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CD44 promotes resistance to apoptosis in human colon cancer cells
Experimental and Molecular Pathology 77 (2004) 18 – 25 www.elsevier.com/locate/yexmp
CD44 promotes resistance to apoptosis in human colon cancer cells Minalini Lakshman, Venkateswaran Subramaniam, Umayal Rubenthiran, and Serge Jothy * Department of Laboratory Medicine and Pathobiology, St. Michael’s Hospital and University of Toronto, Toronto, Ontario, Canada M5B 1W8 Received 19 February 2004
Abstract Overexpression of CD44, especially its variant isoforms, occurs consistently in colon cancer, as compared to autologous normal colon, and this change occurs also in most other types of cancer. One of the basic features of malignant transf ormation is the acquisition of resistance to apoptosis. In this study, we asked whether the expression of CD44 and some of its variant isoforms commonly found in colon cancer participate in resistance to apoptosis and what are the mechanisms involved. A human colon cancer cell line, SW620, which does not express CD44 was stably transfected with standard, v3-10, and v8-10 containing isoforms of CD44. Mock-transfected and CD44-transfected cells were exposed to etoposide to induce apoptosis. Apoptotic and concomitant changes relevant to the mechanisms of apoptosis were monitored by flow cytometry, DNA fragmentation, and immunoblot analyses. It was observed that resistance to apoptosis induced by etoposide is promoted by CD44 expression in SW620, and this resistance is better sustained by the full variant isoform, v3-10. Concomitant alterations in caspase 9, caspase 3, Bcl-xl, and Bak indicated that the resistance to apoptosis in this model involved the mitochondrial pathway. The differential response of CD44 transfectants was associated with a downregulation of pRb and phosphorylated AKT. The results of this study are consistent with the conclusion that expression of variant CD44 isoforms which is characteristic of colon cancer, and most other types of cancer, confers a selective advantage to resist apoptosis, thereby promoting cell transformation into a malignant phenotype, in conjunction with other anti-apoptotic factors. D 2004 Elsevier Inc. All rights reserved. Keywords: CD44; Bcl-2; Caspases; pRb; p21; AKT phosphorylation; Apoptosis; Colon cancer
Introduction Colon cancer is the second leading cause of cancerrelated deaths in North America. The transformation of the normal colonic epithelium follows an adenoma to carcinoma sequence characterized by a large number of molecular alterations, converging to alterations in epithelial cell proliferation and apoptosis. These two processes are tightly regulated in the constantly renewing untransformed colonic epithelium. They involve adhesion molecules and regulators of cell cycle and apoptosis. CD44 is a ubiquitously expressed cell adhesion molecule and is a rare example in that it facilitates both cell – cell and cell – matrix interactions (reviewed in Ponta et al., 2003; Rudzki et al., 1997). Its expression is restricted to the proliferative lower two thirds of the normal colonic crypt * Corresponding author. Department of Laboratory Medicine and Pathobiology, St. Michael’s Hospital and University of Toronto, 30 Bond Street, Toronto, Ontario, Canada M5B 1W8. Fax: +1-416-864-5648. E-mail address: [email protected] (S. Jothy). 0014-4800/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2004.03.002
and is absent in the upper third of the crypt where apoptosis occurs. Hyaluronan is the major ligand of CD44. The cytoplasmic domain of CD44 is known to interact with ERM proteins: ezrin, radixin, moesin, and merlin. A large number of CD44 isoforms are generated by alternative splicing, some of which have been implicated in tumor growth and metastases (Gunthert et al., 1991; Hofmann et al., 1991). One of the key features of tumorigenesis is the aberrant homeostasis between cell cycle and apoptosis, which is otherwise governed by cell cycle and apoptosis regulators and inhibitors. The key cell cycle regulators are a highly conserved family of cyclin-dependent kinases (cdk) whose activation requires binding to cyclins. In turn, cdks are negatively controlled by inhibitory proteins which include the CIP/KIP family like p21. The retinoblastoma protein is a cell cycle regulatory protein that engages transcription factors and blocks cell cycle (Garriga et al., 1998; Weinberg, 1995). On the other hand, apoptosis regulators, predominantly Bcl-2 family and executors, Caspases along with stimulators and inhibitors of these families, are also
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involved in dictating a cell’s fate to survive or to die. Several studies have reported cross talks among the cell cycle and apoptosis regulators (Chau et al., 2002; Li et al., 2002; Tan and Wang, 1998; Woo et al., 2003). The interaction of CD44 with some of these key regulators favors survival of cancer cells (Bates et al., 2001; Tian et al., 2000). Disruption of cell adhesion to neighboring cells and extracellular matrix can result in apoptosis (Frisch and Francis, 1994; Grossman, 2002; Shanmugathasan and Jothy, 2000). Defective apoptosis may be involved in the accumulation of cells, still attached to their matrix and to adjacent cells in colonic adenomas, which coincides with overexpression of discrete CD44 isoforms (Bedi et al., 1995; Partik et al., 1998; Wielenga et al., 1993). Previously, using a CD44 knockout murine model, we have shown evidence of early signs of apoptosis, involving the mitochondrial pathway in the normal colon (Lakshman et al., 2004). In this study, we focused on the role of CD44 in the apoptosis of human colon cancer cells. A human colon cancer cell line, SW620, which does not express CD44 was stably transfected with standard and variant isoforms of CD44 expressed in colonic tumors. We found that CD44 transfectants were resistant to etoposide-induced apoptosis and this resistance was more pronounced with the v3-10 isoform of CD44. Noticeable alterations in Bcl-xl, Bak and caspases 9 and 3 can attribute the resistance to apoptosis by CD44 transfectants. Downregulation of AKT phosphorylation and pRb were also associated with the differential response to etoposide-induced apoptosis by CD44 transfectants. Overall, we conclude that CD44 promotes resistance to apoptosis in the human colon cancer cells studied.
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St Louis, MO). Hyaluronan from rooster comb and etoposide were from Sigma-Aldrich. Hyaluronic acid adhesion assay Adhesion assay for SW620 cells transfected with vector and CD44 isoforms and HT-29 to hyaluronic acid was performed as described previously (Herrera-Gayol and Jothy, 1999). Briefly, flat bottom 96-well plates were coated with 5 Ag/ml of hyaluronan, or 2% BSA overnight at 4jC. The wells were then coated with 0.2% BSA for immobilization for 2 h at 37jC. 1 105 cells were allowed to adhere for 1 h at 37jC. After rinsing twice with phosphate-buffered saline, pH 7.4, adherent cells were quantitated using a hematocytometer and the percentage of adherent cells of the control (adherent cells to HA-adherent cells to BSA/adherent cell to BSA) was determined from three independent experiments, each in triplicates. Induction of apoptosis Cells, 1 106, were plated in a 5-cm culture dish. Fortyeight hours later, cells were treated with fresh medium, and subjected to 8 Gy of g irradiation from a Cs-137 source for 0, 4, 8, 12, 24, and 48 h, or 10 Ag/ml of etoposide, or its control solvent, DMSO for 0, 24, and 48 h. Drug toxicity assay Cells, 5 103, were plated in flat bottom 96-well plates and 48 h later treated with etoposide, or DMSO. MTT assay (Promega, Madison, WI) was performed according to manufacturer’s protocol to assess the cell viability after etoposide treatment at indicated time points. Experiments were repeated twice and in triplicates.
Materials and methods DNA fragmentation Materials Human colon carcinoma lines, HT-29 and SW620 were maintained in culture using DMEM with 10% fetal bovine serum and Penicillin/Streptomycin. SW620 transfected with CD44 isoforms (S, v3-10 and v8-10 and vector construct (V)) were maintained as described in (Wong et al., 2003). Primary antibodies used were rabbit anti-rat Bcl-xl, mouse anti-p21 and -pRb (BD Biosciences, Mississauga, ON, Canada), rabbit anti-human Bak (Upstate Biotech, Charlottesville, VA), rabbit anti-human recombinant caspases 3 and 9, anti-rabbit cyclin D1 (Neomarker Lab Vision Corp., Fremont, CA), rabbit anti-human cleaved caspase 3 (Cell Signaling Technology, Beverly, MA), rabbit anti-cyclin A, cdk-2 and cdk-4 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-AKT-phosphorylated (473Ser) and total AKT (New England Biolabs, Mississauga, ON, Canada) and mouse monoclonal anti-human h-actin (Sigma-Aldrich,
After the induction of apoptosis as described above, DNA from these cells was isolated using a DNeasy kit (Qiagen, Mississauga, ON, Canada) according to manufacturer’s protocol. Lysates from 0, 24, and 48 h after etoposide treatment were electrophoresed on 0.8% agarose gel containing 0.1% ethidium bromide. Detection of apoptosis using annexin V and propidium iodide Flow cytometry analyses with FITC-labeled annexin V and propidium iodide were performed according to manufacturer’s protocol (BD Pharmingen, Mississauga, ON, Canada) on cells treated with 10 Ag/ml of etoposide for 0, 24, and 48 h, or DMSO. Etoposide-induced apoptosis is represented as % apoptosis of control (i.e., apoptosis with etoposide treatment in DMSO/DMSO 100).
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Immunoblotting Immunoblotting protocol was performed as described (Lakshman et al., 2004). Briefly, lysates were prepared using NP-40 lysis buffer with protease inhibitors and subjected to 12% SDS-PAGE under reducing conditions.
Results Functional validation of CD44 transfection We tested whether CD44 transfected in SW620 was functional using a hyaluronic acid-adhesion assay. Adhesive capacity was measured by counting the number of cells attached to hyaluronan after 1 h. Fig. 1 shows that adhesion to hyaluronan was significantly increased in SW620 cells transfected with the v3-10 (67%, P < 0.05) and v8-10 isoform of CD44 (126%, P < 0.001) as compared to vector control. Cells transfected with the standard isoform(S) of CD44 were also more adhesive than the vector control (72%, P = 0.1). HT-29, a human colon cancer line that overexpresses CD44 isoforms, also showed an increased adhesive capacity and served as a positive control for the assay. Thus, adhesion to hyaluronan, a major ligand of CD44, is dramatically increased when CD44 is expressed in SW620 indicating that CD44 is functional in the transfectants. CD44 expression promotes resistance to apoptosis We investigated the role of CD44 on apoptosis, induced either by gamma irradiation or by a chemotherapeutic drug, etoposide (Fujita et al., 2002). Induction of apoptosis by gamma irradiation was detected as shown by DNA fragmentation and caspase 3 activation (Figs. 2A and B), which are markers of apoptotic events. We observed the onset of caspase 3 activation and marked DNA fragmentation in all cell lines at 48 h. Surprisingly, caspase 9 was already activated at 0 h and remained activated up to 48 h following
Fig. 2. Gamma irradiation induced apoptosis in SW620 vector control and CD44 transfectants. Induction of apoptosis by g irradiation (8 Gy) was assessed by DNA fragmentation (A) and by Western blot analyses (B) at indicated time points. Apoptosis caused by irradiation was detected only at 48 h in all cell lines, as shown by DNA fragmentation. Caspase 9 remained active in all cell lines from 0 to 48 h post-irradiation. Strong activation of caspase 3 is observed in all cell lines at 48 h with weak activation in HT-29.
Fig. 1. CD44 transfected in SW620 human colon carcinoma cells is functional. Cells, 1 106, were plated on 2% BSA or hyaluronan (HA) at 5 Ag/ml and allowed to adhere for 1 h. Results are shown as average % adhesion of control (BSA). Adhesive capacity is increased in all CD44 transfectants as compared to the vector control. * indicated that the adhesion in v3-10 and v8-10 transfectants was significant. By Student’s t test, the p values were P < 0.05 for v3-10 and P < 0.001 for v8-10.
gamma irradiation (Fig. 2B). In parallel experiments, we administered a chemotherapeutic drug, etoposide, to induce apoptosis. The toxicity of this drug was assessed by MTT assay for cell viability (Fig. 3A). Viable cell population remained unaltered when treated with etoposide as compared to the control (DMSO) up to 24 h ( P > 0.1). Only
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SW620 cells transfected with variant CD44 isoforms were less apoptotic than control cells and cells expressing the standard CD44 isoform. HT-29 cells displayed a DNA fragmentation pattern comparable to variant CD44 transfectants, as expected (Fig. 4B). Cumulatively, these results indicate that resistance to apoptosis induced by etoposide is promoted by CD44 expression in SW620 after 24 h and this resistance is greatly sustained by the full variant isoform, v3-10. Resistance to apoptosis in SW620 transfected with CD44 involves the mitochondrial pathway To determine the mechanism by which CD44 promoted resistance to apoptosis, we analyzed the expression of regulators and executors of apoptosis by immunoblotting. The expression of CD44 in the transfectants remained unaltered during etoposide-induced apoptosis (Fig. 5A). Before etoposide treatment, expression of anti-apoptotic Bcl-xl and pro-apoptotic Bak remained comparable in vector and variant isoform transfectants (Fig. 5B). At 24 h, the vector control showed undetectable levels of Bak, accompanied by reduced Bcl-xl expression (Fig. 5B). CD44 transfectants, on the other hand, maintained a higher Bcl-xl/Bak ratio, at 24 h, indicating a shift towards survival. At 48 h, decreased expression level of Bcl-xl reverted similar to 0 h. Apoptosis induction did not alter the level of cytochrome c in vector control but its level was reduced in v3-10 among the transfectants at 24 h (Fig. 5C). Activation of caspase 9, an effector of cytochrome c, was lost in vector control at 24 h, though it remained unaltered at 0 and 48 h in all cell lines. CD44 transfectants showed activation of caspase 3 at 24 and 48 h, which was less marked in the cells transfected with CD44 v3-10, resembling therefore the pattern observed in HT29 cells. Hence, alterations in key apoptosis regulators/effectors indicate that the mitochondrial pathway is involved in rendering the CD44 transfectants resistant to etoposide induced apoptosis at 24 h. Fig. 3. Induction of apoptosis by etoposide is not toxic to cells. About 10 Ag/ml of etoposide was administered for 0 (A), 24 (B), and 48 h (C). DMSO served as control. Cell viability was measured by MTT assay. (A and B) Cell viability remained unaltered between etoposide- and DMSOtreated groups at 0 and 24 h, respectively. (C) Cell viability decreased significantly in standard isoform transfectants (P < 0.001) after 48 h.
cells transfected with the standard CD44 isoform had a significant reduction in viable cell population ( P < 0.001) at 48 h. In addition to DNA fragmentation, quantitation of apoptosis was performed by flow cytometry using annexin V as a marker (Fig. 4). At 24 h, all CD44 transfectants showed a marked decrease in apoptosis as compared to vector control. Interestingly, resistance to apoptosis was highest in cells transfected with CD44 v3-10 as compared to the standard (S) and v8-10 isoform. This resistance to apoptosis was also shared by HT-29, which showed that
Role of cell cycle and survival proteins in CD44-mediated anti-apoptosis The role of regulators of cell cycle and cell survival in relation to the anti-apoptotic function of CD44 was analyzed by immunoblotting. We studied the expression levels of prosurvival AKT/PKB, cell cycle inhibitors p21 and pRb, cell cycle regulators cyclins A and D, and their respective inhibitors, cdk-2 and 4. Before etoposide treatment, expression levels of total AKT and its phosphorylated form, pRb, cyclins, and cdk-4 was comparable in vector control and CD44 transfectants (Figs. 6A and B). Also, p21 levels were undetectable in vector control and CD44 transfectants. As compared to SW620 cells, elevated levels of phosphorylated AKT, p21, and pRb were observed in HT-29 at 0 h. After 24 h of etoposide treatment, AKT phosphorylation and pRb were markedly increased in vector control as compared to
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Fig. 4. CD44 promotes resistance to apoptosis induced by etoposide. Etoposide induced apoptosis was detected by flow cytometry with annexin V (A) and by DNA fragmentation analysis (B). In A, % apoptosis of the control (DMSO) was calculated as described in Materials and methods. After 24 h, shown in black bars, vector control showed the highest % of apoptosis (67%). Resistance to apoptosis was marked in CD44 transfectants and v3-10 had pronounced resistance (14%) among the CD44 transfectants. DNA fragmentation was clearly visible at 48 h consistent with late events of apoptosis and absent in 24 h. HT-29 and v3-10 showed the least fragmentation after 48 h.
Fig. 5. Bcl-2 and caspase protein members are involved in the resistance to etoposide-induced apoptosis in CD44 transfectants. (A) CD44 expression remains unaltered by etoposide at 24 and 48 h as shown by immunoblotting. HT-29 shows expression of both standard and variant CD44 isoforms. (B) Twenty-four hours after irradiation, Bcl-xl expression was markedly downregulated in vector control cells accompanied by undetectable expression of Bak. (C) Cytochrome c levels were higher in CD44 v3-10 and CD44 v8-10 than CD44s and control cells at time 0. At 24 h, cytochrome c level is reduced in CD44 v3-10 cells, compared to cells expressing CD44s and v8-10. At 24 h, caspase 9 was inactivated in vector control. Onset of caspase 3 activation was observed at 24 h. Low levels of caspase 3 activation was observed in vector at 24 h. v3-10 showed the least activation of caspase 3 among the CD44 transfectants.
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Fig. 6. Cell cycle regulators/inhibitors stabilize resistance to apoptosis in CD44 transfectants. (A) Total AKT expression remained unaltered in all cell lines before and after treatment up to 48 h. AKT phosphorylation (AKT-P) remained upregulated in cells expressing CD44 v3-10 at 48 h. Cell cycle inhibitor, p21 was not detected before induction of apoptosis and was consistently increased in all SW620 cell lines at 48 h, irrespective of CD44 status. pRb, a transcription agonist was expressed in all cell lines at 0 h but markedly downregulated after 24 h and weakly expressed after 48 h in CD44 transfectants. Cyclin D1 level was decreased in all cell lines except in standard isoform at 24 h. Cdk-4 levels remained unaltered among cell lines. (B) Cyclin A levels remained unaltered among cell lines but was increased after treatment. Cdk-2 levels remained unaltered among cell lines.
CD44 transfectants. However, p21 expression was weak in vector as compared to CD44 transfectants. Twenty-four hours of etoposide treatment resulted in an increase of cyclin A in all cell lines (Fig. 6B), and a decrease in Cyclin D expression (Fig. 6A) is observed in all except standard isoforms. Expression of cdks remained comparable to preetoposide treatment in all cell lines. After 48 h, phosphorylation of AKT decreased in SW620 cells transfected with the standard and v8-10 isoforms of CD44, as compared to other cell lines. In relation to pre-etoposide and 24 h levels, p21 expression was upregulated at 48 h in vector control and CD44 transfectants. However, pRb expression was decreased at 48 h versus previous times in all SW620 lines. No significant alterations in cyclin and cdk levels of expression occurred as a result of etoposide treatment. Hence, expression of CD44 modulates cell cycle regulators pRb and p21, and pro-survival proteins such as AKT. These findings are consistent with the resistance to apoptosis that was observed in CD44 transfectants at 24 h.
Discussion Unregulated homeostasis between cell proliferation and apoptosis is characteristic of tumorigenesis. It is well documented that colonic adenomas are defective in apoptosis (Bedi et al., 1995; Partik et al., 1998). Interestingly,
overexpression of discrete isoforms of CD44 is observed in 80% of colonic adenomas (Wielenga et al., 1993). It is important to note that the standard isoform is ubiquitously expressed and variant isoform, v8-10, is expressed in epithelial cells and activated lymphocytes. Following the discovery that discrete CD44 variant isoforms are determinant in pancreatic tumor metastasis, it was found that expression of discrete isoforms of CD44 such v3, v5, and v6 are implicated in colorectal tumor growth and metastasis (Gunthert et al., 1991; Mulder et al., 1997; Wielenga et al., 1993). It is still not clear how the expression of CD44 variant exons contributes to tumor progression in colon carcinoma. Although cells expressing CD44 have been reported to be resistant to etoposide-induced apoptosis, a major functional role for CD44 and its variant isoforms in apoptosis and colon cancer has not been dealt in depth so far (Fujita et al., 2002). In this study, we looked at the role of CD44 and its variants in apoptosis and cancer, using human colon cancer cells. For this, we used SW620, a human colon cancer cell line that does not express CD44, and stably transfected standard, v3-10 and v8-10 isoforms of CD44 (Wong et al., 2003). We induced apoptosis using g irradiation and etoposide, further compared the resistance to etoposide-induced apoptosis between the vector control and CD44 transfectants, and the mechanisms involved. Based on our previous study using a CD44 knockout mouse model, we looked first at major molecular executors of
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apoptosis of the mitochondrial pathway and cell cycle regulators in cells undergoing apoptosis (Lakshman et al., 2004). Cumulatively, these data indicate that expression of CD44 in human colon carcinoma cells confers resistance to apoptosis, and this involved the mitochondrial pathway in conjunction with cell cycle regulators. The communication between cells and their extracellular matrix is facilitated by adhesion molecules like CD44. Studies with melanoma cell lines have shown that cells expressing CD44 are adherent to hyaluronan, a major ligand of CD44, and are functionally motile and invasive (Birch et al., 1991; Goebeler et al., 1996; Thomas et al., 1992, 1993). Results from our hyaluronan-binding assay showed that SW620 transfected with CD44 standard and variant isoforms were more adhesive than vector control. Therefore, CD44 in the SW620 transfectants used in this study is functional and capable of responding to an adhesive stimulus. The CD44 transfectants showed a delayed onset of apoptosis and in the case of transfectants expressing the v3 to v10 CD44 variant exons, there was a markedly increased resistance to apoptosis. This is consistent with the upregulation of apoptosis molecular markers, such as active caspase 3 which was apparent as early as 24 h. Resistance to apoptotic stimuli observed with the CD44 v3-10 was similar to that observed with the HT-29 cells, which express a high level of variant CD44 isoforms. The results are overall independent of the mode of apoptosis induction as we obtained comparable results with etoposide. Several studies have successfully employed the above concentration of etoposide in colon cancer cells for induction of apoptosis (Fujita et al., 2002; Gunthert et al., 1996). Of note, neither the expression nor adhesion to hyaluronan of CD44 in the various transfectants was affected by etoposide even after 48 h of induction. Previously, we reported that regulation of apoptosis in the normal murine colon operates through the mitochondrial pathway (Lakshman et al., 2004). Hence, in this study, we looked at the some of the major regulators involved in this pathway. We observed that the expression levels of Bak remained steady over the time period of apoptosis induction. However, the other indicator of mitochondrial disturbance, Bcl-xl was being fine tuned in response to etoposide. This resulted in a pro-apoptotic Bcl-xl/Bak ratio in the vector control with complete loss of expression of Bak at 24 h, as opposed to being anti-apoptotic in the CD44 transfectants. Disturbance in mitochondrial integrity resulting in the release of cytochrome c and caspase 9 activation is well documented (Ruemmele et al., 2003; Slee et al., 1999; Zou et al., 1999). In our study using etoposide, where we have so far observed the major changes related to apoptosis occurring at 24 h, the vector control had an appreciable loss in active caspase 9 levels. When SW620 cells were transfected with CD44 v3-10, cytochrome c levels were decreased as compared to CD44s, with accompanying caspase 9 remaining active suggesting that CD44 role in apoptosis in these cancer cells is through the mitochondrial pathway.
Both proliferation and cell death contributes to the normal homeostasis of the cells. To rationalize the differential apoptotic response between CD44 transfectants and CD44 negative control cells, we looked at alterations in cell cycle regulators. Phosphorylated AKT, a pro survival molecule and cell cycle inhibitors, p21, and regulator pRb were upregulated in vector control as compared to CD44 transfectants. In contrast, the cell cycle inhibitor, p21 was weakly expressed in vector control. Previous studies have shown the reduction or absence of p21 expression sensitizes cells to etoposide-induced apoptosis (Tian et al., 2000; Waldman et al., 1996). This may explain the susceptibility of control cells to etoposide-induced apoptosis, in contrast to upregulated levels of p21 in CD44 transfectants at 24 h. Interestingly, even though p21 expression in v3-10 was similar to vector control, the expression of CD44 seems to offer resistance to apoptosis probably by downregulating pRb. The role of CD44 in determining survival/proliferation of colon carcinoma cells has been linked to the involvement of AKT, a substrate of PI3 kinase which was recently reported to be a pivotal player in colonic tumorigenesis (Bates et al., 2001; Sithanandam et al., 2003). In our study, we observed an upregulation in the expression of AKT (Ser 473) phosphorylation in the vector control at 24 h with a steady state remaining in the transfectants. This leads us to speculate that in the absence of appreciable levels of p21 and the loss of protective effect promoted by CD44 against apoptosis, the cells in the vector control compensate apoptotic induction by upregulating AKT-phosphorylation, and thus through a different cell survival pathway. Conversely, in CD44 transfectants, upregulated levels of AKT phosphorylation and downregulation of pRb were indicative of pro-survival which coincides with our earlier observation that by virtue of the cells being transfected with CD44 isoforms, apoptotic regulation channels through the mitochondrial pathway. Overall, the data indicate that expression of CD44 and more importantly, v3-10 isoform, promotes resistance to apoptosis. Key regulators of cell cycle and the mitochondrial pathway were involved in imparting this resistance in SW620 transfected with CD44 isoforms. However, SW620 cells, which lack CD44, are sensitive to apoptosis by an alternate pathway. Of note is the similarity in the marked apoptotic resistance of SW620 colon cancer cells transfected with v3-10 and HT-29, another human colon cancer line overexpressing variant CD44 isoforms. This observation is relevant to the understanding of colonic neoplasia. The mechanistics discussed might lead to looking at CD44 expression as a marker of sensitivity to adjuvant chemotherapy. From this study, we propose that expression of specific variant CD44 isoforms characteristic of colon cancer and most other types of cancer confers a selective advantage to resist apoptosis, therefore promoting cell transformation into a malignant phenotype in conjunction with other antiapoptotic factors.
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Li, Y., Dowbenko, D., Lasky, L.A., 2002. AKT/PKB phosphorylation of p21Cip/WAF1 enhances protein stability of p21Cip/WAF1 and promotes cell survival. J. Biol. Chem. 277, 11352 – 11361. Mulder, J.W., Wielenga, V.J., Pals, S.T., Offerhaus, G.J., 1997. p53 and CD44 as clinical markers of tumor progression in colorectal carcinogenesis. Histochem. J. 29, 439 – 452. Partik, G., Kahl-Rainer, P., Sedivy, R., Ellinger, A., Bursch, W., Marian, B., 1998. Apoptosis in human colorectal tumors: ultrastructure and quantitative studies on tissue localization and association with Bak expression. Virchows Arch. 452, 415 – 420. Ponta, H., Sherman, L., Herrlich, P.A., 2003. CD44: from adhesion molecules to signaling regulators. Nat. Rev., Mol. Cell Biol. 4, 33 – 45. Rudzki, Z., LeDuy, L., Jothy, S., 1997. Changes in CD44 expression during carcinogenesis of the mouse colon. Exp. Mol. Pathol. 64, 114 – 125. Ruemmele, F.M., Schwartz, S., Seidman, E.G., Dionne, S., Levy, E., Lentze, M.J., 2003. Butyrate induced Caco-2 cell apoptosis is mediated via the mitochondrial pathway. Gut 52, 94 – 100. Shanmugathasan, M., Jothy, S., 2000. Apoptosis, anoikis and their relevance to the pathobiology of colon cancer. Pathol. Int. 50, 273 – 279. Sithanandam, G., Smith, G.T., Masuda, A., Takahashi, T., Anderson, L.M., Fornwald, L.W., 2003. Cell cycle activation in lung adenocarcinoma cells by ErbB3/phosphatidylinositol 3-kinase/Akt. Carcinogenesis 24, 1581 – 1592. Slee, E.A., Harte, M.T., Kluck, R.M., Wolf, B.B., Casiano, C.A., Newmeyer, D.D., Wang, H.G., Reed, J.C., Nicholson, D.W., Alnemri, E.S., Green, D.R., Martin, S.J., 1999. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8 and 10 in a caspase 9 dependent manner. J. Cell Biol. 144, 281 – 292. Tan, X., Wang, J.Y., 1998. The caspases-RB connection in cell death. Trends Cell Biol. 8, 116 – 120. Thomas, L., Byers, H.R., Vink, J., Stamenkovic, I., 1992. CD44H regulates tumor migration on hyaluronate coated substrate. Cell Biol. 118, 971 – 977. Thomas, L., Etoh, T., Stamenkovic, I., Mihm Jr., M.C., Byers, H.R., 1993. Migration of human melanoma cells on hyaluronate is related to CD44 expression. J. Invest. Dermatol. 100, 115 – 120. Tian, B., Takasu, T., Henke, C., 2000. Functional role of cyclin A on induction of fibroblast apoptosis due to ligation of CD44 matrix receptor by anti-CD44 antibody. Exp. Cell Res. 257, 135 – 144. Waldman, T., Lengauer, C., Kinzler, K.W., Vogelstein, B., 1996. Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature 381, 713 – 716. Weinberg, R.A., 1995. The retinoblastoma protein and cell cycle control. Cell 81, 323 – 330. Wielenga, V.J., Heider, K.H., Offerhaus, G.J., Adolf, G.R., van den Berg, F.M., Ponta, H., Herrlich, P., Pals, S.T., 1993. Expression of CD44 variant proteins in human colorectal cancer is related to tumor progression. Cancer Res. 53, 4754 – 4756. Wong, K., Rubenthiran, U., Jothy, S., 2003. Motility of colon cancer cells: modulation by CD44 isoform expression. Exp. Mol. Pathol. 75, 124 – 130. Woo, M., Hakem, R., Furlonger, C., Hakem, A., Duncan, G.S., Sasaki, T., Bouchard, D., Lu, L., Wu, G., Paige, C.J., Tak, M., 2003. Caspase 3 regulates cell cycle in B cells: a consequence of substrate specificity. Nat. Immunol. 4, 1016 – 1022. Zou, H., Li, Y., Liu, X., Wang, X., 1999. An APAF-1 cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J. Biol. Chem. 274, 11549 – 11556.
CD44 promotes resistance to apoptosis in human colon cancer cells Minalini Lakshman, Venkateswaran Subramaniam, Umayal Rubenthiran, and Serge Jothy * Department of Laboratory Medicine and Pathobiology, St. Michael’s Hospital and University of Toronto, Toronto, Ontario, Canada M5B 1W8 Received 19 February 2004
Abstract Overexpression of CD44, especially its variant isoforms, occurs consistently in colon cancer, as compared to autologous normal colon, and this change occurs also in most other types of cancer. One of the basic features of malignant transf ormation is the acquisition of resistance to apoptosis. In this study, we asked whether the expression of CD44 and some of its variant isoforms commonly found in colon cancer participate in resistance to apoptosis and what are the mechanisms involved. A human colon cancer cell line, SW620, which does not express CD44 was stably transfected with standard, v3-10, and v8-10 containing isoforms of CD44. Mock-transfected and CD44-transfected cells were exposed to etoposide to induce apoptosis. Apoptotic and concomitant changes relevant to the mechanisms of apoptosis were monitored by flow cytometry, DNA fragmentation, and immunoblot analyses. It was observed that resistance to apoptosis induced by etoposide is promoted by CD44 expression in SW620, and this resistance is better sustained by the full variant isoform, v3-10. Concomitant alterations in caspase 9, caspase 3, Bcl-xl, and Bak indicated that the resistance to apoptosis in this model involved the mitochondrial pathway. The differential response of CD44 transfectants was associated with a downregulation of pRb and phosphorylated AKT. The results of this study are consistent with the conclusion that expression of variant CD44 isoforms which is characteristic of colon cancer, and most other types of cancer, confers a selective advantage to resist apoptosis, thereby promoting cell transformation into a malignant phenotype, in conjunction with other anti-apoptotic factors. D 2004 Elsevier Inc. All rights reserved. Keywords: CD44; Bcl-2; Caspases; pRb; p21; AKT phosphorylation; Apoptosis; Colon cancer
Introduction Colon cancer is the second leading cause of cancerrelated deaths in North America. The transformation of the normal colonic epithelium follows an adenoma to carcinoma sequence characterized by a large number of molecular alterations, converging to alterations in epithelial cell proliferation and apoptosis. These two processes are tightly regulated in the constantly renewing untransformed colonic epithelium. They involve adhesion molecules and regulators of cell cycle and apoptosis. CD44 is a ubiquitously expressed cell adhesion molecule and is a rare example in that it facilitates both cell – cell and cell – matrix interactions (reviewed in Ponta et al., 2003; Rudzki et al., 1997). Its expression is restricted to the proliferative lower two thirds of the normal colonic crypt * Corresponding author. Department of Laboratory Medicine and Pathobiology, St. Michael’s Hospital and University of Toronto, 30 Bond Street, Toronto, Ontario, Canada M5B 1W8. Fax: +1-416-864-5648. E-mail address: [email protected] (S. Jothy). 0014-4800/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2004.03.002
and is absent in the upper third of the crypt where apoptosis occurs. Hyaluronan is the major ligand of CD44. The cytoplasmic domain of CD44 is known to interact with ERM proteins: ezrin, radixin, moesin, and merlin. A large number of CD44 isoforms are generated by alternative splicing, some of which have been implicated in tumor growth and metastases (Gunthert et al., 1991; Hofmann et al., 1991). One of the key features of tumorigenesis is the aberrant homeostasis between cell cycle and apoptosis, which is otherwise governed by cell cycle and apoptosis regulators and inhibitors. The key cell cycle regulators are a highly conserved family of cyclin-dependent kinases (cdk) whose activation requires binding to cyclins. In turn, cdks are negatively controlled by inhibitory proteins which include the CIP/KIP family like p21. The retinoblastoma protein is a cell cycle regulatory protein that engages transcription factors and blocks cell cycle (Garriga et al., 1998; Weinberg, 1995). On the other hand, apoptosis regulators, predominantly Bcl-2 family and executors, Caspases along with stimulators and inhibitors of these families, are also
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involved in dictating a cell’s fate to survive or to die. Several studies have reported cross talks among the cell cycle and apoptosis regulators (Chau et al., 2002; Li et al., 2002; Tan and Wang, 1998; Woo et al., 2003). The interaction of CD44 with some of these key regulators favors survival of cancer cells (Bates et al., 2001; Tian et al., 2000). Disruption of cell adhesion to neighboring cells and extracellular matrix can result in apoptosis (Frisch and Francis, 1994; Grossman, 2002; Shanmugathasan and Jothy, 2000). Defective apoptosis may be involved in the accumulation of cells, still attached to their matrix and to adjacent cells in colonic adenomas, which coincides with overexpression of discrete CD44 isoforms (Bedi et al., 1995; Partik et al., 1998; Wielenga et al., 1993). Previously, using a CD44 knockout murine model, we have shown evidence of early signs of apoptosis, involving the mitochondrial pathway in the normal colon (Lakshman et al., 2004). In this study, we focused on the role of CD44 in the apoptosis of human colon cancer cells. A human colon cancer cell line, SW620, which does not express CD44 was stably transfected with standard and variant isoforms of CD44 expressed in colonic tumors. We found that CD44 transfectants were resistant to etoposide-induced apoptosis and this resistance was more pronounced with the v3-10 isoform of CD44. Noticeable alterations in Bcl-xl, Bak and caspases 9 and 3 can attribute the resistance to apoptosis by CD44 transfectants. Downregulation of AKT phosphorylation and pRb were also associated with the differential response to etoposide-induced apoptosis by CD44 transfectants. Overall, we conclude that CD44 promotes resistance to apoptosis in the human colon cancer cells studied.
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St Louis, MO). Hyaluronan from rooster comb and etoposide were from Sigma-Aldrich. Hyaluronic acid adhesion assay Adhesion assay for SW620 cells transfected with vector and CD44 isoforms and HT-29 to hyaluronic acid was performed as described previously (Herrera-Gayol and Jothy, 1999). Briefly, flat bottom 96-well plates were coated with 5 Ag/ml of hyaluronan, or 2% BSA overnight at 4jC. The wells were then coated with 0.2% BSA for immobilization for 2 h at 37jC. 1 105 cells were allowed to adhere for 1 h at 37jC. After rinsing twice with phosphate-buffered saline, pH 7.4, adherent cells were quantitated using a hematocytometer and the percentage of adherent cells of the control (adherent cells to HA-adherent cells to BSA/adherent cell to BSA) was determined from three independent experiments, each in triplicates. Induction of apoptosis Cells, 1 106, were plated in a 5-cm culture dish. Fortyeight hours later, cells were treated with fresh medium, and subjected to 8 Gy of g irradiation from a Cs-137 source for 0, 4, 8, 12, 24, and 48 h, or 10 Ag/ml of etoposide, or its control solvent, DMSO for 0, 24, and 48 h. Drug toxicity assay Cells, 5 103, were plated in flat bottom 96-well plates and 48 h later treated with etoposide, or DMSO. MTT assay (Promega, Madison, WI) was performed according to manufacturer’s protocol to assess the cell viability after etoposide treatment at indicated time points. Experiments were repeated twice and in triplicates.
Materials and methods DNA fragmentation Materials Human colon carcinoma lines, HT-29 and SW620 were maintained in culture using DMEM with 10% fetal bovine serum and Penicillin/Streptomycin. SW620 transfected with CD44 isoforms (S, v3-10 and v8-10 and vector construct (V)) were maintained as described in (Wong et al., 2003). Primary antibodies used were rabbit anti-rat Bcl-xl, mouse anti-p21 and -pRb (BD Biosciences, Mississauga, ON, Canada), rabbit anti-human Bak (Upstate Biotech, Charlottesville, VA), rabbit anti-human recombinant caspases 3 and 9, anti-rabbit cyclin D1 (Neomarker Lab Vision Corp., Fremont, CA), rabbit anti-human cleaved caspase 3 (Cell Signaling Technology, Beverly, MA), rabbit anti-cyclin A, cdk-2 and cdk-4 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-AKT-phosphorylated (473Ser) and total AKT (New England Biolabs, Mississauga, ON, Canada) and mouse monoclonal anti-human h-actin (Sigma-Aldrich,
After the induction of apoptosis as described above, DNA from these cells was isolated using a DNeasy kit (Qiagen, Mississauga, ON, Canada) according to manufacturer’s protocol. Lysates from 0, 24, and 48 h after etoposide treatment were electrophoresed on 0.8% agarose gel containing 0.1% ethidium bromide. Detection of apoptosis using annexin V and propidium iodide Flow cytometry analyses with FITC-labeled annexin V and propidium iodide were performed according to manufacturer’s protocol (BD Pharmingen, Mississauga, ON, Canada) on cells treated with 10 Ag/ml of etoposide for 0, 24, and 48 h, or DMSO. Etoposide-induced apoptosis is represented as % apoptosis of control (i.e., apoptosis with etoposide treatment in DMSO/DMSO 100).
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Immunoblotting Immunoblotting protocol was performed as described (Lakshman et al., 2004). Briefly, lysates were prepared using NP-40 lysis buffer with protease inhibitors and subjected to 12% SDS-PAGE under reducing conditions.
Results Functional validation of CD44 transfection We tested whether CD44 transfected in SW620 was functional using a hyaluronic acid-adhesion assay. Adhesive capacity was measured by counting the number of cells attached to hyaluronan after 1 h. Fig. 1 shows that adhesion to hyaluronan was significantly increased in SW620 cells transfected with the v3-10 (67%, P < 0.05) and v8-10 isoform of CD44 (126%, P < 0.001) as compared to vector control. Cells transfected with the standard isoform(S) of CD44 were also more adhesive than the vector control (72%, P = 0.1). HT-29, a human colon cancer line that overexpresses CD44 isoforms, also showed an increased adhesive capacity and served as a positive control for the assay. Thus, adhesion to hyaluronan, a major ligand of CD44, is dramatically increased when CD44 is expressed in SW620 indicating that CD44 is functional in the transfectants. CD44 expression promotes resistance to apoptosis We investigated the role of CD44 on apoptosis, induced either by gamma irradiation or by a chemotherapeutic drug, etoposide (Fujita et al., 2002). Induction of apoptosis by gamma irradiation was detected as shown by DNA fragmentation and caspase 3 activation (Figs. 2A and B), which are markers of apoptotic events. We observed the onset of caspase 3 activation and marked DNA fragmentation in all cell lines at 48 h. Surprisingly, caspase 9 was already activated at 0 h and remained activated up to 48 h following
Fig. 2. Gamma irradiation induced apoptosis in SW620 vector control and CD44 transfectants. Induction of apoptosis by g irradiation (8 Gy) was assessed by DNA fragmentation (A) and by Western blot analyses (B) at indicated time points. Apoptosis caused by irradiation was detected only at 48 h in all cell lines, as shown by DNA fragmentation. Caspase 9 remained active in all cell lines from 0 to 48 h post-irradiation. Strong activation of caspase 3 is observed in all cell lines at 48 h with weak activation in HT-29.
Fig. 1. CD44 transfected in SW620 human colon carcinoma cells is functional. Cells, 1 106, were plated on 2% BSA or hyaluronan (HA) at 5 Ag/ml and allowed to adhere for 1 h. Results are shown as average % adhesion of control (BSA). Adhesive capacity is increased in all CD44 transfectants as compared to the vector control. * indicated that the adhesion in v3-10 and v8-10 transfectants was significant. By Student’s t test, the p values were P < 0.05 for v3-10 and P < 0.001 for v8-10.
gamma irradiation (Fig. 2B). In parallel experiments, we administered a chemotherapeutic drug, etoposide, to induce apoptosis. The toxicity of this drug was assessed by MTT assay for cell viability (Fig. 3A). Viable cell population remained unaltered when treated with etoposide as compared to the control (DMSO) up to 24 h ( P > 0.1). Only
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SW620 cells transfected with variant CD44 isoforms were less apoptotic than control cells and cells expressing the standard CD44 isoform. HT-29 cells displayed a DNA fragmentation pattern comparable to variant CD44 transfectants, as expected (Fig. 4B). Cumulatively, these results indicate that resistance to apoptosis induced by etoposide is promoted by CD44 expression in SW620 after 24 h and this resistance is greatly sustained by the full variant isoform, v3-10. Resistance to apoptosis in SW620 transfected with CD44 involves the mitochondrial pathway To determine the mechanism by which CD44 promoted resistance to apoptosis, we analyzed the expression of regulators and executors of apoptosis by immunoblotting. The expression of CD44 in the transfectants remained unaltered during etoposide-induced apoptosis (Fig. 5A). Before etoposide treatment, expression of anti-apoptotic Bcl-xl and pro-apoptotic Bak remained comparable in vector and variant isoform transfectants (Fig. 5B). At 24 h, the vector control showed undetectable levels of Bak, accompanied by reduced Bcl-xl expression (Fig. 5B). CD44 transfectants, on the other hand, maintained a higher Bcl-xl/Bak ratio, at 24 h, indicating a shift towards survival. At 48 h, decreased expression level of Bcl-xl reverted similar to 0 h. Apoptosis induction did not alter the level of cytochrome c in vector control but its level was reduced in v3-10 among the transfectants at 24 h (Fig. 5C). Activation of caspase 9, an effector of cytochrome c, was lost in vector control at 24 h, though it remained unaltered at 0 and 48 h in all cell lines. CD44 transfectants showed activation of caspase 3 at 24 and 48 h, which was less marked in the cells transfected with CD44 v3-10, resembling therefore the pattern observed in HT29 cells. Hence, alterations in key apoptosis regulators/effectors indicate that the mitochondrial pathway is involved in rendering the CD44 transfectants resistant to etoposide induced apoptosis at 24 h. Fig. 3. Induction of apoptosis by etoposide is not toxic to cells. About 10 Ag/ml of etoposide was administered for 0 (A), 24 (B), and 48 h (C). DMSO served as control. Cell viability was measured by MTT assay. (A and B) Cell viability remained unaltered between etoposide- and DMSOtreated groups at 0 and 24 h, respectively. (C) Cell viability decreased significantly in standard isoform transfectants (P < 0.001) after 48 h.
cells transfected with the standard CD44 isoform had a significant reduction in viable cell population ( P < 0.001) at 48 h. In addition to DNA fragmentation, quantitation of apoptosis was performed by flow cytometry using annexin V as a marker (Fig. 4). At 24 h, all CD44 transfectants showed a marked decrease in apoptosis as compared to vector control. Interestingly, resistance to apoptosis was highest in cells transfected with CD44 v3-10 as compared to the standard (S) and v8-10 isoform. This resistance to apoptosis was also shared by HT-29, which showed that
Role of cell cycle and survival proteins in CD44-mediated anti-apoptosis The role of regulators of cell cycle and cell survival in relation to the anti-apoptotic function of CD44 was analyzed by immunoblotting. We studied the expression levels of prosurvival AKT/PKB, cell cycle inhibitors p21 and pRb, cell cycle regulators cyclins A and D, and their respective inhibitors, cdk-2 and 4. Before etoposide treatment, expression levels of total AKT and its phosphorylated form, pRb, cyclins, and cdk-4 was comparable in vector control and CD44 transfectants (Figs. 6A and B). Also, p21 levels were undetectable in vector control and CD44 transfectants. As compared to SW620 cells, elevated levels of phosphorylated AKT, p21, and pRb were observed in HT-29 at 0 h. After 24 h of etoposide treatment, AKT phosphorylation and pRb were markedly increased in vector control as compared to
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Fig. 4. CD44 promotes resistance to apoptosis induced by etoposide. Etoposide induced apoptosis was detected by flow cytometry with annexin V (A) and by DNA fragmentation analysis (B). In A, % apoptosis of the control (DMSO) was calculated as described in Materials and methods. After 24 h, shown in black bars, vector control showed the highest % of apoptosis (67%). Resistance to apoptosis was marked in CD44 transfectants and v3-10 had pronounced resistance (14%) among the CD44 transfectants. DNA fragmentation was clearly visible at 48 h consistent with late events of apoptosis and absent in 24 h. HT-29 and v3-10 showed the least fragmentation after 48 h.
Fig. 5. Bcl-2 and caspase protein members are involved in the resistance to etoposide-induced apoptosis in CD44 transfectants. (A) CD44 expression remains unaltered by etoposide at 24 and 48 h as shown by immunoblotting. HT-29 shows expression of both standard and variant CD44 isoforms. (B) Twenty-four hours after irradiation, Bcl-xl expression was markedly downregulated in vector control cells accompanied by undetectable expression of Bak. (C) Cytochrome c levels were higher in CD44 v3-10 and CD44 v8-10 than CD44s and control cells at time 0. At 24 h, cytochrome c level is reduced in CD44 v3-10 cells, compared to cells expressing CD44s and v8-10. At 24 h, caspase 9 was inactivated in vector control. Onset of caspase 3 activation was observed at 24 h. Low levels of caspase 3 activation was observed in vector at 24 h. v3-10 showed the least activation of caspase 3 among the CD44 transfectants.
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Fig. 6. Cell cycle regulators/inhibitors stabilize resistance to apoptosis in CD44 transfectants. (A) Total AKT expression remained unaltered in all cell lines before and after treatment up to 48 h. AKT phosphorylation (AKT-P) remained upregulated in cells expressing CD44 v3-10 at 48 h. Cell cycle inhibitor, p21 was not detected before induction of apoptosis and was consistently increased in all SW620 cell lines at 48 h, irrespective of CD44 status. pRb, a transcription agonist was expressed in all cell lines at 0 h but markedly downregulated after 24 h and weakly expressed after 48 h in CD44 transfectants. Cyclin D1 level was decreased in all cell lines except in standard isoform at 24 h. Cdk-4 levels remained unaltered among cell lines. (B) Cyclin A levels remained unaltered among cell lines but was increased after treatment. Cdk-2 levels remained unaltered among cell lines.
CD44 transfectants. However, p21 expression was weak in vector as compared to CD44 transfectants. Twenty-four hours of etoposide treatment resulted in an increase of cyclin A in all cell lines (Fig. 6B), and a decrease in Cyclin D expression (Fig. 6A) is observed in all except standard isoforms. Expression of cdks remained comparable to preetoposide treatment in all cell lines. After 48 h, phosphorylation of AKT decreased in SW620 cells transfected with the standard and v8-10 isoforms of CD44, as compared to other cell lines. In relation to pre-etoposide and 24 h levels, p21 expression was upregulated at 48 h in vector control and CD44 transfectants. However, pRb expression was decreased at 48 h versus previous times in all SW620 lines. No significant alterations in cyclin and cdk levels of expression occurred as a result of etoposide treatment. Hence, expression of CD44 modulates cell cycle regulators pRb and p21, and pro-survival proteins such as AKT. These findings are consistent with the resistance to apoptosis that was observed in CD44 transfectants at 24 h.
Discussion Unregulated homeostasis between cell proliferation and apoptosis is characteristic of tumorigenesis. It is well documented that colonic adenomas are defective in apoptosis (Bedi et al., 1995; Partik et al., 1998). Interestingly,
overexpression of discrete isoforms of CD44 is observed in 80% of colonic adenomas (Wielenga et al., 1993). It is important to note that the standard isoform is ubiquitously expressed and variant isoform, v8-10, is expressed in epithelial cells and activated lymphocytes. Following the discovery that discrete CD44 variant isoforms are determinant in pancreatic tumor metastasis, it was found that expression of discrete isoforms of CD44 such v3, v5, and v6 are implicated in colorectal tumor growth and metastasis (Gunthert et al., 1991; Mulder et al., 1997; Wielenga et al., 1993). It is still not clear how the expression of CD44 variant exons contributes to tumor progression in colon carcinoma. Although cells expressing CD44 have been reported to be resistant to etoposide-induced apoptosis, a major functional role for CD44 and its variant isoforms in apoptosis and colon cancer has not been dealt in depth so far (Fujita et al., 2002). In this study, we looked at the role of CD44 and its variants in apoptosis and cancer, using human colon cancer cells. For this, we used SW620, a human colon cancer cell line that does not express CD44, and stably transfected standard, v3-10 and v8-10 isoforms of CD44 (Wong et al., 2003). We induced apoptosis using g irradiation and etoposide, further compared the resistance to etoposide-induced apoptosis between the vector control and CD44 transfectants, and the mechanisms involved. Based on our previous study using a CD44 knockout mouse model, we looked first at major molecular executors of
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apoptosis of the mitochondrial pathway and cell cycle regulators in cells undergoing apoptosis (Lakshman et al., 2004). Cumulatively, these data indicate that expression of CD44 in human colon carcinoma cells confers resistance to apoptosis, and this involved the mitochondrial pathway in conjunction with cell cycle regulators. The communication between cells and their extracellular matrix is facilitated by adhesion molecules like CD44. Studies with melanoma cell lines have shown that cells expressing CD44 are adherent to hyaluronan, a major ligand of CD44, and are functionally motile and invasive (Birch et al., 1991; Goebeler et al., 1996; Thomas et al., 1992, 1993). Results from our hyaluronan-binding assay showed that SW620 transfected with CD44 standard and variant isoforms were more adhesive than vector control. Therefore, CD44 in the SW620 transfectants used in this study is functional and capable of responding to an adhesive stimulus. The CD44 transfectants showed a delayed onset of apoptosis and in the case of transfectants expressing the v3 to v10 CD44 variant exons, there was a markedly increased resistance to apoptosis. This is consistent with the upregulation of apoptosis molecular markers, such as active caspase 3 which was apparent as early as 24 h. Resistance to apoptotic stimuli observed with the CD44 v3-10 was similar to that observed with the HT-29 cells, which express a high level of variant CD44 isoforms. The results are overall independent of the mode of apoptosis induction as we obtained comparable results with etoposide. Several studies have successfully employed the above concentration of etoposide in colon cancer cells for induction of apoptosis (Fujita et al., 2002; Gunthert et al., 1996). Of note, neither the expression nor adhesion to hyaluronan of CD44 in the various transfectants was affected by etoposide even after 48 h of induction. Previously, we reported that regulation of apoptosis in the normal murine colon operates through the mitochondrial pathway (Lakshman et al., 2004). Hence, in this study, we looked at the some of the major regulators involved in this pathway. We observed that the expression levels of Bak remained steady over the time period of apoptosis induction. However, the other indicator of mitochondrial disturbance, Bcl-xl was being fine tuned in response to etoposide. This resulted in a pro-apoptotic Bcl-xl/Bak ratio in the vector control with complete loss of expression of Bak at 24 h, as opposed to being anti-apoptotic in the CD44 transfectants. Disturbance in mitochondrial integrity resulting in the release of cytochrome c and caspase 9 activation is well documented (Ruemmele et al., 2003; Slee et al., 1999; Zou et al., 1999). In our study using etoposide, where we have so far observed the major changes related to apoptosis occurring at 24 h, the vector control had an appreciable loss in active caspase 9 levels. When SW620 cells were transfected with CD44 v3-10, cytochrome c levels were decreased as compared to CD44s, with accompanying caspase 9 remaining active suggesting that CD44 role in apoptosis in these cancer cells is through the mitochondrial pathway.
Both proliferation and cell death contributes to the normal homeostasis of the cells. To rationalize the differential apoptotic response between CD44 transfectants and CD44 negative control cells, we looked at alterations in cell cycle regulators. Phosphorylated AKT, a pro survival molecule and cell cycle inhibitors, p21, and regulator pRb were upregulated in vector control as compared to CD44 transfectants. In contrast, the cell cycle inhibitor, p21 was weakly expressed in vector control. Previous studies have shown the reduction or absence of p21 expression sensitizes cells to etoposide-induced apoptosis (Tian et al., 2000; Waldman et al., 1996). This may explain the susceptibility of control cells to etoposide-induced apoptosis, in contrast to upregulated levels of p21 in CD44 transfectants at 24 h. Interestingly, even though p21 expression in v3-10 was similar to vector control, the expression of CD44 seems to offer resistance to apoptosis probably by downregulating pRb. The role of CD44 in determining survival/proliferation of colon carcinoma cells has been linked to the involvement of AKT, a substrate of PI3 kinase which was recently reported to be a pivotal player in colonic tumorigenesis (Bates et al., 2001; Sithanandam et al., 2003). In our study, we observed an upregulation in the expression of AKT (Ser 473) phosphorylation in the vector control at 24 h with a steady state remaining in the transfectants. This leads us to speculate that in the absence of appreciable levels of p21 and the loss of protective effect promoted by CD44 against apoptosis, the cells in the vector control compensate apoptotic induction by upregulating AKT-phosphorylation, and thus through a different cell survival pathway. Conversely, in CD44 transfectants, upregulated levels of AKT phosphorylation and downregulation of pRb were indicative of pro-survival which coincides with our earlier observation that by virtue of the cells being transfected with CD44 isoforms, apoptotic regulation channels through the mitochondrial pathway. Overall, the data indicate that expression of CD44 and more importantly, v3-10 isoform, promotes resistance to apoptosis. Key regulators of cell cycle and the mitochondrial pathway were involved in imparting this resistance in SW620 transfected with CD44 isoforms. However, SW620 cells, which lack CD44, are sensitive to apoptosis by an alternate pathway. Of note is the similarity in the marked apoptotic resistance of SW620 colon cancer cells transfected with v3-10 and HT-29, another human colon cancer line overexpressing variant CD44 isoforms. This observation is relevant to the understanding of colonic neoplasia. The mechanistics discussed might lead to looking at CD44 expression as a marker of sensitivity to adjuvant chemotherapy. From this study, we propose that expression of specific variant CD44 isoforms characteristic of colon cancer and most other types of cancer confers a selective advantage to resist apoptosis, therefore promoting cell transformation into a malignant phenotype in conjunction with other antiapoptotic factors.
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