br shRNA ACSL cells which may indicate a
231 shRNA-ACSL4 cells, which may indicate a limited capacity of Coelenterazine overexpressing ACSL4 to retain Hoechst 33342. As it is well established that Hoechst 33342 eﬄux partly involves ABCG2 participation , these findings provide support for ABCG2 activation in these cells.
Furthermore, flow cytometry analyses were conducted on calcein AM eﬄux, an anionic fluorescent probe substrate for ATP-binding Biochemical Pharmacology 159 (2019) 52–63
cassette subfamily C (ABCC) to assess transporter activity . Results also showed a right shift in the peak of calcein AM fluorescence in-tensity in MCF-7 Tet-Oﬀ empty vector, MDA-MB-231 shRNA-ACSL4 and MDA-MB-231 mock cells treated with triacsin C as compared to MCF-7 Tet-Oﬀ/ACSL4 and untreated MDA-MB-231 mock cells (Fig. 3E and F), which suggests ABCC transporter activation in cells over-expressing ACSL4. Once again, MDA-MB-231 shRNA-ACSL4 and MDA-MB-231 mock cells treated with triacsin C rendered comparable right shifts (Fig. 3F).
3.3. ACSL4 eﬀects on mRNA and protein expression and energy-dependent transporter regulation
As cells overexpressing ACSL4 showed greater compound eﬄux, we focused on the identification of ACSL4-responsive drug resistance genes in MCF-7 Tet-Oﬀ/ACSL4 cells through a massive in-depth mRNA se-quencing approach . The genes found to be ACSL4-responsive in the transcriptome analysis included members of the ABC family which were significantly and diﬀerentially expressed in MCF-7 Tet-Oﬀ/ACSL4 compared to MCF-7 Tet-Oﬀ empty vector cells (Table 1). ABC subfamily
C members 8 (ABCC8) and 4 (ABCC4), both located in the plasma membrane, were the two molecules rendering the highest log2 fold changes between MCF-7 Tet-Oﬀ/ACSL4 and MCF-7 Tet-Oﬀ empty vector, i.e. 2.95165 and 2.23521, respectively. In addition, ABC sub-family G member 2 (ABCG2), which is located in the mitochondrion, nucleus and plasma membrane, rendered a log2 fold change of 1.89082 (Table 1).
On the basis of the results obtained in RNA-Seq, real-time PCR as-says were conducted on ABCC8, ABCC4 and ABCG2 to compare their expression in MCF-7 Tet-Oﬀ/ACSL4 and MCF-7 Tet-Oﬀ empty vector cells. Results showed gene expression tendencies comparable to those observed through RNA-Seq and further validated the correlation be-tween these genes and ACSL4 expression (Fig. 4).
We next analyzed protein expression level of these three genes by Western blot and measured molecule basal expression levels, along with the eﬀect of ACSL4 up and downregulation. Regarding MCF-7 Tet-Oﬀ cells, ABCG2, ABCC4 and ABCC8 levels were significantly higher in MCF-7 Tet-Oﬀ/ACSL4 than in MCF-7 Tet-Oﬀ empty vector or doxycy-cline-treated MCF-7 Tet-Oﬀ/ACSL4 cells (Fig. 5A). In turn, MDA-MB-231 shRNA-ACSL4 showed sharply reduced ABCG2, ABCC4 and ABCC8 protein levels as compared to MDA-MB-231 mock cells (Fig. 5B). In addition, ACSL4 endogenous activity inhibition in MDA-MB-231 wild type cells using triacsin C (1 µM) rendered ABCG2, ABCC4 and ABCC8 protein levels resembling those of MDA-MB-231 shRNA-ACSL4 (Fig. 5C). In summary, the inhibition of ACSL4 activity or expression markedly decreased the protein levels of energy-dependent transpor-ters.
To further analyze whether the impact of ACSL4 expression on cell survival is mediated by its action on ABC transporters, we studied the eﬀect of chemotherapeutic drugs on each cell line under the inhibition of ABCG2 and ABCC4 through Ko 143  and Ceefourin 1 , re-spectively, the two transporters most widely associated to cancer drug resistance . Worth highlighting, ABCG2 inhibition counteracted both MCF-7 Tet-Oﬀ/ACSL4 (Fig. 6A) and MDA-MB-231 mock (Fig. 6B) cell resistance to the three chemotherapeutic drugs in the concentra-tions used, rendering cell survival inhibition rates comparable to that of MCF-7 Tet-Oﬀ empty vector and MDA-MB-231 shRNA-ACSL4 cells, respectively. In turn, ABCC4 inhibition oﬀset MCF-7 Tet-Oﬀ/ACSL4 (Fig. 6A) and MDA-MB-231 mock (Fig. 6B) cell resistance to cisplatin, but only partially reduced resistance to doxorubicin and paclitaxel. These results suggest greater participation of ABCG2 in ACSL4-medi-ated cell resistance to therapeutic drugs.
To establish the signaling pathway mediating the impact of ACSL4 activity on cell resistance to chemotherapeutic drugs, and considering previous work by our group showing significant inhibition of MCF-7 Tet-Oﬀ/ACSL4 cell growth upon mTOR and ACSL4 inhibition , a
pharmacological approach was used in MCF-7 Tet-Oﬀ/ACSL4 cells consisting of submaximal concentrations of mTOR inhibitor rapamycin (5 nM) and ACSL4 inhibitor triacsin C (0.5 µM) in combination with chemotherapeutic agents. Interestingly, this three-agent combination proved much more eﬃcient in enhancing cell sensitivity to che-motherapeutic drugs and reducing cell survival than single-handed ACSL4 inhibition or chemotherapeutic treatment, or any two-agent combination (Fig. 7A). Worth pointing out, these assays were only conducted in MCF-7 Tet-Oﬀ/ACSL4 and empty vector cells on the basis of previous reports showing MDA-MB-231 cells to be non-responsive to rapamycin treatment .