Tensin-1(TNS1)is a 220 kD focal adhesion protein that binds to actin filaments,integrin receptors,small GTPases,tyrosine-phosphorylated proteins,and lipids.1 These binding activities enable TNS1 to link the actin cytoskeleton to integrins and transduce outside-in and inside-out signals at focal adhesion sites,thereby regulating cell attachment,migration,prolif-eration,and mechanical sensing.

Supplementary Figure Legends 1-3 from DLC1 Negatively Regulates Angiogenesis in a Paracrine Fashion

Supplementary Figure 2 from DLC1 Negatively Regulates Angiogenesis in a Paracrine Fashion

The differentiation of oligodendroglia from oligodendrocyte precursor cells (OPCs) to complex and extensive myelinating oligodendrocytes (OLs) is a multistep process that involves large-scale morphological changes with significant strain on the cytoskeleton. While key chromatin and transcriptional regulators of differentiation have been identified, their target genes responsible for the morphological changes occurring during OL myelination are still largely unknown. Here, we show that the regulator of focal adhesion, Tensin3 (Tns3), is a direct target gene of Olig2, Chd7, and Chd8, transcriptional regulators of OL differentiation. Tns3 is transiently upregulated and localized to cell processes of immature OLs, together with integrin-β1, a key mediator of survival at this transient stage. Constitutive Tns3 loss of function leads to reduced viability in mouse and humans, with surviving knockout mice still expressing Tns3 in oligodendroglia. Acute deletion of Tns3 in vivo, either in postnatal neural stem cells (NSCs) or in OPCs, leads to a twofold reduction in OL numbers. We find that the transient upregulation of Tns3 is required to protect differentiating OPCs and immature OLs from cell death by preventing the upregulation of p53, a key regulator of apoptosis. Altogether, our findings reveal a specific time window during which transcriptional upregulation of Tns3 in immature OLs is required for OL differentiation likely by mediating integrin-β1 survival signaling to the actin cytoskeleton as OL undergo the large morphological changes required for their terminal differentiation.

DLC1 is a RhoGAP-containing tumor suppressor that inhibits angiogenesis by repressing VEGF production in epithelial cells. Here we report the roles of DLC1 in endothelial cells. Silencing of DLC1 (siDLC1) enhances cell migration but reduces tube formation activities of human umbilical vein endothelial cells (HUVECs). Biochemically, RhoA activity and paxillin protein level are markedly increased in siDLC1 HUVECs. Although further silencing of RhoA restores the cell migration phenotype, the tube formation defect and up-regulated paxillin level remain unchanged. On the other hand, paxillin knockdown rescues tube formation and migration phenotypes but not the up-regulated RhoA activity. These results indicate that DLC1 regulates endothelial cell migration through RhoA and paxillin independently and controls tube formation mainly via paxillin. To further determine endothelial DLC1's function, we have generated endothelial specific knockout mice (DLC1-Tek). DLC1-Tek mice appear to be normal and healthy but their angiogenesis processes are compromised as shown in gel plug and aortic ring sprouting assays. Analysis of endothelial cells isolated from DLC1-Tek mice has further affirmed the cellular and biochemical phenotypes established in siDLC1 HUVECs. Our studies have demonstrated a positive regulatory role of endothelial DLC1 in angiogenesis.

Cten is a focal adhesion molecule that is expressed at very low levels in most normal tissues. Nonetheless, its expression has been found to increase dramatically in many types of cancer including colorectal, breast, gastric, and pancreatic cancer, suggesting that cten may play a critical role during tumorigenesis. To study the mechanisms that induce cten expression and the function of up-regulated cten, we examined the effects of several cancer-associated growth factors and cytokines on cten expression. We found that EGF, FGF2, NGF, PDGF, TGF-β, IGF-1, IL-6, and IL-13 were able to induce cten expression in a dose- and time-dependent manner. The Mek-Erk and PI3K-Akt pathways were two main signaling cascades responsible for cten up-regulation, whereas the Jak-Stat pathway could contribute to the increase in some conditions. Since many of these factors are known to promote cell migration, we hypothesized that up-regulated cten might contribute to this process. This hypothesis was investigated in FGF2-mediated cell migration. Silencing of cten not only reduced regular cell motility but also FGF2-mediated cell migration. Overexpression of cten promoted cell migration and FGF2 treatment failed to further enhance cell migration. Our findings that (1) cten is a common downstream molecule of these cancer-associated growth factors and cytokines; and that (2) up-regulated cten modulates cell migration induced by FGF2 and likely other growth factors as well, strongly suggest that cten could be a potential downstream therapeutic target for treating cancers associated with aberrant signaling of these growth factors and cytokines.

Abstract Epidermal growth factor receptor (EGFR) tyrosine kinase engages a vast array of signaling pathways regulating tissue development and homeostasis. The signal is normally induced by ligand binding (such as EGF) that activates the receptors and then the downstream molecules, which mediate cell fate decisions such as proliferation, migration, and differentiation. EGF binding not only turns on the EGFR activity; it also turns on the mechanisms to inactivate EGFR signaling. One way is the activation of c-Cbl, an E3 ubiquitin ligase that binds to tyrosine-phosphorylated EGFR and leads to ubiquitination of EGFR for lysosomal degradation. Dysregulated EGFR signaling either by up-regulation or aberrant activation of the receptor will trigger cellular events that contribute to tumor microenvironments and cancer formation. Focal adhesions connect extracellular matrix to cytoskeletal networks and play critical roles in cell adhesion, migration, proliferation, and survival. They also crosstalk with growth factor receptors, such as EGFR to elicit a wider range of cellular responses, and may function as biosensors for microenvironments. Cten (C-terminal tensin like) is a focal adhesion molecule known to regulate cell adhesion and migration. Previously, we had shown that cten was up-regulated by EGF and mediated EGF induced cell migration. To further investigate cten's role in EGFR signaling pathway, we examined cten's effect on EGFR protein stability. Cten significantly reduced ligand-induced EGFR degradation through decreasing EGFR ubiquitination. This was accomplished by binding to c-Cbl in a phosphotyrosine-SH2 dependent fashion. A functional SH2 domain of cten was essential for its activities in promoting tumor cell migration, invasion, and colony formation. In addition to cell culture systems, EGFR levels were markedly enhanced in a cten tissue-specific knockin mouse model. By analysis of cten and EGFR expressions in human cancer samples, we found a positive association between cten and EGFR protein levels. Since cten regulates the activated EGFR level and plays roles in tumor migration/invasion, targeting of cten might have a beneficial effect on EGFR targeted therapies. Citation Format: Shiao-Ya Hong, Yi-Ping Shih, Tianhong Li, Kermit Carraway, Su Hao Lo. CTEN prolongs EGFR signaling by reducing ligand-induced EGFR degradation. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A40.

Activation of EGF receptor (EGFR) triggers signaling pathways regulating various cellular events that contribute to tissue development and function. Aberrant activation of EGFR contributes to tumor progression as well as therapeutic resistance in patients with cancer. C-terminal tensin-like (CTEN; TNS4) is a focal adhesion molecule that is a member of the tensin family. Its expression is upregulated by EGF and elevated CTEN mediates EGF-induced cell migration. In the presence of CTEN, we found that EGF treatment elevated the level of EGFR protein but not mRNA. The extended half-life of activated EGFR sustained its signaling cascades. CTEN reduced ligand-induced EGFR degradation by binding to the E3 ubiquitin ligase c-Cbl and decreasing the ubiquitination of EGFR. The Src homology 2 domain of CTEN is not only required for binding to the phosphorylated tyrosine residue at codon 774 of c-Cbl, but is also essential for the tumorigenicity observed in the presence of CTEN. Public database analyses indicated that CTEN mRNA levels are elevated in breast, colon, lung, and pancreas cancers, but not correlated with EGFR mRNA levels in these cancers. In contrast, immunohistochemistry analyses of lung cancer specimens showed that CTEN and EGFR protein levels were positively associated, in support of our finding that CTEN regulates EGFR protein levels through a posttranslational mechanism. Overall, this work defines a function for CTEN in prolonging signaling from EGFR by reducing its ligand-induced degradation.

Deleted in liver cancer 1 (DLC1) is a GTPase-activating protein (GAP) domain containing tumor suppressor that localizes to focal adhesions. In cancer cells, loss of DLC1 is known to enhance cancer cell migration. However, the role of DLC1 in normal cell migration has not been well studied. Here, we show that silencing of DLC1 (shDLC1) in normal prostate epithelial cells reduces cell migration in both Transwell and wound-healing assays. This migration defect is mainly due to upregulation of plasminogen activator inhibitor 1 (PAI-1). Silencing of PAI-1 rescues the shDLC1-reduced migration phenotype. Reexpression of DLC1 suppresses PAI-1 and restores the migration defect as well. In contrast, DLC1-K714E (GAP inactive) mutant neither decreases the PAI-1 level nor rescues the shDLC1 migration defect. Interestingly, DLC1-Y442F (tensin-binding and focal adhesion–localizing defective) mutant is able to suppress PAI-1 expression but does not restore the migration defect. Furthermore, PAI-1 upregulation in shDLC1 cells is EGFR-MEK pathway dependent and is able to promote in vitro angiogenesis. Together, our results show that at least the following two new mechanisms are involved in DLC1-mediated normal cell migration: (i) DLC1 modulates the expression of PAI-1, which is a negative regulator for cell migration, in a GAP domain and EGFR-MEK–dependent manner and (ii) Independent of PAI-1, the interaction of DLC1 with tensin members positively regulates cell migration. Mol Cancer Res; 10(1); 34–9. ©2011 AACR.

Integrin clustering, typically nanometers in dimension, is the first and an important step in integrin-mediated cellular signaling processes such as focal adhesion. Engineered nanostructures mimicking extracellular matrices (ECM) provide a new approach for investigation and regulation of this initial step and of downstream cascades of focal adhesion. This work reveals that fibronectin (Fn) nanostructures, even at a small height of 3.2 nm ± 0.5 nm, exhibit high efficacy in guiding cellular orientation and polarization. More interestingly, the Fn nanostructures also impact intracellular structures such as preferential filopodia attachment, and commensurate alignment of intracellular actin stress fibers. The impact can be rationalized by the strong and specific interaction between integrin and Fn, leading to integrin clusters and then focal adhesion assemblies following the underlying nanostructure of Fn. This guided assembly further mediates the downstream behavior, such as actin stress fiber alignment and overall cellular morphology. Our observations collectively demonstrate that engineered nanostructures of Fn provide an alternative and high efficacy biochemical cue for regulation of cellular signaling processes.

Abstract The Rho GTPase-activating protein DLC1 is a tumor suppressor that is often deleted in liver cancer and downregulated in other cancers. DLC1 regulates the actin cytoskeleton, cell shape, adhesion, migration, and proliferation through its Rho GTPase-activating protein activity and focal adhesion localization. In this study, we silenced DLC1 in nonmalignant prostate epithelial cells to explore its tumor suppression functions. Small hairpin RNA-mediated silencing of DLC1 was insufficient to promote more aggressive phenotypes associated with tumor cell growth. In contrast, DLC1 silencing promoted pro-angiogenic responses through vascular endothelial growth factor (VEGF) upregulation, accompanied by the accumulation of hypoxia-inducible factor 1α and its nuclear localization. Notably, modulation of VEGF expression by DLC1 was dependent on epidermal growth factor receptor–MAP/ERK kinase–hypoxia-inducible factor 1 signaling but on RhoA pathways. Clinically, VEGF upregulation is a highly significant event in prostate cancers in which DLC1 is downregulated. Thus, our results strongly suggest that loss of DLC1 may serve as a “second hit” in promoting angiogenesis in a paracrine fashion during tumorigenesis. Cancer Res; 70(21); 8270–5. ©2010 AACR.

Glycine N-methyltransferase (GNMT) is a tumor susceptibility gene for both hepatocellular carcinoma and prostate cancer. We have previously characterized GNMT genomic structure and mapped its chromosomal localization to 6p12. For this study we identified a GNMT transcriptional start site at the 14th position upstream of the ATG codon. Electrophoretic mobility shift assay results indicate binding of the nuclear factor-Y (NF-Y) transcription factor to the CCAAT box (-71/-67) of the GNMT gene. Mutation assay results suggest that the nucleotide sequence in the -56/-47 region is a binding site for a putative transcriptional factor. The TATA-less core promoter (-133/+14) contains three major elements: an Sp1 site, CCAAT box, and a novel box within the CTGTCGGCTG sequence. One functional xenobiotic response element (XRE) located at the -104/-82 region is inducable by benzo[a]pyrene treatment. We believe our results have value for the study of GNMT transcriptional regulation.

Cell tracing dyes are very frequently utilized in cellular biology research because they provide highly sensitive fluorescent tags that do not compromise cellular functions such as growth and proliferation. In many investigations concerning cellular adhesion and mechanics, fluorescent dyes have been employed with the assumption of little impact on the results. Using the single cell compression technique developed by our team, the single cell mechanics of MDA-MB-468 and MLC-SV40 cells were investigated as a function of dye uptake. Cell tracing dyes increase living cell stiffness 3-6 times and cell-to-probe adhesion up to 7 times. These results suggest a more significant effect than toxins, such as thrombin. A simple analytical model was derived to enable the extraction of the Young's moduli of the cell membrane and cytoskeleton from the force-deformation profiles measured for individual cells. The increase in Young's modulus of the membrane is 3-7 times, which is more significant than that of the cytoskeleton (1.1-3.4 times). We propose that changes in cell mechanics upon the addition of fluorescent tracing dye are primarily due to the incorporation of amphiphilic dye molecules into the cellular plasma membrane, which increases the lateral interaction among phospholipid chains and thus enhances their rigidity and adhesion.

C-terminal tensin-like (cten) is a focal adhesion molecule belonging to the tensin family. Previous studies have suggested that cten may function as a prostate-specific tumor suppressor. Here, we show that although cten is expressed at a very low level in normal colon, its expression is significantly up-regulated in colon cancer. Furthermore, a high population of cten is found in the nucleus, where it interacts with beta-catenin, a critical player in the canonical Wnt pathway. This interaction may contribute to the role of cten in enhancing the colony formation, anchorage-independent growth, and invasiveness of colon cancer cells. Our studies have identified cten as a novel nuclear partner of beta-catenin, showed an oncogenic activity of cten in colon cancers, and revealed cten as a potential biomarker and target for colon cancers.

Deleted in liver cancer-1 (DLC-1) is a RhoGTPase-activating protein (RhoGAP) domain containing tumor suppressor that is often down-regulated in various cancer types. Previously, we have shown that DLC-1 is recruited to focal adhesions by binding to the Src homology 2 domains of tensins and the focal adhesion localization is critical for the tumor suppression activity of DLC-1. To investigate whether mutations in the focal adhesion targeting (FAT) region might occur and attenuate the expression, localization, and function of DLC-1, we have first mapped the FAT region to the amino acid residues from 201 to 500, and then sequenced cDNAs and genomic DNAs encoding the FAT region from cancer patients. Several missense and nonsense mutations were detected. All missense mutations were further examined for the potential effect on the function of DLC-1. Although these mutations did not seem to affect the focal adhesion localization of DLC-1, the activities of suppressing tumor cell growth were impaired in two mutants: T301K and S308I. Consistent with the fact that the RhoGAP activity of DLC-1 is essential for inhibiting tumor cell growth, the RhoGAP activities were significantly reduced in these mutants, suggesting that the FAT region also contains a regulatory element for its COOH-terminal RhoGAP domain. Our studies have shown that mutations in DLC-1 may lead to loss of function and contribute to the tumorigenesis, and have revealed an allosteric regulation site for its RhoGAP activity.