CD44 alternative splicing and hnRNP A1 expression are associated with the metastasis of breast cancer

Loh,Tiing  Jen; Moon,Heegyum; Cho,Sunghee; Jang,Hana; Liu,Yong  Chao; Tai,Hongmei; Jung,Da-Woon; Williams,Darren  R.; Kim,Hey-Ran; Shin,Myung-Geun; Liao,D.  Joshua; Zhou,Jianhua; Shi,Wei; Zheng,Xuexiu; Shen,Haihong

doi:10.3892/or.2015.4110

CD44 alternative splicing and hnRNP A1 expression are associated with the metastasis of breast cancer

Authors:
- Tiing Jen Loh
- Heegyum Moon
- Sunghee Cho
- Hana Jang
- Yong Chao Liu
- Hongmei Tai
- Da-Woon Jung
- Darren R. Williams
- Hey-Ran Kim
- Myung-Geun Shin
- D. Joshua Liao
- Jianhua Zhou
- Wei Shi
- Xuexiu Zheng
- Haihong Shen
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Affiliations: School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea, Department of Endocrinology, Yanji Hospital, Jilin, P.R. China, Department of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hwasun Hospital, Hwasun, Republic of Korea, Hormel Institute, University of Minnesota, Austin, MN, USA, Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, Jiangsu, P.R. China, Key Laboratory for Molecular Enzymology and Engineering, The Ministry of Education, College of Life Sciences, Jilin University, Changchun, Jilin, P.R. China
Published online on: July 6, 2015 https://doi.org/10.3892/or.2015.4110
Pages: 1231-1238

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Abstract

CD44 is a transmembrane receptor for hyaluronic acid. CD44 pre-mRNA contains 19 exons, 9 of which are alternatively spliced. Among the CD44 spliced variants, the v4-7 variant, one of the v6 exon-containing isoforms that contains variable exon 4, 5, 6 and 7, confers metastatic potential to non-metastatic cells. Splicing of CD44 and the function of CD44 isoforms are different in breast cancer cells. hnRNP A1 is a ubiquitously expressed protein with an inhibitory function in pre-mRNA splicing. We showed that CD44v6 isoform, which includes all of the v6-containing mRNA isoforms, had the highest expression level in non-metatatic breast cancer cells (MCF7) when compared to the level in metastatic breast cancer cells (MDA-MB-231) and normal breast cells (MCF10A). Furthermore we showed that hnRNP A1 knockdown regulated splicing of CD44 differently in breast cancer cells. We showed here that CD44 isoform expression is completely different in MDA-MB-231 cells than that in MCF7 and MCF10A cells, whereas MCF7 and MCF10A cells had a similar expression pattern of CD44 isoforms. RT-PCR analysis of CD44v6 showed that MCF7 and MCF10A cells predominantly expressed the c5v6v7v8v9v10c6 isoform. However, in addition to this isoform, MDA-MB-231 cells also expressed the c5v6v8v9v10c6 and c5v6c6 isoforms. We also found that knockdown of hnRNP A1 significantly reduced the expression of c5v6v7v8v9v10c6 and c5v6v8v9v10c6, and promoted the expression of c5v6c6. hnRNP A1 knockdown significantly induced cell death. In addition, hnRNP A1 knockdown induced a decrease in cell invasion in the MDA-MB-231 cells. Our results indicate that the knockdown of hnRNP A1 has a specific function on the splicing of CD44 in breast cancer cells.

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1	Modrek B and Lee C: A genomic view of alternative splicing. Nat Genet. 30:13–19. 2002. View Article : Google Scholar
2	Moon H, Cho S, Yang X, Zhou J, Loh TJ, Zheng X and Shen H: Identification of novel splicing variants from RON proto-oncogene pre-mRNA. Oncol Rep. 28:2217–2220. 2012.PubMed/NCBI
3	Shen H, Zheng X, Luecke S and Green MR: The U2AF35-related protein Urp contacts the 3′ splice site to promote U12-type intron splicing and the second step of U2-type intron splicing. Genes Dev. 24:2389–2394. 2010. View Article : Google Scholar : PubMed/NCBI
4	Shen H and Green MR: RS domain-splicing signal interactions in splicing of U12-type and U2-type introns. Nat Struct Mol Biol. 14:597–603. 2007. View Article : Google Scholar : PubMed/NCBI
5	Lee J, Zhou J, Zheng X, Cho S, Moon H, Loh TJ, Jo K and Shen H: Identification of a novel cis-element that regulates alternative splicing of Bcl-x pre-mRNA. Biochem Biophys Res Commun. 420:467–472. 2012. View Article : Google Scholar : PubMed/NCBI
6	Cho S, Moon H, Yang X, Zhou J, Kim HR, Shin MG, Loh TJ, Zheng X and Shen H: Validation of trans-acting elements that promote exon 7 skipping of SMN2 in SMN2-GFP stable cell line. Biochem Biophys Res Commun. 423:531–535. 2012. View Article : Google Scholar : PubMed/NCBI
7	Chen M and Manley JL: Mechanisms of alternative splicing regulation: Insights from molecular and genomics approaches. Nat Rev Mol Cell Biol. 10:741–754. 2009.PubMed/NCBI
8	Matlin AJ, Clark F and Smith CW: Understanding alternative splicing: Towards a cellular code. Nat Rev Mol Cell Biol. 6:386–398. 2005. View Article : Google Scholar : PubMed/NCBI
9	Cáceres JF and Kornblihtt AR: Alternative splicing: Multiple control mechanisms and involvement in human disease. Trends Genet. 18:186–193. 2002. View Article : Google Scholar : PubMed/NCBI
10	Faustino NA and Cooper TA: Pre-mRNA splicing and human disease. Genes Dev. 17:419–437. 2003. View Article : Google Scholar : PubMed/NCBI
11	Cooper TA, Wan L and Dreyfuss G: RNA and disease. Cell. 136:777–793. 2009. View Article : Google Scholar : PubMed/NCBI
12	Kansas GS, Wood GS and Dailey MO: A family of cell-surface glycoproteins defined by a putative anti-endothelial cell receptor antibody in man. J Immunol. 142:3050–3057. 1989.PubMed/NCBI
13	Pals ST, Hogervorst F, Keizer GD, Thepen T, Horst E and Figdor CC: Identification of a widely distributed 90-kDa glycoprotein that is homologous to the Hermes-1 human lymphocyte homing receptor. J Immunol. 143:851–857. 1989.PubMed/NCBI
14	Picker LJ, Nakache M and Butcher EC: Monoclonal antibodies to human lymphocyte homing receptors define a novel class of adhesion molecules on diverse cell types. J Cell Biol. 109:927–937. 1989. View Article : Google Scholar : PubMed/NCBI
15	Fox SB, Fawcett J, Jackson DG, Collins I, Gatter KC, Harris AL, Gearing A and Simmons DL: Normal human tissues, in addition to some tumors, express multiple different CD44 isoforms. Cancer Res. 54:4539–4546. 1994.PubMed/NCBI
16	Screaton GR, Bell MV, Bell JI and Jackson DG: The identification of a new alternative exon with highly restricted tissue expression in transcripts encoding the mouse Pgp-1 (CD44) homing receptor. Comparison of all 10 variable exons between mouse, human, and rat. J Biol Chem. 268:12235–12238. 1993.PubMed/NCBI
17	Ponta H, Wainwright D and Herrlich P: The CD44 protein family. Int J Biochem Cell Biol. 30:299–305. 1998. View Article : Google Scholar : PubMed/NCBI
18	Hofmann M, Rudy W, Günthert U, Zimmer SG, Zawadzki V, Zöller M, Lichtner RB, Herrlich P and Ponta H: A link between ras and metastatic behavior of tumor cells: Ras induces CD44 promoter activity and leads to low-level expression of metastasis-specific variants of CD44 in CREF cells. Cancer Res. 53:1516–1521. 1993.PubMed/NCBI
19	Weg-Remers S, Ponta H, Herrlich P and König H: Regulation of alternative pre-mRNA splicing by the ERK MAP-kinase pathway. EMBO J. 20:4194–4203. 2001. View Article : Google Scholar : PubMed/NCBI
20	Matzke A, Herrlich P, Ponta H and Orian-Rousseau V: A five-amino-acid peptide blocks Met- and Ron-dependent cell migration. Cancer Res. 65:6105–6110. 2005. View Article : Google Scholar : PubMed/NCBI
21	Orian-Rousseau V, Chen L, Sleeman JP, Herrlich P and Ponta H: CD44 is required for two consecutive steps in HGF/c-Met signaling. Genes Dev. 16:3074–3086. 2002. View Article : Google Scholar : PubMed/NCBI
22	Tremmel M, Matzke A, Albrecht I, Laib AM, Olaku V, Ballmer-Hofer K, Christofori G, Héroult M, Augustin HG, Ponta H, et al: A CD44v6 peptide reveals a role of CD44 in VEGFR-2 signaling and angiogenesis. Blood. 114:5236–5244. 2009. View Article : Google Scholar : PubMed/NCBI
23	Günthert U, Hofmann M, Rudy W, Reber S, Zöller M, Haussmann I, Matzku S, Wenzel A, Ponta H and Herrlich P: A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell. 65:13–24. 1991. View Article : Google Scholar : PubMed/NCBI
24	Heider KH, Kuthan H, Stehle G and Munzert G: CD44v6: A target for antibody-based cancer therapy. Cancer Immunol Immunother. 53:567–579. 2004. View Article : Google Scholar : PubMed/NCBI
25	Louderbough JM and Schroeder JA: Understanding the dual nature of CD44 in breast cancer progression. Mol Cancer Res. 9:1573–1586. 2011. View Article : Google Scholar : PubMed/NCBI
26	Hill A, McFarlane S, Mulligan K, Gillespie H, Draffin JE, Trimble A, Ouhtit A, Johnston PG, Harkin DP, McCormick D, et al: Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells. Oncogene. 25:6079–6091. 2006. View Article : Google Scholar : PubMed/NCBI
27	Ouhtit A, Abd Elmageed ZY, Abdraboh ME, Lioe TF and Raj MH: In vivo evidence for the role of CD44s in promoting breast cancer metastasis to the liver. Am J Pathol. 171:2033–2039. 2007. View Article : Google Scholar : PubMed/NCBI
28	Busch A and Hertel KJ: Evolution of SR protein and hnRNP splicing regulatory factors. Wiley Interdiscip Rev RNA. 3:1–12. 2012. View Article : Google Scholar
29	Mayeda A, Munroe SH, Cáceres JF and Krainer AR: Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins. EMBO J. 13:5483–5495. 1994.PubMed/NCBI
30	Martinez-Contreras R, Cloutier P, Shkreta L, Fisette JF, Revil T and Chabot B: hnRNP proteins and splicing control. Adv Exp Med Biol. 623:123–147. 2007. View Article : Google Scholar
31	Zhou ZJ, Dai Z, Zhou SL, Fu XT, Zhao YM, Shi YH, Zhou J and Fan J: Overexpression of HnRNP A1 promotes tumor invasion through regulating CD44v6 and indicates poor prognosis for hepatocellular carcinoma. Int J Cancer. 132:1080–1089. 2013. View Article : Google Scholar
32	van Weering DH, Baas PD and Bos JL: A PCR-based method for the analysis of human CD44 splice products. PCR Methods Appl. 3:100–106. 1993. View Article : Google Scholar : PubMed/NCBI
33	Afify A, Purnell P and Nguyen L: Role of CD44s and CD44v6 on human breast cancer cell adhesion, migration, and invasion. Exp Mol Pathol. 86:95–100. 2009. View Article : Google Scholar : PubMed/NCBI