| Summary: | It is well known now that changes in the cellular microenvironment contribute to tumour genesis through paracrine effects. Fibroblasts, the predominant cells of the stromal breast carcinomas, play an important role in the development and spread of cancer cells. However, the nature, molecular mediators and pathways of the crosstalk between cancer cells and their stromal fibroblasts are still not well defined. There is evidence that normal breast stromal fibroblasts (NBFs) suppress tumor growth while cancer-associated fibroblasts (CAFs) promote tumorogenesis through functional interactions with cancer cells. Little is known about the biology and the carcinogenic potential of stromal fibroblasts present in histologically normal surgical margins (TCFs). In addition, the nature, molecular mediators and pathways of the crosstalk between cancer cells and their stromal fibroblasts are still not well defined. Therefore, we first undertook gene expression analysis on five CAF/TCF pairs from breast cancer patients and three NBFs (derived from mammoplasties). This comparative analysis revealed variation in gene expression between these three categories of cells, with a TCF-specific gene expression profile. This variability was higher in TCFs than in their paired CAFs and also NBFs. Cytokine arrays show that TCFs have a specific secretory cytokine profile. In addition, stromal fibroblasts from surgical margins expressed high levels of α-SMA and SDF-1 and exhibited higher migratory/invasiveness abilities. Indirect co-culturing showed that TCF cells enhance the proliferation of noncancerous mammary epithelial cells, and the epithelial to mesenchymal transition of breast cancer cells. Moreover, TCF and CAF cells increased the level of PCNA, MMP-2 and the phosphorylated/activated form of Akt in normal breast luminal fibroblasts in a paracrine manner. Furthermore, TCFs were able to promote the formation and growth of humanized orthotopic breast tumors in nude mice. Interestingly, these TCF phenotypes and the extent of their effects were intermediate between NBFs and CAFs. Together, these results indicate that stromal fibroblasts located in noncancerous tissues exhibit a tumor-promoting phenotype, indicating that their presence post-surgery may play important roles in cancer recurrence. Moreover, we have shown that the tumor suppressor CHEK2 gene is down-regulated in CAF cells as compared to TCF cells and NBF cells in vitro at both the mRNA and protein levels and in vivo. Using specific siRNA we have shown that CHEK2-down-regulation increases the expression and the secretion of SDF-1, IL-6, VEGF-A and MMP-9 and enhances the proliferation, migration and invasion of breast cancer cells. These results present the Chk2 kinase protein as an important mediator in the cross-talk between breast carcinomas and their stromal fibroblasts.
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