Bone morphogenetic protein 4 and Gremlin-1 communication between mouse breast cancer cells and fibroblasts

Abstract

Breast cancer (BC) metastasis is the leading cause of death in breast cancer patients. Interactions between breast cancer tumors and stromal cells allow the microenvironment surrounding the tumor to co-evolve into an activated state leading to tumor progression and metastasis. The 4T1 breast cancer mouse model which consists of five cell lines with different metastatic propensities was used to identify mechanisms of tumor-stroma communication that might facilitate metastasis. It was hypothesized that the breast cancer cell lines which can leave the primary tumor (168FARN, 4T07, 66cl4 and 4T1) are better equipped to attract and communicate with fibroblasts than the non- metastasizing 67NR cell line. Transcriptome analysis found some fibroblast associated genes and markers as well as the TGF-β family member Bmp4 and its antagonist Grem1 to be upregulated in 66cl4 as compared to 67NR. Immunoblotting and ELISA showed that the micrometastatic cell line 168FARN, had the highest GREM1 protein levels. BMP4 was expressed only by the metastatic 66cl4 at protein level. Meta-analysis identified correlation between high GREM1 expression levels and poor prognosis in BC patients in KM plot and BreastMark. The paracrine crosstalk between the tumor cell lines and fibroblasts was further analyzed in vitro. Treatment of Mouse Embryonic Fibroblasts (MEFs) with condition medium (CM) from the tumor cells showed that, 67NR CM, 168FARN CM and 66cl4 CM increased SMAD 1/5/9 phosphorylation on MEFs, indicating activation of BMP4 signaling. Interestingly, the tumor cells ability to stimulate migration of fibroblasts was in line with the degree of expression of GREM1. In conclusion, this study indicates that, the metastatic cell lines in the 4T1 model have a higher propensity to recruit fibroblasts. Moreover, GREM1 expression might aid the recruitment of fibroblasts that facilitates metastasis. These findings can be further researched into to aid in improving BC therapy.