ECC-1 Cell Research Articles

Abstract B016: Novel curcumin analogues as therapies for endometrial cancer with TP53 mutations

Abstract The protein p53 is designated as the guardian of the genome because it is the principal tumor suppressor and functions to surveil the genome, enforce cell cycle checkpoints, and trigger apoptosis when genomic instability is beyond repair. Mutations in TP53 are found in >50% of human tumors and in 25% of the deadliest endometrial malignancies. Yet, few studies have targeted the mutant protein to restore its wild type (WT) tumor suppressor functions. Curcumin, a turmeric derivative, has been shown to help return WT function to mutant p53. However, curcumin is not bioavailable to most of the body because of its water insolubility, hydrophobic nature and rapid metabolization. Our laboratory is testing novel, more bioavailable analogs of curcumin including HO-3867 and AKT-100. In these studies, we tested these agents in preclinical models of serous endometrial cancer to assess their effects on cell growth and p53-dependent signaling. We used CyQUANT cell proliferation assay to identify IC50 values for HO-3867 and AKT-100 in ECC-1 and KLE endometrial cancer cell lines. We performed RNA sequencing to elucidate mechanisms of action and verified p53 expression changes via Western blotting. IC50 values ranged from 100 nanomolar (AKT-100) to low micromolar (HO-3867). RNA sequencing data revealed the re-enforcement of cell cycle checkpoints including the upregulation of CDKN1A (p21) and GADD45 (Gadd45). Proliferative signaling downstream of ATM, an important protein kinase stimulating cell growth, was downregulated by both agents. Western blotting confirmed the classic negative feedback loop of p53, where total p53 levels decreased after drug treatment. Taken together, our findings show that AKT-100 and HO-3867 are able to restore WT tumor suppressor function in otherwise oncogenic mutant p53 by reducing cell replication and promoting apoptosis. Thus, curcumin analogues such as AKT-100 and HO-3867 show promise in the treatment of a subset of endometrial tumors expressing mutant p53 by returning WT p53 functionality, and future studies will move to in vivo analyses of curcumin analogue function. Citation Format: Kimberly K. Leslie, Alex Goss, Jamie Padilla, Lane E. Smith. Novel curcumin analogues as therapies for endometrial cancer with TP53 mutations [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B016.

Soluble guanylyl cyclase beta1 subunit targets epithelial-to-mesenchymal transition and downregulates Akt pathway in human endometrial and cervical cancer cells

Endometrial and cervical cancer are among the most frequently diagnosed malignancies globally. Nitric oxide receptor-soluble guanylyl cyclase (sGC) is a heterodimeric enzyme composed of two subunits, α1 and β1. Previously we showed that sGCα1 subunit promotes cell survival, proliferation, and migration, but the role of sGCβ1 subunit has not been addressed. The aim of the present work was to study the impact of sGCβ1 restoration in proliferation, survival, migration, and cell signaling in endometrial and cervical cancer cells. We found that sGCβ1 transcript levels are reduced in endometrial and cervical tumors vs normal tissues. We confirmed nuclear enrichment of sGCβ1, unlike sGCα1. Overexpression of sGCβ1 reduced cell viability and augmented apoptotic index. Cell migration and invasion were also negatively affected. All these sGCβ1-driven effects were independent of sGC enzymatic activity. sGCβ1 reduced the expression of epithelial-to-mesenchymal transition factors such as N-cadherin and β-catenin and increased the expression of E-cadherin. sGCβ1 impacted signaling in endometrial and cervical cancer cells through significant downregulation of Akt pathway affecting some of its main targets such as GSK-3β and c-Raf. Our results show for the first time that sGCβ1 exerts several antiproliferative actions in ECC-1 and HeLa cell lines by targeting key regulatory pathways.

Oleic acid, a monounsaturated fatty acid, exhibits antiproliferative and anti-tumorigenic effects in pre-clinical studies for endometrioid endometrial cancer (261)

<b>Objectives:</b> Oleic acid (OA), a monounsaturated fatty acid commonly found in olive oil, has been studied for its potential protective qualities in inflammatory conditions and cancers. In breast cancer cells, OA has been shown to reduce the expression of the oncogene <i>Her-2/ neu</i> and promote apoptosis. The relationship between the intake of olive oil and endometrial cancer risk and progression has been largely unexplored. Thus, we aimed to evaluate the effect of OA on cell proliferation and tumor growth in endometrial cancer cell lines and a transgenic mouse model of endometrial cancer. <b>Methods:</b> The human endometrioid endometrial cancer cell lines, ECC-1 and KLE, were exposed to varying concentrations of OA. Cell proliferation was assessed by MTT assay. Apoptosis was assessed by cleaved caspase-3, -8, and -9 assays. Cell cycle progression was measured by Cellometer. Cellular stress was measured by DCFH-DA assay. Mitochondrial membrane potential was assessed by TMRE assay. Western immunoblotting was performed to determine the effects of OA on BCL-xl, MCL-1, CDK4, CyclinD1, Calnexin, and Bip. The LKB1^fl/flp53^fl/fl mouse model of endometrioid endometrial cancer was used to determine the antitumorigenic activity of OA. The mice were treated with OA (10 mg/kg, oral gavage, daily) versus control for four weeks. <b>Results:</b> OA inhibited proliferation in a dose-dependent manner in both cell lines, with a 60% reduction at a dose of 500 µM in the ECC-1 cell line and 250 µM in the KLE cell line. OA at a dose of 200 µM significantly increased the activity of cleaved caspase-3, 8, and 9 by 2.2, 1.4, and 1.7-fold in the ECC-1 cells (p<0.05), respectively, and 1.4, 1.3, and 1.4-fold in the KLE cells (p<0.05), respectively. OA simultaneously decreased expression of Bcl-xl and MCL-1 in both cell lines. OA inhibited cell cycle progression by G1 phase arrest in both cell lines in a dose-dependent fashion (p=0.02) and decreased expression of the cell cycle-related proteins, CDK4, and cyclin D1. Treatment with OA significantly increased ROS production at doses of 50 and 200 µM (p<0.05) and induced Calnexin and Bip expression in both cell lines. TMRE assay showed decreased mitochondrial membrane potential with OA at doses of 50 and 200 µM (p<0.05). Treatment of LKB1^fl/flp53^fl/fl mice with OA was well-tolerated and reduced tumor weight by 53% compared with control mice (p<0.05). <b>Conclusions:</b> We found that OA, the main monounsaturated fatty acid of olive oil, had antiproliferative and antitumorigenic effects in endometrioid endometrial cancer. These findings provide a preclinical rationale for OA as a potential dietary intervention and therapeutic adjunct in endometrial cancer patients.

Cancer-associated fibroblasts as cellular vehicles in endometrial cancer cell migration.

Cell motility is a critical step in the metastasis cascade. However, the role of cancer-associated fibroblasts (CAFs) in facilitating endometrial cancer (EC) cell motility remains unclear. The present study aimed to investigate the role of CAFs in EC motility in a 3D environment. A co-culture model was established using an EC cell line (ECC-1) and CAFs on a Matrigel® matrix and compared to the respective individual monocultures. It was demonstrated that endometrial CAFs increased the motility of the EC cell line, compared with the monoculture. Using live cell imaging, CAFs were observed to form cell projections that served as contact guidance for ECC-1 cell locomotion in the spheroid formation process. These effects were specific to CAFs, as fibroblasts isolated from benign endometrial tissue samples did not form cell projections. Molecular analysis revealed that RhoA/Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) signaling activation partly contributed to CAF-mediated ECC-1 cell migration. The presence of Matrigel® increased the mRNA expression of RhoA, and the mRNA and protein expression levels of its downstream effectors, ROCK1 and p-MLC, respectively, in the ECC-1 and CAF co-culture, as well as the ECC-1 and CAF monocultures. Interestingly, high phosphorylation levels of myosin light chain mediated the activation of RhoA/ROCK1 signaling in the ECC-1 and CAF co-culture. The ROCK1 inhibitor Y-27632 attenuated the motility of tumor cells in ECC-1 and CAF co-cultures. However, similar treatment led to a significant inhibition in the motility of the CAF monoculture, but not the ECC-1 monoculture. Moreover, tumor spheroid formation was inhibited due to a reduction in stress fiber formation in ECC-1 and CAF co-cultures. Altogether, these findings suggest that the regulation of the RhoA/ROCK1 signaling pathway is required for CAFs to serve as cellular vehicles in order for EC cells to migrate and form spheroids in a 3D environment.

Ipatasertib, an oral AKT inhibitor, exhibits anti-proliferative and anti-tumorigenic effects in pre-clinical studies for endometrioid endometrial cancer: Endometrial Cancer Molecularly Targeted Therapy Consortium

<h3>Objectives:</h3> Ipatasertib (IPAT) is an orally administered selective protein kinase B (AKT) inhibitor that has demonstrated clinical activity in triple-negative breast and metastatic prostate cancer. Given that the AKT/mTOR pathway is altered in the vast majority of endometrioid endometrial cancers (ECs), it is logical that IPAT may also have efficacy in EC. Thus, we aimed to evaluate the anti-proliferative effects of IPAT in human endometrioid cell lines as well as in the <i>LKB1fl/flp53fl/fl</i> genetically engineered mouse model of endometrioid EC. <h3>Methods:</h3> The human endometrioid EC cell lines ECC-1 (PTEN mutant) and HEC-1A (PTEN wild type, PIK3CA mutant, KRAS mutant) were exposed to varying concentrations of IPAT (Genentech). Cell proliferation was assessed by MTT and colony assays. Cell cycle progression was measured by Cellometer. Apoptosis was assessed by cleaved caspase-3 assay. Cellular stress was assessed using tetra-methylrhodamine ester (TMRE) and DCFDA assays. Western immunoblotting determined effects of IPAT on BCL-2, MCL-1, Bip, CDK4, CDK6, cyclin D1, pAKT, pS6, PERK, calnexin, and PDI in both cell <i>lines.LKB1fl/flp53fl/fl</i> mice were used to evaluate the effect of IPAT on endometrial tumor growth. The mice were treated with placebo or IPAT (15mg/kg daily intraperitoneally for 4 weeks) starting 8 weeks after tumor induction via AdCre injection. <h3>Results:</h3> IPAT inhibited cellular proliferation in a dose dependent fashion in both cell lines after 72 hours of treatment. Median IC50 was 0.18µM in ECC-1 and 3.5µM in HEC-1A. IPAT induced the activity of cleaved caspase 3 by 15.6-fold in ECC-1 and 1.41-fold in HEC-1A (p<0.01), while simultaneously reducing expression of the apoptotic proteins MCL-1 and BCL-2. IPAT inhibited cell cycle progression by arrest in G1 phase in both cell lines after 36 hours of treatment and decreased expression of the cell cycle related proteins, CDK4, CDK6, and cyclin D1. DCFDA assay analysis demonstrated that IPAT induced ROS products by 18% and 19% (p<0.01) in the ECC-1 and HEC-1A cell lines, respectively, and increased Bip and PERK protein expression. In addition, IPAT reduced mitochondrial potential by 12% in the HEC-1A cells and 14% in the ECC-1 cells (p<0.01). Treatment of cells with IPAT at 10µM increased phosphorylated-AKT (pAKT) expression and decreased phosphorylated-S6 (p-S6) expression in a time-dependent manner in both cell lines; however, measurable changes in pAKT and pS6 were observed at concentrations as low as 0.1µM. Notably, an increase in phosphorylated-AKT was expected and is indicative of engagement and binding of IPAT with the phospho-AKT complex. Treatment of <i>LKB1fl/flp53fl/fl</i> mice with IPAT was well-tolerated and reduced tumor weight by 52.1% as compared with control mice (p<0.05). <h3>Conclusions:</h3> IPAT significantly inhibited cell proliferation and induced apoptosis and cellular stress via inhibition of the AKT/mTOR pathway in human endometrioid EC cell lines. Furthermore, IPAT reduced tumor growth in a transgenic mouse model of endometrioid EC. These results suggest that IPAT is worthy of further exploration in clinical trials for EC.

Ipatasertib, an oral AKT inhibitor, effectively inhibits cell proliferation and migration, and induces apoptosis in serous endometrial cancer cell lines in vitro: Endometrial Cancer Molecularly Targeted Therapy Consortium

<h3>Objectives:</h3> Ipatasertib (IPAT) is an orally administered selective protein kinase B (AKT) inhibitor that has demonstrated clinical activity in triple-negative breast and metastatic prostate cancer. Given that the AKT/mTOR pathway is altered in the vast majority of endometrioid endometrial cancers (ECs), it is logical that IPAT may also have efficacy in EC. Thus, we aimed to evaluate the anti-proliferative effects of IPAT in human endometrioid cell lines as well as in the <i>LKB1fl/flp53fl/fl</i> genetically engineered mouse model of endometrioid EC. <h3>Methods:</h3> The human endometrioid EC cell lines ECC-1 (PTEN mutant) and HEC-1A (PTEN wild type, PIK3CA mutant, KRAS mutant) were exposed to varying concentrations of IPAT (Genentech). Cell proliferation was assessed by MTT and colony assays. Cell cycle progression was measured by Cellometer. Apoptosis was assessed by cleaved caspase-3 assay. Cellular stress was assessed using tetramethylrhodamine ester (TMRE) and DCFDA assays. Western immunoblotting determined effects of IPAT on BCL-2, MCL-1, Bip, CDK4, CDK6, cyclin D1, pAKT, pS6, PERK, calnexin, and PDI in both cell lines. <i>LKB1fl/flp53fl/fl</i> mice were used to evaluate the effect of IPAT on endometrial tumor growth. The mice were treated with placebo or IPAT (15mg/kg daily intraperitoneally for 4 weeks) starting 8 weeks after tumor induction via AdCre injection. <h3>Results:</h3> IPAT inhibited cellular proliferation in a dose dependent fashion in both cell lines after 72 hours of treatment. Median IC50 was 0.18µM in ECC-1 and 3.5µM in HEC-1A. IPAT induced the activity of cleaved caspase 3 by 15.6-fold in ECC-1 and 1.41-fold in HEC-1A (p<0.01), while simultaneously reducing expression of the apoptotic proteins MCL-1 and BCL-2. IPAT inhibited cell cycle progression by arrest in G1 phase in both cell lines after 36 hours of treatment and decreased expression of the cell cycle related proteins, CDK4, CDK6, and cyclin D1. DCFDA assay analysis demonstrated that IPAT induced ROS products by 18% and 19% (p<0.01) in the ECC-1 and HEC-1A cell lines, respectively, and increased Bip and PERK protein expression. In addition, IPAT reduced mitochondrial potential by 12% in the HEC-1A cells and 14% in the ECC-1 cells (p<0.01). Treatment of cells with IPAT at 10µM increased phosphorylated-AKT (pAKT) expression and decreased phosphorylated-S6 (p-S6) expression in a time-dependent manner in both cell lines; however, measurable changes in pAKT and pS6 were observed at concentrations as low as 0.1µM. Notably, an increase in phosphorylated-AKT was expected and is indicative of engagement and binding of IPAT with the phospho-AKT complex. Treatment of <i>LKB1fl/flp53fl/fl</i> mice with IPAT was well-tolerated and reduced tumor weight by 52.1% as compared with control mice (p<0.05). <h3>Conclusions:</h3> IPAT significantly inhibited cell proliferation and induced apoptosis and cellular stress via inhibition of the AKT/mTOR pathway in human endometrioid EC cell lines. Furthermore, IPAT reduced tumor growth in a transgenic mouse model of endometrioid EC. These results suggest that IPAT is worthy of further exploration in clinical trials for EC.

Abstract PO044: A novel dopamine receptor D2 antagonist (ONC206) potentiates the effects of olaparib in endometrial cancer

Abstract Objectives: Poly ADP-ribose polymerase (PARP) inhibitors are effective therapies for patients with homologous recombination (HR) deficient tumors. While HR mutations are rare in endometrial cancers (ECs), emerging data suggests that loss of function of the tumor suppressor gene PTEN, which is mutated in 80% of endometrioid ECs, may also sensitize tumors to PARP inhibition. The imipridone ONC206 is an orally bioavailable dopamine receptor D2 antagonist that has anti-tumorigenic effects in EC via induction of apoptosis, activation of the integrated stress response and modulation of Akt signaling. Both PARP inhibitors and imipridones are being evaluated in EC clinical trials, but have yet to be explored in combination. We sought to examine the effects of the PARP inhibitor olaparib in combination with ONC206 in human endometrioid EC cell lines and in a mouse model of endometrioid EC. Methods: The ECC-1 and HEC-1 EC cell lines were used. Cell proliferation was assessed after exposure to varying doses of olaparib (MedChemExpress) and ONC206 (Oncoceutics) via the MTT assay. Synergy between olaparib and ONC206 was assessed by the combination index (CI) method of Chou-Talalay. Cellular stress was evaluated using the DCFH-DA assay. Apoptosis was assessed using the cleaved caspase-3 assay. Western immunoblotting was performed to determine effects of olaparib and ONC206 on apoptosis and the mTOR pathway. LKB1fl/flp53fl/fl mice were fed either a low-fat diet (10% of calories from fat, lean) or high-fat diet (60% of calories from fat, obese). Following EC onset, the mice were treated with ONC206 (100 mg/kg weekly orally), olaparib (25 mg/kg daily orally), or the combination for 4 weeks. Immunohistochemistry was used to assess Ki-67 expression in EC. Results: Both drugs inhibited cell proliferation in a dose-dependent manner (IC50 for olaparib of 15 µM for ECC-1 and 25 µM for HEC-1; IC50 for ONC206 of 3.3 µM for ECC-1 and 0.9 µM for HEC-1). Simultaneous exposure of EC cells to ONC206 and olaparib resulted in synergistic anti-proliferative effects (CI&amp;lt;1). Combination treatment with ONC206 and olaparib increased cellular stress and apoptosis in both cell lines (p=0.05-0.001), compared to either drug alone. Dual therapy with olaparib and ONC206 decreased expression of the anti-apoptotic protein Bcl-2 and decreased phosphorylation of S6, a downstream target of the mTOR pathway, with greater effects compared to either drug alone. The combination of ONC206 and olaparib led to a greater reduction in EC tumor weight in obese (91%) and lean (85%) mice compared to ONC 206 alone (obese 84% and lean 77%) or olaparib alone (obese 83% and lean 65%) (p&amp;lt;.05). ONC206 and olaparib significantly reduced Ki-67 expression in obese and lean mice, with more potent effects for the combination treatment. Conclusions: Olaparib and ONC206 had synergistic anti-tumorigenic effects in human endometrioid EC cell lines and in a mouse model of endometrioid EC, suggesting that this novel dual therapy may be worthy of further exploration in clinical trials in EC. Citation Format: Sarah E. Paraghamian, Gabrielle M. Hawkins, Wenchuan Sun, Yali Fan, Xin Zhang, Hongyan Suo, Varun Vijay Prabhu, Joshua E. Allen, Chunxiao Zhou, Victoria Bae-Jump. A novel dopamine receptor D2 antagonist (ONC206) potentiates the effects of olaparib in endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO044.

Predicting FOXM1-Mediated Gene Regulation through the Analysis of Genome-Wide FOXM1 Binding Sites in MCF-7, K562, SK-N-SH, GM12878 and ECC-1 Cell Lines

Forkhead box protein M1 (FOXM1) is a key transcription factor (TF) that regulates a common set of genes related to the cell cycle in various cell types. However, the mechanism by which FOXM1 controls the common gene set in different cellular contexts is unclear. In this study, a comprehensive meta-analysis of genome-wide FOXM1 binding sites in ECC-1, GM12878, K562, MCF-7, and SK-N-SH cell lines was conducted to predict FOXM1-driven gene regulation. Consistent with previous studies, different TF binding motifs were identified at FOXM1 binding sites, while the NFY binding motif was found at 81% of common FOXM1 binding sites in promoters of cell cycle-related genes. The results indicated that FOXM1 might control the gene set through interaction with the NFY proteins, while cell type-specific genes were predicted to be regulated by enhancers with FOXM1 and cell type-specific TFs. We also found that the high expression level of FOXM1 was significantly associated with poor prognosis in nine types of cancer. Overall, these results suggest that FOXM1 is predicted to function as a master regulator of the cell cycle through the interaction of NFY-family proteins, and therefore the inhibition of FOXM1 could be an attractive strategy for cancer therapy.

Abstract 5314: A novel dopamine receptor D2 antagonist (ONC206) potentiates the effects of olaparib in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway

Abstract Objectives: Poly ADP-ribose polymerase (PARP) inhibitors are effective therapies for patients with homologous recombination (HR) deficient tumors. While HR mutations are rare in endometrial cancers (ECs), emerging data suggests that loss of function of the tumor suppressor gene Phosphatase and Tensin homolog (PTEN), which is mutated in 80% of endometrioid ECs, may also sensitize tumors to PARP inhibition. The imipridone ONC206, a chemical derivative of ONC201, is an orally bioavailable dopamine receptor D2 (DRD2) antagonist that has anti-tumorigenic effects in EC via induction of apoptosis and activation of the integrated stress response. ONC206 exhibits enhanced non-competitive DRD2 antagonism, potency, and bioavailability relative to ONC201. Both olaparib and imipridones (i.e. ONC201) are being evaluated in EC clinical trials, but have yet to be explored in combination. Thus, we sought to examine the effects of olaparib in combination with ONC206 in human endometrioid EC cell lines. Methods: The ECC-1 and HEC-1 EC cell lines were used. Cell proliferation was assessed after exposure to varying doses of olaparib (MedChemExpress) and ONC206 (Oncoceutics) via the MTT assay. Synergy between olaparib and ONC206 was assessed by the combination index (CI) method of Chou-Talalay. Cellular stress was evaluated using the DCFH-DA assay. Apoptosis was assessed using the cleaved caspase-3 assay. Western immunoblotting was performed to determine effects of olaparib and ONC206 on apoptosis and the mTOR pathway. Results: Both drugs inhibited cell proliferation in a dose-dependent manner (IC50 for olaparib of 15 uM for ECC-1 and 25 uM for HEC-1; IC50 for ONC206 of 3.3 uM for ECC-1 and 0.9 uM for HEC-1). Simultaneous exposure of OC cells to ONC206 and olaparib at low doses resulted in synergistic anti-proliferative effects (CI&amp;lt;1). Combination treatment with ONC206 and olaparib at low doses increased cellular stress and apoptosis in both cell lines (p=0.05-0.001), compared to either drug alone. Dual therapy with olaparib and ONC206 decreased expression of the anti-apoptotic protein Bcl-2 and decreased phosphorylation of S6, a downstream target of the mTOR pathway, with greater effects compared to either drug alone. Conclusions: Olaparib and ONC206 had synergistic anti-proliferative effects in human endometrioid EC cell lines, suggesting that this novel dual therapy may be worthy of further exploration in clinical trials in EC. Citation Format: Sarah E. Paraghamian, Gabrielle M. Hawkins, Wenchuan Sun, Yali Fan, Xin Zhang, Hongyan Suo, Varun Vijay Prabhu, Joshua E. Allen, Chunxiao Zhou, Victoria Bae-Jump. A novel dopamine receptor D2 antagonist (ONC206) potentiates the effects of olaparib in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5314.

Soluble guanylyl cyclase \u03b11 subunit is a key mediator of proliferation, survival, and migration in ECC-1 and HeLa cell lines

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme constituted by two subunits, α1 and β1. Previously we have shown that 17β-estradiol (E2) exerts opposite effects on these subunits by increasing α1 and decreasing both β1 expression and enzymatic activity. To date, the physiological relevance of E2-induced sGC subunits’ imbalance has not been addressed. Also, increased levels strongly correlate with E2-induced proliferation in E2-dependent tissues. The aim of the present study was to investigate the role of sGCα1 in proliferation, survival, and migration in two E2-responsive and non-responsive tumour cell lines. Here we showed that E2 stimulated sGCα1 expression in ECC-1 endometrial cancer cells. sGCα1 knock-down significantly reduced E2-dependent cell proliferation. Moreover, sGCα1 silencing caused G1 arrest together with an increase in cell death and dramatically inhibited cell migration. Surprisingly, disruption of sGCα1 expression caused a similar effect even in absence of E2. Confirming this effect, sGCα1 knock-down also augmented cell death and decreased proliferation and migration in E2-unresponsive HeLa cervical cancer cells. Our results show that sGCα1 mediated cell proliferation, survival, and migration in ECC-1 and HeLa cells and suggest that sGCα1 can not only mediate E2-tumour promoting effects but can also be involved in hormone-independent tumour progression.

Picropodophyllin inhibits type I endometrial cancer cell proliferation via disruption of the PI3K/Akt pathway.

The type-I insulin-like growth factor receptor (IGF-IR) is overexpressed in endometrial cancer. High IGF-IR expression was considered as an important prognostic factor for tumor progression. The purpose of this study was to investigate the role and molecular mechanism of IGF-IR inhibitor picropodophyllin (PPP) in the growth and development of endometrial cancer. High expression of IGF-IR was observed in endometrial cancer tissues, as well as in ECC-1 and KLE cell lines. PPP suppressed the number of clones of ECC-1 and KLE cell lines; however, it had no significant effect on HEC-1-A cell line, which expressed lower IGF-IR than ECC-1 and KLE cell lines. Furthermore, PPP reduced cell proliferation capacity, inhibited the IGF-IR mRNA expression, and suppressed protein phosphorylation of IGF-IR and Akt in the three cell lines. In addition, PPP inhibited the protein expression of survivin in KLE cell line after 1h of exposure, though this effect did not last for prolonged time. In conclusion, IGF-IR was mostly overexpressed in type I endometrial cancer. High IGF-IR expression was an important prognostic factor of tumor progression. PPP mediated the down-regulation of IGF-IR phosphorylation and inhibited cell proliferation via the PI3K/Akt signal pathway. PPP may have the potential to become a clinical treatment target in endometrial carcinoma.

Abstract 4916: OCT and MATE transporters mediate uptake and antiproliferative activity of the anticancer drug metformin in endometrial cancer cell lines

Abstract Purpose Metformin, a first-line drug for treatment of Type 2 diabetes, is effective against multiple type of cancers including endometrial cancer. The anticancer effects of metformin, in part, are mediated via activation of its intracellular target adenosine monophosphate-activated protein kinase (AMPK) and subsequent modulation of the downstream signaling of Mammalian Target of Rapamycin (mTOR) and P70S6K. Since metformin is positively charged (pKa 12.4) and hydrophilic (logD -6.13 at pH 6.0) it requires organic cation transporters (OCT) 1-3, plasma membrane monoamine transporter (PMAT) and multidrug and toxin extrusion transporters (MATE) 1 and 2 to mediate its cellular uptake/egress. The goal of this study is to assess the expression of these transporters in the endometrial cancer (EC) cell lines ECC-1, Ishikawa and SPEC-2. Methods EC cells (ECC-1, Ishikawa and SPEC-2) were treated with varying concentrations (0.1 mM-100 mM) of metformin for 72 hours, cell proliferation was assessed by MTT assay. Cellular uptake (5 min) of [14C] metformin at 50μM concentration was measured in the presence or absence of cation-selective transporter inhibitors, 1-methyl-4-phenylpyridinium (MPP+) (500 μM), desipiramine (200 μM) and pyrimethamine (2.4 μM). Expression of 6 cation-selective transporters (OCT 1-3, MATE1, MATE2 and PMAT) and phosphorylation of AMPK and P70S6K was evaluated by Western blot analysis. Results Metformin exhibited dose-dependent anti-proliferative activity against ECC-1, Ishikawa and SPEC-2 cell lines with an IC50 of 4.3, 2.2 and 8.8mM respectively. Protein expression analysis showed that OCT 1-3 were highly expressed in ECC-1 and Ishikawa cells whereas MATE1 and 2 were the predominant in SPEC-2 cells. [14C] metformin uptake into these 3 cell lines was mediated by cation-selective transporters as evidenced by the observation that &amp;gt;80% uptake was inhibited by the pan cation inhibitors MPP+ (500 μM). In the ECC-1 and Ishikawa cell lines, ≈50% of [14C]metformin uptake was inhibited by OCT1-3 and PMAT inhibitor desipiramine (200 μM) and ≈50% of uptake was inhibited by MATE1 and 2 inhibitor pyrimethamine (2.4 μM). In contrast, pyramithamine inhibited 72% of [14C]metformin uptake in SPEC-2 cells. Western blot analysis revealed that 5 µM pyrimethamine reversed metformin-mediated phosphorylation of AMPK and inhibition of pP70S6K in all the EC cell lines. Discussion Our results demonstrate that the OCT 1-3 and MATE 1-2 transporters play an approximately equal role in the uptake of metformin in ECC-1 and Ishikawa cells, whereas MATE 1-2 play a major role in metformin uptake into the SPEC-2 cells. As expected, metformin promoted phosphorylation of AMPK (activation) and attenuated phosphorylation of P70S6K (inhibition) in all of the EC cell lines. This work highlights the importance of transporters in the anti-proliferative activity of metformin in EC cells. Citation Format: Aruljothi Muralidharan, Victoria L. Bae-Jump, Dhiren R. Thakker. OCT and MATE transporters mediate uptake and antiproliferative activity of the anticancer drug metformin in endometrial cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4916.

Tumor-suppressor role of miR-139-5p in endometrial cancer

BackgroundEndometrial cancer (EC) is the fourth most common malignancy of the female genital tract worldwide. MicroRNAs are important gene regulators with critical roles in diverse biological processes, including tumorigenesis. Several study’s show that miR-139-5p is involved in the tumorigenesis and metastasis of various cancers. However, its expression and potential biologic role in endometrial cancer remain to be determined. This study aimed to investigate the miR-139-5p expression and to analyze its function and underlying molecular mechanism in endometrial cancer.MethodsExpression of miR-139-5p was measured using qRT-PCR. The expression of HOXA10 was detected by Immunofluorescence staining in endometrial cancer tissues and adjacent normal tissues. CCK-8 and colony formation assays were used to assess the effect of miR-139-5p on ECC1 and Ishikawa cell line proliferation. Transwell migration assay was used to study the effect of miR-139-5p on EC cell migration. Luciferase reporter assay and western blot were used to confirm targeting of HOXA10 by miR-139-5p.ResultWe demonstrated that miR-139-5p was down-regulated in human endometrial cancer compared to their matched adjacent non-tumor tissues. Overexpressed miR-139-5p significantly inhibited endometrial cancer cell viability and migration. Computational algorithm in combination with dual luciferase reporter assays identified HOXA10 as the target of miR-139-5p. HOXA10 expression was downregulated in endometrial cancer cells after miR-139-5p overexpression. The expression level of HOXA10 was significantly increased in endometrial cancer tissues, which was inversely correlated with miR-139-5p expression in clinical endometrial cancer tissues.ConclusionThese findings indicate that miR-139-5p targets the HOXA10 transcript and suppresses endometrial cancer cell growth and migration, suggesting that miR-139-5p acts as a tumor suppressive role in human endometrial cancer pathogenesis.

High throughput silencing identifies novel genes in endometrioid endometrial cancer

ObjectiveTo validate the gene expression profile obtained from the previous microarray analysis and to further study the biological functions of these genes in endometrial cancer. From our previous study, we identified 621 differentially expressed genes in laser-captured microdissected endometrioid endometrial cancer as compared to normal endometrial cells. Among these genes, 146 were significantly up-regulated in endometrial cancer. Materials and MethodsA total of 20 genes were selected from the list of up-regulated genes for the validation assay. The qPCR confirmed that 19 out of the 20 genes were up-regulated in endometrial cancer compared with normal endometrium. RNA interference (RNAi) was used to knockdown the expression of the upregulated genes in ECC-1 and HEC-1A endometrial cancer cell lines and its effect on proliferation, migration and invasion were examined. ResultsKnockdown of MIF, SOD2, HIF1A and SLC7A5 by RNAi significantly decreased the proliferation of ECC-1 cells (p < 0.05). Our results also showed that the knockdown of MIF, SOD2 and SLC7A5 by RNAi significantly decreased the proliferation and migration abilities of HEC-1A cells (p < 0.05). Moreover, the knockdown of SLC38A1 and HIF1A by RNAi resulted in a significant decrease in the proliferation of HEC1A cells (p < 0.05). ConclusionWe have identified the biological roles of SLC38A1, MIF, SOD2, HIF1A and SLC7A5 in endometrial cancer, which opens up the possibility of using the RNAi silencing approach to design therapeutic strategies for treatment of endometrial cancer.

GnRH-(1–5) activates matrix metallopeptidase-9 to release epidermal growth factor and promote cellular invasion

In the extracellular space, the gonadotropin-releasing hormone (GnRH) is metabolized by the zinc metalloendopeptidase EC3.4.24.15 (EP24.15) to form the pentapeptide, GnRH-(1–5). GnRH-(1–5) diverges in function and mechanism of action from GnRH in the brain and periphery. GnRH-(1–5) acts on the orphan G protein-coupled receptor 101 (GPR101) to sequentially stimulate epidermal growth factor (EGF) release, phosphorylate the EGF receptor (EGFR), and facilitate cellular migration. These GnRH-(1–5) actions are dependent on matrix metallopeptidase (MMP) activity. Here, we demonstrated that these GnRH-(1–5) effects are dependent on increased MMP-9 enzymatic activity in the Ishikawa and ECC-1 cell lines. Furthermore, the effects of GnRH-(1–5) mediated by GPR101 and the subsequent increase in MMP-9 enzymatic activity lead to an increase in cellular invasion. These results suggest that GnRH-(1–5) and GPR101 regulation of MMP-9 may have physiological relevance in the metastatic potential of endometrial cancer cells.

The role of WWOX tumor suppressor gene in the regulation of EMT process via regulation of CDH1-ZEB1-VIM expression in endometrial cancer.

This study defines the role of WWOX in the regulation of epithelial to mesenchymal transition. A group of 164 endometrial adenocarcinoma patients was studied as well as an ECC1 well-differentiated steroid-responsive endometrial cell line, which was transducted with WWOX cDNA by a retroviral system. The relationship between WWOX gene and EMT marker (CDH1, VIM, ZEB1, SNAI1) expression on mRNA (RT-qPCR) and protein levels (western blotting) was evaluated. The EMT processes were also analysed in vitro by adhesion of cells to extracellular matrix proteins, migration through a basement membrane, anchorage-independent growth and MMP activity assay. DNA microarrays (HumanOneArray™) were used to determine WWOX-dependent pathways in an ECC1 cell line. A positive correlation was observed between WWOX and ZEB1, and a negative correlation between CDH1 and VIM. WWOX expression was found to inversely correlate with the risk of recurrence of tumors in patients. However, in the WWOX-expressing ECC1 cell line, WWOX expression was found to be inversely related with VIM and positively with CDH1. The ECC1/WWOX cell line variant demonstrated increased migratory capacity, with increased expression of metalloproteinases MMP2/MMP9. However, these cells were not able to form colonies in suspension and revealed decreased adhesion to fibronectin and fibrinogen. Microarray analysis demonstrated that WWOX has an impact on the variety of cellular pathways including the cadherin and integrin signalling pathways. Our results suggest that the WWOX gene plays a role in the regulation of EMT processes in endometrial cancer by controlling the expression of proteins associated with cell motility, thus influencing tissue remodeling, with the suppression of mesenchymal markers.

Abstract 1312: Buformin, an anti-diabetic biguanide, inhibits proliferation, invasion and adhesion, and acts synergistically with paclitaxel in endometrial cancer cell lines

Abstract Objectives: Epidemiologic and pre-clinical studies suggest that anti-diabetic biguanide drugs, such as metformin and buformin, may have anti-tumorigenic effects and behave as chemosensitizers. Thus, we evaluated the effects of buformin alone and in combination with paclitaxel on proliferation in human endometrial cancer (EC) cell lines as well as the impact of buformin on apoptosis and adhesion/invasion. Methods: The ECC-1 and Ishikawa EC cell lines were treated with varying doses of buformin alone and in combination with paclitaxel. Cell growth was determined by MTT assay. Cell cycle progression was assessed by Cellometer. Apoptosis was evaluated by Annexin V-FITC assay. Invasion was demonstrated by transwell invasion assay. Adhesion was assessed by ELISA. Effects on AMPK and S6 expression were documented by Western blotting. Results: Buformin inhibited proliferation in a dose-dependent manner in the EC cell lines (IC50 1000 μM for ECC-1, 10μM for Ishikawa, p=0.00001-0.0010). Treatment with buformin resulted in G1 arrest in the ECC-1 cell line and G2 arrest in the Ishikawa cell line. Buformin induced apoptosis in the Ishikawa cell line, but not in the ECC-1 cell line. Treatment with buformin resulted in inhibition of adhesion (p=0.42-0.0009) and invasion (p=0.00001-0.227) in both EC cell lines. Western blotting analysis demonstrated that buformin increased phosphorylation of AMPK and decreased phosphorylation of S6. Paclitaxel inhibited proliferation in a dose-dependent manner in both cell lines with IC50 values of 0.1-0.5 nM and 1-5 nM for Ishikawa and ECC-1 cells, respectively. To assess synergy of paclitaxel and buformin, the combination index (CI) was calculated by the method of Chou and Talalay. Simultaneous exposure of cells to various doses of paclitaxel in combination with buformin (0.1-1 μM) resulted in a significant synergistic anti-proliferative effect (CI &amp;lt;1, range 0.009-0.85). Conclusions: Buformin inhibited proliferation in EC cell lines, via AMPK activation and mTOR pathway inhibition. Inhibition of adhesion/invasion was also seen with buformin. Buformin acted synergistically with paclitaxel to inhibit cell proliferation. More work is needed to determine if buformin and other biguanides alone or in combination with cytotoxic agents will be beneficial in the treatment of women with EC. Citation Format: Joshua Kilgore, Amanda Jackson, Haifeng Qiu, Chunxiao Zhou, Paola Gehrig, Victoria Bae-Jump. Buformin, an anti-diabetic biguanide, inhibits proliferation, invasion and adhesion, and acts synergistically with paclitaxel in endometrial cancer cell lines. [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 1312. doi:10.1158/1538-7445.AM2014-1312

Simvastatin, an HMG-CoA reductase inhibitor, exhibits anti-metastatic and anti-tumorigenic effects in endometrial cancer

ObjectiveOur goal was to evaluate the effects of simvastatin on endometrial cancer cell lines and primary cultures of endometrial cancer cells. MethodsCell proliferation in the ECC-1 and Ishikawa endometrial cancer cell lines and primary cultures of endometrial cancer cells was assessed by MTT assay. Apoptosis and cell cycle were detected by Annexin V assay and propidium iodide staining, respectively. Reactive oxygen species and cell adhesion were assessed using ELISA assays. Invasion was analyzed using a transwell invasion assay. Mitochondrial DNA damage was confirmed using qPCR. The effects of simvastatin on the AKT/mTOR and MAPK pathways were determined by Western blotting. ResultsSimvastatin inhibited cell proliferation in a dose-dependent manner in both endometrial cancer cell lines and 5/8 primary cultures of endometrial cancer cells. Simvastatin treatment resulted in G1 cell cycle arrest, a reduction in the enzymatic activity of HMG-CoA, induction of apoptosis as well as DNA damage and cellular stress. Treatment with simvastatin resulted in inhibition of the MAPK pathway and exhibited differential effects on the AKT/mTOR pathway in the ECC-1 and Ishikawa cells. Minimal change in AKT phosphorylation was seen in both cell lines. An increase in phosphorylated S6 was seen in ECC-1 and a decrease was seen in Ishikawa. Treatment with simvastatin reduced cell adhesion and invasion (p<0.01) in both cell lines. ConclusionSimvastatin had significant anti-proliferative and anti-metastatic effects in endometrial cancer cells, possibly through modulation of the MAPK and AKT/mTOR pathways, suggesting that statins may be a promising treatment strategy for endometrial cancer.

Gene expression profile of endometrial carcinoma cells exposed to di-(2-ethylhexyl) phthalate

To evaluation of the contribution of DEHP (di-(2-ethylhexyl) phthalate) to the pathophysiology of endometriosis, ECC-1 (human endometrial cancer) cell lines were subjected to DEHP for 48 h with 50 μM DEHP and total RNA was extracted. Quantified spectro-photometrically, and RNA quality was verified via agarose-gel electrophoresis. Gene expression changes were analyzed using oligonucleotide microarray hybridization by comparing the treated and control groups (two-fold change, P<0.05). Three independent samples were analyzed. Data indicated that 522 genes were up-regulated and 379 were down-regulated. These genes were classified according to the GO terms of biological processes and the KEGG pathway. Among biologic processes, regulation of cell death, lipid metabolism, gluconeogenesis, response to hormone stimulus, and regulation of cell differentiation were positively associated with up-regulated genes. Mitosis, meiosis, DNA damage check point, anatomical structure development, reproductive development, and regulation of protein ubiquitination were significantly associated with down-regulated genes. In the KEGG pathway analysis, the MAPK, VEGF, p53, Erbb, mTOR, and phosphatidylinositol signaling pathways were positively associated with up-regulated genes. The cell cycle, oocyte meiosis, spliceosome, and pyrimidine metabolism were positively associated with downregulated genes. In conclusion, After DEHP treatment, some genes associated with cell growth, cell differentiation, meiosis, migration, apoptosis, and the pathophysiology of endometriosis were up regulated.

Tamoxifen Represses miR-200 MicroRNAs and Promotes Epithelial-to-Mesenchymal Transition by Up-Regulating c-Myc in Endometrial Carcinoma Cell Lines

Although tamoxifen (TAM), a selective estrogen receptor modulator, has been widely used in the treatment of hormone-responsive breast cancer, its estrogen-like effect increases the risk of endometrial cancer. However, the molecular mechanisms of TAM-induced endometrial carcinoma still remain unclear. In this report, we explored the role of microRNAs (miRNAs) in TAM-induced epithelial-mesenchymal transition (EMT) in ECC-1 and Ishikawa endometrial cancer cell lines and found miR-200 is involved in this process via the regulation of c-Myc. When treated with TAM, ECC-1 and Ishikawa cells were characterized by higher invasiveness and motility and underwent EMT. miR-200, a miRNA family with tumor suppressive functions in a wide range of cancers, was found reduced in response to TAM treatment. Consistent with zinc finger E-box binding homeobox 2, which was confirmed as a direct target of miR-200b in endometrial cancer cell lines, some other key factors of EMT such as Snail and N-cadherin increased, whereas E-cadherin decreased in the TAM-treated cells, contributing to TAM-induced EMT in these endometrial cancer cells. In addition, we showed that c-Myc directly binds to and represses the promoter of miR-200 miRNAs, and its up-regulation in TAM-treated endometrial cancer cells leads to the down-regulation of miR-200 and eventually to EMT. Collectively, our data suggest that TAM can repress the miR-200 family and induce EMT via the up-regulation of c-Myc in endometrial cancer cells. These findings describe a possible mechanism of TAM-induced EMT in endometrial cancer and provide a potential new therapeutic strategy for it.