Filopodome Mapping Identifies p130Cas as a Mechanosensitive Regulator of Filopodia Stability.

Filopodia are adhesive cellular protrusions specialized in the detection of extracellular matrix (ECM)-derived cues. Although ECM engagement at focal adhesions is known to trigger the recruitment of hundreds of proteins ("adhesome") to fine-tune cellular behavior, the components of the filopodia adhesions remain undefined. Here, we performed a structured-illumination-microscopy-based screen to map the localization of 80 target proteins, linked to cell adhesion and migration, within myosin-X-induced filopodia. We demonstrate preferential enrichment of several adhesion proteins to either filopodia tips, filopodia shafts, or shaft subdomains, suggesting divergent, spatially restricted functions for these proteins. Moreover, proteins with phosphoinositide (PI) binding sites are particularly enriched in filopodia. This, together with the strong localization of PI(3,4)P2 in filopodia tips, predicts critical roles for PIs in regulating filopodia ultra-structure and function. Our mapping further reveals that filopodia adhesions consist of a unique set of proteins, the filopodome, that are distinct from classical nascent adhesions, focal adhesions, and fibrillar adhesions. Using live imaging, we observe that filopodia adhesions can give rise to nascent adhesions, which, in turn, form focal adhesions. We demonstrate that p130Cas (BCAR1) is recruited to filopodia tips via its C-terminal Cas family homology domain (CCHD) and acts as a mechanosensitive regulator of filopodia stability. Finally, we demonstrate that our map based on myosin-X-induced filopodia can be translated to endogenous filopodia and fascin- and IRSp53-mediated filopodia.

Essential role of SH3GL1 in interleukin-6(IL-6)- and vascular endothelial growth factor (VEGF)-triggered p130cas-mediated proliferation and migration of osteosarcoma cells.

We recently demonstrated that interleukin-6 (IL-6)- and vascular endothelial growth factor (VEGF)-induced osteosarcoma (OS) cell proliferation and migration are parallel to significant increased expression of SH3GL1 and the phosphorylation level of P130cas. The expression level of SH3GL1 was widely upregulated in human OS tissues, and their overexpression was significantly correlated with more aggressive clinicopathological features. Conversely, depletion of SH3GL1 by adenovirus shRNA abrogates P130cas phosphorylation and IL-6- and VEGF-induced OS cell proliferation and migration. To further pinpoint the mechanism how SH3GL1 was responsible for cell proliferation and migration, we deleted SH3GL1 in vitro and in vivo. In vitro, depletion of SH3GL1 abrogates P130cas phosphorylation and IL-6- and VEGF-induced OS cell proliferation and migration. SH3GL1 knockdown caused cell cycle arrest in G0/G1 phase via downregulation of cyclin D1, caused activation of p27KIP, and attenuated the activation of p-Rb. Interestingly, SH3GL1 knockdown also markedly attenuated the phosphorylation level of Akt/GSK-3ß/FAK. In vivo, depletion of SH3GL1 by shRNA inhibited the tumor tissue growth and the expression of p-P130cas. Collectively, our results strongly suggest that SH3GL1 is a novel target for anti-osteosarcoma.

A truncated phosphorylated p130Cas substrate domain is sufficient to drive breast cancer growth and metastasis formation in vivo.

Elevated p130Cas (Crk-associated substrate) levels are found in aggressive breast tumors and are associated with poor prognosis and resistance to standard therapeutics in patients. p130Cas signals majorly through its phosphorylated substrate domain (SD) that contains 15 tyrosine motifs (YxxP) which recruit effector molecules. Tyrosine phosphorylation of p130Cas is important for mediating migration, invasion, tumor promotion, and metastasis. We previously developed a Src*/SD fusion molecule approach, where the SD is constitutively phosphorylated. In a polyoma middle T-antigen (PyMT)/Src*/SD double-transgenic mouse model, Src*/SD accelerates PyMT-induced tumor growth and promotes a more aggressive phenotype. To test whether Src*/SD also drives metastasis and which of the YxxP motifs are involved in this process, full-length and truncated SD molecules fused to Src* were expressed in breast cancer cells. The functionality of the Src*/SD fragments was analyzed in vitro, and the active proteins were tested in vivo in an orthotopic mouse model. Breast cancer cells expressing the full-length SD and the functional smaller SD fragment (spanning SD motifs 6-10) were injected into the mammary fat pads of mice. The tumor progression was monitored by bioluminescence imaging and caliper measurements. Compared with control animals, the complete SD promoted primary tumor growth and an earlier onset of metastases. Importantly, both the complete and truncated SD significantly increased the occurrence of metastases to multiple organs. These studies provide strong evidence that the phosphorylated p130Cas SD motifs 6-10 (Y236, Y249, Y267, Y287, and Y306) are important for driving mammary carcinoma progression.

The proto-oncogene c-Src and its downstream signaling pathways are inhibited by the metastasis suppressor, NDRG1.

N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor that plays a key role in regulating signaling pathways involved in mediating cancer cell invasion and migration, including those derived from prostate, colon, etc. However, the mechanisms and molecular targets through which NDRG1 reduces cancer cell invasion and migration, leading to inhibition of cancer metastasis, are not fully elucidated. In this investigation, using NDRG1 over-expression models in three tumor cell-types (namely, DU145, PC3MM and HT29) and also NDRG1 silencing in DU145 and HT29 cells, we reveal that NDRG1 decreases phosphorylation of a key proto-oncogene, cellular Src (c-Src), at a well-characterized activating site (Tyr416). NDRG1-mediated down-regulation of EGFR expression and activation were responsible for the decreased phosphorylation of c-Src (Tyr416). Indeed, NDRG1 prevented recruitment of c-Src to EGFR and c-Src activation. Moreover, NDRG1 suppressed Rac1 activity by modulating phosphorylation of a c-Src downstream effector, p130Cas, and its association with CrkII, which acts as a "molecular switch" to activate Rac1. NDRG1 also affected another signaling molecule involved in modulating Rac1 signaling, c-Abl, which then inhibited CrkII phosphorylation. Silencing NDRG1 increased cell migration relative to the control and inhibition of c-Src signaling using siRNA, or a pharmacological inhibitor (SU6656), prevented this increase. Hence, the role of NDRG1 in decreasing cell migration is, in part, due to its inhibition of c-Src activation. In addition, novel pharmacological agents, which induce NDRG1 expression and are currently under development as anti-metastatic agents, markedly increase NDRG1 and decrease c-Src activation. This study leads to important insights into the mechanism involved in inhibiting metastasis by NDRG1 and how to target these pathways with novel therapeutics.

CAS proteins in health and disease: an update.

The CAS family of scaffolding proteins has increasingly attracted scrutiny as important for regulation of cancer-associated signaling. BCAR1 (also known as p130Cas), NEDD9 (HEF1, Cas-L), EFS (Sin), and CASS4 (HEPL) are regulated by and mediate cell attachment, growth factor, and chemokine signaling. Altered expression and activity of CAS proteins are now known to promote metastasis and drug resistance in cancer, influence normal development, and contribute to the pathogenesis of heart and pulmonary disease. In this article, we provide an update on recently published studies describing signals regulating and regulated by CAS proteins, and evidence for biological activity of CAS proteins in normal development, cancer, and other pathological conditions.

SRChing for the substrates of Src.

By the mid 1980's, it was clear that the transforming activity of oncogenic Src was linked to the activity of its tyrosine kinase domain and attention turned to identifying substrates, the putative next level of control in the pathway to transformation. Among the first to recognize the potential of phosphotyrosine-specific antibodies, Parsons and colleagues launched a risky shotgun-based approach that led ultimately to the cDNA cloning and functional characterization of many of today's best-known Src substrates (for example, p85-Cortactin, p110-AFAP1, p130Cas, p125FAK and p120-catenin). Two decades and over 6000 citations later, the original goals of the project may be seen as secondary to the enormous impact of these protein substrates in many areas of biology. At the request of the editors, this review is not restricted to the current status of the substrates, but reflects also on the anatomy of the project itself and some of the challenges and decisions encountered along the way.

Cas proteins: dodgy scaffolding in breast cancer.

The members of the Cas protein family (p130Cas/BCAR1, Nedd9/HEF1, EFS and CASS4) are scaffold proteins required for the assembly of signal transduction complexes in response to several stimuli, such as growth factors, hormones and extracellular matrix components. Given their ability to integrate and coordinate multiple signalling events, Cas proteins have emerged as crucial players in the control of mammary cell proliferation, survival and differentiation. More importantly, it has been found that alterations of their expression levels result in aberrant signalling cascades, which promote initiation and progression of breast cancer. Based on the increasing data from in vitro, mouse model and clinical studies, in this review we will focus on two Cas proteins, p130Cas/BCAR1 and Nedd9, and their coupled signalling pathways, to examine their role in mammary cell transformation and in the acquirement of invasiveness and drug resistance of breast cancer cells.

Skeletal muscle adaptation in response to mechanical stress in p130cas-/- mice.

Mammalian skeletal muscles undergo adaptation in response to changes in the functional demands upon them, involving mechanical-stress-induced cellular signaling called "mechanotransduction." We hypothesized that p130Cas, which is reported to act as a mechanosensor that transduces mechanical extension into cellular signaling, plays an important role in maintaining and promoting skeletal muscle adaptation in response to mechanical stress via the p38 MAPK signaling pathway. We demonstrate that muscle-specific p130Cas-/- mice express the contractile proteins normally in skeletal muscle. Furthermore, muscle-specific p130Cas-/- mice show normal mechanical-stress-induced muscle adaptation, including exercise-induced IIb-to-IIa muscle fiber type transformation and hypertrophy. Finally, we provide evidence that exercise-induced p38 MAPK signaling is not impaired by the muscle-specific deletion of p130Cas. We conclude that p130Cas plays a limited role in mechanical-stress-induced skeletal muscle adaptation.

The RB family is required for the self-renewal and survival of human embryonic stem cells.

The mechanisms ensuring the long-term self-renewal of human embryonic stem cells are still only partly understood, limiting their use in cellular therapies. Here we found that increased activity of the RB cell cycle inhibitor in human embryonic stem cells induces cell cycle arrest, differentiation and cell death. Conversely, inactivation of the entire RB family (RB, p107 and p130) in human embryonic stem cells triggers G2/M arrest and cell death through functional activation of the p53 pathway and the cell cycle inhibitor p21. Differences in E2F target gene activation upon loss of RB family function between human embryonic stem cells, mouse embryonic stem cells and human fibroblasts underscore key differences in the cell cycle regulatory networks of human embryonic stem cells. Finally, loss of RB family function promotes genomic instability in both human and mouse embryonic stem cells, uncoupling cell cycle defects from chromosomal instability. These experiments indicate that a homeostatic level of RB activity is essential for the self-renewal and the survival of human embryonic stem cells.

E2F4 plays a key role in Burkitt lymphoma tumorigenesis.

Sporadic Burkitt lymphoma (sBL) is a rapidly growing B-cell non-Hodgkin's lymphoma whose treatment requires highly aggressive therapies that often result severely toxic. Identification of proteins whose expression or function is deregulated in sBL and play a role in its formation could facilitate development of less toxic therapies. We have previously shown that E2F1 expression is deregulated in sBL. We have now investigated the mechanisms underlying E2F1 deregulation and found that the E2F sites in its promoter fail to repress its transcriptional activity in BL cells and that the transcriptional repressor E2F4 barely interacts with these sites. We also have found that E2F4 protein levels, but not those of its mRNA, are reduced in sBL cell lines relative to immortal B-cell lines. E2F4 protein expression is also decreased in 24 of 26 sBL tumor samples from patients compared with control tissues. Our data demonstrate that enforced E2F4 expression in BL cells not only diminishes E2F1 levels, but also reduces selectively the tumorigenic properties and proliferation of BL cells, while increasing their accumulation in G(2)/M. Our results therefore point to E2F4 as a target for developing novel and less toxic treatments for sBL.

BCAR1 expression improves prediction of biochemical reccurence after radical prostatectomy.

BACKGROUND: Because prostate cancer exhibits a great variability in clinical outcome, biomarkers that can be used in daily practice are needed to better stratify patients into prognostic groups. Since steroid hormones play a central role in the development and progression of prostate cancer, we aimed to analyze in a matched nested case-control study the value of molecules involved in steroid signaling, to predict recurrence after radical prostatectomy, independently from standard prognostic tools. METHODS: Among 1,200 patients treated by radical prostatectomy with negative margins with at least 4 years follow-up, 121 prostate cancers with biochemical relapse were matched after pathological reassessment with 121 cancers with identical clinicopathological features but without relapse. Immunohistochemistry was performed on tissue microarrays, using antibodies directed against molecules involved in androgen and estrogen signaling, including hormone receptors, enzymes (such as the five alpha reductases 1,2 and 3, aromatase, alpha-keto reductase 1C3 and squalene epoxidase), the breast cancer antiestrogen resistance 1 (BCAR1), and the proliferation marker Ki67. RESULTS: The median follow-up for patients without recurrence was 7 years. Both cell proliferation and BCAR1 expression were significantly associated with biochemical relapse, in univariate and multivariate analysis. In subgroup analysis, the sole predictive marker in patients with well-differentiated prostate cancer was BCAR1 (P = 0.004), whereas only proliferation (P = 0.001) was significantly associated with relapse in less-differentiated prostate cancer patients. CONCLUSIONS: BCAR1 is an independent predictor of recurrence after radical prostatectomy for "low risk" prostate cancer. The use of this biomarker may enable more individualized treatment approaches.

Comparing mechano-transduction in fibroblasts deficient of focal adhesion proteins.

Mechano-transduction was studied in wildtype and focal adhesion (FA) protein-deficient mouse embryonic fibroblasts (MEFs). Using a cell stretcher, we determined the effect of stretch on cell morphology, apoptosis, and phosphorylation of ERK(1/2). After 20% cyclic, uniaxial stretch, FA-deficient MEFs showed morphological changes and levels of apoptosis of the order: focal adhesion kinase>p130Cas>vinculin compared to wildtype cells. ERK(1/2) phosphorylation peaked in wildtype cells at around 10 min, and in all FA-deficient cells at around 5 min. The relative change in strain energy of FA-deficient cells compared to wildtype cells was of the order: vinculin>FAK>p130Cas. Taken together, FAK and p130Cas are more important in the stretch-mediated downstream signaling and cell survival pathway, while vinculin is more critical in maintaining cell contractility.

P130Cas attenuates epidermal growth factor (EGF) receptor internalization by modulating EGF-triggered dynamin phosphorylation.

BACKGROUND: Endocytosis controls localization-specific signal transduction via epidermal growth factor receptor (EGFR), as well as downregulation of that receptor. Extracellular matrix (ECM)-integrin coupling induces formation of macromolecular complexes that include EGFR, integrin, Src kinase and p130Cas, resulting in EGFR activation. In addition, cell adhesion to ECM increases EGFR localization at the cell surface and reduces EGFR internalization. The molecular mechanisms involved are not yet well understood. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the molecular mechanism by which p130Cas affects the endocytic regulation of EGFR. Biochemical quantification revealed that cell adhesion to fibronectin (FN) increases total EGFR levels and its phosphorylation, and that p130Cas is required for this process. Measurements of Texas Red-labeled EGF uptake and cell surface EGFR revealed that p130Cas overexpression reduces EGF-induced EGFR internalization, while p130Cas depletion enhances it. In addition, both FN-mediated cell adhesion and p130Cas overexpression reduce EGF-stimulated dynamin phosphorylation, which is necessary for EGF-induced EGFR internalization. Coimmunoprecipitation and GST pull-down assays confirmed the interaction between p130Cas and dynamin. Moreover, a SH3-domain-deleted form of p130Cas, which shows diminished binding to dynamin, inhibits dynamin phosphorylation and EGF uptake less effectively than wild-type p130Cas. CONCLUSIONS/SIGNIFICANCE: Our results show that p130Cas plays an inhibitory role in EGFR internalization via its interaction with dynamin. Given that the EGFR internalization process determines signaling density and specificity in the EGFR pathway, these findings suggest that the interaction between p130Cas and dynamin may regulate EGFR trafficking and signaling in the same manner as other endocytic regulatory proteins related to EGFR endocytosis.

Systematizing and cloning of genes involved in the cerebellar cortex circuit development.

The cerebellar cortical circuit of mammals develops via a series of magnificent cellular events in the postnatal stage of development to accomplish the formation of functional circuit architectures. The contribution of genetic factors is thought to be crucial to cerebellar development. Therefore, it is essential to analyze the underlying transcriptome during development to understand the genetic blueprint of the cerebellar cortical circuit. In this review, we introduce the profiling of large numbers of spatiotemporal gene expression data obtained by developmental time-series microarray analyses and in situ hybridization cellular mRNA mapping, and the creation of a neuroinformatics database called the Cerebellar Development Transcriptome Database. Using this database, we have identified thousands of genes that are classified into various functional categories and are expressed coincidently with related cellular developmental stages. We have also suggested the molecular mechanisms of cerebellar development by functional characterization of several identified genes (Cupidin, p130Cas, very-KIND, CAPS2) responsible for distinct cellular events of developing cerebellar granule cells. Taken together, the gene expression profiling during the cerebellar development demonstrates that the development of cerebellar cortical circuit is attributed to the complex but orchestrated transcriptome.

P130Cas Src-binding and substrate domains have distinct roles in sustaining focal adhesion disassembly and promoting cell migration.

The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas -/- mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif were able to partially restore the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly attempts. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation.

Relevance of BCAR4 in tamoxifen resistance and tumour aggressiveness of human breast cancer.

BACKGROUND: Breast cancer anti-oestrogen resistance 4 (BCAR4) was identified in a search for genes involved in anti-oestrogen resistance in breast cancer. We explored whether BCAR4 is predictive for tamoxifen resistance and prognostic for tumour aggressiveness, and studied its function. METHODS: BCAR4 mRNA levels were measured in primary breast tumours, and evaluated for association with progression-free survival (PFS) and clinical benefit in patients with oestrogen receptor (ERα)-positive tumours receiving tamoxifen as first-line monotherapy for advanced disease. In a separate cohort of patients with lymph node-negative, ERα-positive cancer, and not receiving systemic adjuvant therapy, BCAR4 levels were evaluated for association with distant metastasis-free survival (MFS). The function of BCAR4 was studied with immunoblotting and RNA interference in a cell model. RESULTS: Multivariate analyses established high BCAR4 mRNA levels as an independent predictive factor for poor PFS after start of tamoxifen therapy for recurrent disease. High BCAR4 mRNA levels were associated with poor MFS and overall survival, reflecting tumour aggressiveness. In BCAR4-expressing cells, phosphorylation of v-erb-b2 erythroblastic leukaemia viral oncogene homolog (ERBB)2, ERBB3, and their downstream mediators extracellular signal-regulated kinase 1/2 and v-akt murine thymoma viral oncogene homolog (AKT) 1/2, was increased. Selective knockdown of ERBB2 or ERBB3 inhibited proliferation, confirming their role in BCAR4-induced tamoxifen resistance. CONCLUSION: BCAR4 may have clinical relevance for tumour aggressiveness and tamoxifen resistance. Our cell model suggests that BCAR4-positive breast tumours are driven by ERBB2/ERBB3 signalling. Patients with such tumours may benefit from ERBB-targeted therapy.

p130Cas, Crk-associated substrate plays essential roles in liver development by regulating sinusoidal endothelial cell fenestration.

UNLABELLED: p130Cas, Crk-associated substrate (Cas), is an adaptor/scaffold protein that plays a central role in actin cytoskeletal reorganization. We previously showed that mice in which Cas was deleted (Cas(-/-)) died in utero because of early cardiovascular maldevelopment. To further investigate the in vivo roles of Cas, we generated mice with a hypomorphic Cas allele lacking the exon 2-derived region (Cas(Deltaex2/Deltaex2)), which encodes Src homology domain 3 (SH3) of Cas. Cas(Deltaex2/Deltaex2) mice again died as embryos, but they particularly showed progressive liver degeneration with hepatocyte apoptosis. Because Cas expression in the liver is preferentially detected in sinusoidal endothelial cells (SECs), the observed hepatocyte apoptosis was most likely ascribable to impaired function of SECs. To address this possibility, we stably introduced a Cas mutant lacking the SH3 domain (Cas DeltaSH3) into an SEC line (NP31). Intriguingly, the introduction of Cas DeltaSH3 induced a loss of fenestrae, the characteristic cell-penetrating pores in SECs that serve as a critical route for supplying oxygen and nutrients to hepatocytes. The disappearance of fenestrae in Cas DeltaSH3-expressing cells was associated with an attenuation of actin stress fiber formation, a marked reduction in tyrosine phosphorylation of Cas, and defective binding of Cas to CrkII. CONCLUSION: Cas plays pivotal roles in liver development through the reorganization of the actin cytoskeleton and formation of fenestrae in SECs.

p130Cas is an essential transducer element in ErbB2 transformation.

The ErbB2 oncogene is often overexpressed in breast tumors and associated with poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration, and proliferation in normal and pathological cells. The functional role of p130Cas in ErbB2-dependent breast tumorigenesis was assessed by its silencing in breast cancer cells derived from mouse mammary tumors overexpressing ErbB2 (N202-1A cells), and by its reexpression in ErbB2-transformed p130Cas-null mouse embryonic fibroblasts. We demonstrate that p130Cas is necessary for ErbB2-dependent foci formation, anchorage-independent growth, and in vivo growth of orthotopic N202-1A tumors. Moreover, intranipple injection of p130Cas-stabilized siRNAs in the mammary gland of Balbc-NeuT mice decreases the growth of spontaneous tumors. In ErbB2-transformed cells, p130Cas is a crucial component of a functional molecular complex consisting of ErbB2, c-Src, and Fak. In human mammary cells, MCF10A.B2, the concomitant activation of ErbB2, and p130Cas overexpression sustain and strengthen signaling, leading to Rac1 activation and MMP9 secretion, thus providing invasive properties. Consistently, p130Cas drives N202-1A cell in vivo lung metastases colonization. These results demonstrate that p130Cas is an essential transducer in ErbB2 transformation and highlight its potential use as a novel therapeutic target in ErbB2 positive human breast cancers.

CAS proteins in normal and pathological cell growth control.

Proteins of the CAS (Crk-associated substrate) family (BCAR1/p130Cas, NEDD9/HEF1/Cas-L, EFS/SIN and CASS4/HEPL) are integral players in normal and pathological cell biology. CAS proteins act as scaffolds to regulate protein complexes controlling migration and chemotaxis, apoptosis, cell cycle, and differentiation, and have more recently been linked to a role in progenitor cell function. Reflecting these complex functions, over-expression of CAS proteins has now been strongly linked to poor prognosis and increased metastasis in cancer, as well as resistance to first-line chemotherapeutics in multiple tumor types including breast and lung cancers, glioblastoma, and melanoma. Further, CAS proteins have also been linked to additional pathological conditions including inflammatory disorders, Alzheimer's and Parkinson's disease, as well as developmental defects. This review will explore the roles of the CAS proteins in normal and pathological states in the context of the many mechanistic insights into CAS protein function that have emerged in the past decade.