Tumor‐suppressive microRNA‐223 inhibits cancer cell migration and invasion by targeting ITGA3/ITGB1 signaling in prostate cancer

Analysis of microRNA (miRNA) expression signatures in prostate cancer (PCa) and castration‐resistant PCa has revealed that miRNA‐223 is significantly downregulated in cancer tissues, suggesting that miR‐223 acts as a tumor‐suppressive miRNA by targeting oncogenes. The aim of this study was to investigate the functional roles of miR‐223 and identify downstream oncogenic targets regulated by miR‐223 in PCa cells. Functional studies of miR‐223 were carried out to investigate cell proliferation, migration, and invasion using PC3 and PC3M PCa cell lines. Restoration of miR‐223 significantly inhibited cancer cell migration and invasion in PCa cells. In silico database and genome‐wide gene expression analyses revealed that ITGA3 and ITGB1 were direct targets of miR‐223 regulation. Knockdown of ITGA3 and ITGB1 significantly inhibited cancer cell migration and invasion in PCa cells by regulating downstream signaling. Moreover, overexpression of ITGA3 and ITGB1 was observed in PCa clinical specimens. Thus, our data indicated that downregulation of miR‐223 enhanced ITGA3/ITGB1 signaling and contributed to cancer cell migration and invasion in PCa cells. Elucidation of the molecular pathways modulated by tumor‐suppressive miRNAs provides insights into the mechanisms of PCa progression and metastasis.     

I‐branching N‐acetylglucosaminyltransferase regulates prostate cancer invasiveness by enhancing α5β1 integrin signaling

Cell surface carbohydrates are important for cell migration and invasion of prostate cancer (PCa). Accordingly, the I‐branching N‐acetylglucosaminyltransferase (GCNT2) converts linear i‐antigen to I‐branching glycan, and its expression is associated with breast cancer progression. In the present study, we identified relationships between GCNT2 expression and clinicopathological parameters in patients with PCa. Paraffin‐embedded PCa specimens were immunohistochemically tested for GCNT2 expression, and the roles of GCNT2 in PCa progression were investigated using cell lines with high GCNT2 expression and low GCNT2 expression. GCNT2‐positive cells were significantly lesser in organ‐confined disease than in that with extra‐capsular extensions, and GCNT2‐negative tumors were associated with significantly better prostate‐specific antigen‐free survival compared with GCNT2‐positive tumors. Subsequent functional studies revealed that knockdown of GCNT2 expression in PCa cell lines significantly inhibited cell migration and invasion. GCNT2 regulated the expression of cell surface I‐antigen on the O‐glycan and glycolipid. Moreover, I‐antigen‐bearing glycolipids were subject to α5β1 integrin–fibronectin mediated protein kinase B phosphorylation. In conclusion, GCNT2 expression is closely associated with invasive potential of PCa.     

Tumor‐suppressive microRNA‐218 inhibits cancer cell migration and invasion via targeting of LASP1 in prostate cancer

Our recent studies of the microRNA (miRNA) expression signature in prostate cancer (PCa) indicated that miRNA‐218 (miR‐218) was significantly downregulated in clinical specimens, suggesting that miR‐218 might act as a tumor‐suppressive miRNA in PCa. The aim of the present study was to investigate the functional significance of miR‐218 in PCa and to identify novel miR‐218‐regulated cancer pathways and target genes involved in PCa oncogenesis and metastasis. Restoration of miR‐218 in PCa cell lines (PC3 and DU145) revealed that this miRNA significantly inhibited cancer cell migration and invasion. Gene expression data and in silico analysis demonstrated that LIM and SH3 protein 1 (LASP1) is a potential target of miR‐218 regulation. LASP1 is a cytoskeletal scaffold protein that plays critical roles in cytoskeletal organization and cell migration. Luciferase reporter assays showed that miR‐218 directly regulated expression of LASP1. Moreover, downregulating the LASP1 gene significantly inhibited cell migration and invasion in cancer cells, and the expression of LASP1 was upregulated in cancer tissues. We conclude that loss of tumor‐suppressive miR‐218 enhanced cancer cell migration and invasion in PCa through direct regulation of LASP1. Our data on pathways regulated by tumor‐suppressive miR‐218 provide new insight into the potential mechanisms of PCa oncogenesis and metastasis.     

BOK displays cell death‐independent tumor suppressor activity in non‐small‐cell lung carcinoma

As the genomic region containing the Bcl‐2‐related ovarian killer (BOK) locus is frequently deleted in certain human cancers, BOK is hypothesized to have a tumor suppressor function. In the present study, we analyzed primary non‐small‐cell lung carcinoma (NSCLC) tumors and matched lung tissues from 102 surgically treated patients. We show that BOK protein levels are significantly downregulated in NSCLC tumors as compared to lung tissues (p < 0.001). In particular, we found BOK downregulation in NSCLC tumors of grades two (p = 0.004, n = 35) and three (p = 0.031, n = 39) as well as in tumors with metastases to hilar (pN1) (p = 0.047, n = 31) and mediastinal/subcarinal lymph nodes (pN2) (p = 0.021, n = 18) as opposed to grade one tumors (p = 0.688, n = 7) and tumors without lymph node metastases (p = 0.112, n = 51). Importantly, in lymph node‐positive patients, BOK expression greater than the median value was associated with longer survival (p = 0.002, Mantel test). Using in vitro approaches, we provide evidence that BOK overexpression is inefficient in inducing apoptosis but that it inhibits TGFβ‐induced migration and epithelial‐to‐mesenchymal transition (EMT) in lung adenocarcinoma‐derived A549 cells. We have identified epigenetic mechanisms, in particular BOK promoter methylation, as an important means to silence BOK expression in NSCLC cells. Taken together, our data point toward a novel mechanism by which BOK acts as a tumor suppressor in NSCLC by inhibiting EMT. Consequently, the restoration of BOK levels in low‐BOK‐expressing tumors might favor the overall survival of NSCLC patients.     

VEGFR‐2 expression in carcinoid cancer cells and its role in tumor growth and metastasis

Carcinoid tumors are slow growing and highly vascular neuroendocrine neoplasms that are increasing in incidence. Previously, we showed that carcinoid tumors express vascular endothelial growth factor receptor 2 (VEGFR‐2) in the epithelial compartment of carcinoid tumor sections; yet, its role is not completely understood. The purpose of our study was to: (i) assess the expression of VEGFR‐2 in the novel human carcinoid cell line BON, (ii) to determine the role of PI3K/Akt signaling on VEGFR‐2 expression and (iii) to assess the effect of VEGFR‐2 on BON cell invasion, migration and proliferation. We found that, although VEGFR‐2 is expressed in BON cells, reduction in VEGFR‐2 expression actually enhanced proliferation, invasion, and migration of the BON cell line. Also, expression of VEGFR‐2 was inversely related to PI3K signaling. Carcinoid liver metastases in mice demonstrated decreased VEGFR‐2 expression. Furthermore, the expression of a truncated, soluble form of VEGFR‐2 (sVEGFR‐2), a protein demonstrated to inhibit cell growth, was detected in BON cells. The presence of VEGFR‐2 in the epithelial component of carcinoid tumors and in the BON cell line suggests an alternate role for VEGFR‐2, in addition to its well‐defined role in angiogenesis. The expression of sVEGFR‐2 may explain the inverse relationship between VEGFR‐2 expression and PI3K/Akt signaling and the inhibitory effect VEGFR‐2 has on BON cell proliferation, migration and invasion.     

Epigenetic inactivation of endothelin‐2 and endothelin‐3 in colon cancer

Endothelin‐1 (ET‐1) and its receptors are overexpressed in human cancers, but much less is known about the roles of ET‐2 and ET‐3 in cancer etiology. We sought to examine human and rat colon tumors for dysregulation of ET‐2 and ET‐3 expression and determine the underlying mechanisms. Human primary colon cancers and carcinogen‐induced rat colon tumors were subjected to real‐time RT‐PCR, immunoblotting and immunohistochemistry; EDN2 and EDN3 genes were examined by methylation‐specific PCR, bisulfite sequencing and pyrosequencing; and forced expression of ET‐2 and ET‐3 was conducted in human colon cancer cells followed by real‐time cell migration and invasion assays. Rat and human colon tumors had markedly reduced expression of ET‐2 and ET‐3 mRNA and protein compared with matched controls. Mechanistic studies revealed hypermethylation of EDN2 and EDN3 genes in human primary colon cancers and in a panel of human colon cancer cell lines. Forced expression of ET‐2 and ET‐3 attenuated significantly the migration and invasion of human colon cancer cells. We conclude that epigenetic inactivation of ET‐2 and ET‐3 occurs frequently in both rat and human colon cancers. Current therapeutic strategies target overexpressed members of the ET axis via small molecule inhibitors and receptor antagonists, but this work supports a complementary approach based on the re‐expression of ET‐2 and ET‐3 as natural antagonists of ET‐1 in colon cancer.     

miR‐203 inhibits cell proliferation,invasion, and migration of non‐small‐cell lung cancer by downregulating RGS17

Involvement of the RGS17 oncogene in the promotion of non‐small‐cell lung cancer (NSCLC) has been reported, but the regulation mechanism in NSCLC remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression, and their dysregulation has been implicated in tumorigenesis. To understand the role of miRNAs in Regulator of G Protein Signaling 17 (RGS17)‐induced NSCLC, we showed that miR‐203 was downregulated during tumorigenesis, and inhibited the proliferation and invasion of lung cancer cells. We then determined whether miR‐203 regulated NSCLC by targeting RGS17. To characterize the regulatory effect of miR‐203 on RGS17, we used lung cancer cell lines, A549 and Calu‐1, and the constructed miR‐203 and RGS17 overexpression vectors. The CCK8 kit was used to determine cell proliferation, and the Transwell® assay was used to measure cell invasion and migration. RT‐PCR, western blots, and immunofluorescence were used to analyze expression of miR‐203 and RGS17, and the luciferase reporter assay was used to examine the interaction between miR‐203 and RGS17. Nude mice were used to characterize in vivo tumor growth regulation. Expression of miR‐203 inhibited proliferation, invasion, and migration of lung cancer cell lines A549 and Calu‐1 by targeting RGS17. The regulatory effect of miR‐203 was inhibited after overexpression of RGS17. The luciferase reporter assay showed that miR‐203 downregulated RGS17 by direct integration into the 3′‐UTR of RGS17 mRNA. In vivo studies showed that expression of miR‐203 significantly inhibited growth of tumors. Taken together, the results suggested that expression of miR‐203 inhibited tumor growth and metastasis by targeting RGS17.     

Long non‐coding RNA RP11‐552M11.4 promotes cells proliferation,migration and invasion by targeting BRCA2 in ovarian cancer

The present study aimed to investigate the effect of long non‐coding RNA (lncRNA) RP11‐552M11.4 on cell proliferation, apoptosis, migration and invasion as well as its targeting genes in epithelial ovarian cancer (EOC) cells. LncRNA RP11‐552M11.4 expression was detected in 67 tumor tissues and paired adjacent tissues obtained from EOC patients. lncRNA RP11‐552M11.4 mimic/inhibitor plasmids were transferred into ovarian cancer cells (SKOV3, A‐2780) and normal ovarian epithelial cells (IOSE80 cells). In addition, rescue experiment was carried out by transferring BRCA2 inhibitor&lncRNA RP11‐552M11.4 inhibitor plasmids into SKOV3 and A‐2780 cells. qPCR, western blot, CKK‐8, Annexin V/propidium iodide (AV/PI), wound‐healing and Matrigel invasion assays were carried out to detect RNA expression, protein expression, cell proliferation, apoptosis, migration, and invasion, respectively. LncRNA RP11‐552M11.4 expression was elevated in tumor tissues compared with paired adjacent tissues and correlated with higher pathological grade, International Federation of Gynecology and Obstetrics stage and worse overall survival in EOC patients. LncRNA RP11‐552M11.4 promoted SKOV3 cell proliferation, migration and invasion whereas it inhibited apoptosis. Rescue experiment and luciferase reporter assay showed that lncRNA RP11‐552M11.4 regulated SKOV3 cells functions through binding BRCA2. Further experiments in A‐2780 cells also validated that lncRNA RP11‐552M11.4 induced A‐2780 cell proliferation while repressing apoptosis by targeting BRCA2. In addition, upregulation of lncRNA RP11‐552M11.4 increased IOSE80 cell proliferation, migration and invasion while decreasing apoptosis. In conclusion, lncRNA RP11‐552M11.4 correlates with worse prognosis, and promotes cell proliferation, migration, invasion, and inhibits cell apoptosis by down‐regulating BRCA2 in EOC.     

Metastasis‐associated in colon cancer‐1 upregulation predicts a poor prognosis of gastric cancer,and promotes tumor cell proliferation and invasion

Metastasis‐associated in colon cancer‐1 (MACC1) is a newly identified oncogene, and little is known about its role in gastric cancer (GC). Our study was performed to investigate whether MACC1 influences the prognosis of GC patients and to explore the potential mechanisms involved. MACC1 expression was verified to be higher in GC tissues than in adjacent nontumorous tissues by Western blotting. A retrospective analysis of 361 GC patients (Stages I–IV) revealed that higher MACC1 expression was associated with more advanced disease, more frequent postoperative recurrence, more metastases and a higher mortality rate. The disease‐free survival of Stage I–III patients and overall survival of Stage‐IV patients were significantly worse when their tumors showed high MACC1 expression. To investigate the underlying mechanisms, MACC1 overexpression and downregulation were established in two GC cell lines (BGC‐823 and MKN‐28 cells). MACC1 overexpression significantly accelerated tumor growth and facilitated metastasis in athymic mice. MACC1 also promoted the proliferation, migration and invasion of both GC cell lines. Moreover, gastric MACC1 mRNA expression levels were significantly correlated with markers of the epithelial‐to‐mesenchymal transition (EMT) in patients with GC. MACC1 overexpression upregulated mesenchymal–epithelial transition factor and induced changes to markers of EMT, whereas silencing of MACC1 reversed all these changes. These findings provide some novel insights into the role of MACC1, a gene that contributes to a poor prognosis of GC by promoting tumor cell proliferation and invasion as well as the EMT.     

Expression of galectin‐1 in carcinoma‐associated fibroblasts promotes gastric cancer cell invasion through upregulation of integrin β1

Increased expression of galectin‐1 (Gal‐1) in carcinoma‐associated fibroblasts (CAFs) has been reported to correlate with progression and prognosis in many cancers. However, rarely have reports sought to determine whether high Gal‐1 expression in CAFs in gastric cancer is involved in the tumor process, and the specific mechanism by which it promotes the evolution of gastric cancer is still unknown. In this study, we cultured gastric cancer CAFs, which showed strong expression of Gal‐1, and established a co‐culture system of CAFs with gastric cancer cells. Specific siRNA and in vitro migration and invasion assays were used to explore the effects of the interaction between Gal‐1 expression of CAFs and gastric cancer cells on cell migration and invasion. We found that the overexpression of Gal‐1 in CAFs enhanced gastric cancer cell migration and invasion, and these stimulatory effects could be blocked by specific siRNA which reduced the Gal‐1 expression level. A set of cancer invasion‐associated genes were then chosen to identify the possible mechanism of Gal‐1‐induced cell invasion. Among these genes, integrin β1 expression in cancer cells was considered to be associated with Gal‐1 expression. Pre‐blocking of the integrin β1 expression in gastric cancer cells with siRNA could interrupt the invasion‐promoting effect of CAFs with high Gal‐1 expression. Furthermore, immunohistochemical assay confirmed a positive correlation between Gal‐1 and integrin β1 expression. Our results showed that high expression of Gal‐1 in CAFs might facilitate gastric cancer cell migration and invasion by upregulating integrin β1 expression in gastric cancer.     

Inositol hexaphosphate downregulates both constitutive and ligand‐induced mitogenic and cell survival signaling,and causes caspase‐mediated apoptotic death of human prostate carcinoma PC‐3 cells

Constitutively active mitogenic and prosurvival signaling cascades due to aberrant expression and interaction of growth factors and their receptors are well documented in human prostate cancer (PCa). Epidermal growth factor (EGF) and insulin‐like growth factor‐1 (IGF‐1) are potent mitogens that regulate proliferation and survival of PCa cells via autocrine and paracrine loops involving both mitogen‐activated protein kinase (MAPK)‐ and Akt‐mediated signaling. Accordingly, here we assessed the effect of inositol hexaphosphate (IP6) on constitutive and ligand (EGF and IGF‐1)‐induced biological responses and associated signaling cascades in advanced and androgen‐independent human PCa PC‐3 cells. Treatment of PC‐3 cells with 2 mM IP6 strongly inhibited both growth and proliferation and decreased cell viability; similar effects were also observed in other human PCa DU145 and LNCaP cells. IP6 also caused a strong apoptotic death of PC‐3 cells together with caspase 3 and PARP cleavage. Mechanistic studies showed that biological effects of IP6 were associated with inhibition of both constitutive and ligand‐induced Akt phosphorylation together with a decrease in total Akt levels, but a differential inhibitory effect on MAPKs extra cellular signal‐regulated kinase 1/2 (ERK1/2), c‐Jun N‐terminal protein kinase (JNK1/2), and p38 under constitutive and ligand‐activated conditions. Under similar condition, IP6 also inhibited AP‐1 DNA‐binding activity and decreased nuclear levels of both phospho and total c‐Fos and c‐Jun. Together, these findings for the first time establish IP6 efficacy in inhibiting aberrant EGF receptor (EGFR) or IGF‐1 receptor (IGF‐1R) pathway‐mediated sustained growth promoting and survival signaling cascades in advanced and androgen‐independent human PCa PC‐3 cells, which might have translational implications in advanced human PCa control and management. © 2009 Wiley‐Liss, Inc.     

Regulation of spindle and kinetochore‐associated protein 1 by antitumor miR‐10a‐5p in renal cell carcinoma

Analysis of our original microRNA (miRNA) expression signature of patients with advanced renal cell carcinoma (RCC) showed that microRNA‐10a‐5p (miR‐10a‐5p) was significantly downregulated in RCC specimens. The aims of the present study were to investigate the antitumor roles of miR‐10a‐5p and the novel cancer networks regulated by this miRNA in RCC cells. Downregulation of miR‐10a‐5p was confirmed in RCC tissues and RCC tissues from patients treated with tyrosine kinase inhibitors (TKI). Ectopic expression of miR‐10a‐5p in RCC cell lines (786‐O and A498 cells) inhibited cancer cell migration and invasion. Spindle and kinetochore‐associated protein 1 (SKA1) was identified as an antitumor miR‐10a‐5p target by genome‐based approaches, and direct regulation was validated by luciferase reporter assays. Knockdown of SKA1 inhibited cancer cell migration and invasion in RCC cells. Overexpression of SKA1 was observed in RCC tissues and TKI‐treated RCC tissues. Moreover, analysis of The Cancer Genome Atlas database demonstrated that low expression of miR‐10a‐5p and high expression of SKA1 were significantly associated with overall survival in patients with RCC. These findings showed that downregulation of miR‐10a‐5p and overexpression of the SKA1 axis were highly involved in RCC pathogenesis and resistance to TKI treatment in RCC.     

MicroRNA‐224 inhibits progression of human prostate cancer by downregulating TRIB1

Our previous microarray data showed that microRNA‐224 (miR‐224) was downregulated in human prostate cancer (PCa) tissues compared with adjacent benign tissues. However, the underlying mechanisms by which miR‐224 is involved in PCa remain unclear. In this study, we identified TRIB1 as a target gene of miR‐224. Forced expression of miR‐224 suppressed PCa cell proliferation, invasion and migration, and promoted cell apoptosis by downregulating TRIB1. Moreover, the expression level of miR‐224 in PCa tissues was negatively correlated with that of TRIB1. miR‐224 downregulation was frequently found in PCa tissues with metastasis, higher PSA level and clinical stage, whereas TRIB1 upregulation was significantly associated with metastasis. Both miR‐224 downregulation and TRIB1 upregulation were significantly associated with poor biochemical recurrence‐free survival of patients with PCa. In conclusion, these findings reveal that the aberrant expression of miR‐224 and TRIB1 may promote PCa progression and have potentials to serve as novel biomarkers for PCa prognosis.     

ATP‐P2Y2‐β‐catenin axis promotes cell invasion in breast cancer cells

Extracellular adenosine 5′‐triphosphate (ATP), secreted by living cancer cells or released by necrotic tumor cells, plays an important role in tumor invasion and metastasis. Our previous study demonstrated that ATP treatment in vitro could promote invasion in human prostate cancer cells via P2Y2, a preferred receptor for ATP, by enhancing EMT process. However, the pro‐invasion mechanisms of ATP and P2Y2 are still poorly studied in breast cancer. In this study, we found that P2Y2 was highly expressed in breast cancer cells and associated with human breast cancer metastasis. ATP could promote the in vitro invasion of breast cancer cells and enhance the expression of β‐catenin as well as its downstream target genes CD44, c‐Myc and cyclin D1, while P2Y2 knockdown attenuated above ATP‐driven events in vitro and in vivo. Furthermore, iCRT14, a β‐catenin/TCF complex inhibitor, could also suppress ATP‐driven migration and invasion in vitro. These results suggest that ATP promoted breast cancer cell invasion via P2Y2‐β‐catenin axis. Thus blockade of the ATP‐P2Y2‐β‐catenin axis could suppress the invasive and metastatic potential of breast cancer cells and may serve as potential targets for therapeutic interventions of breast cancer.