Disturbed Expression of Splicing Factors in Renal Cancer Affects Alternative Splicing of Apoptosis Regulators, Oncogenes, and Tumor Suppressors

Background Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may lead to tumoral transformation due to synthesis of impaired splice variants with oncogenic potential. In this paper we hypothesized that disturbed alternative splicing in ccRCC may result from improper expression of splicing factors, mediators of splicing reactions. Methodology/Principal Findings Using real-time PCR and Western-blot analysis we analyzed expression of seven splicing factors belonging to SR proteins family (SF2/ASF, SC35, SRp20, SRp75, SRp40, SRp55 and 9G8), and one non-SR factor, hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) in 38 pairs of tumor-control ccRCC samples. Moreover, we analyzed splicing patterns of five genes involved in carcinogenesis and partially regulated by analyzed splicing factors: RON, CEACAM1, Rac1, Caspase-9, and GLI1. Conclusions/Significance We found that the mRNA expression of splicing factors was disturbed in tumors when compared to paired controls, similarly as levels of SF2/ASF and hnRNP A1 proteins. The correlation coefficients between expression levels of specific splicing factors were increased in tumor samples. Moreover, alternative splicing of five analyzed genes was also disturbed in ccRCC samples and splicing pattern of two of them, Caspase-9 and CEACAM1 correlated with expression of SF2/ASF in tumors. We conclude that disturbed expression of splicing factors in ccRCC may possibly lead to impaired alternative splicing of genes regulating tumor growth and this way contribute to the process of carcinogenesis.

[1]  G. Dreyfuss,et al.  In vivo and in vitro arginine methylation of RNA-binding proteins , 1995, Molecular and cellular biology.

[2]  John G. Collard,et al.  Rac Affects Invasion of Human Renal Cell Carcinomas by Up-regulating Tissue Inhibitor of Metalloproteinases (TIMP)-1 and TIMP-2 Expression* , 2001, The Journal of Biological Chemistry.

[3]  G. Gyapay,et al.  Alterations of the DNA repair gene OGG1 in human clear cell carcinomas of the kidney. , 2000, Cancer research.

[4]  M. McFarlane,et al.  RNA splicing factors regulated by HPV16 during cervical tumour progression , 2009, The Journal of pathology.

[5]  T. Boulikas,et al.  Induction of poly(ADP-ribosyl)ation in the kidney after in vivo application of renal carcinogens. , 1994, Toxicology.

[6]  P. Jordan,et al.  Increased Rac1b Expression Sustains Colorectal Tumor Cell Survival , 2008, Molecular Cancer Research.

[7]  Johanne Toutant,et al.  Small interfering RNA-mediated reduction in heterogeneous nuclear ribonucleoparticule A1/A2 proteins induces apoptosis in human cancer cells but not in normal mortal cell lines. , 2003, Cancer research.

[8]  Lorenzo Marconi,et al.  Guidelines on Renal Cell Carcinoma , 2007 .

[9]  A. Krainer,et al.  Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors. , 1994, Science.

[10]  A. Sureau,et al.  hnRNP A1 and the SR Proteins ASF/SF2 and SC35 Have Antagonistic Functions in Splicing of β-Tropomyosin Exon 6B* , 2004, Journal of Biological Chemistry.

[11]  B. Hartmann,et al.  Decrypting the genome's alternative messages. , 2009, Current opinion in cell biology.

[12]  K. Schulze-Osthoff,et al.  Regulation of apoptosis by alternative pre-mRNA splicing. , 2005, Molecular cell.

[13]  N. Stoecklein,et al.  Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer , 2002, The Lancet.

[14]  V. Darras,et al.  Untranslated regions of thyroid hormone receptor beta 1 mRNA are impaired in human clear cell renal cell carcinoma. , 2010, Biochimica et biophysica acta.

[15]  C. Suschek,et al.  Distinct in vivo expression patterns of survivin splice variants in renal cell carcinomas , 2002, International journal of cancer.

[16]  M. Kasper,et al.  Novel Human Glioma-associated Oncogene 1 (GLI1) Splice Variants Reveal Distinct Mechanisms in the Terminal Transduction of the Hedgehog Signal , 2008, Journal of Biological Chemistry.

[17]  Antonio Lopez-Beltran,et al.  2004 WHO classification of the renal tumors of the adults. , 2006, European urology.

[18]  B. Chabot,et al.  A proteomic approach to the identification of heterogeneous nuclear ribonucleoproteins as a new family of poly(ADP-ribose)-binding proteins. , 2003, The Biochemical journal.

[19]  A. Krainer,et al.  The gene encoding the splicing factor SF2/ASF is a proto-oncogene , 2007, Nature Structural &Molecular Biology.

[20]  K. Furge,et al.  Gene expression profiling of clear cell renal cell carcinoma: Gene identification and prognostic classification , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[21]  P. Comoglio,et al.  The MET receptor tyrosine kinase in invasion and metastasis , 2007, Journal of cellular physiology.

[22]  A. Piekiełko-Witkowska,et al.  Disturbed expression of type 1 iodothyronine deiodinase splice variants in human renal cancer. , 2009, Thyroid : official journal of the American Thyroid Association.

[23]  B. Brinkman,et al.  Splice variants as cancer biomarkers. , 2004, Clinical biochemistry.

[24]  S. Datta,et al.  Gene expression profiling of epithelial ovarian tumours correlated with malignant potential , 2004, Molecular Cancer.

[25]  Michael Q. Zhang,et al.  An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers. , 2006, Human molecular genetics.

[26]  T. Roskams,et al.  Emerging role of tyrosine kinase inhibitors in the treatment of advanced renal cell cancer: a review. , 2006, Annals of oncology : official journal of the European Society for Medical Oncology.

[27]  C. Dinney,et al.  Suppression of human bladder cancer growth by increased expression of C-CAM1 gene in an orthotopic model. , 1996, Cancer research.

[28]  P. Jordan,et al.  Cloning of a novel human Rac1b splice variant with increased expression in colorectal tumors , 1999, Oncogene.

[29]  Michael R Green,et al.  Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene. , 2005, Molecular cell.

[30]  L. Corcos,et al.  E2F1 controls alternative splicing pattern of genes involved in apoptosis through upregulation of the splicing factor SC35 , 2008, Cell Death and Differentiation.

[31]  J. Venables.  Unbalanced alternative splicing and its significance in cancer , 2006, BioEssays : news and reviews in molecular, cellular and developmental biology.

[32]  E. Petricoin,et al.  Proteomic analysis of human prostate cancer † , 2002, Molecular carcinogenesis.

[33]  P. Jordan,et al.  Antagonistic SR proteins regulate alternative splicing of tumor-related Rac1b downstream of the PI3-kinase and Wnt pathways. , 2009, Human molecular genetics.

[34]  Y. Yen,et al.  Altered splicing of CEACAM1 in breast cancer: Identification of regulatory sequences that control splicing of CEACAM1 into long or short cytoplasmic domain isoforms , 2008, Molecular Cancer.

[35]  M. Fernandez-Zapico.  Primers on Molecular Pathways GLI: More than Just Hedgehog? , 2008, Pancreatology.

[36]  C. Chalfant,et al.  SRp30a (ASF/SF2) regulates the alternative splicing of caspase-9 pre-mRNA and is required for ceramide-responsiveness Published, JLR Papers in Press, February 27, 2006. , 2006, Journal of Lipid Research.

[37]  J. Cheng,et al.  Molecular and Genetic Studies Imply Akt-mediated Signaling Promotes Protein Kinase CβII Alternative Splicing via Phosphorylation of Serine/Arginine-rich Splicing Factor SRp40* , 2005, Journal of Biological Chemistry.

[38]  D. Jacqmin,et al.  The sonic hedgehog signaling pathway is reactivated in human renal cell carcinoma and plays orchestral role in tumor growth , 2009, Molecular Cancer.

[39]  J. Cáceres,et al.  The SR protein family of splicing factors: master regulators of gene expression. , 2009, The Biochemical journal.

[40]  G. Deikus,et al.  Antagonistic Roles of Four Sr Proteins in the Biosynthesis of Alternatively Spliced Tissue Factor Transcripts in Monocytic Cells , 2009 .

[41]  M. V. Panchenko,et al.  Jade-1, a candidate renal tumor suppressor that promotes apoptosis. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[42]  A. Bianco,et al.  Covering the base-pairs in iodothyronine deiodinase-1 biology: holes remain in the lineup. , 2009, Thyroid : official journal of the American Thyroid Association.

[43]  Robert Tibshirani,et al.  Gene Expression Profiling Predicts Survival in Conventional Renal Cell Carcinoma , 2005, PLoS medicine.

[44]  C. Wood,et al.  Suppression of tumorigenicity of breast cancer cells by an epithelial cell adhesion molecule (C-CAM1): the adhesion and growth suppression are mediated by different domains , 1997, Oncogene.

[45]  B R Franza,et al.  Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors. , 1998, RNA.

[46]  Walter Artibani,et al.  Grading systems in renal cell carcinoma. , 2007, The Journal of urology.

[47]  J. Köllermann,et al.  Expression of Mcl-1 Splicing Variants in Clear-Cell Renal Cancer and Their Correlation with Histopathological Parameters and Prognosis , 2009, Tumor Biology.

[48]  M. Malanga,et al.  Poly(ADP-ribose) Binds to the Splicing Factor ASF/SF2 and Regulates Its Phosphorylation by DNA Topoisomerase I* , 2008, Journal of Biological Chemistry.

[49]  B. Öbrink.  On the role of CEACAM1 in cancer. , 2008 .

[50]  D. Botstein,et al.  Gene expression patterns in human liver cancers. , 2002, Molecular biology of the cell.

[51]  Terukazu Nakamura,et al.  Overexpression of XIAP expression in renal cell carcinoma predicts a worse prognosis. , 2007, International journal of oncology.

[52]  Yaowu He,et al.  Nuclear functions of heterogeneous nuclear ribonucleoproteins A/B , 2008, Cellular and Molecular Life Sciences.

[53]  O. Vorontsova,et al.  Homophilic adhesion and CEACAM1-S regulate dimerization of CEACAM1-L and recruitment of SHP-2 and c-Src , 2009, The Journal of cell biology.

[54]  Dong Wang,et al.  Altered expression of the RON receptor tyrosine kinase in primary human colorectal adenocarcinomas: generation of different splicing RON variants and their oncogenic potential , 2003, Oncogene.

[55]  P. Comoglio,et al.  Truncated RON Tyrosine Kinase Drives Tumor Cell Progression and Abrogates Cell-Cell Adhesion Through E-Cadherin Transcriptional Repression , 2004, Cancer Research.

[56]  W. Zimmermann,et al.  The tumour suppressor gene CEACAM1 is completely but reversibly downregulated in renal cell carcinoma , 2004, The Journal of pathology.

[57]  A. Tulin,et al.  Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins modulates splicing , 2009, Nucleic acids research.

[58]  C. Porta,et al.  Phosphatidylinositol-3-kinase/Akt signaling pathway and kidney cancer, and the therapeutic potential of phosphatidylinositol-3-kinase/Akt inhibitors. , 2009, The Journal of urology.

[59]  E. Bosco,et al.  Rac1 GTPase: A “Rac” of All Trades , 2009, Cellular and Molecular Life Sciences.

[60]  F. Blasi,et al.  Proteomics Analysis of Nucleolar SUMO-1 Target Proteins upon Proteasome Inhibition* , 2009, Molecular & Cellular Proteomics.

[61]  Christopher J Kane,et al.  The human T-cell factor-4 gene splicing isoforms, Wnt signal pathway, and apoptosis in renal cell carcinoma. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[62]  A. Krainer,et al.  Arginine Methylation Controls the Subcellular Localization and Functions of the Oncoprotein Splicing Factor SF2/ASF , 2010, Molecular and Cellular Biology.

[63]  Mala Sinha,et al.  Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma , 2010, PloS one.

[64]  E. Lengyel,et al.  Rac1b, a tumor associated, constitutively active Rac1 splice variant, promotes cellular transformation , 2004, Oncogene.

[65]  Jinhua Wang,et al.  ESEfinder: a web resource to identify exonic splicing enhancers , 2003, Nucleic Acids Res..

[66]  M. Puzianowska-Kuźnicka,et al.  Overexpression of E2F1 in clear cell renal cell carcinoma: a potential impact of erroneous regulation by thyroid hormone nuclear receptors. , 2007, Thyroid : official journal of the American Thyroid Association.

[67]  J. Fackenthal,et al.  Aberrant RNA splicing and its functional consequences in cancer cells , 2008, Disease Models & Mechanisms.

[68]  Christian A. Rees,et al.  Molecular portraits of human breast tumours , 2000, Nature.

[69]  Martin Vingron,et al.  Gene expression in kidney cancer is associated with cytogenetic abnormalities, metastasis formation, and patient survival. , 2005, Clinical cancer research : an official journal of the American Association for Cancer Research.

[70]  Thomas,et al.  Consistent expression of an epithelial cell adhesion molecule (C-CAM) during human prostate development and loss of expression in prostate cancer: implication as a tumor suppressor. , 1995, Cancer research.