562 Lysophosphatidic acid-induced breast cancer metastasis depends on LPA1/ZEB1/miR-21-activation pathway

Lysophosphatidic acid (LPA) is a natural bioactive lipid that promotes metastasis of many types of cancer cells. We have shown that blocking LPA receptor type 1 (LPA1) activity inhibits early stage of bone metastasis by inhibiting motility and invasion of breast cancer cells. However, the signaling pathways and gene activation involved in this process have not been well characterized. Micro-RNAs (miR) are well known master regulators of gene expression. Based on the complete miR expression profile in human MDAMB-231 breast cells stimulated by LPA, we found that miR-21 was one of the highest up-regulated miR. Using the Taqman RT-QPCR system, we found that LPA induced miR-21 expression in MDA-MB-231 cells and in their highly osteotropic sub-clone MDA-B02 cells. MiR-21 is well known to act as an oncomiR promoting metastasis in multiple cancers. Also, it is known that miR-21 expression is controlled by several transcription factors. The full transcriptomic analysis of our breast cancer cell lines showed that among those transcription factors, ZEB1, STAT3 and cFos were up regulated by LPA. Interestingly, silencing ZEB1 expression in these cells using synthetic ZEB1-siRNAs abolished LPA-induced miR-21 expression whereas silencing STAT3 or cFos had no effect. Hence, silencing ZEB1 up-regulated the expression of miR-21 target genes PDCD4, PTEN and SPRY2 in MDA-MB-231 cells stimulated by LPA. RT-QPCR analyses also showed that LPA1 was the most abundant LPA receptor in MDA-MB-231 cells (LPA1>LPA2>>LPA6=LPA7>LPA5) whereas LPA3 and LPA4 were not detectable. We found that the treatment of our breast cancer cells with Ki16425, a LPA1/LPA3 antagonist, inhibited LPA-induced ZEB1 and miR-21 expression. Silencing LPA1 expression in these cells using synthetic LPA1siRNAs also abolished LPA-induced ZEB1 and miR-21 expression. MirVana miR-21 inhibitor, silencing LPA1 or ZEB1 totally blocked in vitro LPAinduced cell migration and invasion, and in vivo tumor bone colonization. In all cases the in vitro and in vivo breast cancer functions were rescued with miR-21 mimic. All together our results demonstrate that miR-21 controls the pro-metastatic activity of LPA involving LPA1-dependent activation of ZEB1 in breast cancer cells.