CHEK2 represses breast stromal fibroblasts and their paracrine tumor-promoting effects through suppressing SDF-1 and IL-6

Background: Active fibroblasts, the predominant and the most active cells of breast cancer stroma, are responsible for tumor growth and spread. However, the molecular mediators and pathways responsible for stromal fibroblast activation, and their paracrine pro-carcinogenic effects are still not well defined. The CHEK2 tumor suppressor gene codes for a protein kinase, which plays important roles in the cellular response to various genotoxic stresses. Methods: Immunoblotting, quantitative RT-PCR and Immunofluorescence were used to assess the expression of CHEK2 in different primary breast fibroblasts and in tissues. The effect of CHEK2 on the expression and secretion of SDF-1 and IL-6 was evaluated by immunoblotting and ELISA. The WST-1 colorimetric assay was used to assess cell proliferation, while the BD BioCoat Matrigel invasion chambers were utilized to determine the effects of CHEK2 on the migratory and the invasiveness capacities of breast stromal fibroblasts as well as breast cancer cells. Results: We have shown that CHEK2 is down-regulated in most cancer-associated fibroblasts (CAFs) as compared to their corresponding tumor counterpart fibroblasts (TCFs) at both the mRNA and protein levels. Interestingly, CHEK2 down-regulation using specific siRNA increased the expression/secretion of both cancer-promoting cytokines SDF-1 and IL-6, and transdifferentiated stromal fibroblasts to myofibroblasts. These cells were able to enhance the proliferation of non-cancerous epithelial cells, and also boosted the migration/invasion abilities of breast cancer cells in a paracrine manner. The later effect was SDF-1/IL-6-dependent. Importantly, ectopic expression of CHEK2 in active CAFs converted these cells to a normal state, with lower migration/invasion capacities and reduced paracrine pro-carcinogenic effects. Conclusion: These results indicate that CHEK2 possesses non-cell-autonomous tumor suppressor functions, and present the Chk2 protein as an important mediator in the functional interplay between breast carcinomas and their stromal fibroblasts.