Promotion of hepatocellular carcinoma metastasis through matrix metalloproteinase activation by epithelial‐mesenchymal transition regulator Twist1

E‐cadherin loss is a key biological mechanism in tumour invasion. As a main regulator of epithelial‐mesenchymal transition (EMT) mechanism‐mediated invasion and metastasis, Twist1 plays an important role through its regulation of E‐cadherin expression. However, whether or not Twist2 has the same function in tumour metastasis remains unclear. The purpose of this study is to investigate the expressions and different roles of Twist1 and Twist2 in human hepatocellular carcinoma (HCC). The expressions of Twist1 and Twist2 in HCC tissue were evaluated by immunohistochemical staining. The role of Twist1 and Twist2 in invasiveness was also evaluated in vitro by using HCC cell lines. Twist1 nuclear overexpression is found to be correlated with HCC metastasis, and its expression is negatively correlated with E‐cadherin expression in human tissue. Twist2, a Twist1 homology protein, only expresses in the cytoplasm and shows no significant correlation with HCC metastasis. By ectopic transfection of Twist1 and Twist2 into the HCC cells, HepG2 and PLC, Twist1 is able to down‐regulate E‐cadherin expression and promote matrix metalloproteinase (MMP) activation, specifically in MMP2 and MMP9. In functional assays, Twist1 is found to promote invasion in HepG2 and PLC cells, but the invasion ability of the groups is not affected Twist2. Our findings indicate that Twist1 induces HCC invasion via increased activity in MMPs, leading to poor clinical prognoses. The results of this study also demonstrate a novel cogitation in Twist2, which has no effect on HCC invasion and metastasis. Twist1 may contribute to HCC invasion and metastasis and may be used as a novel therapeutic target for the inhibition of HCC metastasis.     

The lncRNA TP73‐AS1 promotes ovarian cancer cell proliferation and metastasis via modulation of MMP2 and MMP9

Ovarian cancer is one of the most common gynecologic malignancy with poor prognosis. Recently, long noncoding RNAs (lncRNAs) have been identified as key regulators in cancer development. The current study investigated the role of lncRNA P73 antisense RNA 1T (TP73‐AS1) in ovarian cancer. Quantitative real‐time polymerase chain reaction determined the expression levels of TP‐73AS1, matrix metallopeptidases (MMPs) messenger RNA. Cell proliferative ability, cell invasion, and migration were CCK‐8 and colony formation, and transwell invasion and migration assays, respectively. The protein levels of matrix metallopeptidase 2 (MMP2) and MMP9 were measured by Western blot. TP73‐AS1 was upregulated in the ovarian cancer tissues and ovarian cancer cells, and upregulation of TP73‐AS1 was associated with poor prognosis. Knockdown of TP73‐AS1 significantly suppressed cell proliferation, invasion, and migration of SKOV3 cells, and overexpression of TP73‐AS1 promoted cell proliferation, invasion, and migration of OVCA429 cells. In addition, knockdown of TP73‐AS1 suppressed the in vivo tumor growth. Tumor metastasis RT² profiler polymerase chain reaction array showed that MMP2 and MMP9 was significantly upregulated by TP73‐AS1 overexpression in ovarian cancer cells. TP73‐AS1 overexpression enhanced the expression of MMP2 and MMP9 in ovarian cancer cells. Knockdown of MMP2 and MMP9 attenuated the effects of TP73‐AS1 overexpression on cell invasion and migration. The clinical data showed that MMP2 and MMP9 were upregulated and positively correlated with TP73‐AS1 expression in ovarian cancer tissues. Collectively, our results demonstrated the oncogenic role of TP73‐AS1 in ovarian cancer, and targeting TP73‐AS1 may represent a novel approach in battling against ovarian cancer.     

Epithelial‐mesenchymal transition softens head and neck cancer cells to facilitate migration in 3D environments

The biological impact and signalling of epithelial‐mesenchymal transition (EMT) during cancer metastasis has been established. However, the changes in biophysical properties of cancer cells undergoing EMT remain elusive. Here, we measured, via video particle tracking microrheology, the intracellular stiffness of head and neck cancer cell lines with distinct EMT phenotypes. We also examined cells migration and invasiveness in different extracellular matrix architectures and EMT‐related signalling in these cell lines. Our results show that when cells were cultivated in three‐dimensional (3D) environments, the differences in cell morphology, migration speed, invasion capability and intracellular stiffness were more pronounced among different head and neck cancer cell lines with distinct EMT phenotypes than those cultivated in traditional plastic dishes and/or seated on top of a thick layer of collagen. An inverse correlation between intracellular stiffness and invasiveness in 3D culture was revealed. Knock‐down of the EMT regulator Twist1 or Snail or inhibition of Rac1 which is a downstream GTPase of Twist1 increased intracellular stiffness. These results indicate that the EMT regulators, Twist1 and Snail and the mediated signals play a critical role in reducing intracellular stiffness and enhancing cell migration in EMT to promote cancer cells invasion.     

SPOCK1 promotes the invasion and metastasis of gastric cancer through Slug‐induced epithelial‐mesenchymal transition

Metastasis is a crucial impediment to the successful treatment for gastric cancer. SPOCK1 has been demonstrated to facilitate cancer metastasis in certain types of cancers; however, the role of SPOCK1 in the invasion and metastasis of gastric cancer remains elusive. SPOCK1 and epithelial‐mesenchymal transition (EMT)‐related biomarkers were detected by immunohistochemistry and Western blot in gastric cancer specimens. Other methods including stably transfected against SPOCK1 into gastric cancer cells, Western blot, migration and invasion assays in vitro and metastasis assay in vivo were also performed. The elevated expression of SPOCK1 correlates with EMT‐related markers in human gastric cancer tissue, clinical metastasis and a poor prognosis in patients with gastric cancer. In addition, knockdown of SPOCK1 expression significantly inhibits the invasion and metastasis of gastric cancer cells in vitro and in vivo, inversely, SPOCK1 overexpression results in the opposite effect. Interestingly, SPOCK1 expression has no effect on cell proliferation in vitro and in vivo. Regarding the mechanism(s) of SPOCK1‐induced cells invasion and metastasis, we prove that Slug‐induced EMT is involved in SPOCK1‐facilitating gastric cancer cells invasion and metastasis. The elevated SPOCK1 expression is closely correlated with cancer metastasis and patient survival, and SPOCK1 promotes the invasion and metastasis of gastric cancer through Slug‐mediated EMT, thereby possibly providing a novel therapeutic target for gastric cancer.     

The calcium pump PMCA4 prevents epithelial-mesenchymal transition by inhibiting NFATc1-ZEB1 pathway in gastric cancer

Epithelial-mesenchymal transition (EMT) is considered as the key mechanism involved in cancer metastasis. Several studies showed that various cell membrane calcium channels play different roles in cancer metastasis. In the present study, the potential role of ATPase plasma membrane Ca²⁺ transporting 4 (PMCA4) in regulating EMT in gastric cancer (GC) was investigated. GC patients who underwent radical surgery were enrolled in this study. In vitro human GC cell lines MKN45 and NCI-N87 were used, and MKN45 cells were injected in nude mice to evaluate tumor development. Our results showed that low PMCA4 expression was associated with advanced TNM stage and poor prognosis in GC patients. Knockdown of PMCA4 suppressed E-cadherin, grainyhead like 2 (GRHL2) and ovo-like 1 (OVOL1) expression, up-regulated vimentin expression, increased migration and invasion ability, and promoted the resistance to cytotoxic drug. Furthermore, GC cells displayed an elongated fibroblastoid morphology when PMCA4 was knockdown. PMCA4 overexpression resulted in an up-regulated E-cadherin expression and decreased migration and invasion ability. In vivo metastasis assay showed that PMCA4 overexpression resulted in a decreased incidence of lung metastasis. PMCA4 inhibition increased ZEB1 expression and nuclear accumulation of nuclear factor of activated T-cell isoform c1 (NFATc1). EMT induced by PMCA4 inhibition could be prevented by the knockdown of NFATc1 or ZEB1. In addition, cyclosporine A prevented EMT induced by PMCA4 inhibition by suppressing the NFATc1-ZEB1 pathway. Our data identified a novel mechanism in the regulation of EMT in GC, and provided a novel target in the treatment of EMT subtype in GC.     

TIPE1 suppresses invasion and migration through down‐regulating Wnt/β‐catenin pathway in gastric cancer

Epithelial–mesenchymal transition (EMT) plays an important role in the invasiveness and metastasis of gastric cancer. Therefore, identifying key molecules involved in EMT will provide new therapeutic strategy for treating patients with gastric cancer. TIPE1 is a newly identified member of the TIPE (TNFAIP8) family, and its contributions to progression and metastasis have not been evaluated. In this study, we found that the levels of TIPE1 were significantly reduced and inversely correlated with differentiation status and distant metastasis in primary gastric cancer tissues. We further observed overexpression of TIPE1 in aggressive gastric cancer cell lines decreased their metastatic properties both in vitro and in vivo as demonstrated by markedly inhibiting EMT and metastasis of gastric cancer cells in nude mice. Consistently, gene silencing of TIPE1 in well‐differentiated gastric cancer cell line (AGS) inhibited these processes. Mechanistically, we found that TIPE1‐medicated Wnt/β‐catenin signalling was one of the critical signal transduction pathways that link TIPE1 to EMT inhibition. Importantly, TIPE1 dramatically restrained the expression and activities of MMP2 and MMP9 which are demonstrated to promote tumour progression and are implicated in EMT. Collectively, these findings provide new evidence for a better understanding of the biological activities of TIPE1 in progression and metastasis of gastric cancer and suggest that TIPE1 may be an innovative diagnostic and therapeutic target of gastric cancer.     

二氢杨梅素对人胃癌MKN45细胞迁移和侵袭的影响及其机制*

目的:探讨二氢杨梅素(DHM )对人胃癌MKN45细胞迁移和侵袭的作用及其分子机制。方法:培养人低分化胃癌MKN45细胞,用不同浓度的DHM(0,10,20,30,40,50 μmol/L)分别处理细胞24及48 h,每组实验重复3次,采用CCK8实验检测癌细胞增殖活力;划痕实验检测细胞迁移能力;Transwell小室检测细胞侵袭能力;免疫印迹分析细胞迁移和侵袭相关蛋白表达情况。结果:不同浓度DHM干预可降低MKN45细胞活力。20,30及40 μmol/L的DHM处理48 h可明显抑制细胞的迁移能力(P＜0.01)和侵袭能力(P＜0.05及0.01)。20及30 μmol/L的DHM处理48 h可增加E-cadherin蛋白表达(P＜0.01)、降低Vimentin表达(P＜0.01),从而逆转EMT过程;10,20及30 μmol/L的DHM处理48 h可明显降低pJNK的活性表达水平(P＜0.05及0.01),及MMP-2蛋白表达(P＜ 0.01);JNK通路特异性抑制剂SP600125预处理可明显促进DHM对癌细胞侵袭能力的抑制作用(P＜0.01)及降低MMP-2表达(P＜0.01)。结论:DHM具有抑制人胃癌MKN45细胞的迁移及侵袭的作用,其机制可能与通过JNK通路下调MMP-2蛋白表达水平、逆转上皮间质转化有关。     

Alpha B‐crystallin promotes the invasion and metastasis of gastric cancer via NF‐κB‐induced epithelial‐mesenchymal transition

Alpha B‐crystallin (CRYAB) is overexpressed in a variety of cancers. However, little is known about its specific function and regulatory mechanism in gastric cancer. Here, we first explore the role of CRYAB in gastric cancer progression and metastasis. The expression of CRYAB was determined by western blot and immunohistochemistry in gastric cancer tissues. Besides, methods including stably transfected against CRYAB into gastric cancer cells, western blot, migration and invasion assays in vitro and metastasis assay in vivo were also conducted. The expression of CRYAB is up‐regulated in gastric cancer tissues compared with matched normal tissues. High expression level of CRYAB is closely correlated with cancer metastasis and shorter survival time in patients with gastric cancer. Additionally, CRYAB silencing significantly suppresses epithelial‐mesenchymal transition (EMT), migration and invasion of gastric cancer cells in vitro and in vivo, whereas CRYAB overexpression dramatically reverses these events. Mechanically, CRYAB facilitates gastric cancer cells invasion and metastasis via nuclear factor‐κ‐gene binding (NF‐κB)‐regulated EMT. These findings suggest that CRYAB expression predicts a poor prognosis in patients with gastric cancer. Besides, CRYAB contributes to gastric cancer cells migration and invasion via EMT, mediated by the NF‐κB signalling pathway, thus possibly providing a novel therapeutic target for gastric cancer.     

Inhibition of PRL-3 gene expression in gastric cancer cell line SGC7901 via microRNA suppressed reduces peritoneal metastasis

High expression of PRL-3, a protein tyrosine phosphatase, is proved to be associated with lymph node metastasis in gastric carcinoma from previous studies. In this paper, we examined the relationship between PRL-3 expression and peritoneal metastasis in gastric carcinoma. We applied the artificial miRNA (pCMV-PRL3miRNA), which is based on the murine miR-155 sequence, to efficiently silence the target gene expression of PRL-3 in SGC7901 gastric cancer cells at both mRNA and protein levels. Then we observed that, in vitro, pCMV-PRL3miRNA significantly depressed the SGC7901 cell invasion and migration independent of cellular proliferation. In vivo, PRL-3 knockdown effectively suppressed the growth of peritoneal metastases and improved the prognosis in nude mice. Therefore, we concluded that artificial miRNA can depress the expression of PRL-3, and that PRL-3 might be a potential therapeutic target for gastric cancer peritoneal metastasis.     

Naringenin Decreases Invasiveness and Metastasis by Inhibiting TGF-β-Induced Epithelial to Mesenchymal Transition in Pancreatic Cancer Cells

Epithelial to mesenchymal transition (EMT) promotes cellular motility, invasiveness and metastasis during embryonic development and tumorigenesis. Transforming growth factor-β (TGF-β) signaling pathway is a key regulator of EMT. A lot of evidences suggest that this process is Smad3-dependent. Herein we showed that exposure of aspc-1 and panc-1 pancreatic cancer cells to TGF-β1 resulted in characteristic morphological alterations of EMT, and enhancement of cell motility and gemcitabine (Gem) resistance along with an up-regulation of EMT markers genes such as vimentin, N-cadherin, MMP2 and MMP9. Naringenin (Nar) down-regulated EMT markers expression in both mRNA and protein levels by inhibiting TGF-β1/Smad3 signal pathway in the pancreatic cancer cells. Consequently, Nar suppressed the cells migration and invasion and reversed their resistance to Gem.     

Silencing of lncRNA ZFAS1 inhibits malignancies by blocking Wnt/β-catenin signaling in gastric cancer cells

Gastric cancer is a common malignancy with high mortality. Long noncoding RNA (lncRNA) zinc finger antisense (ZFAS)1 is upregulated in gastric cancer specimens compared with the para-carcinoma tissues. The silencing of ZFAS1 inhibited the growth, proliferation, cell cycle progress, migration, invasion and epithelial-mesenchymal transition (EMT), and enhanced the sensitivity to cis-platinum or paclitaxel in SGC7901 cells, as evidenced by the expression changes of proliferating cell nuclear antigen, Cyclin D1, Cyclin E, Cyclin B1, E-cadherin, N-cadherin, vimentin, matrix metalloproteinase (MMP)-2 and MMP-14. The ZFAS1 also activated the Wnt/β-catenin signaling. Subsequently, the ZFAS1 knockdown-induced the inhibition of migration, invasion, EMT and resistance to chemotherapeutic reagens was reversed by the overexpression of β-catenin. In summary, the silencing of ZFAS1 inhibited the growth, proliferation, cell cycle progress, migration, invasion, EMT and chemotherapeutic tolerance by blocking the Wnt/β-catenin signaling in gastric cancer cells.     

A novel biphenyl urea derivate inhibits the invasion of breast cancer through the modulation of CXCR4

The increased migration and invasion of breast carcinoma cells are key events in the development of metastasis to the lymph nodes and distant organs. CXCR4, the receptor for stromal‐derived factor‐1, is reportedly involved in breast carcinogenesis and invasion. In this study, we investigated a novel biphenyl urea derivate, TPD7 for its ability to affect CXCR4 expression as well as function in breast cancer cells. We demonstrated that TPD7 inhibited the breast cancer proliferation and down‐regulated the CXCR4 expression on breast cancer cells both over‐expressing and low‐expressing HER2, an oncogene known to induce the chemokine receptor. Treatments with pharmacological proteasome inhibitors partial suppressed TPD7‐induced decrease in CXCR4 expression. Real‐time PCR analysis revealed that down‐regulation of CXCR4 by TPD7 also occurred at the translational level. Inhibition of CXCR4 expression by TPD7 further correlated with the suppression of SDF‐1α‐induced migration and invasion in breast tumour cells, knockdown of CXCR4 attenuated TPD7‐inhibitory effects. In addition, TPD7 treatment significantly suppressed matrix metalloproteinase (MMP)‐2 and MMP‐9 expression, the downstream targets of CXCR4, perhaps via inactivation of the ERK signaling pathway. Overall, our results showed that TPD7 exerted its anti‐invasive effect through the down‐regulation of CXCR4 expression and thus had the potential for the treatment of breast cancer.