The chemokine CCL2/CCR2 signaling mediates cancer associated fibroblasts-cancer cells interaction and promotes basal-like breast cancer progression

Abstract

Breast cancer is the most common cancer diagnosed in women in the United States. The basal-like breast cancer subtype represents about 12% of breast cancer and has the worst outcome in patients due to lack of effective therapies. Tumor stroma plays an important role in cancer progression. Cancer associated fibroblasts (CAFs) are the most abundant stromal cells in breast cancer, but their function in cancer progression has not been fully understood. We previously identified that the chemokine CCL2 was highly expressed in breast cancer associated fibroblasts. CCL2 is known to recruit monocyte/macrophage and promotes cancer progression. We previously found that recombinant CCL2 can directly signal to breast cancer cells and promote cell survival and invasion in vitro. In this study, we aimed to determine the functional importance of CCL2 signaling mediated fibroblast-cancer cell interactions in the breast cancer progression. We evaluated the expression of CCL2 and fibroblast marker Fsp1 in breast cancer tissue microarray containing 427 samples by immunohistochemistry. Expression of CCL2 and Fsp1 were co-upregulated in invasive ductal carcinoma compared to normal breast. High stromal CCL2 expression associated with reduced recurrence free survival in the basal-like subtype. Further data mining in the breast cancer TCGA and METABRIC datasets revealed that the CCL2/CCR2 pair expressed highest in the basal-like subtype. We confirmed high CCL2 expression from CAFs isolated from primary human and mouse breast cancer samples. CAFs with high CCL2 expression significantly promoted xenograft growth when co-grated with human basal-like breast cancer MCF10A-CA1D cell line. Reduction of CCL2 expression by CRISPR mutation or shRNA knockdown in human or mouse CAFs significantly reduced tumor growth in the co-graft model. Fibroblasts condition medium or recombinant CCL2 directly promoted CA1D cancer cell growth in vitro. Mutation of the CCL2 receptor CCR2 receptor in CA1D cancer cell significantly reduced cancer cells growth in vitro and in vivo in response to CCL2 stimulation. The CCL2/CCR2 signaling activated PKC and SRC pathways in cancer cells, and contributed to growth promotion. We aimed to test the therapeutic efficacy by blocking CCL2 in breast cancer mouse model through continuous delivery of CCL2 neutralizing antibody. Despite the steady delivery of the antibody, CCL2 blocking did not significantly affect tumor progression. Examination of CCL2 level in blood and tumor sites revealed that CCL2 blocking significantly induced higher free CCL2 level, which may contribute to lack of efficacy. In summary, our studies have showed that CCL2 derived from cancer associated fibroblasts plays an important role in basal-like breast cancer progression. Direct signaling to cancer cells via CCR2 is an important mechanism in CCL2 function. Thus, the CCL2/CCR2 signaling may serve as potential therapeutic targets in basal-like breast cancer. CCL2 blocking by neutralizing antibody can lead to undesired increased CCL2 level, and alternative targeting approaches are needed to achieve therapeutic efficacy.