br We have previously described the development of stably
We have previously described the development of stably transfected MCF-7 Tet-Oﬀ/ACSL4 JNJ-42153605 using the tetracycline-oﬀ system and MDA-MB-231 cells using short hairpin RNA (shRNA)-mediated gene silencing of ACSL4. The MCF-7 Tet-Oﬀ/ACSL4 cell line shows significantly higher levels of ACSL4 mRNA and protein than MCF-7 Tet-Oﬀ empty vector cells, which parallels a higher rate of cell survival . Moreover, the inhibition of ACSL4 expression by doxycycline or shRNA significantly inhibits cell growth, indicating that the system is fully functional in these cell lines . In the current work, both MCF-7 Tet-Oﬀ/ACSL4 and MDA-MB-231 mock cells oﬀered stronger resistance to che-motherapeutic drugs, which was then partially oﬀset when either ACSL4 expression or ACSL4 activity was inhibited. Furthermore, ACSL4 inhibition proved eﬀective with the three drugs used, i.e. cisplatin, doxorubicin and paclitaxel (Figs. 1 and 2). In our interpretation of these data, the inhibition of ACSL4 might force the tumor to recover che-mosensitivity signaling pathways. Thus, the blockade of ACSL4 in combination with chemotherapeutic drugs might significantly amelio-rate tumor growth.
Tumoral cells develop resistance to chemotherapeutic drugs through several mechanisms and signaling pathways. Resistance ac-quisition may involve alterations in drug transport and usually responds to the expression of one or more energy-dependent transporters which eject anti-cancer drugs from cells [28,50,51]. For instance, increased ABC transporter expression and activity have been implicated in triple-negative breast cancer tumor growth, while their inhibition has been shown to restore cell sensitivity to chemotherapeutic drugs [52–55]. In this context, the increase in drug eﬄux observed in our current assays (Fig. 3) and results obtained from transcriptome analyses hint at the participation of ABC transporters in anti-cancer drug resistance in cells overexpressing ACSL4 (Table 1).
Further assays unveiled the role of ACSL4 as a novel regulator of some energy-dependent transporters, as the overexpression of ACSL4 increased the expression of ABCC8, ABCC4 and ABCG2 (Fig. 4), while the inhibition of its expression or activity reduced protein levels (Fig. 5). The specificity of ACSL4 action on the expression of energy-dependent transporters was verified through ACSL4 expression inhibi-tion by doxycycline in the MCF-7 Tet-Oﬀ/ACSL4 model, as well as
through the inhibition of ACSL4 expression and activity in the highly aggressive MDA-MB-231 cells. These results are in agreement with our observations that ACSL4 overexpression reduces cell sensitivity to chemotherapeutic agents, while ACSL4 inhibition restores it.
Given that the ABCC8 transporter is strongly associated to sensi-tivity to treatment of diabetes mellitus [56–58] but has not been linked to anti-cancer drug eﬄux, subsequent assays focused on ABCG2 and ABCC4. ABCG2 has been implicated not only in therapeutic drug eﬄux, including cisplatin, doxorubicin and paclitaxel [59–62], but also in physiological compound eﬄux . ABCG2 is overexpressed in highly aggressive breast cancer [64,65] and is considered critically responsible for drug resistance in mammalian cells . Moreover, Zhang et al. (2011) have reported the development of a chemotherapy-resistant cell line through the overexpression of Erb-B2 receptor tyrosine kinase 2 (ERBB2 or HER2) in MCF-7 cells, with higher expression of ABCG2 . Previous results by our group provide further support, as the overexpression of ACSL4 in MCF-7 cells rendered an increase in the expression of HER2 .
In turn, ABCC4 has been linked to cisplatin eﬄux in diﬀerent types of cancer [66–68] but seems to have no eﬀects on paclitaxel or dox-orubicin treatment [69,70]. Furthermore, ABCC4 has been shown to promote PGE2 eﬄux and to contribute to triple-negative cell metastasis , which is in agreement with previous reports by our group showing ACSL4 participation in COX-2 overexpression and an increase in extracellular PGE2 levels .
Our current results in cell functionality studies are in agreement with the evidence reported above, as the inhibition of ABCG2 activity restored sensitivity to cisplatin, doxorubicin and paclitaxel in cells overexpressing ACSL4. Along the same line, ABCC4 activity inhibition succeeded in oﬀsetting ACSL4-overexpressing cell resistance to cis-platin but only partially counteracted resistance to paclitaxel or dox-orubicin (Fig. 6). These functional assays demonstrate the eﬀects of ACSL4 overexpression on ABCG2 and ABCC4 levels and their impact on cell resistance to chemotherapeutic drugs.