CXCR4 expression is highly correlated with decreased breast carcinoma patient survival [14
] and the SDF-1-CXCR4 axis is a known regulator of cancer metastasis [34
]. Consistent with the role of CXCR4 in cancer cell proliferation, survival and metastasis, patient data now show that persons diagnosed with CXCR4(+) tumors have a significantly worse survival prognosis than those with CXCR4(−) tumors, independent of ER status [14
]. Studies examining CXCR4 expression in clinical breast carcinoma samples revealed CXCR4 expression in both primary invasive breast carcinomas as well as ductal carcinoma in situ
(DCIS), suggesting a role for the SDF-1-CXCR4 axis at all stages of the disease [4
]. Despite numerous studies on the SDF-1-CXCR4 axis, its role in primary tumorigenesis is not fully understood. Using the endogenously CXCR4 and ER-α-positive MDA-MB-361 breast carcinoma cell line in addition to our MCF-7-CXCR4 cell line, we have shown here that inhibition of SDF-1-CXCR4 signaling reduces both hormone-independent and estrogen-induced tumorigenesis in vivo
. Furthermore, we show the ability of SDF-1 treatment to induce phosphorylation of the ER-α at both S118 and S167, indicating ligand-independent activation, and further supporting the existence of SDF-1/CXCR4-ER-α crosstalk in the regulation of hormone-independence.
Analysis of miRNAs regulated by SDF-1 using qPCR revealed key miRNAs involved in ER-α regulation. miRNAs miR-222, miR-206, and miR-18b, miRNAs previously shown by us and others to target ER-α [48
], were aberrantly expressed following stimulation with SDF-1. Importantly, miR-222 is not only a known repressor of ER-α gene expression, but has also been implicated in establishing resistance to both tamoxifen [51
] and, more recently, fulvestrant [41
]. It has been suggested that miR-222 is involved in the transition from an ER-α (+) status to an ER-α (−) one, indicative of the progression to a more advanced phenotype [49
]. Furthermore, SDF-1 induced the expression of miRNAs commonly shown to be upregulated in ER-α (−) breast cancer profiles, including miR-222, miR-206, and miR-181d [49
]. Taken together, these data suggest that the SDF-1-CXCR4 axis mediates hormone independence and the progression to a more aggressive phenotype through mechanisms including miRNA expression changes and ER-α regulation.
While progression to hormone independence is a hallmark of advanced breast carcinomas which ultimately progress to more invasive and metastatic phenotypes [53
], and the link between SDF-1/CXCR4 expression and breast cancer metastasis has been clearly documented [3
], we demonstrate here that SDF-1 also stimulates the EMT, a key feature of metastatic cells, through decreased expression of the epithelial marker CDH1. Furthermore, inhibition of CXCR4 signaling with AMD3100 revealed inverse effects with increased CDH1 gene expression and decreased expression of CDH2, a mesenchymal cell marker. While the precise mechanism is currently unknown, we believe these data, along with our previously published work [14
], suggest SDF-1/CXCR4 regulation of EMT.
Regulation of miRNA expression might represent one possible mechanism underlying SDF-1 regulation of EMT. qPCR miRNA analyses revealed significant SDF-1-induced changes in expression of miRs associated with the regulation of cellular invasion and metastasis. Well established metastatic-inducing miRNAs miR-214, miR-222, and miR-373 were increased following treatment with SDF-1 [54
]. SDF-1 also upregulated miRNAs miR-143 and miR-142-5p, which have been reported to be increased in profiling studies of metastatic versus non-metastatic tumor samples [58
]. These data suggest that altered miRNA expression may play a role in the ability of SDF-1 to induce a more mesenchymal phenotype in MDA-MB-361 and MCF-7-CXCR4 cells.
The current study demonstrates a role for SDF-1-CXCR4 signaling in both estrogen-induced and hormone-independent tumorigenesis of the endogenously CXCR4-positive, ER-α (+) breast cancer cell line MDA-MB-361. Furthermore, SDF-1 treatment induced changes in miRNA expression, demonstrated here for the first time, consistent with hormone independence and metastasis. In addition to furthering our knowledge of the role of the SDF-1-CXCR4 axis in the progression of breast carcinoma, these data provide insight into the effects of SDF-1-CXCR4 signaling on miRNA expression, a novel alternative mechanism which may provide future therapeutic targets for the treatment of ER-α (+), CXCR4 (+), hormone-independent breast disease.