Role In Cancer Cell Migration Research Articles

The regulatory roles of ROCK and MRCK kinases in the plasticity of cancer cell migration

Metastatic cancer cells show great plasticity in their migratory mechanisms. In this review we briefly describe the signal transduction pathways associated with the ROCK and MRCK kinases and their roles in cancer cell migration and in its plasticity. With respect to therapeutic strategies targeting metastatic cancers, selectively blocking a single target, such as ROCK or MRCK, can induce alternate modes of cancer cell migration (i.e. plasticity) making the treatment ineffective. To address the problem of plasticity, we will discuss the strategy of simultaneous targeting of both ROCK and MRCK as an effective anti-metastatic therapeutics.

Conditioned medium from mesenchymal stem cells enhances the migration of hepatoma cells through CXCR4 up-regulation and F-actin remodeling.

Interactions between tumors and mesenchymal stem cells (MSCs) can regulate cancer cell behavior and cancer progression. Rat bone marrow-derived MSCs (rMSCs) were isolated and purified by Percoll density gradient centrifugation. Conditioned media from rMSCs (MSC-CM) was prepared, and its role in cancer cell migration and the underlying molecular mechanism were investigated. MSC-CM increased the migration and up-regulated the expression of CXC chemokine receptor 4 (CXCR4) in rat hepatoma cells (CBRH-7919). F-actin remodeling was observed, and the Young's modulus was decreased in CBRH-7919 cells. A CXCR4 inhibitor suppressed the MSC-CM-induced CXCR4 expression and migration, restored the decrease in the Young's modulus and disrupted the formation of F-actin. MSC-CM thus promotes CBRH-7919 cell migration by lessening cell stiffness and increasing F-actin formation through up-regulation of CXCR4 expression. MSC-CM may therefore have a positive impact on cancer metastases and underlines a potential safety issue associated with clinical applications of MSCs.

Abstract LB-70: BRCA1 impacts ovarian cancer cell migration through modulating Pfn1 expression

Abstract Functional loss of BRCA1 has been implicated in genomic instability and cancer progression. A recent study demonstrated that BRCA1 plays a role in cancer cell migration. Using a random cell motility assay, we showed that BRCA1 promotes ovarian cancer cell migration. A proteomics study of BRCA1 +/- cell lines and tissues identified Profilin1 (Pfn1) as one of the differentially expressed proteins. This was further confirmed by Western blot and IHC analyses with tissue samples and cell lines. Pfn1 is an actin cytoskeleton-associated protein which promotes breast cancer cell motility/invasion and dissemination when downregulated. To further study whether BRCA1 impacts cell migration through Pfn1, Pfn1 was silenced which revealed enhanced motility in multiple BRCA1-WT ovarian cancer cell lines. Furthermore, overexpression of Pfn1 in BRCA1 cell lines reduced random motility which further implicates the importance of Pfn1 in ovarian cancer motility. This is the first evaluation of both motility and the functional role of Pfn1 in ovarian cancer cells. While BRCA1 has been extensively studied in the context of DNA repair, there is emerging evidence that BRCA1 proteins may play a role in cell migration. The increased motility seen in wild-type cells compared to BRCA1-mutated cells may portend increased metastatic potential in wild-type cancers. Our findings demonstrate that 1) BRCA1 positively influences ovarian cancer cell motility, and 2) BRCA1 status determines Pfn1's role in ovarian cancer cell motility suggesting a possible novel mechanism association between Pfn1 and BRCA1 and ovarian cancer cell motility. Future studies should explore whether Pfn1 downregulation enhances the metastatic potential of ovarian cancer cells with intact BRCA1 function and whether Pfn1 may serve as a possible therapeutic target. Citation Format: David M. Gau, Jamie Lesnock, Thomas Krivak, Robert Edwards, Partha Roy. BRCA1 impacts ovarian cancer cell migration through modulating Pfn1 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-70. doi:10.1158/1538-7445.AM2014-LB-70

Abstract 5292: The functional role of Tensin4 in hepatocellular carcinoma

Abstract The focal adhesion protein Tensin4 (also known as cten: c-terminal tensin like) is structurally distinct from the three other members in the Tensin family and has been reported to play critical roles in cancer cell migration and invasion in breast and colorectal cancers. However, its expression and potential function in other cancers including hepatocellular carcinoma are not well understood. By quantitative real time PCR and western blotting with Tensin4 specific antibodies, Tensin4 was found to be expressed in a panel of HCC cell lines with a number of cells showing high levels of expression. In these cells, treatment with growth factors (e.g. EGF) significantly induced Tensin4 protein expression; while treatment of pharmacological inhibitors against the kinases in the MAPK signaling cascade abolished this induction. These observations suggested that Tensin4 expression was Ras/MAPK signaling dependent. Intriguingly, the growth factor-induced Tensin4 expression could not be observed in HCC cell lines with low basal Tensin4 expression, implying that other regulatory mechanisms do exist. By immunofluorescence microscopy, the focal adhesion localization of Tensin4 was confirmed in HCC cells. Detail examination of a panel of Tensin4 deletion constructs revealed the requirement of the C-terminal SH2 domain for this specific localization. To examine the functional role of Tensin4, SMMC and BEL7402 HCC cells with high basal expression of Tensin4 were subjected to stable Tensin4 knockdown followed by cell proliferation and cell migration assays. Consistently, we observed that both stable Tensin4 knockdown cells proliferated and migrated in slower rates in vitro. However, the molecular mechanisms regarding this reduction remains to be explored. Taken together, our data suggested that Tensin4 may play a pro-oncogenic role in HCC, possibly function as a downstream effector of Ras/MAPK signaling. The study was funded by a Hong Kong Research Grants Council GRF grant (775110). Citation Format: Lo Kong Chan. The functional role of Tensin4 in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5292. doi:10.1158/1538-7445.AM2014-5292

Enhanced breast cancer cell adherence to the lung endothelium via PAF acetylhydrolase inhibition: a potential mechanism for enhanced metastasis in smokers.

Cancer deaths are primarily caused by distant metastases, rather than by primary tumor growth; however, the role of smoking in metastasis remains unclear. We demonstrated previously that endothelial cell platelet-activating factor (PAF) production results in enhanced inflammatory cell recruitment to the lung. We propose that endothelial cell PAF accumulation plays a role in cancer cell migration to distal locations. We used cigarette smoke extract (CSE) to inhibit the activity of endothelial cell PAF acetylhydrolase (PAF-AH), which hydrolyzes and inactivates PAF, and determined whether this results in increased endothelial cell PAF accumulation and breast cancer adherence. Incubation of human lung microvascular endothelial cells (HMVEC-L) with CSE resulted in a significant inhibition of PAF-AH activity that was accompanied by increased PAF production and adherence of highly invasive MDA-MB-231 breast cancer cells. Pretreatment of HMVEC-L with (S)-bromoenol lactone to inhibit calcium-independent phospholipase A2β (iPLA2β, which initiates endothelial cell PAF production) prior to CSE exposure resulted in complete inhibition of MDA-MB-231 cell adherence. Similarly, pretreatment of MDA-MB-231 cells with the PAF receptor antagonist Ginkgo biloba resulted in inhibition of adherence to the endothelium. Immunoblot analysis indicated an increase in MDA-MB-231 cell PAF receptor expression with CSE exposure. Taken together, our data indicate that CSE exposure increases endothelial cell PAF production, resulting in enhanced adherence of tumor cells to the endothelium. Our in vitro data indicate that increased tumor cell adherence would lead to enhanced metastasis formation in smokers. Potential therapeutic targets include endothelial cell iPLA2β or the tumor cell PAF receptor.

CCR7: Roles in cancer cell dissemination, migration and metastasis formation

The CC-chemokine receptor 7 (CCR7) coordinates the migration of cancer cells as well as immune cells towards lymphatic organs where its two ligands CCL19 and CCL21 are constitutively expressed. Here we provide a topological model of CCR7, which belongs to the class A of G-protein coupled, seven-transmembrane spanning receptors, and describe how CCR7 expression is regulated. We focus on its role in cancer cell migration and metastasis formation and discuss how cancer cells can utilize CCR7 or its ligands to escape from immune surveillance.

Role of LPAR3, PKC and EGFR in LPA-induced cell migration in oral squamous carcinoma cells.

BackgroundOral squamous cell carcinoma is an aggressive neoplasm with serious morbidity and mortality, which typically spreads through local invasive growth. Lysophosphatidic acid (LPA) is involved in a number of biological processes, and may have a role in cancer cell migration and invasiveness. LPA is present in most tissues and can activate cells through six different LPA receptors (LPAR1-6). Although LPA is predominantly promigratory, some of the receptors may have antimigratory effects in certain cells. The signalling mechanisms of LPA are not fully understood, and in oral carcinoma cells the specific receptors and pathways involved in LPA-stimulated migration are unknown.MethodsThe oral carcinoma cell lines E10, SCC-9, and D2 were investigated. Cell migration was studied in a scratch wound assay, and invasion was demonstrated in organotypic three dimensional co-cultures. Protein and mRNA expression of LPA receptors was studied with Western blotting and qRT-PCR. Activation of signalling proteins was examined with Western blotting and isoelectric focusing, and signalling mechanisms were further explored using pharmacological agents and siRNA directed at specific receptors and pathways.ResultsLPA stimulated cell migration in the two oral carcinoma cell lines E10 and SCC-9, but was slightly inhibitory in D2. The receptor expression profile and the effects of specific pharmacological antagonist and agonists indicated that LPA-stimulated cell migration was mediated through LPAR3 in E10 and SCC-9. Furthermore, in both these cell lines, the stimulation by LPA was dependent on PKC activity. However, while LPA induced transactivation of EGFR and the stimulated migration was blocked by EGFR inhibitors in E10 cells, LPA did not induce EGFR transactivation in SCC-9 cells. In D2 cells, LPA induced EGFR transactivation, but this was associated with slowing of a very high inherent migration rate in these cells.ConclusionThe results demonstrate LPA-stimulated migration in oral carcinoma cells through LPAR3, mediated further by PKC, which acts either in concert with or independently of EGFR transactivation.

A mechanically-induced colon cancer cell population shows increased metastatic potential.

BackgroundMetastasis accounts for the majority of deaths from cancer. Although tumor microenvironment has been shown to have a significant impact on the initiation and/or promotion of metastasis, the mechanism remains elusive. We previously reported that HCT-8 colon cancer cells underwent a phenotypic transition from an adhesive epithelial type (E-cell) to a rounded dissociated type (R-cell) via soft substrate culture, which resembled the initiation of metastasis. The objective of current study was to investigate the molecular and metabolic mechanisms of the E-R transition.MethodsGlobal gene expressions of HCT-8 E and R cells were measured by RNA Sequencing (RNA-seq); and the results were further confirmed by real-time PCR. Reactive oxygen species (ROS), anoikis resistance, enzyme activity of aldehyde dehydrogenase 3 family, member A1 (ALDH3A1), and in vitro invasion assay were tested on both E and R cells. The deformability of HCT-8 E and R cells was measured by atomic force microscopy (AFM). To study the in vivo invasiveness of two cell types, athymic nude mice were intra-splenically injected with HCT-8 E or R cells and sacrificed after 9 weeks. Incidences of tumor development and metastasis were histologically evaluated and analyzed with Fisher’s exact test.ResultsBesides HCT-8, E-R transition on soft substrates was also seen in three other cancer cell lines (HCT116, SW480 colon and DU145 prostate cancer). The expression of some genes, such as ALDH3A1, TNS4, CLDN2, and AKR1B10, which are known to play important roles in cancer cell migration, invasion, proliferation and apoptosis, were increased in HCT-8 R cells. R cells also showed higher ALDH3A1 enzyme activity, higher ROS, higher anoikis resistance, and higher softness than E cells. More importantly, in vitro assay and in vivo animal models revealed that HCT-8 R cells were more invasive than E cells.ConclusionsOur comprehensive comparison of HCT-8 E and R cells revealed differences of molecular, phenotypical, and mechanical signatures between the two cell types. To our knowledge, this is the first study that explores the molecular mechanism of E-R transition, which may greatly increase our understanding of the mechanisms of cancer mechanical microenvironment and initiation of cancer metastasis.

Conformational changes and translocation of tissue-transglutaminase to the plasma membranes: role in cancer cell migration

BackgroundTissue-transglutaminase (TG2), a dual function G-protein, plays key roles in cell differentiation and migration. In our previous studies we reported the mechanism of TG2-induced cell differentiation. In present study, we explored the mechanism of how TG2 may be involved in cell migration.MethodsTo study the mechanism of TG2-mediated cell migration, we used neuroblastoma cells (SH-SY5Y) which do not express TG2, neuroblastoma cells expressing exogenous TG2 (SHYTG2), and pancreatic cancer cells which express high levels of endogenous TG2. Resveratrol, a natural compound previously shown to inhibit neuroblastoma and pancreatic cancer in the animal models, was utilized to investigate the role of TG2 in cancer cell migration. Immunofluorescence assays were employed to detect expression and intracellular localization of TG2, and calcium levels in the migrating cells. Native gel electrophoresis was performed to analyze resveratrol-induced cellular distribution and conformational states of TG2 in migrating cells. Data are presented as the mean and standard deviation of at least 3 independent experiments. Comparisons were made among groups using one-way ANOVA followed by Tukey-Kramer ad hoc test.ResultsTG2 containing cells (SHYTG2 and pancreatic cancer cells) exhibit increased cell migration and invasion in collagen-coated and matrigel-coated transwell plate assays, respectively. Resveratrol (1 μM-10 μM) prevented migration of TG2-expressing cells. During the course of migration, resveratrol increased the immunoreactivity of TG2 without affecting the total TG2 protein level in migrating cells. In these cells, resveratrol increased calcium levels, and depletion of intracellular calcium by a calcium chelator, BAPTA, attenuated resveratrol-enhanced TG2 immunoreactivity. In native-polyacrylamide gels, we detected an additional TG2 protein band with slower migration in total cell lysates of resveratrol treated cells. This TG2 form is non-phosphorylated, exclusively present in plasma membrane fractions and sensitive to intracellular Ca2+ concentration suggesting a calcium requirement in TG2-regulated cell migration.ConclusionsTaken together, we conclude that resveratrol induces conformational changes in TG2, and that Ca2+-mediated TG2 association with the plasma membrane is responsible for the inhibitory effects of resveratrol on cell migration.

Increased platelet‐activating factor accumulation in the endothelium in response to cigarette smoke may contribute to breast cancer metastasis (405.5)

Cancer deaths are more often caused by distant metastases than by growth of the primary tumor. We have previously demonstrated that endothelial cell platelet‐activating factor (PAF) production results in increased adherence of breast cancer cells to the endothelium and may play a role in cancer cell migration to locations distant to the primary tumor. In this study, we used cigarette smoke extract (CSE) to inhibit endothelial cell PAF‐acetylhydrolase (PAF‐AH, hydrolyzes and inactivates PAF) activity and determined whether this results in increased endothelial cell PAF production and breast cancer cell adherence. Incubation of human lung microvascular endothelial cells (HMVEC‐L) with CSE (20 µg/ml) for up to 24 hours resulted in a significant inhibition of PAF‐AH activity that was accompanied by increased PAF production and adherence of highly invasive MDA‐MB‐231 breast cancer cells. Pretreatment of HMVEC‐L with (S)‐bromoenol lactone to inhibit calcium‐independent phospholipase A2β (iPLA2β, initiates endothelial cell PAF production) prior to CSE exposure resulted in complete inhibition of MDA‐MB‐231 cells. Likewise, pretreatment of MDA‐MB‐231 cells with the PAF receptor antagonist Ginkgo biloba resulted in inhibition of adherence to the endothelium. Western blot analysis revealed increased PAF receptor expression in MDA‐MB‐231 cells with CSE exposure. Histological samples of human breast carcinoma show that PAF receptor expression is increased in tumor regions when compared to the surrounding epithelium. Taken together, our data indicate that CSE exposure increases endothelial cell PAF production resulting in enhanced adherence of breast tumor cells. Our in vitro data indicate that increased tumor cell adherence would lead to enhanced metastasis formation in smokers. Potential therapeutic targets include endothelial cell iPLA2β or the tumor cell PAF receptor.

Receptor-interacting protein kinase 2 promotes triple-negative breast cancer cell migration and invasion via activation of nuclear factor-kappaB and c-Jun N-terminal kinase pathways

IntroductionMetastasis is the main cause of breast cancer morbidity and mortality. Processes that allow for tumor cell migration and invasion are important therapeutic targets. Here we demonstrate that receptor-interacting protein kinase 2 (RIP2), a kinase known to be involved in inflammatory processes, also has novel roles in cancer cell migration and invasion.MethodsA total of six breast cancer expression databases, including The Cancer Genome Atlas, were assessed for RIP2 expression among various clinical subtypes and its role as a prognostic biomarker. mRNA fluorescence in situ hybridization (FISH) for RIP2 was performed on 17 stage III breast cancers to determine if there was a correlation between RIP2 expression and lymph node involvement. RNA-interference was used to knock-down RIP2 expression in MDA-MB-231, Htb126, SUM149PT, MCF7, T47D, and HCC1428 cells. Cell migration and invasion were measured in vitro by scratch/wound healing and transwell migration assays. A xenograft mouse model was used to assess tumor growth and chemosensitivity to docetaxel in vivo in MDA-MB-231 cells with and without RIP2 small hairpin RNA knockdown. Western blot and immunofluorescence imaging were used to evaluate protein expressions.ResultsInterrogation of expression databases showed that RIP2 expression is significantly over-expressed in triple-negative breast cancers (TNBC: estrogen-receptor (ER) negative, progesterone-receptor (PR) negative, Her2/neu- (Her2) negative), compared to other clinical subtypes. High RIP2 expression correlates with worse progression-free survival using a combined breast cancer expression array dataset consisting of 946 patients. Multivariate analysis shows RIP2 as an independent prognostic biomarker. Knock-down of RIP2 significantly decreases migration in both scratch/wound healing and transwell migration assays in MDA-MB-231, Htb126, SUM149PT, MCF7, and T47D cells and is correlated with decreased Nuclear Factor-kappaB and c-Jun N-terminal kinase (JNK) activation. Finally, RIP2 knock-down leads to increased sensitivity to docetaxel and decreased tumor mass and lung metastases in a xenograft mouse model.ConclusionThese results highlight RIP2 as a pro-metastasis kinase in patients with advanced breast cancer. These results also illustrate a novel role for this kinase in addition to its known role in inflammation, and suggest that targeting RIP2 may improve outcomes in advanced breast cancer patients, in which it is overexpressed.

Capsosiphon fulvescens glycoprotein reduces AGS gastric cancer cell migration by downregulating transforming growth factor-β1 and integrin expression

Seaweeds are commonly used as functional foods and drugs. A glycoprotein (GP) from the green alga Capsosiphonfulvescens (Cf) has been reported to have antitumor activity toward various cancer cells. We previously observed that Cf-GP induced different pathways of apoptosis in AGS human gastric cancer cells. Transforming growth factor (TGF)-β1 plays an important role in cancer cell migration. Increased TGF-β1 levels increase the expression of the small GTPases and activate the FAK/PI3K/AKT pathways, resulting in the upregulation of integrin receptor proteins, which mediate the attachment of cells to surrounding tissues, cells or extracellular matrix. Thus, the inhibition of TGF-β1 signaling would downregulate integrin expression and thereby effectively decrease cell growth and migration. In the present study, we determined the effect of Cf-GP treatment on the proliferation, migration and apoptosis of AGS human gastric cancer cells. To investigate the mechanism by which Cf-GP exerts its anticancer actions, we examined the effect of Cf-GP on the expression levels of TGF-β1, FAK, PI3K, AKT, the small GTPases and integrins in AGS cells. Our findings indicate that Cf-GP inhibits AGS cell proliferation and migration by downregulating integrin expression via the TGF-β1-activated FAK/PI3K/AKT pathways. These results suggest that Cf-GP may be an important factor in the development of functional foods and therapeutic agents.

Silencing the YB‐1 Gene Inhibits Cell Migration in Gastric Cancer In Vitro

The Y-Box-Binding Protein-1 (YB-1) is known to regulate the processes of transcription, translation, cellular response to drug treatment and viral infection as well as DNA repair among others. As gastric cancer is a common cancer with a high incidence in countries in Asia, we evaluated the association of YB-1 with the malignant potential of gastric cancer cells in vitro. YB-1 mRNA expression levels were first determined by real-time RT-PCR in two adherent gastric cancer cell lines (viz., MKN7 and NUGC3 gastric cancer cells) and a normal GES-1 gastric epithelial cell line. Poorly differentiated NUGC3 gastric cancer cells were found to have the highest YB-1 gene expression among the adherent cells. YB-1 gene expression was also observed to be higher in non-adherent SNU5 gastric cancer cells compared to more aggressive SNU16 cells. Silencing of the YB-1 gene by siRNA in NUGC3 cells was associated with a significant reduction of the YB-1 protein by more than 55% as verified by Western blot analysis. Down-regulation of YB-1 protein expression was further demonstrated qualitatively by immunocytochemistry and immunofluorescence staining. Silencing of the YB-1 gene induced significant inhibition of cell migration in NUGC3 cells by 60% but did not influence cell invasion. Although epithelial-mesenchymal-transition (EMT) is known to be associated with the migratory phenotype in cancer cells, there was no change in the expression of EMT genes when YB-1 expression was modulated. YB-1 appears to have an integral role in cancer cell migration, a process which is important for gastric cancer metastasis.

Abstract 3845: Developing a novel 3D model to study colon cancer cell invasion.

Abstract Introduction: Colon cancer is the fifth most diagnosed in the UK, with nearly 22,000 cases diagnosed in 20101 with a five-year survival rate of 54.75%2. The formation of distant metastases also influences patient survival, causing a decline from 93% for the earliest stage tumours down to below 7% for tumours with distant metastases3. Previous literature has highlighted the limitations of two-dimensional (2D) cancer cell migration assays4. These include alterations in gene expression of cells and the provision of a migration substrate that does not closely reflect that of the in vivo environment. This project aims to mimic the in vivo situation to investigate factors that affect cancer cell invasion; including the expression of EMT markers and signalling by developing a three-dimensional (3D) model of colon cancer. Methods: This project utilises commercial scaffold known as Alvetex®5 and two colon cancer cell lines (SW480 and SW620) were used to look at the differences between pre- and post-metastatic colon cancer respectively. Both cell lines were cultured prior to analysis using various biochemical assays and histological processing. Results: Our data show a difference in cell penetration between the two cell lines. The later stage SW620 cells, which have a more motile phenotype in 2D, migrate and penetrate the scaffold to a greater extent than the earlier stage, less invasive, SW480 cells. This reflects a known difference in the level of EMT markers between the two cell lines, a result of the Wnt signalling pathway. This model also allows for the use of the known chemoattractant Insulin-like Growth Factor (IGF) to encourage cell penetration. IGF has been shown to play an important role in cancer cell migration. The use of IGF results in a 54% increase in cell penetration by the SW480 cell line, but not the SW620 cell line. Discussion: Our data shows great potential for further development of a 3D cancer invasion model using these scaffolds. This model provides a more realistic 3D environment and will be a tool for chemoattractant and co-culture studies to determine the mechanism of invasion utilised by these cells and to develop this model further to obtain an assay which is closer to the in vivo situation. Acknowledgements: This work was carried out with funding from the Grevillea Trust, Reinnervate Limited and BBSRC. Citation Format: Rosie Adams. Developing a novel 3D model to study colon cancer cell invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3845. doi:10.1158/1538-7445.AM2013-3845

RhoA, RhoB and RhoC have different roles in cancer cell migration

Rho GTPases are well known to regulate cell motility through activation of a variety of downstream effector proteins, including enzymes, adaptor proteins and actin nucleators. The three closely related Rho GTPases RhoA, RhoB and RhoC all have the potential to interact with the same downstream effectors, yet they have substantially different effects on cell shape and migratory properties. Here I review the different ways in which RhoA, RhoB and RhoC expression is regulated in cancer and how they play distinct roles in cancer progression. I describe their main effectors known to contribute to cell motility. Recent results from our laboratory and others indicate that RhoA, RhoB and RhoC can be activated by specific stimuli and act through different effectors to control distinct aspects of cancer cell migration and invasion. This suggests that they each make unique contributions to cancer by participating in different protein complexes.

Membrane potential and cancer progression

Membrane potential (Vm), the voltage across the plasma membrane, arises because of the presence of different ion channels/transporters with specific ion selectivity and permeability. Vm is a key biophysical signal in non-excitable cells, modulating important cellular activities, such as proliferation and differentiation. Therefore, the multiplicities of various ion channels/transporters expressed on different cells are finely tuned in order to regulate the Vm. It is well-established that cancer cells possess distinct bioelectrical properties. Notably, electrophysiological analyses in many cancer cell types have revealed a depolarized Vm that favors cell proliferation. Ion channels/transporters control cell volume and migration, and emerging data also suggest that the level of Vm has functional roles in cancer cell migration. In addition, hyperpolarization is necessary for stem cell differentiation. For example, both osteogenesis and adipogenesis are hindered in human mesenchymal stem cells (hMSCs) under depolarizing conditions. Therefore, in the context of cancer, membrane depolarization might be important for the emergence and maintenance of cancer stem cells (CSCs), giving rise to sustained tumor growth. This review aims to provide a broad understanding of the Vm as a bioelectrical signal in cancer cells by examining several key types of ion channels that contribute to its regulation. The mechanisms by which Vm regulates cancer cell proliferation, migration, and differentiation will be discussed. In the long term, Vm might be a valuable clinical marker for tumor detection with prognostic value, and could even be artificially modified in order to inhibit tumor growth and metastasis.

Hypoxia induces CXCR4 expression and biological activity in gastric cancer cells through activation of hypoxia-inducible factor-1α

Given the important role of CXCR4 in cancer metastasis, microenvironmental factors that modulate CXCR4 may have an impact on the process of tumor expansion. Hypoxia is a common feature of solid tumors and a significant microenvironmental factor that drives aggressive behavior. CXCR4 is upregulated in several cancer cells under hypoxic conditions, suggesting a relationship between tumor hypoxia and CXCR4. However, the role of hypoxia in regulating CXCR4 in gastric cancer remains poorly understood. KATO III gastric cancer cells were exposed to hypoxia or normoxia. CXCR4 expression in cells transfected with shRNA specific for HIF-1α was investigated by western blotting and flow cytometry. Wound healing, migration and invasion assays were used to assess cell motility and the chemotactic response to CXCL12, a major CXCR4 ligand. CXCR4 expression at the protein level and in the cell membrane was significantly increased in KATOIII cells following exposure to hypoxia. This upregulation of CXCR4 was implicated in increased cell motility and enhanced chemotactic responses (migration and invasion) to CXCL12 treatment in vitro. The increases in CXCR4 expression and metastatic potential in gastric cancer cells exposed to hypoxia were blocked by HIF-1α-specific shRNA. Our results indicate that hypoxia upregulates CXCR4 in gastric cancer cells in a HIF-1α-dependent manner, and that upregulation of CXCR4 plays a role in cancer cell migration and invasion. Thus, disrupting the hypoxia-HIF-1α-CXCR4 axis could be an attractive therapeutic strategy for the treatment of gastric cancer.

Enhanced membrane-type 1 matrix metalloproteinase expression by hyaluronan oligosaccharides in breast cancer cells facilitates CD44 cleavage and tumor cell migration

Hyaluronan (HA), a component of the extracellular matrix, plays an important role in cell-cell adhesion and cell migration. Membrane type 1-matrix metalloproteinase (MT1‑MMP) is often expressed in invasive cancer cells. CD44, a transmembrane receptor for HA, is implicated in various adhesion-dependent cellular processes including cell migration, tumor cell metastasis and invasion. Previous studies have shown that CD44 is highly expressed in cancer cells and may be proteolytically cleaved at the ectodomain by MT1-MMP; this process of inducing CD44 cleavage plays a critical role in cancer cell migration. We hypothesized that HA modulates MT1-MMP expression to facilitate breast cancer cell migration. Flow cytometry, real-time PCR, western blotting and immunofluorescence staining were used to quantify HA-induced MT1-MMP expression in breast cancer cells. In order to validate the relevance of cell migration and HA-induced MT1-MMP, we analyzed the cell migration via matrigel-coated transwell. We found that after HA oligosaccharide (6.5 kDA) stimulation, MT1-MMP expression in the membrane of breast cancer cells was increased. In response to HA oligosaccharide stimulation, significant upregulation of MT1-MMP mRNA occurred. Our data also provide evidence that HA oligosaccharide enhances MT1-MMP; the elevated expression of MT1-MMP confers enhanced CD44 cleavage and cell migration. In conclusion, we have identified a new function of HA in the induction of MT1-MMP expression in breast cancer cell lines and CD44 cleavage to increase cell migration during the invasion process. The HA oligosaccharide-induced MT1-MMP expression in breast cancer cells may be a critical step in the formation of metastatic colonies.

MLK3 Regulates Paxillin Phosphorylation in Chemokine-Mediated Breast Cancer Cell Migration and Invasion to Drive Metastasis

MLK3 kinase activates multiple mitogen-activated protein kinases and plays a critical role in cancer cell migration and invasion. In the tumor microenvironment, prometastatic factors drive breast cancer invasion and metastasis, but their associated signaling pathways are not well-known. Here, we provide evidence that MLK3 is required for chemokine (CXCL12)-induced invasion of basal breast cancer cells. We found that MLK3 induced robust phosphorylation of the focal adhesion scaffold paxillin on Ser 178 and Tyr 118, which was blocked by silencing or inhibition of MLK3-JNK. Silencing or inhibition of MLK3, inhibition of JNK, or expression of paxillin S178A all led to enhanced Rho activity, indicating that the MLK3-JNK-paxillin axis limits Rho activity to promote focal adhesion turnover and migration. Consistent with this, MLK3 silencing increased focal adhesions and stress fibers in breast cancer cells. MLK3 silencing also decreased the formation of breast cancer lung metastases in vivo, and breast cancer cells derived from mouse lung metastases showed enhanced Ser 178 paxillin phosphorylation. Taken together, our findings suggest that the MLK3-JNK-paxillin signaling axis may represent a potential therapeutic target and/or prognostic marker in breast cancer metastasis.

Restoration of caveolin‐1 expression suppresses growth, membrane‐type‐4 metalloproteinase expression and metastasis‐associated activities in colon cancer cells

Caveolin-1 (cav-1) and flotillin-1 are two major structural proteins associated with lipid rafts in mammalian cells. The membrane-type matrix metalloproteinases (MT-MMPs) are expressed at the cell surface, hydrolyze extracellular matrix, and play an important role in cancer cell migration and metastasis. Expression of cav-1, flotillin-1, and MT4-MMP in lysates and lipid rafts of LS174T and HM-7 colon cancer cells was determined. The impact of restoration of cav-1 expression on proliferation, adhesion, motility in vitro, and growth of implanted tumors in vivo was characterized. Cav-1 is not expressed in lipid rafts of the highly metastatic colon cancer cell line (HM-7), but expressed in cytosolic fractions of the parental lower metastatic cell line (LS174T). In contrast, MT4-MMP was expressed in lipid rafts of HM-7 cells but not in LS174T cells. Overexpression of cav-1 in HM-7 cells down-regulate proliferation, viability, wound closure, adhesion to laminin, invasion, and development of filopodial and lamellipodial structures in a dose-dependent manner. Cav-1 positive HM-7 clones ceased to express MT4-MMP in their lipid rafts. Comparative proteomic analyses of lipid rafts from cav-1 positive and cav-1 negative cells demonstrated de novo expression of flotillin-1 only on the cells expressing cav-1. Xenografting control cells devoid of cav-1 in nude mice induced development of bigger tumors expressing higher levels of proliferating cell nuclear antigen as compared to mice injected with cells expressing the highest cav-1 levels. We conclude that cav-1 orchestrates and reorganize several proteins in lipid rafts, activities directly associated with reduced tumorigenic and metastatic ability of colon cancer cells.