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p53 expression in human colon cancer tumors in nude mice after siRNA CD44 gene therapy Shao Tiffany, Venkateswaran Subramaniam, Serge Jothy * Department of Laboratory Medicine, St. Michael’s Hospital, Toronto, ON, Canada M5B 1W8 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Abstract Colorectal cancer is the second leading cause of cancer related deaths in North America. CD44, a ubiquitously expressed transmembrane adhesion protein, is involved in fundamental aspects of cancer cell biology such as tumor stem cell phenotype, cell adhesion and invasion, and resistance to apoptosis. It is over-expressed in most human malignant tumors including human colon cancer. Another protein with a major implication in colorectal carcinogenesis is p53, a tumor suppressor protein. It is widely known to play an important role in the control of cell cycle and apoptosis and is often referred to as a “guardian of the genome”. Mutations in the p53 gene result in decreased genetic stability, reduced apoptosis, and increased survival and propagation of cells with damaged DNA. The nuclear accumulation of the p53 protein has been reported in a variety of malignant tumors, including colon cancer. The number of mutations in p53 was found to increase as colorectal adenomas progress to carcinomas to metastatic carcinoma. To further delineate the role of p53 and CD44 in colon cancer, immunohistochemistry studies were carried out using tumors generated by HT-29 human colon cancer cells in nude mice after siRNA CD44 gene therapy, as compared to untreated control xenografts. Using this xenograft mouse model with reduced CD44 expression after siRNA CD44 gene therapy, we were able to demonstrate a significant decrease in p53 expression that is associated with the inhibition of CD44 expression in tumors derived from human colon cancer cells. This study suggests that the association between decreased CD44 expression and decreased p53 expression could be a possible manifestation of a return to a less aggressive tumor phenotype after siRNA CD44 gene therapy, providing insights into the likely roles of CD44 and p53 in the regulation of apoptosis and cancer progression. Keywords: siRNA; CD44; p53; Colon cancer; Gene therapy; xenograft model; HT-29 cells (Subramaniam et al., 2005). Epithelial cell Introduction turn over in the colon involves a 6-day Colorectal cancer is the second leading migration of the cells generated by stem cause of cancer related mortality in North cells from the base of the crypt to cell America. Transformation of the normal detachment, and apoptosis at the surface colonic epithelium to an adenoma and then epithelium. to a carcinoma is the result of a sequence of multiple steps. is CD44, a ubiquitously expressed cell of adhesion molecule, facilitates both cell-cell molecular alterations, including alterations and cell-matrix interactions in normal and in epithelial cell differentiation, proliferation, transformed epithelial cells (Ponta et al., and apoptosis. The latter two processes are 2003; Rudzki et al., 1997). Dysregulations highly regulated in the constantly renewing in cell adhesion to neighbouring cells and untransformed The extracellular matrix can result in apoptosis maintenance of homeostasis of the colonic (Frisch et al., 1994; Grossman et al., 2002; epithelium involves adhesion molecules and Shanmugathasan and regulators of cell cycle and apoptosis normal characterized by This a colonic sequence large number epithelium. colonic Jothy, 2000). epithelium, CD44 In is expressed in dividing cells at the base of the RNAi. Intratumoral RNAi of CD44 has been crypt but not the nonproliferating epithelium shown to suppress tumor growth and of the upper crypt and luminal surface increased (Lakshman et al., 2005). It is frequently (Subramanian et al., 2007). Based on this overexpressed at both the mRNA and previous mouse xenograft model, siRNA protein level in colon cancer and other types targeting of a discrete sequence of human of carcinomas (Fox et al., 1994; Wielenga et CD44 provides a potential therapeutic al., 1993, 2000; Woodman et al., 1996). approach for colon cancer. apoptosis in nude mice CD44 is also found to be associated with the characteristic tumor stem cell phenotype and One of the most common genetic changes the modulation of the directional motility in colorectal cancer involves the p53 tumour and migration of human colon cancer cells. suppressor gene (Greenblatt et al., 1994). (Jin et al., 2006; Lakshman et al., 2004a,b, The p53 gene is located on the short arm of 2005; Shipitsin et al., 2007; Wong et al., chromosome 17, and its protein product acts 2003). Recently, it has been suggested that as a transcription factor to maintain genetic CD44 provides resistance to apoptosis in integrity. It does so by arresting cell cycle at human colon tumors xenotrasplanted in mice the G1-S transition for DNA repair and after siRNA mediated CD44 knock down induction of apoptosis, resulting in tumor (Subramaniam et al., 2007). growth suppression. (Kastan et al., 1991). Conversely, allelic loss and mutations in the Previous studies in our laboratory p53 gene result in decreased genetic stability, employed the strategy of inhibiting CD44 reduced apoptosis, and increased survival mRNA expression using a method based on and propagation of cells with damaged DNA (Soong wt al., 1997). The number of this mutations in p53 thus increases as colorectal demonstrated that a significant decrease in adenomas to p53 expression is associated with the metastatic carcinoma (Goh et al., 1994; inhibition of CD44 expression in tumors Scott et al., 1995). The wild type p53 protein derived from human colon cancer cells. progress to carcinomas mouse xenograft model, we has a short half-life and is removed rapidly from the nucleus, while the mutant p53 has a prolonged half-life and accumulates in the nucleus, where it is detectable Materials and Methods siRNA CD44 gene therapy tumor samples by immunohistochemistry (Leahy et al., 1996). The nuclear accumulation of the p53 protein has been reported in a variety of malignant tumors, including colon cancer (Bartek et al., 1991). Furthermore, a relation between p53 overexpression and poor prognosis has been shown in many types of human cancers, Human colon cancer tumors derived from the HT29 colon cancer cell line, were xenotransplanted into nude mice and treated by siRNA CD44 gene therapy were obtained according to the published protocol of Subramaniam,V., et al., 2007. Antibodies such as colon cancer (Manne et al., 1997). Rabbit anti-human p53 antibody and rabbit IgG control serum (Santa Cruz In the present study, we performed Biotechnology, Santa Cruz, CA) were used immunohistochemical experiments to further for immunohistochemistry experiments at a investigate p53 expression of human colon concentration of 4 µg/mL. The secondary cancer tumors xenotransplanted in nude antibody used was Polymer-HRP labelled mice after siRNA CD44 gene therapy. Using goat anti-rabbit IgG (Dako, Carpinteria, CA). Immunohistochemistry diaminobenzidine (DAB) substrate (Dako, Carpinteria, CA) as chromogen. Sections Paraffin embedded sections of human were then dehydrated and mounted with colon cancer tumors in nude mice (either Permount (Fisher Scientific, Ottawa, ON). after mock transfection or after siRNA As CD44 gene therapy) were dewaxed and immunostaining, a human breast cancer rehydrated. Antigen retrieval was performed tissue was used. Negative controls for all with sodium citrate buffer (pH6.0) in a immunostaining pressure cooker for 30 min followed by substituting the primary antibody with rabbit cooling for 20 min at room temperature. IgG Endogenous peroxidase was blocked with concentration. Each slide was first scanned 1% H2O2 in methanol for 10 min at room with a low magnification, and 3 tumor fields temperature. Sections were incubated in 5% were normal goat serum to block non-specific measurements at 20X power magnification. binding sites, followed by incubation with Quantification of the p53 immunostaining primary p53 antibody for 1 hr at room was performed by calculating the percentage temperature and then overnight at 4°C. After of positive nuclei of tumour cells in relation washing with 1X PBS, sections were to the area of the representative fields (40 incubated with secondary antibody for 1 hr 000 um2). Statistical analysis was carried out at room temperature. Immunoperoxidase using the Student’s t-test. detection was achieved using a positive at the selected control were same for carried for out p53 by immunoglobulin observation and Results Reduction of p53 expression after siRNA nuclearimmunostaining for p53 was CD44 gene therapy of human colon cancer significantly reduced by 16% in the tumors tumors in nude mice that had received the siRNA CD44 gene therapy, when compared to the mock We measured the expression of p53 in our xenograft mouse model and found that A transfected vector control group (Figure 1, Figure 2, p<0.05). B Fig.1. p53 expression is reduced in human colon cancer tumors in nude mice after siRNA CD44 gene therapy: Immunohistochemical staining showed a reduction of p53 expression in siRNA CD44 treatment group (A), when compared to the mock transfected vector control group (B). Positive dark brown staining indicates p53 expression (p53 immunostaining, no counterstaining, x20 magnification). Fig.2. Human colon cancer tumors in nude mice after siRNA CD44 gene therapy shows decreased p53 expression: A significant (*p<0.05) reduction in the number of p53 positive nuclei occurs after siRNA CD44 knock down, when compared to the vector control. Data represents the average of 4 independent experiments. plays an anti-apoptotic role as an oncogenic Discussion It is well documented that apoptosis is defective in colonic adenomas and carcinomas, and it can be induced by disrupting cellular interaction with protein in cancer metastasis (Wielenga et al., 1993; Mulder et al., 1997; Lakshman et al., 2004a,b, 2005 ). In our previous study, we neighbouring cells and the matrix of demonstrated that the expression of CD44 basement membranes (Bedi et al., 1995; can be significantly suppressed in HT-29 Partik et al., 1998). Thus, upregulation of human colon cancer cells by siRNA CD44 CD44 expression, a cell adhesion protein, inhibition in a xenograft mouse model facilitates these cellular interactions and (Subramaniam et al, 2007). HT-29 cells express one of the highest levels of CD44 suggest that the main mechanism of the among human colon cancer cell lines. The therapeutic effect of CD44 siRNA inhibition CD44 sequence susceptible for siRNA is through increased apoptosis of tumour inhibition is located at the junction of cells by reversing the anti-apoptotic effect of transcripts encoded by standard exons 16 CD44, involving caspase 3 and poly (ADP- and 17 (Screaton et al., 1992). In this study, ribose) intratumoral delivery of siRNA CD44 was caspase 3 is found to mediate the cleavage accomplished using polyethyleneimine (PEI). of PARP and apoptosis in human colon PEI is a cationic, nontoxic polymer, which cancer cells. In our previous study, we retains its physical characteristics without observed a significant increase in levels of eliciting a significant immune response active caspase 3 and PARP in the tumors (Guan et al., 2005). The injection of after siRNA CD44 gene therapy when PEI/siRNA CD44 resulted in significant compared to vector control. Taken together, reduction in tumor growth compared with the data above and the anti-apoptotic effect tumor with of CD44 suggest that the tumor growth PEI/siRNA vector. The siRNA CD44 clones suppression after siRNA CD44 inhibition were less adhesive than vector control, might be due to the promotion of tumor cell suggesting a loss of function for CD44. apoptosis (Subramaniam et al., 2007). bearing mice injected polymersase (PARP). Cleaved These xenografted tumors after siRNA CD44 gene therapy had reduced level of CD44, decreased tumor mass and tumor growth, and increased tumor apoptosis. These results from this xenograft model Due to the fundamental role of p53 in cancer cell biology, it has been studied by different methods such as xenografts and tumor cell lines and tissues in a variety of human malignancies (Kaklamanis et al., The significant correlation between the 1993). Abnormalities in p53 expression have overexpression of p53 protein and the been studied in particular detail in colorectal histologic grade, tumor size, serosal invasion, cancer. The normal p53 protein has a short lymphatic invasion, venous invasion, lymph intracellular half-life of 6-20 minutes, node whereas many missense mutations found in remains controversial (Chang et al., 1996). cancer cells induce conformational changes Numerous studies have investigated the resulting in a mutant p53 protein with a significance of p53 expression and its longer half-life of up to 6 h (Clarke et al., relation to CD44 expression in human 1988; Dermot et al., 1996; Rogel et al., 1985; cancer, providing contradictory results on Sturzbecher et al., 1987). This functionally the conflicting expressions between CD44 inactive and stabilized p53 protein can be and p53 (Darai et al., 1998; Formby et al., detected by 1999; Kuncova et al., 2007; Endo et al., immunohistochemical methods (Roviello et 2000; Kim et al., 1994; Mulder et al., 1995; al., 1999). Zavrides et al., 2005). For example, it has in the cell nucleus metastasis, or colon carcinomas been shown that there is a correlation of In previous immunohistochemistry studies, CD44v6 expression with a higher incidence there have been conflicting results on the of p53 expression in various forms of human prognostic cancers, including colon cancer (Darai et al., significance of p53 overexpression and its association with poor 1998; Kanenori et al., 2000). CD44 patient survival in colorectal cancer (Leahy expression in colorectal adenomas is an et al., 1996; Diez et al., 2000; Soong et al, early event prior to p53 gene mutation and it 1997; Ofner et al., 1995; Wolf et al., 1997). has been suggested that aberrant p53 expression may on p53 expression of human colon cancer upregulation of CD44v6 isoform (Kim et al., tumors in nude mice after siRNA CD44 1994). However, other studies have provided gene therapy. We exploited our previous contradicting results with respect to the observation correlation p53 expression by siRNA CD44 gene therapy, expression (Formby et al., 1999; Jitka et al., results in increased apoptosis (Subramaniam 2007). These studies have raised the et al., 2007). Using this mouse xenograft questions of the true frequency of p53 model, we demonstrate in this study that a expression, its relationship to CD44, what significant decrease in the frequency of p53 association it has with concurrent colon expression is associated with a reduction of carcinomas, and its mechanistic role in the CD44 after siRNA CD44 gene therapy, context of apoptosis in colon cancer. consistent with the therapeutic pressure that However, few studies have analyzed the was used. A possible explanation for the correlation of p53 and CD44 expression in observed decrease in p53 expression in the context of apoptosis or looked into the xenografted human tumors could be that it is possible role of p53 in relation to CD44 in caused by decreased signalling from CD44. the mechanistic regulation of apoptosis. Globally, p53 expression in human colon between be dependent CD44 and that reduction of CD44 cancer cells might be dependent on CD44, in The present study has undertaken to addition to other cellular signals altering p53. examine one of these questions: the specific Furthermore, in the case of a complete role of p53 in relation to CD44 in the reversion of the tumor cell phenotype to a context of apoptosis. Immunohistochemistry normal cell phenotype, there would be close experiments were performed to investigate to no CD44 expression or p53 expression. Therefore, the association between decreased CD44 expression and decreased p53 expression could be a manifestation of a return to a less aggressive tumor phenotype due to the therapeutic effect of siRNA CD44 gene therapy. Overall, this study was the first to look at p53 expression after siRNA CD44 gene therapy in xenografted human tumors in nude mice and it provides insights into the possible roles of CD44 and p53 in the regulation of apoptosis. However, more studies are necessary to further delineate the possible linking of p53 and CD44 in the regulation of apoptotic process in colorectal cancer. References Bártek J, Bártková J, Vojtĕsek B, Stasková Z, Lukás J, Rejthar A, Kovarík J, Midgley CA, Gannon JV, Lane DP. Aberrant expression of the p53 oncoprotein is a common feature of a wide spectrum of human malignancies. Oncogene. 1991. 6,1699-703. Bedi A, Pasricha PJ, Akhtar AJ, Barber JP, Bedi GC, Giardiello FM, Zehnbauer BA, Hamilton SR, Jones RJ. Inhibition of apoptosis during development of colorectal cancer. Cancer Res. 1995. 55, 1811-6. Chang KJ, Lee TT, Linares-Cruz G, Fournier S, de Ligniéres B Influences of percutaneous administration of estradiol and progesterone on human breast epithelial cell cycle in vivo. Fertil Steril. 1995. 63,785-9. Clarke CF, Cheng K, Frey AB, Stein R, Hinds PW, Levine AJ. Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP. Mol Cell Biol. 1988. 8, 1206-1215. Acknowledgement I thank Dr. Venkateswaran Sergy Jothy Subramaniam guidance and support. Conflict of Interest There was no conflict of interest. and for Dr. their Darai E, Walker-Combrouze F, Fauconnier A, Madelenat P, Potet F, Scoazec JY. Analysis of CD44 expression in serous and mucinous borderline tumours of the ovary: comparison with cystadenomas and overt carcinomas. Histopathology. 1998. 2,151-9. Diez M, Pollan M, Müguerza JM, Gaspar MJ, Duce AM, Alvarez MJ, Ratia T, Herñandez P, Ruiz A, Granell J, 2000. Time-dependency of the prognostic effect of carcinoembryonic antigen and p53 protein in colorectal adenocarcinoma. Cancer. 88, 3541. Endo K, Terada T, 2000. Protein expression of CD44 (standard and variant isoforms) in hepatocellular carcinoma: relationships with tumor grade, clinicopathologic parameters, p53 expression, and patient survival. J Hepatol. 32,78-84. Formby B, Wiley TS, 1998. Progesterone inhibits growth and induces apoptosis in breast cancer cells: inverse effects on Bcl-2 and p53. Ann Clin Lab Sci. 28, 360-9. Fox, S., Fawcett, J., Jackson, D., Collins, I., Gatter, K., Harris, A., Gearing, A., Simmons, D., 1994. Normal human tissues, in addition to some tumors, express multiple different CD44 isoforms. Cancer Res. 54, 4539–4546. Frisch, S.M., Francis, H., 1994. Disruption of epithelial cell–matrix interactions induces apoptosis. J. Cell Biol. 124, 619–626. Ghatak, S., Misra, S., Toole, B.P. Hyaluronan constitutively regulates ErbB2 phosphorylation and signaling complex formation in carcinoma cells. J. Biol. Chem. 2005. 280, 8875–8883. Goh HS, Yao J, Smith DR. p53 point mutation and survival in colorectal cancer patients. Cancer Res. 1995. 55, 5217-21. Grossman, J. Molecular mechanisms of detachment induced apoptosis– Anoikis. Apoptosis. 2002. 7, 247– 260. Jin, L., Hope, K.J., Zhai, Q., Smadja-Joffe, F., Dick, J.E. Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat. Med. 2006. 12, 1167–1174. Kaklamanis L, Gatter KC, Mortensen N, Baigrie RJ, Heryet A, Lane DP, Harris AL,1993. p53 expression in colorectal adenomas. Am J Pathol. 142, 87-93. Kastan MB, Onyekwere O, Sidransky D, Vogelstein B, Craig RW,1991.Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 51, 6304-11. Kim, H., Xiao-Ling, Y., Rosada, C., Stanley, R., August, J. CD44 expression in colorectal adenomas is an early event occurring prior to K-ras and p53 mutation. Arch. Biochem. Biophys. 1994. 310, 504– 507. Krause, D.S., Lazarides, K., von Andrian, U.H., Van Etten, R.A., 2006. Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells. Nat. Med. 12, 1175–1180. Kuncová J, Urban M, Mandys V, 2007. Expression of CD44s and CD44v6 in transitional cell carcinomas of the urinary bladder: comparison with tumour grade, proliferative activity and p53 immunoreactivity of tumour cells. APMIS.115,1194-205. Lakshman, M., Subramaniam, V., Jothy, S., 2004a. CD44 negatively regulates apoptosis in murine colonic epithelium via the mitochondrial pathway. Exp.Mol. Pathol. 76, 196–204. Lakshman, M., Subramaniam, V., Rubenthiran, U., Jothy, S., 2004b. CD44 promotes resistance to apoptosis in human colon cancer cells. Exp. Mol. Pathol. 77, 18– 25. Lakshman, M., Subramaniam, V., Wong, S., Jothy, S., 2005. CD44 promotes resistance to apoptosis in murine colonic epithelium. J. Cell. Physiol. 203, 583–588. Leahy DT, Salman R, Mulcahy H, Sheahan K, O'Donoghue DP, Parfrey NA, 1996.Prognostic significance of p53 abnormalities in colorectal carcinoma detected by PCR-SSCP and immunohistochemical analysis. J Pathol. 180, 364-70. Manne U, Myers RB, Moron C, Poczatek RB, Dillard S, Weiss H, Brown D, Srivastava S, Grizzle WE, 1997. Prognostic significance of Bcl-2 expression and p53 nuclear accumulation in colorectal adenocarcinoma. Int J Cancer. 74, 346-58. Ofner D, Maier H, Riedmann B, Holzberger P, Nogler M, Tötsch M, Bankfalvi A, Winde G, Böcker W, Schmid KW, 1995. Immunohistochemically detectable p53 and mdm-2 oncoprotein expression in colorectal carcinoma: prognostic significance. Clin Mol Pathol. 48, 12-16. Pan G, Greenblatt J, 1994. Initiation of transcription by RNA polymerase II is limited by melting of the promoter DNA in the region immediately upstream of the initiation site. J Biol Chem. 269, 30101-4. Partik G, Kahl-Rainer P, Sedivy R, Ellinger A, Bursch W, Marian B, 1998. Apoptosis in human colorectal tumours: ultrastructure and quantitative studies on tissue localization and association with bak expression. Virchows Arch.432, 415-26. Ponta, H., Sherman, L., Herrlich, P.A., 2003. CD44: from adhesion molecules to signalling regulators. Nat. Rev., Mol. Cell Biol. 4, 33–45. Porter, A.G., Janicke, R.U., 1999. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 6, 99–104. Rogel A, Popliker M, Webb CG, Oren M, 1985.p53 cellular tumor antigen: analysis of mRNA levels in normal adult tissues, embryos and tumors. Mol Cell Biol. 5,28512855. Rudzki, Z., LeDuy, L., Jothy, S., 1997. Changes in CD44 expression during carcinogenesis of the mouse colon. Exp. Mol. Pathol. 64, 114–125. Scott N, Bell SM, Sagar P, Blair GE, Dixon MF, Quirke P, 1993.p53 expression and Kras mutation in colorectal adenomas. Gut. 34,621-4. Screaton, G.R., Bell, M.V., Jackson, D.G., Cornelis, F.B., Gerth, U., Bell, J.I., 1992. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc. Natl. Acad. Sci. U. S. A. 89, 12160–12164. Shipitsin, M., Campbell, L.L., Argani, P., Weremowicz, S., Bloushtain-Qimron, N., Yao, J., Nikolskaya, T., Serebryiskaya, T., Beroukhim, R., Hu, M., Halushka, M.K., Sukumar, S., Parker, L.M., Anderson, K.S., Harris, L.N., Garber, J.E., Richardson, A.L., Schnitt, S.J., Nikolsky, Y., Gelman, R.S., Polyak, K., 2007. Molecular definition of breast tumor heterogeneity. Cancer Cells 11, 259–273. Shirasawa, S., Furuse, M., Yokoyama, N., Sasazuki, T., 1993. Altered growth of human colon cancer cell lines disrupted at activated Ki-ras. Science 260, 85–88. Song, E., Zhu, P., Lee, S.K., Chowdhury, D., Kussman, S., Dykxhoorn, D.M., Feng, Y., Palliser, D.,Weiner, D.B., Shankar, P., Marasco,W.A., Lieberman, J., 2005. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol. 23, 709–717. Soong R, Iacopetta BJ, 1997. A rapid and nonisotopic method for the screening and sequencing of p53 gene mutations in formalin-fixed, paraffin-embedded tumors. Mod Pathol. 10,252-8. Sturzbecher HW, Chumakov P, Welch WJ, Jenkins JR, 1987. Mutant p53 proteins bind hsp 72/73 cellular heat shock-related proteins in SV40-transformed monkey cells. Oncogene. 1, 201-211. Subramaniam V, Vincent IR, Jothy S, 2005. Upregulation and dephosphorylation of cofilin: modulation by CD44 variant isoform in human colon cancer cells. Exp Mol Pathol. 79, 187-93. Subramaniam V, Vincent IR, Gilakjan M, Jothy S, 2007. Suppression of human colon cancer tumors in nude mice by siRNA CD44 gene therapy. Exp Mol Pathol. 83,332-40 Wielenga, V., Heider, K., Offerhaus, G., Adolf, G., van den Berg, F., Ponta, H., Herrlich, P., Pals, S., 1993. Expression of CD44 variant proteins in human colorectal cancer is related to tumor progression. Cancer Res. 53, 4754– 4756. Wolf JC, Ginn PE, Homer B, Fox LE, Kurzman ID, 1997. Immunohistochemical detection of p53 tumor suppressor gene protein in canine epithelial colorectal tumors. Vet Pathol. 34, 394-404. Wong, K., Rubenthiran, U., Jothy, S., 2003. Motility of colon cancer cells: modulation by CD44 isoform expression. Exp. Mol. Pathol. 75, 124–130. Woodman, A., Sugiyama, M., Yoshida, K., Sugino, T., Borgya, A., Goodison, S., Matsumura, Y., Tarin, D., 1996. Analysis of anomalous CD44 gene expression in human breast, bladder and colon cancer and correlation of observed mRNA and protein isoforms. Am. J. Pathol. 149, 1519– 1530.