Su Hao Lo

A reciprocal tensin-3-cten switch mediates EGF-driven mammary cell migration

Cell migration driven by the epidermal growth factor receptor (EGFR) propels morphogenesis and involves reorganization of the actin cytoskeleton. Although de novo transcription precedes migration, transcript identity remains largely unknown. Through their actin-binding domains, tensins link the cytoskeleton to integrin-based adhesion sites. Here we report that EGF downregulates tensin-3 expression, and concomitantly upregulates cten, a tensin family member that lacks the actin-binding domain. Knockdown of cten or tensin-3, respectively, impairs or enhances mammary cell migration. Furthermore, cten displaces tensin-3 from the cytoplasmic tail of integrin β1, thereby instigating actin fibre disassembly. In invasive breast cancer, cten expression correlates not only with high EGFR and HER2, but also with metastasis to lymph nodes. Moreover, treatment of inflammatory breast cancer patients with an EGFR/HER2 dual-specificity kinase inhibitor significantly downregulated cten expression. In conclusion, a transcriptional tensin-3–cten switch may contribute to the metastasis of mammary cancer.

The phosphotyrosine-independent interaction of DLC-1 and the SH2 domain of cten regulates focal adhesion localization and growth suppression activity of DLC-1

The tensin family member cten (C-terminal tensin like) is an Src homology 2 (SH2) and phosphotyrosine binding domain–containing focal adhesion molecule that may function as a tumor suppressor. However, the mechanism has not been well established. We report that cten binds to another tumor suppressor, deleted in liver cancer 1 (DLC-1), and the SH2 domain of cten is responsible for the interaction. Unexpectedly, the interaction between DLC-1 and the cten SH2 domain is independent of tyrosine phosphorylation of DLC-1. By site-directed mutagenesis, we have identified several amino acid residues on cten and DLC-1 that are essential for this interaction. Mutations on DLC-1 perturb the interaction with cten and disrupt the focal adhesion localization of DLC-1. Furthermore, these DLC-1 mutants have lost their tumor suppression activities. When these DLC-1 mutants were fused to a focal adhesion targeting sequence, their tumor suppression activities were significantly restored. These results provide a novel mechanism whereby the SH2 domain of cten-mediated focal adhesion localization of DLC-1 plays an essential role in its tumor suppression activity.

Tensin1 and a previously undocumented family member, tensin2, positively regulate cell migration

Tensin is a focal adhesion molecule that binds to actin filaments and participates in signaling pathways. In this study, we have characterized a previously undocumented tensin family member, tensin2/KIAA 1075. Human tensin2 cDNA encodes a 1,285-aa sequence that shares extensive homology with tensin1 at its amino- and carboxyl-terminal ends, which include the actin-binding domain, the Src homology 2 (SH2) domain, and the phosphotyrosine binding (PTB) domain. Analysis of the genomic structures of tensin1 and tensin2 further confirmed that they represent a single gene family. Examination of tensin2 mRNA distribution revealed that heart, kidney, skeletal muscle, and liver were tissues of high expression. The endogenous and recombinant tensin2 were expressed as a 170-kDa protein in NIH 3T3 cells. The subcellular localization of tensin2 was determined by transfection of green fluorescence protein (GFP)–tensin2 fusion construct. The results indicated that tensin2 is also localized to focal adhesions. Finally, functional analysis of tensin genes has demonstrated that expression of tensin genes is able to promote cell migration on fibronectin, indicating that the tensin family plays a role in regulating cell motility.

Molecular characterization of human tensin.

Tensin is a focal-adhesion molecule that binds to actin filaments and interacts with phosphotyrosine-containing proteins. To analyse tensins function in mammals, we have cloned tensin cDNAs from human and cow. The isolated approx. 7.7-kb human cDNA contains an open reading frame encoding 1735 amino acid residues. The amino acid sequence of human tensin shares 60% identity with chicken tensin, and contains all the structural features described previously in chicken tensin. This includes the actin-binding domains, the Src homology domain 2, and the region similar to a tumour suppressor, PTEN. Two major differences between human and chicken tensin are (i) the lack of the first 54 residues present in chicken tensin, and (ii) the addition of 34- and 38-residue inserts in human and bovine tensin. In addition, our interspecies sequencing data have uncovered the presence of a glutamine/CAG repeat that appears to have expanded in the course of evolution. Northern-blot analysis reveals a 10-kb message in most of the human tissues examined. An additional 9-kb message is detected in heart and skeletal muscles. The molecular mass predicted from the human cDNA is 185 kDa, although both endogenous and recombinant human tensin migrate as 220-kDa proteins on SDS/PAGE. The discrepancy is due to the unusually low electrophoretic mobility of the central region of the tensin polypeptide (residues 306-981). A survey of human prostate and breast cancer cell lines by Western-blot analysis shows a lack of tensin expression in most cancer cell lines, whereas these lines express considerable amounts of focal-adhesion molecules such as talin and focal-adhesion kinase. Finally, tensin is rapidly cleaved by a focal-adhesion protease, calpain II. Incubation of cells with a calpain inhibitor, MDL, prevented tensin cleavage and induced morphological change in these cells, suggesting that cleavage of tensin and other focal-adhesion constituents by calpain disrupts maintenance of normal cell shape.

Regulation of tensin-promoted cell migration by its focal adhesion binding and Src homology domain 2

Tensin1 is an actin- and phosphotyrosine-binding protein that localizes to focal adhesions. Recently, we have shown that both tensin1 and a new family member, tensin2, promote cell migration [Chen, Duncan, Bozorgchami and Lo (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 733-738]. Since localization of proteins to particular intracellular compartments often regulates their functions, and Src homology domain 2 may mediate signals related to cell migration, we hypothesize that tensin-mediated cell migration is regulated by the focal adhesion localization and the Src homology domain 2 of tensin. To test this hypothesis, we have analysed the effects of a series of tensin1 mutants on cell migration. Our results have shown that (1) tensin1 contains two focal adhesion-binding sites, (2) the wild-type tensin1 significantly promotes cell migration, (3) mutants with one focal adhesion-binding site do not promote cell migration, (4) the non-focal adhesion localized mutant suppresses cell migration and (5) the mutant that is not able to bind to phosphotyrosine-containing proteins has no effect on cell migration. These results have indicated that focal adhesion localization of tensin1 and the phosphotyrosine-binding activity are two critical factors in regulating tensin-mediated cell migration.

Cell Tracing Dyes Significantly Change Single Cell Mechanics

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 Youngs moduli of the cell membrane and cytoskeleton from the force-deformation profiles measured for individual cells. The increase in Youngs 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.

Interleukin-17 receptor-like gene is a novel antiapoptotic gene highly expressed in androgen-independent prostate cancer.

We have recently identified a new gene, interleukin-17 receptor-like (IL-17RL), which is expressed in normal prostate and prostate cancer. This investigation is focused on the role of IL-17RL in prostate cancer. We found that IL-17RL was expressed at significantly higher levels in several androgen-independent prostate cancer cell lines (PC3, DU145, cds1, cds2, and cds3) and tumors compared with the androgen-dependent cell lines (LNCaP and MLC-SV40) and tumors. In an in vivo model of human prostate tumor growth in nude mice (CWR22 xenograft model), IL-17RL expression in tumors was induced by androgen deprivation. The relapsed androgen-independent tumors expressed higher levels of IL-17RL compared with the androgen-dependent tumors. Overexpression of IL-17RL in tumor necrosis factor alpha (TNFalpha)-sensitive LNCaP cells inhibited TNFalpha-induced apoptosis by blocking activation of caspase-3 downstream to caspase-2 and caspase-8. Reciprocally, knocking down IL-17RL expression by small interfering RNA induced apoptosis in all the prostate cancer cell lines studied. Taken together, these results show that IL-17RL is a novel antiapoptotic gene, which may confer partially the property of androgen-independent growth of prostate cancer by promoting cell survival. Thus, IL-17RL is a potential therapeutic target in the treatment of prostate cancer.

Up-regulation of C-Terminal Tensin-like Molecule Promotes the Tumorigenicity of Colon Cancer through β-Catenin

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.

Mutations in the focal adhesion targeting region of deleted in liver cancer-1 attenuate their expression and function.

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.

Genetic association analyses highlight biological pathways underlying mitral valve prolapse

Nonsyndromic mitral valve prolapse (MVP) is a common degenerative cardiac valvulopathy of unknown etiology that predisposes to mitral regurgitation, heart failure and sudden death. Previous family and pathophysiological studies suggest a complex pattern of inheritance. We performed a meta-analysis of 2 genome-wide association studies in 1,412 MVP cases and 2,439 controls. We identified 6 loci, which we replicated in 1,422 cases and 6,779 controls, and provide functional evidence for candidate genes. We highlight LMCD1 (LIM and cysteine-rich domains 1), which encodes a transcription factor and for which morpholino knockdown of the ortholog in zebrafish resulted in atrioventricular valve regurgitation. A similar zebrafish phenotype was obtained with knockdown of the ortholog of TNS1, which encodes tensin 1, a focal adhesion protein involved in cytoskeleton organization. We also showed expression of tensin 1 during valve morphogenesis and describe enlarged posterior mitral leaflets in Tns1−/− mice. This study identifies the first risk loci for MVP and suggests new mechanisms involved in mitral valve regurgitation, the most common indication for mitral valve repair.