KLE Cells Research Articles

Nestin suppression attenuates invasive potential of endometrial cancer cells by downregulating TGF-β signaling pathway.

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 G0/G1 phase of the cell cycle, whereas overexpression in Ishikawa decreased cells in G0/G1 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.

MiR-10b Inhibits Apoptosis and Promotes Proliferation and Invasion of Endometrial Cancer Cells via Targeting HOXB3.

MicroRNAs are small RNA that are tightly interrelated with the initiation, development, and metastasis of cancers. Studies have shown that miR-10b is increased in various cancers. However, the underlying mechanisms of miR-10b in the occurrence and metastasis of endometrial cancer are poorly understood. To investigate its roles and correlations with Homeobox box 3 (HOXB3) in endometrial cancer, cancer tissues and adjacent normal endometrium tissues from 20 patients with endometrial cancer were studied. miR-10b expression was significantly up-regulated (p < 0.01) in endometrial cancer tissue, whereas HOXB3 was lowly expressed. The silence of miR-10b resulted in significantly enhanced cell apoptosis, and remarkably reduced cell proliferation, migration, and invasion (p < 0.05). Moreover, the protein levels of HOXB3 were increased in KLE cells with silenced miR-10b, and dual-luciferase reporter assay suggested that miR-10b could directly target HOXB3. Furthermore, overexpression of HOXB3 promoted cell apoptosis but inhibited cell proliferation, migration, and invasion (p < 0.01). To conclude, miR-10b might control cell apoptosis, proliferation, migration, and invasion in endometrial cancer via regulation of HOXB3 expression.

Visfatin stimulates endometrial cancer cell proliferation via activation of PI3K/Akt and MAPK/ERK1/2 signalling pathways

ObjectiveEndometrial carcinoma is one of the most common malignancies of the female reproductive system, but the aetiology and pathogenesis are not well understood, although adipokines such as visfatin may be involved. Our study provides insight into the mechanism underlying the tumorigenic effects of visfatin in endometrial carcinoma. MethodsWe investigated the effect of visfatin on endometrial carcinoma cell proliferation, cell cycle, and apoptosis using well-differentiated Ishikawa cells and poorly differentiated KLE cells. We also assessed the effect of visfatin on tumour growth in vivo. ResultsVisfatin stimulated the proliferation of both Ishikawa and KLE cells, and visfatin treatment promoted G1/S phase progression and inhibited endometrial carcinoma cell apoptosis. Visfatin promoted endometrial carcinoma tumour growth in BALB/c-nu mice. Transplanted tumour tissues from an endometrial carcinoma mouse model were analysed using immunohistochemical staining, which revealed much stronger positive signals for Ki-67 with over-abundant visfatin. Western blot analysis revealed that insulin receptor (IR), insulin receptor substrate (IRS)1/2 and key components of the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)1/2 signalling pathways were highly expressed in endometrial carcinoma cells exposed to visfatin. Treated cells showed increased C-MYC and cyclin D1 and reduced caspase-3 expression. The effects of visfatin on proliferation and apoptosis were abrogated by treatment with the PI3K inhibitor LY294002 and MEK inhibitor U0126. ConclusionsVisfatin promotes the malignant progression of endometrial carcinoma via activation of IR and PI3K/Akt and MAPK/ERK signalling. Visfatin may serve as a therapeutic target in the treatment of endometrial carcinoma.

Abstract 2994: Orlistat decreases endometrial cancer cell proliferation: implications for a novel treatment strategy

Abstract Objectives: Fatty acid synthase (FAS) is a key enzyme involved in fatty acid synthesis. It also plays an important role in tumorigenesis and is highly expressed in many cancers, including endometrial cancer. Orlistat is a weight loss medication that has been shown to be a potent inhibitor of FAS. The goal of this study was to evaluate the anti-tumorigenic potential of orlistat in endometrial cancer cell lines. Methods: The endometrial cancer cell lines ECC-1 and KLE were treated with varying doses of orlistat (0.01-500 μM). Cell proliferation was assessed using the MTT assay. Cell cycle progression was evaluated by Cellometer and apoptosis was assessed using the Annexin V assay. Reactive oxygen species (ROS) was measured using the DCFH-DA assay. Western immunoblotting was performed to determine changes in FAS, cellular stress, cell cycle progression and the AMPK/mTOR pathways. Results: Orlistat inhibited cell proliferation in a dose-dependent manner in both cell lines (IC50 = 81.7 μM in ECC-1; IC50 = 145.5 μM in KLE). Treatment with orlistat resulted in G1 arrest but did not affect apoptosis. Orlistat increased ROS at high doses (500 uM, p = 0.008 for ECC-1 cells and p = 0.012 for KLE cells) and induced expression of BIP and PERK. Western immunoblot analysis demonstrated that orlistat decreased expression of FAS, acetyl-CoA carboxylase (ACC), and carnitine palmitoyltransferase 1A (CPT1A), important proteins in fatty acid metabolism. Treatment with orlistat also increased expression of phosphorylated AMPK and decreased expression of the downstream S6 protein. Conclusions: Orlistat suppressed cell growth in endometrial cancer cell lines through inhibition of fatty acid metabolism, induction of cell cycle G1 arrest, and modulation of the AMPK/mTOR pathway. Given that patients with endometrial cancer have high rates of obesity, orlistat should be further investigated as a novel strategy for endometrial cancer treatment and prevention. Citation Format: Weiya Z. Wysham, Dario R. Roque, Jianjun Han, Hui Guo, Lu Zhang, Paola A. Gehrig, Chunxiao Zhou, Victoria L. Bae-Jump. Orlistat decreases endometrial cancer cell proliferation: implications for a novel treatment strategy. [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 2994.

Abstract 4428: Nestin suppression attenuates invasive potential of endometrial cancer cells by down regulating TGF-β signaling pathway

Abstract 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 reduced PDCD5 protein is correlated with the degree of tumor differentiation in endometrioid endometrial carcinoma.

Endometrial cancer is one of the most common malignancies in the female genital tract. Programmed cell death 5 (PDCD5) is a newly identified apoptosis related gene and plays an important role in the development of some human tumors. However, the expression and clinical significance of PDCD5 in endometrial cancer have not been fully elucidated. Here, we evaluated the expression of PDCD5 in endometrioid endometrial carcinoma and control endometrium by qRT-PCR, western blot and immunohistochemistry, and analyzed the associations of PDCD5 expression with clinicopathological parameters of patients. In addition, we detected the expression of PDCD5 in control endometrial glandular epithelial cells and endometrioid endometrial carcinoma-derived cell line KLE by immunocytochemistry. The results showed that PDCD5 protein mainly expressed in the cytoplasm of glandular epithelial cells and endometrial carcinoma cells, and there was a low level of PDCD5 expression in the nuclei of the above cells. Furthermore, PDCD5 protein level was significantly lower in endometrial carcinoma samples than that in control endometrium. The decreased PDCD5 expression was correlated with the tumor differentiation degree. It is clear that PDCD5 protein expression was lower in middle and low differentiated endometrial carcinoma compared with control endometrium and high differentiated endometrial carcinoma. However, there were no significant differences of PDCD5 expression between the proliferative phase and the secretory phase of control endometrium, as well as between high differentiated endometrial carcinoma and controls. The results were verified in control glandular epithelial cells and KLE cells by immunocytochemistry. Therefore, PDCD5 may play a key role in the pathogenesis of endometrial cancer and may be a novel target for diagnosis and treatment of endometrial cancer.

SPAG9 promotes endometrial carcinoma cell invasion through regulation of genes related to the epithelial-mesenchymal transition.

To investigate the impact of sperm-associated antigen 9 (SPAG9) on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in endometrial cancer. The present authors' previous study demonstrated that SPAG9 is highly expressed in endometrial cancer tissues. They analyzed correlation between the levels of SPAG9 and mRNA of EMT-related genes in endometrial carcinoma tissue by using quantitative real-time PCR. They induced EMT process in ECC endometrial cancer cell lines by TGF-beta1 treatment and spheroids formation assay, and analyzed SPAG9 expression as well as correlation with EMT-related genes. In addition, they performed SPAG9 gene silencing in KLE and ECC endometrial cancer cells and evaluated the expression of genes involved in EMT, using real time PCR and Western blot analysis. Cell proliferation, colony formation, and transwell assays were employed to evaluate the functional role of SPAG9 in endometrial cancer. The results showed that SPAG9 expression was positively correlated with Slug and N-cadherin (NcaD) in human endometrial cancer tissues. The expression of SPAG9 in ECC cells with TGF-β1 treatment and spheroids formation was increased, which was correlated with EMT-related genes. SPAG9 knockdown significantly inhibited cell growth and proliferation and reduced the motility and invasion of endometrial cancer cells. These phenotypes may partly be explained by decreased expression of EMT-related genes, including Twist, Slug, and Vimentin, after SPAG9 depletion. SPAG9 may be required for cellular invasion and migration in endometrial cancer through regulation of EMT-related genes.

Activin B promotes endometrial cancer cell migration by down-regulating E-cadherin via SMAD-independent MEK-ERK1/2-SNAIL signaling

High-risk type II endometrial cancers account for ~30% of cases but ~75% of deaths due, in part, to their tendency to metastasize. Histopathological studies of type II endometrial cancers (non-endometrioid, mostly serous) suggest overproduction of activin B and down-regulation of E-cadherin, both of which are associated with reduced survival. Our previous studies have shown that activin B increases the migration of type II endometrial cancer cell lines. However, little is known about the relationship between activin B signaling and E-cadherin in endometrial cancer. We now demonstrate that activin B treatment significantly decreases E-cadherin expression in both a time- and concentration-dependent manner in KLE and HEC-50 cell lines. Interestingly, these effects were not inhibited by knockdown of SMAD2, SMAD3 or SMAD4. Rather, the suppressive effects of activin B on E-cadherin were mediated by MEK-ERK1/2-induced production of the transcription factor SNAIL. Importantly, activin B-induced cell migration was inhibited by forced-expression of E-cadherin or pre-treatment with the activin/TGF-β type I receptor inhibitor SB431542 or the MEK inhibitor U0126. We have identified a novel SMAD-independent pathway linking enhanced activin B signaling to reduced E-cadherin expression and increased migration in type II endometrial cancer.

Effects of Fatty Acid Synthase Inhibition by Orlistat on Proliferation of Endometrial Cancer Cell Lines.

Fatty acid synthase (FAS) is a key lipogenic enzyme that is highly expressed in endometrial cancer. Orlistat is a weight loss medication that has been shown to be a potent inhibitor of FAS. The goal of this study was to evaluate the anti-tumorigenic potential of orlistat in endometrial cancer cell lines. The endometrial cancer cell lines ECC-1 and KLE were used. Cell proliferation was assessed by MTT assay after treatment with orlistat. Cell cycle progression was evaluated by Cellometer and apoptosis was assessed using the Annexin V assay. Reactive oxygen species (ROS) was measured using the DCFH-DA assay. Western immunoblotting was performed to determine changes in FAS, cellular stress, cell cycle progression, and the AMPK/mTOR pathways. Orlistat inhibited cell proliferation by 61% in ECC-1 cells and 57% in KLE cells at a dose of 500μM. Treatment with orlistat at this concentration resulted in G1 arrest (p < 0.05) but did not affect apoptosis. Orlistat increased ROS and induced the expression of BIP (1.28-fold in ECC-1 compared to control, p < 0.05; 1.92-fold in KLE, p < 0.05) and PERK (2.25-fold in ECC-1, 1.4-fold in KLE, p < 0.05). Western immunoblot analysis demonstrated that orlistat decreased expression of important proteins in fatty acid metabolism including FAS (67% in ECC-1, 15% in KLE), acetyl-CoA carboxylase (40% in ECC-1, 35% in KLE), and carnitine palmitoyltransferase 1A (CPT1A) (65% in ECC-1, 25% in KLE) in a dose-dependent manner. In addition, orlistat at a dose of 500μM increased expression of phosphorylated-AMPK (1.9-fold in ECC-1, p < 0.01; 1.5-fold in KLE, p < 0.05) and decreased expression of phosphorylated-Akt (25% in ECC-1, p < 0.05; 37% in KLE, p < 0.05) and phosphorylated-S6 (68% in ECC-1, 56% in KLE). Orlistat inhibits cell growth in endometrial cancer cell lines through inhibition of fatty acid metabolism, induction of cell cycle G1 arrest, activation of AMPK and inhibition of the mTOR pathway. Given that patients with endometrial cancer have high rates of obesity, orlistat should be further investigated as a novel strategy for endometrial cancer treatment.

Metformin inhibits 17β-estradiol-induced epithelial-to-mesenchymal transition via βKlotho-related ERK1/2 signaling and AMPKα signaling in endometrial adenocarcinoma cells.

The potential role of metformin in treating endometrial cancer remains to be explored. The current study investigated the role of metformin in 17β-estradiol-induced epithelial-mesenchymal transition (EMT) in endometrial adenocarcinoma cells. We found that 17β-estradiol promoted proliferation and migration, attenuated apoptosis in both estrogen receptor (ER) positive and ER negative endometrial adenocarcinoma cells (Ishikawa and KLE cells, respectively). Metformin abolished 17β-estradiol-induced cell proliferation and reversed 17β-estradiol-induced EMT in Ishikawa cells. In addition, metformin increased the expression of βKlotho, a fibroblast growth factors (FGFs) coreceptor, and decreased ERK1/2 phosphorylation in both Ishikawa and KLE cells. Decreased expression of βKlotho was noted in human endometrial adenocarcinomas, and plasmid-driven expression of βKlotho in Ishikawa cells abolished 17β-estradiol-induced EMT via inhibiting ERK1/2 signaling. βKlotho expression and metformin show synergetic effects on the proliferation and the EMT in Ishikawa cells. Furthermore, we demonstrated that the anti-EMT effects of metformin could be partly abolished by introducing Compound C, a specific AMPKα signaling inhibitor. In conclusion, metformin abolishes 17β-estradiol-induced cell proliferation and EMT in endometrial adenocarcinoma cells by upregulating βKlotho expression, inhibiting ERK1/2 signaling, and activating AMPKα signaling. Our study provides novel mechanistic insight into the anti-tumor effects of metformin.

Progestin treatment decreases CD133 + cancer stem cell populations in endometrial cancer

ObjectivesEndometrial cancer is a hormonally responsive malignancy. Response to progestins is associated with estrogen receptor (ER) and progesterone receptor (PR) status. CD133 is a marker of endometrial cancer stem cells. We postulated that CD133+ cells express ER and PR and that progestin therapy differentially regulates CD133+ cells. MethodsThe Ishikawa (ER/PR positive) and KLE (ER/PR negative) cell lines were examined for the presence of CD133 populations. Cell lines were treated with 30.4μM medroxyprogesterone 17-acetate (MPA) for 6days. After treatment, cell counts, apoptosis assays and CD133+ populations were examined. In a clinical project, we identified 12 endometrial cancer patients who were treated with progestin drugs at our institution. Using immunohistochemistry, CD133, ER, PR, and androgen receptor (AR) expression was scored and evaluated for change over time on serial biopsies. ResultsCD133+ populations were identified in Ishikawa and KLE cell lines. MPA treatment resulted in a significant reduction in the percentage of live cells (Ishikawa, P=0.036; KLE, P=0.0002), significant increase in apoptosis (Ishikawa, P=0.01; KLE, P=0.0006) and significant decrease in CD133+ populations (Ishikawa, P<0.0001; KLE, P=0.0001). ER, PR, AR and CD133 were present in 96.4%, 96.4%, 89.3% and 100% of patient samples respectively. Paralleling the in vitro results, CD133 expression decreased in patients who had histologic response to progestin treatment. ConclusionCD133+ populations decreased after treatment with MPA in an in vitro model and in patients responding to treatment with progestins. Progestin treatment differentially decreases CD133+ cells.

Effect of the molecular targeted drug, erlotinib, against endometrial cancer expressing high levels of epidermal growth factor receptor.

BackgroundThe epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib, has been clinically applied for the treatment of a variety of tumors with EGFR overexpression. A phase II clinical study of erlotinib (NCIC IND-148) for recurrent or metastatic endometrial carcinoma (EC) resulted in an unfavorable result. However, in that study, the expression levels of EGFR were not accurately analyzed. Thus, the aim of this study was to re-examine the efficacy of erlotinib in EC cells by utilizing in vitro and in vivo models.MethodsTissue samples obtained from patients histologically diagnosed with EC of the uterine corpus were subjected to immunohistochemistry and RT-PCR to determine the protein and mRNA expression levels of EGFR. Western blot and WST-1 assays of EGFR siRNA-transfected HEC-1A, KLE, and Ishikawa cells were used to evaluate the efficacy of erlotinib in tumor cell lines expressing different EGFR levels. Furthermore, HEC-1A and Ishikawa cells were implanted into athymic mice treated with either erlotinib or trastuzumab.ResultsAt our institution, 20.9 % of endometrial cancer patients with low grade endometrioid histology have been diagnosed as stage III and IV. Immunohistochemical analysis and RT-PCR revealed the presence of significant EGFR and EGFR mRNA expression in low-grade endometrioid carcinoma in comparison with high-grade endometrioid carcinoma. In vitro study, WST-1 assay and Western blot analysis revealed that EGFR expression levels were correlated with tumor cell viability. Erlotinib reduced the proliferation of HEC-1A expressing high levels of EGFR, while trastuzumab showed similar effect in Ishikawa cells dominantly expressing human epidermal growth factor receptor type2 (HER2). In vivo erlotinib decreased tumor growth in mice xenografted with HEC-1A cells, whereas this tumor-growth inhibition was not observed in trastuzumab-treated mice xenografted with Ishikawa cell.ConclusionsEGF contributed to tumor proliferation in EC cell lines along with EGFR expression in vitro. Erlotinib also demonstrated anti-tumor effects in xenograft mice models. Our results suggest that erlotinib continues to have clinical usefulness in specific cases, after taking into consideration the EGFR expression levels.

Mechanism of thioridazine plus medroxyprogesterone in the treatment of endometrial cancer

To explore the in vitro effects of thioridazine (THIO) plus medroxyprogesterone (MPA) on the proliferation and apoptosis of endometrial cancer cell lines (Ishikawa & KLE) and examine the mechanism in the treatment of endometrial cancer. CCK-8 assay was employed for detecting the influence of THIO plus MPA on the proliferation and apoptosis of endometrial cancer cells (ISK & KLE). And the concentration and timepoints of drugs were determined according to the results. Real-time polymerase chain reaction (PCR) and Western blot were used to detect the expression levels of PRB, DRD2 and p-AKT/AKT in PI3K/AKT signal pathway. Flow cytometry was applied for detecting the apoptosis of combination (THIO+MPA) and MPA groups. Compared to MPA group, the proliferation inhibiting effect of combination group increased significantly in ISK and KLE cells (52.5% ± 2.6% vs 37.3% ± 0.3%, P < 0.05; 97.7% ± 0.7% vs 50.0% ± 0.4%, P < 0.001); the apoptotic rates increased (34.0% ± 1.4% vs 50.5% ± 2.4%, P < 0.01; 5.5% ± 3.6% vs 11.3% ± 0.7%, P < 0.01); the expression levels of PRB and DRD2 were up-regulated (all P < 0.05). And the ratio of p-AKT/AKT decreased obviously. Thioridazine significantly enhances the effects of MPA on proliferative inhibition and apoptotic promotion in endometrial cancer cells. And it may be associated with the PRB/DRD2-mediated PI3K/AKT signal pathway.

Fisetin induces G2/M phase cell cycle arrest by inactivating cdc25C-cdc2 via ATM-Chk1/2 activation in human endometrial cancer cells

&lt;p&gt;Endometrial cancer is one of the most prevalent gynaecological malignancies where, currently available therapeutic options remain limited. Recently phytochemicals are exploited for their efficiency in cancer therapy. The present study investigates the anti-proliferative effect of fisetin, a flavonoid on human endometrial cancer cells (KLE and Hec1 A). Fisetin (20-100 µM) effectively reduced the viability of Hec1 A and KLE cells and potentially altered the cell population at G2/M stage. Expression levels of the cell cycle proteins (cyclin B1, p-Cdc2, p-Cdc25C, p-Chk1, Chk2, p-ATM, cyclin B1, H2AX, p21 and p27) were analyzed. Fisetin suppressed cyclin B1 expression and caused inactiva-tion of Cdc25C and Cdc2 by increasing their phosphorylation levels and further activated ATM, Chk1 and Chk2. Increased levels of p21 and p27 were observed as well. These results suggest that fisetin induced G2/M cell cycle arrest via inactivating Cdc25c and Cdc2 through activation of ATM, Chk1 and Chk2.&lt;/p&gt;&lt;p&gt; &lt;/p&gt;&lt;p&gt; &lt;/p&gt;

Stimulation of peroxisome proliferator-activated receptor γ inhibits estrogen receptor α transcriptional activity in endometrial carcinoma cells.

Peroxisome proliferator-activated receptor γ (PPARγ) and estrogen receptor (ER) belong to a family of nuclear hormone receptors that have been demonstrated to affect each other's transcriptional activity. At present, little is known regarding the effect of PPARγ on ER-mediated transcriptional activity in endometrial carcinoma. In the present study, we aimed to demonstrate the correlation between PPARγ and ER in endometrial carcinoma and to elucidate the biological effects of abnormal expression of PPARγ on endometrial carcinoma cell lines. Immunohistochemical and western blotting methods were used to detect the expression of PPARγ, ERα and ERβ in normal and malignant endometrium. Next, we performed transient transfection to assess the interaction between PPARγ and ER in vitro. Furthermore, we examined cell migration, invasion and proliferation as a biological counterpart. PPARγ and ERα expression levels were significantly associated with pathological grade and clinical stage in endometrial carcinoma (P<0.05). Pearson correlation analysis revealed that PPARγ expression was positively correlated with ERα expression (P<0.05). Using KLE and ERα-positive cells (ECC-1), we demonstrated that the PPARγ regulation of ER expression occurred predominantly through ERα. Moreover, our findings suggest that PPARγ activation inhibited the migration, invasion and proliferation of endometrial carcinoma cells; ECC-1 cells were more sensitive to this inhibition. The present study demonstrated that PPARγ activation inhibited ERα expression in ERα-positive endometrial carcinoma cell lines. This crosstalk may facilitate the development of novel therapeutic methods targeting PPARγ in endometrial carcinoma treatment, particularly ERα-positive carcinomas.

Reversible inhibition of lysine specific demethylase 1 is a novel anti-tumor strategy for poorly differentiated endometrial carcinoma.

BackgroundEndometrial cancer is the most common gynecologic malignancy. Type II endometrial carcinoma is often poorly differentiated and patients diagnosed with Type II disease (~11%) are disproportionately represented in annual endometrial cancer deaths (48%). Recent genomic studies highlight mutations in chromatin regulators as drivers in Type II endometrial carcinoma tumorigenesis, suggesting the use of epigenetic targeted therapies could provide clinical benefit to these patients. We investigated the anti-tumor efficacy of the LSD1 inhibitor HCI2509 in two poorly differentiated Type II endometrial cancer cell lines AN3CA and KLE.MethodsThe effects of HCI2509 on viability, proliferation, anchorage-independent growth, global histone methylation, LSD1 target gene induction, cell cycle, caspase activation and TUNEL were assayed. KLE cells were used in an orthotopic xenograft model to assess the anti-tumor activity of HCI2509.ResultsBoth AN3CA and KLE cells were sensitive to HCI2509 treatment with IC50s near 500 nM for cell viability. Inhibition of LSD1 with HCI2509 caused decreased proliferation and anchorage independent growth in soft agar, elevated global histone methylation, and perturbed the cell cycle in both cell lines. These effects were largely dose-dependent. HCI2509 treatment also caused apoptotic cell death. Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen. Tumor burden was distributed log-normally. Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p = 0.034).ConclusionsOur findings demonstrate the anti-cancer properties of the LSD1 inhibitor HCI2509 on poorly differentiated endometrial carcinoma cell lines, AN3CA and KLE. HCI2509 showed single-agent efficacy in orthotopic xenograft studies. Continued studies are needed to preclinically validate LSD1 inhibition as a therapeutic strategy for endometrial carcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-752) contains supplementary material, which is available to authorized users.

Abstract 4693: Inhibition of BET bromodomain targets PTEN positive endometrioid endometrial cancers

Abstract Introduction: Endometrioid endometrial cancers (EEC) are commonly characterized by PTEN mutations and loss of function. JQ-1 is a selective small-molecule BET bromodomain inhibitor, and BET inhibition by JQ-1 leads to the downregulation of c-Myc transcription. Thus, JQ-1 functions as a novel c-Myc inhibitor and has been found to potently suppress tumor growth in many human cancers. Given the known interactions between c-Myc and the PI3K/Akt/mTOR pathway, we aim to assess the impact of JQ-1 on human EEC cell lines and further explore the mechanism underlying cellular resistance to JQ-1 treatment. Methods: Six EEC cancer cell lines were used in this study. Cell proliferation was assessed by MTT assay and colony formation assay after exposure to JQ-1. Cell cycle progression was evaluated by flow cytometry. Apoptosis was assessed by Annexin V-FITC assay. Expression of c-Myc, PTEN, phosphorylated-Akt, phosphorylated-S6, cyclins and cyclin-dependent kinases (CDKs) was detected by Western blotting analysis. Up- or down-regulation of PTEN was achieved by stable transfection with pcDNA3.1-PTEN or shRNA-PTEN, respectively. Results: JQ-1 significantly suppressed proliferation in the EEC cell lines that were PTEN positive (IC50=75 nM for HEC-1A, 550 nM for KLE and 1000 nM for ECC-1), but not in those that were PTEN negative (IC50&amp;gt;10000 nM for Ishikawa, AN3CA and RL 95-2). JQ-1 induced G1 cell cycle arrest in the PTEN positive HEC-1A and KLE cell lines, but had minimal effects on cell cycle progression in the PTEN negative Ishikawa and AN3CA cell lines. Western blotting analysis demonstrated that JQ-1 suppressed the expression of c-Myc, cyclins and CDKs. JQ-1 up-regulated PTEN expression and decreased phosphorylation of downstream targets of the PI3K/Akt/mTOR signaling pathway, including Akt and S6. Knock-down of PTEN led to cellular resistance to JQ-1 in the HEC-1A and KLE cells, while over-expression of PTEN sensitized Ishikawa and AN3CA cells to JQ-1 treatment. Conclusions: We find that JQ-1 significantly suppressed cellular proliferation through G1 cell cycle arrest in the EEC cell lines with abundant PTEN expression. Knock-down of PTEN caused cellular resistance while over-expression of PTEN sensitized EEC cells to JQ-1. This evidence suggests that JQ-1 may be a promising targeted therapy for EEC patients with PTEN positive tumors. Note: This abstract was not presented at the meeting. Citation Format: Haifeng Qiu, Amanda Jackson, Joshua Kilgore, Chunxiao Zhou, Victoria Lin Bae-Jump. Inhibition of BET bromodomain targets PTEN positive endometrioid endometrial cancers. [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 4693. doi:10.1158/1538-7445.AM2014-4693

Correlation between the overexpression of epidermal growth factor receptor and mesenchymal makers in endometrial carcinoma

ObjectiveThe objective of this study was to evaluate the effect of overexpression of epidermal growth factor receptor (EGFR) on the expression of epithelial cell markers (E-cadherin and α-catenin) and mesenchymal cell markers (N-cadherin and vimentin) in endometrial carcinoma.MethodsThe expression of all 4 markers was evaluated in EGFR overexpressing Ishikawa cells, control Ishikawa cells, and KLE cells using reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. The expression of these 4 markers was also determined in cancerous tissues of patients with endometrial carcinoma using immunohistochemical staining.ResultsIshikawa cells transfected with EGFR showed decreased expression of E-cadherin and α-catenin and increased expression of N-cadherin and vimentin compared with control Ishikawa cells (p<0.01 for all). The expression of N-cadherin and vimentin was higher and the expression of E-cadherin and α-catenin was lower in stage II-III than stage I and in grade II-III than grade I endometrial carcinoma tissue (p<0.01 for all).ConclusionDecreased expression of epithelial markers (E-cadherin and α-catenin) and increased expression of mesenchymal markers (N-cadherin and vimentin) were observed in human endometrial carcinoma tissue. These findings correlate with high EGFR expression in cultured endometrial carcinoma cells.

Consideration of EphA2 in relation to epithelial-mesenchymal transition in uterine endometrial cancer.

To the editor: For decades, the mechanisms of cancer cell metastasis have been critical subject in the field of cancer research. Among them, the epithelial-mesenchymal transition (Emt) and mesenchymal-epithelial transition (MET) have been, currently, acknowledged as a crucial process by which carcinoma cells are acquired metastatic phenotype such as invasion of vessels and distant colonization, as well as a physiologic process during embryonic development. In the volume 25 January, a research article regarding the correlation between the overexpression of epidermal growth factor receptor (EGFR) and EMT makers in endometrial cancer gave us the result that mesenchymal markers (N-cadherin and vimentin) was overexpressed in advanced stage and high grade tissue specimens of patients and also the upregulation of mesenchymal markers was observed in KLE cells and Ishikawa cells transfected with EGFR [1]. EphA2, a member of the biggest subfamily of RTKs, could be recognized as a molecular target based on the growing evidence that high levels of it promote various aspects of malignant phenotype, including proliferation, invasion, angiogenesis and survival of cancer cells. My previous research showed that high EphA2 expression and high angiogenesis makers were significantly associated with shorter disease-specific survival in tissue specimens of patients with endometrial cancer, and also the therapy using EphA2 agonist monoclonal antibody led to tumor inhibition over controls in murine orthotopic xenograft model made by Hec1A and Ishikawa cells [2]. Interestingly, several research papers regarding the correlation between EphA2 and EMT were currently published. Huang et al. [3] reported that EphA2 promotes EMT in gastric cancer cells and demonstrated that the promoting effect of EphA2 on EMT was made through the Wnt/β-catenin pathway based on the experiments employing the inhibitor and activator of that pathway. Additionally, Giannoni et al. [4] reported that mesenchymal-amoeboid transition, a second type of motility shifting process in cancer cells induced by endothelial progenitor cells, was mediated through the bidirectional ephrinA1/EphA2 signaling. Thereby, EphA2 could be also considered as a regulator in relation to epithelialmesenchymal transition in endometrial cancer.

Upregulation of estrogen receptor mediates migration, invasion and proliferation of endometrial carcinoma cells by regulating the PI3K/AKT/mTOR pathway

The precise mechanism through which the two estrogen receptor subtypes, ERα and ERβ, are linked to endometrial malignant progression is not fully understood. The aim of the present study was to examine their role in endometrial carcinoma cell migration, invasion and proliferation. We also explored the correlation between the ERs and phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathways in endometrial carcinoma cells. Using Ishikawa and KLE cells as model systems, we performed transient transfection to upregulate ERα and ERβ expression; fluorescence microscopy analysis was then employed to evaluate transfection efficiencies, RT-PCR and western blot assays were used to evaluate the mRNA and protein levels. We further examined the effects on cell migration, invasion and proliferation. We showed that ERα raised the phosphorylation levels of PI3K p85α, activated the phosphorylation of AKT and mTOR in Ishikawa and KLE cells, but ERβ had no effect on PI3K p85α phosphorylation. Moreover, the overexpression of ERs enhanced cell migration, invasion and proliferation. The effect on the activation of the PI3K/AKT/mTOR transduction cascade by ERα explains, at least in part, the enhancement on cell invasion and proliferation exerted by overexpression of ERα. This crosstalk could be taken into account in developing novel therapeutic methods by targeting the ERα and PI3K/AKT/mTOR pathways in endometrial carcinoma.