Staphylococcal Nuclease Domain-containing 1 Research Articles

Abstract 2628: The multifunctional protein staphylococcal nuclease domain containing-1 (SND1) promotes migration and invasion of hepatocellular carcinoma (HCC) cells by modulating angiotensin II type 1 receptor (AT1R) and transforming growth factor-β (TGF-β) s

Abstract Staphylococcal nuclease domain containing-1 (SND1) is a multifunctional protein that modulates transcription, mRNA splicing and stability, and miRNA function. We previously documented that SND1 is markedly overexpressed in human hepatocellular carcinoma (HCC) patients in a stage-specific manner and inhibition of SND1 in human HCC cells significantly abrogates in vitro proliferation and in vivo tumorigenesis in nude mice. In the present study we document that migration and invasion of human HCC cells are significantly augmented in SND1-overexpressing clones and significantly inhibited in SND1 shRNA-expressing clones. Differential gene expression analysis between control clone and SND1 shRNA expressing clones by Affymetrix microarray revealed downregulation of TGF-β downstream genes upon SND1 inhibition. Additionally, a human angiogenesis array identified plasminogen activator inhibitor-1 (PAI-1), a TGF-β downstream gene, to be upregulated by SND1 and this upregulation was confirmed by ELISA. TGF-β expression is regulated by angiotensin II type 1 receptors (AT1R) and previous studies have documented that SND1 increases AT1R expression by increasing AT1R mRNA stability. We hypothesized that increased AT1R expression, conferred by SND1 overexpression, might facilitate activation of TGF-β signaling and subsequently PAI-1-mediated increase in migration and invasion. Indeed, we observed that AT1R and TGF-β expression was significantly increased in SND1-overexpressing clones and decreased in SND1 knockdown clones. The half-life of AT1R mRNA was significantly longer in SND1-overexpressing clones compared to the control clone confirming that SND1 modulates AT1R post-transcriptionally. The AT1R blocker Losartan Potassium and AT1R siRNA significantly inhibited SND1-induced TGF-β and PAI-1 expression with associated inhibition in migration and invasion. As a corollary, inhibition of PAI-1 by siRNA significantly abrogated migration and invasion of SND1-overexpressing cells. TGF-β induces epithelial-mesenchymal transition (EMT) and we observed increased expression of EMT markers vimentin, snail and slug and decreased expression of E-cadherin in SND1-overexpressing clones and vice versa in SND1-knockdown clones. Our studies thus unravel a novel mechanism in which post-transcriptional regulation of AT1R by SND1, with subsequent activation of TGF-β signaling, promotes invasion of human HCC cells thereby conferring an aggressive phenotype. Small molecule inhibitors of SND1, thus, might be effective therapeutic strategy for late-stage aggressive HCC. Citation Format: Prasanna K. Santhekadur, Rachel Gredler, Maaged Akiel, Paul Dent, Paul B. Fisher, Devanand Sarkar. The multifunctional protein staphylococcal nuclease domain containing-1 (SND1) promotes migration and invasion of hepatocellular carcinoma (HCC) cells by modulating angiotensin II type 1 receptor (AT1R) and transforming growth factor-β (TGF-β) s [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2628. doi:10.1158/1538-7445.AM2013-2628

Multifunction Protein Staphylococcal Nuclease Domain Containing 1 (SND1) Promotes Tumor Angiogenesis in Human Hepatocellular Carcinoma through Novel Pathway That Involves Nuclear Factor κB and miR-221

Staphylococcal nuclease domain-containing 1 (SND1) is a multifunctional protein that is overexpressed in multiple cancers, including hepatocellular carcinoma (HCC). Stable overexpression of SND1 in Hep3B cells expressing a low level of SND1 augments, whereas stable knockdown of SND1 in QGY-7703 cells expressing a high level of SND1 inhibits establishment of xenografts in nude mice, indicating that SND1 promotes an aggressive tumorigenic phenotype. In this study we analyzed the role of SND1 in regulating tumor angiogenesis, a hallmark of cancer. Conditioned medium from Hep3B-SND1 cells stably overexpressing SND1 augmented, whereas that from QGY-SND1si cells stably overexpressing SND1 siRNA significantly inhibited angiogenesis, as analyzed by a chicken chorioallantoic membrane assay and a human umbilical vein endothelial cell differentiation assay. We unraveled a linear pathway in which SND1-induced activation of NF-κB resulted in induction of miR-221 and subsequent induction of angiogenic factors Angiogenin and CXCL16. Inhibition of either of these components resulted in significant inhibition of SND1-induced angiogenesis, thus highlighting the importance of this molecular cascade in regulating SND1 function. Because SND1 regulates NF-κB and miR-221, two important determinants of HCC controlling the aggressive phenotype, SND1 inhibition might be an effective strategy to counteract this fatal malady.

Multifunction Protein Staphylococcal Nuclease Domain Containing 1 (SND1) Promotes Tumor Angiogenesis in Human Hepatocellular Carcinoma through Novel Pathway That Involves Nuclear Factor κB and miR-221

Multifunction Protein Staphylococcal Nuclease Domain Containing 1 (SND1) Promotes Tumor Angiogenesis in Human Hepatocellular Carcinoma through Novel Pathway That Involves Nuclear Factor κB and miR-221

Identification of Staphylococcal Nuclease Domain-containing 1 (SND1) as a Metadherin-interacting Protein with Metastasis-promoting Functions

Metastasis is the deadliest and most poorly understood feature of malignant diseases. Recent work has shown that Metadherin (MTDH) is overexpressed in over 40% of breast cancer patients and promotes metastasis and chemoresistance in experimental models of breast cancer progression. Here we applied mass spectrometry-based screen to identify staphylococcal nuclease domain-containing 1 (SND1) as a candidate MTDH-interacting protein. After confirming the interaction between SND1 and MTDH, we tested the role of SND1 in breast cancer and found that it strongly promotes lung metastasis. SND1 was further shown to promote resistance to apoptosis and to regulate the expression of genes associated with metastasis and chemoresistance. Analyses of breast cancer clinical microarray data indicated that high expression of SND1 in primary tumors is strongly associated with reduced metastasis-free survival in multiple large scale data sets. Thus, we have uncovered SND1 as a novel MTDH-interacting protein and shown that it is a functionally and clinically significant mediator of metastasis.

Increased RNA-induced silencing complex (RISC) activity contributes to hepatocellular carcinoma

There is virtually no effective treatment for advanced hepatocellular carcinoma (HCC) and novel targets need to be identified to develop effective treatment. We recently documented that the oncogene Astrocyte elevated gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis. Employing yeast two-hybrid assay and coimmunoprecipitation followed by mass spectrometry, we identified staphylococcal nuclease domain containing 1 (SND1), a nuclease in the RNA-induced silencing complex (RISC) facilitating RNAi-mediated gene silencing, as an AEG-1 interacting protein. Coimmunoprecipitation and colocalization studies confirmed that AEG-1 is also a component of RISC and both AEG-1 and SND1 are required for optimum RISC activity facilitating small interfering RNA (siRNA) and micro RNA (miRNA)-mediated silencing of luciferase reporter gene. In 109 human HCC samples SND1 was overexpressed in ≈74% cases compared to normal liver. Correspondingly, significantly higher RISC activity was observed in human HCC cells compared to immortal normal hepatocytes. Increased RISC activity, conferred by AEG-1 or SND1, resulted in increased degradation of tumor suppressor messenger RNAs (mRNAs) that are target of oncomiRs. Inhibition of enzymatic activity of SND1 significantly inhibited proliferation of human HCC cells. As a corollary, stable overexpression of SND1 augmented and siRNA-mediated inhibition of SND1 abrogated growth of human HCC cells in vitro and in vivo, thus revealing a potential role of SND1 in hepatocarcinogenesis. We unravel a novel mechanism that overexpression of AEG-1 and SND1 leading to increased RISC activity might contribute to hepatocarcinogenesis. Targeted inhibition of SND1 enzymatic activity might be developed as an effective therapy for HCC.

Abstract 4706: Overexpression of the components of RNA-induced silencing complex: Contribution to hepatocellular carcinoma

Abstract Objectives: There is virtually no treatment for advanced hepatocellular carcinoma (HCC). As such novel targets need to be identified to develop effective therapies to combat this fatal disease. The oncogene Astrocyte elevated gene-1 (AEG-1) is overexpressed in more than 90% of human HCC patients and plays a seminal role in hepatocarcinogenesis. AEG-1 is localized on the cell membrane as well as in the cytoplasm, nucleus and endoplasmic reticulum. In each location AEG-1 interacts with specific proteins thereby modulating diverse intracellular processes which contributes pleotrophic oncogenic properties to AEG-1. Identification of AEG-1 interacting proteins would facilitate better understanding of AEG-1 function and help develop novel and targeted therapies for HCC. Methods: Yeast two-hybrid assay and co-immunoprecipitation (co-ip) followed by mass spectrometry were used to identify AEG-1-interacting proteins. Co-ip and double immunofluorescence analyses were performed to confirm protein-protein interaction. A Renilla luciferase reporter plasmid containing a miRNA-recognition site in the 3’-untranslated region was used in a transfection based assay to monitor RNA-induced silencing complex (RISC) activity. Immunohistochemistry in tissue microarray was performed to check protein expression. Nude mice xenograft studies were done to check tumorigenesis. Results: We identified Staphylococcal nuclease domain containing 1 (SND1), a nuclease functioning in the RNA-induced silencing complex (RISC), as an AEG-1 interacting protein. We documented that AEG-1 interacts with SND1 and Argonaute 2 (Ago2), the major nuclease in RISC, and thus AEG-1 itself is a component of RISC. Knocking down AEG-1 or SND1 abrogates and overexpression of AEG-1 or SND1 augments RISC activity in human HCC cells. In 109 human HCC samples SND1 was overexpressed in ∼74% cases compared to normal liver. Correspondingly, significantly higher RISC activity was observed in human HCC cells compared to normal hepatocytes. We hypothesized that increased RISC activity, conferred by AEG-1 or SND1 overexpression, might facilitate function of oncomiRs resulting in increased degradation of tumor suppressor mRNAs. Indeed, overexpression of AEG-1 or SND1 downregulated while knockdown of AEG-1 or SND1 upregulated the mRNA level of tumor suppressor PTEN, a target of several oncomiRs overexpressed in HCC. Inhibition of SND1 enzymatic activity significantly abrogated cell growth and proliferation of human HCC cells. Stable overexpression of SND1 augmented while stable knockdown of SND1 inhibited tumor growth by human HCC cells in nude mice xenograft assay. Conclusion: We unravel a novel mechanism that overexpression of AEG-1 and SND1 leading to increased RISC activity might contribute to hepatocarcinogenesis. Targeted inhibition of SND1 enzymatic activity might be developed as an effective therapy for HCC. 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 4706. doi:10.1158/1538-7445.AM2011-4706

Structural basis for recognition of arginine methylated Piwi proteins by the extended Tudor domain

Arginine methylation modulates diverse cellular processes and represents a molecular signature of germ-line-specific Piwi family proteins. A subset of Tudor domains recognize arginine methylation modifications, but the binding mechanism has been lacking. Here we establish that, like other germ-line Tudor proteins, the ancestral staphylococcal nuclease domain-containing 1 (SND1) polypeptide is expressed and associates with PIWIL1/Miwi in germ cells. We find that human SND1 binds PIWIL1 in an arginine methylation-dependent manner with a preference for symmetrically dimethylated arginine. The entire Tudor domain and a bifurcated SN domain are required for this binding activity, whereas the canonical Tudor domain alone is insufficient for methylarginine ligand binding. Crystal structures show that the intact SND1 extended Tudor domain forms a wide and negatively charged binding groove, which can accommodate distinct symmetrically dimethylated arginine peptides from PIWIL1 in different orientations. This analysis explains how SND1 preferentially recognizes symmetrical dimethylarginine via an aromatic cage and conserved hydrogen bonds, and provides a general paradigm for the binding mechanisms of methylarginine-containing peptides by extended Tudor domains.