CD151 Maintains Endolysosomal Protein Quality to Inhibit Vascular Inflammation.

BACKGROUND: Tetraspanin CD151 is highly expressed in endothelia and reinforces cell adhesion, but its role in vascular inflammation remains largely unknown. METHODS: In vitro molecular and cellular biological analyses on genetically modified endothelial cells, in vivo vascular biological analyses on genetically engineered mouse models, and in silico systems biology and bioinformatics analyses on CD151-related events. RESULTS: Endothelial ablation of Cd151 leads to pulmonary and cardiac inflammation, severe sepsis, and perilous COVID-19, and endothelial CD151 becomes downregulated in inflammation. Mechanistically, CD151 restrains endothelial release of proinflammatory molecules for less leukocyte infiltration. At the subcellular level, CD151 determines the integrity of multivesicular bodies/lysosomes and confines the production of exosomes that carry cytokines such as ANGPT2 (angiopoietin-2) and proteases such as cathepsin-D. At the molecular level, CD151 docks VCP (valosin-containing protein)/p97, which controls protein quality via mediating deubiquitination for proteolytic degradation, onto endolysosomes to facilitate VCP/p97 function. At the endolysosome membrane, CD151 links VCP/p97 to (1) IFITM3 (interferon-induced transmembrane protein 3), which regulates multivesicular body functions, to restrain IFITM3-mediated exosomal sorting, and (2) V-ATPase, which dictates endolysosome pH, to support functional assembly of V-ATPase. CONCLUSIONS: Distinct from its canonical function in strengthening cell adhesion at cell surface, CD151 maintains endolysosome function by sustaining VCP/p97-mediated protein unfolding and turnover. By supporting protein quality control and protein degradation, CD151 prevents proteins from (1) buildup in endolysosomes and (2) discharge through exosomes, to limit vascular inflammation. Also, our study conceptualizes that balance between degradation and discharge of proteins in endothelial cells determines vascular information. Thus, the IFITM3/V-ATPase-tetraspanin-VCP/p97 complexes on endolysosome, as a protein quality control and inflammation-inhibitory machinery, could be beneficial for therapeutic intervention against vascular inflammation.

Single-cell genomics analysis reveals complex genetic interactions in an in vivo model of acquired BRAF inhibitor resistance.

The evolution of therapeutic resistance is a major obstacle to the success of targeted oncology drugs. While both inter- and intratumoral heterogeneity limit our ability to detect resistant subpopulations that pre-exist or emerge during treatment, our ability to analyze tumors with single-cell resolution is limited. Here, we utilized a cell-based transposon mutagenesis method to identify mechanisms of BRAF inhibitor resistance in a model of cutaneous melanoma. This screen identified overexpression of NEDD4L and VGLL3 as significant drivers of BRAF inhibitor resistance in vivo. In addition, we describe a novel single-cell genomics profiling method to genotype thousands of individual cells within tumors driven by transposon mutagenesis. This approach revealed a surprising genetic diversity among xenograft tumors and identified recurrent co-occurring mutations that emerge within distinct tumor subclones. Taken together, these observations reveal an unappreciated genetic complexity that drives BRAF inhibitor resistance.

Abl kinases can function as suppressors of tumor progression and metastasis.

Introduction: Abl family kinases function as proto-oncogenes in various leukemias, and pro-tumor functions have been discovered for Abl kinases in many solid tumors as well. However, a growing body of evidence indicates that Abl kinases can function to suppress tumor cell proliferation and motility and tumor growth in vivo in some settings. Methods: To investigate the role of Abl kinases in tumor progression, we used RNAi to generate Abl-deficient cells in a model of androgen receptor-indifferent, metastatic prostate cancer. The effect of Abl kinase depletion on tumor progression and metastasis was studied in an in vivo orthotopic model, and tumor cell motility, 3D growth, and signaling was studied in vitro. Results: Reduced Abl family kinase expression resulted in a highly aggressive, metastatic phenotype in vivo that was associated with AKT pathway activation, increased growth on 3D collagen matrix, and enhanced cell motility in vitro. Inhibiting AKT pathway signaling abolished the increased 3D growth of Abl-deficient cells, while treatment with the Abl kinase inhibitor, imatinib, promoted 3D growth of multiple additional tumor cell types. Moreover, Abl kinase inhibition also promoted soft-agar colony formation by pre-malignant fibroblasts. Conclusions: Collectively, our data reveal that Abl family kinases can function to suppress malignant cell phenotypes in vitro, and tumor progression and metastasis in vivo.

SRC-RAC1 signaling drives drug resistance to BRAF inhibition in de-differentiated cutaneous melanomas.

Rare gain-of-function mutations in RAC1 drive drug resistance to targeted BRAF inhibition in cutaneous melanoma. Here, we show that wildtype RAC1 is a critical driver of growth and drug resistance, but only in a subset of melanomas with elevated markers of de-differentiation. Similarly, SRC inhibition also selectively sensitized de-differentiated melanomas to BRAF inhibition. One possible mechanism may be the suppression of the de-differentiated state, as SRC and RAC1 maintained markers of de-differentiation in human melanoma cells. The functional differences between melanoma subtypes suggest that the clinical management of cutaneous melanoma can be enhanced by the knowledge of differentiation status. To simplify the task of classification, we developed a binary classification strategy based on a small set of ten genes. Using this gene set, we reliably determined the differentiation status previously defined by hundreds of genes. Overall, our study informs strategies that enhance the precision of BRAFi by discovering unique vulnerabilities of the de-differentiated cutaneous melanoma subtype and creating a practical method to resolve differentiation status.

PRC2 loss drives MPNST metastasis and matrix remodeling.

The histone methyltransferase PRC2 plays a complex role in cancer. Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with frequent loss-of-function mutations in PRC2 that are associated with poor outcome. Here, we identify a critical role for PRC2 loss in driving MPNST metastasis. PRC2-dependent metastatic phenotypes included increased collagen-dependent invasion, upregulation of matrix-remodeling enzymes, and elevated lung metastasis in orthotopic mouse models. Furthermore, clinical sample analysis determined that PRC2 loss correlated with metastatic disease, increased fibrosis, and decreased survival in patients with MPNSTs. These results may have broad implications for PRC2 function across multiple cancers and provide a strong rationale for investigating potential therapies targeting ECM-remodeling enzymes and tumor fibrosis to improve outcomes in patients with MPNSTs.

Functional Genomic Screening Independently Identifies CUL3 as a Mediator of Vemurafenib Resistance via Src-Rac1 Signaling Axis.

Patients with malignant melanoma have a 5-year survival rate of only 15-20% once the tumor has metastasized to distant tissues. While MAP kinase pathway inhibitors (MAPKi) are initially effective for the majority of patients with melanoma harboring BRAFV600E mutation, over 90% of patients relapse within 2 years. Thus, there is a critical need for understanding MAPKi resistance mechanisms. In this manuscript, we performed a forward genetic screen using a whole genome shRNA library to identify negative regulators of vemurafenib resistance. We identified loss of NF1 and CUL3 as drivers of vemurafenib resistance. NF1 is a known driver of vemurafenib resistance in melanoma through its action as a negative regulator of RAS. However, the mechanism by which CUL3, a key protein in E3 ubiquitin ligase complexes, is involved in vemurafenib resistance was unknown. We found that loss of CUL3 was associated with an increase in RAC1 activity and MEKS298 phosphorylation. However, the addition of the Src family inhibitor saracatinib prevented resistance to vemurafenib in CUL3KD cells and reversed RAC1 activation. This finding suggests that inhibition of the Src family suppresses MAPKi resistance in CUL3KD cells by inactivation of RAC1. Our results also indicated that the loss of CUL3 does not promote the activation of RAC1 through stabilization, suggesting that CUL3 is involved in the stability of upstream regulators of RAC1. Collectively, our study identifies the loss of CUL3 as a driver of MAPKi resistance through activation of RAC1 and demonstrates that inhibition of the Src family can suppress the MAPKi resistance phenotype in CUL3KD cells by inactivating RAC1 protein.

Cancer Cells Resist Mechanical Destruction in Circulation via RhoA/Actomyosin-Dependent Mechano-Adaptation.

During metastasis, cancer cells are exposed to potentially destructive hemodynamic forces including fluid shear stress (FSS) while en route to distant sites. However, prior work indicates that cancer cells are more resistant to brief pulses of high-level FSS in vitro relative to non-transformed epithelial cells. Herein, we identify a mechano-adaptive mechanism of FSS resistance in cancer cells. Our findings demonstrate that cancer cells activate RhoA in response to FSS, which protects them from FSS-induced plasma membrane damage. We show that cancer cells freshly isolated from mouse and human tumors are resistant to FSS, that formin and myosin II activity protects circulating tumor cells (CTCs) from destruction, and that short-term inhibition of myosin II delays metastasis in mouse models. Collectively, our data indicate that viable CTCs actively resist destruction by hemodynamic forces and are likely to be more mechanically robust than is commonly thought.

Src-Dependent DBL Family Members Drive Resistance to Vemurafenib in Human Melanoma.

The use of selective BRAF inhibitors (BRAFi) has produced remarkable outcomes for patients with advanced cutaneous melanoma harboring a BRAFV600E mutation. Unfortunately, the majority of patients eventually develop drug-resistant disease. We employed a genetic screening approach to identify gain-of-function mechanisms of BRAFi resistance in two independent melanoma cell lines. Our screens identified both known and unappreciated drivers of BRAFi resistance, including multiple members of the DBL family. Mechanistic studies identified a DBL/RAC1/PAK signaling axis capable of driving resistance to both current and next-generation BRAFis. However, we show that the SRC inhibitor, saracatinib, can block the DBL-driven resistance. Our work highlights the utility of our straightforward genetic screening method in identifying new drug combinations to combat acquired BRAFi resistance. SIGNIFICANCE: A simple, rapid, and flexible genetic screening approach identifies genes that drive resistance to MAPK inhibitors when overexpressed in human melanoma cells.

Chronic liver injury alters driver mutation profiles in hepatocellular carcinoma in mice.

Most hepatocellular carcinomas (HCCs) develop in a chronically injured liver, yet the extent to which this microenvironment promotes neoplastic transformation or influences selective pressures for genetic drivers of HCC remains unclear. We sought to determine the impact of hepatic injury in an established mouse model of HCC induced by Sleeping Beauty transposon mutagenesis. Chemically induced chronic liver injury dramatically increased tumor penetrance and significantly altered driver mutation profiles, likely reflecting distinct selective pressures. In addition to established human HCC genes and pathways, we identified several injury-associated candidates that represent promising loci for further study. Among them, we found that FIGN is overexpressed in human HCC and promotes hepatocyte invasion. We also validated Gli2's oncogenic potential in vivo, providing direct evidence that Hedgehog signaling can drive liver tumorigenesis in the context of chronic injury. Finally, we show that a subset of injury-associated candidate genes identifies two distinct classes of human HCCs. Further analysis of these two subclasses revealed significant trends among common molecular classification schemes of HCC. The genes and mechanisms identified here provide functional insights into the origin of HCC in a chronic liver damage environment. CONCLUSION: A chronically damaged liver microenvironment influences the genetic mechanisms that drive hepatocarcinogenesis. (Hepatology 2018;67:924-939).

α3ß1 Integrin Suppresses Prostate Cancer Metastasis via Regulation of the Hippo Pathway.

Existing anticancer strategies focused on disrupting integrin functions in tumor cells or tumor-involved endothelial cells have met limited success. An alternative strategy is to augment integrin-mediated pathways that suppress tumor progression, but how integrins can signal to restrain malignant behavior remains unclear. To address this issue, we generated an in vivo model of prostate cancer metastasis via depletion of α3ß1 integrin, a correlation observed in a significant proportion of prostate cancers. Our data describe a mechanism whereby α3ß1 signals through Abl family kinases to restrain Rho GTPase activity, support Hippo pathway suppressor functions, and restrain prostate cancer migration, invasion, and anchorage-independent growth. This α3ß1-Abl kinase-Hippo suppressor pathway identified α3 integrin-deficient prostate cancers as potential candidates for Hippo-targeted therapies currently under development, suggesting new strategies for targeting metastatic prostate cancer based on integrin expression. Our data also revealed paradoxical tumor suppressor functions for Abl kinases in prostate cancer that may help to explain the failure of Abl kinase inhibitor imatinib in prostate cancer clinical trials. Cancer Res; 76(22); 6577-87. ©2016 AACR.

TAZ and YAP are frequently activated oncoproteins in sarcomas.

TAZ (WWTR1) and YAP are transcriptional coactivators and oncoproteins inhibited by the Hippo pathway. Herein we evaluate 159 sarcomas representing the most prevalent sarcoma types by immunohistochemistry for expression and activation (nuclear localization) of TAZ and YAP. We show that 50% of sarcomas demonstrate activation of YAP while 66% of sarcomas demonstrate activated TAZ. Differential activation of TAZ and YAP are identified in various sarcoma types. At an RNA level, expression of WWTR1 or YAP1 predicts overall survival in undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma. Immunohistochemistry demonstrates that TAZ and YAP expression and activation are positively correlated with grade in the well-differentiated liposarcoma to dedifferentiated liposarcoma tumor progression sequence as well as conventional chondrosarcomas. TAZ and YAP are constitutively activated oncoproteins in sarcoma cell lines. Knock-down of TAZ and YAP demonstrate differential activity for the two proteins. Verteporfin decreases colony formation in soft agar as well as CTGF expression in sarcoma cell lines harboring activated TAZ and YAP.

CD151 promotes α3ß1 integrin-dependent organization of carcinoma cell junctions and restrains collective cell invasion.

Integrins function in collective migration both as major receptors for extracellular matrix and by crosstalk to adherens junctions. Despite extensive research, important questions remain about how integrin signaling mechanisms are integrated into collective migration programs. Tetraspanins form cell surface complexes with a subset of integrins and thus are good candidates for regulating the balance of integrin functional inputs into cell-matrix and cell-cell interactions. For example, tetraspanin CD151 directly associates with α3ß1 integrin in carcinoma cells and promotes rapid α3ß1-dependent single cell motility, but CD151 also promotes organized adherens junctions and restrains collective carcinoma cell migration on 2D substrates. However, the individual roles of CD151s integrin partners in CD151s pro-junction activity in carcinoma cells were not well understood. Here we find that CD151 promotes organized carcinoma cell junctions via α3ß1 integrin, by a mechanism that requires the a3b1 ligand, laminin-332. Loss of CD151 promotes collective 3D invasion and growth in vitro and in vivo, and the enhanced invasion of CD151-silenced cells is α3 integrin dependent, suggesting that CD151 can regulate the balance between α3ß1s pro-junction and pro-migratory activities in collective invasion. An analysis of human cancer cases revealed that changes in CD151 expression can be linked to either better or worse clinical outcomes depending on context, including potentially divergent roles for CD151 in different subsets of breast cancer cases. Thus, the role of the CD151-α3ß1 complex in carcinoma progression is context dependent, and may depend on the mode of tumor cell invasion.

Integrin α3ß1 can function to promote spontaneous metastasis and lung colonization of invasive breast carcinoma.

UNLABELLED: Significant evidence implicates α3ß1 integrin in promoting breast cancer tumorigenesis and metastasis-associated cell behaviors in vitro and in vivo. However, the extent to which α3ß1 is actually required for breast cancer metastasis remains to be determined. We used RNA interference to silence α3 integrin expression by approximately 70% in 4T1 murine mammary carcinoma cells, a model of aggressive, metastatic breast cancer. Loss of α3 integrin reduced adhesion, spreading, and proliferation on laminin isoforms, and modestly reduced the growth of orthotopically implanted cells. However, spontaneous metastasis to lung was strikingly curtailed. Experimental lung colonization after tail vein injection revealed a similar loss of metastatic capacity for the α3-silenced (α3si) cells, suggesting that critical, α3-dependent events at the metastatic site could account for much of α3ß1's contribution to metastasis in this model. Reexpressing α3 in the α3si cells reversed the loss of metastatic capacity, and silencing another target, the small GTPase RhoC, had no effect, supporting the specificity of the effect of silencing α3. Parental, α3si, and α3-rescued cells, all secreted abundant laminin α5 (LAMA5), an α3ß1 integrin ligand, suggesting that loss of α3 integrin might disrupt an autocrine loop that could function to sustain metastatic growth. Analysis of human breast cancer cases revealed reduced survival in cases where α3 integrin and LAMA5 are both overexpressed. IMPLICATIONS: α3 integrin or downstream effectors may be potential therapeutic targets in disseminated breast cancers, especially when laminin α5 or other α3 integrin ligands are also over-expressed.

The CD9/CD81 tetraspanin complex and tetraspanin CD151 regulate α3ß1 integrin-dependent tumor cell behaviors by overlapping but distinct mechanisms.

Integrin α3ß1 potently promotes cell motility on its ligands, laminin-332 and laminin-511, and this may help to explain why α3ß1 has repeatedly been linked to breast carcinoma progression and metastasis. The pro-migratory functions of α3ß1 depend strongly on lateral interactions with cell surface tetraspanin proteins. Tetraspanin CD151 interacts directly with the α3 integrin subunit and links α3ß1 integrin to other tetraspanins, including CD9 and CD81. Loss of CD151 disrupts α3ß1 association with other tetraspanins and impairs α3ß1-dependent motility. However, the extent to which tetraspanins other than CD151 are required for specific α3ß1 functions is unclear. To begin to clarify which aspects of α3ß1 function require which tetraspanins, we created breast carcinoma cells depleted of both CD9 and CD81 by RNA interference. Silencing both of these closely related tetraspanins was required to uncover their contributions to α3ß1 function. We then directly compared our CD9/CD81-silenced cells to CD151-silenced cells. Both CD9/CD81-silenced cells and CD151-silenced cells showed delayed α3ß1-dependent cell spreading on laminin-332. Surprisingly, however, once fully spread, CD9/CD81-silenced cells, but not CD151-silenced cells, displayed impaired α3ß1-dependent directed motility and altered front-rear cell morphology. Also unexpectedly, the CD9/CD81 complex, but not CD151, was required to promote α3ß1 association with PKCα in breast carcinoma cells, and a PKC inhibitor mimicked aspects of the CD9/CD81-silenced cell motility defect. Our data reveal overlapping, but surprisingly distinct contributions of specific tetraspanins to α3ß1 integrin function. Importantly, some of CD9/CD81's α3ß1 regulatory functions may not require CD9/CD81 to be physically linked to α3ß1 by CD151.

Integrin α3ß1 regulates tumor cell responses to stromal cells and can function to suppress prostate cancer metastatic colonization.

Integrin α3ß1 promotes tumor cell adhesion, migration, and invasion on laminin isoforms, and several clinical studies have indicated a correlation between increased tumoral α3ß1 integrin expression and tumor progression, metastasis, and poor patient outcomes. However, several other clinical and experimental studies have suggested that α3ß1 can possess anti-metastatic activity in certain settings. To help define the range of α3ß1 functions in tumor cells in vivo, we used RNAi to silence the α3 integrin subunit in an aggressive, in vivo-passaged subline of PC-3 prostate carcinoma cells. Loss of α3 integrin impaired adhesion and proliferation on the α3ß1 integrin ligand, laminin-332 in vitro. Despite these deficits in vitro, the α3-silenced cells were significantly more aggressive in a lung colonization model in vivo, with a substantially increased rate of tumor growth that significantly reduced survival. In contrast, silencing the related α6 integrin subunit delayed metastatic growth in vivo. The increased colonization of α3-silenced tumor cells in vivo was recapitulated in 3D collagen co-cultures with lung fibroblasts or pre-osteoblast-like cells, where α3-silenced cells showed dramatically enhanced growth. The increased response of α3-silenced tumor cells to stromal cells in co-culture could be reproduced by fibroblast conditioned medium, which contains one or more heparin-binding factors that selectively favor the growth of α3-silenced cells. Our new data suggest a scenario in which α3ß1 regulates tumor-host interactions within the metastatic tumor microenvironment to limit growth, providing some of the first direct evidence that specific loss of α3 function in tumor cells can have pro-metastatic consequences in vivo.

Loss of LARGE2 disrupts functional glycosylation of α-dystroglycan in prostate cancer.

Dystroglycan (DG) is a cell surface receptor for extracellular matrix proteins and is involved in cell polarity, matrix organization, and mechanical stability of tissues. Previous studies documented loss of DG protein expression and glycosylation in a variety of cancer types, but the underlying mechanisms and the functional consequences with respect to cancer progression remain unclear. Here, we show that the level of expression of the ßDG subunit as well as the glycosylation status of the αDG subunit inversely correlate with the Gleason scores of prostate cancers; furthermore, we show that the functional glycosylation of αDG is substantially reduced in prostate cancer metastases. Additionally, we demonstrate that LARGE2 (GYLTL1B), a gene not previously implicated in cancer, regulates functional αDG glycosylation in prostate cancer cell lines; knockdown of LARGE2 resulted in hypoglycosylation of αDG and loss of its ability to bind laminin-111 while overexpression restored ligand binding and diminished growth and migration of an aggressive prostate cancer cell line. Finally, our analysis of LARGE2 expression in human cancer specimens reveals that LARGE2 is significantly down-regulated in the context of prostate cancer, and that its reduction correlates with disease progression. Our results describe a novel molecular mechanism to account for the commonly observed hypoglycosylation of αDG in prostate cancer.

Integrin-associated CD151 drives ErbB2-evoked mammary tumor onset and metastasis.

ErbB2+ human breast cancer is a major clinical problem. Prior results have suggested that tetraspanin CD151 might contribute to ErbB2-driven breast cancer growth, survival, and metastasis. In other cancer types, CD151 sometimes supports tumor growth and metastasis. However, a definitive test of CD151 effects on de novo breast cancer initiation, growth, and metastasis has not previously been done. We used CD151 gene-deleted mice expressing the MMTV-ErbB2 transgene to show that CD151 strongly supports ErbB2+ mammary tumor initiation and metastasis. Delayed tumor onset (by 70-100 days) in the absence of CD151 was accompanied by reduced survival of mammary epithelial cells and impaired activation of FAK- and MAPK-dependent pathways. Both primary tumors and metastatic nodules showed smooth, regular borders, consistent with a less invasive phenotype. Furthermore, consistent with impaired oncogenesis and decreased metastasis, CD151-targeted MCF-10A/ErbB2 cells showed substantial decreases in three-dimensional colony formation, EGF-stimulated tumor cell motility, invasion, and transendothelial migration. These CD151-dependent functions were largely mediated through α6ß4 integrin. Moreover, CD151 ablation substantially prevented PKC- and EGFR/ERK-dependent α6ß4 integrin phosphorylation, consistent with retention of epithelial cell polarity and intermediate filament cytoskeletal connections, which helps to explain diminished metastasis. Finally, clinical data analyses revealed a strong correlation between CD151 and ErbB2 expression and metastasis-free survival of breast cancer patients. In conclusion, we provide strong evidence that CD151 collaborates with LB integrins (particularly α6ß4 and ErbB2 (and EGFR) receptors to regulate multiple signaling pathways, thereby driving mammary tumor onset, survival, and metastasis. Consequently, CD151 is a useful therapeutic target in malignant ErbB2+ breast cancer.

ALCAM regulates motility, invasiveness, and adherens junction formation in uveal melanoma cells.

ALCAM, a member of the immunoglobulin superfamily, has been implicated in numerous developmental events and has been repeatedly identified as a marker for cancer metastasis. Previous studies addressing ALCAM's role in cancer have, however, yielded conflicting results. Depending on the tumor cell type, ALCAM expression has been reported to be both positively and negatively correlated with cancer progression and metastasis in the literature. To better understand how ALCAM might regulate cancer cell behavior, we utilized a panel of defined uveal melanoma cell lines with high or low ALCAM levels, and directly tested the effects of manipulating these levels on cell motility, invasiveness, and adhesion using multiple assays. ALCAM expression was stably silenced by shRNA knockdown in a high-ALCAM cell line (MUM-2B); the resulting cells displayed reduced motility in gap-closure assays and a reduction in invasiveness as measured by a transwell migration assay. Immunostaining revealed that the silenced cells were defective in the formation of adherens junctions, at which ALCAM colocalizes with N-cadherin and ß-catenin in native cells. Additionally, we stably overexpressed ALCAM in a low-ALCAM cell line (MUM-2C); intriguingly, these cells did not exhibit any increase in motility or invasiveness, indicating that ALCAM is necessary but not sufficient to promote metastasis-associated cell behaviors. In these ALCAM-overexpressing cells, however, recruitment of ß-catenin and N-cadherin to adherens junctions was enhanced. These data confirm a previously suggested role for ALCAM in the regulation of adherens junctions, and also suggest a mechanism by which ALCAM might differentially enhance or decrease invasiveness, depending on the type of cadherin adhesion complexes present in tissues surrounding the primary tumor, and on the cadherin status of the tumor cells themselves.

Structure-function analysis of tetraspanin CD151 reveals distinct requirements for tumor cell behaviors mediated by α3ß1 versus α6ß4 integrin.

The basement membrane protein laminin-332 (laminin-5) mediates both stable cell adhesion and rapid cell migration and thus has the potential to either restrain or promote tumor cell metastasis. The major cellular receptors for laminin-332 are integrin α3ß1, which mediates rapid tumor cell migration, and integrin α6ß4, which often mediates stable cell attachment. Tetraspanin protein CD151 interacts directly with both α3ß1 and α6ß4 integrins and with other tetraspanins, thereby promoting α3ß1 and α6ß4 association with tetraspanin-enriched microdomains on the cell surface. To explore the possibility of selectively modulating tumor cell responses to laminin-332, we re-expressed a series of CD151 mutants in epidermoid carcinoma cells with near total, RNAi-mediated silencing of endogenous CD151. The interactions of CD151 with its integrin partners or its interactions with other tetraspanins were selectively disrupted by specific mutations in the CD151 large extracellular loop (EC2 domain) or in intracellular CD151 palmitoylation sites, respectively. CD151-integrin association and CD151-tetraspanin association were both important for α3ß1 integrin-dependent initial adhesion and rapid migration on laminin-332. Remarkably, however, only CD151-integrin association was required for stable, α6ß4 integrin-dependent cell attachment on laminin-332. In addition, we found that a QRD amino acid motif in the CD151 EC2 domain, which had been thought to be crucial for CD151-integrin interaction, is not essential for CD151-integrin association or for the ability of CD151 to promote several different integrin functions. These new data suggest potential strategies for selectively modulating migratory cell responses to laminin-332, while leaving stable cell attachment on laminin-332 intact.

ZEB1 coordinately regulates laminin-332 and {beta}4 integrin expression altering the invasive phenotype of prostate cancer cells.

Metastasis involves the invasion of cancer cells across both the extracellular matrix and cellular barriers, and an evolving theme is that epithelial-to-mesenchymal transition (EMT) may mediate invasive cellular behavior. Previously, we isolated and analyzed a subpopulation of PC-3 prostate cancer cells, TEM4-18, and found that these cells both invaded an endothelial barrier more efficiently and exhibited enhanced metastatic colonization in vivo. Transendothelial migration of these cells depended on expression of ZEB1, a known regulator of EMT. Surprisingly, these cells were much less invasive than parental PC-3 cells in assays that involve matrix barriers. Here, we report that TEM4-18 cells express significantly reduced levels of two subunits of laminin-332 (ß3 and γ2) and that exogenous laminin-332, or co-culture with laminin-332-expressing cells, rescues the in vitro invasion phenotype in these cells. Stable knockdown of ZEB1 in prostate cancer cells up-regulated LAMC2 and ITGB4 mRNA and protein and resulted in a concomitant increase in Transwell migration. Using chromatin immunoprecipitation (ChIP), we show that ZEB1 directly interacts with the promoters of LAMC2 and ITGB4. These results provide a novel molecular basis for reduced laminin-332 observed in clinical prostate cancer specimens and demonstrate a context-dependent role for EMT in invasive cellular behavior.