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// Amber A. Bokhari 1 , Laura R. Lee 1 , Dewayne Raboteau 1 , Jane Turbov 2 , Isabel V. Rodriguez 2 , John Wesley Pike 3 , Chad A. Hamilton 1, 4, 5 , George Larry Maxwell 5, 6 , Gustavo C. Rodriguez 2 , Viqar Syed 1, 5, 7 1 Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA 2 Division of Gynecologic Oncology, North Shore University Health System, University of Chicago, Evanston, IL, USA 3 Department of Biochemistry, University of Wisconsin, Madison, WI, USA 4 Division of Gynecologic Oncology, and Gynecologic Cancer Translational Research Center of Excellence, Walter Reed National Military Medical Center, Bethesda, MD, USA 5 John P. Murtha Cancer Center at Water Reed National Military Medical Center, Bethesda, MD, USA 6 Department of Obstetrics and Gynecology and Women’s Health Integrated Research Center, Inova Fairfax Hospital, Falls Church, VA, USA 7 Department of Molecular and Cell Biology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA Correspondence to: Viqar Syed, email: viqar.syed@usuhs.edu Keywords: chemoprevention, cell proliferation, progesterone, calcitriol, vitamin D receptor Received: June 01, 2016 Accepted: October 03, 2016 Published: October 18, 2016 ABSTRACT 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.

Transforming growth factor-β (TGF-β), regulates cell proliferation, angiogenesis, metastasis, and is an inducer of epithelial-mesenchymal transition (EMT). Cancer cells exhibit activated TGF-β/SMAD signaling pathway and its inhibition is an attractive strategy for cancer treatment. The Chinese Herbs Scutellaria baicalensis (SB) and Fritillaria cirrhosa (FC) have been shown to be beneficial to cancer patients, but the mechanisms by which the extracts of two herbs elicit the beneficial effects are unclear. In this study, we have used human endometrial cancer cells to assess the anticancer efficacy of SB and FC on TGF-β signaling pathway components. SB and FC treatment of cancer cells resulted in a significant decrease in expression of TGF-β isoforms, TGF-β receptors, and SMADs. Both herbs effectively inhibited basal and TGF-β1-induced cancer cell proliferation and invasion, which was accompanied with abrogation of Snail, Slug, matrix metalloproteinases (MMPs), αvβ3 integrin, focal adhesion kinase (FAK), and p-FAK expression. An inhibitor of TGF-βRI blocked TGF-β1-induced cell invasion and significantly diminished antitumor effects of SB and FC. These results suggest that SB and FC block endometrial cancer growth by downregulating TGF-β/SMAD signaling pathway.

Increased expression of TGFβ 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 TGFβ. The goal of this study was to characterize the effect of progesterone on TGFβ signaling pathway components and on TGFβ-induced protumorigenic activities in endometrial cancer cell lines. Progesterone significantly decreased expression of three TGFβ isoforms at 72 hours after treatment except for TGFβ2 in HEC-1B and TGFβ3 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 TGFβ receptors in HEC-1B cells and all but TGFβR1 in Ishikawa cells. Progesterone reduced TGFβR3 expression in RL-95 cells at 72 hours, but TGFβR1 and βR2 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 TGFβ1-induced cancer cell viability and invasion, which was accompanied by increased E-cadherin and decreased vimentin expression. An inhibitor of TGFβRI blocked TGFβ1-induced effects on cell viability and invasion and attenuated antitumor effects of progesterone. These results suggest that downregulation of TGFβ signaling is a key mechanism underlying progesterone inhibition of endometrial cancer growth. Cancer Prev Res; 7(10); 1045–55. ©2014 AACR.

The herbs Scutellaria baicalensis (SB) and Fritillaria cirrhosa (FC) are widely used in Chinese medicine to treat several aliments and as an adjuvant to chemotherapy of lung cancer. No information is available regarding the two herbs' influence on ovarian and endometrial cancer. To fill this data gap we compared cell growth responses to SB and FC in ovarian and endometrial cancer cell lines. Dose-dependent cell growth inhibition was observed following higher doses in all cell lines while lower doses stimulated growth in only endometrial cell lines. Higher doses of SB and FC significantly decreased cell growth on soft agar and decreased the invasive potential of cancer cells. Treatment of cells with both herbs resulted in activation of caspase-3, G0/G1 phase cell cycle arrest, downregulation of cyclins D1 and D3 and induction of p27. Both herbs decreased NFκB DNA binding, reduced expression of phosphorylated IκBα, abrogated NFκB activation, and downregulated NFκB-regulated metastasis-promoting proteins in cancer cells. Furthermore, knockdown of NFκB attenuated SB- and FC-induced cell growth inhibition. These results suggest that inhibition of NFκB activation may be an important mechanism for growth suppression by SB and FC. Data indicate that these herbs may represent a new source of agents for NFκB inhibition in cancer therapy. © 2013 Wiley Periodicals, Inc.

// Amber A. Bokhari 1 , Tabari M. Baker 1 , Batsukh Dorjbal 1 , Sana Waheed 1 , Christopher M. Zahn 2 , Chad A. Hamilton 1, 3, 4 , G. Larry Maxwell 3, 4, 5 , Viqar Syed 1, 4, 6 1 Uniformed Services University, Department of Obstetrics and Gynecology, Bethesda, MD 20814, USA 2 American College of Obstetricians and Gynecologists, Washington, DC 20024, USA 3 Women’s Health Integrated Research Center at Inova Health System, Department of Defense Gynecologic Cancer Center of Excellence, Annandale, VA 22003, USA 4 John P. Murtha Cancer Center at Water Reed National Military Medical Center, Bethesda, MD 20889, USA 5 Inova Fairfax Hospital, Department of Obstetrics and Gynecology, Falls Church, VA 22042, USA 6 Uniformed Services University, Department of Molecular and Cell Biology, Bethesda, MD 20814, USA Correspondence to: Viqar Syed, email: viqar.syed@usuhs.edu Keywords: cell proliferation, progesterone, angiogenesis, matrix metalloproteinases, epithelial-mesenchymal-transition Received: April 25, 2016 Accepted: September 02, 2016 Published: September 10, 2016 ABSTRACT Nestin, an intermediate filament protein and a stem cell marker is expressed in several tumors. Until recently, little was known about the expression levels and the role of Nestin in endometrial cancer. Compared to the immortalized endometrial epithelial cell line EM-E6/E7-TERT, endometrial cancer cell lines express high to moderate levels of Nestin. Furthermore, endometrial tumors and tumor cell lines have a cancer stem-like cell subpopulation expressing CD133. Among the cancer lines, AN3CA and KLE cells exhibited both a significantly higher number of CD133+ cells and expressed Nestin at higher levels than Ishikawa cells. Knockdown of Nestin in AN3CA and KLE increased cells in G 0 /G 1 phase of the cell cycle, whereas overexpression in Ishikawa decreased cells in G 0 /G 1 phase and increased cells in S-phase. Nestin knockdown cells showed increased p21, p27, and PNCA levels and decreased expression of cyclin-D1 and D3. In contrast, Nestin overexpression revealed an inverse expression pattern of cell cycle regulatory proteins. Nestin knockdown inhibited cancer cell growth and invasive potential by downregulating TGF-β signaling components, MMP-2, MMP-9, vimentin, SNAIL, SLUG, Twist, N-cadherin, and upregulating the epithelial cell marker E-cadherin whereas the opposite was observed with Nestin overexpressing Ishikawa cells. Nestin knockdown also inhibited, while overexpression promoted invadopodia formation and pFAK expression. Knockdown of Nestin significantly reduced tumor volume in vivo . Finally, progesterone inhibited Nestin expression in endometrial cancer cells. These results suggest that Nestin can be a therapeutic target for cancer treatment.

Transforming growth factor-β (TGF-β), regulates cell proliferation, angiogenesis, metastasis, and is an inducer of epithelial-mesenchymal transition (EMT). Cancer cells exhibit activated TGF-β/SMAD signaling pathway and its inhibition is an attractive strategy for cancer treatment. The Chinese Herbs Scutellaria baicalensis (SB) and Fritillaria cirrhosa (FC) have been shown to be beneficial to cancer patients, but the mechanisms by which the extracts of two herbs elicit the beneficial effects are unclear. In this study, we have used human endometrial cancer cells to assess the anticancer efficacy of SB and FC on TGF-β signaling pathway components. SB and FC treatment of cancer cells resulted in a significant decrease in expression of TGF-β isoforms, TGF-β receptors, and SMADs. Both herbs effectively inhibited basal and TGF-β1-induced cancer cell proliferation and invasion, which was accompanied with abrogation of Snail, Slug, matrix metalloproteinases (MMPs), αvβ3 integrin, focal adhesion kinase (FAK), and p-FAK expression. An inhibitor of TGF-βRI blocked TGF-β1-induced cell invasion and significantly diminished antitumor effects of SB and FC. These results suggest that SB and FC block endometrial cancer growth by downregulating TGF-β/SMAD signaling pathway.

Nestin, an intermediate filament protein and a stem cell marker is expressed in several cancers. Little is known about the expression level and role of Nestin in endometrial cancer. Compared to immortalized endometrial epithelial cell line EM-E6/E7/TERT, endometrial cancer lines express high to moderate levels of Nestin. Endometrial tumors and tumor cell lines have a cancer stem-like cells population expressing CD133. The AN3CA and KLE cells showed more CD133+ cells and expressed Nestin at higher levels than Ishikawa cells. Knockdown of Nestin in AN3CA and KLE, and overexpression in Ishikawa cells was associated respectively with attenuation and enhancement of CD133+ cells. Knockdown of Nestin increased cells in G0/G1 phase and decreased in S phase, whereas overexpression decreased cells in G0/G1 phase and increased in S phase of cell cycle. Nestin knockdown cells showed increased expression of p21, p27 and PNCA and decreased expression of cyclin-D1 and D3. Nestin overexpression revealed an inverse expression pattern of cell cycle regulatory proteins. Nestin knockdown inhibited cancer cell growth and invasive potential by down regulating TGF-β signaling components, MMP-2, MMP-9, vimentin, SNAIL, SLUG, Twist, N-cadherin, and upregulating E-cadherin. Conversely, Nestin overexpression enhanced cell invasiveness by upregulating TGF-β signaling components, MMP-2, MMP-9, mesenchymal markers and downregulaing epithelial marker, E-cadherin. Nestin knockdown inhibited and overexpression promoted invadopodia formation and pFAK expression. Knockdown of Nestin significantly reduced tumor volume in vivo. Finally, progesterone and a nitric oxide donor inhibited Nestin expression in endometrial cancer cells. These results suggest that Nestin can be a therapeutic target for cancer treatment. Citation Format: Amber A. Bokhari, Batsukh Dorjbal, Tabari M. Baker, Sana Waheed, Christopher M. Zahn, Chad A. Hamilton, George L. Maxwell, Viqar Syed. Nestin suppression attenuates invasive potential of endometrial cancer cells by down regulating TGF-β signaling pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4428.

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