Richard Braverman

The tuberous sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination

We report here that overexpression of the tuberous sclerosis-1 (TSC1) gene product hamartin results in the inhibition of growth, as well as changes in cell morphology. Growth inhibition was associated with an increase in the endogenous level of the product of the tuberous sclerosis-2 (TSC2) gene, tuberin. As overexpression of tuberin inhibits cell growth, and hamartin is known to bind tuberin, these results suggested that hamartin stabilizes tuberin and this contributes to the inhibition of cell growth. Indeed, transient transfection of TSC1 increased the endogenous level of tuberin, and transient co-transfection of TSC1 with TSC2 resulted in higher tuberin levels. The stabilization was explained by the finding that tuberin is highly ubiquitinated in cells, while the fraction of tuberin that is bound to hamartin is not ubiquitinated. Co-expression of tuberin stabilized hamartin, which is weakly ubiquitinated, in transiently transfected cells. The amino-terminal two-thirds of tuberin was responsible for its ubiquitination and for stabilization of hamartin. A mutant of tuberin from a patient missense mutation of TSC2 was also highly ubiquitinated, and was unable to stabilize hamartin. We conclude that hamartin is a growth inhibitory protein whose biological effect is likely dependent on its interaction with tuberin.

Oncogenic inhibition by a deleted in liver cancer gene requires cooperation between tensin binding and Rho-specific GTPase-activating protein activities

The three deleted in liver cancer genes (DLC1–3) encode Rho-GTPase-activating proteins (RhoGAPs) whose expression is frequently down-regulated or silenced in a variety of human malignancies. The RhoGAP activity is required for full DLC-dependent tumor suppressor activity. Here we report that DLC1 and DLC3 bind to human tensin1 and its chicken homolog. The binding has been mapped to the tensin Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains at the C terminus of tensin proteins. Distinct DLC1 sequences are required for SH2 and PTB binding. DCL binding to both domains is constitutive under basal conditions. The SH2 binding depends on a tyrosine in DCL1 (Y442) but is phosphotyrosine-independent, a highly unusual feature for SH2 binding. DLC1 competed with the binding of other proteins to the tensin C terminus, including β3-integrin binding to the PTB domain. Point mutation of a critical tyrosine residue (Y442F) in DLC1 rendered the protein deficient for binding the tensin SH2 domain and binding full-length tensin. The Y442F protein was diffusely cytoplasmic, in contrast to the localization of wild-type DLC1 to focal adhesions, but it retained the ability to reduce the intracellular levels of Rho-GTP. The Y442F mutant displayed markedly reduced biological activity, as did a mutant that was RhoGAP-deficient. The results suggest that DLC1 is a multifunctional protein whose biological activity depends on cooperation between its tensin binding and RhoGAP activities, although neither activity depends on the other.

E-cadherin negatively regulates neoplastic growth in non-small cell lung cancer: role of Rho GTPases

Non-small cell lung cancers (NSCLC) that express the cell surface adhesion protein E-cadherin may carry a better prognosis than E-cadherin-negative tumors. Here, we found substantial inhibition of anchorage-independent growth in soft agar and cell migration in each of four NSCLC lines stably transfected with E-cadherin. The inhibitory effects were independent of the EGFR and β-catenin/Wnt-signaling pathways. However, E-cadherin expression was associated with an adhesion-dependent reduction in the activity of Rho family proteins, RhoA in two lines and Cdc42 in the other two. The reduction of RhoA activity was dependent on DLC-1 Rho-GAP and p190 Rho-GAP and associated with an increase in a membrane-associated p190 Rho-GAP/p120 Ras-GAP complex. In parental cells with high levels of RhoA-GTP, siRNA-mediated knock-down of RhoA reduced cell migration and agar growth in a manner analogous to E-cadherin. In parental cells with high levels of Cdc42-GTP, transfection of a Cdc42 dominant-negative mutant reduced cell growth and migration similarly to cells expressing E-cadherin. Thus, E-cadherin can negatively regulate cell proliferation and migration in NSCLC by reducing the level of the predominant active form of Rho family protein, RhoA or Cdc42. These proteins can be considered downstream effectors of E-cadherin and might represent therapeutic targets in some NSCLC.

The mechanism by which actinomycin D inhibits protein synthesis in animal cells

IN the course of studies on the regulation of protein synthesis during the activation of human lymphocytes by phytohaemagglutinin (PHA), it became necessary to determine whether an effect which occurred following treatment with actinomycin D (AMD) was secondary to the action of the drug on transcription, or resulted from a direct effect on translation. Using enucleated lymphocytes, we have shown that AMD has no effect on protein synthesis in the absence of the cell nucleus and conclude that AMD interferes with protein synthesis by its effect on transcription of RNA.

Functional interaction of tumor suppressor DLC1 and caveolin-1 in cancer cells.

Deleted in liver cancer 1 (DLC1), a tumor suppressor gene frequently inactivated in non-small cell lung cancer (NSCLC) and other malignancies, encodes a multidomain protein with a RhoGTPase-activating (RhoGAP) domain and a StAR-related lipid transfer (START) domain. However, no interacting macromolecule has been mapped to the DLC1 START domain. Caveolin-1 (CAV-1) functions as a tumor suppressor in most contexts and forms a complex with DLC1. Here, we have mapped the region of DLC1 required for interaction with CAV-1 to the DLC1 START domain. Mutation of the DLC1 START domain disrupted the interaction and colocalization with CAV-1. Moreover, DLC1 with a START domain mutation failed to suppress neoplastic growth, although it negatively regulated active Rho. CAV-1 and DLC1 expression levels were correlated in two public datasets of NSCLC lines and in two independent publicly available mRNA expression datasets of NSCLC tumors. Clinically, low DLC1 expression predicted a poor clinical outcome in patients with lung cancer. Together, our findings indicate that complex formation between the DLC1 START domain and CAV-1 contributes to DLC1 tumor suppression via a RhoGAP-independent mechanism, and suggest that DLC1 inactivation probably contributes to cancer progression.

Regulation of cell morphology and adhesion by the tuberous sclerosis complex (TSC1/2) gene products in human kidney epithelial cells through increased E-cadherin/β-catenin activity

We investigated the effects of overexpression of the tuberous sclerosis‐1 and ‐2 (TSC1/2) gene products (hamartin and tuberin, respectively) in the human kidney epithelial cell line 293 with an inducible expression system. As we had observed previously in fibroblasts, 293 cells overexpressing hamartin and/or tuberin grew more slowly in vitro. However, here we also observed that the 293 overexpressing cells underwent a dramatic morphological change in which groups of cells formed compact clusters. The overexpressing cells also displayed decreased dissociation and increased reaggregation in vitro. These changes were found to be associated with an increased level of E‐cadherin, which is known to regulate cell‐cell interactions in epithelial cells, and of its binding partner β‐catenin. Consistent with the role of E‐cadherin in these effects, we found that the observed changes in 293 cell morphology, dissociation, and adhesion were calcium‐dependent, and were reproduced by overexpression of E‐cadherin. In contrast, overexpression of TSC1 in rat embryo fibroblasts, which lack E‐cadherin, failed to elicit the same changes as in 293 cells. We conclude that the hamartin/tuberin complex exerted a direct effect on the morphology and adhesive properties of 293 cells through regulation of the level and/or activity of cellular E‐cadherin/β‐catenin.

Protein synthesis in resting and growth-stimulated human peripheral lymphocytes: Evidence for regulation by a non-messenger RNA

Abstract Stimulation of lymphocyte growth is accompanied by an early increase in the rate of protein synthesis. This increase is dependent upon the flow of inactive free ribosomes into polysomes, which is limited by a rate-controlling step at initiation [2]. Addition of actinomycin D (actD) to lymphocytes caused a gradual reduction in protein synthesis in resting cells, but rapidly inhibited both the elevation of protein synthesis and the activation of free ribosomes which normally follow exposure to mitogens. Since actD does not affect protein synthesis in enucleated lymphocytes [4], the effect in intact cells must be mediated by a nuclear event, which available data indicate is RNA synthesis. ActD prevented the accumulation of 80S initiation complexes which normally occurs in resting lymphocytes treated with pactamycin and cycloheximide, showing that its locus of action was at some point in initiation. The decline in rate of protein synthesis began without detectable lag when resting lymphocytes were treated with actD. However, after growth stimulation, a delay of ca 50 min occurred before the protein synthetic rate declined in response to actD. These observations agree with the hypothesis that the concentration of some moderately short-lived RNA is rate-limiting for protein synthesis in resting lymphocytes, and that an early event in growth stimulation is a rise in the amount of this component to levels which are no longer rate-limiting. This permits an increased flow of ribosomes into polysomes and a consequent rise in protein synthesis. Available evidence indicates that the regulatory RNA is neither mRNA nor rRNA, but may either be one of the small cytoplasmic RNAs whose function is unknown, or tRNA i met .

Abstract 2184: Complex formation between the DLC1 START domain and Cav1 contributes to the tumor suppressor function of DLC1

Deleted in live cancer (DLC1), a tumor suppressor gene with Rho GTPase-activating (RhoGAP) activity, is frequently inactivated in a variety of cancers. DLC1 encodes a 1091 amino acid multi-domain protein whose RhoGAP catalytic domain (aa 609-878) is flanked by an amino-terminal sterile alpha motif (SAM; aa 17-76) and a carboxy-terminal steroidogenic acute regulatory (StAR)-related lipid transfer (START; aa 879-1080) domain. Caveolin-1 (Cav1), a key constituent of caveolae, plays an important role in signal transduction, endocytosis, and cholesterol transport, behaves as a tumor suppressor in most contexts, and is reported to form a complex with DLC1. Here, we have sought to assess the biological relevance of the DLC1-Cav1 interaction and to determine the region of DLC1 that interacts with Cav1. After confirming that endogenous DLC1 and Cav1 co-localize with each other by immunomicroscopy and form a complex in vivo as assessed by co-immunoprecipitation, we determined the DLC1 sequences that were required for complex formation. Using GST-Pull down of a GST-Cav1 fusion protein that had been co-transfected with a series of GFP-DLC1 fragments, we identified a polypeptide of DLC1 (aa 899-996), composed of residues from the START domain that was necessary and sufficient for complex formation. A full-length GFP-DLC1 mutant lacking some of these START residues (aa 929-957) was deficient for complex formation with endogenous Cav1. When stably expressed in a human lung cancer cell line that did not express endogenous DLC1, the mutant was severely compromised in suppressing cell migration, colony formation, and anchorage-independent growth in soft agar compared to cells stably expressing the wild type GFP-DLC1. We conclude that the START domain of DLC1 is required for its complex formation with Cav1, and that this interaction contributes to the tumor suppressor activity of DLC1. To our knowledge, this is the first identified function for the DLC1 START domain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2184. doi:10.1158/1538-7445.AM2011-2184