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A Novel Rho Gtpase-Activating Protein, Is Downregulated in Cancer and Inhibits Tumor Cell Growth

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A Novel Rho Gtpase-Activating Protein, Is Downregulated in Cancer and Inhibits Tumor Cell Growth Oncogene (2007) 26, 4580–4589 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Deleted in liver cancer 3 (DLC-3), a novel Rho GTPase-activating protein, is downregulated in cancer and inhibits tumor cell growth ME Durkin1, V Ullmannova1, M Guan and NC Popescu Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Two related Rho GTPase-activating proteins, DLC-1 wide range of cellular processes (Ridley, 2001; Jaffe and (deleted in liver cancer 1) and DLC-2, are emerging as Hall, 2005). Rho activity is frequently increased in bona fide tumor suppressor genes that inhibit cancer cell human tumors, but as activating mutations in Rho growth. In this report, we characterized a gene on protein genes are rare, the regulators of Rho GTPases chromosome Xq13 that encodes DLC-3 (also known as may be targeted during oncogenesis (Gomez del Pulgar KIAA0189 and STARD8), a third member of the DLC et al., 2005). Rho proteins transmit signals in the active, family. The DLC-3 gene has transcripts with alternative GTP-bound state, which is positively controlled by 50 ends, one of which, DLC-3a, encodes an 1103-amino guanine nucleotide exchange factors that catalyse the acid polypeptide highly similar to DLC-1 and DLC-2. A exchange of bound GDP for GTP and negatively second isoform (DLC-3b) would yield a protein lacking controlled by Rho GTPase-activating proteins (GAPs) the N-terminal sterile alpha motif domain. The DLC-3 that stimulate GTP hydrolysis (Van Aelst and D’Souza- gene is widely expressed in normal tissues, but DLC-3 Schorey, 1997). Decreased GAP activity could result in mRNA levels were low or absent in a significant number of sustained Rho signaling that facilitates the growth and breast, ovarian, liver and prostate cancer cell lines. Using metastasis of tumor cells. a cancer profiling array to compare matched tumor and The human RhoGAPs are a diverse family of proteins normal human tissues, downregulation of DLC-3 mRNA that share a conserved, 150–200 amino acid GAP was observed in kidney, lung, ovarian, uterine and breast domain that contains the catalytic activity (Bernards, cancer samples. By quantitative reverse transcriptase– 2003; Moon and Zheng, 2003). One subgroup of the polymerase chain reaction, DLC-3 expression was re- human RhoGAPs includes DLC-1 (deleted in liver duced in primary prostate carcinomas relative to normal cancer 1), the human homologue of rat p122RhoGAP prostate tissue. Transfection of human breast and prostate (Homma and Emori, 1995; Yuan et al., 1998), and cancer cells with a DLC-3a expression vector inhibited DLC-2, also known as STARD13 (Ching et al., 2003). cell proliferation, colony formation and growth in soft The DLC-1 and DLC-2 genes encode polypeptides of agar. These results indicate that deregulation of DLC-3 approximately 1100 amino acids (aa) with a character- may contribute to breast and prostate tumorigenesis. istic modular architecture, consisting of an N-terminal Oncogene (2007) 26, 4580–4589; doi:10.1038/sj.onc.1210244; sterile a motif (SAM) domain, a serine-rich domain, published online 5 February 2007 a RhoGAP domain and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) Keywords: tumor suppressor gene; Rho GTPase-activating domain (Homma and Emori, 1995; Yuan et al., 1998; protein; prostate cancer; breast cancer Ponting and Aravind, 1999; Ching et al., 2003). DLC-1 and DLC-2 were shown to have in vitro GAP activity for RhoA and Cdc42 and to influence cell morphology and cytoskeletal organization (Homma and Emori, 1995; Sekimata et al., 1999; Ching et al., 2003; Wong et al., Introduction 2003, 2005). DLC-1 inhibits the growth, colony-forming ability, tumorigenicity and invasiveness of human liver, The development of neoplasia is associated with breast, ovarian, nasopharyngeal, esophageal and non- alterations in signal transduction pathways that regulate small cell lung cancer cells (Ng et al., 2000; Yuan et al., cell proliferation, survival and invasiveness (Hanahan 2003, 2004; Zhou et al., 2004; Goodison et al., 2005; and Weinberg, 2000). The members of the Rho family of Syed et al., 2005; Wong et al., 2005; Seng et al., 2006), small GTPases act as molecular switches that influence a and DLC-2 has been shown to inhibit the ras-induced transformation of rodent cells and the growth of breast Correspondence: Dr NC Popescu, Laboratory of Experimental and liver cancer cells in culture (Nagaraja and Kandpal, Carcinogenesis, Center for Cancer Research, National Cancer Institute, 2004; Leung et al., 2005). Bethesda, MD 20892, USA. A third member of the deleted in liver cancer gene E-mail: [email protected] 1These two authors contributed equally to this work. family is present in the human genome. The KIAA0189 Received 13 September 2006; revised 17 October 2006; accepted 17 cDNA clone, isolated from a human myeloid cell line October 2006; published online 5 February 2007 library, was noted to encode a protein similar to DLC-3 and cancer ME Durkin et al 4581 p122RhoGAP (Nagase et al., 1996). The gene encoding genomic DNA sequence revealed the exon structure of KIAA0189 (official gene name, STARD8, for ‘START the gene (Figure1b and Table 1) and showed that the domain protein 8’) has been localized to the X heterogeneity at the 50 ends of the DLC-3 transcripts chromosome and is distinct from the DLC-1 and appears to arise through the use of alternative promo- DLC-2 genes on 8p22 and 13q13.1, respectively. In this ters and exon skipping. communication, we have characterized additional tran- A transcript represented by a cDNA from a human scripts of the STARD8 locus that have alternative 50 endometrium carcinoma cell line (GenBank CR749411, ends and analysed the sequence of the predicted protein clone DKFZp686H1668), designated DLC-3a, shares product, which we term DLC-3. We also present exons 2–14with KIAA0189, now termed DLC-3 b. evidence that DLC-3 expression is altered in some Nucleotides 1–160 and nt 161–194of the DLC-3 a human cancers and that DLC-3 can suppress tumor cell sequence are present in exons 1A and 1B, respectively, growth. located upstream of the first exon of DLC-3b (exon 1C, nt 1–176 of D80011). A third transcript, DLC-3g, represented by a cDNA from a uterine leiomyosarcoma Results library (IMAGE 5518429, GenBank BC035587) appears to use the same transcription start site as DLC-3a Analysis of DLC-3 transcripts but lacks exon 3, causing a reading frame shift that The STARD8 gene encoding DLC-3 is closely linked to would result in premature translation termination the androgen receptor (AR), oligophrenin 1 (OPHN1) after 52 aa. Reverse transcriptase–polymerase chain and ephrin-B1 (EFNB1) genes on chromosome Xq13.1 reaction (RT–PCR) analysis of normal human liver, (Figure 1a). Database searches identified cDNAs that prostate and mammary gland RNA using primers were nearly identical to the original KIAA0189 sequence in exon 1A and exon 5 indicated that the DLC-3a throughout most of their lengths but differed at the transcript, and not the DLC-3g isoform, is present in 50 ends. Comparison of the cDNA sequences to the these tissues (Figure 1d). Figure 1 Structure of the DLC-3/STARD8 gene. (a) Map of the human chromosome Xq13 region, with boxes representing STARD8 and neighboring genes encoding the androgen receptor (AR), oligophrenin-1 (OPHN1) and ephrin-B1 (EFNB1). The numbers above the line refer to the sequence coordinates, in megabases. (b) Exon organization of the DLC-3 gene. Boxes represent exons and are numbered to correspond with exons 2–14of the homologous DLC-1 gene (Durkin et al., 2002). Arrows indicate the potential transcription start sites upstream of exons 1A and 1C. (c) Schematic representation of the exon sequences present at the 50 ends of the three DLC-3 transcripts. The KIAA0189 cDNA sequence is designated as the DLC-3b isoform, and the GenBank accession numbers of the cDNAs corresponding to the DLC-3a and g isoforms are given in parentheses. The locations of the putative ATG translation start codons in the three transcripts and the premature TGA stop codon in the DLC-3g isoform are marked. (d) Amplification of cDNA prepared from normal human liver (L), mammary gland (M) and prostate (P) with DLC-3 primers 1F and 5R2. The major RT–PCR product is a 343 bp band containing exon 3 sequences, and not a 261 bp band missing exon 3. The identity of the PCR product was verified by sequencing. Oncogene DLC-3 and cancer ME Durkin et al 4582 Table 1 Exon organization of the human DLC-3 (STARD8) gene Numbera Size (bp) cDNAb (nt) Genomic DNAc (nt) DLC-1/DLC-2 exon sizes (bp) Exon 1A X259 12–270 85290–85548 Exon 1B 34271–304 103120–103153 Exon 1C X176 1–176 17211–17386 Exon 2 72 177–248 36474–36545 72/72 Exon 3 82 249–330 38854–38935 82/82 Exon 464331–39439928–39991 64/64 Exon 5 1418 395–1812 40772–42189 1424/1361 Exon 6 1741813–1986 177/174 42785–42958 Exon 7 160 1987–2146 43824–43983 160/160 Exon 8 199 2147–2145 44484–44682 199/199 Exon 9 211 2146–2556 45096–45306 214/211 Exon 10 115 2557–2671 45607–45721 115/115 Exon 11 225 2672–2896 45982–46206 219/222 Exon 12 218 2897–3114 47186–47403 218/218 Exon 13 177 3115–3291 47505–47681 174/177 Exon 14115 d 3292–4824 47865–49393 118/115d aExons are numbered to correspond to the numbers of the equivalent exons of the DLC-1 gene.
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