Xiaoping Wan

miR-130b is an EMT-related microRNA that targets DICER1 for aggression in endometrial cancer

Endometrial cancer (EC) is the most common gynecologic malignancy, but the molecular events involved in the development and progression of EC remain unclear. Certain microRNAs (miRNAs) and DICER1 play important roles in cell motility and survival. This study investigated the role of miR-130b and DICER1 in EC. We profiled miR-130b and DICER1 expression in clinical samples explored its relationship with clinical parameters. A luciferase reporter assay assessed the miR-130b targeting potential of DICER1. We show both in vitro and in vivo that miR-130b overexpression along with DICER1 dysfunction leads to tumor aggression and miRNA synthesis abnormalities that are related to cancer hallmarks through DICER1–miRNAs axis modulation. We also identify the mechanism related to this potential tumor predisposing phenotype: miR-130b and loss of DICER1 induced abnormal expression of EMT-related genes, which constitutes a loop regulation of the miR-130b–DICER1–EMT axis.

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.

miR-205 promotes tumor proliferation and invasion through targeting ESRRG in endometrial carcinoma

Increasing evidence suggests that miR‑205 is frequently dysregulated in many types of human cancers, suggesting its important roles in the initiation and progression of cancer. However, the functions of miR‑205 in human endometrial endometrioid carcinoma (EEC) are still unknown. In this study, we investigated the expression of miR‑205 in both normal endometrium and EEC tissues using TaqManxa0PCR. Compared to normal tissues, miR‑205 was significantly upregulated in EEC (P<0.001). After transfection of miR‑205 inhibitors into Ishikawa cells (or transfection of miR‑205 mimics into AN3CA cells), we demonstrated that the cellular proliferation, migration and invasion properties were negatively regulated by miR‑205. Moreover, by combination of microRNA target prediction algorithms and luciferase reporter system, we identified estrogen-related receptor‑γ (ESRRG) as a target of miR‑205. In conclusion, we demonstrated frequent upregulation of miR‑205 in EEC. In gain‑of‑function and loss‑of‑function assays, inhibition of miR‑205 reduced cellular proliferation, migration and invasion; vice versa, increased levels of miR‑205 led to upregulated cellular proliferation, migration and invasion. Nonetheless, we identified the ESRRG gene to be a novel target, which could be helpful to elucidate mechanisms underlying the tumorigenesis of EEC.

Upregulation of TrkB Promotes Epithelial-Mesenchymal Transition and Anoikis Resistance in Endometrial Carcinoma

Mechanisms governing the metastasis of endometrial carcinoma (EC) are poorly defined. Recent data support a role for the cell surface receptor tyrosine kinase TrkB in the progression of several human tumors. Here we present evidence for a direct role of TrkB in human EC. Immunohistochemical analysis revealed that TrkB and its secreted ligand, brain-derived neurotrophic factor (BDNF), are more highly expressed in EC than in normal endometrium. High TrkB levels correlated with lymph node metastasis (p<0.05) and lymphovascular space involvement (p<0.05) in EC. Depletion of TrkB by stable shRNA-mediated knockdown decreased the migratory and invasive capacity of cancer cell lines in vitro and resulted in anoikis in suspended cells. Conversely, exogenous expression of TrkB increased cell migration and invasion and promoted anoikis resistance in suspension culture. Furthermore, over-expression of TrkB or stimulation by BDNF resulted in altered the expression of molecular mediators of the epithelial-to-mesenchymal transition (EMT). RNA interference (RNAi)-mediated depletion of the downstream regulator, Twist, blocked TrkB-induced EMT-like transformation. The use of in vivo models revealed decreased peritoneal dissemination in TrkB-depleted EC cells. Additionally, TrkB-depleted EC cells underwent mesenchymal-to-epithelial transition and anoikis in vivo. Our data support a novel function for TrkB in promoting EMT and resistance to anoikis. Thus, TrkB may constitute a potential therapeutic target in human EC.

FOXA1 promotes tumor cell proliferation through AR involving the Notch pathway in endometrial cancer.

BackgroundIncreasing evidence suggests that forkhead box A1 (FOXA1) is frequently dysregulated in many types of human cancers. However, the exact function and mechanism of FOXA1 in human endometrial cancer (EC) remains unclear.MethodsFOXA1 expression, androgen receptor (AR) expression, and the relationships of these two markers with clinicopathological factors were determined by immunohistochemistry analysis. FOXA1 and AR were up-regulated by transient transfection with plasmids, and were down-regulated by transfection with siRNA or short hairpin RNA (shRNA). The effects of FOXA1 depletion and FOXA1 overexpression on AR-mediated transcription as well as Notch pathway and their impact on EC cell proliferation were examined by qRT-PCR, western blotting, co-immunoprecipitation, ChIP-PCR, MTT, colony-formation, and xenograft tumor–formation assays.ResultsWe found that the expression of FOXA1 and AR in ECs was significantly higher than that in a typical hyperplasia and normal tissues. FOXA1 expression was significantly correlated with AR expression in clinical tissues. High FOXA1 levels positively correlated with pathological grade and depth of myometrial invasion in EC. High AR levels also positively correlated with pathological grade in EC. Moreover, the expression of XBP1, MYC, ZBTB16, and UHRF1, which are downstream targets of AR, was promoted by FOXA1 up-regulation or inhibited by FOXA1 down-regulation. Co-immunoprecipitation showed that FOXA1 interacted with AR in EC cells. ChIP-PCR assays showed that FOXA1 and AR could directly bind to the promoter and enhancer regions upstream of MYC. Mechanistic investigation revealed that over-expression of Notch1 and Hes1 proteins by FOXA1 could be reversed by AR depletion. In addition, we showed that down-regulation of AR attenuated FOXA1-up-regulated cell proliferation. However, AR didn’t influence the promotion effect of FOXA1 on cell migration and invasion. In vivo xenograft model, FOXA1 knockdown reduced the rate of tumor growth.ConclusionsThese results suggest that FOXA1 promotes cell proliferation by AR and activates Notch pathway. It indicated that FOXA1 and AR may serve as potential gene therapy in EC.

Acrp30 inhibits leptin-induced metastasis by downregulating the JAK/STAT3 pathway via AMPK activation in aggressive SPEC-2 endometrial cancer cells

Obesity is a well-established risk factor for endometrial cancer, due in part to the adipokines generated by adipose tissue, such as adiponectin (also known as Acrp30) and leptin, which are associated with many endocrine-related cancers. Recent reports suggested that Acrp30 inhibits leptin-stimulated cell proliferation in HEC-1A and RL95-2 endometrial cancer cell lines, and that the serum leptin/Acrp30 ratio plays an important role in endometrial cancer development. We explored whether Acrp30 could reverse the leptin-induced metastasis phenotype in the SPEC-2 endometrial cancer cell line. Transcripts for Acrp30 receptors (AdipoR1 and AdipoR2) and leptin receptor (Ob-Rb) were detected by quantitative real-time RT-PCR (qRT-PCR) in six endometrial cancer cell lines. Leptin (1xa0µg/ml) treatment stimulated SPEC-2 cell proliferation by inducing cell cycle arrest and apoptosis, while Acrp30 (10xa0µg/ml) treatment inhibited the growth of SPEC-2 cells. Importantly, Acrp30 was able to inhibit leptin-induced SPEC-2 cell proliferation. Leptin promoted SPEC-2 cell invasion in a Matrigel transwell assay, while Acrp30 partly suppressed the invasion stimulated by leptin. To investigate the molecular mechanism underlying this phenomenon, we monitored the AMPK and JAK/STAT3 signaling pathways by western blotting and cell immunofluorescence. Acrp30 reduced leptin-induced STAT3 phosphorylation and nuclear translocation via activation of the MAPK pathway. AG490 (JAK/STAT3 inhibitor) reduced MMP-2 and MMP-9 protein levels in cells treated with leptin, while IL-6 (JAK/STAT3 stimulator) and Compound C (AMPK inhibitor) elevated MMP-2 and MMP-9 protein levels in cells treated with Acrp30. In conclusion, we demonstrated that Acrp30 effectively reversed the invasion stimulated by leptin, and AMPK and JAK/STAT3 pathways mediated the invasive phenotype of SPEC-2 cells.

MicroRNA-361-5p facilitates cervical cancer progression through mediation of epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) promotes cervical cancer progression, and microRNAs have been found to be master regulators of EMT. The aim of the present study was to investigate the functional roles of miR-361-5p in EMT and cervical cancer progression. Differentially expressed miRNAs were screened with microarray analysis in SiHa and CasKi cells; cellular and animal studies were used to observe the impact of miR-361-5p on cell proliferation; invasion and migration ability of cervical cancer cells were investigated by Transwell and wound-healing studies; enzyme-linked immunosorbent assay and Western blot methods were used to test protein levels; miR-361-5p level in cervical cancer specimens was detected with in situ hybridization. MicroRNA-361-5p (miR-361-5p) was found to be the most upregulated microRNA in transferred cervical cancer cells. MiR-361-5p acts as an oncogene to enhance cell proliferation and promote cell invasion, and these changes were accompanied by the characteristics of EMT. miR-361-5p is increasingly elevated during cervical carcinoma progression and inversely correlated with E-cadherin, a marker of EMT. These findings suggest that miR-361-5p is an oncomicroRNA and an important factor in the progression of cervical cancer.

Follicle-stimulating hormone inhibits apoptosis in ovarian cancer cells by regulating the OCT4 stem cell signaling pathway.

OCT4, a stem cell marker, is overexpressed in several types of human cancer and can induce resistance to chemotherapy and inhibition of apoptosis. We previously demonstrated that human follicle stimulating hormone (FSH) can inhibit ovarian cancer cell apoptosis. However, the role of OCT4 in FSH-induced inhibition of apoptosis has not been reported in detail. Here, we profiled OCT4 protein expression in ovarian epithelial cancer (OEC) with benign cystadenoma, borderline tumor and carcinoma tissues as well as different ovarian cancer cell lines and normal ovarian epithelial cells. Furthermore, the effects of FSH on OCT4 expression and related signaling pathways were evaluated. The overexpression of OCT4 in ovarian carcinoma and OEC cell lines suggest that OCT4 plays a critical role in OEC carcinogenesis. Moreover, FSH-induced apoptosis inhibition was confirmed and FSH stimulation induced the expansion of CD44+CD117+ cells with a stem cell-like phenotype. Re-expression of OCT4 enhanced the expression of Notch, Sox2 and Nanog molecules that play critical roles in cancer stem cell proliferation and differentiation. FSH upregulated the expression of Notch, Sox2 and Nanog and these effects were abolished by knocking down OCT4, suggesting that several cancer stem cell pathways are involved in FSH regulation. We also examined OCT4 expression in surgical specimens of ovarian cancer. Immunohistostaining revealed that OCT4 expression was increased in ovarian carcinoma compared with benign cystadenomas and borderline tumors, and OCT4 expression was significantly correlated with histological grade. Staining for OCT4 was increased in serous cystadenocarcinoma, when compared with clear cell carcinoma. In summary, the OCT4 cancer stem cell signaling pathway may mediate FSH-induced inhibition of apoptosis and could provide a target for treatment of ovarian cancer.

CpG island hypermethylation-associated silencing of microRNAs promotes human endometrial cancer

BackgroundEndometrial cancer (EC) is the most common gynecologic malignancy, but the molecular events involved in the development and progression of EC remain unclear. This study aimed to explore epigenetic modification of genes and miRNAs involved in EC development.MethodsIshikawa and AN3CA cells were treated with 5’-Aza-2-deoxycytidine or histone deacetylase inhibitor. The expression of miRNAs and related genes were detected by PCR and Western blot. Promoter methylation was detected by bisulfite specific PCR sequencing. The proliferation, colony formation, cell cycle progression, migration and invasion of EC cells were evaluated by MTT, soft agar assay, flow cytometry, wound healing and invasion assay, respectively.ResultsAberrant expression of miRNAs including miR-200b, miR-130a/b, miR-625 and miR-222 was associated with tumorigenesis and metastasis in endometrial cancer. Silencing of miR-130b induced E-cadherin expression, while ectopic expression of miR-130b and knockdown of DICER1 increased the expression of Vimentin, zeb2, N-cadherin, Twist and Snail in EC cells. Furthermore, 5’-Aza-2-deoxycytidine and Histone deacetylase (HDAC) inhibitor inhibited the proliferation, colony formation, migration and invasion of EC cells, accompanied by reduced MMP secretion.ConclusionsOur study provides the first description of epigenetic modification of epithelial mesenchymal transition associated genes and miRNAs in EC cells, which are extensively involved in the regulation of gene expression and subsequent accumulation of malignant features of EC cells.

WWOX induces apoptosis and inhibits proliferation in cervical cancer and cell lines.

Cervical cancer is the second most common gynecological malignancy, but the molecular events involved in its development remain unclear. The tumor‑suppressor gene, WWxa0domain-containing oxidoreductase (WWOX), has been found to be lost in various types of cancers. Few studies have been reported detailing the function of WWOX in human cervical cancer; therefore we aimed to investigate the role played by WWOX in human cervical cancer. Immunohistochemistry was used to study preinvasive and invasive primary cervical cancer. Full length cDNA was transfected into HeLa cells to overexpress WWOX, and short hairpin RNA (shRNA) was transfected into SiHa cells to deplete its expression, respectively. The cellular levels of WWOX RNA and protein were detected by real-time PCR and western immunoblotting. Proliferation rates were assessed by methyl thiazolyl tetrazolium (MTT), plate colony formation and soft agar colony assays. Cellular apoptosis was measured by flow cytometry and TdT-mediated dUTP nick-end labeling (TUNEL) assay. The activity of caspase-3 and its protein levels were determined by caspase-3 activity assay and western blot analysis. Xenografts were established by injecting cells into nude mice. The results showed that WWOX expression was decreased in human cervical cancer and cervical cancer cell lines. Reconstitution of WWOX in HeLa cells inhibited their proliferation and induced apoptosis, while knockdown of WWOX in SiHa cells promoted proliferation and inhibited apoptosis. Xenografts in groups of mice verified the effect inxa0vivo. These data suggest that underexpression of WWOX is associated with cervical cancer development. Modulation of WWOX expression may be an effective and novel method for the treatment of cervical cancer.