reading of the manuscript and María M. Rancez for providing language help. We are especially grateful to Monica Costas, Estrella Levy and Roxana Peroni for their collaboration with different reagents and materials.
    Conflict of interest
    The authors declare no conflict of interest.
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