Qi Che

The long non-coding RNA HOTAIR is upregulated in endometrial carcinoma and correlates with poor prognosis

Long non-coding RNAs (lncRNAs) are emerging as key molecules in human cancer. Homeobox (HOX) transcript antisense intergenic RNA (HOTAIR), a long non-coding RNA (lncRNA), is associated with a variety of human cancers, such as breast, liver and lung cancer. However, whether HOTAIR can function as a molecular marker in endometrial carcinoma (EC) remains unknown. In the present study, the expression of HOTAIR in 66 EC tissues from patients with EC and 30 normal tissues from healthy age-matched control subjects was determined using quantitative reverse transcription PCR. Furthermore, using in situ hybridization, we measured HOTAIR expression in 129 formalin-fixed paraffin-embedded (FFPE) tissue sections, which included 96 tissues that matched the frozen cases, 21 other EC tissues and 12 atypical hyperplasia tissues. Correlations between HOTAIR expression and the clinicopathological characteristics of patients were analyzed. Our results revealed that HOTAIR expression in the EC tissues was significantly upregulated compared with normal tissues (p<0.001). In addition, we observed a significant association between HOTAIR expression and the EC grade (p<0.05) and lymph node metastasis (p<0.05). Moreover, in the FFPE tissues, but not the frozen tissues, we found that a higher HOTAIR expression also correlated with the depth of myometrial invasion (p=0.019) and lymphovascular space invasion (p=0.015). More importantly, patients with a higher HOTAIR expression showed significantly poorer overall survival than those with lower HOTAIR expression (p<0.05). In conclusion, our results suggest that a high expression of HOTAIR is involved in the progression of cancer and may be a novel biomarker of poor prognosis in patients with EC.

Activation of a positive feedback loop involving IL‐6 and aromatase promotes intratumoral 17β‐estradiol biosynthesis in endometrial carcinoma microenvironment

Tumor–stroma interactions contribute greatly to intratumoral estrogen biosynthesis in endometrial carcinoma, but the mechanisms involved remain largely unknown. Previous study demonstrated that intratumoral aromatase upregulation in stromal cells participated in this process, but the specific aromatase‐regulators have not been reported. In the present study, we found that aromatase expression in intratumoral stroma, but not in tumor epithelium, correlated positively with interleukin 6 (IL‐6) expression in cancer epithelial cells by immunohistochemistry, which was confirmed using laser capture microdissection/real‐time reverse transcription‐PCR. With stimulation by exogenous IL‐6, aromarase expression was increased in stromal cells not but not in cancer cells. Aromatase mRNA levels in endometrial cancer cells were not influenced by cocultivation with intratumoral stromal cells. When cocultured with 17β‐estradiol (E2)‐treated cancer cells, aromatase mRNA in stromal cells was significantly elevated and increased IL‐6 protein levels were detected in E2‐treated culture medium. Next, we demonstrated that E2‐induced IL‐6 production was through cooperation between estrogen receptor α and nuclear factor‐kappa B. Furthermore, an IL‐6 receptor blocking antibody could attenuate the upregulation of aromatase expression in stromal cells and the E2 concentration in coculture systems of cancer and stromal cells. The results were confirmed by an orthotopic nude endometrial carcinoma model in vivo. These studies elucidated the activation of a positive feedback loop, that is, IL‐6 stimulated by E2 in endometrial cancer cells induced aromatase expression in stromal cells, promoting enhanced intratumoral E2 synthesis. Blocking of this tumor–stroma interaction may be a therapeutic strategy to overcome in situ estrogen biosynthesis in endometrial carcinoma.

Mutant p53 (p53-R248Q) functions as an oncogene in promoting endometrial cancer by up-regulating REGγ

P53 mutation plays a pivotal role in tumorigenesis of endometrial cancer (EC), here we report that the gain-of-function mutant p53-R248Q targets the proteasome activator REGγ to promote EC progression. Increased p53 expression significantly correlated with high pathological grade and lymph node metastasis in EC specimens. Manipulation of p53-R248Q in EC cells caused coincident changes in REGγ expression, and chromatin immunoprecipitation coupled with PCR further indicated that p53-R248Q bound to the REGγ gene promoter at a p53 responsive element. Silencing of REGγ in EC cells attenuated the cell proliferation, migration and invasion abilities, whereas overexpression of p53-R248Q rescued these activities. Overexpression of REGγ also induced an epithelial-mesenchymal transition phenotype. Moreover, a mouse xenograft tumor model showed that REGγ promoted tumor growth, further demonstrating a p53-R248Q-REGγ oncogenic pathway. Finally, examination of EC and normal endometrium specimens confirmed the oncogenic role of REGγ, in that REGγ was more highly overexpressed in p53-positive specimens than in p53-negative specimens. Our data suggest that REGγ is a promising therapeutic target for EC with the p53-R248Q mutation.

Piwil1 causes epigenetic alteration of PTEN gene via upregulation of DNA methyltransferase in type I endometrial cancer.

Piwil1, a member of the Piwi family, has been well demonstrated to mediate tumorigenesis associated with DNA hypermethylation. It has been reported that Piwil1 is overexpressed in various types of cancer, including endometrial cancer. However, the underlying mechanism of Piwil1 in endometrial cancer remains largely unclear. PTEN exerts an important tumor suppressor role in endometrial carcinogenesis. The present study aimed to investigate whether Piwil1 could regulate the expression of PTEN. Herein, we found that Piwil1 could promote the loss of PTEN expression and increase aberrant hypermethylation of PTEN gene promoter in Ishikawa cells. We also found that Piwil1 could regulate the expression of DNA methyltransferase 1 (DNMT1). Silencing DNMT1 gene could upregulate the PTEN gene expression and change the methylation status of PTEN gene promoter in Ishikawa cells. These results suggested that Piwil1 caused the loss of PTEN expression through DNMT1-mediated PTEN hypermethylation. Taken together, these data provide a novel regulatory mechanism of Piwil1 in endometrial cancer.

Elevated miR-222-3p promotes proliferation and invasion of endometrial carcinoma via targeting ERα

MicroRNAs play key roles in tumor proliferation and invasion. Here we show distinct expression of miR-222-3p between ERα-positive and ERα-negative endometrial carcinoma (EC) cell lines and primary tumors, and investigation of its relationship with ERα and other clinical parameters. In vitro, the function of miR-222-3p was examined in RL95-2 and AN3CA cell lines. MiR-222-3p expression was negatively correlated with ERα. Over-expressed miR-222-3p in RL95-2 cells promoted cell proliferation, enhanced invasiveness and induced a G1 to S phase shift in cell cycle. Furthermore, the miR-222-3p inhibitor decreased the activity of AN3CA cells to proliferate and invade. In vivo, down-regulated miR-222-3p of AN3CA cells inhibited EC tumor growth in a mouse xenograft model. Additionally, miR-222-3p increased raloxifene resistance through suppressing ERα expression in EC cells. In conclusion, miR-222-3p plays a significant role in the regulation of ERα expression and could be potential targets for restoring ERα expression and responding to antiestrogen therapy in a subset of ECs.

Oncostatin M activates STAT3 to promote endometrial cancer invasion and angiogenesis

Oncostatin M (OSM), a pleiotropic cytokine, can either promote or inhibit the growth of tumors derived from specific tissues. However, little is known about the activity and expression pattern of OSM in endometrial cancers (ECs). Herein we show that expression of OSM in human ECs was significantly higher than that in hyperplastic or normal tissues. In EC tissues, high OSM levels were positively correlated with tumor stage, histological grade, myometrial invasion, and lymph node metastasis. Additionally, we demonstrated that recombinant human OSM (rhOSM) promoted tumor angiogenesis in EC cell lines by activating STAT3 (signal transducer and activator of transcription 3) and enhanced both cell migration and cell invasion. rhOSM did not, however, influence the proliferation of EC cells in vitro. In contrast, in our in vivo xenograft model, overexpression of rhOSM promoted cell proliferation, tumor growth, and angiogenesis in nude mice. Collectively, these experiments suggest that OSM may be a tumor promoter that encourages EC progression. OSM may thus serve as a potential target of antiangiogenic therapy for endometrial cancer.

Forkhead-box A1 suppresses the progression of endometrial cancer via crosstalk with estrogen receptor α.

Mechanisms governing the function of Forkhead-box A1 (FOXA1), a member of the FOX class of transcription factors, have been extensively studied. However, little is known about the activities and expression pattern of FOXA1 in endometrial cancer (EC). In the present study, we investigated the level of FOXA1 in multiple human EC cell lines and clinical samples by immunohistochemistry, qRT-PCR and Western blot analysis. FOXA1 overexpression was observed in estrogen receptor (ER)α-positive EC cell lines (P=0.0048). In endometrial tissues, FOXA1 was significantly upregulated in both normal endometrium and well-differentiated endometrial cancer tissues (P<0.001). Functional analyses of FOXA1 were evaluated by MTT, plate colony formation and Transwell assay. The results revealed that forced expression of FOXA1 inhibited EC cell proliferation, whereas FOXA1 depletion promoted cell viability and was associated with tumorigenesis. The nude mouse tumor xenograft assay also confirmed that ablation of FOXA1 expression promoted cell proliferation. Furthermore, we found that knockdown of FOXA1 decreased the expression of ERα, and FOXA1 interacted with this receptor in the EC cell lines. Collectively, these experiments suggest that FOXA1 is a tumor suppressor in EC and has a possible interaction with ERα.

Mutant p53 induces EZH2 expression and promotes epithelial-mesenchymal transition by disrupting p68-Drosha complex assembly and attenuating miR-26a processing.

The tumor suppressor p53 and the transcriptional repressor Enhancer of Zeste Homolog 2 (EZH2) have both been implicated in the regulation of epithelial-mesenchymal transition (EMT) and tumor metastasis via their impacts on microRNA expression. Here, we report that mutant p53 (mutp53) promotes EMT in endometrial carcinoma (EC) by disrupting p68-Drosha complex assembly. Overexpression of mutp53 has the opposite effect of wild-type p53 (WTp53), repressing miR-26a expression by reducing pri-miR-26a-1 processing in p53-null EC cells. Re-expression of miR-26a in mutp53 EC cells decreases cell invasion and promotes mesenchymal-epithelial transition (MET). Rescuing miR-26a expression also inhibits EZH2, N-cadherin, Vimentin, and Snail expression and induces E-cadherin expression both in vitro and in vivo. Moreover, patients with higher serum miR-26a levels have a better survival rate. These results suggest that p53 gain-of-function mutations accelerate EC tumor progression and metastasis by interfering with Drosha and p68 binding and pri-miR-26a-1 processing, resulting in reduced miR-26a expression and EZH2 overexpression.

SHARP1 Suppresses Angiogenesis of Endometrial Cancer by Decreasing Hypoxia-Inducible Factor-1α Level

Recent data support a role for SHARP1, a basic helix-loop-helix transcription repressor, in the regulation of malignant cell behavior in several human cancers. However, the expression and role of SHARP1 during the development of endometrial cancer (EC) remain unclear. Here we show that upregulation of SHARP1 suppressed tumor angiogenesis by decreasing hypoxia-inducible factor-1α (HIF-1α), inhibited cell viability and tumor growth in EC. Immunohistochemical staining showed that the expression of SHARP1 was negatively correlated with tumor stage, histological grade, myometrial invasion, lymph node metastasis, blood vessel permeation in the myometrium and HIF-1α expression. Mechanistic studies showed that SHARP1 interacted with HIF-1α physically, and the protein level of HIF-1α and the mRNA level of its target genes (VEGFA, ANGPTL4 and CA9) were decreased by SHARP1 under hypoxia. Upregulation of SHARP1 in EC impeded hypoxia-induced angiogenesis by reducing VEGF secretion. Immunohistochemical analysis verified a correlation between decreased SHARP1 expression and increased microvessel density in EC tissues. Additionally, SHARP1 inhibited cell viability in EC cell lines. Overexpression of SHARP1 in vivo inhibited tumor growth and angiogenesis, and decreased HIF-1α expression. In this study, we established SHARP1 as a novel tumor suppressor of EC and shed light on the mechanisms by how SHARP1 inhibited EC progression. Therefore, SHARP1 may be a valuable prognostic biomarker for EC progression and shows promise as a new potential target for antiangiogenic therapeutics in human EC.

Interleukin 6 promotes endometrial cancer growth through an autocrine feedback loop involving ERK-NF-κB signaling pathway.

Interleukin (IL)-6 as an inflammation factor, has been proved to promote cancer proliferation in several human cancers. However, its role in endometrial cancer has not been studied clearly. Previously, we demonstrated that IL-6 promoted endometrial cancer progression through local estrogen biosynthesis. In this study, we proved that IL-6 could directly stimulate endometrial cancer cells proliferation and an autocrine feedback loop increased its production even after the withdrawal of IL-6 from the medium. Next, we analyzed the mechanism underlying IL-6 production in the feedback loop and found that its production and IL-6-stimulated cell proliferation were effectively blocked by pharmacologic inhibitors of nuclear factor-kappa B (NF-κB) and extra-cellular signal-regulated kinase (ERK). Importantly, activation of ERK was upstream of the NF-κB pathways, revealing the hierarchy of this event. Finally, we used an orthotopic nude endometrial carcinoma model to confirm the effects of IL-6 on the tumor progression. Taken together, these data indicate that IL-6 promotes endometrial carcinoma growth through an expanded autocrine regulatory loop and implicate the ERK-NF-κB pathway as a critical mediator of IL-6 production, implying IL-6 to be an important therapeutic target in endometrial carcinoma.