Panoply™ Human CHEK2 Knockdown Stable Cell Line

Active fibroblasts are the most abundant and active cells in the breast cancer stroma, responsible for tumor growth and spread. However, the molecular mediators and pathways responsible for stromal fibroblast activation, and the mechanisms by which they exert paracrine oncogenic effects, remain poorly understood. The CHEK2 tumor suppressor gene encodes a protein kinase that plays a crucial role in cellular responses to various genotoxic stresses. Here, researchers found that both the mRNA and protein levels of CHEK2 were downregulated in most cancer-associated fibroblasts (CAFs) compared to their corresponding tumor fibroblasts (TCFs). Interestingly, downregulating CHEK2 using specific siRNAs increased the expression/secretion of the pro-cancer cytokines SDF-1 and IL-6 and induced the transformation of stromal fibroblasts into myofibroblasts. These cells were able to promote the proliferation of non-cancerous epithelial cells and enhance the migration/invasion of breast cancer cells in a paracrine manner. The latter effect depends on the SDF-1/IL-6 signaling pathway. Importantly, ectopic expression of CHEK2 in active CAFs restored these cells to a normal state, reducing their migration/invasion capabilities and weakening their paracrine oncogenic effects. These results demonstrate that CHEK2 possesses non-cell-autonomous tumor-suppressive functions and reveal the important mediating role of the Chk2 protein in the functional interactions between breast cancer and its stromal fibroblasts.

After confirming that CHEK2 levels were reduced in CAF cells compared to the corresponding TCF cells from the same patient, researchers sought to investigate the potential impact of CHEK2-specific downregulation on the activation of these cells. To this end, they knocked down CHEK2 in TCF-169 cells (Figure 1a). Interestingly, although the three markers α-SMA, IL-6, and SDF-1 were not detected in either TCF-169 cells or control siRNA cells, their expression was significantly upregulated after CHEK2 downregulation, reaching levels similar to those in CAF-169 cells (Figure 1a). On the other hand, p53 expression was downregulated (6-fold) in CHEK2 knockdown cells compared to control cells, reaching levels similar to those in CAF-169 cells (Figure 1a). Next, the researchers used qRT-PCR to examine the effect of CHEK2 downregulation on SDF-1 and IL-6 mRNA expression levels. Compared to the control group, SDF-1 levels were increased 2-fold and IL-6 levels were increased 3-fold in CHEK2 knockdown TCF-169 cells. This is similar to the levels of SDF-1 and IL-6 in TCF-169 cells and their corresponding CAF-169 cells (Figure 1b). This indicates that CHEK2 inhibits the expression of these genes in mammary stromal fibroblasts, suggesting that CHEK2 downregulation may be an important step in activating these cells. To further demonstrate the effect of CHEK2 downregulation on the expression of important secretory cytokines, the researchers evaluated the effect of CHEK2 knockdown on the secretion of SDF-1 and IL-6. The secretion of SDF-1 and IL-6 in CHEK2 knockdown cells increased by 3.3-fold and 2-fold, respectively. This is consistent with the secretion levels of SDF-1 and IL-6 in TCF-169 cells and their corresponding CAF-169 cells (Figure 1c).

Figure 1. CHEK2 represses the expression/secretion of SDF-1 and IL-6. (Al-Rakan M A, et al., 2016)