Regulate Tumor Cell Migration Research Articles

P3-03-02: Higher Expression Levels of Circulating miR-21, miR-19a and miR-10b Are Associated with High Risk Features in Breast Cancer.

Abstract Background: MicroRNAs (miRs) have oncogenic and tumor-suppressor functions. MiR-21, miR-19a and miR-10b are overexpressed in breast cancer and regulate tumor cell migration, invasion and angiogenesis. We assessed the levels of miR-21, -19a, and -10b in sera of breast cancer patients and their association with the stage, histological type, hormonal receptor (HR) status and HER2 amplification in the primary tumor. Since circulating tumor cells (CTC) detected by CellSearch® are an independent and strong predictor of overall survival in metastatic breast cancer (MBC), we assessed the relationship between circulating miRs and CTCs. Methods: The study consisted of 30 healthy donors (HD) and 95 breast cancer (BC) patients. Patients’ sera were collected before starting a new line of treatment. Total RNA was isolated, reverse transcribed to cDNA and then subjected to qRT-PCR for the detection of miR-21, -19a, -10b and -192 using the TaqMan MicroRNA Assay (Applied Biosystems, Foster City, CA). Mir-192 was used to normalize the expression levels of the other miRs. Fold-changes in expression of miRs were calculated using the 2−DCt method, where DCt= mean CTtarget-miRNA -mean CTmiR-192. CTCs were enumerated using CellSearch™ (Veridex LLC, Warren, NJ). Mann-Whitney U test was used to determine differences in serum miR expression levels between patients and HD. Results: Of the 95 BC patients, 39 were non-MBC and 56 MBC. Patients grouped according to the receptor expression by immunohistochemical staining consisted of 27 HR+HER2−, 30 HR+HER2+, 20 HRHER2+, and 18 HRHER2− triple negative BC (TNBC). MiR-21 and miR-19a were higher in non-MBC patients than in HD (p=.001, p<.001, respectively). MiR-21, miR-19a and miR-10b levels were higher in metastatic patients than in HD (p<.001, p<.001, p=.038, respectively). MBC patients had a higher median level of miR-21 than that of non-MBC patients (p=.02). Patients with (HR+HER2+, HR−HER2+, TNBC) had significantly higher median levels of both miR-21 (p=.018; p=.009; p=.045) and miR-10b (p=.011; p=.014; p=.03) compared with HR+HER2− BC. HER2+ patients had higher median levels of both miR-21 and miR-10b than those of HER2− BC (p=.033; p=.01) and HD (p<.001; p=.009). Further, median miR-19a expression was higher in IBC patients than in non-IBC patients (p=.025). Finally, patients with <5 CTCs had a higher median expression level of miR-10b than that of patients with ≥5 CTCs (p=.042). Discussion: High expression levels of miR-21, miR-19a and miR-10b in sera are observed in breast cancer patients, especially with advanced disease. HER2+ BC patients had higher serum levels of miR-21 and miR-10b than HER2−. IBC patients had a higher serum level of miR-19a than non-IBC patients. Moreover, patients with <5 CTCs had high serum levels of miR-10b that can be induced by Twist1 during the epithelial-mesenchymal transition (EMT) and, in part, explain the inability of CellSearch® to detect CTCs undergoing EMT. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-03-02.

Berberine-induced AMPK activation inhibits the metastatic potential of melanoma cells via reduction of ERK activity and COX-2 protein expression

Berberine is clinically important natural isoquinoline alkaloid that affects various biological functions, such as cell proliferation, migration and survival. The activation of AMP-activated protein kinase (AMPK) regulates tumor cell migration. However, the specific role of AMPK on the metastatic potential of cancer cells remains largely unknown. The present study investigated whether berberine induces AMPK activation and whether this induction directly affects mouse melanoma cell migration, adhesion and invasion. Berberine strongly increased AMPK phosphorylation via reactive oxygen species (ROS) production. 5-Aminoimidazole-4-carboxamide-1-β- d-ribofuranoside (AICAR), a well-known AMPK activator, also inhibited tumor cell adhesion and invasion and reduced the expression of epithelial to mesenchymal transition (EMT)-related genes. Knockdown of AMPKα subunits using siRNAs significantly abated the berberine-induced inhibition of tumor cell invasion. Furthermore, berberine inhibited the metastatic potential of melanoma cells through a decrease in ERK activity and protein levels of cyclooxygenase-2 (COX-2) by a berberine-induced AMPK activation. These data were confirmed using specific MEK inhibitor, PD98059, and a COX-2 inhibitor, celecoxib. Berberine- and AICAR-treated groups demonstrated significantly decreased lung metastases in the pulmonary metastasis model in vivo. Treatment with berberine also decreased the metastatic potential of A375 human melanoma cells. Collectively, our results suggest that berberine-induced AMPK activation inhibits the metastatic potential of tumor cells through a reduction in the activity of the ERK signaling pathway and COX-2 protein levels.

A novel functional site of extracellular matrix metalloproteinase inducer (EMMPRIN) that limits the migration of human uterine cervical carcinoma cells

EMMPRIN (extracellular matrix metalloproteinase inducer)/CD147, a membrane-bound glycoprotein with two extracellular loop domains (termed loops I and II), progresses tumor invasion and metastasis by increasing the production of matrix metalloproteinase (MMP) in peritumoral stoma cells. EMMPRIN has also been associated with the control of migration activity in some tumor cells, but little is known about how EMMPRIN regulates tumor cell migration. In the present study, EMMPRIN siRNA suppressed the gene expression and production of EMMPRIN in human uterine cervical carcinoma SKG-II cells. An in vitro scratch wound assay showed enhancement of migration of EMMPRIN-knockdown SKG-II cells. In addition, the SKG-II cell migration was augmented by adding an E. coli-expressed human EMMPRIN mutant with two extracellular loop domains (eEMP-I/II), which bound to the cell surface of SKG-II cells. However, eEMP-I/II suppressed the native EMMPRIN-mediated augmentation of proMMP-1/procollagenase-1 production in a co-culture of the SKG-II cells and human uterine cervical fibroblasts, indicating that the augmentation of SKG-II cell migration resulted from the interference of native EMMPRIN functions by eEMP-I/II on the cell surface. Furthermore, a systematic peptide screening method using nine synthetic EMMPRIN peptides coding the loop I and II domains (termed EM1-9) revealed that EM9 (170HIENLNMEADPGQYR184) facilitated SKG-II cell migration. Moreover, SKG-II cell migration was enhanced by administration of an antibody against EM9, but not EM1 which is a crucial site for the MMP inducible activity of EMMPRIN. Therefore, these results provide novel evidence that EMMPRIN on the cell surface limits the cell migration of human uterine cervical carcinoma cells through 170HIENLNMEADPGQYR184 in the loop II domain. Finally, these results should provide an increased understanding of the functions of EMMPRIN in malignant cervical carcinoma cells, and could contribute to the development of clinical strategies for cervical cancer therapy.

Metastasis-associated phosphatase PRL-2 regulates tumor cell migration and invasion

The Phosphatase of Regenerating Liver (PRL) family, comprising PRL-1, PRL-2, and PRL-3, is a group of prenylated phosphatases, which are candidate cancer biomarkers and therapeutic targets. Although several studies have documented that altered expression of PRL-1 or PRL-3 can influence cell proliferation, migration and invasion, there is a dearth of knowledge about the biological functions of PRL-2. Thus, in the current study we have evaluated the role of PRL-2 in cell migration and invasion in human cancer cells. We found four human lung cancer cells, including A549 cells, over-express PRL-2 when compared with normal lung cells. PRL-2 knockdown by RNA interference markedly inhibited cell migration and invasion and this inhibition can be restored by over-expressing the siRNA resistant vector HA-PRL-2m. PRL-2 suppression by siRNA decreased p130Cas and vinculin expression, and decreased ERK phosphorylation, while increasing the phosphorylation of ezrin on tyrosine 146. We found no significant changes in total p53, Akt and c-Src expression levels or their phosphorylation status, suggesting PRL-2 knockdown could inhibit tumor cell migration and invasion through a Src-independent p130Cas signaling pathway. Ectopic expression of wild type PRL-2, a catalytic inactive C101S mutant and a C-terminal CAAX deletion revealed a requirement for both the PRL-2 catalytic functionality and prenylation site. Expression of wild type but not mutant forms of PRL-2 caused ERK phosphorylation and nuclear translocation. These results support a model in which PRL-2 promotes cell migration and invasion through an ERK-dependent signaling pathway.

Abstract 126: Characterization of microRNA-10b in human glioblastoma

Abstract Glioblastoma is the most common and deadly malignant brain tumor. One main reason of resistance of glioblastoma to therapy is the ability of the tumor cells to migrate and invade the surrounding brain, rendering total surgical removal of the tumor practically impossible. MicroRNAs are recently discovered small noncoding regulatory RNA molecules with emerging critical roles in cancer. MicroRNAs modulate protein expression by binding to the 3′-untranslated region of mRNA and promoting RNA degradation or inhibiting transcription. By acting on oncogenes and tumor suppressors, microRNAs can behave as tumor suppressors or oncogenes, respectively. In this study, we show that microRNA-10b (miR-10b) is deregulated in glioblastoma where it regulates tumor cell migration and invasion. While analyzing The Cancer Genome Atlas (TCGA) data, we noticed that miR-10b is associated with poor prognosis of glioblastoma patients. We therefore embarked on a comprehensive characterization of miR-10b in glioblastoma. We first assessed the expression of miR-10b in human glioblastoma cells and cancer stem cells using quantitative RT-PCR. We found that miR-10b expression is up-regulated in all human glioblastoma cell lines and glioblastoma stem cells tested as compared to normal human astrocytes. We then tested the effects of miR-10b inhibition on cell cycle status, cell proliferation, cell invasion, cell migration, clonogenicity and apoptosis by propidium iodide flow cytometry, cell counting, transwell invasion assay, scratch assay, soft agar assay and annexin V flow cytometry, respectively. Glioblastoma cells, stem cells or normal human astrocytes were transfected with a specific anti-miR-10b antagomir and assessed for the above functional endpoints. Inhibition of miR-10b had variable effects on cell proliferation. It only mildly decreased the proliferation of established glioblastoma cell lines but exhibited greater inhibition on the proliferation of glioblastoma stem cells. Inhibition of miR-10b did not affect apoptosis or death of any of the tested cells. Importantly, inhibition of miR-10b strongly inhibited cell invasion, cell migration and anchorage-independent growth in glioblastoma cells and stem cells. Conversely, overexpression of miR-10b by transfection of pre-miR-10b in glioblastoma cells promoted tumor cell invasion and migration in vitro. We are currently in the process of researching potential targets of miR-10b that mediate its effects on glioblastoma cell migration and invasion. We are also studying the potential correlation between the levels of miR-10b in human glioblastoma tissues and tumor grade. Altogether, our findings suggest that miR-10b is a tumor promoter that is upregulated in glioblastoma where it induces tumor cell migration and invasion. These preliminary results suggest that targeting miR-10b could be a promising new strategy for glioblastoma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 126. doi:10.1158/1538-7445.AM2011-126

MicroRNA-151 and Its Hosting Gene FAK (Focal Adhesion Kinase) Regulate Tumor Cell Migration and Spreading of Hepatocellular Carcinoma†

Recurrent chromosomal aberrations are often observed in hepatocellular carcinoma (HCC), but little is known about the functional non-coding sequences, particularly microRNAs (miRNAs), at the chromosomal breakpoints in HCC. Here we show that 22 miRNAs are often amplified or deleted in HCC. MicroRNA-151 (miR-151), a frequently amplified miRNA on 8q24.3, is correlated with intrahepatic metastasis of HCC. We further show that miR-151, which is often expressed together with its host gene FAK, encoding focal adhesion kinase, significantly increases HCC cell migration and invasion in vitro and in vivo, mainly through miR-151-5p, but not through miR-151-3p. Moreover, miR-151 exerts this function by directly targeting RhoGDIA, a putative metastasis suppressor in HCC, thus leading to the activation of Rac1, Cdc42 and Rho GTPases. In addition, miR-151 can function synergistically with FAK to enhance HCC cell motility and spreading. Thus, our findings indicate that chromosome gain of miR-151 is a crucial stimulus for tumour invasion and metastasis of HCC.

Tetraspanin CD151 Regulates Growth of Mammary Epithelial Cells in Three-Dimensional Extracellular Matrix: Implication for Mammary Ductal CarcinomaIn situ

Tetraspanin CD151 is associated with laminin-binding integrins (i.e., alpha(3)beta(1), alpha(6)beta(1), and alpha(6)beta(4)) and regulates tumor cell migration and invasion. Here, we examined the role of CD151 in proliferation of mammary epithelial cells using in vitro and in vivo models. Depletion of CD151 suppressed growth of HB2 cells, a nontumorigenic breast epithelial cell line, in three-dimensional (3D) extracellular matrices (ECM) and in Matrigel-based xenografts. Whereas the presence of alpha(3)beta(1) (but not alpha(6) integrins) was necessary to support growth of HB2 cells in 3D ECM, the pro-proliferative activity of CD151 did not require direct interaction with integrins. Furthermore, depletion of CD151 potentiated formation of the internal lumen and partial restoration of polarity when HB2 cells were cultured in 3D ECM. This correlated with a decrease in phosphorylation levels of extracellular signal-regulated kinase 1/2 and cAkt in CD151-negative cells and increase in activation of caspase-3. Accordingly, the number of CD151-positive colonies with internal lumen was increased by approximately 5-fold when cells were cultured in the presence of MAP/ERK kinase (U0126) and phosphoinositide 3-kinase (LY29004) inhibitors. To establish the physiologic relevance of pro-proliferative and morphogenetic activities of CD151, we analyzed the expression of this tetraspanin in ductal carcinoma in situ (DCIS), which is characterized by neoplastic proliferation of mammary epithelial cells. Strong homogeneous membrane expression of CD151 was found to be associated with a high grade of DCIS (P = 0.004). Taken together, these results strongly suggest that CD151 complexes play a crucial role in the development of hyperproliferative diseases in the mammary gland.

Protein C inhibitor regulates both cathepsin L activity and cell-mediated tumor cell migration

Background Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation including thrombin and activated protein C. However, the physiological role of PCI remains under investigation. The cysteine protease, cathepsin L, has a role in many physiological processes including cardiovascular diseases, blood vessel remodeling, and cancer. Methods and results We found that PCI inhibits cathepsin L with an inhibition rate (k 2) of 3.0 × 10 5 M − 1 s − 1. Whereas, the PCI P1 mutant (R354A) inhibits cathepsin L at rates similar to wild-type PCI, mutating the P2 residue results in a slight decrease in the rate of inhibition. We then assessed the effect of PCI and cathepsin L on the migration of human breast cancer (MDA-MB-231) cells. Cathepsin L was expressed in both the cell lysates and conditioned media of MDA-MB-231 cells. Wound-induced and transwell migration of MDA-MB-231 cells was inhibited by exogenously administered wtPCI and PCI P1 but not PCI P14 mutant. In addition, migration of MDA-MB-231 cells expressing wtPCI was significantly decreased compared to non-expressing MDA-MB-231 cells or MDA-MB-231 cells expressing the PCI P14 mutant. Downregulation of cathepsin L by either a specific cathepsin L inhibitor or siRNA technology also resulted in a decrease in the migration of MDA-MB-231 cells. Conclusions Overall, our data show that PCI regulates tumor cell migration partly by inhibiting cathepsin L. General significance Consequently, inhibiting cathepsin L by serpins like PCI may be a new pathway of regulating hemostasis, cardiovascular and metastatic diseases.

Locking Src/Abl Tyrosine Kinase Activities Regulate Cell Differentiation and Invasion of Human Cervical Cancer Cells Expressing E6/E7 Oncoproteins of High-Risk HPV

In this study, we compared the effects of SKI-606 with Iressa, Src/Abl and EGF-R kinase inhibitors, respectively, on selected parameters in HeLa and SiHa cervical cancer cell lines, which express E6/E7 oncoproteins of high-risk HPV types 18 and 16, respectively. Our results show that SKI-606 and Iressa inhibit cell proliferation and provoke G0-G1 cell cycle arrest and reduction of S and G2-M phase using 2 and 5 μM concentrations of these inhibitors. In contrast, SKI-606 induces differentiation to an epithelial phenotype “mesenchymal-epithelial transition”; thus SKI-606 causes a dramatic decrease in cell motility and invasion abilities of HeLa and SiHa cancer cells, in comparison to untreated cells and Iressa-treated cells in which these parameters are only slightly affected. These changes are accompanied by a regulation of the expression patterns of E-cadherin and catenins. The molecular pathway analysis of Src/Abl inhibitor revealed that SKI-606 blocks the phosphorylation of β-catenin and consequently converts its role from a transcriptional regulator to a cell-cell adhesion molecule. Our findings indicate that SKI-606 inhibits signaling pathways involved in regulating tumor cell migration and invasion genes via β-catenin alteration, suggesting that Src inhibitor, in comparison to EGF-R, is a promising therapeutic agent for human cervical cancer.

Fibroblast hepatocyte growth factor promotes invasion of human mammary ductal carcinoma in situ.

Stromal-derived hepatocyte growth factor (HGF) acting through its specific proto-oncogene receptor c-Met has been suggested to play a paracrine role in the regulation of tumor cell migration and invasion. The transition from preinvasive ductal carcinoma in situ (DCIS) to invasive breast carcinoma is marked by infiltration of stromal fibroblasts and the loss of basement membrane. We hypothesized that HGF produced by the infiltrating fibroblasts may alter proteolytic pathways in DCIS cells, and, to study this hypothesis, established three-dimensional reconstituted basement membrane overlay cocultures with two human DCIS cell lines, MCF10.DCIS and SUM102. Both cell lines formed large dysplastic structures in three-dimensional cultures that resembled DCIS in vivo and occasionally developed invasive outgrowths. In coculture with HGF-secreting mammary fibroblasts, the percentage of DCIS structures with invasive outgrowths was increased. Activation of c-Met with conditioned medium from HGF-secreting fibroblasts or with recombinant HGF increased the percentage of DCIS structures with invasive outgrowths, their degradation of collagen IV, and their secretion of urokinase-type plasminogen activator and its receptor. In agreement with the in vitro findings, coinjection with HGF-secreting fibroblasts increased invasiveness of MCF10.DCIS xenografts in severe combined immunodeficient mice. Our study shows that paracrine HGF/c-Met signaling between fibroblasts and preinvasive DCIS cells enhances the transition to invasive carcinomas and suggests that three-dimensional cocultures are appropriate models for testing therapeutics that target tumor microenvironment-enhanced invasiveness.

The planar cell polarity protein Van Gogh-Like 2 regulates tumor cell migration and matrix metalloproteinase-dependent invasion

Van Gogh-Like 2 (VANGL2) is a planar cell polarity protein essential for collective migration during embryonic development, yet its contribution to tumor cell motility and invasion are unknown. We report for the first time that loss of VANGL2 in human cancer cells promotes efficient collective and directed migration and matrix metalloproteinase (MMP)-dependent ECM invasion. We show that VANGL2 knockdown cells exhibit increased activation of secreted MMP2, higher levels of membrane-localized MMP14, and decreased cell-surface fibronectin. These important findings support the notion that planar cell polarity proteins act in coordination with known regulators of cancer cell migration to influence invasion and perhaps metastasis.

The Immunoregulatory Protein Human B7H3 is a Tumor-Associated Antigen that Regulates Tumor Cell Migration and Invasion

The monoclonal antibody (mAb) 376.96 has been used for detection of micrometastatic tumor cells due to its high binding specificity for a wide range of tumor cells, but the identity and function of its target antigen have not been known. Here, using immunoprecipitation and siRNA technology, we demonstrate that the antigen is the human 4Ig-B7H3 (4Ig-hB7H3) protein, previously known as an immunoregulatory protein in immune cells. Immunoblots of whole cell lysates, subcellular fractionation and tunicamycin treatment of human tumor cells indicated that 4Ig-hB7H3 is a approximately 100-kDa N-linked glycosylated membrane protein. The tumor promoter phorbol 12-myristate 13-acetate (PMA) enhanced the expression of 4Ig-hB7H3 in FEMX-I (melanoma), MA11 (breast cancer), and OHS (osteosarcoma) cells, suggesting that 4Ig-hB7H3 may be implicated in tumorigenesis. Most importantly, siRNA-downregulation of hB7H3 reduced cell adhesion to fibronectin of melanoma and breast cancer cells by up to 50 %, and migration and matrigel-invasion by more than 70 %, but surprisingly had no apparent impact on cell proliferation. In conclusion, our data present 4Ig-hB7H3 as a tumor-associated antigen and suggests a novel biological role of 4Ig-hB7H3 in tumor progression and metastasis.

Migration of renal carcinoma cells is dependent on protein kinase Cδ via β1 integrin and focal adhesion kinase

Migration and adhesion of tumor cells are essential prerequisites for the formation of metastases in malignant diseases. Protein kinase C (PKC) has been shown to regulate cell migration, adhesion and proliferation. In order to identify a connection between PKC isoforms and tumor progression in renal cell carcinoma (RCC), the influence of PKC isoforms on cell migration, adhesion and proliferation and possible influences of the activity of integrins and focal adhesion kinase (FAK) were analyzed in RCC cells. The experiments were performed in the RCC cell line CCF-RC1 after pre-incubation of the cells with the PKC inhibitors GF109203X, GO6976, RO31-8220 and rottlerin. Cell migration and adhesion were assessed through chemotaxis analysis and adhesion to an endothelial monolayer, respectively. Cell proliferation was analysed by a BrdU incorporation assay. The expression and activity of beta1 integrins and FAK were analysed by Western blot analysis. GF109203X reduced cell migration to 69%, the activity of beta1 integrins to 63% and FAK expression to 82% compared to untreated cells. Rottlerin reduced cell migration in a concentration-dependent manner to 36%, cell proliferation to 81%, expression and activity of beta1 integrins to 72 and 79%, and expression and activity of FAK to 56 and 76% of untreated cells, respectively. RO31-8220 also reduced the expression and activity of beta1 integrins as well as the expression of FAK to 84, 66 and 66% of untreated cells, respectively. GO6976 reduced the expression of FAK to 60% of untreated cells. Cell migration was only slightly reduced by GO6976 to 84% of untreated cells, and cell adhesion remained uninfluenced. These findings show a critical role of PKCdelta in the regulation of tumor cell migration, which seems to be caused by affecting the expression and activity of beta1 integrins and FAK. These results can provide a basis for new strategies in preventing metastases of renal cell carcinoma.

Suppression of ClC-3 channel expression reduces migration of nasopharyngeal carcinoma cells

Recent studies suggest that chloride (Cl −) channels regulate tumor cell migration. In this report, we have used antisense oligonucleotides specific for ClC-3, the most likely molecular candidate for the volume-activated Cl − channel, to investigate the role of ClC-3 in the migration of nasopharyngeal carcinoma cells (CNE-2Z) in vitro. We found that suppression of ClC-3 expression inhibited the migration of CNE-2Z cells in a concentration-dependent manner. Whole-cell patch-clamp recordings and image analysis further demonstrated that ClC-3 suppression inhibited the volume-activated Cl − current ( I Cl,vol) and regulatory volume decrease (RVD) of CNE-2Z cells. The expression of ClC-3 positively correlated with cell migration, I Cl,vol and RVD. These results strongly suggest that ClC-3 is a component or regulator of the volume-activated Cl − channel. ClC-3 may regulate CNE-2Z cell migration by modulating cell volume. ClC-3 may be a new target for cancer therapies.

Melanoma-Associated Antigen Family Protein-D1 Regulation of Tumor Cell Migration, Adhesion to Endothelium, and Actin Structures Reorganization in Response to Hypoxic Stress

Melanoma-associated antigen family protein-D1 (MAGE-D1) is a recently identified p75 neurotrophin receptor intracellular binding protein and functions as an adaptor that mediates multiple signaling pathways, including Dlx/Msx-mediated transcription. Here, a new regulatory function for MAGE-D1 in tumor cell motility and adhesion to endothelium is described. MAGE-D1 over-expression suppressed HeLa cell and BEL7402 cell migration, invasion, and adhesion to the monolayer of ECV304 cells. We also report that MAGE-D1 over-expression disrupted actin cytoskeleton rearrangement induced by hypoxia and down-regulated hypoxia inducible factor 1-dependent luciferase gene expression. These findings provide new insight into the ability of MAGE-D1 to suppress the motility and adhesion response of tumor cells by interfering with actin cytoskeleton reorganization and hypoxia inducible factor 1-dependent gene expression.

Chapter 17 - MscCa Regulation of Tumor Cell Migration and Metastasis

The acquisition of cell motility is a required step in order for a cancer cell to migrate from the primary tumor and spread to secondary sites (metastasize). For this reason, blocking tumor cell migration is considered a promising approach for preventing the spread of cancer. However, cancer cells just as normal cells can migrate by several different modes referred to as "amoeboid," "mesenchymal," and "collective cell." Under appropriate conditions, a single cell can switch between modes. A consequence of this plasticity is that a tumor cell may be able to avoid the effects of an agent that targets only one mode by switching modes. Therefore, a preferred strategy would be to target mechanisms that are shared by all modes. This chapter reviews the evidence that Ca(2+) influx via the mechanosensitive Ca(2+)-permeable channel (MscCa) is a critical regulator of all modes of cell migration and therefore represents a very good therapeutic target to block metastasis.

The The Role of Thrombomodulin in Prostate Tumor Cell Migration

The Role of Thrombomodulin in Prostate Tumor Cell Migration Thrombomodulin (TM) is an endothelial transmembrane receptor that regulates coagulation by binding to thrombin and activating the protein C system. TM is also expressed by prostate cancer (CaP) cells. CaP associated TM activates the protein C system. We investigated if TM expressed by CaP cells may also be involved in tumor progression by determining its ability to regulate tumor cell migration. PC-3 and DU-145 CaP cell lines were incubated in a Boyden chamber in the presence and absence of monoclonal antibodies to each extracellular domain of TM and amount of cell migration determined. The extracellular domains of TM include an N-terminal lectin-like domain, six epidermal growth factor-like (EGF) domains, and a serine-threonine rich domain, which is differentially glycosylated with a chondroiten sulphate moiety. We determined that monoclonal antibodies to the chondroiten sulphate domain increased the ability of both PC-3 and DU-145 cell lines to migrate. We also investigated if CaP cell invasion was regulated by thrombin, protein C, and activated protein C. We determined that PC-3 and DU-145 cell migration was not regulated by these TM-associated proteins. We conclude that the chondroiten sulphate domain of CaP cell TM regulates cell migration. Funding Support: South Carolina NIH INBRE grant, Freedland Foundation, McKay Urology Endowment Fund

Wnt 5a signaling is critical for macrophage-induced invasion of breast cancer cell lines

Interactions between neoplastic and stromal cells contribute to tumor progression. Wnt genes, involved in cell migration and often deregulated in cancers, are attractive candidates to regulate these effects. We have recently shown that coculture of breast cancer cells with macrophages enhances invasiveness via matrix metalloproteases and TNF-alpha. Here we demonstrate that coculture of MCF-7 cells and macrophages leads to up-regulation of Wnt 5a in the latter. This was accompanied by activation of AP-1/c-Jun in MCF-7. Recombinant Wnt 5a mimicked the coculture effect. Wnt 5a was also detectable in tumor-associated macrophages in primary breast cancers. Experiments with agonists and antagonists of Wnt signaling revealed that a functional canonical pathway in the tumor cells was a necessary prerequisite; however, noncanonical signaling via Wnt 5a and the Jun-N-terminal kinase pathway was critical for invasiveness. It was also responsible for induction of matrix metalloprotease-7, known to release TNF-alpha. All these effects could be antagonized by dickkopf-1. Our results indicate that Wnt 5a is essential for macrophage-induced invasiveness, because it regulates tumor cell migration as well as proteolytic activity of the macrophages. The function of Wnt 5a as either a suppressor or promoter of malignant progression seems to be modulated by intercellular interactions. Wnt 5a detection in tumor-associated macrophages in breast cancer biopsies supports the assumption that similar events play a role in vivo.

A Critical Role for Tetraspanin CD151 in α3β1 and α6β4 Integrin–dependent Tumor Cell Functions on Laminin-5

The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.

The Cytoplasmic Domain of Tissue Factor Inhibits Migration and Enhances Adhesion of Human Breast Cancer Cells.

Tissue factor (TF) is a 47 kDa transmembrane glycoprotein that when complexed with its cofactor, factor VIIa (FVIIa), initiates blood coagulation. Apart from hemostasis, TF has been shown to have roles in cellular signaling, development, inflammation, metastasis and angiogenesis. We showed previously that both the cytoplasmic and extracellular domains of TF are required for the full metastatic effect of TF. Recently, we showed that TF-FVIIa-FXa complex induces cellular signaling in human breast cancer cells and is associated with enhanced cell migration and prevention of apoptosis. However, the role of the cytoplasmic domain of TF in tumor cell function is not fully known. In the present study, the role of the cytoplasmic domain of TF in cell migration and adhesion was investigated using the Adr-MCF-7 cell line, a multidrug resistant subline of the human breast cancer cell line, MCF-7. The Adr-MCF-7 cell line has high endogenous expression of TF and expression of PAR1 and PAR2. Adr-MCF-7 cells were retrovirally transfected with either a cDNA construct encoding a FLAG epitope tag fused to the transmembrane and cytoplasmic domains of TF (known as FLAG-TFCD) or vector (LXSN) alone as a control, and stable, polyclonal cell lines selected using G418. Expression of the FLAG-TFCD construct was verified by RT-PCR, Western blot analysis and flow cytometry. To test the effect of overexpression of the FLAG-TFCD construct on cell motility a modified Boyden chamber chemotaxis assay was used. The control LXSN cell line had a nearly 9 fold increase in cell migration [33.5± 3.2 cells/hpf (mean± SEM)]using the combination of rFVIIa (10 nM) and FX (150 nM) as the chemoattractant compared with 0.1% bovine serum albumin (BSA) [3.9± 1.2 cells/hpf]. In contrast, the FLAG-TFCD cell line had no increase in migration of using the combination of rFVIIa and FX [6.6± 0.57 cells/hpf] compared with BSA [5.4± 0.67 cells/hpf]. We then examined the ability of the transfected cell lines to adhere to type IV collagen. The number of adherent cells for the transfected cell line, FLAG-TFCD, was nearly 3 fold higher than that for the LXSN line using a colorimetric MTS assay (0.295± 0.041 vs 0.119± 0.011). Moreover, treatment of the FLAG-TFCD cells with the combination of rFVIIa and FX increased the adhesion by nearly 2 fold compared with untreated FLAG-TFCD cells (0.561±0.055 vs 0.296±0.044). In summary, overexpression of TF cytoplasmic domain leads to inhibition of tumor cell migration and enhancement of cell adhesion and potentially acts as a dominant negative in these cellular processes. These data suggest that a function of the cytoplasmic domain of TF in metastasis is to regulate tumor cell migration and adhesion.