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Douglas R Lowy - One of the best experts on this subject based on the ideXlab platform.

  • Abstract 3431: Epigenetic reactivation of the DLC1 tumor suppressor protein is a new approach for cancer treatment
    Cancer Chemistry, 2020
    Co-Authors: Brajendra K Tripathi, Xiaolan Qian, Meghan F. Anderman, Luciarita Boccuzzi, Kylie J. Walters, James H. Doroshow, Douglas R Lowy
    Abstract:

    DLC1 is a potent tumor suppressor protein, which is frequently downregulated in cancer. In addition, several types of solid tumors express DLC1 mRNA but have lost DLC1 protein through its destabilization. Here, we identify a methyltransferase, EZH2, that directly methylates the DLC1 protein, leading to its ubiquitin-dependent proteasomal degradation, and explore the clinical relevance of this finding. Our studies indicate that although the canonical activity of EZH2 is on histone H3 in the nucleus, the methylation of DLC1 protein occurs in the cytoplasm, where EZH2 protein is readily detectible and is active. EZH2-dependent methylation of DLC1 leads to its degradation induced by the Cullin-4 ubiquitin ligase. In several tumor cell lines, including NSCLC A549, that carry mutant KRAS and express DLC1 mRNA but lack detectable DLC1 protein, treatment with the EZH2 inhibitor Tazemetostat stabilized the DLC1 protein. As previous studies have indicated that activated KRAS can induce EZH2, we asked whether there might be a link between the mutant KRAS and DLC1 protein stability. Consistent with this hypothesis, siRNA knockdown of KRAS leads to substantial stabilization of the DLC1 protein in A549 cells and other tumor lines with mutant KRAS. Although EZH2 inhibitors can stabilize the DLC1 protein, this restoration of DLC1 has limited tumor suppressor activity because AKT and SRC kinases, which we found directly phosphorylate DLC1 and cooperatively attenuate its tumor suppressor functions, tend to be activated in solid tumors, thereby reducing the tumor suppressor activity of the stabilized DLC1 protein. Treatment of tumor xenografts that carry mutant KRAS with AKT or SRC kinase inhibitors in conjunction with EZH2 inhibition has much greater antitumor activity than treatment with any one of the three inhibitors, and the combined treatment with all three drugs has even greater therapeutic activity, which is correlated with the induction of greater cellular senescence and apoptosis, as measured by the beta-galactosidase and annexin V expression, respectively. Remarkably, these combinations are well-tolerated, and their therapeutic efficacy against xenografts with mutant KRAS make them a candidate intervention against this important unmet clinical need. We have made similar observations with proteasomal inhibition together with AKT and/or SRC inhibition in A549 cells and other tumor lines that carry mutant KRAS and express DLC1 mRNA, but lack DLC1 protein. Inhibitory RNA knockdown of DLC1 expression reduces the antitumor activity of the drug combination, strongly suggesting that DLC1 plays a critical role in the therapeutic response to the combination. In summary, our studies provide a mechanistic rationale for combining EZH2 or proteasomal inhibition with AKT and/or SRC inhibition in treating tumors that carry mutant KRAS and express DLC1 mRNA, but lack DLC1 protein. Thus, this study is a novel example where epigenetic reactivation of the tumor suppressor can be a key therapeutic target, in contrast to the usual situation where the focus is principally on inhibiting pro-oncogenic factors. Citation Format: Brajendra K. Tripathi, Luciarita Boccuzzi, Meghan Anderman, Xiaolan Qian, Kylie J. Walters, James H. Doroshow, Douglas R. Lowy. Epigenetic reactivation of the DLC1 tumor suppressor protein is a new approach for cancer treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3431.

  • Cancer-Associated Point Mutations in the DLC1 Tumor Suppressor and Other Rho-GAPs Occur Frequently and Are Associated with Decreased Function
    Cancer research, 2020
    Co-Authors: Dunrui Wang, Xiaolan Qian, Marian E. Durkin, Brajendra K Tripathi, Beatriz Sanchez-solana, Douglas R Lowy
    Abstract:

    In advanced cancer, the RHOA GTPase is often active together with reduced expression of genes encoding Rho-specific GTPase-accelerating proteins (Rho-GAP), which negatively regulate RHOA and related GTPases. Here we used the The Cancer Genome Atlas dataset to examine 12 tumor types (including colon, breast, prostate, pancreas, lung adenocarcinoma, and squamous cell carcinoma) for the frequency of codon mutations of 10 Rho-GAP and experimentally tested biochemical and biological consequences for cancer-associated mutants that arose in the DLC1 tumor suppressor gene. DLC1 was the Rho-GAP gene mutated most frequently, with 5%-8% of tumors in five of the tumor types evaluated having DLC1 missense mutations. Furthermore, 20%-26% of the tumors in four of these five tumor types harbored missense mutations in at least one of the 10 Rho-GAPs. Experimental analysis of the DLC1 mutants indicated 7 of 9 mutants whose lesions were located in the Rho-GAP domain were deficient for Rho-GAP activity and for suppressing cell migration and anchorage-independent growth. Analysis of a DLC1 linker region mutant and a START domain mutant showed each was deficient for suppressing migration and growth in agar, but their Rho-GAP activity was similar to that of wild-type DLC1. Compared with the wild-type, the linker region mutant bound 14-3-3 proteins less efficiently, while the START domain mutant displayed reduced binding to Caveolin-1. Thus, mutation of Rho-GAP genes occurs frequently in some cancer types and the majority of cancer-associated DLC1 mutants evaluated were deficient biologically, with various mechanisms contributing to their reduced activity. SIGNIFICANCE: These findings indicate that point mutation of Rho-GAP genes is unexpectedly frequent in several cancer types, with DLC1 mutants exhibiting reduced function by various mechanisms.

  • DLC1 deficiency and YAP signaling drive endothelial cell contact inhibition of growth and tumorigenesis
    Oncogene, 2019
    Co-Authors: Lisa Ritchey, Douglas R Lowy, Dunrui Wang, Taekyu Ha, Atsushi Otsuka, Kenji Kabashima, Yuyi Wang, Giovanna Tosato
    Abstract:

    Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene deleted in many cancers, including angiosarcoma, an aggressive malignancy of endothelial cell derivation. DLC1-deficiency in primary endothelial cells causes the loss of cell contact inhibition of growth through incompletely defined mechanisms. We report that DLC1 is a regulator of YAP, a transcriptional coactivator of proliferation-promoting and tumor-promoting genes; when confluent, active/nuclear YAP was significantly more abundant in DLC1-deficient endothelial cells compared with control cells. We also found that YAP is a required effector of the loss of cell contact inhibition of growth manifested by DLC1-deficient endothelial cells, as the silencing of YAP prevents this loss. Consistently, human angiosarcomas specimens contained a significantly greater proportion of DLC1^− tumor cells with nuclear YAP compared with the DLC1^+ normal cells in the adjacent tissue. Verteporfin, an inhibitor of YAP, significantly reduced angiosarcoma growth in mice. These results identify YAP as a previously unrecognized effector of DLC1 deficiency-associated loss of cell contact growth inhibition in endothelial cells and a potential therapeutic target in angiosarcoma.

  • SRC and ERK cooperatively phosphorylate DLC1 and attenuate its Rho-GAP and tumor suppressor functions.
    The Journal of cell biology, 2019
    Co-Authors: Brajendra K Tripathi, Alex G Papageorge, Xiaolan Qian, Dunrui Wang, Meghan F. Anderman, Ming Zhou, Douglas R Lowy
    Abstract:

    SRC and ERK kinases control many cell biological processes that promote tumorigenesis by altering the activity of oncogenic and tumor suppressor proteins. We identify here a physiological interaction between DLC1, a focal adhesion protein and tumor suppressor, with SRC and ERK. The tumor suppressor function of DLC1 is attenuated by phosphorylation of tyrosines Y451 and Y701 by SRC, which down-regulates DLC1’s tensin-binding and Rho-GAP activities. ERK1/2 phosphorylate DLC1 on serine S129, which increases both the binding of SRC to DLC1 and SRC-dependent phosphorylation of DLC1. SRC inhibitors exhibit potent antitumor activity in a DLC1-positive transgenic cancer model and a DLC1-positive tumor xenograft model, due to reactivation of the tumor suppressor activities of DLC1. Combined treatment of DLC1-positive tumors with SRC plus AKT inhibitors has even greater antitumor activity. Together, these findings indicate cooperation between the SRC, ERK1/2, and AKT kinases to reduce DLC1 Rho-GAP and tumor suppressor activities in cancer cells, which can be reactivated by the kinase inhibitors.

  • Abstract 1762: The START domain of the DLC1 tumor suppressor binds phosphatidylserine and two proteins, PLCD1 and Caveolin-1
    Molecular and Cellular Biology Genetics, 2019
    Co-Authors: Beatriz Sanchez-solana, Alex G Papageorge, Xiaolan Qian, Dunrui Wang, Parthibane Velayoudame, Jairaj Acharya, Douglas R Lowy
    Abstract:

    The tumor suppressor gene DLC1 encodes a multi-domain protein, which includes a Rho-GAP domain (Rho GTPase activating protein domain) that negatively regulates the activity of RhoA, B, and C, and has been hypothesized to be the basis of its tumor suppressor functions. DLC1 also contains a less well characterized START (StAR-related lipid transfer) domain in its C-terminus, whose overall contribution to DLC1 function remains incompletely understood. START domains in other proteins have been shown to bind lipids, but no lipid has yet been identified that binds the DLC1 START domain. In the present study, we attempted to gain further insight into the molecular function and lipid binding properties of the DLC1 START domain. We previously determined that Caveolin-1, the main structural component and marker of caveolae, interacts with the DLC1 START domain, and this interaction contributes to the full tumor suppressor activity of DLC1. In unpublished studies, we additionally identified Phospholipase C delta 1 (PLCD1), which participates in intracellular Ca2+ mobilization, forming a complex with the DLC1 START domain and with Caveolin-1. We have now developed evidence that, in addition to binding Caveolin-1 and PLCD1, the DLC1 START domain binds a lipid, phosphatidylserine (PS), and have identified a colon cancer-associated DLC1-START domain mutant (R947C) that results in a protein deficient in forming a complex with all three binding partners and displays reduced tumor suppressor activity but intact RhoGAP activity. The interaction between the DLC1-START domain and PS can occur independently of PLCD1 and/or Caveolin-1, but PS promotes complex formation between the DLC1 START domain and PLCD1 or Caveolin-1, without altering PLCD1 to Caveolin-1 binding. Biological assays with wild type DLC1 show cooperation between DLC1 and PLCD1 or Caveolin-1 to inhibit cell migration, but no cooperation is observed when all three genes are overexpressed, suggesting that Caveolin-1 and PLCD1 occupy the same migration signaling pathway. Thus, we have identified a biologically relevant complex between DLC1 and three macromolecules (PLCD1, PS and Caveolin-1) that contributes to the full tumor suppressor function of DLC1 independently of its RhoGAP activity, and are testing the hypothesis that PS serves as a linker between the DLC1 START domain and PLCD1 or Caveolin-1. Citation Format: Beatriz Sanchez-Solana, Dunrui Wang, Xiaolan Qian, Alex Papageorge, Parthibane Velayoudame, Jairaj Acharya, Douglas R. Lowy. The START domain of the DLC1 tumor suppressor binds phosphatidylserine and two proteins, PLCD1 and Caveolin-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1762.

Nicholas C. Popescu - One of the best experts on this subject based on the ideXlab platform.

  • Cooperative antiproliferative effect of coordinated ectopic expression of DLC1 tumor suppressor protein and silencing of MYC oncogene expression in liver cancer cells: Therapeutic implications.
    Oncology letters, 2016
    Co-Authors: Xu-yu Yang, Marian E. Durkin, Xiaoling Zhou, Paul Tone, Nicholas C. Popescu
    Abstract:

    Human hepatocellular carcinoma (HCC) is one of the most common types of cancer and has a very poor prognosis; thus, the development of effective therapies for the treatment of advanced HCC is of high clinical priority. In the present study, the anti-oncogenic effect of combined knockdown of c-Myc expression and ectopic restoration of deleted in liver cancer 1 (DLC1) expression was investigated in human liver cancer cells. Expression of c-Myc in human HCC cells was knocked down by stable transfection with a Myc-specific short hairpin (sh) RNA vector. DLC1 expression in Huh7 cells was restored by adenovirus transduction, and the effects of DLC1 expression and c-Myc knockdown on Ras homolog gene family, member A (RhoA) levels, cell proliferation, soft agar colony formation and cell invasion were measured. Downregulation of c-Myc or re-expression of DLC1 led to a marked reduction in RhoA levels, which was associated with decreases in cell proliferation, soft agar colony formation and invasiveness; this inhibitory effect was augmented with a combination of DLC1 transduction and c-Myc suppression. To determine whether liver cell-specific delivery of DLC1 was able to enhance the inhibitory effect of c-Myc knockdown on tumor growth in vivo, DLC1 vector DNA complexed with galactosylated polyethylene glycol-linear polyethyleneimine was administered by tail vein injection to mice bearing subcutaneous xenografts of Huh7 cells transfected with shMyc or control shRNA. A cooperative inhibitory effect of DLC1 expression and c-Myc knockdown on the growth of Huh7-derived tumors was observed, suggesting that targeted liver cell delivery of DLC1 and c-Myc shRNA may serve as a possible gene therapy modality for the treatment of human HCC.

  • DLC1 suppresses NF-κB activity in prostate cancer cells due to its stabilizing effect on adherens junctions.
    SpringerPlus, 2014
    Co-Authors: Veenu Tripathi, Nicholas C. Popescu, Drazen B. Zimonjic
    Abstract:

    DLC1 (Deleted in Liver Cancer 1) gene encodes a RhoGTPase-activating protein (RhoGAP), which exerts most of its tumor suppressor functions through suppression of small Rho GTPases proteins RhoA, RhoB, RhoC and to some degree Cdc42, but not Rac. RhoGTPases are implicated in NF-κB activation in highly invasive prostate carcinoma (PCA), with consequences on cell proliferation, survival and metastatic capacity. Here we demonstrate that DLC1 transduction in two androgen-independent (AI) and highly metastatic PCA cell lines negatively regulates NF-κB activity in a GAP- and α-catenin-dependent manner. Expressed DLC1 protein suppresses the phosphorylation of NF-κB inhibitor, IκBα, causes its relocation from membrane ruffles into cytoplasm and attenuates its ubiquitination and subsequent degradation. DLC1-mediated NF-kB suppression and its effects are comparable to NF-κB inhibition using either shRNA knockdown or peptide inhibitor. Expression of transduced DLC1 suppressed the expression of NF-κB mediated genes. Such effects were found to be reliant on presence of calcium, indicating that the observed modifications are dependent on, and enabled by DLC-mediated stabilization of adherens junctions. These results expand the multitude of DLC1 interactions with other genes that modulate its oncosuppressive function, and may have potential therapeutic implications.

  • DLC1 induces expression of E-cadherin in prostate cancer cells through Rho pathway and suppresses invasion
    Oncogene, 2013
    Co-Authors: Veenu Tripathi, Nicholas C. Popescu, Drazen B. Zimonjic
    Abstract:

    E-cadherin is a cell-cell adhesion molecule that acts as a suppressor of cancer cell invasion and its expression is downregulated in many advanced, poorly differentiated, human cancers. In this study, we found that the expression of DLC1 (deleted in liver cancer 1) tumor-suppressor gene in metastatic prostate carcinoma (PCA) cells increased the expression of E-cadherin and resulted in an elevated rate of cell-cell aggregation as measured by aggregation assay. DLC1-mediated increase in E-cadherin expression was not dependent on α-catenin, a DLC1-binding protein associated with E-cadherin, and/or cellular density. The increase of E-cadherin expression occurred at mRNA level and relied on DLC1 RhoGAP function, leading to suppression of high level of RhoA-GTP and RhoC-GTP activity in metastatic PCA cells. Application of Rho/ROCK inhibitors produced the same effect as introduction of DLC1. Knocking down of RhoA produced a moderate increase in E-cadherin whereas knocking down of RhoC resulted in a significant increase of E-cadherin. Downregulation of E-cadherin caused by constitutively active RhoA(V14) and RhoC(V14) could not be reversed by expression of DLC1 in DLC1-negative cell line. DLC1-mediated suppression of metastatic PCA cells invasion was comparable with the one associated with ectopic E-cadherin expression, or caused by suppression of Rho pathway either by Rho/ROCK inhibitors, or by shRNA repression. This study demonstrates that DLC1 expression positively regulates E-cadherin and suppresses highly metastatic PCA cell invasion by modulating Rho pathway, which appears as a feasible therapeutic target in cancers with high activity of RhoGTPases.

  • DLC1 interaction with α-catenin stabilizes adherens junctions and enhances DLC1 antioncogenic activity.
    Molecular and cellular biology, 2012
    Co-Authors: Veenu Tripathi, Nicholas C. Popescu, Drazen B. Zimonjic
    Abstract:

    The DLC1 (for deleted in liver cancer 1) tumor suppressor gene encodes a RhoGAP protein that inactivates Rho GTPases, which are implicated in regulation of the cytoskeleton and adherens junctions (AJs), a cell-cell adhesion protein complex associated with the actin cytoskeleton. Malignant transformation and tumor progression to metastasis are often associated with changes in cytoskeletal organization and cell-cell adhesion. Here we have established in human cells that the AJ-associated protein α-catenin is a new binding partner of DLC1. Their binding was mediated by the N-terminal amino acids 340 to 435 of DLC1 and the N-terminal amino acids 117 to 161 of α-catenin. These proteins colocalized in the cytosol and in the plasma membrane, where together they associated with E-cadherin and β-catenin, constitutive AJ proteins. Binding of DLC1 to α-catenin led to their accumulation at the plasma membrane and required DLC1 GAP activity. Knocking down α-catenin in DLC1-positive cells diminished DLC1 localization at the membrane. The DLC1-α-catenin complex reduced the Rho GTP level at the plasma membrane, increased E-cadherin's mobility, affected actin organization, and stabilized AJs. This process eventually contributed to a robust oncosuppressive effect of DLC1 in metastatic prostate carcinoma cells. Together, these results unravel a new mechanism through which DLC1 exerts its strong oncosuppressive function by positively influencing AJ stability.

  • Preclinical evaluation of combined antineoplastic effect of DLC1 tumor suppressor protein and suberoylanilide hydroxamic acid on prostate cancer cells.
    Biochemical and biophysical research communications, 2012
    Co-Authors: Xiaoling Zhou, Xu-yu Yang, Nicholas C. Popescu
    Abstract:

    Deleted in liver cancer (DLC1), a tumor suppressor gene in multiple cancers, is recurrently down regulated or inactivated by epigenetic mechanisms in primary prostate carcinomas (PCAs). In this study the methylation and acetylation profile of the DLC1 promoter region was examined in three PCA cell lines with low or undetectable DLC1 expression: LNCaP, its derivative C4-2B-2, and 22Rv1. Two histone deacetylase inhibitors (HDAC), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) induced histone acetylation of the DLC1 promoter in all three lines. DLC1 promoter methylation and deacetylation were detected in LNCaP and C4-2B-2 cells while in 22Rv1 cells DLC1 is silenced by deacetylation. Treatment with SAHA or TSA efficiently increased DLC1 expression in all lines, particularly in 22Rv1 cells, and activated the DLC1 promoter through the same Sp1 sites. The 22Rv1 cell line was selected to evaluate the efficacy of combined DLC1 transduction and SAHA treatment on tumor growth in athymic mice. Individually, DLC1 transduction and SAHA exposure reduced the tumor size by 75–80% compared to controls and in combination almost completely inhibited tumor growth. The antitumor effect was associated with the induction of apoptosis and inhibition of RhoA activity. SAHA alone significantly reduced RhoA activity, showing that this RhoGTPase is a target for SAHA. These results, obtained with a reliable preclinical in vivo test, predict that combined therapeutic agents targeting the pathways governing DLC1 function and HDAC inhibitors may be beneficial in management of prostate cancer.

Judy Wai Ping Yam - One of the best experts on this subject based on the ideXlab platform.

  • Abstract 1565: Novel RhoGAP independent pathway of tumor suppressor DLC1 regulates cancer invasion and metastasis
    Molecular and Cellular Biology, 2014
    Co-Authors: Yin-shan Yeung, Xiaowen Mao, Judy Wai Ping Yam
    Abstract:

    Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene critically involved in hepatocarcinogenesis. DLC1 is a Rho GTPase activating protein (RhoGAP), which serves as the negative regulator of Rho proteins. Rho proteins are important in remodeling of actin cytoskeleton, transcription regulation, cell proliferation, tumorigenesis and metastasis. Apart from RhoGAP activity, the inhibitory activity of DLC1 is also dependent on its proper focal adhesion localization. It has been found that tensin proteins are responsible for directing DLC1 to the focal adhesions via their interaction which is important to the tumor suppression activity of DLC1. Recently, we identified that cAMP/Protein Kinase A (PKA) could induce DLC1 dimerization by phosphorylation for activation. PKC and PKD had also been demonstrated to phosphorylate DLC1 and modulate the nuclear shuttling and RhoGAP activity of DLC1. Our previous study also revealed that PKB/Akt phosphorylates DLC1 and subsequently attenuates the tumor suppressive activity of DLC1 through a RhoGAP independent pathway. Our data revealed that DLC1 Ser-567 phosphorylation affects its binding with Akt, prompting us to further search for other DLC1 interacting partners which are specifically regulated by Ser-567 phosphorylation. We performed differential co-immunoprecipitation assay to compare protein expressions of phospho-defective mutant (S567A) and phosphomimetic mutant (S567D) of DLC1. Co-immunoprecipitated proteins were then subjected to mass spectrometry for protein identity. Among all interacting candidates, we are interested to characterize the midline family genes: midline-1 (MID-1) and midline-2 (MID-2). MID-1 mutation is associated with embryonic developmental defect, X-linked Opitz G/BBB syndrome (XLOS). It is important for epithelial-mesenchymal differentiation, cell migration and adhesion, and apoptosis during embryogenesis. MID1 protein is a microtubule-associated ubiquitin E3 ligase. MID1 targets catalytic subunit of protein phosphatase 2A (PP2Ac) for ubiquitination by binding with α4. In HCC, α4 is highly expressed. From our analysis, MID-1/2 proteins interact with S567A but not with S567D, suggesting that Akt phosphorylation abolishes the interaction between DLC1 and MID-1/2. Thus, it would be tempting to investigate how DLC1 affects MID/α4/PP2A complex in controlling epithelial-mesenchymal transition, cell-cycle checkpoint and inducing apoptosis. Our initial investigation has provided evidence that MID-1/2 are novel interacting proteins of DLC1 and their interaction is mediated by phosphorylation of DLC1 at Ser-567. In this study, we aim to investigate the functional significance of DLC1 and MID-1/2 and how DLC1 modulates the functions of MID/α4/PP2A complex in liver cancer. The functional roles of DLC1 and MID-1/2 interaction in HCC metastasis will also be discussed. Citation Format: Frankie Chi Fat Ko, Yin-Shan Yeung, Xiaowen Mao, Judy Wai Ping Yam. Novel RhoGAP independent pathway of tumor suppressor DLC1 regulates cancer invasion and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1565. doi:10.1158/1538-7445.AM2014-1565

  • PKA-induced dimerization of the RhoGAP DLC1 promotes its inhibition of tumorigenesis and metastasis
    Nature communications, 2013
    Co-Authors: Lo-kong Chan, Karen Man-fong Sze, Edith Yuk Ting Tse, Yin-shan Yeung, Judy Wai Ping Yam
    Abstract:

    Deleted in Liver Cancer 1 (DLC1) is a tumour suppressor that inhibits metastasis by inactivating the small GTPase, RhoA. Here the authors reveal that DLC1 function is stimulated by protein kinase A, which induces phosphorylation-dependent DLC1 dimerization.

  • solution structure of the phosphotyrosine binding ptb domain of human tensin2 protein in complex with deleted in liver cancer 1 DLC1 peptide reveals a novel peptide binding mode
    Journal of Biological Chemistry, 2012
    Co-Authors: Lihong Chen, Judy Wai Ping Yam, Changdong Liu, Guang Zhu
    Abstract:

    Abstract The protein deleted in liver cancer 1 (DLC1) interacts with the tensin family of focal adhesion proteins to play a role as a tumor suppressor in a wide spectrum of human cancers. This interaction has been proven to be crucial to the oncogenic inhibitory capacity and focal adhesion localization of DLC1. The phosphotyrosine binding (PTB) domain of tensin2 predominantly interacts with a novel site on DLC1, not the canonical NPXY motif. In this study, we characterized this interaction biochemically and determined the complex structure of tensin2 PTB domain with DLC1 peptide by NMR spectroscopy. Our HADDOCK-derived complex structure model elucidates the molecular mechanism by which tensin2 PTB domain recognizes DLC1 peptide and reveals a PTB-peptide binding mode that is unique in that peptide occupies the binding site opposite to the canonical NPXY motif interaction site with the peptide utilizing a non-canonical binding motif to bind in an extended conformation and that the N-terminal helix, which is unique to some Shc- and Dab-like PTB domains, is required for binding. Mutations of crucial residues defined for the PTB-DLC1 interaction affected the co-localization of DLC1 and tensin2 in cells and abolished DLC1-mediated growth suppression of hepatocellular carcinoma cells. This tensin2 PTB-DLC1 peptide complex with a novel binding mode extends the versatile binding repertoire of the PTB domains in mediating diverse cellular signaling pathways as well as provides a molecular and structural basis for better understanding the tumor-suppressive activity of DLC1 and tensin2.

  • Abstract 2141: cAMP/PKA signaling enhances activity of Deleted in Liver Cancer 1 (DLC1) tumor suppressor in suppressing liver cancer tumorigenesis
    Molecular and Cellular Biology, 2012
    Co-Authors: Lo-kong Chan, Karen Man-fong Sze, Edith Yuk Ting Tse, Yin-shan Yeung, Judy Wai Ping Yam
    Abstract:

    Deleted in Liver Cancer 1 (DLC1) which encodes a RhoGTPase-activating protein (RhoGAP) is a tumor suppressor frequently inactivated in a wide spectrum of human cancers. The RhoGAP activity has been shown to play a predominant role in the biological activities of DLC1. It has been shown that cells with silenced DLC1 exhibit increased active RhoA level. This finding provides evidence about the activation of RhoA as the consequence of deregulated DLC1 and points to the importance of RhoGAP activity in the biological activity of DLC1. In this regard, it is important to comprehend how RhoGAP activity of DLC1 is related. Here, we show that DLC1 was robustly phosphorylated by cyclic AMP (cAMP)/Protein kinase A (PKA) signaling in cells. Phosphorylation of DLC1 was enhanced by forskolin, a known activator of PKA while suppressed when H-89, an inhibitor of PKA was added. Direct phosphorylation of DLC1 by PKA was further confirmed by the in vitro kinase assay. Using specific phospho-DLC1 antibodies, PKA was shown to phosphorylate DLC1 at S431 and S549. Functional assays demonstrate that phosphorylation at S549 plays a critical role in provoking the inhibitory activity of DLC1 in suppressing growth and motility of Ras-transduced p53 null mouse hepatoblasts. When compared with the stable clone of wild-type DLC1, stable clone of DLC1 phosphomimetic mutant, S549D, displayed a largely reduced growth of subcutaneous and orthotopic liver implanted tumors and an enhanced apoptosis. The migration and invasion rates of S549D cells were also significantly inhibited. Furthermore, S549D expression abolished stress fiber formation but failed to alter filopodia protrusions. These functional effects exerted by S549D were ascribed to the enhanced RhoGAP activity against RhoA. To further investigate the mechanism through which the RhoGAP activity is enhanced, we found that DLC1 dimerized upon S549 phosphorylation. Our findings have revealed for the first time about the regulation of RhoGAP activity of DLC1 via dimerization. Our study suggests a molecular link between PKA and DLC1/Rho pathways and underscores the importance of S549 phosphorylation in the regulation of RhoGAP activity of DLC1. (This study was funded by the Small Project Funding Program, The University of Hong Kong 200907176125) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2141. doi:1538-7445.AM2012-2141

  • abstract 2141 camp pka signaling enhances activity of deleted in liver cancer 1 DLC1 tumor suppressor in suppressing liver cancer tumorigenesis
    Cancer Research, 2012
    Co-Authors: Lo-kong Chan, Karen Man-fong Sze, Edith Yuk Ting Tse, Yin-shan Yeung, Judy Wai Ping Yam
    Abstract:

    Deleted in Liver Cancer 1 (DLC1) which encodes a RhoGTPase-activating protein (RhoGAP) is a tumor suppressor frequently inactivated in a wide spectrum of human cancers. The RhoGAP activity has been shown to play a predominant role in the biological activities of DLC1. It has been shown that cells with silenced DLC1 exhibit increased active RhoA level. This finding provides evidence about the activation of RhoA as the consequence of deregulated DLC1 and points to the importance of RhoGAP activity in the biological activity of DLC1. In this regard, it is important to comprehend how RhoGAP activity of DLC1 is related. Here, we show that DLC1 was robustly phosphorylated by cyclic AMP (cAMP)/Protein kinase A (PKA) signaling in cells. Phosphorylation of DLC1 was enhanced by forskolin, a known activator of PKA while suppressed when H-89, an inhibitor of PKA was added. Direct phosphorylation of DLC1 by PKA was further confirmed by the in vitro kinase assay. Using specific phospho-DLC1 antibodies, PKA was shown to phosphorylate DLC1 at S431 and S549. Functional assays demonstrate that phosphorylation at S549 plays a critical role in provoking the inhibitory activity of DLC1 in suppressing growth and motility of Ras-transduced p53 null mouse hepatoblasts. When compared with the stable clone of wild-type DLC1, stable clone of DLC1 phosphomimetic mutant, S549D, displayed a largely reduced growth of subcutaneous and orthotopic liver implanted tumors and an enhanced apoptosis. The migration and invasion rates of S549D cells were also significantly inhibited. Furthermore, S549D expression abolished stress fiber formation but failed to alter filopodia protrusions. These functional effects exerted by S549D were ascribed to the enhanced RhoGAP activity against RhoA. To further investigate the mechanism through which the RhoGAP activity is enhanced, we found that DLC1 dimerized upon S549 phosphorylation. Our findings have revealed for the first time about the regulation of RhoGAP activity of DLC1 via dimerization. Our study suggests a molecular link between PKA and DLC1/Rho pathways and underscores the importance of S549 phosphorylation in the regulation of RhoGAP activity of DLC1. (This study was funded by the Small Project Funding Program, The University of Hong Kong 200907176125) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2141. doi:1538-7445.AM2012-2141

Xiaolan Qian - One of the best experts on this subject based on the ideXlab platform.

  • Abstract 3431: Epigenetic reactivation of the DLC1 tumor suppressor protein is a new approach for cancer treatment
    Cancer Chemistry, 2020
    Co-Authors: Brajendra K Tripathi, Xiaolan Qian, Meghan F. Anderman, Luciarita Boccuzzi, Kylie J. Walters, James H. Doroshow, Douglas R Lowy
    Abstract:

    DLC1 is a potent tumor suppressor protein, which is frequently downregulated in cancer. In addition, several types of solid tumors express DLC1 mRNA but have lost DLC1 protein through its destabilization. Here, we identify a methyltransferase, EZH2, that directly methylates the DLC1 protein, leading to its ubiquitin-dependent proteasomal degradation, and explore the clinical relevance of this finding. Our studies indicate that although the canonical activity of EZH2 is on histone H3 in the nucleus, the methylation of DLC1 protein occurs in the cytoplasm, where EZH2 protein is readily detectible and is active. EZH2-dependent methylation of DLC1 leads to its degradation induced by the Cullin-4 ubiquitin ligase. In several tumor cell lines, including NSCLC A549, that carry mutant KRAS and express DLC1 mRNA but lack detectable DLC1 protein, treatment with the EZH2 inhibitor Tazemetostat stabilized the DLC1 protein. As previous studies have indicated that activated KRAS can induce EZH2, we asked whether there might be a link between the mutant KRAS and DLC1 protein stability. Consistent with this hypothesis, siRNA knockdown of KRAS leads to substantial stabilization of the DLC1 protein in A549 cells and other tumor lines with mutant KRAS. Although EZH2 inhibitors can stabilize the DLC1 protein, this restoration of DLC1 has limited tumor suppressor activity because AKT and SRC kinases, which we found directly phosphorylate DLC1 and cooperatively attenuate its tumor suppressor functions, tend to be activated in solid tumors, thereby reducing the tumor suppressor activity of the stabilized DLC1 protein. Treatment of tumor xenografts that carry mutant KRAS with AKT or SRC kinase inhibitors in conjunction with EZH2 inhibition has much greater antitumor activity than treatment with any one of the three inhibitors, and the combined treatment with all three drugs has even greater therapeutic activity, which is correlated with the induction of greater cellular senescence and apoptosis, as measured by the beta-galactosidase and annexin V expression, respectively. Remarkably, these combinations are well-tolerated, and their therapeutic efficacy against xenografts with mutant KRAS make them a candidate intervention against this important unmet clinical need. We have made similar observations with proteasomal inhibition together with AKT and/or SRC inhibition in A549 cells and other tumor lines that carry mutant KRAS and express DLC1 mRNA, but lack DLC1 protein. Inhibitory RNA knockdown of DLC1 expression reduces the antitumor activity of the drug combination, strongly suggesting that DLC1 plays a critical role in the therapeutic response to the combination. In summary, our studies provide a mechanistic rationale for combining EZH2 or proteasomal inhibition with AKT and/or SRC inhibition in treating tumors that carry mutant KRAS and express DLC1 mRNA, but lack DLC1 protein. Thus, this study is a novel example where epigenetic reactivation of the tumor suppressor can be a key therapeutic target, in contrast to the usual situation where the focus is principally on inhibiting pro-oncogenic factors. Citation Format: Brajendra K. Tripathi, Luciarita Boccuzzi, Meghan Anderman, Xiaolan Qian, Kylie J. Walters, James H. Doroshow, Douglas R. Lowy. Epigenetic reactivation of the DLC1 tumor suppressor protein is a new approach for cancer treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3431.

  • Cancer-Associated Point Mutations in the DLC1 Tumor Suppressor and Other Rho-GAPs Occur Frequently and Are Associated with Decreased Function
    Cancer research, 2020
    Co-Authors: Dunrui Wang, Xiaolan Qian, Marian E. Durkin, Brajendra K Tripathi, Beatriz Sanchez-solana, Douglas R Lowy
    Abstract:

    In advanced cancer, the RHOA GTPase is often active together with reduced expression of genes encoding Rho-specific GTPase-accelerating proteins (Rho-GAP), which negatively regulate RHOA and related GTPases. Here we used the The Cancer Genome Atlas dataset to examine 12 tumor types (including colon, breast, prostate, pancreas, lung adenocarcinoma, and squamous cell carcinoma) for the frequency of codon mutations of 10 Rho-GAP and experimentally tested biochemical and biological consequences for cancer-associated mutants that arose in the DLC1 tumor suppressor gene. DLC1 was the Rho-GAP gene mutated most frequently, with 5%-8% of tumors in five of the tumor types evaluated having DLC1 missense mutations. Furthermore, 20%-26% of the tumors in four of these five tumor types harbored missense mutations in at least one of the 10 Rho-GAPs. Experimental analysis of the DLC1 mutants indicated 7 of 9 mutants whose lesions were located in the Rho-GAP domain were deficient for Rho-GAP activity and for suppressing cell migration and anchorage-independent growth. Analysis of a DLC1 linker region mutant and a START domain mutant showed each was deficient for suppressing migration and growth in agar, but their Rho-GAP activity was similar to that of wild-type DLC1. Compared with the wild-type, the linker region mutant bound 14-3-3 proteins less efficiently, while the START domain mutant displayed reduced binding to Caveolin-1. Thus, mutation of Rho-GAP genes occurs frequently in some cancer types and the majority of cancer-associated DLC1 mutants evaluated were deficient biologically, with various mechanisms contributing to their reduced activity. SIGNIFICANCE: These findings indicate that point mutation of Rho-GAP genes is unexpectedly frequent in several cancer types, with DLC1 mutants exhibiting reduced function by various mechanisms.

  • SRC and ERK cooperatively phosphorylate DLC1 and attenuate its Rho-GAP and tumor suppressor functions.
    The Journal of cell biology, 2019
    Co-Authors: Brajendra K Tripathi, Alex G Papageorge, Xiaolan Qian, Dunrui Wang, Meghan F. Anderman, Ming Zhou, Douglas R Lowy
    Abstract:

    SRC and ERK kinases control many cell biological processes that promote tumorigenesis by altering the activity of oncogenic and tumor suppressor proteins. We identify here a physiological interaction between DLC1, a focal adhesion protein and tumor suppressor, with SRC and ERK. The tumor suppressor function of DLC1 is attenuated by phosphorylation of tyrosines Y451 and Y701 by SRC, which down-regulates DLC1’s tensin-binding and Rho-GAP activities. ERK1/2 phosphorylate DLC1 on serine S129, which increases both the binding of SRC to DLC1 and SRC-dependent phosphorylation of DLC1. SRC inhibitors exhibit potent antitumor activity in a DLC1-positive transgenic cancer model and a DLC1-positive tumor xenograft model, due to reactivation of the tumor suppressor activities of DLC1. Combined treatment of DLC1-positive tumors with SRC plus AKT inhibitors has even greater antitumor activity. Together, these findings indicate cooperation between the SRC, ERK1/2, and AKT kinases to reduce DLC1 Rho-GAP and tumor suppressor activities in cancer cells, which can be reactivated by the kinase inhibitors.

  • Abstract 1762: The START domain of the DLC1 tumor suppressor binds phosphatidylserine and two proteins, PLCD1 and Caveolin-1
    Molecular and Cellular Biology Genetics, 2019
    Co-Authors: Beatriz Sanchez-solana, Alex G Papageorge, Xiaolan Qian, Dunrui Wang, Parthibane Velayoudame, Jairaj Acharya, Douglas R Lowy
    Abstract:

    The tumor suppressor gene DLC1 encodes a multi-domain protein, which includes a Rho-GAP domain (Rho GTPase activating protein domain) that negatively regulates the activity of RhoA, B, and C, and has been hypothesized to be the basis of its tumor suppressor functions. DLC1 also contains a less well characterized START (StAR-related lipid transfer) domain in its C-terminus, whose overall contribution to DLC1 function remains incompletely understood. START domains in other proteins have been shown to bind lipids, but no lipid has yet been identified that binds the DLC1 START domain. In the present study, we attempted to gain further insight into the molecular function and lipid binding properties of the DLC1 START domain. We previously determined that Caveolin-1, the main structural component and marker of caveolae, interacts with the DLC1 START domain, and this interaction contributes to the full tumor suppressor activity of DLC1. In unpublished studies, we additionally identified Phospholipase C delta 1 (PLCD1), which participates in intracellular Ca2+ mobilization, forming a complex with the DLC1 START domain and with Caveolin-1. We have now developed evidence that, in addition to binding Caveolin-1 and PLCD1, the DLC1 START domain binds a lipid, phosphatidylserine (PS), and have identified a colon cancer-associated DLC1-START domain mutant (R947C) that results in a protein deficient in forming a complex with all three binding partners and displays reduced tumor suppressor activity but intact RhoGAP activity. The interaction between the DLC1-START domain and PS can occur independently of PLCD1 and/or Caveolin-1, but PS promotes complex formation between the DLC1 START domain and PLCD1 or Caveolin-1, without altering PLCD1 to Caveolin-1 binding. Biological assays with wild type DLC1 show cooperation between DLC1 and PLCD1 or Caveolin-1 to inhibit cell migration, but no cooperation is observed when all three genes are overexpressed, suggesting that Caveolin-1 and PLCD1 occupy the same migration signaling pathway. Thus, we have identified a biologically relevant complex between DLC1 and three macromolecules (PLCD1, PS and Caveolin-1) that contributes to the full tumor suppressor function of DLC1 independently of its RhoGAP activity, and are testing the hypothesis that PS serves as a linker between the DLC1 START domain and PLCD1 or Caveolin-1. Citation Format: Beatriz Sanchez-Solana, Dunrui Wang, Xiaolan Qian, Alex Papageorge, Parthibane Velayoudame, Jairaj Acharya, Douglas R. Lowy. The START domain of the DLC1 tumor suppressor binds phosphatidylserine and two proteins, PLCD1 and Caveolin-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1762.

  • Effects of DLC1 Deficiency on Endothelial Cell Contact Growth Inhibition and Angiosarcoma Progression
    Journal of the National Cancer Institute, 2017
    Co-Authors: David Sánchez-martín, Xiaolan Qian, Douglas R Lowy, Dunrui Wang, Atsushi Otsuka, Kenji Kabashima, Giovanna Tosato
    Abstract:

    Background Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene frequently deleted in cancer. However, DLC1 is not known to be deleted in angiosarcoma, an aggressive malignancy of endothelial cell derivation. Additionally, the physiologic functions of DLC1 protein in endothelial cells are poorly defined.

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  • DLC1 suppresses NF-κB activity in prostate cancer cells due to its stabilizing effect on adherens junctions.
    SpringerPlus, 2014
    Co-Authors: Veenu Tripathi, Nicholas C. Popescu, Drazen B. Zimonjic
    Abstract:

    DLC1 (Deleted in Liver Cancer 1) gene encodes a RhoGTPase-activating protein (RhoGAP), which exerts most of its tumor suppressor functions through suppression of small Rho GTPases proteins RhoA, RhoB, RhoC and to some degree Cdc42, but not Rac. RhoGTPases are implicated in NF-κB activation in highly invasive prostate carcinoma (PCA), with consequences on cell proliferation, survival and metastatic capacity. Here we demonstrate that DLC1 transduction in two androgen-independent (AI) and highly metastatic PCA cell lines negatively regulates NF-κB activity in a GAP- and α-catenin-dependent manner. Expressed DLC1 protein suppresses the phosphorylation of NF-κB inhibitor, IκBα, causes its relocation from membrane ruffles into cytoplasm and attenuates its ubiquitination and subsequent degradation. DLC1-mediated NF-kB suppression and its effects are comparable to NF-κB inhibition using either shRNA knockdown or peptide inhibitor. Expression of transduced DLC1 suppressed the expression of NF-κB mediated genes. Such effects were found to be reliant on presence of calcium, indicating that the observed modifications are dependent on, and enabled by DLC-mediated stabilization of adherens junctions. These results expand the multitude of DLC1 interactions with other genes that modulate its oncosuppressive function, and may have potential therapeutic implications.

  • DLC1 induces expression of E-cadherin in prostate cancer cells through Rho pathway and suppresses invasion
    Oncogene, 2013
    Co-Authors: Veenu Tripathi, Nicholas C. Popescu, Drazen B. Zimonjic
    Abstract:

    E-cadherin is a cell-cell adhesion molecule that acts as a suppressor of cancer cell invasion and its expression is downregulated in many advanced, poorly differentiated, human cancers. In this study, we found that the expression of DLC1 (deleted in liver cancer 1) tumor-suppressor gene in metastatic prostate carcinoma (PCA) cells increased the expression of E-cadherin and resulted in an elevated rate of cell-cell aggregation as measured by aggregation assay. DLC1-mediated increase in E-cadherin expression was not dependent on α-catenin, a DLC1-binding protein associated with E-cadherin, and/or cellular density. The increase of E-cadherin expression occurred at mRNA level and relied on DLC1 RhoGAP function, leading to suppression of high level of RhoA-GTP and RhoC-GTP activity in metastatic PCA cells. Application of Rho/ROCK inhibitors produced the same effect as introduction of DLC1. Knocking down of RhoA produced a moderate increase in E-cadherin whereas knocking down of RhoC resulted in a significant increase of E-cadherin. Downregulation of E-cadherin caused by constitutively active RhoA(V14) and RhoC(V14) could not be reversed by expression of DLC1 in DLC1-negative cell line. DLC1-mediated suppression of metastatic PCA cells invasion was comparable with the one associated with ectopic E-cadherin expression, or caused by suppression of Rho pathway either by Rho/ROCK inhibitors, or by shRNA repression. This study demonstrates that DLC1 expression positively regulates E-cadherin and suppresses highly metastatic PCA cell invasion by modulating Rho pathway, which appears as a feasible therapeutic target in cancers with high activity of RhoGTPases.

  • DLC1 interaction with α-catenin stabilizes adherens junctions and enhances DLC1 antioncogenic activity.
    Molecular and cellular biology, 2012
    Co-Authors: Veenu Tripathi, Nicholas C. Popescu, Drazen B. Zimonjic
    Abstract:

    The DLC1 (for deleted in liver cancer 1) tumor suppressor gene encodes a RhoGAP protein that inactivates Rho GTPases, which are implicated in regulation of the cytoskeleton and adherens junctions (AJs), a cell-cell adhesion protein complex associated with the actin cytoskeleton. Malignant transformation and tumor progression to metastasis are often associated with changes in cytoskeletal organization and cell-cell adhesion. Here we have established in human cells that the AJ-associated protein α-catenin is a new binding partner of DLC1. Their binding was mediated by the N-terminal amino acids 340 to 435 of DLC1 and the N-terminal amino acids 117 to 161 of α-catenin. These proteins colocalized in the cytosol and in the plasma membrane, where together they associated with E-cadherin and β-catenin, constitutive AJ proteins. Binding of DLC1 to α-catenin led to their accumulation at the plasma membrane and required DLC1 GAP activity. Knocking down α-catenin in DLC1-positive cells diminished DLC1 localization at the membrane. The DLC1-α-catenin complex reduced the Rho GTP level at the plasma membrane, increased E-cadherin's mobility, affected actin organization, and stabilized AJs. This process eventually contributed to a robust oncosuppressive effect of DLC1 in metastatic prostate carcinoma cells. Together, these results unravel a new mechanism through which DLC1 exerts its strong oncosuppressive function by positively influencing AJ stability.

  • Abstract 4017: DLC1 and α-catenin protein interaction enhances DLC1 antioncogenic activity by stabilizing adherens junctions and suppressing NFκB signaling
    Cellular and Molecular Biology, 2011
    Co-Authors: Veenu Tripathi, Drazen B. Zimonjic, Nicholas C. Popescu
    Abstract:

    Adherens junctions (AJs), which are cell-cell adhesion complexes closely associated with actin cytoskeleton, play an important role in maintaining epithelial tissue homeostasis. Both malignant transformation, and tumor progression to metastasis are often associated with major changes in cytoskeletal organization, the cell-cell adhesion, and aberrant adhesion-mediated signaling. The tumor suppressor gene DLC1 (Deleted in Liver Cancer 1) encodes a RhoGTPase activating protein (GAP) that acts as a negative regulator of the Rho family of small GTPases, which are implicated in cytoskeleton organization and regulation of AJs. A yeast two-hybrid screening identified α-catenin, a component of AJs, as a potential DLC19s binding partner. We relied on this finding and investigated the role of DLC1 in the regulation of AJs and in adhesion-mediated signaling. Using human embryonic kidney (HEK 293) cells we demonstrated that DLC1 interacted with α-catenin and associated with E-cadherin and β-catenin, both constitutive AJs proteins. Co-immunoprecipitation using various deletion constructs of DLC1 and of α-catenin established that the N-terminal (340-435 aa) of DLC1 interacted with the N-terminal (39-161 aa) of α-catenin. Immuno-fluorescence staining and western blot of cellular membrane and cytoplasmic fractions, demonstrated that binding of DLC1 to α-catenin recruited α-catenin to the plasma membrane and required DLC1 GAP activity. In contrast, the DLC1 GAP mutant (R718E) failed to produce the same effect. Fluorescence Recovery After Photobleaching (FRAP) analysis demonstrated that DLC1-α-catenin interaction increased the GFP-E-cadherin mobility at cell-cell junctions, whereas phalloidin staining showed intact actin cable formation around the cell periphery, resulting in the stabilization of AJs. The DLC1-α-catenin interaction is instrumental for a maximal oncosuppressive effects of DLC1 (i.e., by inhibiting proliferation and abolishing colony formation of prostate carcinoma cells); DLC1 mutant defective for α-catenin binding failed to reproduce such an effect. Loss of α-catenin is usually accompanied by an increase in the activation of NFkB, which is associated with increased cell proliferation and resistance to apoptosis. We demonstrated that, by immobilizing NFkB to the AJ complex, DLC1-α-catenin interaction effectively inhibited NFkB phosphorylation and, therefore, its activation. Together, these results unravel a new mechanism through which DLC1 exerts its oncosuppressive function by stabilizing AJs and suppressing NFkB signaling. 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 4017. doi:10.1158/1538-7445.AM2011-4017

  • DLC1 interaction with S100A10 mediates inhibition of in vitro cell invasion and tumorigenicity of lung cancer cells through a RhoGAP-independent mechanism
    Cancer Research, 2011
    Co-Authors: Xu-yu Yang, Nicolae C. Popescu, Drazen B. Zimonjic
    Abstract:

    The DLC1 gene encodes a Rho GTPase-activating protein (RhoGAP) that functions as a tumor suppressor in several common human cancers. The multidomain structure of DLC1 enables interaction with a number of other proteins. Here we report that the proinflammatory protein S100A10 (also known as p11), a key cell surface receptor for plasminogen which regulates pericellular proteolysis and tumor cell invasion, is a new binding partner of DLC1 in human cells. We determined that the 2 proteins colocalize in the cell cytoplasm and that their binding is mediated by central sequences in the central domain of DLC1 and the C-terminus of S100A10. Because the same S100A10 sequence also mediates binding to Annexin 2, we found that DLC1 competed with Annexin 2 for interaction with S100A10. DLC1 binding to S100A10 did not affect DLC1's RhoGAP activity, but it decreased the steady-state level of S100A10 expression in a dose-dependent manner by displacing it from Annexin 2 and making it accessible to ubiquitin-dependent degradation. This process attenuated plasminogen activation and resulted in inhibition of in vitro cell migration, invasion, colony formation, and anchorage-independent growth of aggressive lung cancer cells. These results suggest that a novel GAP-independent mechanism contributes to the tumor suppressive activity of DLC1, and highlight the importance and complexity of protein-protein interactions involving DLC1 in certain cancers.