Abstract
Src associated in mitosis (Sam68; 68 kDa) is a KH domain RNA-binding protein that belongs to the signal transduction and activation of RNA family, and has been implicated in the oncogenesis and progression of several human cancers. Our study aimed to investigated the clinicopathologic significance of Sam68 expression and its role in cell proliferation and the underlying molecular mechanism in hepatocellular carcinoma (HCC). We demonstrated that Sam68 expression was significantly increased in HCC and high expression of Sam68 was significantly associated with Edmondson grade, tumor size, tumor nodule number, HBsAg status and Ki-67 expression. The Kaplan–Meier survival curves showed that increased expression of Sam68 was correlated with poor prognosis in HCC patients and served as an independent prognostic marker of overall survival in a multivariable analysis. In addition, through serum starvation and refeeding assay, we demonstrated that Sam68 was lowly expressed in serum-starved HCC cells, and was progressively increased after serum-additioning. Furthermore, siRNA knockdown of endogenous Sam68 inhibited cell proliferation and tumourigenicity of HCC cells in vitro, through blocking the G1 to S phase transition. Moreover, we reported that the anti-proliferative effect of silencing Sam68 was accompanied with up-regulated expression of cyclin-dependent kinase inhibitors, p21Cip1 and p27Kip1, enhanced transactivation of FOXO factors (FOXO4), and dysreuglation of Akt/GSK-3β signaling. Taken together, these findings provide a rational framework for the progression of HCC and thereby indicated that Sam68 might be a novel and useful prognostic marker and a potential target for human HCC treatment.
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References
Altekruse SF, Henley SJ, Cucinelli JE, McGlynn KA (2014) Changing hepatocellular carcinoma incidence and liver cancer mortality rates in the United States. Am J Gastroenterol 109:542–553. doi:10.1038/ajg.2014.11
Bedford MT, Frankel A, Yaffe MB, Clarke S, Leder P, Richard S (2000) Arginine methylation inhibits the binding of proline-rich ligands to Src homology 3, but not WW, domains. J Biol Chem 275:16030–16036. doi:10.1074/jbc.M909368199
Bielli P, Busa R, Paronetto MP, Sette C (2011) The RNA-binding protein Sam68 is a multifunctional player in human cancer. Endocr Relat Cancer 18:R91–R102. doi:10.1530/ERC-11-0041
Brenkman AB, van den Broek NJ, de Keizer PL, van Gent DC, Burgering BM (2010) The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response. FASEB J 24:4271–4280. doi:10.1096/fj.10-158717
Brunet A et al (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857–868
Busa R et al (2007) The RNA-binding protein Sam68 contributes to proliferation and survival of human prostate cancer cells. Oncogene 26:4372–4382. doi:10.1038/sj.onc.1210224
Cardozo T, Pagano M (2004) The SCF ubiquitin ligase: insights into a molecular machine. Nat Rev Mol Cell Biol 5:739–751. doi:10.1038/nrm1471
Chang R et al (2015) Upregulated expression of ubiquitin-conjugating enzyme E2Q1 (UBE2Q1) is associated with enhanced cell proliferation and poor prognosis in human hapatocellular carcinoma. J Mol Histol 46:45–56. doi:10.1007/s10735-014-9596-x
Chawla G, Lin CH, Han A, Shiue L, Ares M Jr, Black DL (2009) Sam68 regulates a set of alternatively spliced exons during neurogenesis. Mol Cell Biol 29:201–213. doi:10.1128/MCB.01349-08
Chen T, Boisvert FM, Bazett-Jones DP, Richard S (1999) A role for the GSG domain in localizing Sam68 to novel nuclear structures in cancer cell lines. Mol Biol Cell 10:3015–3033
Cooper TA, Wan L, Dreyfuss G (2009) RNA and disease. Cell 136:777–793. doi:10.1016/j.cell.2009.02.011
Cui X et al (2014) Decreased expression of SERPINB1 correlates with tumor invasion and poor prognosis in hepatocellular carcinoma. J Mol Histol 45:59–68. doi:10.1007/s10735-013-9529-0
Derry JJ et al (2000) Sik (BRK) phosphorylates Sam68 in the nucleus and negatively regulates its RNA binding ability. Mol Cell Biol 20:6114–6126
Ding Y et al (2009) Overexpression of Tiam1 in hepatocellular carcinomas predicts poor prognosis of HCC patients. Int J Cancer 124:653–658. doi:10.1002/ijc.23954
Espejo A, Cote J, Bednarek A, Richard S, Bedford MT (2002) A protein-domain microarray identifies novel protein-protein interactions. Biochem J 367:697–702. doi:10.1042/BJ20020860
Fumagalli S, Totty NF, Hsuan JJ, Courtneidge SA (1994) A target for Src in mitosis. Nature 368:871–874. doi:10.1038/368871a0
Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H (1984) Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:1710–1715
Gramantieri L et al (2009) MicroRNA-221 targets Bmf in hepatocellular carcinoma and correlates with tumor multifocality. Clin Cancer Res 15:5073–5081. doi:10.1158/1078-0432.CCR-09-0092
Hu TH et al (2003) Expression and prognostic role of tumor suppressor gene PTEN/MMAC1/TEP1 in hepatocellular carcinoma. Cancer 97:1929–1940. doi:10.1002/cncr.11266
Johnson DL, Johnson SA (2008) Cell biology. RNA metabolism and oncogenesis. Science 320:461–462. doi:10.1126/science.1158680
Lan X et al (2014) The effect of CXCL9 on the invasion ability of hepatocellular carcinoma through up-regulation of PREX2. J Mol Histol 45:689–696. doi:10.1007/s10735-014-9593-0
Lee HK et al (2007) beta-catenin regulates multiple steps of RNA metabolism as revealed by the RNA aptamer in colon cancer cells. Cancer Res 67:9315–9321. doi:10.1158/0008-5472.CAN-07-1128
Li Z et al (2012) Sam68 expression and cytoplasmic localization is correlated with lymph node metastasis as well as prognosis in patients with early-stage cervical cancer. Ann Oncol 23:638–646. doi:10.1093/annonc/mdr290
Liao WT et al (2013) High expression level and nuclear localization of Sam68 are associated with progression and poor prognosis in colorectal cancer. BMC Gastroenterol 13:126. doi:10.1186/1471-230X-13-126
Liu K, Li L, Nisson PE, Gruber C, Jessee J, Cohen SN (2000) Neoplastic transformation and tumorigenesis associated with sam68 protein deficiency in cultured murine fibroblasts. J Biol Chem 275:40195–40201. doi:10.1074/jbc.M006194200
Liu Z, Wu X, Zhang F, Han L, Bao G, He X, Xu Z (2013) AhR expression is increased in hepatocellular carcinoma. J Mol Histol 44:455–461. doi:10.1007/s10735-013-9495-6
Lu M, Wu J, He F, Wang XL, Li C, Chen ZN, Bian H (2015) Cell expression patterns of CD147 in N-diethylnitrosamine/phenobarbital-induced mouse hepatocellular carcinoma. J Mol Histol 46:79–91. doi:10.1007/s10735-014-9602-3
Lukong KE, Richard S (2003) Sam68, the KH domain-containing superSTAR. Biochim Biophys Acta 1653:73–86
Matter N, Herrlich P, Konig H (2002) Signal-dependent regulation of splicing via phosphorylation of Sam68. Nature 420:691–695. doi:10.1038/nature01153
Michlewski G, Sanford JR, Caceres JF (2008) The splicing factor SF2/ASF regulates translation initiation by enhancing phosphorylation of 4E-BP1. Mol Cell 30:179–189. doi:10.1016/j.molcel.2008.03.013
Modem S, Chinnakannu K, Bai U, Reddy GP, Reddy TR (2011) Hsp22 (HspB8/H11) knockdown induces Sam68 expression and stimulates proliferation of glioblastoma cells. J Cell Physiol 226:2747–2751. doi:10.1002/jcp.22868
Paronetto MP et al (2010) Alternative splicing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68. Cancer Res 70:229–239. doi:10.1158/0008-5472.CAN-09-2788
Perrotti D, Neviani P (2007) From mRNA metabolism to cancer therapy: chronic myelogenous leukemia shows the way. Clin Cancer Res 13:1638–1642. doi:10.1158/1078-0432.CCR-06-2320
Richard S, Yu D, Blumer KJ, Hausladen D, Olszowy MW, Connelly PA, Shaw AS (1995) Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1. Mol Cell Biol 15:186–197
Richard S et al (2005) Ablation of the Sam68 RNA binding protein protects mice from age-related bone loss. PLoS Genet 1:e74. doi:10.1371/journal.pgen.0010074
Richard S, Vogel G, Huot ME, Guo T, Muller WJ, Lukong KE (2008) Sam68 haploinsufficiency delays onset of mammary tumorigenesis and metastasis. Oncogene 27:548–556. doi:10.1038/sj.onc.1210652
Ross J, Lemm I, Berberet B (2001) Overexpression of an mRNA-binding protein in human colorectal cancer. Oncogene 20:6544–6550. doi:10.1038/sj.onc.1204838
Seoane J, Le HV, Shen L, Anderson SA, Massague J (2004) Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 117:211–223
Song L, Wang L, Li Y, Xiong H, Wu J, Li J, Li M (2010) Sam68 up-regulation correlates with, and its down-regulation inhibits, proliferation and tumourigenicity of breast cancer cells. J Pathol 222:227–237. doi:10.1002/path.2751
Taylor SJ, Shalloway D (1994) An RNA-binding protein associated with Src through its SH2 and SH3 domains in mitosis. Nature 368:867–871. doi:10.1038/368867a0
Taylor SJ, Resnick RJ, Shalloway D (2004) Sam68 exerts separable effects on cell cycle progression and apoptosis. BMC cell biology 5:5. doi:10.1186/1471-2121-5-5
Wan C et al (2015) MIF4G domain containing protein regulates cell cycle and hepatic carcinogenesis by antagonizing CDK2-dependent p27 stability. Oncogene 34:237–245. doi:10.1038/onc.2013.536
Wang Y et al (2013) Interaction with cyclin H/cyclin-dependent kinase 7 (CCNH/CDK7) stabilizes C-terminal binding protein 2 (CtBP2) and promotes cancer cell migration. J Biol Chem 288:9028–9034. doi:10.1074/jbc.M112.432005
Wu YY et al (2013) Inhibition of hepatocellular carcinoma growth and angiogenesis by dual silencing of NET-1 and VEGF. J Mol Histol 44:433–445. doi:10.1007/s10735-012-9480-5
Zhang Z et al (2009) Expression and cytoplasmic localization of SAM68 is a significant and independent prognostic marker for renal cell carcinoma. Cancer Epidemiol Biomarkers Prev 18:2685–2693. doi:10.1158/1055-9965.EPI-09-0097
Zhang Z, Xu Y, Sun N, Zhang M, Xie J, Jiang Z (2014) High Sam68 expression predicts poor prognosis in non-small cell lung cancer. Clin Transl Oncol 16:886–891. doi:10.1007/s12094-014-1160-3
Zhao JJ et al (2011) Identification of LZAP as a new candidate tumor suppressor in hepatocellular carcinoma. PloS One 6:e26608. doi:10.1371/journal.pone.0026608
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This work was supported by the National Natural Science Foundation of China (No. 81272708).
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Zhang, T., Wan, C., Shi, W. et al. The RNA-binding protein Sam68 regulates tumor cell viability and hepatic carcinogenesis by inhibiting the transcriptional activity of FOXOs. J Mol Hist 46, 485–497 (2015). https://doi.org/10.1007/s10735-015-9639-y
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DOI: https://doi.org/10.1007/s10735-015-9639-y