Increased expression of TGFb isoforms in human endometrial cancer correlates with decreased survival and poor prognosis. Progesterone has been shown to exert a chemoprotective effect against endometrial cancer, and previous animal models have suggested that these effects are accompanied by changes in TGFb. The goal of this study was to characterize the effect of progesterone on TGFb signaling pathway components and on TGFb-induced protumorigenic activities in endometrial cancer cell lines. Progesterone significantly decreased expression of three TGFb isoforms at 72 hours after treatment except for TGFb2 in HEC-1B and TGFb3 in Ishikawa cells. Progesterone treatment for 120 hours attenuated expression of the three isoforms in all cell lines. Progesterone exposure for 72 hours reduced expression of TGFb receptors in HEC-1B cells and all but TGFbR1 in Ishikawa cells. Progesterone reduced TGFbR3 expression in RL-95 cells at 72 hours, but TGFbR1 and bR2 expression levels were not affected by progesterone at any time point. SMAD2/3 and pSMAD2/3 were substantially reduced at 72 hours in all cell lines. SMAD4 expression was reduced in RL-95 cells at 24 hours and in HEC-1B and Ishikawa cells at 72 hours following progesterone treatment. Furthermore, progesterone effectively inhibited basal and TGFb1-induced cancer cell viability and invasion, which was accompanied by increased E-cadherin and decreased vimentin expression. An inhibitor of TGFbRI blocked TGFb1-induced effects on cell viability and invasion and attenuated antitumor effects of progesterone. These results suggest that downregulation of TGFb signaling is a key mechanism underlying progesterone inhibition of endometrial cancer growth. Cancer Prev Res; 7(10); 1045-55. Ó2014 AACR.
Here, we evaluated the expression of CYP24A1, a protein that inactivates vitamin D in tissues. CYP24A1 expression was increased in advanced-stage endometrial tumors compared to normal tissues. Similarly, endometrial cancer cells expressed higher levels of CYP24A1 than immortalized endometrial epithelial cells. RT-PCR and Western blotting were used to examine CYP24A1 mRNA and protein levels in endometrial cancer cells after 8, 24, 72, and 120 h of exposure to progesterone, progestin derivatives and calcitriol, either alone or in combination. Progestins inhibited calcitriol-induced expression of CYP24A1 and splice variant CYP24SV mRNA and protein in cancer cells. Furthermore, actinomycin D, but not cycloheximide, blocked calcitriol-induced CYP24A1 splicing. siRNA-induced knockdown of CYP24A1 expression sensitized endometrial cancer cells to calcitriol-induced growth inhibition. These data suggest that CYP24A1 overexpression reduces the antitumor effects of calcitriol in cancer cells and that progestins may be beneficial for maintaining calcitriol's anti-endometrial cancer activity.
The cytochrome P450 enzyme, 24-hydroxylase, encoded by CYP24A1 is critical for the catabolism of 1,25(OH)2D3 (calcitriol). The unbalanced high levels of CYP24A1 seem to be a determinant of calcitriol resistance in tumors. We have previously shown that progesterone enhances calcitriol antitumor activity by upregulating vitamin D receptor expression and promoting apoptosis in endometrial cancer cells. In the present study, we evaluated CYP24A1 protein expression in normal and endometrial tumor tissues, assessed the effect of progesterone and calcitriol on CYP24A1 and its spliced variant expression in endometrial cancer cell lines and correlated this with tumor cell growth. Expression of CYP24A1 was assessed in tissue microarrays by immunohistochemistry. A grade-dependent increase of CYP24A1 expression was found in endometrial carcinomas. Endometrial cancer cells expressed high levels of CYP24A1 compared to immortalized endometrial epithelial cells. Furthermore, CYP24A1 is induced in cells in response to calcitriol. Regulation of CYP24A1 by progesterone, progestin derivatives, calcitriol and their combination was examined by RT-PCR and Western blotting following 8, 24, 72 and 120 h exposure of cells to hormones. In all cancer cell lines, progesterone, medroxyprogesterone acetate and norgestrol attenuated calcitriol-induced CYP24A1, spliced variant CYP24SV transcripts and protein expression at 72 and 120 h of treatment. Furthermore, knockdown of CYP24A1 gene expression by siRNA sensitized endometrial cancer cells to the growth suppressive effect of calcitriol. The data suggest that CYP24A1 overexpression limits calcitriol anti-proliferative signaling in cancer cells, and provide evidence that progestins may be beneficial in preserving calcitriol action in endometrial cancer. Citation Format: Amber A. Bokhari, Laura R. Lee, Raboteau Dewayne, Chad A. Hamilton, George L. Maxwell, Gustavo C. Rodriguez, Viqar Syed. Role and regulation of CYP24A1 in endometrial cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 37. doi:10.1158/1538-7445.AM2015-37
Objectives: Our group has shown previously that progesterone may exert a chemoprotective effect against endometrial cancer through modulation of TGF-β signaling and concomitant activation of apoptosis in the endometrium. In addition, it has been shown that increased expression of TGF-β isoforms in human endometrial cancer correlates with decreased survival and poor prognosis. These data suggest that the TGF-β pathway may be an attractive target for chemoprevention strategies. The goal of this study was to further characterize the effect of progesterone on TGF-β signaling pathway components (TGF-β isoform and Smads) and on TGF- β-induced pro-tumorigenic activities in endometrial cancer cell lines. Methods: The expression levels of TGF-β ligands (TGF-β1, TGF-β2 and TGF-β3), TGF-β receptors (TGF-βR1, TGF-βR2, and TGF-βR3) and SMADs (pSMAD2/3, SMAD2/3 and SMAD-4) were determined by immunoblotting in HEC-1B endometrial cancer cells exposed to progesterone for 24, 72 and 120 h. Proliferation and cellular invasion assays were used to access the functional effects of progestin exposure in the HEC-1B and Ishikawa endometrial cancer cell lines. Results: A marked decrease in TGF-β1and TGF-β3 expression was observed at 72 h after treatment with progesterone. Expression of TGF-βR1, TGF-βR2, SMAD2/3 and pSMAD2/3 were substantially reduced at 72 h while levels of SMAD4 and TGF-β2 expression were reduced at 120 h following progesterone. TGF-βR3 expression levels were not affected by any treatment at any time point. Furthermore, TGF-β1-induced cancer cell proliferation and invasion was effectively inhibited by progesterone. Cellular proliferation and invasion were significantly reduced by progestin compared to controls even in the presence of exogenous TGF-β. Conclusions: These results suggest that the down-regulation of TGF-β signaling may be a key mechanism underlying progestin inhibition of endometrial carcinogenesis. Citation Format: Amber A. Bokhari, Laura R. Lee, Dewayne Raboteau, Chad A. Hamilton, George L. Maxwell, Jane M. Turbov, Larry G. Thaete, Gustavo C. Rodriguez, Viqar Syed. Progesterone inhibits endometrial cancer growth and invasiveness by modulating the TGF-ß pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3467. doi:10.1158/1538-7445.AM2014-3467
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