CXCR7 signaling promotes breast cancer survival in response to mesenchymal stromal stem cell-derived factors

Abstract

The interaction between cancer cells and molecular cues provided by tumor stromal cells plays a crucial role in cancer growth and progression. We have recently reported that the outcome of interaction between tumor cells and stromal cells is dependent on the gene expression signature of tumor cells. In the current study, we observed that several cancer cell lines, e.g., MCF7 breast cancer line, exhibited growth advantage when cultured in the presence of conditioned media (CM) derived from human bone marrow stromal stem cells (hBMSCs). Regarding the underlying molecular mechanism, we have identified CXCR7 as highly expressed by MCF7 cells and that it mediated the enhanced growth in response to hBMSC CM. Regarding the clinical relevance, we found an inverse correlation between the level of tumor gene expression of CXCR7 in bladder, breast, cervical, kidney, liver, lung, pancreatic, stomach, and uterine cancers, and patients' overall survival. Interestingly, significant positive correlation between CXCR7 and CXCL12 gene expression (Pearson = 0.3, p = 2.0 × 10^-16) was observed in breast cancer patients, suggesting a biological role for the CXCR7/CXCL12 genetic circuit in breast cancer biology. Our data provide insight into the molecular mechanisms by which stromal-derived microenvironmental cues mediate CXCR7 signaling and growth enhancement of breast cancer cells. Therapeutic targeting of this circuit might provide novel therapeutic opportunity for breast cancer.

Fig. 1. Effect of hBMSC-derived conditioned media on tumor growth.

Bright field (upper panels) images of FaDu, MCF7, MDA-MB-231, PC-3, HT-29, and MDA-MB-468 tumor cell lines cultured in MCM or FM for 2, 4, or 6 days (10×). Representative image of H and E-stained tumor cells cultured in MCM or FM (lower panels). Images were taken on day 6 (20× (left) and 60× (right) panel). MCM human bone marrow stromal stem cells conditioned media, FM fresh media

Fig. 2. Effect of hBMSC-derived conditioned media on tumor viability.

FaDu, MCF7, MDA-MB-231, PC-3, HT-29, and MDA-MB-468 tumor cells were cultured in FM, TCM, or MCM for 2, 4, and 6 days. Cell viability on the indicated days was assessed using alamarBlue. Data are presented as mean ± S.E.M. from a minimum of three experiments, n ≥ 20. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005. p values were calculated using two-tailed Student test with equal variance. Black bars indicate compared experimental groups. MCM human bone marrow stromal stem cells conditioned media, TCM tumor-derived conditioned media, FM fresh media

Fig. 3. The effect of secreted factors from hBMSCs on tumor growth using the co-culture system.

Cell viability of the indicated tumor cell line cultured under different experimental conditions using the transwell system (0.4 µm). Tumor cells were cultured in the lower chamber, while the other treatment was in the upper chamber. Cell viability was assessed using alamarBlue assay on day 6. Data are presented as mean ± S.E.M. from a minimum of three experiments, n ≥ 40. *P ≤ 0.05, **P ≤0.005, ***P≤0.0005. p values were calculated using two-tailed Student test with equal variance. Black bars indicate compared experimental groups

Fig. 4. Gene expression analysis of tumor cell lines as a function of response to hBMSC-derived CM.

a Hierarchical clustering based on differentially expressed genes between tumor cell lines that exhibited growth advantage (MCF7, FaDu, MDA-MB-231, and PC-3) compared to those that did not exhibit growth advantage (HT-29 and MDA-MB-468). b Bar chart depicting the expression of CXCR7 and CXCR4 on the indicated tumor cell lines. c Effect of inhibition of CXCR4 (using WZ811) or inhibition of CXCR7 on tumor cell growth in the presence of recombinant CXCL12 (SDF1) or hBMSC-derived CM. Data are presented as mean ± S.E.M. from three experiments

Fig. 5. Expression of CXCR7 is associated with poor prognosis in several cancer types.

Kaplan–Meier plots illustrate the duration of overall survival according to the expression of CXCR7 in bladder, breast, cervical, kidney, liver, lung, pancreatic, stomach, and uterine cancer. Log-rank test was used for curve comparison

Fig. 6. CXCR7 and CXCL12-dependent network interactions in breast cancer.

a Scatter plot depicting the correlation between CXCR7 and CXCL12 expression in breast cancer. Pearson and Spearman correlations and associated p values are indicated. b Network analysis illustrating the interaction between CXCL12, CXCR7, and various members of the G-protein family. c Schema illustrating CXCL12 released by stromal cell to promote tumor cell survival and proliferation via binding to CXCR7 on breast cancer cells