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.     

Vasohibin‐1 expression inhibits advancement of ovarian cancer producing various angiogenic factors

Vasohibin‐1 (VASH1) is a negative feedback regulator of angiogenesis, the first to be discovered, and was identified in vascular endothelial growth factor (VEGF)‐stimulated vascular endothelial cells. Vasohibin‐1 inhibits abnormal vascularization induced by various angiogenic factors including fibroblast growth factor and platelet‐derived growth factor (PDGF), in addition to VEGF. By focusing on this characteristic of VASH1, we investigated the antitumor effects of VASH1 expression on ovarian cancer cells that produce different angiogenic factors. By using a high VEGF‐producing ovarian cancer cell line, SHIN‐3, and a high PDGF‐producing ovarian cancer cell line, KOC‐2S, the cells were transfected with either a VEGF antagonist, soluble VEGF receptor‐1 (sVEGFR‐1, or sFlt‐1), or VASH1 genes to establish their respective cellular expression. The characteristics of these transfectants were compared with controls. We previously reported that the expression of sFlt‐1 inhibited tumor vascularization and growth of high VEGF‐producing ovarian cancer cells, reduced peritoneal dissemination and ascites development, and prolonged the survival time of the host. However, in the current study, the expression of sFlt‐1 had no such effect on the high PDGF‐producing ovarian cancer cells used here, whereas VASH1 expression inhibited tumor vascularization and growth, not only in high VEGF‐producing cells, but also in high PDGF‐producing cells, reduced their peritoneal dissemination and ascites, and prolonged the survival time of the host. These results suggest that VASH1 is an effective treatment for ovarian cancer cells that produce different angiogenic factors.     

Androgen‐induced lncRNA POTEF‐AS1 regulates apoptosis‐related pathway to facilitate cell survival in prostate cancer cells

Although long non‐coding RNAs (lncRNAs) have been associated with a variety of cancers, the interplay between lncRNAs and androgen receptor signaling in prostate cancer is still unclear. We identified an androgen‐dependent lncRNA, POTEF‐AS1, whose expression was regulated by androgen receptor in two androgen‐dependent cells by using directional RNA sequencing analysis. POTEF‐AS1 promoted cell growth, repressed genes related to the Toll‐like receptor signaling and apoptosis pathways, and inhibited apoptosis in docetaxel‐treated LNCaP cells. These findings suggest that POTEF‐AS1 would play a key role in the progression of prostate cancer by repressing Toll‐like receptor signaling.     

Protein kinase C alpha‐dependent signaling mediates endometrial cancer cell growth and tumorigenesis

Endometrial cancer is the most common invasive gynecologic malignancy, yet molecular mechanisms and signaling pathways underlying its etiology and pathophysiology remain poorly characterized. We sought to define a functional role for the protein kinase C (PKC) isoform, PKCα, in an established cell model of endometrial adenocarcinoma. Ishikawa cells depleted of PKCα protein grew slower, formed fewer colonies in anchorage‐independent growth assays and exhibited impaired xenograft tumor formation in nude mice. Consistent with impaired growth, PKCα knockdown increased levels of the cyclin‐dependent kinase (CDK) inhibitors p21^Cip1/WAF1 (p21) and p27^Kip1 (p27). Despite the absence of functional phosphatase and tensin homolog (PTEN) protein in Ishikawa cells, PKCα knockdown reduced Akt phosphorylation at serine 473 and concomitantly inhibited phosphorylation of the Akt target, glycogen synthase kinase‐3β (GSK‐3β). PKCα knockdown also resulted in decreased basal ERK phosphorylation and attenuated ERK activation following EGF stimulation. p21 and p27 expression was not increased by treatment of Ishikawa cells with ERK and Akt inhibitors, suggesting that PKCα regulates CDK expression independently of Akt and ERK. Immunohistochemical analysis of Grade 1 endometrioid adenocarcinoma revealed aberrant PKCα expression, with foci of elevated PKCα staining, not observed in normal endometrium. These studies demonstrate a critical role for PKCα signaling in endometrial tumorigenesis by regulating expression of CDK inhibitors p21 and p27 and activation of Akt and ERK‐dependent proliferative pathways. Thus, targeting PKCα may provide novel therapeutic options in endometrial tumors. © 2009 UICC     

Regulation of ITGA3 by the anti‐tumor miR‐199 family inhibits cancer cell migration and invasion in head and neck cancer

For patients with head and neck squamous cell carcinoma (HNSCC), survival rates have not improved due to local recurrence and distant metastasis. Current targeted molecular therapies do not substantially benefit HNSCC patients. Therefore, it is necessary to use advanced genomic approaches to elucidate the molecular mechanisms underlying the aggressiveness of HNSCC cells. Analysis of our microRNA (miRNA) expression signature by RNA sequencing showed that the miR‐199 family (miR‐199a‐5p, miR‐199a‐3p, miR‐199b‐5p and miR‐199b‐3p) was significantly reduced in cancer tissues. Ectopic expression of mature miRNA demonstrated that all members of the miR‐199 family inhibited cancer cell migration and invasion by HNSCC cell lines (SAS and HSC3). These findings suggested that both passenger strands and guide strands of miRNA are involved in cancer pathogenesis. In silico database and genome‐wide gene expression analyses revealed that the gene coding for integrin α3 (ITGA3) was regulated by all members of the miR‐199 family in HNSCC cells. Knockdown of ITGA3 significantly inhibited cancer cell migration and invasion by HNSCC cells. Moreover, overexpression of ITGA3 was confirmed in HNSCC specimens, and high expression of ITGA3 predicted poorer survival of the patients (P = 0.0048). Our data revealed that both strands of pre‐miR‐199a (miR‐199a‐5p and miR‐199a‐3p) and pre‐miR‐199b (miR‐199b‐5p and miR‐199b‐3p) acted as anti‐tumor miRNA in HNSCC cells. Importantly, the involvement of passenger strand miRNA in the regulation of cellular processes is a novel concept in RNA research. Novel miRNA‐based approaches for HNSCC can be used to identify potential targets for the development of new therapeutic strategies.     

Combination of IFITM1 knockdown and radiotherapy inhibits the growth of oral cancer

This research aimed to analyze the effect of IFITM1 on the radioresistance of oral neoplasm. Using a multi‐group heat map from GSE9716 analysis of the GEO database, IFITM1 was determined to be a relevant radioresistance gene. The TCGA database was analyzed before the expression of IFITM1 was analyzed. IFITM1 expression was quantified by quantitative RT‐PCR and immunohistochemistry in 19 paired oral neoplasm cases. The effects of time and dose of radiation on IFITM1 expression level in CAL27 and TSCC1 cell lines were tested by quantitative RT‐PCR. Oral neoplasm cells were transfected with siRNA after radiotherapy to disturb IFITM1 expression. After this, the survival rates, cell apoptosis, caspase‐3 viability, expression and γ‐H2AX were detected using colony formation, flow cytometry, western blot and immunofluorescence, respectively. Western blot was used for STAT1/2/3/p21‐related protein and phosphorylation changes. Finally, an in vivo nude mice tumor model was established to verify the effect of IFITM1 on oral neoplasm cells radioresistance. Through microarray analysis, the head and neck neoplasm radioresistance‐related gene IFITM1 was found to be overexpressed. IFITM1 overexpression was verified not only using the TCGA database but also in 19 paired cases of oral neoplasm tissues and cells. With increases of dose and time of radiation, the expression of IFITM1 was increased in CAL27 and TSCC1 cell lines. Furthermore, si‐IFITM1 may restrain cell proliferation, DNA damage and cell apoptosis in oral neoplasm cell lines. Finally, pSTAT1/2/p21 was found to be upregulated while pSTAT3/p‐p21 was downregulated due to IFITM1 inhibition after radiotherapy. The evidence suggested that IFITM1 in combination with radiotherapy can inhibit oral neoplasm cells.     

Downregulation of CPA4 inhibits non small–cell lung cancer growth by suppressing the AKT/c‐MYC pathway

Carboxypeptidase A4 (CPA4) is a member of the metallocarboxypeptidase family. A previous study indicated that CPA4 may participate in the modulation of peptide hormone activity and hormone‐regulated tissue growth and differentiation. However, the role of CPA4 in lung tumorigenesis remains unclear. Our study revealed that CPA4 expression was higher in both lung cancer cells and tumor tissues. We performed 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assays, colony‐formation assays, and Cellomics ArrayScan Infinity analysis to demonstrate that CPA4 knockdown inhibited non small–cell lung cancer (NSCLC) cell proliferation. Conversely, ectopic expression of CPA4 enhanced lung cancer cell proliferation. Consistent with these observations, we generated xenograft tumor models to confirm that CPA4 downregulation suppressed NSCLC cell growth. Mechanistically, we revealed that CPA4 downregulation may induce apoptosis and G1‐S arrest by suppressing the protein kinase B/c‐MYC pathway. These results suggest that CPA4 has an oncogenic effect on lung cancer growth. Taken together, we identified a novel gene in lung cancer that might provide a basis for new therapeutic targets.     

Vasohibin‐2 is required for epithelial–mesenchymal transition of ovarian cancer cells by modulating transforming growth factor‐β signaling

Vasohibin‐2 (VASH2) is a homolog of VASH1, an endothelium‐derived angiogenesis inhibitor. Vasohibin‐2 is mainly expressed in cancer cells, and has been implicated in the progression of cancer by inducing angiogenesis and tumor growth. Although VASH2 has been recently reported to be involved in epithelial–mesenchymal transition (EMT), its precise roles are obscure. The aim of the present study was to clarify the role of VASH2 in the EMT of cancer cells in relation to transforming growth factor‐β (TGF‐β) signaling, which is a major stimulator of EMT. Decreased expression of VASH2 in ovarian cancer cells significantly repressed the expression of TGF‐β type I receptor, namely activin receptor‐like kinase 5. Transforming growth factor‐β1‐induced phosphorylation of Smad2 and Smad3 was markedly decreased in VASH2 knockdown cells while the expression of Smad2 and Smad3 was unchanged. Accordingly, the responses to TGF‐β1 shown by promoter assay and plasminogen activator inhibitor type 1 expression were significantly attenuated in VASH2 knockdown cells. Furthermore, knockdown of VASH2 in cancer cells abrogated the TGF‐β1‐induced reduced expression of epithelial markers including E‐cadherin, and the elevated expression of mesenchymal markers including fibronectin, ZEB2, and Snail2, suggesting that endogenous VASH2 is required for TGF‐β1‐induced EMT. In accordance with these results, the effects of TGF‐β1 on cell morphology, migration, invasion, and MMP2 expression were also abrogated when VASH2 was knocked down. These results indicate that VASH2 played a significant role in the EMT by modulating the TGF‐β signaling. We propose that VASH2 would be a novel molecular target for the prevention of EMT in cancers.     

Endoplasmic reticulum stress pathway PERK‐eIF2α confers radioresistance in oropharyngeal carcinoma by activating NF‐κB

Endoplasmic reticulum stress (ERS) plays an important role in the pathogenesis and development of malignant tumors, as well as in the regulation of radiochemoresistance and chemoresistance in many malignancies. ERS signaling pathway protein kinase RNA‐like endoplasmic reticulum kinase (PERK)‐eukaryotic initiation factor‐2 (eIF2α) may induce aberrant activation of nuclear factor‐κB (NF‐κB). Our previous study showed that NF‐κB conferred radioresistance in lymphoma cells. However, whether PERK‐eIF2α regulates radioresistance in oropharyngeal carcinoma through NF‐κB activation is unknown. Herein, we showed that PERK overexpression correlated with a poor prognosis for patients with oropharyngeal carcinoma (P < 0.01). Meanwhile, the percentage of the high expression level of PERK in oropharyngeal carcinoma patients resistant to radiation was higher than in patients sensitive to radiation (77.7 and 33.3%, respectively; P < 0.05). Silencing PERK and eIF2α increased the radiosensitivity in oropharyngeal carcinoma cells and increased radiation‐induced apoptosis and G2/M phase arrest. PERK‐eIF2α silencing also inhibited radiation‐induced NF‐κB phosphorylation and increased the DNA double strand break‐related proteins ATM phosphorylation. NF‐κB activator TNF‐α and the ATM inhibitor Ku55933 offset the regulatory effect of eIF2α on the expression of radiation‐induced cell apoptosis‐related proteins and the G2/M phase arrest‐related proteins. These data indicate that PERK regulates radioresistance in oropharyngeal carcinoma through NF‐kB activation‐mediated phosphorylation of eIF2α, enhancing X‐ray‐induced activation of DNA DSB repair, cell apoptosis inhibition and G2/M cell cycle arrest.     

53BP1 suppresses epithelial–mesenchymal transition by downregulating ZEB1 through microRNA‐200b/429 in breast cancer

Epithelial–mesenchymal transition (EMT) is an important mechanism of cancer invasion and metastasis. Although p53 binding protein 1 (53BP1) has been implicated in several biological processes, its function in EMT of human cancers has not yet been reported. Here, we show that 53BP1 negatively regulated EMT by modulating ZEB1 through targeting microRNA (miR)‐200b and miR‐429. Furthermore, 53BP1 promoted ZEB1‐mediated upregulation of E‐cadherin and also inhibited the expressions of mesenchymal markers, leading to increased migration and invasion in MDA‐MB‐231 breast cancer cells. Consistently, in MCF‐7 breast cancer cells, low 53BP1 expression reduced E‐cadherin expression, resulting in increased migration and invasion. These effects were reversed by miR‐200b and miR‐429 inhibition or overexpression. Sections of tumor xenograft model showed increased ZEB1 expression and decreased E‐cadherin expression with the downregulation of 53BP1. In 18 clinical tissue samples, expression of 53BP1 was positively correlated with miR‐200b and mir‐429 and negatively correlated with ZEB1. It was also found that 53BP1 was associated with lymph node metastasis. Taken together, these results suggest that 53BP1 functioned as a tumor suppressor gene by its novel negative control of EMT through regulating the expression of miR‐200b/429 and their target gene ZEB1.     

UNC5D,suppressed by promoter hypermethylation,inhibits cell metastasis by activating death‐associated protein kinase 1 in prostate cancer

Prostate cancer (PCa) death primarily occurs due to metastasis of the cells, but little is known about the underlying molecular mechanisms. This study aimed to evaluate the expression of UNC5D, a newly identified tumor suppressor gene, analyze its epigenetic alterations, and elucidate its functional relevance to PCa metastasis. Meta‐analysis of publicly available microarray datasets revealed that UNC5D expression was frequently downregulated in PCa tissues and inversely associated with PCa metastasis. These results were verified in clinical specimens by real‐time PCR and immunohistochemistry assays. Through methylation analysis, the downregulated expression of UNC5D in PCa tissues and cell lines was found to be attributable to the hypermethylation of the promoter. A negative correlation was observed between methylation and UNC5D mRNA expression in PCa samples. The ectopic expression of UNC5D in PCa cells effectively reduced their ability to migrate and invade both in vitro and in vivo, and siRNA‐mediated knockdown of UNC5D yielded consistent results. UNC5D can recruit and activate death‐associated protein kinase 1, which remained to be essential for its metastatic suppressor function. In conclusion, these results suggested that UNC5D as a novel putative metastatic suppressor gene that is commonly down‐regulated by hypermethylation in PCa.     

Inactivated Sendai virus particle upregulates cancer cell expression of intercellular adhesion molecule‐1 and enhances natural killer cell sensitivity on cancer cells

We have already reported that the inactivated Sendai virus (hemagglutinating virus of Japan; HVJ) envelope (HVJ‐E) has multiple anticancer effects, including induction of cancer‐selective cell death and activation of anticancer immunity. The HVJ‐E stimulates dendritic cells to produce cytokines and chemokines such as β‐interferon, interleukin‐6, chemokine (C‐C motif) ligand 5, and chemokine (C‐X‐C motif) ligand 10, which activate both CD8⁺ T cells and natural killer (NK) cells and recruit them to the tumor microenvironment. However, the effect of HVJ‐E on modulating the sensitivity of cancer cells to immune cell attack has yet to be investigated. In this study, we found that HVJ‐E induced the production of intercellular adhesion molecule‐1 (ICAM‐1, CD54), a ligand of lymphocyte function‐associated antigen 1, in several cancer cell lines through the activation of nuclear factor‐κB downstream of retinoic acid‐inducible gene I and the mitochondrial antiviral signaling pathway. The upregulation of ICAM‐1 on the surface of cancer cells increased the sensitivity of cancer cells to NK cells. Knocking out expression of ICAM‐1 in MDA‐MB‐231 cells using the CRISPR/Cas9 method significantly reduced the killing effect of NK cells on ICAM‐1‐depleted MDA‐MB‐231 cells. In addition, HVJ‐E suppressed tumor growth in MDA‐MB‐231 tumor‐bearing SCID mice, and the HVJ‐E antitumor effect was impaired when NK cells were depleted by treatment with the anti‐asialo GM1 antibody. Our findings suggest that HVJ‐E enhances NK cell sensitivity against cancer cells by increasing ICAM‐1 expression on the cancer cell surface.     

NeuroD1 promotes neuroblastoma cell growth by inducing the expression of ALK

Neuroblastoma is derived from the sympathetic neuronal lineage of neural crest cells, and is the most frequently observed of the extracranial pediatric solid tumors. The neuronal differentiation factor, NeuroD1, has previously been shown to promote cell motility in neuroblastoma by suppressing the expression of Slit2. Here we report that NeuroD1 is also involved in the proliferation of neuroblastoma cells, including human cell lines and primary tumorspheres cultured from the tumor tissues of model mice. Interestingly, the growth inhibition of neuroblastoma cells induced by knockdown of NeuroD1 was accompanied by a reduction of ALK expression. ALK is known to be one of the important predisposition genes for neuroblastoma. The phenotype resulting from knockdown of NeuroD1 was suppressed by forced expression of ALK and, therefore, NeuroD1 appears to act mainly through ALK to promote the proliferation of neuroblastoma cells. Furthermore, we showed that NeuroD1 directly bound to the promoter region of ALK gene. In addition, the particular E‐box in the promoter was responsible for NeuroD1‐mediated ALK expression. These results indicate that ALK should be a direct target gene of NeuroD1. Finally, the expressions of NeuroD1 and ALK in the early tumor lesions of neuroblastoma model mice coincided in vivo. We conclude that the novel mechanism would regulate the expression of ALK in neuroblastoma and that NeuroD1 should be significantly involved in neuroblastoma tumorigenesis.     

Putative tumor suppressor miR‐145 inhibits colon cancer cell growth by targeting oncogene friend leukemia virus integration 1 gene

BACKGROUND:

Tumor suppressor microRNA miR‐145 is commonly down‐regulated in colon carcinoma tissues, but its specific role in tumors remains unknown.

METHODS:

In this study, the authors identified the Friend leukemia virus integration 1 gene (FLI1) as a novel target of miR‐145. FLI1 is involved in t(11;22)(q24:q12) reciprocal chromosomal translocation in Ewing sarcoma, and its expression appears to be associated with biologically more aggressive tumors.

RESULTS:

The authors demonstrated that miR‐145 targets a putative microRNA regulatory element in the 3′‐untranslated region (UTR) of FLI1, and its abundance is reversely associated with FLI1 expression in colon cancer tissues and cell lines. By using a luciferase/FLI1 3′‐UTR reporter system, they found that miR‐145 down‐regulated the reporter activity, and this down‐regulation was reversed by anti–miR‐145. Mutation of the miR‐145 microRNA regulatory element sequence in the FLI1 3′‐UTR abolished the activity of miR‐145. miR‐145 decreased FLI1 protein but not FLI1 mRNA, suggesting a mechanism of translational regulation. Furthermore, the authors demonstrated that miR‐145 inhibited cell proliferation and sensitized LS174T cells to 5‐fluorouracil–induced apoptosis.

CONCLUSIONS:

Taken together, these results suggest that miR‐145 functions as a tumor suppressor by down‐regulating oncogenic FLI1 in colon cancer. Cancer 2011. © 2010 American Cancer Society.     

Induction of human endometrial cancer cell senescence through modulation of HIF-1alpha activity by EGLN1

Previous observations indicate that transfer of human chromosome (chr.) 1 induces senescence of endometrial cancer cells. To identify the gene(s) responsible for the senescence, we first analyzed the structural integrity of the introduced chr. 1 in immortal revertant from chr.1-transferred HHUA cells. The data demonstrated a correlation between nonrandom deletions within the 1q31-qter region and reversion to immortality. Next, by using a panel of 12 microsatellite markers, we found high frequencies of loss of heterozygosity in the particular 1q region (1q41-42), in surgically removed samples. Then, we screened the genetic mutation of the genes involved in this region, with endometrial cancer panel. Among them, EGLN1, that is a member of prolyl hydroxylase and can facilitate HIF-1 degradation by ubiquitination through the hydroxylation of HIF-1, was mutated at significantly higher frequencies (12/20, 60%). Introduction of wild-type EGLN1 into endometrial cancer cell lines (HHUA, Ishikawa and HWCA), that carry EGLN1 gene mutations induced senescence. This was invoked through the negative regulation of HIF-1 expression. In addition, alternative way of negative regulation of HIF-1 by Factor inhibiting HIF-1(FIH), SiRNA against HIF-1, and HIF-1 inhibitor, YC-1, could also induce senescence. Thus, EGLN1 can be considered as a candidate tumor suppressor on chr. 1q, and our observation could open the new aspect in exploring the machinery of senescence induction associated with HIF-1 signal transduction. These results also suggested the availability of negative regulation of HIF-1 signals for uterine cancer treatment, especially for uterine sarcomas that have worse prognosis and show a high frequency of EGLN1 gene abnormality.