Fangyuan Wang

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.

Fibroblast activation protein regulates tumor-associated fibroblasts and epithelial ovarian cancer cells

The fibroblast activation protein (FAP) is a cell surface serine protease which has emerged as a specific marker of tumor-associated fibroblasts (TAFs). FAP has been shown to have both inxa0vitro dipeptidyl peptidase and collagenase activity. However, the biological function of FAP in the tumor microenvironment is largely unknown. In this study, we first show that TAFs isolated from ovarian cancer samples have the characteristics of stem cells. To explore the functional role of FAP, the protein was silenced by siRNA lentiviral vector transfection. FAP silencing inhibited the growth of TAFs inxa0vitro, accompanied with cell cycle arrest at the G2 and Sxa0phase in TAFs. FAP silencing also reduced the stem cell marker gene expression in TAFs. SKOV3 cells do not express FAP. Although FAP-silenced SKOV3 cells induced ovarian tumors, the rate of tumor growth was significantly decreased, as shown in the xenograft mouse model. TAF phenotypes in the xenograft tumor tissues were further assayed by immunohistochemistry. The expression of TAF markers, including fibroblast-specific protein, FAP, smooth muscle actin, desmin, vascular endothelial growth factor and fibroblast growth factor was decreased in the tumor stroma induced by FAP-silenced SKOV3 cells. In conclusion, FAP is an important regulator of the microenvironment in tumor formation and targeting FAP is a potential therapeutic strategy to combat ovarian cancer.

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.

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.

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.

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.

Stem cell protein Piwil1 endowed endometrial cancer cells with stem-like properties via inducing epithelial-mesenchymal transition.

BackgroundStem cell protein Piwil1 functions as an oncogene in various tumor types. However, the exact function and mechanism of Piwil1 in endometrial cancer remains unclear.MethodsThe expression of Piwil1 and its relationships with clinicopathological factors were investigated using immunohistochemistry. Up- or down-regulation of Piwil1 were achieved by stable or transient transfection with plasmids or short hairpin RNA (shRNA). Effects of Piwil1 on cancer cells viability, invasion and migration were evaluated by MTT, plate colony formation, transwell assay and nude mouse tumor xenograft assay. The stem-like properties of endometrial cancer cells was detected by spheroid formation assay. Effects of Piwil1 on expression levels of target genes were detected by qRT-PCR, western blotting and Immunofluorescence.ResultsCompared with atypical hyperplasia and normal tissues, Piwil1 was much higher in endometrial carcinoma tissues. We found that Piwil1 expression was significantly correlated with FIGO stage, lymphovascular space involvement, lymph node metastasis and level of myometrial invasion. Overexpression of Piwil1 functioned to maintain stem-like characteristics, including enhancing tumor cell viability, migration, invasion and sphere-forming activity. Conversely, Piwil1 knockdown inhibited cell viability, migration, invasion, sphere-forming activity in vitro and tumor formation in xenograft model in vivo. Furthermore, study of the expression of epithelial and mesenchymal markers showed that Piwil1 was responsible for an EMT-like phenotype associated with an increase in mesenchymal markers and suppression of E-cadherin. Moreover, Piwil1 augmented expression levels of CD44 and ALDH1 expression, two known endometrial CSC markers, as well as other stemness-associated genes.ConclusionsOur results suggested that stem cell protein Piwil1 play important roles in regulating EMT and the acquisition of stem-like properties of endometrial cancer cells. Therefore, it indicated that Piwil1 may represent a promising target for developing a novel treatment strategy for endometrial cancer.

Suppression of the epithelial-mesenchymal transition by SHARP1 is linked to the NOTCH1 signaling pathway in metastasis of endometrial cancer

BackgroundMechanisms governing the metastasis of endometrial cancer (EC) are poorly defined. Recent data support a role for Enhancer-of-split and hairy-related protein 1 (SHARP1), a basic helix-loop-helix transcription repressor, in regulating invasiveness and angiogenesis of several human cancers. However, the role of SHARP1 in metastasis of EC remains unclear.MethodsHuman EC cell lines (Ishikawa and HEC-1B) were used. SHARP1 was upregulated by lentivirus transduction, while intracellular domain of NOTCH1 (ICN) were upregulated by transient transfection with plasmids. Effects of SHARP1 on cell migration and invasion were evaluated by wound healing assay and transwell invasion assay. Experimental metastasis assay were performed in nude mice. Effects of SHAPR1 on protein levels of target genes were detected by western blotting. Furthermore, the association between SHARP1 and the NOTCH1/EMT pathway was further verified in EC tissue specimens by immunohistochemical analysis.ResultsOverexpression of SHARP1 in EC cells inhibited cell migration, invasion, and metastasis. Exogenous SHARP1 overexpression affected the proteins levels of genes involved in EMT process and NOTCH1 signaling pathway. Upregulation of ICN in SHARP1-overexpressing Ishikawa cells induced cell migration and an EMT phenotype. Additionally, immunohistochemical analysis demonstrated that SHARP1 protein levels were lower in metastatic EC than in primary tumors, and statistical analysis revealed correlations between levels of SHARP1 and markers of EMT and NOTCH1 signaling pathway in human EC tissue specimen.ConclusionsThis work supports a role for SHARP1 in suppressing EMT and metastasis in EC by attenuating NOTCH1 signaling. Therefore, SHARP1 may be a novel marker for lymphatic metastasis in EC patients.

Prostaglandin E2 (PGE2) promotes proliferation and invasion by enhancing SUMO-1 activity via EP4 receptor in endometrial cancer.

Prostaglandin E2 (PGE2), a derivative of arachidonic acid, has been identified as a tumorigenic factor in many cancers in recent studies. Prostaglandin E synthase 2 (PTGES2) is an enzyme that in humans is encoded by the PTGES2 gene located on chromosome 9, and it synthesizes PGE2 in human cells. In our study, we selected 119 samples from endometrial cancer patients, with 50 normal endometrium tissue samples as controls, in which we examined the expression of PTGES2. Both immunohistochemistry (IHC) and Western blot analyses demonstrated that synthase PTGES2, which is required for PGE2 synthesis, was highly expressed in endometrium cancer tissues compared with normal endometrium. Stable PTGES2-shRNA transfectants were generated in Ishikawa and Hec-1B endometrial cancer cell lines, and transfection efficiencies were confirmed by RT-PCR and Western blot analyses. We found that PGE2 promoted proliferation and invasion of cells in Ishikawa and Hec-1B cells by cell counting kit-8 tests (CCK8) and transwell assays, respectively. PGE2 stimulation enhanced the expression of SUMO-1, via PGE2 receptor subtype 4 (EP4). Further analysis implicated the Wnt/β-catenin signaling pathway function as the major mediator of EP4 and SUMO-1. The increase in SUMO-1 activity prompted the SUMOlyation of target proteins which may be involved in proliferation and invasion. These findings suggest SUMO-1 and EP4 as two potential targets for new therapeutic or prevention strategies for endometrial cancers.