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.     

AEG‐1 induces gastric cancer metastasis by upregulation of eIF4E expression

Gastric cancer is the third leading cause of cancer‐related deaths worldwide, and patients with lymph node, peritoneal and distant metastasis have a poor prognosis. Overexpression of Astrocyte‐elevated gene‐1 (AEG‐1) has been reported to be correlated with the progression and metastasis of gastric cancer. However, its mechanisms are quite unclear. In this study, we found that elevated expression of AEG‐1 was correlated with metastasis in human gastric cancer tissues. Moreover, gain‐ or loss‐of‐function of AEG‐1, respectively, promoted or suppressed epithelial–mesenchymal transition (EMT), migration and invasion of gastric cancer cells. AEG‐1 positively regulated eIF4E, MMP‐9 and Twist expression. Manipulating eIF4E expression by transfection of overexpression constructs or siRNAs partially eliminated AEG‐1‐regulated EMT, cell migration and invasion. In addition, overexpression or knockdown of eIF4E promoted or suppressed EMT, cell migration and invasion in parallel with upregulation of MMP‐9 and Twist expression, while manipulating eIF4E expression partially abrogated AEG‐1‐induced MMP‐9 and Twist. Finally, silencing of AEG‐1 expression not only inhibited tumour growth in parallel with downregulation of eIF4E, MMP‐9 and Twist expression in a xenograft nude mouse model, but also suppressed lymph node and peritoneal metastasis of gastric cancer in an orthotopic nude mouse model. These findings suggest that AEG‐1 promotes gastric cancer metastasis through upregulation of eIF4E‐mediated MMP‐9 and Twist, which provides new diagnostic markers and therapeutic targets for cancer metastasis.     

Decrease in claudin‐2 expression enhances cell migration in renal epithelial madin–darby canine kidney cells

Migration of renal epithelial cells increases after renal tubular damage, but its mechanism has not been clarified in detail. Hyperosmotic stress increased a cellular injury concomitant with a decrease in mRNA and protein expression of claudin‐2 in renal tubular epithelial Madin–Darby canine kidney cells. We hypothesized that claudin‐2 is involved in the regulation of cell migration. To knockdown claudin‐2 expression, we made the cells expressing doxycycline‐inducible claudin‐2 shRNA vector. Claudin‐2 knockdown affected neither the endogenous expression levels of claudin‐1, ‐3, ‐4, and ‐7 nor the Triton X‐100 solubility of these claudins. Transepithelial electrical resistance was increased by claudin‐2 knockdown without affecting permeability to FITC‐dextran (4,000 Da). BrdU incorporation assay and cell counting revealed that cell proliferation and viability are unaffected by claudin‐2 knockdown. In the wound‐healing assay, the recovery rate of wound area was increased by claudin‐2 knockdown. The mRNA expression and activity of matrix metalloproteinase‐9 (MMP‐9) were increased by claudin‐2 knockdown. A selective MMP‐9 inhibitor suppressed cell migration in the claudin‐2 knockdown cells. Hyperosmotic stress increased the expression and activity of MMP‐9, which were inhibited by claudin‐2 overexpression. These results suggest that the decrease in claudin‐2 expression enhances cell migration mediated by the increase in the expression and activity of MMP‐9. J. Cell. Physiol. 226: 1471–1478, 2011. © 2010 Wiley‐Liss, Inc.     

LINC00961 restrains cancer progression via modulating epithelial–mesenchymal transition in renal cell carcinoma

Recently, long noncoding RNA have been identified as new gene regulators and prognostic biomarkers in various cancers, including renal cell carcinoma (RCC). The expression and biological roles of LINC00961 have been reported in many human cancers. However, up to date, no study of LINC00961 has been shown in RCC. Currently, we aimed to investigate the function of LINC00961 in RCC progression. Interestingly, we observed that LINC00961 could act as a novel biomarker in predicting the diagnosis of RCC. Then, we found that LINC00961 was greatly downregulated in RCC cell lines (Caki-1, Caki-2, 786-O, A498, and ACHN cells) compared with normal renal cell lines (HK-2 cells). Then, 786-O cells and ACHN cells were infected with LV-LINC00961. As displayed in our current study, LINC00961 overexpression could obviously suppress the proliferation and survival of RCC cells in vitro. In addition, RCC cell apoptosis was greatly induced and cell cycle progression was blocked in G1 phase by upregulation of LINC00961 in 786-O cells and ACHN cells. Subsequently, we found that LV-LINC00961 was able to restrain RCC cell migration and cell invasion capacity. Meanwhile, the messenger RNA and protein expression levels of epithelial–mesenchymal transition (EMT)-associated markers Slug and N-cadherin in RCC cell lines were dramatically inhibited by overexpressing LINC00961. Finally, the in vivo experiment was carried out and we observed that LINC00961 could inhibit RCC development through modulating EMT process. Taken these together, it was indicated in our study that LINC00961 was involved in RCC progression through targeting EMT pathway.     

TRAF6 regulates tumour metastasis through EMT and CSC phenotypes in head and neck squamous cell carcinoma

Epithelial–mesenchymal transition (EMT) is associated with metastasis formation, generation and maintenance of cancer stem cells (CSCs). However, the regulatory mechanisms of CSCs have not been clarified. This study aims to investigate the role of TNF receptor‐associated factor 6 (TRAF6) on EMT and CSC regulation in squamous cell carcinoma of head and neck (SCCHN). We found TRAF6 was overexpressed in human SCCHN tissues, and high TRAF6 expression was associated with lymphatic metastasis and resulted in poor prognosis in patients with SCCHN. In addition, elevated TRAF6 expression was observed in several HNSCC cell lines, and wound healing and transwell assay results showed that TRAF6 knockdown inhibited the migration and invasion ability of the SCCHN cells. Moreover, the expression of Vimentin, Slug and N‐cadherin was down‐regulated and that of E‐cadherin was elevated after TRAF6 knockdown but decreased by transforming growth factor beta 1 (TGF‐β1) and CAL27 similar to mesenchymal cells formed after TGF‐β1 induction. In addition, the expression levels of CD44, ALDH1, KLF4 and SOX2 were inhibited after TRAF6 knockdown, and the anchor‐dependent colony formation number and sphere number were remarkably reduced. Flow cytometry showed TRAF6 knockdown reduced ALDH1‐positive cancer stem cells. We also demonstrated that TRAF6 is closely associated with EMT process and cancer stem cells using a Tgfbr1/Pten 2cKO mice SCCHN model and human SCCHN tissue microarray. Our findings indicate that TRAF6 plays a role in EMT phenotypes, the generation and maintenance of CSCs in SCCHN, suggesting that TRAF6 is a potential therapeutic target for SCCHN.     

A novel matrine derivative WM622 inhibits hepatocellular carcinoma by inhibiting PI3K/AKT signaling pathways

Nasopharyngeal carcinoma (NPC) is a unique subtype of head and neck cancer, with tendency to spread to regional lymph nodes and distant organs at early stage. Vimentin, a major cytoskeletal protein constituent of the intermediate filament, plays a critical role in the epithelial–mesenchymal transition. Overexpression of vimentin is considered to be a critical prerequisite for metastasis in numerous human cancers. Therefore, targeting vimentin for cancer therapy has gained a lot of interest. In the present study, we detected vimentin expression in NPC tissues and found that overexpression of vimentin is associated with poor prognosis of NPC patients. Silencing of vimentin in NPC CNE2 cells by RNAi suppresses cells migration and invasion in vitro. However, blocking vimentin did not affect cell proliferation of CNE2 cells. In addition, the in vivo metastatic potential of CNE2 cells transfected with Vimentin shRNA was suppressed in a nude mouse model of pulmonary metastasis. Silencing of Vimentin in CNE2 cells leads to a decrease of microvessel density and VEGF, CD31, MMP2, and MMP9 expressions in pulmonary metastatic tumors. Importantly, we found that it is easier for the tumor cells from the high vimentin-expressing pulmonary metastatic tumors to reinvade the microvessel and to form stable tumor plaques attached to the endothelial cells, which resemble the resource of circulating tumor cells and are very hard to eliminate. However, depletion of vimentin inhibits the formation of vascular tumor plaques. Our findings suggest that RNAi-based vimentin silencing may be a potential and promising anti-metastatic therapeutic strategy for NPC.     

Ivermectin suppresses tumour growth and metastasis through degradation of PAK1 in oesophageal squamous cell carcinoma

Oesophageal squamous cell carcinoma (ESCC), the most common form of oesophageal malignancies in the Asia‐Pacific region, remains a major clinical challenge. In this study, we found that ivermectin, an effective antiparasitic drug that has been approved for patients to orally treat onchocerciasis for over 30 years, displayed potent antitumour activity against ESCC cells in vitro and in nude mice. We demonstrated that ivermectin significantly inhibited cell viability and colony formation, and induced apoptosis through a mitochondrial‐dependent manner in ESCC cells. Ivermectin also abrogated ESCC cell migration, invasion, as well as the protein levels of MMP‐2 and MMP‐9. Mechanistically, ivermectin strongly inhibited the expression of PAK1; by further gain‐ and loss‐of‐function experiments, we confirmed that PAK1 played a crucial role in ivermectin‐mediated inhibitory effects on ESCC cells. In addition, the data indicated that ivermectin promoted PAK1 degradation through the proteasome‐dependent pathway. Additionally, ivermectin synergized with chemotherapeutic drugs including cisplatin and 5‐fluorouracil to induce apoptosis of ESCC cells. Interestingly, the in vivo experiments also confirmed that ivermectin effectively suppressed tumour growth and lung metastasis of ESCC. Collectively, these results indicate that ivermectin exerts a potent antitumour activity against ESCC and is a promising therapeutic candidate drug for ESCC patients, even those carrying metastasis.     

FKBP51 promotes invasion and migration by increasing the autophagic degradation of TIMP3 in clear cell renal cell carcinoma

The occurrence of metastasis is a serious risk for renal cell carcinoma (RCC) patients. In order to develop novel therapeutic approaches to control the progression of metastatic RCC, it is of urgent need to understand the molecular mechanisms underlying RCC metastasis and identify prognostic markers of metastatic risk. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been known to be closely associated with extracellular matrix (ECM) turnover, which plays a highly active role in tumor metastasis. Recent studies have shown that immunophilin FK-506-binding protein 51 (FKBP51) may be important for the regulation of ECM function, and exert effects on the invasion and migration of tumor cells. However, the mechanisms underlying these activities remain unclear. The present study detected the role of FKBP51 in clear cell renal cell carcinoma (ccRCC), the most common subtype of RCC, and found that FKBP51 significantly promotes ccRCC invasion and migration by binding with the TIMP3, connecting TIMP3 with Beclin1 complex and increasing autophagic degradation of TIMP3. Given the important roles that TIMPs/MMPs play in ECM regulation and remodeling, our findings will provide new perspective for future investigation of the regulation of metastasis of kidney cancer and other types of cancer.Subject terms: Renal cell carcinoma, Extracellular matrix     

Chelerythrine induces apoptosis via ROS‐mediated endoplasmic reticulum stress and STAT3 pathways in human renal cell carcinoma

Renal cell carcinoma (RCC) is a heterogeneous histological disease and it is one of the most common kidney cancer. The treatment of RCC has been improved for the past few years, but its mortality still remains high. Chelerythrine (CHE) is a natural benzo[c]phenanthridine alkaloid and a widely used broad‐range protein kinase C inhibitor which has anti‐cancer effect on various types of human cancer cells. However, its effect on RCC has not been fully elucidated. In this study, we evaluated the effect and mechanism of CHE on RCC cells. Our study showed that CHE induced colony formation inhibition and G2/M cell cycle arrest in a dose‐dependent manner in RCC cells. In addition, CHE increased cellular ROS level, leading to endoplasmic reticulum (ER) stress, inactivating STAT3 activities and inducing apoptosis in RCC cells which were suppressed by NAC, a special ROS inhibitor. We further found that both knockdown of ATF4 protein and overexpression of STAT3 protein could reduce CHE‐induced apoptosis in Caki cells. These results demonstrated that the apoptosis induced by CHE was mediated by ROS‐caused ER stress and STAT3 inactivation. Collectively, our studies provided support for CHE as a potential new therapeutic agent for the management of RCC.     

Matrix metalloproteinase 2 promotes cell growth and invasion in colorectal cancer

Colorectal cancer (CRC) is the third leading cause of cancer-related death in the western world. In this study, we evaluated the expression of matrix metalloproteinase 2 gene (MMP2) in CRC and analyzed its correlation with clinicopathological features. We found that the expression of MMP2 was significantly higher in CRC tissues than in the colorectal tissues. In addition, high levels of MMP2 protein were positively correlated with the status of tumor size, lymph node metastasis, distant metastasis, Dukes' stage, and tumor invasion. Moreover, patients with higher MMP2 levels had markedly shorter overall survivals than those with low MMP2 levels. Multivariate analysis results suggested that the level of MMP2 expression is an independent prognostic indicator for the survival of patients with CRC. Silencing MMP2 expression in CRC cell lines with lentiviral-mediated shRNA markedly suppressed cell proliferation, colony formation, and invasion. Furthermore, we observed that vascular endothelial growth factor (VEGF) and membrane type 1 (MT1)-MMP protein levels were decreased in MMP2-down-regulated colorectal cells. Therefore, our study demonstrated that MMP2 is an important factor related to carcinogenesis and metastasis of CRC, and MMP2 promotes CRC cell growth and invasion by up-regulating VEGF and MT1-MMP expression, which makes this pathway a potential target for cancer treatment.     

Hypoxia Induces EMT in Low and Highly Aggressive Pancreatic Tumor Cells but Only Cells with Cancer Stem Cell Characteristics Acquire Pronounced Migratory Potential

Tumor hypoxia induces epithelial-mesenchymal transition (EMT), which induces invasion and metastasis, and is linked to cancer stem cells (CSCs). Whether EMT generates CSCs de novo, enhances migration of existing CSCs or both is unclear. We examined patient tissue of pancreatic ductal adenocarcinoma (PDA) along with carcinomas of breast, lung, kidney, prostate and ovary. For in vitro studies, five established PDA cell lines classified as less (CSC^low) and highly aggressive CSC-like cells (CSC^high) were examined by single and double immunofluorescence microscopy, wound-, transwell-, and time-lapse microscopy. HIF-1α and Slug, as well as HIF-2α and CD133 were co-expressed pointing to a putative co-existence of hypoxia, EMT and CSCs in vivo. CSC^high cells exhibited high basal expression of the mesenchymal Vimentin protein but low or absent expression of the epithelial marker E-cadherin, with the opposite result in CSC^low cells. Hypoxia triggered altering of cell morphology from an epithelial to a mesenchymal phenotype, which was more pronounced in CSC^high cells. Concomitantly, E-cadherin expression was reduced and expression of Vimentin, Slug, Twist2 and Zeb1 enhanced. While hypoxia caused migration in all cell lines, velocity along with the percentage of migrating, polarized and pseudopodia-forming cells was significantly higher in CSC^high cells. These data indicate that hypoxia-induced EMT occurs in PDA and several other tumor entities. However although hypoxia-induced EMT signaling occurs in all tumor cell populations, only the stem-like cells acquire high migratory potential and thus may be responsible for invasion and metastasis.     

A novel synthetic small molecule YH‐306 suppresses colorectal tumour growth and metastasis via FAK pathway

Cell migration and invasion are key processes in the metastasis of cancer, and suppression of these steps is a promising strategy for cancer therapeutics. The aim of this study was to explore small molecules for treating colorectal cancer (CRC) and to investigate their anti‐metastatic mechanisms. In this study, six CRC cell lines were used. We showed that YH‐306 significantly inhibited the migration and invasion of CRC cells in a dose‐dependent manner. In addition, YH‐306 inhibited cell adhesion and protrusion formation of HCT116 and HT‐29 CRC cells. Moreover, YH‐306 potently suppressed uninhibited proliferation in all six CRC cell lines tested and induced cell apoptosis in four cell lines. Furthermore, YH‐306 inhibited CRC colonization in vitro and suppressed CRC growth in a xenograft mouse model, as well as hepatic/pulmonary metastasis in vivo. YH‐306 suppressed the activation of focal adhesion kinase (FAK), c‐Src, paxillin, and phosphatidylinositol 3‐kinases (PI3K), Rac1 and the expression of matrix metalloproteases (MMP) 2 and MMP9. Meanwhile, YH‐306 also inhibited actin‐related protein (Arp2/3) complex‐mediated actin polymerization. Taken together, YH‐306 is a candidate drug in preventing growth and metastasis of CRC by modulating FAK signalling pathway.     

Knockdown of GPC3 inhibits the proliferation of Huh7 hepatocellular carcinoma cells through down‐regulation of YAP

Glypican‐3 (GPC3), a membrane‐associated heparan sulfate proteoglycan, is frequently upregulated in hepatocellular carcinoma (HCC). Yes‐associated protein (YAP) is also found over‐expressed in HCC and has been identified as a key effector molecule in Hippo pathway, which could control the organ size in animals through the regulation of cell proliferation and apoptosis and plays an important role in the development of malignant tumors. Studies have reported that GPC3 and YAP might collaborate to regulate the development of HCC. To elucidate the role of GPC3 in the development of HCC and its relationship with YAP, siRNA technique was employed to knock down GPC3 in Huh7 HCC cells. Moreover, recombinant human YAP‐1 was used to examine the effects of GPC3 on Huh7 cells. The results of flow cytometric analysis and Annexin‐V‐FLUOS apoptosis assay showed that knockdown of GPC3‐induced apoptosis in Huh7 cells, resulting in inhibition of cell proliferation as examined by EdU incorporation assay, migration, and invasion. GPC3 knockdown also suppressed the expression of YAP in mRNA and protein levels, as examined by fluorescence quantitative PCR and Western blot analysis. Moreover, addition of recombinant human YAP‐1 effectively rescued the cells from apoptosis triggered by GPC3 knockdown. Taken together, our findings suggest that GPC3 regulates HCC cell proliferation with the involvement of Hippo pathway. J. Cell. Biochem. 114: 625–631, 2013. © 2012 Wiley Periodicals, Inc.     

Down-regulation of Gli-1 inhibits hepatocellular carcinoma cell migration and invasion

Glioma-associated oncogene homolog-1 (Gli-1) is considered a marker of Hedgehog pathway activation and is associated with the progression of several cancers. We have previously reported that Gli-1 was correlated with invasion and metastasis in hepatocellular carcinoma (HCC). However, the exact roles and mechanisms of Gli-1 in HCC invasion are unclear. In this study, we found that small interfering RNA mediated down-regulation of Gli-1 expression significantly suppressed adhesion, motility, migration, and invasion of both SMMC-7721 and SK-Hep1 cells. Furthermore, down-regulation of Gli-1 significantly reduced expressions and activities of both matrix metalloproteinase (MMP)-2 and MMP-9. In addition, we found that down-regulation of Gli-1 resulted in up-regulation of E-cadherin and concomitant down-regulation of Snail and Vimentin, consistent with inhibition of epithelial-mesenchymal transition (EMT). Taken together, our results suggest that down-regulation of Gli-1 suppresses HCC cell migration and invasion likely through inhibiting expressions and activations of MMP-2, 9 and blocking EMT.     

Intracellular reduction in ATP levels contributes to CYT997‐induced suppression of metastasis of head and neck squamous carcinoma

The incidence rate of head and neck squamous cell carcinoma (HNSCC) has steadily increased over the past decade. However, treatment options for metastatic HNSCC are often limited and the 5‐year survival rate has remained static. Therefore, the development and assessment of more efficient but less toxic therapeutic strategies is an unmet need for treatment of more extensive HNSCC. Here, we report that CYT997, a novel microtubule‐disrupting agent, exerts strong activity in inhibiting HNSCC cell invasion and metastasis. The loss of invasion capacity by CYT997 was accompanied by an associated increase in cell adhesion and the reversal of epithelial‐mesenchymal transition (EMT). Increased expression of E‐cadherin protein and decreased expression of Vimentin protein became evident in HNSCC cells following CYT997 exposure, which were consistently observed in HNSCC xenografts from the mice receiving CYT997. Moreover, the capacity of invasive HNSCC cells to form pulmonary metastases was significantly blocked with CYT997 treatment, indicating that the diminishment of EMT traits contributes to CYT997‐suppressed metastasis. Intriguingly, CYT997 impaired intracellular ATP levels in HNSCC cells, at least in part, through its inhibitory effect on the mitochondrial protein IF1. The addition of ATP attenuated CYT997‐induced suppression of cell invasion, coupled with down‐regulation of E‐Cadherin and up‐regulation of Vimentin. These findings support a critical role of ATP levels in cell invasion and metastasis under the influence of CYT997. Collectively, our data unveil the mechanism involved in mediating CYT997 action, and provide preclinical rationale for possible clinical application of CYT997 as a novel therapeutic strategy against aggressive HNSCC.