Atlas of Genetics and Cytogenetics

in Oncology and Haematology

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Gene Section Review

SIVA1 (SIVA1, Apoptosis-Inducing Factor) João Agostinho Machado-Neto, Fabiola Traina Hematology and Hemotherapy Center-University of Campinas/Hemocentro-Unicamp, Instituto Nacional de Ciencia e Tecnologia do Sangue, Campinas, Sao Paulo, Brazil (JAMN, FT), Hematology/Oncology Division, Department of Internal Medicine, Medical School of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil (FT)

Published in Atlas Database: October 2013 Online updated version : http://AtlasGeneticsOncology.org/Genes/SIVA1ID42301ch14q32.html DOI: 10.4267/2042/53646 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2014 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Two alternatively-spliced transcript variants Abstract encoding distinct proteins have been described, Review on SIVA1, with data on DNA/RNA, on the SIVA1 transcript variant 1, which is the protein encoded and where the gene is implicated. predominant transcript variant with a cDNA containing 790 bp (codifying the SIVA1 protein), Identity and the SIVA1 transcript variant 2 lacking the exon 2 with a cDNA containing 595 bp (codifying the Other names: CD27BP, SIVA, Siva-1, Siva-2 SIVA2 protein). HGNC (Hugo): SIVA1 Protein Location: 14q32.33 Description DNA/RNA SIVA1 contains a unique amphipathic helical region (SAH) in the N-terminal region, a death Description domain homology region (DDHR) in the central The entire SIVA1 gene is about 15.3 Kb and part of the protein, and a Zinc finger-like structure contains 4 exons (Start: 105219437 bp and End: at its C-terminal region. The SIVA2 isoform lacks 105234831; Orientation: plus strand). the DDHR domain (Figure 1).

Figure 1. Schematic structure of SIVA1 and SIVA2 proteins. The amphipathic helical region (SAH) at the N-terminal region, a death domain homology region (DDHR) in the central section, and a Zinc finger-like (ZF) structure at its C-terminal region are illustrated. The amino acids (aa) positions are indicated.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(5) 334 SIVA1 (SIVA1, Apoptosis-Inducing Factor) Machado-Neto JA, Traina F

Figure 2. Intracellular localization of SIVA1 protein in a prostate cancer cell line. Confocal analysis of LNCaP cells displaying SIVA (green), DAPI (blue) and Actin (red) staining; MERGE shows the overlapped images. Scale bar, 10 µm. Note the cytoplasmatic and nuclear localization of SIVA1. Anti-SIVA1 (sc-7436) was from Santa Cruz Biotechnology, (Santa Cruz, CA, USA), Phalloidin (A12379) and DAPI (P-36931) were from Invitrogen (Carlsbad, CA, USA). Personal data.

Expression upregulated under stress or following DNA damage (Ray et al., 2011; Fortin et al., 2004). Ubiquitous. Recently, novel partners and functions have been Localisation attributed to SIVA1. SIVA1 binds to and regulates SIVA1 is found in the nucleus and cytoplasm p53 stability by acting as an adapter protein (Figure 2). between p53 and MDM2, and participates in an auto-regulatory feedback loop between p53 and Function SIVA1 (Du et al., 2009; Mei and Wu, 2012). The proapoptotic function of SIVA1 is well SIVA1 associates with ARF, enabling its elucidated and characterized. SIVA1 binds to death ubiquitination and degradation; this mechanisms receptors, including CD27 and TNFRSF18, and also regulates the p53/MDM2 signaling pathway plays a role in the transduction of the proapoptotic (Wang et al., 2013). signal by the extrinsic pathway (Prasad et al., 1997; Finally, SIVA1 is a novel adaptor protein that Spinicelli et al., 2002). SIVA1 interacts with BCL2 promotes Stathmin 1/CaMKII interaction. and BCL-XL, abrogates their antiapoptotic SIVA1 inhibits Stathmin 1 activity through functions and modulates the intrinsic apoptosis Stathmin 1 phosphorylation at serine 16, which pathway (Chu et al., 2004; Chu et al., 2005). In results in reduced cell migration and metastasis by addition, SIVA1 associates with XIAP and stabilizing microtubules of tumor cells (Li et al., regulates the apoptosis mediated by NFkB and JNK 2011). signaling (Resch et al., 2009). The SIVA gene is a The main functions and signaling pathways of transcription target of p53, p73 and E2F1 and is SIVA1 are illustrated in Figure 3.

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(5) 335 SIVA1 (SIVA1, Apoptosis-Inducing Factor) Machado-Neto JA, Traina F

Figure 3. SIVA1 signaling pathway. (1) SIVA1 binds to death receptors and modulates the extrinsic apoptosis pathway. (2) SIVA 1 binds to BCL2 proteins family, inhibits the antiapoptotic proteins, BCL2 and BCL-XL, and leads to proapoptotic BAD protein oligomerization, and modulates the intrinsic apoptosis pathway. (3) SIVA1 binds to the XIAP protein and balances the proapoptotic and antiapoptotic signaling through the JNK and NFkB pathway, respectively, and modulates the extrinsic apoptosis pathway. (4) SIVA1 promotes Stathmin 1/CaMKII interaction, Stathmin 1 phosphorylation and inhibition, and modulates microtubule dynamics. (5) The SIVA1 gene is a transcription target of p53, p73 and E2F1. (6) SIVA1 protein acts as an adapter protein between p53 and MDM2, and promotes p53 ubiquitination. (7) SIVA1 acts as an ARF E3 ubiquitin ligase and regulates cell proliferation by the ARF/p53/MDM2 pathway. Abbreviations: P, phosphorylation; Ac, acetylation; Ub, ubiquitination. Figure was produced using Servier Medical Art.

The binding partners of SIVA1 are: cells expressing the GFP-BCL-XL protein (Xue et CD27: SIVA1 was initially identified by two- al., 2002). Later on, Chu et al. reported that the hybrid (Y2H) screening using CD27 as a bait, and SAH region of SIVA1 was sufficient to specifically its interaction was confirmed by interact with BCL-XL (Chu et al., 2004). immunoprecipitation (IP) of 293 cells co- B-cell CLL/lymphoma 2 (BCL2): The association expressing both proteins (Prasad et al., 1997). In of BCL2 and SIVA1 was verified using GST pull agreement, Yoon et al. found that murine Siva1 and down assays with GST-SIVA in Cos-7 cells Siva2 also bind to CD27 (Yoon et al., 1999). overexpressing full-length BCL2 protein, and this c-abl oncogene 2, non-receptor tyrosine kinase interaction occurred at the SAH region of SIVA1 (ABL2): Y2H screening using ABL2 (previously (Chu et al., 2004). known as ARG) as the bait identified SIVA1 as a CD4: Y2H screen using cytoplasmic domain of binding partner. This protein association was CD4 as the bait identified SIVA1. This protein confirmed by IP of MCF7 cells co-expressing interaction was confirmed by in vitro binding FLAG-ABL2 and GFP-SIVA1 (Cao et al., 2001). assays with GST-SIVA1. The interaction was Tumor necrosis factor receptor superfamily, mapped through GST pull-down assay using GST member 18 (TNFRSF18): TNFRSF18 (previously tagged deletion mutants of SIVA1; the C-terminal known as GITR) presents high homology with region of SIVA1 binds to the cytoplasmic domain CD27. The interaction between TNFRSF18 and of CD4 (Py et al., 2007). SIVA1 was identified using GST pull down and IP Lysophosphatidic acid receptor 2 (LPAR2): Y2H assays (Spinicelli et al., 2002). screening using the C-terminal region of LPAR2 as BCL2-like 1 (BCL-XL): The association of BCL- the bait identified SIVA1. GST pull-down assays XL and SIVA1 was first identified using purified confirmed this protein association and the SIVA1 GST-SIVA and BCL-XL proteins and confirmed by C-terminal region (aa 139-175) is required for this GST pull down assays using GST-SIVA1 in 293 interaction (Lin et al., 2007).

Atlas Genet Cytogenet Oncol Haematol. 2014; 18(5) 336 SIVA1 (SIVA1, Apoptosis-Inducing Factor) Machado-Neto JA, Traina F

Table 1. Comparative identity of human SIVA1 with other species. Source: homologene.

Pyrin (MEFV): Y2H screening using Pyrin as the exogenous or endogenous SIVA1 and Stathmin 1 bait identified SIVA1 binding, and this association proteins (Li et al., 2011). was confirmed by IP. Using deletion mutants of Cyclin-dependent kinase inhibitor 2A Pyrin and of SIVA1 or SIVA2, the C-terminal, rfp (CDKN2A) , also known as ARF: The ARF and and SRPY domain of pyrin were found to interact SIVA interaction was tested by IP assays of H1229 with the N-terminal region of SIVA (Balci- cells containing FLAG-SIVA1 and GFP-ARF, and Peynircioglu et al., 2008). purified recombinant proteins were used for X-linked inhibitor of apoptosis (XIAP): Y2H confirmation. The protein interaction mapping was screening using XIAP as the bait identified SIVA1 performed by GST pull down assays using deletion binding, and this protein association was confirmed mutants of SIVA1 and ARF overexpressed in 293 by IP of 293 cells co-expressing both proteins cells. SIVA1 binds to ARF by its N-terminal region (Resch et al., 2009). and DDHR, while the residue aa 21-64 of ARF is FHL1 four and a half LIM domains 1 (FHL1): required (Wang et al., 2013). Y2H screening using the SLIMMER isoform of Homology FHL1 as the bait identified SIVA; and this protein association was confirmed by IP. Three different SIVA1 shares high homology (around 40%) in its isoforms of FHL1 were used in a Y2H assay for DDHR domain with the FADD and RIP proteins. protein interaction mapping, SIVA1 binds only SIVA1 also shares a high homology with different with the SLIMMER and not with FHL1 and KyoT2 species (Table 1). isoforms (Cottle et al., 2009). p53: The interaction between p53 and SIVA1 was Mutations tested by IP using H1229 cells co-expressing FLAG-p53 and GFP-SIVA1 and confirmed by IP Mutations in the SIVA1 gene are rare, only six using endogenous proteins from A549 cells. GST missense and one nonsense mutations are reported pull-down assays indicate that SIVA1 binds to p53 at COSMIC (Catalogue of somatic mutations in using its N-terminal region and DDHR, while p53 cancer). binds to SIVA1 via its DBD domain (Du et al., 2009). Implicated in Tyrosine kinase 2 (TYK2): Y2H screening using Breast cancer TYK2 as the bait identified SIVA1 binding, and this association was confirmed by IP of 293 cells Note co-expressing FLAG-SIVA1 and full-length TYK2. In breast cancer cells, SIVA1 acts synergistically The SIVA1 N-terminal region binds to TYK2, as with cisplatin in inducing apoptosis (Chu et al., demonstrated by IP of 293T cells overexpressing 2005). Recently, SIVA1 protein has been reported GFP tagged deletion mutants of SIVA1 and FLAG- to be downregulated in primary and metastatic TYK2 (Shimoda et al., 2010). breast cancer tumors and SIVA 1 negatively Stathmin 1: Y2H screening using SIVA1 as bait correlates with Stathmin 1 activity (Li et al., 2011). identified Stathmin 1, and this association was SIVA1 silencing augments metastasis in a confirmed by IP of U2OS cells co-expressing xenograft breast cancer model (Li et al., 2011).

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Acute lymphoid leukemia Lin S, Ying SY. Differentially expressed genes in activin- induced apoptotic LNCaP cells. Biochem Biophys Res Note Commun. 1999 Apr 2;257(1):187-92 In acute lymphoid leukemia cell lines, SIVA1 Yoon Y, Ao Z, Cheng Y, Schlossman SF, Prasad KV. overexpression induces apoptosis (Py et al., 2004), Murine Siva-1 and Siva-2, alternate splice forms of the while SIVA1 inhibition reduces apoptosis (Resch et mouse Siva gene, both bind to CD27 but differentially al., 2009). transduce apoptosis. Oncogene. 1999 Nov 25;18(50):7174-9 Colorectal cancer Cao C, Ren X, Kharbanda S, Koleske AJ, Prasad KV, Kufe Note D. The ARG tyrosine kinase interacts with Siva-1 in the apoptotic response to oxidative stress. J Biol Chem. 2001 In colorectal cancer samples, using a DNA array Apr 13;276(15):11465-8 approach, SIVA1 has been shown to be downregulated when compared to normal tissue Okuno K, Yasutomi M, Nishimura N, Arakawa T, Shiomi M, Hida J, Ueda K, Minami K. Gene expression analysis in (Okuno et al., 2001). In colon cancer cell lines, colorectal cancer using practical DNA array filter. Dis SIVA1 was found to be a transcriptional target of Colon Rectum. 2001 Feb;44(2):295-9 p53 and E2F1 and to participate in the apoptosis Spinicelli S, Nocentini G, Ronchetti S, Krausz LT, Bianchini induced by MDM2 inhibition (Ray et al., 2011). In R, Riccardi C. GITR interacts with the pro-apoptotic protein addition, SIVA1 silencing reduces apoptosis in a Siva and induces apoptosis. Cell Death Differ. 2002 p53-dependent manner in colon cancer cells treated Dec;9(12):1382-4 with cisplatin (Barkinge et al., 2009). Xue L, Chu F, Cheng Y, Sun X, Borthakur A, Ramarao M, Pandey P, Wu M, Schlossman SF, Prasad KV. Siva-1 Osteosarcoma binds to and inhibits BCL-X(L)-mediated protection against Note UV radiation-induced apoptosis. Proc Natl Acad Sci U S A. 2002 May 14;99(10):6925-30 In a xenograft osteosarcoma model, SIVA1 silencing increases p53 stability and augments the Chu F, Borthakur A, Sun X, Barkinge J, Gudi R, Hawkins tumor growth (Du et al., 2009). In osteosarcoma S, Prasad KV. The Siva-1 putative amphipathic helical region (SAH) is sufficient to bind to BCL-XL and sensitize cell lines, SIVA1 silencing increases cell migration cells to UV radiation induced apoptosis. Apoptosis. 2004 and metastasis, while SIVA1 overexpression has an Jan;9(1):83-95 opposite effect (Li et al., 2011). Fortin A, MacLaurin JG, Arbour N, Cregan SP, Kushwaha Prostate cancer N, Callaghan SM, Park DS, Albert PR, Slack RS. The proapoptotic gene SIVA is a direct transcriptional target for Note the tumor suppressors p53 and E2F1. J Biol Chem. 2004 In a study focused on the identification of genes Jul 2;279(27):28706-14 modulated in prostate cancer cells under apoptosis, Py B, Slomianny C, Auberger P, Petit PX, Benichou S. SIVA1 transcripts were found to be upregulated. Siva-1 and an alternative splice form lacking the death This finding indicates a possible role of SIVA1 in domain, Siva-2, similarly induce apoptosis in T lymphocytes via a caspase-dependent mitochondrial the apoptotic pathway of prostate cancer cells (Lin pathway. J Immunol. 2004 Apr 1;172(7):4008-17 and Ying, 1999). Chu F, Barkinge J, Hawkins S, Gudi R, Salgia R, Kanteti PV. Expression of Siva-1 protein or its putative To be noted amphipathic helical region enhances cisplatin-induced apoptosis in breast cancer cells: effect of elevated levels of Note BCL-2. Cancer Res. 2005 Jun 15;65(12):5301-9 SIVA1 was initially identified as a potent protein in Py B, Bouchet J, Jacquot G, Sol-Foulon N, the induction of apoptosis, which led it to be given Basmaciogullari S, Schwartz O, Biard-Piechaczyk M, a similar name to the Hindu god of destruction, Benichou S. The Siva protein is a novel intracellular ligand Shiva (Prasad et al., 1997). In 2009, the paradigm of the CD4 receptor that promotes HIV-1 envelope-induced apoptosis in T-lymphoid cells. Apoptosis. 2007 that SIVA1 has a function strictly related to Oct;12(10):1879-92 apoptosis was broken when its role in p53 stability was reported. More recently, among the new roles Lin FT, Lai YJ, Makarova N, Tigyi G, Lin WC. The lysophosphatidic acid 2 receptor mediates down-regulation proposed for SIVA1 are cell proliferation, of Siva-1 to promote cell survival. J Biol Chem. 2007 Dec migration and metastasis (Du et al., 2009; Mei and 28;282(52):37759-69 Wu, 2012). Balci-Peynircioglu B, Waite AL, Hu C, Richards N, Staubach-Grosse A, Yilmaz E, Gumucio DL. Pyrin, product References of the MEFV locus, interacts with the proapoptotic protein, Siva. J Cell Physiol. 2008 Sep;216(3):595-602 Prasad KV, Ao Z, Yoon Y, Wu MX, Rizk M, Jacquot S, Schlossman SF. CD27, a member of the tumor necrosis Barkinge JL, Gudi R, Sarah H, Chu F, Borthakur A, factor receptor family, induces apoptosis and binds to Siva, Prabhakar BS, Prasad KV. The p53-induced Siva-1 plays a proapoptotic protein. Proc Natl Acad Sci U S A. 1997 Jun a significant role in cisplatin-mediated apoptosis. J 10;94(12):6346-51 Carcinog. 2009;8:2

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Cottle DL, McGrath MJ, Wilding BR, Cowling BS, Kane metastasis of tumor cells by inhibiting stathmin and JM, D'Arcy CE, Holdsworth M, Hatzinisiriou I, Prescott M, stabilizing microtubules. Proc Natl Acad Sci U S A. 2011 Brown S, Mitchell CA. SLIMMER (FHL1B/KyoT3) interacts Aug 2;108(31):12851-6 with the proapoptotic protein Siva-1 (CD27BP) and delays skeletal myoblast apoptosis. J Biol Chem. 2009 Sep Ray RM, Bhattacharya S, Johnson LR. Mdm2 inhibition 25;284(39):26964-77 induces apoptosis in p53 deficient human colon cancer cells by activating p73- and E2F1-mediated expression of Du W, Jiang P, Li N, Mei Y, Wang X, Wen L, Yang X, Wu PUMA and Siva-1. Apoptosis. 2011 Jan;16(1):35-44 M. Suppression of p53 activity by Siva1. Cell Death Differ. 2009 Nov;16(11):1493-504 Mei Y, Wu M. Multifaceted functions of Siva-1: more than an Indian God of Destruction. Protein Cell. 2012 Resch U, Schichl YM, Winsauer G, Gudi R, Prasad K, de Feb;3(2):117-22 Martin R. Siva1 is a XIAP-interacting protein that balances NFkappaB and JNK signalling to promote apoptosis. J Cell Wang X, Zha M, Zhao X, Jiang P, Du W, Tam AY, Mei Y, Sci. 2009 Aug 1;122(Pt 15):2651-61 Wu M. Siva1 inhibits p53 function by acting as an ARF E3 ubiquitin ligase. Nat Commun. 2013;4:1551 Shimoda HK, Shide K, Kameda T, Matsunaga T, Shimoda K. Tyrosine kinase 2 interacts with the proapoptotic protein This article should be referenced as such: Siva-1 and augments its apoptotic functions. Biochem Biophys Res Commun. 2010 Sep 17;400(2):252-7 Machado-Neto JA, Traina F. SIVA1 (SIVA1, Apoptosis- Inducing Factor). Atlas Genet Cytogenet Oncol Haematol. Li N, Jiang P, Du W, Wu Z, Li C, Qiao M, Yang X, Wu M. 2014; 18(5):334-339. Siva1 suppresses epithelial-mesenchymal transition and

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