Background and aims: The majority of colorectal cancers arise from detectable adenomatous or serrated lesions. Here we demonstrate how deregulated alternative splicing of CD44 gene in diseased colon mucosa results in downregulation of standard isoform of CD44 gene (CD44s) and upregulation of variant isoform CD44v8-10. Our aim is to show that upregulation of CD44v8-10 isoform is a possible marker of precancerous lesion in human colon. Methods: We analysed pairs of fresh biopsy specimen of large intestine in a cohort of 50 patients. We studied and compared alternative splicing profile of CD44 gene in colon polyps and adjoined healthy colon mucosa. We performed end-point and qRT PCR, western blotting, IHC staining and flow cytometry analyses. Results: We detected more than five-fold overexpression of CD44v8-10 isoform and almost twenty-fold downregulation of standard isoform CD44s in colon polyps compared to adjoined healthy tissue with p = 0.018 and p < 0.001 in a cohort of 50 patients. Our results also show that aberrant splicing of CD44 occurs in both biologically distinct subtypes of colorectal adenoma possibly in ESRP-1 specific manner. Conclusion: 92% of the colon polyp positive patients overexpressed CD44v8-10 isoform in their colon polyps while only 36% of them had positive fecal occult blood test which is currently a standard non-invasive screening technique. Impact: We believe that our results are important for further steps leading to application of CD44v8-10 isoform as a biomarker of colorectal precancerosis in non-invasive detection. Early detection of colon precancerosis means successful prevention of colorectal carcinoma.

C2P NEXARS Campus Science Park Brno Czech Republic

Department of Gastroenterology and Internal Medicine University Hospital Brno and Faculty of Medicine Masaryk University Brno Brno Czech Republic

Department of Immunology Faculty of Medicine and Dentistry Palacky University Olomouc Olomouc Czech Republic

Department of Pharmacology and Toxicology Veterinary Research Institute Brno Czech Republic

Faculty of Medicine in Hradec Kralove Institute of Hygiene and Preventive Medicine Charles University Hradec Kralove Czech Republic

Institute of Physics of the Czech Academy of Sciences Prague 8 Czech Republic

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Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, Rosso S, Coebergh JWW, Comber H, et al. Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. Eur J Cancer (2013). 49(6):1374–403. 10.1016/j.ejca.2012.12.027 PubMed DOI

Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin (2012). 65(2):87–108. 10.3322/caac.21262 PubMed DOI

Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, et al. Genetic alterations during colorectal-tumor development. N Engl J Med (1988). 319(9):525–32. 10.1056/nejm198809013190901 PubMed DOI

Tomasetti C, Marchionni L, Nowak MA, Parmigiani G, Vogelstein B. Only three driver gene mutations are required for the development of lung and colorectal cancers. Proc Natl Acad Sci USA (2015). 112(1):118–23. 10.1073/pnas.1421839112 PubMed DOI PMC

Zavoral M, Suchanek S, Majek O, Fric P, Minarikova P, Minarik M, et al. Colorectal cancer screening: 20 years of development and recent progress. World J. Gastroenterol (2014). 20(14):3825–34. 10.3748/wjg.v20.i14.3825 PubMed DOI PMC

Prochazka L, Tesarik R, Turanek J. Regulation of alternative splicing of CD44 in cancer. Cell Signal (2014). 26(10):2234–9. 10.1016/j.cellsig.2014.07.011 PubMed DOI

Toole BP. Hyaluronan-CD44 interactions in cancer: paradoxes and possibilities. Clin Cancer Res (2009). 15(24):7462–8. 10.1158/1078-0432.ccr-09-0479 PubMed DOI PMC

Toole BP. Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer (2004). 4(7):528–39. 10.1038/nrc1391 PubMed DOI

Orian-Rousseau V, Ponta H. Perspectives of CD44 targeting therapies. Arch Toxicol (2015). 89(1):3–14. 10.1007/s00204-014-1424-2 PubMed DOI

Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci (2003). 100(7):3983–8. 10.1073/pnas.0530291100 PubMed DOI PMC

Takeo K, Kawai T, Nishida K, Masuda K, Teshima-Kondo S, Tanahashi T, et al. Oxidative stress-induced alternative splicing of transformer 2β (SFRS10) and CD44 pre-mRNAs in gastric epithelial cells. Am J Physiology-Cell Physiol (2009). 297(2):C330–C338. 10.1152/ajpcell.00009.2009 PubMed DOI

Batsché E, Yaniv M, Muchardt C. The human SWI/SNF subunit Brm is a regulator of alternative splicing. Nat Struct Mol Biol (2006). 13(1):22–9. 10.1038/nsmb1030 PubMed DOI

Cappellari M, Bielli P, Paronetto MP, Ciccosanti F, Fimia GM, Saarikettu J, et al. The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells. Oncogene (2014). 33(29):3794–802. 10.1038/onc.2013.360 PubMed DOI

Ameyar-Zazoua M, Rachez C, Souidi M, Robin P, Fritsch L, Young R, et al. Argonaute proteins couple chromatin silencing to alternative splicing. Nat Struct Mol Biol (2012). 19(10):998–1004. 10.1038/nsmb.2373 PubMed DOI

Orian-Rousseau V, Chen L, Sleeman JP, Herrlich P, Ponta H. CD44 is required for two consecutive steps in HGF/c-Met signaling. Genes Dev (2002). 16(23):3074–86. 10.1101/gad.242602 PubMed DOI PMC

Brown RL, Reinke LM, Damerow MS, Perez D, Chodosh LA, Yang J, et al. CD44 splice isoform switching in human and mouse epithelium is essential for epithelial-mesenchymal transition and breast cancer progression. J Clin Invest (2011). 121(3):1064–74. 10.1172/jci44540 PubMed DOI PMC

Rudy W, Hofmann M, Schwartz-Albiez R, Zöller M, Heider KH, Ponta H, et al. The two major CD44 proteins expressed on a metastatic rat tumor cell line are derived from different splice variants: each one individually suffices to confer metastatic behavior. Cancer Res (1993). 53(6):1262–8. PubMed

Seiter S, Arch R, Reber S, Komitowski D, Hofmann M, Ponta H, et al. Prevention of tumor metastasis formation by anti-variant CD44. J Exp Med (1993). 177(2):443–55. 10.1084/jem.177.2.443 PubMed DOI PMC

Tanabe KK, Ellis LM, Saya H. Expression of CD44R1 adhesion molecule in colon carcinomas and metastases. The Lancet (1993). 341(8847):725–6. 10.1016/0140-6736(93)90490-8 PubMed DOI

Günthert U, Hofmann M, Rudy W, Reber S, Zöller M, Hauβmann I, et al. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell (1991). 65(1):13–24. 10.1016/0092-8674(91)90403-l PubMed DOI

Lau WM, Teng E, Chong HS, Lopez KAP, Tay AYL, Salto-Tellez M, et al. CD44v8-10 is a cancer-specific marker for gastric cancer stem cells. Cancer Res (2014). 74(9):2630–41. 10.1158/0008-5472.can-13-2309 PubMed DOI

Jiang H, Zhao W, Shao W. Prognostic value of CD44 and CD44v6 expression in patients with non-small cell lung cancer: meta-analysis. Tumor Biol (2014). 35(8):7383–9. 10.1007/s13277-014-2150-3 PubMed DOI

Yan Y, Zuo X, Wei D. Concise review: emerging role of CD44 in cancer stem cells: a promising biomarker and therapeutic target. Stem Cell Transl Med (2015). 4(9):1033–43. 10.5966/sctm.2015-0048 PubMed DOI PMC

Gao AC, Lou W, Dong JT, Isaacs JT. CD44 is a metastasis suppressor gene for prostatic cancer located on human chromosome 11p13. Cancer Res (1997). 57(5):846–9. PubMed

Neumayer R, Rosen HR, Reiner A, Sebesta C, Schmid A, Tüchler H, et al. CD44 expression in benign and malignant colorectal polyps. Dis Colon Rectum (1999). 42(1):50–5. 10.1007/bf02235182 PubMed DOI

Asao T, Nakamura J-i., Shitara Y, Tsutsumi S, Mochiki E, Shimura T, et al. Loss of standard type of CD44 expression in invaded area as a good indicator of lymph-node metastasis in colorectal carcinoma. Dis Colon Rectum (2000). 43(9):1250–4. ;discussion 1254-5. 10.1007/bf02237430 PubMed DOI

Miyake H, Eto H, Arakawa S, Kamidono S, Hara I. Over expression of CD44V8-10 in urinary exfoliated cells as an independent prognostic predictor in patients with urothelial cancer. J Urol (2002). 167(3):1282–7. 10.1016/s0022-5347(05)65282-2 PubMed DOI

Levine JS, Ahnen DJ. Adenomatous polyps of the colon. N Engl J Med (2006). 355(24):2551–7. 10.1056/nejmcp063038 PubMed DOI

Risio M. Reprint of: the natural history of adenomas. Best Pract Res Clin Gastroenterol (2010). 24(4):397–406. 10.1016/j.bpg.2010.08.002 PubMed DOI

Lech G, Słotwiński R, Słodkowski M, Krasnodębski IW. Colorectal cancer tumour markers and biomarkers: recent therapeutic advances. World J. Gastroenterol (2016). 22(5):1745–55. 10.3748/wjg.v22.i5.1745 PubMed DOI PMC

Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res (2001). 29(9):45e–45. 10.1093/nar/29.9.e45 PubMed DOI PMC

Procházka L, Turánek J, Tesařík R, Knotigová P, Polášková P, Andrysík Z, et al. Apoptosis and inhibition of gap-junctional intercellular communication induced by LA-12, a novel hydrophobic platinum(IV) complex. Arch Biochem Biophys (2007). 462(1):54–61. 10.1016/j.abb.2007.03.021 PubMed DOI

Iida J, Clancy R, Dorchak J, Somiari RI, Somiari S, Cutler ML, et al. DNA aptamers against exon v10 of CD44 inhibit breast cancer cell migration. PLoS One (2014). 9(2):e88712. 10.1371/journal.pone.0088712 PubMed DOI PMC

Warzecha CC, Sato TK, Nabet B, Hogenesch JB, Carstens RP. ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing. Mol Cel (2009). 33(5):591–601. 10.1016/j.molcel.2009.01.025 PubMed DOI PMC

Bartheldyová E, Effenberg R, Mašek J, Procházka L, Knötigová PT, Kulich P, et al. Hyaluronic acid surface modified liposomes prepared via orthogonal aminoxy coupling: synthesis of nontoxic aminoxylipids based on symmetrically α-branched fatty acids, preparation of liposomes by microfluidic mixing, and targeting to cancer cells expressing CD44. Bioconjug Chem. (2018). 29(7):2343–56. 10.1021/acs.bioconjchem.8b00311 PubMed DOI

Wielenga VJ, Heider KH, Offerhaus GJ, Adolf GR, van den Berg FM, Ponta H, et al. Expression of CD44 variant proteins in human colorectal cancer is related to tumor progression. Cancer Res (1993). 53(20):4754–6. PubMed

Kopp R, Fichter M, Schalhorn G, Danescu J, Classen S. Frequent expression of the high molecular, 673-bp CD44v3,v8-10 variant in colorectal adenomas and carcinomas. Int J Mol Med (2009). 24(5):677–83. 10.3892/ijmm_00000279 PubMed DOI

Mäkinen MJ. Colorectal serrated adenocarcinoma. Histopathology (2007). 50(1):131–50. 10.1111/j.1365-2559.2006.02548.x PubMed DOI

Konishi K, Yamochi T, Makino R, Kaneko K, Yamamoto T, Nozawa H, et al. Molecular differences between sporadic serrated and conventional colorectal adenomas. Clin Cancer Res (2004). 10(9):3082–90. 10.1158/1078-0432.ccr-03-0334 PubMed DOI

Muinuddin A, Aslahi R, Hopman WM, Paterson WG. Relationship between the number of positive fecal occult blood tests and the diagnostic yield of colonoscopy. Can J Gastroenterol (2013). 27(2):90–4. 10.1155/2013/612314 PubMed DOI PMC

Wei SC, Tsao PN, Wang YT, Lin BR, Wu DC, Tsai WS, et al. Using serum placenta growth factor could improve the sensitivity of colorectal cancer screening in fecal occult blood negative population: a multicenter with independent cohort validation study. Cancer Med (2019). 8(7):3583–91. 10.1002/cam4.2216 PubMed DOI PMC

Mashayekhi F, Aryaee H, Mirzajani E, Yasin AA, Fathi A. Soluble CD44 concentration in the serum and peritoneal fluid samples of patients with different stages of endometriosis. Arch Gynecol Obstet (2015). 292(3):641–5. 10.1007/s00404-015-3654-9 PubMed DOI

Palagani V, El Khatib M, Krech T, Manns MP, Malek NP, Plentz RR. Decrease of CD44-positive cells correlates with tumor response to chemotherapy in patients with gastrointestinal cancer. Anticancer Res (2012). 32(5):1747–55. PubMed

Seyedmajidi S, Seyedmajidi M, Foroughi R, Zahedpasha A, Zolfaghari Saravi Z, Pourbagher R, et al. Comparison of salivary and serum soluble CD44 levels between patients with oral SCC and healthy controls. Asian Pac J Cancer Prev (2018). 19(11):3059–63. 10.31557/apjcp.2018.19.11.3059 PubMed DOI PMC

Zeilstra J, Joosten SPJ, Dokter M, Verwiel E, Spaargaren M, Pals ST. Deletion of the WNT target and cancer stem cell marker CD44 in Apc(Min/+) mice attenuates intestinal tumorigenesis. Cancer Res (2008). 68(10):3655–61. 10.1158/0008-5472.can-07-2940 PubMed DOI

Zeilstra J, Joosten SPJ, van Andel H, Tolg C, Berns A, Snoek M, et al. Stem cell CD44v isoforms promote intestinal cancer formation in Apc(min) mice downstream of Wnt signaling. Oncogene (2014). 33(5):665–70. 10.1038/onc.2012.611 PubMed DOI

Guo W, Frenette PS. Alternative CD44 splicing in intestinal stem cells and tumorigenesis. Oncogene (2014). 33(5):537–8. 10.1038/onc.2013.260 PubMed DOI

Zhang S, Wu CCN, Fecteau J-F, Cui B, Chen L, Zhang L, et al. Targeting chronic lymphocytic leukemia cells with a humanized monoclonal antibody specific for CD44. Proc Natl Acad Sci (2013). 110(15):6127–32. 10.1073/pnas.1221841110 PubMed DOI PMC

Birzele F, Voss E, Nopora A, Honold K, Heil F, Lohmann S, et al. CD44 isoform status predicts response to treatment with anti-CD44 antibody in cancer patients. Clin Cancer Res (2015). 21(12):2753–62. 10.1158/1078-0432.ccr-14-2141 PubMed DOI

Sy MS, Guo YJ, Stamenkovic I. Distinct effects of two CD44 isoforms on tumor growth in vivo . J Exp Med (1991). 174(4):859–66. 10.1084/jem.174.4.859 PubMed DOI PMC

Bartolazzi A, Peach R, Aruffo A, Stamenkovic I. Interaction between CD44 and hyaluronate is directly implicated in the regulation of tumor development. J Exp Med (1994). 180(1):53–66. 10.1084/jem.180.1.53 PubMed DOI PMC