Date Published: July 26, 2013
Publisher: Public Library of Science
Author(s): Kavita S. Subramaniam, Seng Tian Tham, Zahurin Mohamed, Yin Ling Woo, Noor Azmi Mat Adenan, Ivy Chung, John W Glod.
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.
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 . It is the most common gynecologic malignancy in the United States with an estimate of 47,100 new cases diagnosed in 2012 . Of significance, the incidence and mortality rates for EC have been rising in the developed and developing countries and is expected to rise further with the increasing ageing population and prevalence of obesity . Although the five-year survival for EC is >85%, a subset of endometrial tumors exhibit an aggressive phenotype, characterized by high histological grade, regional lymphovascular invasion and distant metastasis. The prognosis for such tumors is relatively poor, with five-year survival ranging from 16–66% .
While cancer-associated fibroblasts (CAFs) have been implicated in the progression of many cancer types [12,18], their role in EC have not been defined. It has not been previously described whether CAFs in EC exhibit pro-malignant characteristics or anti-malignant properties. To study this, a relatively pure cancer-associated fibroblast (CAFs) cell population was established from human endometrial cancer tissues and compared to normal fibroblasts. In contrast to the effects of normal fibroblasts, these CAFs exerted growth-promoting effects on endometrial cancer cells. The analysis revealed that PI3K/Akt and MAPK/Erk may be the common key pathways by which both normal and cancer fibroblasts regulate cancer cell proliferation. High secretion of one or more cytokines by CAFs (IL-6, IL-8, VEGF, RANTES and MCP-1) may potentially mediate the activation of these pathways to induce EC cell proliferation. This study provides evidence to support the notion that the underlying fibroblasts may directly influence the progression of endometrial cancer.
This study demonstrates that CAFs derived from endometrial cancer tissue are able to promote endometrial cancer cell proliferation, partly by activating PI3K and MAPK signaling pathways.