Cancer-associated fibroblasts promote proliferation of endometrial cancer cells
Authors: Seng Tian Tham
Kavita S Subramaniam
Zahurin Mohamed
Ivy Chung
Yin Ling Woo
Noor Azmi Mat Adenan
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: Endometrial cancer; fibroblast cells; cancer-associated fibroblasts; proliferation; PI3K/Akt pathway
Affiliations: Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.

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&lt;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&lt;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. 

Retracted: No

PubPeer comments:
 Concern about Figure 6. * Red box: A strange u201csmudgeu201d is visible in the third lane of the phopho-Akt panel, and possibly also in the third lane of the phospho-Erk panel.  This issue was reported to the journal in August 2014. This week, more than six years later, the journal posted a  Reader Comment on the paper s landing page. It reads:  *After publication of this article, a question was raised about the blurred appearance of data in lane 3 of the phospho-Akt and phospho-ERK panels of Figure 6A. The authors were unable to clarify the reason for these irregularities, but the figure accurately represents the original blot images provided to the journal office.* *The authors noted that results reported in Figure 6A are representative of three experiments. For each, control data were obtained by stripping the blot used to detect phospho-proteins and then reprobing it with antibodies for u03b2-actin and either total Akt or ERK. The u03b2-actin blot shown in Figure 6A is a representative control from these experiments but is not a matched control for all panels in the figure.* *The original image data provided in support of Figure 6A are available at https: doi.org 10.6084 m.... Blots shown in Figure 6A include images labelled in the repository files as WB33 (phospho-ERK) and WB34 (phospho-Akt, Akt). The matched u03b2-actin blot for the Akt experiment is included in the posted data file named u2018Fig 6 AKTu2019.* *Questions arose during the journalu2019s editorial review of the raw image data and published figures which we have been unable to resolve based on the available data and lab records. To provide assurance as to the results, the authors have agreed to replicate the full Figure 6A experiment, but this work has been delayed in 2020 due to lab closures. The journal will reassess this matter when the replication data are available.* *The corresponding author stated that the underlying data to support other results in this article are available upon request.* ,