BRMS1 Suppresses Glioma Progression by Regulating Invasion,Migration and Adhesion of Glioma Cells

Breast cancer metastasis suppressor 1 (BRMS1) is a metastasis suppressor gene in several solid tumors. However, the expression and function of BRMS1 in glioma have not been reported. In this study, we investigated whether BRMS1 play a role in glioma pathogenesis. Using the tissue microarray technology, we found that BRMS1 expression is significantly decreased in glioma compared with tumor adjacent normal brain tissue (P<0.01, χ² test) and reduced BRMS1 staining is associated with WHO stages (P<0.05, χ² test). We also found that BRMS1 was significantly downregulated in glioma cell lines compared to normal human astrocytes (P<0.01, χ² test). Furthermore, we demonstrated that BRMS1 overexpression inhibited glioma cell invasion by suppressing uPA, NF-κB, MMP-2 expression and MMP-2 enzyme activity. Moreover, our data showed that overexpression of BRMS1 inhibited glioma cell migration and adhesion capacity compared with the control group through the Src-FAK pathway. Taken together, this study suggested that BRMS1 has a role in glioma development and progression by regulating invasion, migration and adhesion activities of cancer cells.     

Src42A modulates tumor invasion and cell death via Ben/dUev1a-mediated JNK activation in Drosophila

Loss of the cell polarity gene could cooperate with oncogenic Ras to drive tumor growth and invasion, which critically depends on the c-Jun N-terminal Kinase (JNK) signaling pathway in Drosophila. By performing a genetic screen, we have identified Src42A, the ortholog of mammalian Src, as a key modulator of both Ras^V12/lgl^−/− triggered tumor invasion and loss of cell polarity gene-induced cell migration. Our genetic study further demonstrated that the Bendless (Ben)/dUev1a ubiquitin E2 complex is an essential regulator of Src42A-induced, JNK-mediated cell migration. Furthermore, we showed that ectopic Ben/dUev1a expression induced invasive cell migration along with increased MMP1 production in wing disc epithelia. Moreover, Ben/dUev1a could cooperate with Ras^V12 to promote tumor overgrowth and invasion. In addition, we found that the Ben/dUev1a complex is required for ectopic Src42A-triggered cell death and endogenous Src42A-dependent thorax closure. Our data not only provide a mechanistic insight into the role of Src in development and disease but also propose a potential oncogenic function for Ubc13 and Uev1a, the mammalian homologs of Ben and dUev1a.     

MicroRNA-212 displays tumor-promoting properties in non-small cell lung cancer cells and targets the hedgehog pathway receptor PTCH1

Dysexpression of microRNAs has been found in many tumors, including lung cancer. The hedgehog (Hh) signaling pathway plays an important role during normal development, and the abnormal regulation of its members has also been related to many tumors. However, little is known about the relationship between microRNA and the Hh pathway. In this paper, we report microRNA-212 (miR-212) playing a role in non-small cell lung cancer (NSCLC) and targeting PTCH1, a receptor of the Hh pathway. We found that miR-212 was up-regulated when cells were treated with 4ß-12-O-tetradecanoylphorbol-13-acetate (TPA). We ectopically expressed miR-212 in NSCLC cell lines to examine the influence of miR-212 overexpression. The results showed that overexpression of miR-212 in NSCLC cells promoted cell cycle progression and cell proliferation, migration, and invasion. The promoting effects of miR-212 on cell proliferation, migration, and invasion were partially reversed by the miR-212 inhibitor anti-miR-212. These results suggested that miR-212 might have tumor-promoting properties. Potential targets of miR-212 were predicted, and we showed tumor suppressor PTCH1 was a functional target of miR-212. PTCH1 may be responsible for the effect of miR-212 on cell proliferation. Altogether, our results indicated that miR-212 was involved in tumorigenesis, and the oncogenic activity of miR-212 in NSCLC cells was due, in part, to suppression of PTCH1.     

Autocrine/Paracrine Human Growth Hormone-stimulated MicroRNA 96-182-183 Cluster Promotes Epithelial-Mesenchymal Transition and Invasion in Breast Cancer

Human growth hormone (hGH) plays critical roles in pubertal mammary gland growth, development, and sexual maturation. Accumulated studies have reported that autocrine/paracrine hGH is an orthotopically expressed oncoprotein that promotes normal mammary epithelial cell oncogenic transformation. Autocrine/paracrine hGH has also been reported to promote mammary epithelial cell epithelial-mesenchymal transition (EMT) and invasion. However, the underlying mechanism remains largely obscure. MicroRNAs (miRNAs) are reported to be involved in regulation of multiple cellular functions of cancer. To determine whether autocrine/paracrine hGH promotes EMT and invasion through modulation of miRNA expression, we performed microarray profiling using MCF-7 cells stably expressing wild type or a translation-deficient hGH gene and identified miR-96-182-183 as an autocrine/paracrine hGH-regulated miRNA cluster. Forced expression of miR-96-182-183 conferred on epithelioid MCF-7 cells a mesenchymal phenotype and promoted invasive behavior in vitro and dissemination in vivo. Moreover, we observed that miR-96-182-183 promoted EMT and invasion by directly and simultaneously suppressing BRMS1L (breast cancer metastasis suppressor 1-like) gene expression. miR-96 and miR-182 also targeted GHR, providing a potential negative feedback loop in the hGH-GHR signaling pathway. We further demonstrated that autocrine/paracrine hGH stimulated miR-96-182-183 expression and facilitated EMT and invasion via STAT3 and STAT5 signaling. Consistent with elevated expression of autocrine/paracrine hGH in metastatic breast cancer tissue, miR-96-182-183 expression was also remarkably enhanced. Hence, we delineate the roles of the miRNA-96-182-183 cluster and elucidate a novel hGH-GHR-STAT3/STAT5-miR-96-182-183-BRMS1L-ZEB1/E47-EMT/invasion axis, which provides further understanding of the mechanism of autocrine/paracrine hGH-stimulated EMT and invasion in breast cancer.     

Knockdown of LncRNA PVT1 inhibits tumorigenesis in non-small-cell lung cancer by regulating miR-497 expression

Plasmacytoma variant translocation1 (PVT1) was reported to be upregulated in non-small-cell lung cancer (NSCLC) tissues, serve as a promising biomarker for diagnosis and prognosis of NSCLC, and promoted NSCLC cell proliferation. However, the detailed molecular mechanism of PVT1 involved in the pathogenesis and development of NSCLC remains largely unknown. In this study, the expression levels of PVT1 and miR-497 in NSCLC cells were determined by qRT-PCR. Cell viability, invasion and apoptosis were detected by MTT assay, cell invasion assay and flow cytometry analysis, respectively. RNA immunoprecipitation (RIP) and luciferase reporter assay were performed to confirm whether PVT1 directly interacts with miR-497. A xenograft mouse model was established to confirm the effect of PVT1 on tumor growth in vivo and the underlying molecular mechanism. Our findings indicated that PVT1 was significantly upregulated and miR-497 was markedly downregulated in NSCLC cell lines. si-PVT1 effectively decreased the expression of PVT1 and increased the expression of miR-497. PVT1 knockdown remarkably inhibited cell viability, invasion and promoted apoptosis in NSCLC cells. RIP and luciferase reporter assay demonstrated that PVT1 could directly interact with miR-497. Moreover, PVT1 overexpression reversed the inhibitory effect of miR-497 on cell viability, invasion and promotion effect on apoptosis of NSCLC cells. Furthermore, in vivo experiment showed that knockdown of PVT1 inhibited tumor growth in vivo and promoted miR-497 expression. In conclusion, knockdown of PVT1 inhibited cell viability, invasion and induced apoptosis in NSCLC by regulating miR-497 expression, elucidating the molecular mechanism of the oncogenic role of PVT1 in NSCLC and providing an lncRNA-directed target for NSCLC.     

Live Imaging and Gene Expression Analysis in Zebrafish Identifies a Link between Neutrophils and Epithelial to Mesenchymal Transition

Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear. Here, we have exploited zebrafish to visualize and quantify the earliest events during epithelial cell transformation induced by oncogenic HRas^V12. Live imaging revealed that expression of HRas^V12 in the epidermis results in EMT and chronic neutrophil and macrophage infiltration. We have developed an in vivo system to probe and quantify gene expression changes specifically in transformed cells from chimeric zebrafish expressing oncogenic HRas^V12 using translating ribosomal affinity purification (TRAP). We found that the expression of genes associated with EMT, including slug, vimentin and mmp9, are enriched in HRas^V12 transformed epithelial cells and that this enrichment requires the presence of neutrophils. An early signal induced by HRas^V12 in epithelial cells is the expression of il-8 (cxcl8) and we found that the chemokine receptor, Cxcr2, mediates neutrophil but not macrophage recruitment to the transformed cells. Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation. Taken together, these findings implicate both autocrine and paracrine signaling through Cxcr2 in the regulation of inflammation and gene expression in transformed epithelial cells.     

Brain cancer stem-like cell genesis from p53-deficient mouse astrocytes by oncogenic Ras

Here, we show that H-ras^V12 causes the p53-knockout mouse astrocytes (p53−/− astrocytes) to be transformed into brain cancer stem-like cells. H-ras^V12 triggers the p53−/− astrocytes to express a Nestin and a Cd133, which are expressed in normal and cancer neural stem cells. H-ras^V12 also induces the formation of a single cell-derived neurosphere under neural stem cell culture conditions. Furthermore, H-ras^V12-overexpressing p53−/− astrocytes (p53−/−ast-H-ras^V12) possess an in vitro self-renewal capacity, and are aberrantly differentiated into Tuj1-positve neurons both in vitro and in vivo. Amongst a variety of Ras-mediated canonical signaling pathways, we demonstrated that the MEK/ERK signaling pathway is responsible for neurosphere formation in p53-deficient astrocytes, whereas the PI3K/AKT signaling pathway is involved in oncogenic transformation in these cells. These findings suggest that the activation of Ras signaling pathways promotes the generation of brain cancer stem-like cells from p53-deficient mouse astrocytes by changing cell fate and transforming cell properties.     

miR-137 impairs the proliferative and migratory capacity of human non-small cell lung cancer cells by targeting paxillin

Human lung cancer is the leading cause of cancer motility worldwide, with nearly 1.4 million deaths each year, among which non-small cell lung cancer (NSCLC) accounts for almost 85 % of this disease. The discovery of microRNAs (miRNAs) provides a new avenue for NSCLC diagnostic and treatment regiments. Currently, a large number of miRNAs have been reported to be associated with the progression of NSCLC, among which serum miR-137 has been examined to be down-regulated in NSCLC patients. However, the function of miR-137 on NSCLC cells migration and invasion and the relative mechanisms were less known. Here, we found that ectopic expression of miR-137 could inhibit cell proliferation, induce cell apoptosis, and suppress cell migration and invasion in NSCLC cell line A549. Moreover, we found that paxillin (PXN) was a target gene of miR-137 in NSCLC cells and restored expression of PXN abolished the miR-137-mediated suppression of cell migration and invasion. Taken together, our results showed that miR-137 acted as a tumor suppressor in NSCLC by targeting PXN, and it may provide novel diagnostic and therapeutic options for human NSCLC clinical operation in future.     

Deguelin attenuates non-small cell lung cancer cell metastasis through inhibiting the CtsZ/FAK signaling pathway

Lung cancer is the leading cause of cancer-related death among both men and women every year, mainly due to metastasis. Although natural compound deguelin has been reported to inhibited cell migration and invasion in various cancer cells, the details of this regulation progress remain to be fully elucidated. In this study, we investigated the underlying mechanism of deguelin-suppressed metastasis of non-small cell lung cancer (NSCLC) cells. Our results demonstrate that deguelin inhibits NSCLC cell migration, invasion, and metastasis both in vitro and in vivo. These inhibitory effects of deguelin were mediated by suppressing of Cathepsin Z (CtsZ) expression and interrupting the interaction of CtsZ with integrin β3. Moreover, deguelin inhibits the activation of CtsZ downstream FAK/Src/Paxillin signaling. Knockdown of CtsZ mimicked the effect of deguelin on NSCLC cells migration and invasion. Our study reveals that deguelin exerts its anti-metastatic effect both in vitro and in vivo is partly dependent on the suppression of CtsZ signaling. Deguelin would be a potential anti-metastasis agent against NSCLC.     

羽扇豆醇介导MDM2-p53通路对胃癌细胞生物学行为的影响

目的:探讨羽扇豆醇介导鼠双微基因2(Mouse double microgene 2,MDM2)-p53通路对胃癌细胞生物学行为的影响及相关机制。方法:对数生长期的胃癌小鼠MFC细胞株随机分为三组。实验1组与实验2组给予10 mg/L和20 mg/L的羽扇豆醇处理,对照组以等体积的1×磷酸盐缓冲液处理。对比三组MFC细胞细胞增殖、凋亡、迁移与侵袭,及MDM2-p53通路蛋白表达。结果:细胞处理后6 h与12 h,实验1组与实验2组的细胞增殖指数、细胞迁移与侵袭指数、MDM2蛋白相对表达水平显著低对于对照组,实验2组也低于实验1组,对比差异都有统计学意义(P<0.05)。细胞处理后6 h与12 h,实验1组与实验2组的细胞凋亡指数、p53蛋白相对表达水平显著高于对照组,实验2组也高于实验1组,对比差异都有统计学意义(P<0.05)。结论:羽扇豆醇能促进胃癌细胞p53蛋白的表达,抑制MDM2蛋白的表达,从而促进细胞凋亡,抑制胃癌的增殖、侵袭与转移,且具有剂量依赖性。     

MIR517C inhibits autophagy and the epithelial-to-mesenchymal (-like) transition phenotype in human glioblastoma through KPNA2-dependent disruption of TP53 nuclear translocation

The epithelial-to-mesenchymal (-like) transition (EMT), a crucial embryonic development program, has been linked to the regulation of glioblastoma (GBM) progression and invasion. Here, we investigated the role of MIR517C/miR-517c, which belongs to the C19MC microRNA cluster identified in our preliminary studies, in the pathogenesis of GBM. We found that MIR517C was associated with improved prognosis in patients with GBM. Furthermore, following treatment with the autophagy inducer temozolomide (TMZ) and low glucose (LG), MIR517C degraded KPNA2 (karyopherin alpha 2 [RAG cohort 1, importin alpha 1]) and subsequently disturbed the nuclear translocation of TP53 in the GBM cell line U87 in vitro. Interestingly, this microRNA could inhibit autophagy and reduce cell migration and infiltration in U87 cells harboring wild-type (WT) TP53, but not in U251 cells harboring mutant (MU) TP53. Moreover, the expression of epithelial markers (i.e., CDH13/T-cadherin and CLDN1 [claudin 1]) increased, while the expression of mesenchymal markers (i.e., CDH2/N-cadherin, SNAI1/Snail, and VIM [vimentin]) decreased, indicating that the EMT status was blocked by MIR517C in U87 cells. Compared with MIR517C overexpression, MIR517C knockdown promoted infiltration of U87 cells to the surrounding structures in nude mice in vivo. The above phenotypic changes were also observed in TP53^+/+ and TP53^-/- HCT116 colon cancer cells. In summary, our study provided support for a link between autophagy and EMT status in WT TP53 GBM cells and provided evidence for the signaling pathway (MIR517C-KPNA2-cytoplasmic TP53) involved in attenuating autophagy and eliminating the increased migration and invasion during the EMT.     

The Different Role of Notch1 and Notch2 in Astrocytic Gliomas

It is well known that Notch signaling plays either oncogenic or tumor suppressive role in a variety of tumors, depending on the cellular context. However, in our previous study, we found that Notch1 was overexpressed while Notch2 downregulated in the majority of astrocytic gliomas with different grades as well as in glioblastoma cell lines U251 and A172. We had knocked down Notch1 by siRNA in glioblastoma cells, and identified that the cell growth and invasion were inhibited, whereas cell apoptosis was induced either in vitro or in vivo. For further clarification of the role of Notch2 in pathogenesis of gliomas, enforced overexpression of Notch2 was carried out with transfection of Notch2 expression plasmid in glioma cells and the cell growth, invasion and apoptosis were examined in vitro and in vivo in the present study, and siRNA targeting Notch1 was used as a positive control in vivo. The results showed that upregulating Notch2 had the effect of suppressing cell growth and invasion as well as inducing apoptosis, just the same as the results of knocking down Notch1. Meanwhile, the activity of core signaling pathway–EGFR/PI3K/AKT in astrocytic glioma cells was repressed. Thus, the present study reveals, for the first time, that Notch1 and Notch2 play different roles in the biological processes of astrocytic gliomas. Knocking down the Notch1 or enforced overexpression of Notch2 both modulate the astrocytic glioma phenotype, and the mechanism by which Notch1 and 2 play different roles in the glioma growth should be further investigated.     

An L314Q mutation in Map3k1 gene results in failure of eyelid fusion in the N-ethyl-N-nitrosourea-induced mutant line

In this study, we describe an N-ethyl-N-nitrosourea-induced mouse model with a corneal opacity phenotype that was associated with “eye open at birth” (EOB). Histological and immunohistochemistry staining analysis showed abnormal differentiation of the corneal epithelial cells in the mutant mice. The EOB phenotype was dominantly inherited on a C57BL/6 (B6) background. This allele carries a T941A substitution in exon 4 that leads to an L314Q amino acid change in the open reading frame of MAP3K1 (MEEK1). We named this novel Map3k1 allele Map3k1^L314Q. Phalloidin staining of F-actin was reduced in the mutant epithelial leading edge cells, which is indicative of abnormality in epithelial cell migration. Interestingly enough, not only p-c-Jun and p-JNK but also c-Jun levels were decreased in the mutant epithelial leading edge cells. This study identifies a novel mouse Map3k1 allele causing EOB phenotype and the EOB phenotype in Map3k1^L314Q mouse may be associated with the reduced level of p-JNK and c-Jun.     

EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival,Cell Invasion,Focal Adhesions,and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130^Cas. Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130^Cas. WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.     

The p53-p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21^WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21^WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21^WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G₁ arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating that the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G₁ arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21^WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on the stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21^WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1.     

KCa3.1 K+ Channel Expression and Function in Human Bronchial Epithelial Cells

The K_Ca3.1 K⁺ channel has been proposed as a novel target for pulmonary diseases such as asthma and pulmonary fibrosis. It is expressed in epithelia but its expression and function in primary human bronchial epithelial cells (HBECs) has not been described. Due to its proposed roles in the regulation of cell proliferation, migration, and epithelial fluid secretion, inhibiting this channel might have either beneficial or adverse effects on HBEC function. The aim of this study was to assess whether primary HBECs express the K_Ca3.1 channel and its role in HBEC function. Primary HBECs from the airways of healthy and asthmatic subjects, SV-transformed BEAS-2B cells and the neoplastic H292 epithelial cell line were studied. Primary HBECs, BEAS-2B and H292 cells expressed K_Ca3.1 mRNA and protein, and robust K_Ca3.1 ion currents. K_Ca3.1 protein expression was increased in asthmatic compared to healthy airway epithelium in situ, and K_Ca3.1 currents were larger in asthmatic compared to healthy HBECs cultured in vitro. Selective K_Ca3.1 blockers (TRAM-34, ICA-17043) had no effect on epithelial cell proliferation, wound closure, ciliary beat frequency, or mucus secretion. However, several features of TGFβ1-dependent epithelial-mesenchymal transition (EMT) were inhibited by K_Ca3.1 blockade. Treatment with K_Ca3.1 blockers is likely to be safe with respect to airway epithelial biology, and may potentially inhibit airway remodelling through the inhibition of EMT.     

miR‐346 promotes migration and invasion of nasopharyngeal carcinoma cells via targeting BRMS1

The aim of this study is to determine the expression and roles of miR‐346 in nasopharyngeal carcinoma (NPC). We showed that miR‐346 was upregulated in NPC tissues compared with adjacent non‐tumorous nasopharyngeal tissues. Inhibition of miR‐346 significantly attenuated the migration and invasion of NPC cells. Luciferase reporter assay showed that miR‐346 targeted the 3′‐untranslated region (3′‐UTR) of breast cancer metastasis suppressor 1 (BRMS1). Overexpression of miR‐346 suppressed the endogenous expression of BRMS1 in NPC cells. There was a significant negative correlation between miR‐346 and BRMS1 protein expression in NPC tissues (r = ?0.372, P = 0.008). Rescue experiments demonstrated that overexpression of BRMS1 lacking the 3′‐UTR impaired the invasiveness of NPC cells transfected with miR‐346 mimic. Taken together, miR‐346 shows the ability to promote the migration and invasion of nasopharyngeal cancer cells via targeting BRMS1 and represents a potential therapeutic target for NPC.