Inhibition of Motile and Invasive Properties of Ovarian Cancer Cells by ASODN Against Rho-associated Protein Kinase

p160ROCK, a kinase effector of Rho GTPase mediating RhoA-induced assembly of focal adhesions and stress fibers, plays an important role in the invasive process of various tumor cells. The purpose of this study was to investigate the role of p160ROCK in the invasive behaviors of human ovarian cancer cells and their metastasis. Transfection with a dominant-active form of p160ROCK mutant (p160ROCKΔ 3) enhanced cell migration and invasion of ovarian cancer cells, while antisense oligodeoxynucleotide (ASODN) against p160ROCK inhibited the motile and invasive properties of the cells. Our data suggested that p160ROCK was involved in ovarian cancer cell invasion and metastasis by facilitating cancer cell migration, and that p160ROCK might be a potential new effective target for preventing metastasis of ovarian cancer.     

Inhibition of motile and invasive properties of ovarian cancer cells by ASODN against Rho-associated protein kinase

p160ROCK, a kinase effector of Rho GTPase mediating RhoA-induced assembly of focal adhesions and stress fibers, plays an important role in the invasive process of various tumor cells. The purpose of this study was to investigate the role of p160ROCK in the invasive behaviors of human ovarian cancer cells and their metastasis. Transfection with a dominant-active form of p160ROCK mutant (p160ROCKDelta 3) enhanced cell migration and invasion of ovarian cancer cells, while antisense oligodeoxynucleotide (ASODN) against p160ROCK inhibited the motile and invasive properties of the cells. Our data suggested that p160ROCK was involved in ovarian cancer cell invasion and metastasis by facilitating cancer cell migration, and that p160ROCK might be a potential new effective target for preventing metastasis of ovarian cancer.     

ODAM inhibits RhoA‐dependent invasion in breast cancer

Odontogenic ameloblast‐associated protein (ODAM) contributes to cell adhesion. In human cancer, ODAM is down‐regulated, and the overexpression of ODAM results in a favourable prognosis; however, the molecular mechanisms underlying ODAM‐mediated inhibition of cancer invasion and metastasis remain unclear. Here, we identify a critical role for ODAM in inducing cancer cell adhesion. ODAM induced RhoA activity and the expression of downstream factors, including Rho‐associated kinase (ROCK). ODAM‐mediated RhoA signalling resulted in actin filament rearrangement by activating PTEN and inhibiting the phosphorylation of AKT. When ODAM is overexpressed in MCF7 breast cancer cells and AGS gastric cancer cells that activate RhoA at high levels, it decreases motility, increases adhesion and inhibits the metastasis of MCF7 cells. Conversely, depletion of ODAM in cancer cells inhibits Rho GTPase activation, resulting in increased cancer migration and invasion. These results suggest that ODAM expression in cells maintains their adhesion, resulting in the prevention of their metastasis via the regulation of RhoA signalling in breast cancer cells. Copyright © 2015 John Wiley & Sons, Ltd. SIGNIFICANCE Breast cancer represents the first most frequent cancer, and the ratio of mortality is high in women. Of utmost importance for reducing risk by breast cancer are their anti‐invasion mechanisms, particularly in the non‐invasive cancer cells because metastasis is the principal cause of death among cancer patients. ODAM induced RhoA activity. ODAM‐mediated RhoA signalling resulted in actin filament rearrangement, increased cell adhesion and inhibited the migration/invasion of MCF7 cells. These results suggest that ODAM expression maintains their adhesion, resulting in the prevention of their metastasis via the regulation of RhoA signalling in breast cancer cells.     

Hypomethylation‐activated cancer‐testis gene SPANXC promotes cell metastasis in lung adenocarcinoma

Many studies have shown that there were similarity between tumorigenesis and gametogenesis. Our previous work found that cancer‐testis (CT) genes could serve as a novel source of candidate of cancer. Here, by analysing The Cancer Genome Atlas (TCGA) database, we characterized a CT gene, SPANXC, which is expressed only in testis. The SPANXC was reactivated in lung adenocarcinoma (LUAD) tissues. And the expression of SPANXC was associated with prognosis of LUAD. We also found that the activation of SPANXC was due to the promoter hypomethylation of SPANXC. Moreover, SPANXC could modulate cell metastasis both in vitro and in vivo. Mechanistically, we found that SPANXC could bind to ROCK1, a metastasis‐related gene, and thus SPANXC may regulate cell metastasis partly through interaction with ROCK1 in LUAD. Together, our results demonstrated that the CT expression pattern of SPANXC served as a crucial role in metastasis of LUAD. And these data further corroborated the resemblance between processes of germ cell development and tumorigenesis, including migration and invasion.     

Melanoma‐initiating cells: a compass needed

Most tumours contain a heterogeneous population of cancer cells, which harbour a range of genetic mutations and have probably undergone deregulated differentiation programmes that allow them to adapt to tumour microenvironments. Another explanation for tumour heterogeneity might be that the cells within a tumour are derived from tumour‐initiating cells through diverse differentiation programmes. Tumour‐initiating cells are thought to constitute one or more distinct subpopulations within a tumour and to drive tumour initiation, development and metastasis, as well as to be responsible for their recurrence after therapy. Recent studies have raised crucial questions about the nature, frequency and importance of melanoma‐initiating cells. Here, we discuss our current understanding of melanoma‐initiating cells and outline several approaches that the scientific community might consider to resolve the controversies surrounding these cells.     

Tissue inhibitor of metalloproteinases‐1‐induced scattered liver metastasis is mediated by host‐derived urokinase‐type plasminogen activator

Paradoxically, not only proteinases but also their inhibitors can correlate with bad prognosis of cancer patients, underlining the evolving concept of the protease web as the complex interplay between proteinases, their inhibitors and effector molecules. Elevated levels of tissue inhibitor of metalloproteinases‐1 (TIMP‐1) render the liver more susceptible to metastasis by triggering urokinase plasminogen activator (uPA) expression as well as hepatocyte growth factor (HGF) signalling, thereby leading to the fatal scattered infiltration of metastasizing tumour cells throughout the parenchyma of the target organ. Here, we investigated whether host uPA is a crucial protagonist for the TIMP‐1‐induced modulation of a pro‐metastatic microenvironment in the liver. Indeed, in livers of uPA‐ablated mice elevated TIMP‐1 levels did not trigger HGF signalling and did not promote metastasis of a murine T‐lymphoma cell line. In contrast, lack of tumour cell‐derived uPA induced by gene silencing did not interfere with this pro‐metastatic pathway. Furthermore, host uPA was necessary for the recruitment of neutrophilic granulocytes and the associated increase of HGF in livers with elevated TIMP‐1 levels. This newly identified co‐operation between TIMP‐1 and host uPA suggests that therapies, simultaneously interfering with pro‐ and anti‐proteolytic pathways may be beneficial for patients with metastatic disease.     

miR‐4732‐5p promotes breast cancer progression by targeting TSPAN13

MiR‐4732‐5p was previously found to be dysregulated in nipple discharge of breast cancer. However, the expression and function of miR‐4732‐5p in breast cancer remain largely unknown. Here, the expression of miR‐4732‐5p was detected using quantitative real‐time PCR in breast cancer tissues and cell lines. Cell proliferation, apoptosis, migration and invasion assays were performed to examine the effects of miR‐4732‐5p in breast cancer. In addition, mRNA sequencing, bioinformatics analysis, Western blot and luciferase assays were performed to identify the target of miR‐4732‐5p. Overall, miR‐4732‐5p was significantly down‐regulated in breast cancer tissues, especially in lymph node metastasis (LNM)‐negative tissues, compared with adjacent normal tissues. However, it was more highly expressed in LNM‐positive breast cancer tissues, compared with LNM‐negative ones. Expression of miR‐4732‐5p was positively correlated with lymph node metastasis, larger tumour size, advanced clinical stage, high Ki‐67 levels and poor prognosis. MiR‐4732‐5p promoted cell proliferation, migration and invasion in breast cancer. MiR‐4732‐5p directly targeted the 3′‐UTR of tetraspanin 13 (TSPAN13) and suppressed TSPAN13 expression at the mRNA and protein levels. These results suggested that miR‐4732‐5p may serve as a tumour suppressor in the initiation of breast cancer, but as a tumour promoter in breast cancer progression by targeting TSPAN13.     

Naturally occurring compounds acting as potent anti‐metastatic agents and their suppressing effects on Hedgehog and WNT/β‐catenin signalling pathways

Despite numerous remarkable achievements in the field of anti‐cancer therapy, tumour relapse and metastasis still remain major obstacles in improvement of overall cancer survival, which may be at least partially owing to epithelial‐mesenchymal transition (EMT). Multiple signalling pathways have been identified in EMT; however, it appears that the role of the Hedgehog and WNT/β‐catenin pathways are more prominent than others. These are well‐known preserved intracellular regulatory pathways of different cellular functions including proliferation, survival, adhesion and differentiation. Over the last few decades, several naturally occurring compounds have been identified to significantly obstruct several intermediates in Hedgehog and WNT/β‐catenin signalling, eventually resulting in suppression of signal transduction. This article highlights the current state of knowledge associated with Hedgehog and WNT/β‐catenin, their involvement in metastasis through EMT processes and introduction of the most potent naturally occurring agents with capability of suppressing them, eventually overcoming tumour relapse, invasion and metastasis.     

Mesenchymal stem cells promote cell invasion and migration and autophagy‐induced epithelial‐mesenchymal transition in A549 lung adenocarcinoma cells

Mesenchymal stem cells (MSCs) are recruited into the tumour microenvironment and promote tumour growth and metastasis. Tumour microenvironment‐induced autophagy is considered to suppress primary tumour formation by impairing migration and invasion. Whether these recruited MSCs regulate tumour autophagy and whether autophagy affects tumour growth are controversial. Our data showed that MSCs promote autophagy activation, reactive oxygen species production, and epithelial‐mesenchymal transition (EMT) as well as increased migration and invasion in A549 cells. Decreased expression of E‐cadherin and increased expression of vimentin and Snail were observed in A549 cells cocultured with MSCs. Conversely, MSC coculture‐mediated autophagy positively promoted tumour EMT. Autophagy inhibition suppressed MSC coculture‐mediated EMT and reduced A549 cell migration and invasion slightly. Furthermore, the migratory and invasive abilities of A549 cells were additional increased when autophagy was further enhanced by rapamycin treatment. Taken together, this work suggests that microenvironments containing MSCs can promote autophagy activation for enhancing EMT; MSCs also increase the migratory and invasive abilities of A549 lung adenocarcinoma cells. Mesenchymal stem cell‐containing microenvironments and MSC‐induced autophagy signalling may be potential targets for blocking lung cancer cell migration and invasion.     

MicroRNA-1280 Inhibits Invasion and Metastasis by Targeting ROCK1 in Bladder Cancer

MicroRNAs (miRNAs) are non-protein-coding sequences that can function as oncogenes or tumor suppressor genes. This study documents the tumor suppressor role of miR-1280 in bladder cancer. Quantitative real-time PCR and in situ hybridization analyses showed that miR-1280 is significantly down-regulated in bladder cancer cell lines and tumors compared to a non-malignant cell line or normal tissue samples. To decipher the functional significance of miR-1280 in bladder cancer, we ectopically over-expressed miR-1280 in bladder cancer cell lines. Over-expression of miR-1280 had antiproliferative effects and impaired colony formation of bladder cancer cell lines. FACS (fluorescence activated cell sorting) analysis revealed that re-expression of miR-1280 in bladder cancer cells induced G2-M cell cycle arrest and apoptosis. Our results demonstrate that miR-1280 inhibited migration and invasion of bladder cancer cell lines. miR-1280 also attenuated ROCK1 and RhoC protein expression. Luciferase reporter assays demonstrated that oncogene ROCK1 is a direct target of miR-1280 in bladder cancer. This study also indicates that miR-1280 may be of diagnostic and prognostic importance in bladder cancer. For instance, ROC analysis showed that miR-1280 expression can distinguish between malignant and normal bladder cancer cases and Kaplan-Meier analysis revealed that patients with miR-1280 high expression had higher overall survival compared to those with low miR-1280 expression. In conclusion, this is the first study to document that miR-1280 functions as a tumor suppressor by targeting oncogene ROCK1 to invasion/migration and metastasis. Various compounds are currently being used as ROCK1 inhibitors; therefore restoration of tumor suppressor miR-1280 might be therapeutically useful either alone or in combination with these compounds in the treatment of bladder 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.     

TFPI‐2 silencing increases tumour progression and promotes metalloproteinase 1 and 3 induction through tumour‐stromal cell interactions

Tissue factor pathway inhibitor‐2 (TFPI‐2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI‐2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI‐2 is frequently down‐regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI‐2 down‐regulation in the National Cancer Institute (NCI)‐H460 non‐small cell lung cancer cell line using specific micro interfering micro‐interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI‐2 down‐regulation enhanced cell adhesion to collagen IV and laminin via an increase in α₁ integrin on cell surface, and increased MMP expression (mainly MMP‐1 and ‐3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI‐2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP‐1, ‐3 and ‐7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.     

The RhoA pathway mediates MMP‐2 and MMP‐9‐independent invasive behavior in a triple‐negative breast cancer cell line

Breast cancer is a heterogeneous disease that varies in its biology and response to therapy. A foremost threat to patients is tumor invasion and metastasis, with the greatest risk among patients diagnosed with triple‐negative and/or basal‐like breast cancers. A greater understanding of the molecular mechanisms underlying cancer cell spreading is needed as 90% of cancer‐associated deaths result from metastasis. We previously demonstrated that the Tamoxifen‐selected, MCF‐7 derivative, TMX2‐28, lacks expression of estrogen receptor α (ERα) and is highly invasive, yet maintains an epithelial morphology. The present study was designed to further characterize TMX2‐28 cells and elucidate their invasion mechanism. We found that TMX2‐28 cells do not express human epidermal growth factor receptor 2 (HER2) and progesterone receptor (PR), in addition to lacking ERα, making the cells triple‐negative. We then determined that TMX2‐28 cells lack expression of active matrix metalloproteinases (MMPs)‐1, MMP‐2, MMP‐9, and other genes involved in epithelial–mesenchymal transition (EMT) suggesting that TMX2‐28 may not utilize mesenchymal invasion. In contrast, TMX2‐28 cells have high expression of Ras Homolog Gene Family Member, A (RhoA), a protein known to play a critical role in amoeboid invasion. Blocking RhoA activity with the RhoA pathway specific inhibitor H‐1152, or a RhoA specific siRNA, resulted in inhibition of invasive behavior. Collectively, these results suggest that TMX2‐28 breast cancer cells exploit a RhoA‐dependent, proteolytic‐independent invasion mechanism. Targeting the RhoA pathway in triple‐negative, basal‐like breast cancers that have a proteolytic‐independent invasion mechanism may provide therapeutic strategies for the treatment of patients with increased risk of metastasis. J. Cell. Biochem. 114: 1385–1394, 2013. © 2013 Wiley Periodicals, Inc.     

Extracellular vesicle‐associated organotropic metastasis

Metastasis refers to the progressive dissemination of primary tumour cells and their colonization of other tissues and is associated with most cancer‐related mortalities. The disproportional and systematic distribution pattern of distant metastasis in different cancers has been well documented, as is termed metastatic organotropism, a process orchestrated by a combination of anatomical, pathophysiological, genetic and biochemical factors. Extracellular vesicles (EVs), nanosized cell‐derived membrane‐bound particles known to mediate intercellular communication, are now considered crucial in organ‐specific metastasis. Here, we review and summarize recent findings regarding EV‐associated organotropic metastasis as well as some of the general mechanisms by which EVs contribute to this important process in cancer and provide a future perspective on this emerging topic. We highlight studies that demonstrate a role of tumour‐derived EVs in organotropic metastasis via pre‐metastatic niche modulation. The bioactive cargo carried by EVs is of diagnostic and prognostic values, and counteracting the functions of such EVs may be a novel therapeutic strategy targeting metastasis. Further investigations are warranted to better understand the functions and mechanisms of EVs in organotropic metastasis and accelerate the relevant clinical translation.     

The long non‐coding RNA‐ROR promotes osteosarcoma progression by targeting miR‐206

The long intergenic non‐protein coding RNA regulator of reprogramming (lncRNA‐ROR) has been reported to play crucial regulatory roles in the pathogenesis and progression of multiple cancers. However, whether ROR is associated with the initiation and development of osteosarcoma (OS) remains unclear. Here, we found that ROR expression level was significantly up‐regulated in OS tissue samples compared to adjacent normal tissues, and the elevated ROR was closely correlated with advanced tumour‐node‐metastasis (TNM) stage and lymph node metastasis and poor overall survival rate. Functional assays showed that ROR knockdown suppressed the OS cell proliferation, colony formation, migration and invasion in vitro, and retarded tumour growth in vivo. In addition, miR‐206 was verified to be a target miRNA of ROR using bioinformatics online program and luciferase report assay. miR‐206 inhibition partially rescued the inhibitory effects on OS cells induced by ROR knockdown. In conclusion, these results suggested that ROR function as an oncogene in OS by sponging miR‐206 and might be a potential therapeutic target for patients with OS.     

Paired box 5 is a frequently methylated lung cancer tumour suppressor gene interfering β‐catenin signalling and GADD45G expression

Recent studies suggest that paired box 5 (PAX5) is down‐regulated in multiple tumours through its promoter methylation. However, the role of PAX5 in non‐small cell lung cancer (NSCLC) pathogenesis remains unclear. The aim of this study is to examine PAX5 expression, its methylation status, biological functions and related molecular mechanism in NSCLC. We found that PAX5 was widely expressed in normal adult tissues but silenced or down‐regulated in 88% (7/8) of NSCLC cell lines. PAX5 expression level was significantly lower in NSCLC than that in adjacent non‐cancerous tissues (P = 0.0201). PAX5 down‐regulation was closely associated with its promoter hypermethylation status and PAX5 expression could be restored by demethylation treatment. Frequent PAX5 promoter methylation in primary tumours (70%) was correlated with lung tumour histological types (P = 0.006). Ectopic expression of PAX5 in silenced lung cancer cell lines (A549 and H1975) inhibited their colony formation and cell viability, arrested cell cycle at G2 phase and suppressed cell migration/invasion as well as tumorigenicity in nude mice. Restoration of PAX5 expression resulted in the down‐regulation of β‐catenin and up‐regulation of tissue inhibitors of metalloproteinase 2, GADD45G in lung tumour cells. In summary, PAX5 was found to be an epigenetically inactivated tumour suppressor that inhibits NSCLC cell proliferation and metastasis, through down‐regulating the β‐catenin pathway and up‐regulating GADD45G expression.     

Estrogen‐related receptor alpha triggers the proliferation and migration of human non‐small cell lung cancer via interleukin‐6

Human non‐small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths worldwide. Estrogenic signals have been suggested to be important for the growth and metastasis of NSCLC cells. Our present data showed that estrogen‐related receptor alpha (ERRα), while not ERRβ or ERRγ, was significantly elevated in NSCLC cell lines as compared with that in normal bronchial epithelial cell line BEAS‐2B. The expression of ERRα in clinical NSCLC tissues was significantly greater than that in their matched normal adjacent tissues. Over expression of ERRα can trigger the proliferation, migration, and invasion of NSCLC cells, while si‐ERRα or ERRα inhibitor showed opposite effects. ERRα can increase the mRNA and protein expression of IL‐6, while not IL‐8, IL‐10, IL‐22, VEGF, TGF‐β, or TNF‐α, in NSCLC cells. Silence of IL‐6 attenuated ERRα induced proliferation and cell invasion. Furthermore, our data revealed the inhibition of NF‐κB, while not ERK1/2 or PI3K/Akt, abolished ERRα induced production of IL‐6. This might be due to that overexpression of ERRα can increase the expression and nuclear translocation of p65 in NSCLC cells. Collectively, our data showed that activation of NF‐κB/IL‐6 is involved in ERRα induced migration and invasion of NSCLC cells. It suggested that ERRα might be a potential target for NSCLC treatment.     

Pan‐cancer RNA‐seq data stratifies tumours by some hallmarks of cancer

Numerous genetic and epigenetic alterations cause functional changes in cell biology underlying cancer. These hallmark functional changes constitute potentially tissue‐independent anticancer therapeutic targets. We hypothesized that RNA‐Seq identifies gene expression changes that underly those hallmarks, and thereby defines relevant therapeutic targets. To test this hypothesis, we analysed the publicly available TCGA‐TARGET‐GTEx gene expression data set from the University of California Santa CruzToil recompute project using WGCNA to delineate co‐correlated ‘modules’ from tumour gene expression profiles and functional enrichment of these modules to hierarchically cluster tumours. This stratified tumours according to T cell activation, NK‐cell activation, complement cascade, ATM, Rb, angiogenic, MAPK, ECM receptor and histone modification signalling. These correspond to the cancer hallmarks of avoiding immune destruction, tumour‐promoting inflammation, evading growth suppressors, inducing angiogenesis, sustained proliferative signalling, activating invasion and metastasis, and genome instability and mutation. This approach did not detect pathways corresponding to the cancer enabling replicative immortality, resisting cell death or deregulating cellular energetics hallmarks. We conclude that RNA‐Seq stratifies tumours along some, but not all, hallmarks of cancer and, therefore, could be used in conjunction with other analyses collectively to inform precision therapy.