Abstract
Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs) using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin) and hormonal (estrogen and progesterone) receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175%) when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51%) (P<0.0001). These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001), suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR), also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, RANTES and vascular endothelial growth factor (VEGF) than normal fibroblasts. Our data suggests that in contrast to normal fibroblasts, CAFs may exhibit a pro-tumorigenic effect in the progression of endometrial cancer, and PI3K/Akt and MAPK/Erk signaling may represent critical regulators in how endometrial cancer cells respond to their microenvironment.
Highlights
Endometrial cancer (EC) is the sixth most commonly diagnosed cancer among women globally, with approximately 288,000 new cases and 50,327 deaths occurring worldwide each year [1]
To establish primary fibroblast cells from endometrial tissues, human endometrial cancer (EC) tissues (EC6, 7, 11 and 14) were digested with collagenase, followed by cell isolation using magnetic beads conjugated with anti-fibroblast antibody
Epithelial cells isolated from EC6 and 14 (EC6-Ep and EC14-Ep) showed moderate expression of EpCAM (55%) with no evidence of CD90 expression, indicating that this epithelial culture was not contaminated with fibroblast cells (Figure 2B, C)
Summary
Endometrial cancer (EC) is the sixth most commonly diagnosed cancer among women globally, with approximately 288,000 new cases and 50,327 deaths occurring worldwide each year [1]. Contribution of cancer-associated fibroblasts (CAFs) in the progression of various cancer types has been studied, for example, in prostate cancer [16,17,18], pancreatic cancer [12], head and neck cancer [19] and breast cancer [20] In these tumor models, CAFs enhanced tumor cell proliferation, invasion and chemoresistance. Using primary cultures from endometrial tissues, Arnold et al demonstrated that the secretion from normal endometrial fibroblast cells inhibited the proliferation of Ishikawa cells, a human EC cell line [26] This observation was further supported by Zhao’s group in which they suggested that such anti-proliferative effect could be due to inhibition of PI3K signaling [27]. The study provides new evidence elucidating the pro-tumorigenic role of fibroblasts in the tumorigenesis of EC
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