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Glycoprotein nmb (GPNMB) promotes breast tumor growth and metastasis and its expression in tumor epithelium correlates with poor prognosis in breast cancer patients. Despite its biological and clinical significance, little is known regarding the molecular mechanisms engaged by GPNMB. Herein, we show that GPNMB engages distinct functional domains and mechanisms to promote primary tumor growth and metastasis. We demonstrate that neuropilin-1 (NRP-1) expression is increased in breast cancer cells that overexpress GPNMB. Interestingly, the GPNMB-driven increase in NRP-1 expression potentiated vascular endothelial growth factor signaling in breast cancer cells and was required for the growth, but not metastasis, of these cells in vivo. Interrogation of RNAseq data sets revealed a positive correlation between GPNMB and NRP-1 levels in human breast tumors. Furthermore, we ascribe pro-growth and pro-metastatic functions of GPNMB to its ability to bind α5β1 integrin and increase downstream signaling in breast cancer cells. We show that GPNMB enhances breast cancer cell adhesion to fibronectin, increases α5β1 expression and associates with this receptor through its RGD motif. GPNMB recruitment into integrin complexes activates Src and Fak signaling pathways in an RGD-dependent manner. Importantly, both the RGD motif and cytoplasmic tail of GPNMB are required to promote primary mammary tumor growth; however, only mutation of the RGD motif impaired the formation of lung metastases. Together, these findings identify novel and distinct molecular mediators of GPNMB-induced breast cancer growth and metastasis. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV)-encoded Latent Membrane Protein 2A (LMP2A) is an EBV latency-associated protein regularly expressed in nasopharyngeal carcinoma (NPC). In B cells, LMP2A activity resembles that of a constitutively activated antigen receptor, which recruits the Syk tyrosine kinase to activate a set of downstream signaling pathways. LMP2A also downregulates cellular Syk levels. In the present study, we demonstrate that Syk interacts with the integrin β4 subunit (ITGβ4) of integrin α6β4 in epithelial cells and that concurrent LMP2A expression interferes with this interaction by competitive binding to Syk. We find that both Syk and LMP2A have an effect on ITGβ4 cell surface expression. However, in LMP2A expressing cells, ITGβ4 remains concentrated at the cellular protrusions, an expression pattern characteristic of motile cells, including NPC-derived epithelial cells. This effect of LMP2A on ITGβ4 localization is associated with a greater propensity for migration and invasion in-vitro, and may contribute to the invasive property of LMP2A-expressing NPC. [ABSTRACT FROM AUTHOR]

Talins are adaptor proteins that regulate focal adhesion signaling by conjugating integrins to the cytoskeleton. Talins directly bind integrins and are essential for integrin activation. We previously showed that β1 integrins are activated in metastatic prostate cancer (PCa) cells, increasing PCa metastasis to lymph nodes and bone. However, how β1 integrins are activated in PCa cells is unknown. In this study, we identified a novel mechanism of β1 integrin activation. Using knockdown experiments, we first demonstrated that talin1, but not talin2, is important in β1 integrin activation. We next showed that talin1 S425 phosphorylation, but not total talin1 expression, correlates with metastatic potential of PCa cells. Expressing a non-phosphorylatable mutant, talin1S425A, in talin1-silenced PC3-MM2 and C4-2B4 PCa cells, decreased activation of β1 integrins, integrin-mediated adhesion, motility and increased the sensitivity of the cells to anoikis. In contrast, reexpression of the phosphorylation-mimicking mutant talin1S425D led to increased β1 integrin activation and generated biologic effects opposite to talin1S425A expression. In the highly metastatic PC3-MM2 cells, expression of a non-phosphorylatable mutant, talin1S425A, in talin1-silenced PC3-MM2 cells, abolished their ability to colonize in the bone following intracardiac injection, while reexpression of phosphorylation-mimicking mutant talin1S425D restored their ability to metastasize to bone. Immunohistochemical staining demonstrated that talin S425 phosphorylation is significantly increased in human bone metastases when compared with normal tissues, primary tumors or lymph node metastases. We further showed that p35 expression, an activator of Cdk5, and Cdk5 activity were increased in metastatic tumor cells, and that Cdk5 kinase activity is responsible for talin1 phosphorylation and subsequent β1 integrin activation. Together, our study reveals Cdk5-mediated phosphorylation of talin1 leading to β1 integrin activation is a novel mechanism that increases metastatic potential of PCa cells. [ABSTRACT FROM AUTHOR]

SIPA1 (signal-induced proliferation-associated protein 1) is a GTPase activation protein that can catalyze the hydrolysis of Rap1 bound GTP to GDP. Recently attention has been paid to a potential role for SIPA1 in cancer metastasis; however, the underlying mechanism of how changes in SIPA1 levels may lead to increased metastasis remains poorly understood. In this study, we showed that SIPA1 was mainly localized to the nuclei in highly invasive breast cancer tumor tissue and MDA-MB-231 cells. Knockdown of SIPA1 in MDA-MB-231 altered cell morphology and cell proliferation ability. Furthermore, this study is the first to establish that nuclear SIPA1 can interact with the integrin β1 promoter and activate its transcription; this interaction appears to be important for SIPA1-dependent MDA-MB-231 cell adhesion and invasion. We also demonstrated that the phosphorylation of FAK, Akt and the expression of MMP9, downstream signaling molecules of integrin β1, were decreased upon SIPA1 knockdown, and MDA-MB-231 cell invasion was impaired. Taken together, these results suggest nuclear SIPA1 contributes to breast cancer cell invasion through the regulation of integrin β1 signaling. [ABSTRACT FROM AUTHOR]

Intratumoral heterogeneity (ITH) represents an obstacle for cancer diagnosis and treatment, but little is known about its functional role in cancer progression. The A Desintegrin And Metalloproteinase 23 (ADAM23) gene is epigenetically silenced in different types of tumors, and silencing is often associated with advanced disease and metastasis. Here, we show that invasive breast tumors exhibit significant ADAM23-ITH and that this heterogeneity is critical for tumor growth and metastasis. We demonstrate that while loss of ADAM23 expression enhances invasion, it causes a severe proliferative deficiency and is not itself sufficient to trigger metastasis. Rather, we observed that, in ADAM23-heterotypic environments, ADAM23-negative cells promote tumor growth and metastasis by enhancing the proliferation and invasion of adjacent A23-positive cells through the production of LGI4 (Leucine-rich Glioma Inactivated 4) and nitric oxide (NO). Ablation of LGI4 and NO in A23-negative cells significantly attenuates A23-positive cell proliferation and invasion. Our work denotes a driving role of ADAM23-ITH during disease progression, shifting the malignant phenotype from the cellular to the tissue level. Our findings also provide insights for therapeutic intervention, enforcing the need to ascertain ITH to improve cancer diagnosis and therapy. [ABSTRACT FROM AUTHOR]

Fibroblasts in the tumour stroma (cancer-associated fibroblasts) influence tumour progression and response to therapeutics; little is known about the mechanisms through which the tumour cell co-opts a normal fibroblast. To study the activation of fibroblasts by tumour cells, a panel of non-small cell lung cancer (NSCLC) cell lines and normal human dermal fibroblasts were co-cultured. A subset of the NSCLC cells induced an activated cancer-associated fibroblast-like fibroblast phenotype defined by induction of fibroblast α-smooth muscle actin expression. Tumour cells that activated fibroblasts were associated with E-Cadherin and EpCAM expression and expression of integrin αvβ6. Co-culture of activating tumour cells with fibroblasts resulted in induction of transcripts associated with tumour cell invasion and growth, TGFβ1 and TGFBR1, SERPINE-1, BMP6, SPHK1 and MMP9. Fibroblast activation was inhibited by an αvβ6/8 integrin blocking antibody (264RAD) and a small molecule inhibitor of the TGF-beta type I receptor activin-like kinase (ALK5) (SB431542), demonstrating that transactivation of the TGFβ pathway initiates fibroblast activation. Both integrin and ALK5 antagonists inhibited initiation. Only ALK5 was effective when added after 3 days of co-culture. This suggests that although activation is αvβ6-dependent, once fibroblasts are activated alternative TGFβ pathway regulators maintain an activation loop. In co-culture activating cells had reduced sensitivity to selumetinib, AZD8931 and afatinib compared with mono-culture. In contrast, non-activating cells were insensitive to selumetinib and AZD8931 in both mono-culture and co-culture. In conclusion NSCLC cell lines, positive for E-Cadherin, EpCAM and αvβ6 expression, activate normal fibroblasts through avβ6/TGFβ signalling in vitro, and influence both gene expression and response to therapeutic agents. [ABSTRACT FROM AUTHOR]

Metastasis is the predominant cause of death in breast cancer patients. Several lines of evidence have shown that microRNAs (miRs) can have an important role in cancer metastasis. Using isogenic pairs of low and high metastatic lines derived from a human breast cancer line, we have identified miR-149 to be a suppressor of breast cancer cell invasion and metastasis. We also identified GIT1 (G-protein-coupled receptor kinase-interacting protein 1) as a direct target of miR-149. Knockdown of GIT1 reduced migration/invasion and metastasis of highly invasive cells. Re-expression of GIT1 significantly rescued miR-149-mediated inhibition of cell migration/invasion and metastasis. Expression of miR-149 impaired fibronectin-induced focal adhesion formation and reduced phosphorylation of focal adhesion kinase and paxillin, which could be restored by re-expression of GIT1. Inhibition of GIT1 led to enhanced protein degradation of paxillin and α5β1 integrin via proteasome and lysosome pathways, respectively. Moreover, we found that GIT1 depletion in metastatic breast cancer cells greatly reduced α5β1-integrin-mediated cell adhesion to fibronectin and collagen. Low level of miR-149 and high level of GIT1 was significantly associated with advanced stages of breast cancer, as well as with lymph node metastasis. We conclude that miR-149 suppresses breast cancer cell migration/invasion and metastasis by targeting GIT1, suggesting potential applications of the miR-149-GIT1 pathway in clinical diagnosis and therapeutics. [ABSTRACT FROM AUTHOR]

The constitutive activation of β-catenin signaling in the mammary basal epithelial cell layer in transgenic K5ΔNβcat mice leads to basal-type tumor development. Integrins of the β1 family and integrin-mediated signaling events have an important role in breast tumor growth and progression. We show here that the deletion of α3β1 integrin, a major laminin receptor, from the basal layer of the mammary epithelium of K5ΔNβcat mice completely prevented the tumorigenesis induced by β-catenin signaling. Moreover, the depletion of α3β1 integrin from a spontaneously transformed mouse mammary basal epithelial cell line (MEC) prevented the cells from forming colonies in soft agar and greatly reduced tumor development in orthotopic grafts. Inhibition of the integrin signaling intermediates Rac1 or PAK1 (P21-activated Kinase 1) in MEC affected tumor cell growth in soft agar, whereas the expression of activated forms of these effectors in α3-depleted cells rescued the capacity of these cells to grow in non-adherent conditions. Similarly, the tumorigenic potential of α3-depleted cells was restored by the expression of activated PAK1, as assessed by orthotopic transplantation assay. In three-dimensional Matrigel culture, MEC survival and proliferation were affected by the depletion of α3β1 integrin, which also significantly decreased the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK). Our data suggest that the activation of signaling cascades downstream from α3β1 and involving the Rac1/PAK1 pathway, MAPK and JNK, promotes prosurvival and proproliferative signals required for the malignant growth of basal mammary epithelial cells, providing further insight into the molecular mechanisms underlying breast cancer initiation and progression. [ABSTRACT FROM AUTHOR]

Integrin α3β1 regulates adhesive interactions of cells with laminins and have a critical role in adhesion-dependent cellular responses. Here, we examined the role of α3β1-integrin in ErbB2-dependent proliferation of breast cancer cells in three-dimensional laminin-rich extracellular matrix (3D lr-ECM). Depletion of α3β1 in ErbB2-overexpressing breast cancer cells suppressed growth and restore cell polarity in 3D lr-ECM. The phenotype of α3β1-depleted cells was reproduced upon depletion of tetraspanin CD151 and mirrored that of the cells treated with Herceptin, an established ErbB2 antagonist. Breast cancer cells expressing the α3β1-CD151 complex have higher steady-state phosphorylation of ErbB2 and show enhanced dimerization of the protein when compared with α3β1-/CD151-depleted cells. Furthermore, Herceptin-dependent dephosphorylation of ErbB2 was only observed in α3β1-CD151-expressing cells. Importantly, the inhibitory activity of Herceptin was more pronounced when cells expressed both α3β1 and CD151. We also found that the level of active RhoA was increased in α3β1- and CD151-depleted cells and that Rho controls dimerization of ErbB2. Expression of α3β1 alone did not have significant prognostic value in patients with invasive ductal carcinoma of the breast. However, expression of α3β1 in combination with CD151 represented a more stringent indicator of poor survival than CD151 alone. Taken together, these results demonstrate that the α3β1-CD151 complex has a critical regulatory role in ErbB2-dependent signalling and thereby may be involved in breast cancer progression. [ABSTRACT FROM AUTHOR]

Integrin expression and activity are altered in tumors, and aberrant integrin signaling promotes malignancy. However, how integrins become altered in tumors remains poorly understood. We discovered that oncogenic activation of MEK signaling induces cell growth and survival, and promotes the malignant phenotype of mammary epithelial cells (MECs) by increasing α5 integrin expression. We determined that MEK activates c-Myc to reduce the transcription of the SWI/SNF chromatin remodeling enzyme Brahma (BRM). Our studies revealed that reduced BRM expression and/or activity drives the malignant behavior of MECs by epigenetically promoting C/EBPβ expression to directly induce α5 integrin transcription. Consistently, we could show that restoring BRM levels normalized the malignant behavior of transformed MECs in culture and in vivo by preventing C/EBPβ-dependent α5 integrin transcription. Our findings identify a novel mechanism whereby oncogenic signaling promotes malignant transformation by regulating transcription of a key chromatin remodeling molecule that regulates integrin-dependent stromal-epithelial interactions. [ABSTRACT FROM AUTHOR]

Tumor-associated macrophages (TAMs) have multifaceted roles in tumor development, particularly linked with tumor angiogenesis and invasion, but the molecular mechanism underlying this association remains unclear. In this study, we report that lack of osteopontin (OPN) suppresses melanoma growth in opn−/− mice and macrophages are the crucial component responsible for OPN-regulated melanoma growth. In tumor microenvironment, OPN activates macrophages and influences angiogenesis by enhancing cyclooxygenase-2 (COX-2)-dependent prostaglandin E2 (PGE2) production in an autocrine manner. Furthermore, we identify α9β1 integrin as a functional receptor for OPN that mediates its effect and activates ERK and p38 signaling, which ultimately leads to COX-2 expression in macrophages. The major role played by OPN and PGE2 in angiogenesis are further amplified by upregulation of MMP-9. OPN-activated macrophages promote the migration of endothelial and cancer cells via PGE2. These findings provide evidence that TAMs serve as source of key components such as OPN and COX-2-derived PGE2 and MMP-9 in melanoma microenvironment. Clinical specimens analyses revealed that increased infiltration of OPN-positive TAMs correlate with melanoma growth and angiogenesis. These data provide compelling evidence that OPN and COX-2 expressing macrophages are obligatory factors in melanoma growth. We conclude that OPN signaling is involved in macrophage recruitment into tumor, and our results emphasize the potential role of macrophage in modulation of tumor microenvironment via secretion of OPN, PGE2 and MMP-9, which trigger angiogenesis and melanoma growth. Thus, blockade of OPN and its regulated signaling network provides unique strategy to eradicate melanoma by manipulating TAMs. [ABSTRACT FROM AUTHOR]

Liver metastasis is the major cause of death associated to colorectal cancer. Cadherin-17 (CDH17) is a non-classical, seven domain, cadherin lacking the conserved cytoplasmic domain of classical cadherins. CDH17 was overexpressed in highly metastatic human KM12SM and present in many other colorectal cancer cells. Using tissue microarrays, we observed a significant association between high expression of CDH17 with liver metastasis and poor survival of the patients. On the basis of these findings, we decided to study cellular functions and signaling mechanisms mediated by CDH17 in cancer cells. In this report, loss-of-function experiments demonstrated that CDH17 caused a significant increase in KM12SM cell adhesion and proliferation. Coimmunoprecipitation experiments demonstrated an interaction between CDH17 and α2β1 integrin with a direct effect on β1 integrin activation and talin recruitment. The formation of this complex, together with other proteins, was confirmed by mass spectrometry analysis. CDH17 modulated integrin activation and signaling to induce specific focal adhesion kinase and Ras activation, which led to the activation of extracellular signal-regulated kinase and Jun N-terminal kinase and the increase in cyclin D1 and proliferation. In vivo experiments showed that CDH17 silencing in KM12 cells suppressed tumor growth and liver metastasis after subcutaneous or intrasplenic inoculation in nude mice. Collectively, our data reveal a new function for CDH17, which is to regulate α2β1 integrin signaling in cell adhesion and proliferation in colon cancer cells for liver metastasis. [ABSTRACT FROM AUTHOR]

αvβ6 integrin expression is upregulated on a wide range of epithelial tumours, and is thought to play a role in modulating tumour growth. Here we describe a human therapeutic antibody 264RAD, which binds and inhibits αvβ6 integrin function. 264RAD cross-reacts with human, mouse and cynomolgus monkey αvβ6, and inhibits binding to all ligands including the latency-associated peptide of TGF-β. Screening across a range of integrins revealed that 264RAD also binds and inhibits the related integrin αvβ8, but not the integrins α5β1, αvβ3, αvβ5 and α4β1. In vitro 264RAD inhibited invasion of VB6 and Detroit 562 cells in a Matrigel invasion assay and αvβ6 mediated production of matrix metalloproteinase-9 in Calu-3 cells. It inhibited TGF-β-mediated activation of dermal skin fibroblasts by preventing local activation of TGF-β by NCI-H358 tumour cells in a tumour cell−fibroblast co-culture assay. In vivo 264RAD showed dose-dependent inhibition of Detroit 562 tumour growth, regressing established tumours when dosed at 20 mg/kg once weekly. The reduction in growth associated with 264RAD was related to a dose-dependent inhibition of Ki67 and phospho-ERK and a reduction of αvβ6 expression in the tumour cells, coupled to a reduction in fibronectin and alpha smooth muscle actin expression in stromal fibroblasts. 264RAD also reduced the growth and metastasis of orthotopic 4T1 tumours. At 20 mg/kg growth of both the primary tumour and the number of metastatic deposits in lung were reduced. The data support the conclusion that 264RAD is a potent inhibitor of αvβ6 integrin, with some activity against αvβ8 integrin, that reduces both tumour growth and metastasis. [ABSTRACT FROM AUTHOR]

Integrins are extracellular matrix (ECM) receptors that are key players in the regulation of tumour cell invasion. The laminin-binding integrin α3β1 has previously been shown to regulate adhesion and migration of carcinoma cells in part through co-operative signalling with the tetraspanin family of transmembrane proteins. However, the spatial and temporal regulation of crosstalk between these families of transmembrane proteins in intact cells remains poorly understood. Here we have used fluorescence resonance energy transfer (FRET) to demonstrate for the first time that α3β1 and the tetraspanin CD151 directly associate at the front and retracting rear of polarised migrating breast carcinoma cells in both two-dimentional (2D) and three-dimentional (3D)matrices. Furthermore, localised α3β1-CD151 binding correlates with lower CD151 homodimerisation in cells migrating on laminin or within matrigel. Loss of α3β1 integrin leads to increased CD151 homodimer formation, increased activation of Rho GTPase, loss of cell polarity and decreased invasion in 3D ECM. As a result, α3-silenced cells show decreased actin-based membrane protrusion and retraction in both 2D and 3D environments. These data demonstrate that associations between α3β1 and CD151 occur dynamically within discrete subcellular compartments and act to establish local GTPase signalling to promote tumour cell invasion. These novel findings shed light on the complex crosstalk and switching between receptor complexes in response to different extracellular cues during cell invasion in 3D environments. [ABSTRACT FROM AUTHOR]

Cancer progression, response to therapy and metastasis depend on tumor microenvironment. Integrins are cell-adhesion receptors that mediate interactions of cells with extracellular matrix. The αv-β-family of integrins contributes to tumorigenesis, response to therapy and cancer stem cell biology. Thus, understanding the function of specific integrins in cancer is critical for the development of therapeutic approaches targeting integrins. The study investigated the role of integrin β5 in breast carcinomas by depleting integrin β5 using RNA interference and reexpression of integrin β5. Depletion of integrin β5 in triple-negative breast carcinoma cells markedly reduced tumor take, growth and tumor angiogenesis, whereas reexpression of integrin β5 rescued this phenotype. Reduction in tumor angiogenesis is associated with lower expression of vascular endothelial growth factor-A in integrin β5-depleted tumors. Tumor cells deficient in integrin β5 have lower migration and proliferative capacities. Biochemical assays revealed that integrin β5 mediates the Src-focal adhesion kinase and MEK-extracellular signal-regulated kinase signaling events that operate independently, and inhibition of these pathways phenocopies integrin β5 deficiency. Breast carcinoma cells express high levels of integrin β5, whereas expression of integrin β3 is limited to stromal compartments and integrin β6 is lost in metastatic cells. Together, these findings show a critical role for integrin β5 in the tumorigenic potential of breast carcinoma cells and therapeutic targeting of integrin β5 is especially attractive for triple-negative breast carcinomas, which are refractory to most of the current therapies. [ABSTRACT FROM AUTHOR]

Junctional adhesion molecule-A (JAM-A) is a membranous cell-cell adhesion protein involved in tight-junction formation in epithelial and endothelial cells. Its overexpression in breast tumors has recently been linked with increased risk of metastasis. We sought to identify if JAM-A overexpression was associated with specific subtypes of breast cancer as defined by the expression of human epidermal growth factor receptor-2 (HER2), estrogen receptor (ER) and progesterone receptor. To this end, JAM-A immunohistochemistry was performed in two breast cancer tissue microarrays. In parallel, cross-talk between JAM-A, HER2 and ER was examined in several breast cell lines, using complementary genetic and pharmacological approaches. High JAM-A expression correlated significantly with HER2 protein expression, ER negativity, lower patient age, high-grade breast cancers, and aggressive luminal B, HER2 and basal subtypes of breast cancer. JAM-A and HER2 were co-expressed at high levels in vitro in SKBR3, UACC-812, UACC-893 and MCF7-HER2 cells. Knockdown or functional antagonism of HER2 did not alter JAM-A expression in any cell line tested. Interestingly, however, JAM-A knockdown decreased HER2 and ER-α expression, resulting in reduced levels of phospho-(active) AKT without an effect on the extracellular signal-related kinase phosphorylation. The downstream effects of JAM-A knockdown on HER2 and phospho-AKT were partially reversed upon treatment with the proteasomal inhibitor MG132. We conclude that JAM-A is co-expressed with HER2 and associates with aggressive breast cancer phenotypes. Furthermore, we speculate that JAM-A may regulate HER2 proteasomal degradation and activity, potentially offering a promise as a therapeutic target in HER2-positive breast cancers. [ABSTRACT FROM AUTHOR]

Here we provide the first evidence that tetraspanin CD151 can support de novo carcinogenesis. During two-stage mouse skin chemical carcinogenesis, CD151 reduces tumor lag time and increases incidence, multiplicity, size and progression to malignant squamous cell carcinoma (SCC), while supporting both cell survival during tumor initiation and cell proliferation during the promotion phase. In human skin SCC, CD151 expression is selectively elevated compared with other skin cancer types. CD151 support of keratinocyte survival and proliferation may depend on activation of transcription factor STAT3 (signal transducers and activators of transcription), a regulator of cell proliferation and apoptosis. CD151 also supports protein kinase C (PKC)α-α6β4 integrin association and PKC-dependent β4 S1424 phosphorylation, while regulating α6β4 distribution. CD151-PKCα effects on integrin β4 phosphorylation and subcellular localization are consistent with epithelial disruption to a less polarized, more invasive state. CD151 ablation, while minimally affecting normal cell and normal mouse functions, markedly sensitized mouse skin and epidermoid cells to chemicals/drugs including 7,12-dimethylbenz[α]anthracene (mutagen) and camptothecin (topoisomerase inhibitor), as well as to agents targeting epidermal growth factor receptor, PKC, Jak2/Tyk2 and STAT3. Hence, CD151 'co-targeting' may be therapeutically beneficial. These findings not only support CD151 as a potential tumor target, but also should apply to other cancers utilizing CD151/laminin-binding integrin complexes. [ABSTRACT FROM AUTHOR]

The type III TGF-β receptor (TβRIII) is a ubiquitous co-receptor for TGF-β superfamily ligands with roles in suppressing cancer progression, in part through suppressing cell motility. Here we demonstrate that TβRIII promotes epithelial cell adhesion to fibronectin in a β-arrestin2 dependent and TGF-β/BMP independent manner by complexing with active integrin α5β1, and mediating β-arrestin2-dependent α5β1 internalization and trafficking to nascent focal adhesions. TβRIII-mediated integrin α5β1 trafficking regulates cell adhesion and fibronectin fibrillogenesis in epithelial cells, as well as α5 localization in breast cancer patients. We further demonstrate that increased TβRIII expression correlates with increased α5 localization at sites of cell-cell adhesion in breast cancer patients, while higher TβRIII expression is a strong predictor of overall survival in breast cancer patients. These data support a novel, clinically relevant role for TβRIII in regulating integrin α5 localization, reveal a novel crosstalk mechanism between the integrin and TGF-β superfamily signaling pathways and identify β-arrestin2 as a regulator of α5β1 trafficking. [ABSTRACT FROM AUTHOR]

Tumour-derived mutant p53 proteins promote invasion, in part, by enhancing Rab coupling protein (RCP)-dependent receptor recycling. Here we identified MET as an RCP-binding protein and showed that mutant p53 promoted MET recycling. Mutant p53-expressing cells were more sensitive to hepatocyte growth factor, the ligand for MET, leading to enhanced MET signalling, invasion and cell scattering that was dependent on both MET and RCP. In cells expressing the p53 family member TAp63, inhibition of TAp63 also lead to cell scattering and MET-dependent invasion. However, in cells that express very low levels of TAp63, the ability of mutant p53 to promote MET-dependent cell scattering was independent of TAp63. Taken together, our data show that mutant p53 can enhance MET signalling to promote cell scattering and invasion through both TAp63-dependent and -independent mechanisms. MET has a predominant role in metastatic progression and the identification of mechanisms through which mutations in p53 can drive MET signalling may help to identify and direct therapy. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase-2 (MMP-2) has pivotal role in the degradation of extracellular matrix, and thereby enhances the invasive, proliferative and metastatic potential in cancer. Knockdown of MMP-2 using MMP-2 small interfering RNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared with mock and pSV (scrambled vector) treatments, as determined by 5-bromo-2′-deoxyuridine incorporation, Ki-67 staining and clonogenic survival assay. Cytokine array and western blotting using tumor-conditioned media displayed modulated secretory levels of various cytokines including granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor-α, angiogenin, vascular endothelial growth factor and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of Janus kinase/Stat3 pathway in pM-treated cells. Mechanistically, MMP-2 is involved in complex formation with α5 and β1 integrins and MMP-2 downregulation inhibited α5β1 integrin-mediated Stat3 phosphorylation and nuclear translocation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed inhibited Stat3 DNA-binding activity and recruitment at CyclinD1 and c-Myc promoters in pM-treated cells. In individual experiments, IL-6 or siRNA-insensitive MMP-2 overexpression by pM-FL-A141G counteracted and restored the pM-inhibited Stat3 DNA-binding activity, suggesting IL-6/Stat3 signaling suppression in pM-treated 4910 and 5310 cells. MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters. Activation of α5β1 signaling by Fibronectin adhesion elevated pM-inhibited Stat3 phosphorylation whereas blocking α5β1 abrogated constitutive Stat3 activation. In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments. Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels. Our data indicate the possible role of MMP-2/α5β1 interaction in the regulation of α5β1-mediated IL-6/Stat3 signaling activation and signifies the therapeutic potential of blocking MMP-2/α5β1 interaction in glioma treatment. [ABSTRACT FROM AUTHOR]

The integrin α9β1 binds a number of extracellular matrix components to mediate cell adhesion, migration and tissue invasion. Although expressed in a variety of normal human cells including endothelium, it is also expressed in cancer cells. We have previously shown that α9β1 binds VEGF-A to facilitate angiogenesis, an important component of the tumor microenvironment. As α9β1 induces accelerated cancer cell migration, we wished to determine what role it played in cancer growth and metastasis. In this study, we show that α9β1 expression induces molecular changes consistent with epithelial-mesenchymal transition. In addition, we found that α9β1 forms a tri-partite protein complex with β-catenin and E-cadherin, which dissociates following integrin activation and subsequent src and β-catenin phosphorylation. These findings were consistent in cells in which: α9β1 was exogenously over-expressed, or when its expression was suppressed in cancer cells endogenously expressing α9β1. These in vitro results are biologically significant as α9β1-expressing cancer cells induce greater tumor growth and metastases in mice as compared to the cells without α9β1 expression or when integrin expression is suppressed. Furthermore, integrin α9β1 is expressed in primary human small cell lung cancer and patients having a high expression of α9β1 demonstrated significantly worse long-term survival compared with patients with low α9β1 expression. These findings highlight a novel mechanism of integrin α9β1 function in human cancer. [ABSTRACT FROM AUTHOR]

Aneuploidy is frequently detected in solid tumors but the mechanisms regulating the generation of aneuploidy and their relevance in cancer initiation remain under debate and are incompletely characterized. Spatial and temporal regulation of integrin traffic is critical for cell migration and cytokinesis. Impaired integrin endocytosis, because of the loss of Rab21 small GTPase or mutations in the integrin β-subunit cytoplasmic tail, induces failure of cytokinesis in vitro. Here, we describe that repeatedly failed cytokinesis, because of impaired traffic, is sufficient to trigger the generation of aneuploid cells, which display characteristics of oncogenic transformation in vitro and are tumorigenic in vivo. Furthermore, in an in vivo mouse xenograft model, non-transformed cells with impaired integrin traffic formed tumors with a long latency. More detailed investigation of these tumors revealed that the tumor cells were aneuploid. Therefore, abnormal integrin traffic was linked with generation of aneuploidy and cell transformation also in vivo. In human prostate and ovarian cancer samples, downregulation of Rab21 correlates with increased malignancy. Loss-of-function experiments demonstrate that long-term depletion of Rab21 is sufficient to induce chromosome number aberrations in normal human epithelial cells. These data are the first to demonstrate that impaired integrin traffic is sufficient to induce conversion of non-transformed cells to tumorigenic cells in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor receptor 2 (HER2)/Neu is overexpressed in 20-30% of breast cancers and associated with aggressive phenotypes and poor prognosis. For deciphering the role of HER2/Neu in breast cancer, mouse mammary tumor virus (MMTV)-Her2/neu transgenic mice that develop mammary tumors resembling human HER2-subtype breast cancer have been established. Several recent studies have revealed that HER2/Neu is overexpressed in and regulates self renewal of breast tumor-initiating cells (TICs). However, in the MMTV-Her2/neu transgenic mouse model, the identity of TICs remains elusive, despite previous studies showing supportive evidence for existence of TICs in Her2/neu-induced mammary tumors. Through systematic screening and characterization, we identified that surface markers CD49f, CD61 and ESA were aberrantly overexpressed in Her2-overexpressing mammary tumor cells. Analysis of these markers and CD24 detected anomalous expansion of the luminal progenitor population in preneoplastic mammary glands of Her2/neu transgenic mice, indicating that aberrant luminal progenitors originated in Her2-induced mammary tumors. The combined markers, CD49f and CD61, further delineated the CD49fhighCD61high-sorted fraction as a TIC-enriched population, which displayed increased tumorsphere formation ability, enhanced tumorigenicity both in vitro and in vivo and drug resistance to pacitaxel and doxorubicin. Moreover, the TIC-enriched population manifested increased transforming growth factor-β (TGFβ) signaling and exhibited gene expression signatures of stemness, TGFβ signaling and epithelial-to-mesenchymal transition. Our findings that self-renewal and clonogenicity of TICs were suppressed by pharmacologically inhibiting the TGFβ signaling further indicate that the TGFβ pathway is vital for maintenance of the TIC population. Finally, we showed that the integrin-β3 (CD61) signaling pathway was required for sustaining active TGFβ signaling and self-renewal of TICs. We for the first time developed a technique to highly enrich TICs from mammary tumors of Her2/neu transgenic mice, unraveled their properties and identified the cooperative integrin-β3-TGFβ signaling axis as a potential therapeutic target for HER2-induced TICs. [ABSTRACT FROM AUTHOR]

Proteins containing a caveolin-binding domain (CBD), such as the Rho-GTPases, can interact with caveolin-1 (Cav1) through its caveolin scaffold domain. Rho-GTPases are important regulators of p130Cas, which is crucial for both normal cell migration and Src kinase-mediated metastasis of cancer cells. However, although Rho-GTPases (particularly RhoC) and Cav1 have been linked to cancer progression and metastasis, the underlying molecular mechanisms are largely unknown. To investigate the function of Cav1-Rho-GTPase interaction in metastasis, we disrupted Cav1-Rho-GTPase binding in melanoma and mammary epithelial tumor cells by overexpressing CBD, and examined the loss-of-function of RhoC in metastatic cancer cells. Cancer cells overexpressing CBD or lacking RhoC had reduced p130Cas phosphorylation and Rac1 activation, resulting in an inhibition of migration and invasion in vitro. The activity of Src and the activation of its downstream targets FAK, Pyk2, Ras and extracellular signal-regulated kinase (Erk)1/2 were also impaired. A reduction in α5-integrin expression, which is required for binding to fibronectin and thus cell migration and survival, was observed in CBD-expressing cells and cells lacking RhoC. As a result of these defects, CBD-expressing melanoma cells had a reduced ability to metastasize in recipient mice, and impaired extravasation and survival in secondary sites in chicken embryos. Our data indicate that interaction between Cav1 and Rho-GTPases (most likely RhoC but not RhoA) promotes metastasis by stimulating α5-integrin expression and regulating the Src-dependent activation of p130Cas/Rac1, FAK/Pyk2 and Ras/Erk1/2 signaling cascades. [ABSTRACT FROM AUTHOR]

Myopodin is a tumor-suppressor gene that suppresses growth of prostate and urothelial carcinomas. However, the mechanism of myopodin tumor-suppressor activity or signaling that leads to activation of myopodin remains unclear. In this report, we showed that the N-terminus of myopodin binds integrin-linked kinase (ILK) both in vivo and in vitro. An ILK interaction motif of 78 amino acids (amino acids 82-157) was identified in the N-terminus region of myopodin. Induction of ILK-dependent kinase activity by integrin α7 led to phosphorylation of myopodin both in vivo and in vitro. Knocking down ILK dramatically reduced the inhibition of cell growth and motility mediated by myopodin. A mutant of myopodin lacking the ILK interaction motif is inactive in suppressing the growth and motility of PC3 cells. As a result, this study showed a novel and critical signaling pathway that leads to activation of myopodin. [ABSTRACT FROM AUTHOR]

Integrin-linked kinase (ILK) is a highly evolutionarily conserved intracellular protein that was originally identified as an integrin-interacting protein, and extensive genetic and biochemical studies have shown that ILK expression is vital during both embryonic development and tissue homeostasis. At the cellular and tissue levels, ILK regulates signaling pathways for cell adhesion-mediated cell survival (anoikis), apoptosis, proliferation and mitosis, migration, invasion, and vascularization and tumor angiogenesis. ILK also has central roles in cardiac and smooth-muscle contractility, and ILK dysregulation causes cardiomyopathies in humans. ILK protein levels are increased in several human cancers and often the expression level predicts poor patient outcome. Abundant evidence has accumulated suggesting that, of the diverse functions of ILK, some may require kinase activity whereas others depend on protein-protein interactions and are, therefore, independent of kinase activity. However, the past several years have seen an ongoing debate about whether ILK indeed functions as a protein serine/threonine kinase. This debate centers on the atypical protein kinase domain of ILK, which lacks some amino-acid residues thought to be essential for phosphotransferase activity. However, similar deficiencies are present in the catalytic domains of other kinases now known to possess protein kinase activity. Numerous studies have shown that ILK phosphorylates peptide substrates in vitro, corresponding to ILK-mediated phosphorylations in intact cells, and a recent report characterizing in vitro phosphotransferase activity of highly purified, full-length ILK, accompanied by detailed enzyme kinetic analyses, shows that, at least in vitro, ILK is a bona fide protein kinase. However, several genetic studies suggest that, not all biological functions of ILK require kinase activity, and that it can function as an adaptor/scaffold protein. Here, we review evidence for and against ILK being an active kinase, and provide a framework for strategies to further analyze the kinase and adaptor functions of ILK in different cellular contexts. [ABSTRACT FROM AUTHOR]

Development of targeted therapy for hepatocellular carcinoma (HCC) remains a major challenge. We have recently identified an elevated expression of the fifth subunit of COP9 signalosome (CSN5) in early HCC as compared with dysplastic stage. In the present study, we explored the possibility of CSN5 being a potential therapeutic target for HCC. Our results show that CSN5 knockdown by small-interfering (si) RNA caused a strong induction of apoptosis and inhibition of cell-cycle progression in HCC cells in vitro. The down-regulation of CSN5 was sufficient to interfere with CSN function as evidenced by the accumulation of neddylated Cullin 1 and changes in the protein levels of CSN-controlled substrates SKP2, p53, p27 and nuclear factor-κB, albeit to a different degree depending on the HCC cell line, which could account for the CSN5 knockdown phenotype. The transcriptomic analysis of CSN5 knockdown signature showed that the anti-proliferative effect was driven by a common subset of molecular alterations including down-regulation of cyclin-dependent kinase 6 (CDK6) and integrin β1 (ITGB1), which were functionally interconnected with key oncogenic regulators MYC and TGFβ1 involved in the control of proliferation, apoptotic cell death and HCC progression. Consistent with microarray analysis, western blotting revealed that CSN5 depletion increased phosphorylation of Smad 2/3, key mediators of TGFβ1 signaling, decreased the protein levels of ITGB1, CDK6 and cyclin D1 and caused reduced expression of anti-apoptotic Bcl-2, while elevating the levels of pro-apoptotic Bak. A chemically modified variant of CSN5 siRNA was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response. Systemic delivery of the CSN5 3/8 variant by stable-nucleic-acid-lipid particles significantly suppressed the tumor growth in Huh7-luc+ orthotopic xenograft model. Taken together, these results indicate that CSN5 has a pivotal role in HCC pathogenesis and maybe an attractive molecular target for systemic HCC therapy. [ABSTRACT FROM AUTHOR]

Lung cancer is the leading cause of cancer death worldwide. The epidermal growth factor receptor (EGFR) represents the main target for non-small cell lung cancer (NSCLC) therapy, as its overexpression or constitutive activation contributes to malignancy and correlates with poor prognosis. Our previous work demonstrated that in epithelial cells β1 integrin is required for propagating EGFR signaling from the plasma membrane to the nucleus. In this study, we silenced β1 integrin in human NSCLC A549 cells. The β1 integrin-silenced cells show a defective activation of the EGFR signaling cascade, leading to decreased in vitro proliferation, enhanced sensitivity to cisplatin and Gefitinib, impaired migration and invasive behavior. Inhibitory effects on tumor growth and on the EGFR pathway were also observed in in vivo experiments. Moreover, β1 integrin silencing increases the amount of EGFR on the cell surface, suggesting that β1 integrin is required for efficient constitutive EGFR turnover at the cell membrane. Although the rate of EGF internalization and recycling is not affected in silenced cells, EGFR signaling is recovered only by expression of the Rab-coupling protein RCP, indicating that β1 integrin sustains the endocytic machinery required for EGFR signaling. Overall, these results show that β1 integrin is an essential regulator of EGFR signaling and tumorigenic properties of lung cancer cells, and that its silencing might represent an adjuvant approach to anti-EGFR therapy. [ABSTRACT FROM AUTHOR]

CTEN/TNS4 is an oncogene in colorectal cancer (CRC), which can induce cell motility although its mechanistic basis of activity and the clinical implications of Cten expression are unknown. As Cten is in complex with integrins at focal adhesions, we hypothesised that it may interact with integrin-linked kinase (ILK). Through forced expression and knockdown of Cten in HCT116 and SW620 (respectively, showing low and high Cten expression), we showed that Cten could regulate ILK. However, inhibition of ILK after forced expression of Cten abrogated the motility-inducing effects of Cten, thereby demonstrating that the Cten-ILK interaction was functionally relevant. Combined knockdown of Cten and ILK had no additive effects on cell motility compared with knockdown of each individually. In order to investigate the clinical implications of Cten expression, a series of 462 CRCs were evaluated by immunohistochemistry. High expression of Cten was associated with advanced Dukes' stage (P<0.001), poor prognosis (P<0.001) and distant metastasis (P=0.008). The role of Cten in metastasis was tested by (a) intrasplenic injection of CRC cells stably transfected with a Cten expression vector into nude mice and (b) testing a series of primary human CRCs and their metastases by immunohistochemistry. Compared with controls, mice injected with cells expressing Cten developed larger tumours in the spleen (P<0.05) and liver (P<0.05). In the human cases, compared with primary tumours, the metastatic deposits had a significantly higher frequency of nuclear localisation of Cten (P=0.002). We conclude that Cten expression is of prognostic significance in CRC, and we delineate a Cten-ILK pathway controlling cell motility and possibly promoting metastasis. [ABSTRACT FROM AUTHOR]

Dynamic interactions between cells and extracellular matrix (ECM) through integrins influence most cellular functions. Normal cells, but even more, tumor cells are subjected to different forms of stress, including ischemia, radical oxygen species production, starvation, mechanical stress or genotoxic insults due to anti-cancer drugs or irradiation. In these situations, an adaptative cellular response occurs, integrating a complex network of intracellular signaling modules, which, depending on stress intensity, may result to either damage repair followed by complete restitution of cellular functions, or programmed cell death. Because of its implication in oncogenesis and anti-cancer therapy, cellular stress response has been thoroughly investigated. However, most of these studies have been performed in the context of isolated cells without taking into consideration that most cells are part of the tissue within which they interact with ECM through integrin. Few studies have described the influence of stress on cell-to-ECM interaction. However, one can speculate that, in these conditions, cells could functionally interact with protein microenvironment either to create positive interactions to survive (for example by facilitating protective pathways) or negative interaction to die (for example by facilitating detachment). In this review, we summarize the knowledge relative to the influence of different stress modalities on ECM remodeling, integrin expression and/or function modifications, and possible functional consequences, independently from the cellular model as these findings came from a large variety of cells (mesenchymal, endothelial, muscular, epithelial and glandular) and fields of application (cancer, vascular biology and tissue engineering). Most studies support the general notion that non-lethal stress favors ECM stiffness, integrin activation and enhanced survival. This field opens large perspectives not only in tumor biology but also in anti-cancer therapy by targeting one or several steps of the integrin-mediated signaling pathway, including integrin ligation, or activation of integrin-linked enzymes or integrin adaptors. [ABSTRACT FROM AUTHOR]

The role of the fibronectin receptor, α5β1-integrin, as an adhesion receptor and in angiogenesis is well established. However, its role in cancer cell invasion and metastasis is less clear. We describe a novel mechanism by which fibronectin regulates ovarian cancer cell signaling and promotes metastasis. Fibronectin binding to α5β1-integrin led to a direct association of α5-integrin with the receptor tyrosine kinase, c-Met, activating it in a hepatocyte growth factor/scatter factor (HGF/SF) independent manner. Subsequently, c-Met associated with Src, and activated Src and focal adhesion kinase (FAK). Inhibition of α5β1-integrin decreased the phosphorylation of c-Met, FAK and Src, both in vitro and in vivo. Independent activation of c-Met by its native ligand, HGF/SF, or overexpression of a constitutively active FAK in HeyA8 cells could overcome the effect of α5β1-integrin inhibition on tumor cell invasion, indicating that α5β1-integrin is upstream of c-Met, Src and FAK. Inhibition of α5β1-integrin on cancer cells in two xenograft models of ovarian cancer metastasis resulted in a significant decrease of tumor burden, which was independent of the effect of α5β1-integrin on angiogenesis. These data suggest that fibronectin promotes ovarian cancer invasion and metastasis through an α5β1-integrin/c-Met/FAK/Src-dependent signaling pathway, transducing signals through c-Met in an HGF/SF-independent manner. [ABSTRACT FROM AUTHOR]

Expression of the integrin αvβ6 is upregulated in a variety of carcinomas where it appears to be involved in malignant progression, although the biology of this integrin is not fully explored. We have generated oral carcinoma cells that express αvβ6 composed of wild-type αv and a mutant β6 that lacks the unique C-terminal 11 amino acids (aa). We found that these residues, although not required for αvβ6-dependent adhesion or migration, are essential for αvβ6-dependent invasive activity. We have used a proteomic approach to identify novel binding partners for the β6 subunit cytoplasmic tail and report that psoriasin (Psor) (S100A7) bound preferentially to the recombinant β6 cytoplasmic domain, though not in the absence of the unique C-terminal 11aa. Endogenous cellular Psor co-precipitated with endogenous β6 and colocalised with αvβ6 at the cell membrane and intracellular vesicles. Knockdown of Psor, with small interfering RNA, had no effect on αvβ6-dependent adhesion or migration but abrogated αvβ6-mediated oral carcinoma cell invasion both in Transwell and, the more physiologically relevant, organotypic invasion assays, recapitulating the behaviour of the β6-mutant cell line. Membrane-permeant Tat-peptides encoding the unique C-terminal residues of β6, bound directly to recombinant Psor and inhibited cellular Psor binding to β6; this blocked αvβ6-dependent, but not αvβ6-independent, invasion. These data identify a novel interaction between Psor and β6 and demonstrate that it is required for αvβ6-dependent invasion by carcinoma cells. Inhibition of this interaction may represent a novel therapeutic strategy to target carcinoma invasion. [ABSTRACT FROM AUTHOR]

The liver represents the third most frequent site of metastasis in patients with breast cancer. We performed in vivo selection using 4T1 breast cancer cells to identify genes associated with the liver metastatic phenotype. Coincident with the loss of numerous tight-junctional proteins, we observe claudin-2 overexpression, specifically in liver-aggressive breast cancer cells. We further demonstrate that claudin-2 is both necessary and sufficient for the ability of 4T1 breast cancer cells to colonize and grow in the liver. The liver-aggressive breast cancer cells display a claudin-2-mediated increase in their ability to adhere to extracellular matrix (ECM) components, such as fibronectin and type IV collagen. Claudin-2 facilitates these cell/matrix interactions by increasing the cell surface expression of α2β1- and α5β1-integrin complexes in breast cancer cells. Indeed, claudin-2-mediated adhesion to fibronectin and type IV collagen can be blocked with neutralizing antibodies that target α5β1 and α2β1 complexes, respectively. Immunohistochemical analyses reveal that claudin-2, although weakly expressed in primary human breast cancers, is readily detected in all liver metastasis samples examined to date. Together, these results uncover novel roles for claudin-2 in promoting breast cancer adhesion to the ECM and define its importance during breast cancer metastasis to the liver. [ABSTRACT FROM AUTHOR]

It has been reported that the miR-106b∼25 cluster, a paralog of the miR-17∼92 cluster, possesses oncogenic activities. However, the precise role of each microRNA (miRNA) in the miR-106b∼25 cluster is not yet known. In this study, we examined the function of miR-93, one of the microRNAs within the miR-106b∼25 cluster, in angiogenesis and tumor formation. We found that miR-93 enhanced cell survival, promoted sphere formation and augmented tumor growth. Most strikingly, when miR-93-overexpressing U87 cells were co-cultured with endothelial cells, they supported endothelial cell spreading, growth, migration and tube formation. In vivo studies revealed that miR-93-expressing cells induced blood vessel formation, allowing blood vessels to extend to tumor tissues in high densities. Angiogenesis promoted by miR-93 in return facilitated cell survival, resulting in enhanced tumor growth. We further showed that integrin-β8 is a target of miR-93. Higher levels of integrin-β8 are associated with cell death in tumor mass and in human glioblastoma. Silencing of integrin-β8 expression using small interfering RNA promoted cell proliferation, whereas ectopic expression of integrin-β8 decreased cell growth. These findings showed that miR-93 promotes tumor growth and angiogenesis by suppressing, at least in part, integrin-β8 expression. Our results suggest that inhibition of miR-93 function may be a feasible approach to suppress angiogenesis and tumor growth. [ABSTRACT FROM AUTHOR]

Many cancer cells contain more than two centrosomes, which imposes a potential for multipolar mitoses, leading to cell death. To circumvent this, cancer cells develop mechanisms to cluster supernumerary centrosomes to form bipolar spindles, enabling successful mitosis. Disruption of centrosome clustering thus provides a selective means of killing supernumerary centrosome-harboring cancer cells. Although the mechanisms of centrosome clustering are poorly understood, recent genetic analyses have identified requirements for both actin and tubulin regulating proteins. In this study, we demonstrate that the integrin-linked kinase (ILK), a protein critically involved in actin and mitotic microtubule organization, is required for centrosome clustering. Inhibition of ILK expression or activity inhibits centrosome clustering in several breast and prostate cancer cell lines that have centrosome amplification. Furthermore, cancer cells with supernumerary centrosomes are significantly more sensitive to ILK inhibition than cells with two centrosomes, demonstrating that inhibiting ILK offers a selective means of targeting cancer cells. Live cell analysis shows ILK perturbation leads cancer cells to undergo multipolar anaphases, mitotic arrest and cell death in mitosis. We also show that ILK performs its centrosome clustering activity in a focal adhesion-independent, but centrosome-dependent, manner through the microtubule regulating proteins TACC3 and ch-TOG. In addition, we identify a specific TACC3 phosphorylation site that is required for centrosome clustering and demonstrate that ILK regulates this phosphorylation in an Aurora-A-dependent manner. [ABSTRACT FROM AUTHOR]

Focal adhesion kinase (FAK) has a crucial role in integration of signals from integrins and growth factor receptors. In this study, we demonstrate that growth factor receptors including hepatocyte growth factor receptor Met, epidermal growth factor receptor, and platelet-derived growth factor receptor directly phosphorylate FAK on Tyr194 in the FERM domain (band 4.1 and ezrin/radixin/moesin homology domain). Upon binding to Met or phosphoinositides, FAK may undergo conformational changes, which renders Tyr194 accessible for phosphorylation. Substitution of Tyr194 with Phe significantly suppresses the activation of FAK by Met. In contrast, substitution of Tyr194 with Glu (Y194E substitution) leads to constitutive activation of FAK. The phosphorylation of FAK on Tyr194 may cause conformational changes in the FERM domain, which disrupts the intramolecular inhibitory interaction between the FERM and kinase domains of FAK. Moreover, substitution of the basic residues in the 216KAKTLRK222 patch in the FERM domain with Ala antagonizes the effect of the Y194E substitution on FAK activation, thus suggesting that the interactions between the phosphorylated Tyr194 and the basic resides in the 216KAKTLRK222 patch may allow FAK to be activated through relief of its autoinhibition. Collectively, this study provides the first example to explain how FAK is activated by receptor tyrosine kinases. [ABSTRACT FROM AUTHOR]

Integrin/cytokine receptor interaction provides permissive signals leading to neoangiogenesis, and integrins are crucial for differentiation of endothelial progenitor cells (EPCs). It is known that the inflammatory interleukin-3 (IL-3), released in the tumoral microenvironment, contributes to both angiogenesis and vasculogenic processes. Herein, we generated IL-3 receptor beta common (IL-3Rβc) extracellular domain-derived fusion proteins (Fc) to elucidate the molecular mechanisms regulating these processes. Three different Fc were generated, containing the entire extracellular domain of IL-3Rβc (Fc1.4), a fragment corresponding to domains 1-3 (Fc1.3) and a fragment corresponding to domain 4 (Fc4), respectively. The ability of the fusion proteins to interfere with IL-3Rβc/β1 integrin interaction was assessed on endothelial cells (ECs), EPCs and murine-derived ECs. Pull-down experiments showed that Fc1.4 and Fc4 fusion proteins specifically interacted with β1 integrin. Fc4 and Fc1.4 fragments prevented IL-3-mediated EPC expansion, arterial morphogenesis and tumour-derived EC migration, without affecting cell adhesion. Fc4 in vivo inhibited the IL-3-mediated vasculogenic process, as well as inflammatory and tumour vascular growth. In conclusion, these data identify the β1 integrin-interacting domain in the juxta-membrane IL-3Rβc extracellular domain, and provide the rational for targeting this interaction to impair vascular growth. [ABSTRACT FROM AUTHOR]

The majority of mortality associated with cancer is due to formation of metastases from the primary tumor. Adhesion mediated by different integrin heterodimers has an important role during cell migration and invasion. Protein interactions with the β1-integrin cytoplasmic tail are known to influence integrin affinity for extracellular ligands, but regulating binding partners for the α-subunit cytoplasmic tails have remained elusive. In this study, we show that mammary-derived growth inhibitor (MDGI) (also known as FABP-3 or H-FABP) binds directly to the cytoplasmic tail of integrin α-subunits and its expression inhibits integrin activity. In breast cancer cell lines, MDGI expression correlates with suppression of the active conformation of integrins. This results in reduced integrin adhesion to type I collagen and fibronectin and inhibition of cell migration and invasion. In tissue microarray of 1331 breast cancer patients, patients with MDGI-positive tumors had more favorable 10-year distant disease-free survival compared with patients with MDGI-negative tumors. Our data indicate that MDGI is a novel interacting partner for integrin α-subunits, and its expression modulates integrin activity and suppresses cell invasion in breast cancer patients. Retained MDGI expression is associated with favorable prognosis. [ABSTRACT FROM AUTHOR]

In a previous study it was found that the therapeutic effects of QLT0267, a small molecule inhibitor of integrin-linked kinase (ILK), were influenced by Her2/neu expression. To understand how inhibition or silencing of ILK influences Her2/neu expression, Her2/neu signaling was evaluated in six Her2/neu-positive breast cancer cell lines (LCC6Her2, MCF7Her2, SKBR3, BT474, JIMT-1 and KPL-4). Treatment with QLT0267 engendered suppression (32-87%) of total Her2/neu protein in these cells. Suppression of Her2/neu was also observed following small interfering RNA-mediated silencing of ILK expression. Time course studies suggest that ILK inhibition or silencing caused transient decreases in P-AKTser473, which were not temporally related to Her2/neu downregulation. Attenuation of ILK activity or expression was, however, associated with decreases in YB-1 (Y-box binding protein-1) protein and transcript levels. YB-1 is a known transcriptional regulator of Her2/neu expression, and in this study it is demonstrated that inhibition of ILK activity using QLT0267 decreased YB-1 promoter activity by 50.6%. ILK inhibition was associated with changes in YB-1 localization, as reflected by localization of cytoplasmic YB-1 into stress granules. ILK inhibition also suppressed TWIST (a regulator of YB-1 expression) protein expression. To confirm the role of ILK on YB-1 and TWIST, cells were engineered to overexpress ILK. This was associated with a fourfold increase in the level of YB-1 in the nucleus, and a 2- and 1.5-fold increase in TWIST and Her2/neu protein levels, respectively. Taken together, these data indicate that ILK regulates the expression of Her2/neu through TWIST and YB-1, lending support to the use of ILK inhibitors in the treatment of aggressive Her2/neu-positive tumors. [ABSTRACT FROM AUTHOR]

Large-scale genetic analyses of human tumor samples have been used to identify novel oncogenes, tumor suppressors and prognostic factors, but the functions and molecular interactions of many individual genes have not been determined. In this study we examined the cellular effects and molecular mechanism of the arrestin family member, ARRDC3, a gene preferentially lost in a subset of breast cancers. Oncomine data revealed that the expression of ARRDC3 decreases with tumor grade, metastases and recurrences. ARRDC3 overexpression represses cancer cell proliferation, migration, invasion, growth in soft agar and in vivo tumorigenicity, whereas downregulation of ARRCD3 has the opposite effects. Mechanistic studies showed that ARRDC3 functions in a novel regulatory pathway that controls the cell surface adhesion molecule, β-4 integrin (ITGβ4), a protein associated with aggressive tumor behavior. Our data indicates ARRDC3 directly binds to a phosphorylated form of ITGβ4 leading to its internalization, ubiquitination and ultimate degradation. The results identify the ARRCD3-ITGβ4 pathway as a new therapeutic target in breast cancer and show the importance of connecting genetic arrays with mechanistic studies in the search for new treatments. [ABSTRACT FROM AUTHOR]

L1 cell adhesion molecule (L1CAM) overexpression is often associated with bad prognosis in various human carcinomas. Recent studies also suggest a role of L1CAM in pancreatic ductal adenocarcinomas (PDAC). To further address its contribution, we expressed functional domains of L1CAM in PT45-P1 PDAC cells. We found that L1CAM that is full length (L1-FL), but neither the soluble ectodomain (L1ecto) nor the cytoplasmic part (L1cyt), could enhance cell proliferation or tumour growth in mice. Expression of L1-FL resulted in constitutive activation of NF-κB, which was abolished by L1CAM knockdown. We showed that the expression of IL-1β was selectively upregulated by L1-FL, and increased IL-1β levels were instrumental for sustained NF-κB activation. IL-1β production and NF-κB activation were abolished by knockdown of α5-integrin and integrin-linked kinase, but insensitive to depletion of L1CAM cleavage proteinases. Supporting these data, PT45-P1 cells transduced with an L1CAM mutant deficient in integrin binding (L1-RGE) did not support the described L1-FL functions. Our results suggest that membranous L1CAM interacts with RGD&!minus;-binding integrins, leading to sustained NF-κB activation by IL-1β production and autocrine/paracrine signalling. The unravelling of this novel mechanism sheds new light on the important role of L1CAM expression in PDAC cells. [ABSTRACT FROM AUTHOR]

Interactions between ovarian cancer cells and the surrounding tumor microenvironment are not well characterized. We have earlier shown that ovarian cancer ascites induces Akt activation and protect tumor cells from TRAIL-induced apoptosis. Here, we investigated the mechanism by which ascites activates Akt. The ability of ovarian cancer ascites to activate Akt and inhibit TRAIL-induced cell death and caspase activity was decreased by heat inactivation, but was retained in ascites fractions >5 kDa. The survival promoting activity of ascites was not affected by inhibitors of growth factor receptor including epidermal growth factor receptor (EGFR), VEGFR, FGFR, Her2/neu, and IGF-R1. However, this activity was inhibited by an αvβ5 integrin-blocking antibody, but not by blocking antibodies against αvβ3, β1, or β3 integrins. αvβ5 integrin-blocking antibodies also inhibited ascites-induced Akt phosphorylation and c-FLIPs up-regulation. Ovarian cancer ascites induced a rapid phosphorylation of focal adhesion kinase (FAK), which closely correlated with the phosphorylation of Akt overtime. FAK phosphorylation was strongly inhibited by αvβ5 integrin-blocking antibodies. Depletion of FAK content by RNA interference was also associated with inhibition of ascites-mediated Akt activation and survival. These results suggest that ovarian cancer ascites induces FAK and Akt activation in an αvβ5 integrin-dependent pathway, which confers protection from TRAIL-induced cell death and caspase activation. [ABSTRACT FROM AUTHOR]

Elevated expression of the integrin-linked kinase (ILK) has been observed in a variety of cancers and has been further correlated with poor clinical outcome. Here, we show that mammary epithelial disruption of ILK results in a profound block in mammary tumor induction. Consistent with these observations, inhibition of ILK function in ErbB2-expressing cells with small molecule inhibitor or RNA interference resulted in profound block in their in vitro invasive properties due to the induction of apoptotic cell death. The rare ILK-deficient tumors that eventually arose overcame this block in tumor induction by an upregulation of ErB3 phosphorylation. These observations provide direct evidence that ILK has a critical role in the initiation phase of ErbB2 tumor induction. [ABSTRACT FROM AUTHOR]

The development of pulmonary metastasis is the major cause of death in osteosarcoma, and its molecular basis is poorly understood. In this study, we show that β4 integrin is highly expressed in human osteosarcoma cell lines and tumor samples. Furthermore, highly metastatic MNNG-HOS cells have increased levels of β4 integrin. Suppression of β4 integrin expression by shRNA and disruption of β4 integrin function by transfection of dominant-negative β4 integrin was sufficient to revert this highly metastatic phenotype in the MNNG-HOS model without significantly affecting primary tumor growth. These findings suggest a role for β4 integrin expression in the metastatic phenotype in human osteosarcoma cells. In addition, we identified a previously uncharacterized interaction between β4 integrin and ezrin, a membrane-cytoskeletal linker protein that is implicated in the metastatic behavior of osteosarcoma. The β4 integrin–ezrin interaction appears to be critical for maintenance of β4 integrin expression. These data begin to integrate ezrin and β4 integrin expression into a model of action for the mechanism of osteosarcoma metastases. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC) is a lethal disease and early death usually occurs as a result of local invasion and regional lymph node metastases. Current treatment regimens are, to a certain degree, inadequate, with a 5-year mortality rate of around 50% and novel therapeutic targets are urgently required. Using expression microarrays, we identified the eps8 gene as being overexpressed in OSCC cell lines relative to normal oral keratinocytes, and confirmed these findings using RT–PCR and western blotting. In human tissues, we found that Eps8 was upregulated in OSCC (32% of primary tumors) compared with normal oral mucosa, and that expression correlated significantly with lymph node metastasis (P=0.032), suggesting a disease-promoting effect. Using OSCC cell lines, we assessed the functional role of Eps8 in tumor cells. Although suppression of Eps8 produced no effect on cell proliferation, both cell spreading and migration were markedly inhibited. The latter cell functions may be modulated through the small GTP-ase, Rac1 and we used pull-down assays to investigate the role of Eps8 in Rac1 signaling. We found that αvβ6- and α5β1-integrin-dependent activation of Rac1 was mediated through Eps8. Knockdown of either Eps8 or Rac1, inhibited integrin-dependent cell migration similarly and transient expression of constitutively active Rac1 restored migration of cells in which Eps8 expression had been suppressed. We also showed that knockdown of Eps8 inhibited tumor cell invasion in an organotypic model of OSCC. These data suggest that Eps8 and Rac1 are part of an integrated signaling pathway modulating integrin-dependent tumour cell motility and identify Eps8 as a possible therapeutic target.Oncogene (2009) 28, 2524–2534; doi:10.1038/onc.2009.105; published online 18 May 2009 [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is incurable, mainly because of cell adhesion-mediated drug resistance (CAM-DR). In this study, we performed functional screening using short hairpin RNA (shRNA) to define the molecule(s) responsible for CAM-DR of MM. Using four bona fide myeloma cell lines (KHM-1B, KMS12-BM, RPMI8226 and U266) and primary myeloma cells, we identified CD29 (β1-integrin), CD44, CD49d (α4-integrin, a subunit of VLA-4), CD54 (intercellular adhesion molecule-1 (ICAM-1)), CD138 (syndecan-1) and CD184 (CXC chemokine receptor-4 (CXCR4)) as major adhesion molecules expressed on MM. shRNA-mediated knockdown of CD49d but not CD44, CD54, CD138 and CD184 significantly reversed CAM-DR of myeloma cells to bortezomib, vincristine, doxorubicin and dexamethasone. Experiments using blocking antibodies yielded almost identical results. Bortezomib was relatively resistant to CAM-DR because of its ability to specifically downregulate CD49d expression. This property was unique to bortezomib and was not observed in other anti-myeloma drugs. Pretreatment with bortezomib was able to ameliorate CAM-DR of myeloma cells to vincristine and dexamethasone. These results suggest that VLA-4 plays a critical role in CAM-DR of MM cells. The combination of bortezomib with conventional anti-myeloma drugs may be effective in overcoming CAM-DR of MM.Oncogene (2009) 28, 231–242; doi:10.1038/onc.2008.385; published online 13 October 2008 [ABSTRACT FROM AUTHOR]