All-trans-retinoic acid activates the pro-invasive Src-YAP-Interleukin 6 axis in triple-negative MDA-MB-231 breast cancer cells while cerivastatin reverses this action

All-trans-retinoic acid (RA), the active metabolite of vitamin A, can reduce the malignant phenotype in some types of cancer and paradoxically also can promote cancer growth and invasion in others. For instance, it has been reported that RA induces tumor suppression in tumor xenografts of MDA-MB-468 breast cancer cells while increasing tumor growth and metastases in xenografts of MDA-MB-231 breast cancer cells. The signaling pathways involved in the pro-invasive action of retinoic acid remain mostly unknown. We show here that RA activates the pro-invasive axis Src-YAP-Interleukin 6 (Src-YAP-IL6) in triple negative MDA-MB-231 breast cancer cells, yielding to increased invasion of these cells. On the contrary, RA inhibits the Src-YAP-IL6 axis of triple-negative MDA-MB-468 cells, which results in decreased invasion phenotype. In both types of cells, inhibition of the Src-YAP-IL6 axis by the Src inhibitor PP2 drastically reduces migration and invasion. Src inhibition also downregulates the expression of a pro-invasive isoform of VEGFR1 in MDA-MB-231 breast cancer cells. Furthermore, interference of YAP nuclear translocation using the statin cerivastatin reverses the upregulation of Interleukin 6 (IL-6) and the pro-invasive effect of RA on MDA-MB-231 breast cancer cells and also decreases invasion and viability of MDA-MB-468 breast cancer cells. These results altogether suggest that RA induces pro-invasive or anti-invasive actions in two triple-negative breast cancer cell lines due to its ability to activate or inhibit the Src-YAP-IL6 axis in different cancer cells. The pro-invasive effect of RA can be reversed by the statin cerivastatin.

Triple-negative breast cancers (TNBC) represent 10-17% of all breast cancers and are associated with increased risk of metastasis 1 . Effective treatment for metastatic TNBC is not yet available 2,3 . All-trans-retinoic acid (RA), the active metabolite of vitamin A, which is considered an anti-cancer agent, also can promote cancer growth and invasion [4][5][6][7] . It has been reported that in tumor xenografts of MDA-MB-231 breast cancer cells RA increases growth and metastasis 7 . Also, in vivo experiments show that an RA-enriched diet promotes tumor growth and invasion of T47D403 breast cancer cells and in vitro treatment with supraphysiological doses of exogenous RA (10 −6 M) significantly enhances T47D403 invasion 4 . However, RA acts as a tumor-suppressor in xenografts of MDA-MB-468 breast cancer cells 7 .
The signaling pathways involved in the pro-invasive action of retinoic acid in MDA-MB-231 cells have not been identified. The Src-YAP-IL6 axis controls invasion, metastasis, resistance to therapy, and stemness of MDA-MB-231 breast cancer cells 8,9 . An autoregulatory Src-YAP-IL6-Src loop also operates in colon cancer 10,11 . IL-6 is the first universal transcriptional target of YAP involved in promoting stemness conserved from flies RA has been previously shown to be tumor suppressive in xenografts of MDA-MB-468 breast cancer cells 7 . Thus, we tested the effect of RA on the Src-YAP-IL6 axis in these cells. In MDA-MB-468 breast cancer cells, the presence of exogenous RA (5 μM) for 48 h decreased nuclear Src activity, nuclear PY-YAP and downregulated IL-6 ( Fig. 1B). The observed opposite effects of RA in xenografts of MDA-MB-231 and MDA-MB-468 breast cancer cells 7 could be  ascribed to the different actions of RA on the Src-YAP-IL6 axis we have observed in these cell lines. Src-family kinases are found in the plasma membrane and the nuclear compartment 15,16 . RA treatment of MDA-MB-231 breast cancer cells increases phosphorylation of both membrane and nuclear Src and upregulates Src expression in the cell membrane, cytosol and nuclear compartment ( Fig. 2A).
Src activation required cell culture with μM concentrations of RA for 48 h (Fig. 2B). No activation was detected for incubation periods of 90 min or 24 h (Fig. 2C). The long incubation times and the μM concentrations of RA required to observe the effect suggest that it is mediated by the expression of RA-dependent genes that are repressed at physiological levels of RA in breast cancer cells but are transcriptionally responsive at supraphysiological concentrations, as previously reported 4 .
Nuclear YAP phosphorylation in MDA-MB-231 breast cancer cells was dependent on Src activity. Until recently, activation of YAP was believed to solely depend on the inhibition of the Hippo signaling pathway that retains YAP in the cytoplasm 17 . To assess if YAP activation in MDA-MB-231 breast cancer cells depends on Src activity as observed in other cancer cells 10,11,18 , we used Src inhibition by PP2, Src interference by siRNA and transfection of Src into MDA-MB-231 breast cancer cells. Src inhibition by PP2 and Src interference decreased YAP activity and downregulated IL-6 expression, while Src transfection activated YAP and upregulated IL-6 ( Fig. 2D).

Migration, invasion and cell viability of MDA-MB-231 and MDA-MB-468 breast cancer cells decreased after inhibition of the Src-YAP-IL6 axis by Src inhibitor PP2. Inhibition of the
Src-YAP-IL6 axis by Src inhibitor PP2 reduced cell viability, migration, and invasion in both MDA-MB-231 and MDA-MB-468 breast cancer cells ( Fig. 3A-D). Also, Src inhibition by PP2 induced downregulation, at transcription and protein levels, of an intracellular isoform of VEGFR1 that initiates transcription in intron 21 (i 21 VEGFR1) (Fig. 3E). In a previous study, we have shown that silencing of i 21 VEGFR1 decreases migration and invasiveness of MDA-MB-231 cells, while transfection of i 21 VEGFR1 increases migration and invasiveness of these cells 19 . No isoforms of VEGFR1, including i 21 VEGFR1, are expressed in MDA-MB-468 breast cancer cells (results not shown). Inhibition of Src by PP2 also decreased the expression of the full-length receptor (VEGFR1), the soluble extracellular isoforms (sVEGFR1) and the truncated intracellular isoforms of VEGFR1 in endothelial cells, both at the transcription and protein levels (Fig. 3E). It has been proposed that VEGFR1 mediates the VEGF-triggered migration of endothelial cells during angiogenesis 20 .

Discussion
We report here that treatment of triple-negative breast cancer cells with RA in vitro activated the pro-invasive Other pro-invasive actions of RA have been reported using in vivo experiments with an RA-enriched diet that promoted tumor growth and invasion of T47D403 breast cancer cells, and in vitro treatment with supraphysiological doses of exogenous RA (10 −6 M) that significantly promoted T47D403 breast cancer cell invasion 4 . Promotion of migration and invasion was also reported in neuroblastoma cells treated with retinoic acid 21 . An association of YAP activity and RA signaling with an increase in migration also has been observed in human neural crest cells 22 .
Our observation of pro-invasive actions of RA in MDA-MB-231 breast cancer cells is in apparent contradiction with our previous finding that RA downregulated putative pro-invasive molecules such as i21VEGFR-1 and Notch-3, and upregulated putative anti-invasive molecules as miR-200c 19,23,24 . Despite these actions, we report here that the effect of exogenous RA activating the Src-YAP-IL6 axis in MDA-MB-231 breast cancer cells and therefore increasing their invasion overrides putative anti-invasive mechanisms produced by RA in these cells. Activation of Src should be relevant for activation of YAP and upregulation of IL-6 in MDA-MB-231 breast cancer cells since inhibition of Src by PP2 or silencing Src expression in these cells decreases PY-YAP and downregulates IL-6 expression (Fig. 2D). On the contrary, transfection of Src increases nuclear PY-YAP and IL-6 expression (Fig. 2D).
The mechanism of Src activation induced by RA is not known at present. Mechanisms independent of transcription have been reported 25   breast cancer cells augmented cell migration in vitro and metastasis, while its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo 26 . MUC4 is an attracting candidate for Src activation because cell knockdown of MUC4 in pancreatic carcinoma decreased Src tyrosine phosphorylation significantly 27 .
Our results indicate that Src inhibition by PP2 inactivates the Src-YAP-IL6 axis in triple-negative MDA-MB-231 and MDA-MB-468 breast cancer cells and suppresses cell migration, invasion, and viability of these cells (Fig. 3A-D). Also, we report that Src inhibition downregulates pro-invasive VEGFR1 molecules 19,20,23,24 in MDA-MB-231 breast cancer cells and endothelial cells (Fig. 3E). Src inhibitors have been suggested as a therapeutic approach for triple-negative breast cancer 28 . Our observation that the statin cerivastatin can reverse the pro-invasive phenotype induced by RA in MDA-MB-231 breast cancer cells by decreasing nuclear PY-YAP and IL-6 production (Fig. 4) is in line with the action recently reported of statins inhibiting the YAP-dependent transcription of the pro-metastatic gene RHAMM in these cells [29][30][31] . Although the concentration of statins used to inhibit YAP nuclear translocation (1 μM) largely exceeds the plasma concentrations of statins used for cardiovascular disease prevention (10 to 200 nM) 32 , statins are the object of intensive studies in cancer recurrence and mortality 33,34 . Further work will show whether combined therapy using RA and statins can reverse the malignant phenotype induced by RA in specific breast cancer cells while preserving the anti-tumor effects of retinoids as differentiation enhancers.