Catecholestrogens induce oxidative stress and malignant transformation in human endometrial glandular cells: protective effect of catechol-O-methyltransferase

Prolonged exposure to unopposed estrogens is a major risk factor for the development of endometrial cancer. Oxidative metabolism of estradiol (E(2)) into the catecholestrogens (CEs), 4-hydroxyestradiol (4-OHE(2)) and 2-hydroxyestradiol (2-OHE(2)), may play an important role in estrogen carcinogenicity. CEs can be oxidized to the corresponding ortho-quinone derivatives with concomitant formation of the reactive oxygen species (ROS). Catechol-O-methyltransferase (COMT) is the major enzyme involved in the detoxification of CEs in extrahepatic tissues. We investigated the potential of E(2), 2-OHE(2) and 4-OHE(2) to induce microsatellite instability (MSI) and neoplastic transformation of immortalized human endometrial glandular (EM) cells. We also investigated the functional significance of COMT gene expression on modulating the effects of E(2) and CEs in EM cells. Our data indicated that E(2) and 4-OHE(2) induce MSI, ROS and neoplastic transformation in EM cells. The capacity of E(2) and its catechol metabolites to induce MSI, ROS and neoplastic transformation in EM cells is ranked as follows: 4-OHE(2) > E(2) > 2-OHE(2). Knockdown of COMT expression in EM cells resulted in increased estrogenic milieu and increased estrogen-induced cell proliferation. More importantly, knockdown of COMT increased the propensity of E(2) or CEs to induce ROS, MSI and neoplastic transformation of EM cells. In contrast, overexpression of COMT in EM cells significantly reduced the cellular estrogenic milieu and protected against E(2)- or CEs-induced, ROS, MSI and neoplastic transformation. The capacity of E(2) or CEs to induce neoplastic transformation of human endometrial glandular cells in vitro may suggest that E(2)-induced endometrial cancer is mediated by its metabolism into CEs. Our study clearly indicates that COMT gene expression plays a critical role in modulating the hormonal and carcinogenic effects of E(2) and CEs and, consequently, modifies the risk for E(2)-induced endometrial cancer. To the best of our knowledge, this is the first study to (i) demonstrate the potential capacity of estrogen and its catechol metabolites to induce neoplastic transformation of immortalized human endometrial glandular cells; and (ii) illustrate the important role of COMT gene expression in protecting against E(2)-induced endometrial cancer.     

FTO promotes the progression of cervical cancer by regulating the N6-methyladenosine modification of ZEB1 and Myc

Cervical cancer is a malignant tumor of the cervix in women. However, the pathogenesis of cervical cancer has not been fully understood. N6-methyladenosine (m6A) is a kind of RNA modification that plays a critical role in cancer development. We aim to find out the possible m6A regulatory mechanism of the fat mass and obesity-associated protein (FTO) on the development of cervical cancer. The proliferative capacity of cervical cancer cells was detected by 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl-2H-tetrazolium bromide (MTT), colony formation and 5-ethynyl-20-deoxyuridine (EdU) staining. The migration and invasion of cervical cancer cells were determined by transwell assay. The function of FTO on tumor growth was evaluated by a xenograft model. We found that FTO was highly expressed in cervical cancer tissues and cell lines. FTO silencing suppressed the proliferation, migration, and invasion of cervical cancer cells. Mechanistically, FTO modulated the m6A modification of Zinc finger E-box binding homeobox 1 (ZEB1) and Myelocytomatosis oncogene (Myc). Furthermore, ZEB1 and Myc overexpression reverse the effect of FTO knockdown on the malignant behaviors of cervical cancer cells. FTO may be a novel therapeutic target for cervical cancer.     

The role of hepatocyte growth factor activator inhibitor (HAI)‐1 and HAI‐2 in endometrial cancer

Hepatocyte growth factor activator inhibitors (HAI‐1 and HAI‐2) are Kunitz‐type serine protease inhibitors that have a broad inhibitory spectrum against serine proteases. This is the first study to investigate the role of HAI‐1 and HAI‐2 in endometrial cancer. We investigated the biological functions of HAI‐1 and HAI‐2 using KLE and HEC‐251 endometrial cancer cell lines, thus HAI‐1 and HAI‐2 were examined in uterine normal endometrium, endometrial hyperplasia and cancer specimens by immunohistochemistry. HAI‐1 and HAI‐2 showed potential inhibitory effects on cell proliferation, migration and cellular invasion by reduction of matriptase and hepsin expression. This in turn led to an increase in the levels of E‐cadherin and Slug, and a reduction in the levels of Vimentin, SIP1, Snail and Twist, and hence ER and PR signal transduction in endometrial cancer cells. The levels of HAI‐1 and HAI‐2 expression were significantly decreased in endometrial cancer specimens relative to the corresponding normal endometrium specimens. Low HAI‐1 and HAI‐2 expression was a significant predictor for a poor prognosis compared with high HAI‐1 and HAI‐2 expression. These findings indicate that HAI‐1 and HAI‐2 could be considered as therapeutic targets and used as favorable prognosis markers for endometrial cancer.     

肿瘤相关巨噬细胞相关性miR-99a对子宫内膜癌细胞生长和侵袭的调控作用

背景与目的：肿瘤相关巨噬细胞（tumor-associated macrophage，TAM）浸润与肿瘤进展密切相关，但作用机制尚不明确，因此，探索miR-99a对单核巨噬细胞极化的影响及其对子宫内膜癌（endometrial cancer，EC）细胞生长、侵袭的影响。方法：检测EC组织中巨噬唾液酸蛋白（macrosialin）CD68表达并分析其与临床病理学特征之间的关系；运用人EC细胞系HEC-1B、RL95-2培养上清液诱导人单核细胞U937向TAM（M2型巨噬细胞）分化；将人工合成的miR-99a模拟物片段转染至诱导后的巨噬细胞，转染后运用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）及流式细胞术检测巨噬细胞相关因子CD68、CD163以及巨噬细胞甘露糖受体（mcrophage mannose receptor）CD206表达量变化，并运用酶联免疫吸附法（enzyme-linked immunosorbent assay，ELISA）检测巨噬细胞分泌相关细胞因子IL-12、IL-4和IL-10分泌量变化；将转染miR-99a的诱导后巨噬细胞与EC细胞共培养，运用细胞计数试剂盒（cell counting kit-8，CCK-8）和transwell法检测其对EC细胞增殖和侵袭能力的影响，并初步分析其可能的作用机制。结果：EC组织CD68高表达并与肿瘤肌层浸润及血管生成呈正相关；肿瘤细胞培养上清液成功诱导单核细胞向M2型TAM极化。转染miR-99a后单核细胞组CD68及CD163表达较对照组下降（P＜0.01），而CD206表达差异无统计学意义（P＞0.05），流式细胞术进一步证实上述表达变化；ELISA结果发现，转染miR-99a诱导后巨噬细胞中IL-12分泌增多（P＜0.01），而IL-4、IL-10分泌减少（P＜0.01），提示巨噬细胞向M2型极化受抑制。将诱导后巨噬细胞与EC细胞共培养，共培养后EC细胞增殖侵袭能力较对照组增加，而转染miR-99a模拟片段至诱导后巨噬细胞能够抑制其对增殖（P＜0.01）及侵袭能力的促进作用（P＜0.05）。诱导后巨噬细胞中过表达miR-99a后细胞中mTOR及其通路受到抑制。结论：EC间质巨噬细胞浸润与肿瘤肌层浸润及血管新生相关，miR-99a能够逆转单核细胞向M2表型极化，并抑制EC细胞介导TAM的促EC细胞生长和侵袭作用，其作用可能通过调控mTOR通路产生。     

Inhibition of gastric cancer cell growth and invasion through siRNA-mediated knockdown of guanine nucleotide exchange factor Vav3

Vav3, a Rho GTPase guanine nucleotide exchange factor, is associated with tumor growth, apoptosis, invasion and metastasis, and angiogenesis. However, the role of Vav3 in gastric cancer remains unclear. In this study, Vav3 expression was blocked by specific siRNA in gastric cancer cell line MGC803. MTT was used to assay cell proliferation activity; wound healing assay and transwell assay were applied to detect cell migration and invasion ability; and qRT-PCR and Western blot were employed to detect expression levels of Vav3 as well as proliferation, migration, and invasion-related genes. The results showed that Vav3 expression in gastric cancer tissues and cell lines was significantly upregulated and was higher than that in adjacent tissues of cancer and normal gastric mucosal cell lines. Vav3 knockdown inhibited proliferation, migration, and invasion of MGC803 gastric cancer cells. The expression of P21, P27, TIMP-1, and TIMP-2 was upregulated, while proliferating cell nuclear antigen, cyclin E1, matrix metalloproteinase (MMP)-2, and MMP-7 were downregulated by Vav3 knockdown in MGC803 gastric cells. In conclusion, Vav3 is involved in the proliferation, migration, and invasion of gastric cancer cell as a tumor oncogene.     

Interleukin 11 regulates endometrial cancer cell adhesion and migration via STAT3

Endometrial carcinoma is the most common gynaecological malignancy. There is however a lack of curative therapies, especially for patients diagnosed with late stage, recurrent or aggressive disease, who have a poor prognosis. Interleukin (IL) 11 is a pleiotropic cytokine that has a role in a number of cancers including colon and breast cancer. IL11 was recently found to be upregulated in endometrial cancers, however the function of IL11 in endometrial cancer is not known. This study aimed to determine the effects of IL11 on endometrial cancer cell proliferation, adhesion and migration. Three endometrial cancer cell lines, Ishikawa, HEC-1A and AN3CA (derived from endometrial cancers grade I, II and III, respectively), were used to determine the effect of IL11 on endometrial cancer cell function. Cell proliferation and viability were assessed by BrdU and Wst-1 assays. Cell adhesion to the extracellular matrix proteins fibronectin, collagen I and IV, vitronectin and laminin was assessed. Modified boyden chambers were utilized to access IL11 action on migration and invasion, respectively. The specific effect of IL11 action on these processes was determined using a unique IL11 inhibitor. IL11 phosphorylated (p)-STAT3 protein abundance in all 3 cell lines but had no effect on pERK and pAKT abundance. Similarly, IL11 had no effect on cell proliferation and viability but increased adhesion of ANC3A cells to fibronectin while having no effect on the other extracellular matrix proteins. IL11 did not alter the adhesive properties of the Ishikawa and HEC-1A cells. In the AN3CA cells, IL11 treatment resulted in a 50% increase in migration and co-treatment with the specific IL11 inhibitor or a STAT3 inhibitor abolished the effect. This study shows a role for IL11 in endometrial cancer and suggests IL11 may be involved in endometrial cancer development and thus may be useful as a therapeutic target.     

AMF/PGI-mediated tumorigenesis through MAPK-ERK signaling in endometrial carcinoma

Autocrine motility factor (AMF), which is also known as phosphoglucose isomerase (PGI), enhances tumor cell growth and motility. In this study, we found that AMF and its receptor were both highly expressed in Endometrial Carcinoma (EC) tissues compared to normal tissues. Levels of AMF were increased in serum of endometrial cancer patients. Downregulation of AMF by shRNA inhibited invasion, migration and proliferation as well as growth in a three-dimensional culture. AMF cytokine function, but not enzymatic activity of PGI, regulated tumorigenic activities of AMF. The MAPK-ERK1/2 pathway contributed to AMF-induced effects in EC cells. In agreement, Mek inhibitor decreased AMF-induced invasion, migration and proliferation of EC cells. In addition, in two mouse tumor metastasis models (EC cells delivered through left ventricle or intraperitoneally) AMF-silenced EC cells showed decreased tumor proliferative and metastatic capacities. We suggest that AMF/PGI is a potential therapeutic target in endometrial carcinoma.     

PRUNE2点突变影响前列腺癌DU145细胞增殖、凋亡、侵袭和迁移

背景与目的：PRUNE2是成神经细胞瘤的一个特异性预后相关基因,在调节成神经细胞瘤的细胞分化、增殖和侵袭方面发挥着重要作用。PRUNE2低表达与前列腺癌的不良预后密切相关。探讨PRUNE2点突变对前列腺癌DU145细胞增殖、凋亡、侵袭和迁移的影响。方法：通过构建PRUNE2基因野生型和突变型过表达的重组载体并将其转染到DU145细胞中构建相应稳转株。利用细胞计数试剂盒-8（cell counting kit-8,CCK-8）实验和克隆形成实验检测细胞恶性增殖能力,通过细胞凋亡实验检测细胞凋亡能力,采用transwell小室法检测细胞侵袭和迁移能力。采用蛋白质印迹法（Western blot）检测蛋白的表达水平,通过免疫荧光和免疫共沉淀实验检测蛋白之间的相互作用。结果：转染突变型PRUNE2 E370K基因的DU145细胞恶性增殖、侵袭和迁移能力均较对照组显著增强,细胞凋亡率显著降低,差异有统计学意义（P<0.05）。PRUNE2与RhoA之间可以相互结合,但是PRUNE2 E370K突变体与RhoA之间的相关作用明显减弱。同时,PRUNE2 E370K突变体促进Rho通路下游ROCK蛋白和FAK蛋白的表达,促进与细胞增殖相关的Bcl-2和cyclin D1蛋白的表达,同时抑制与侵袭、迁移相关抑制蛋白E-cadherin的表达。结论：前列腺癌DU145细胞中PRUNE2基因点突变可以促进细胞增殖、侵袭和迁移,并抑制细胞凋亡。     

miR-152 is a tumor suppressor microRNA that is silenced by DNA hypermethylation in endometrial cancer

The etiology and development of human cancers that remain little understood might be enlightened by defining tumor suppressor microRNAs (TS-miRNA). In this study, we identified TS-miRNAs silenced by aberrant DNA hypermethylation in endometrial cancer. Functional screening of 327 synthetic miRNAs in an endometrial cancer cell proliferation assay identified 103 miRNAs that inhibited cell growth. We then determined the sequence, DNA methylation status, and expression levels of these miRNAs in endometrial cancer cell lines and primary tumors. These determinations led to the identification of miR-152 as a candidate TS-miRNA gene in endometrial cancer. Epigenetic silencing documented in miR-152 was consistent with its location at 17q21.32 in intron 1 of the COPZ2 gene, which is also silenced often in endometrial cancer by DNA hypermethylation, and also with evidence that miR-152 targets the DNA methyltransferase DNMT1. Notably, restoration of miR-152 expression in endometrial cancer cell lines was sufficient to inhibit tumor cell growth in vitro and in vivo. We identified E2F3, MET, and Rictor as novel candidate targets of miR-152, suggesting how its epigenetic silencing can drive endometrial carcinogenesis. Our findings define a central role for miR-152 in endometrial cancer, and they also suggest its use in new therapeutic strategies to treat this cancer.     

Ligand-Activated Peroxisome Proliferator-Activated Receptor β/δ Modulates Human Endometrial Cancer Cell Survival

Endometrial cancer is the fourth most common malignancy among women and is a major cause of morbidity contributing to approximately 8,200 annual deaths in the USA. Despite advances to the understanding of endometrial cancer, novel interventions for the disease are necessary given that many tumors become refractory to therapy. As a strategy to identify novel therapies for endometrial carcinoma, in this study, we examined the contribution of the peroxisome proliferator-activated receptor β/δ (PPARβ/δ) to endometrial cancer cell proliferation and apoptosis. We found that when activated with the highly selective PPARβ/δ agonists, GW0742 and GW501516, PPARβ/δ inhibited the proliferation and markedly induced the apoptosis of three endometrial cancer cell lines. The specificity of the PPARβ/δ-induced effects on cell proliferation and apoptosis was demonstrated using PPARβ/δ-selective antagonists and PPARβ/δ small interfering RNA in combination with PPARβ/δ-selective agonists. Furthermore, we showed that PPARβ/δ activation increased phosphatase and tensin homolog expression, which led to protein kinase B (AKT) and glycogen synthase kinase-3β (GSK3β) dephosphorylation, and increased β-catenin phosphorylation associated with its degradation. Overall, our data suggest that the antitumorigenic effect of PPARβ/δ activation in endometrial cancer is mediated through the negative regulation of the AKT/GSK3β/β-catenin pathway. These findings warrant further investigation of PPARβ/δ as a therapeutic target in endometrial cancer.     

The stem cell factor/c-kit receptor pathway enhances proliferation and invasion of pancreatic cancer cells

Background  

The transmembrane protein c-kit is a receptor tyrosine kinase (KIT) and KIT is expressed in solid tumors and hematological malignancies such as gastrointestinal stromal tumor (GIST), small-cell lung cancer and chronic myelogenous leukemia (CML). KIT plays a critical role in cell proliferation and differentiation and represents a logical therapeutic target in GIST and CML. In pancreatic cancer, c-kit expression has been observed by immunohistochemical techniques. In this study, we examined the influence of c-kit expression on proliferation and invasion using five pancreatic cancer cell lines. In addition, the inhibitory effect of imatinib mesylate on stem cell factor (SCF)-induced proliferation and invasion was evaluated. Finally, we also analyzed KIT and SCF expression in pancreatic cancer tissues using immunohistochemistry and correlated the results with clinical features.