Glutathione S-Transferase P1 Promotes Tumorigenicity in HCT116 Human Colon Cancer Cells

Research Article

Duyen T. Dang,1,3 Fang Chen,2 Manu Kohli,4 Carlo Rago,4 Jordan M. Cummins,4 and Long H. Dang2,3

Divisions of 1Gastroenterology and 2Hematology/Oncology, Department of Internal Medicine, University of Michigan Medical Center; and 3University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan; and 4The Sidney Kimmel Comprehensive Cancer Center at the Johns Hopkins University School of Medicine, Baltimore, Maryland

Abstract all stages of colorectal cancer, from aberrant crypt foci to GSTP1 is a member of the glutathione S-transferase enzyme advanced carcinomas (4, 6, 11, 12). However, the biological superfamily, which catalyzes the conjugation of electrophiles function of GSTP1 overexpression in colorectal neoplasms remains with glutathione in the process of detoxification. GSTP1 is unclear (13, 14). widely overexpressed in colorectal cancer, from aberrant crypt In their enzymatic role, GSTP1 dimers catalyze the conjugation foci to advanced carcinomas. Increased expression of GSTP1 is of the sulfur atom of glutathione to electrophiles, such as reactive associated with multidrug resistance and a worse clinical oxygen species (ROS), xenobiotics, and carcinogens (15). In most prognosis. However, GSTP1-null mice have an increased risk of experimental systems, overexpression of GSTP1 in cancer cells is tumor formation. Thus, the biological function of GSTP1 in associated with increased resistance to anticancer agents (16, 17). colorectal cancer biology remains speculative. In an effort to However, there are contrasting reports that associate GSTP1 gain further insights into the role of GSTP1 in tumorigenesis, expression with protective agents. For example, increased GSTP1 we disrupted the GSTP1 gene in HCT116 human colorectal expression is associated with butyrate induction of differentiation cancer cells using targeted homologous recombination. We and apoptosis in HT29 cells (18, 19). Although these studies have find that loss of GSTP1 resulted in impaired clonogenic shown the effects of GSTP1 on tumor responses to exogenous survival and proliferation. Specifically, under growth-limiting compounds, they have not addressed the direct effect of GSTP1 on conditions, (a) GSTP1 protected HCT116 cells from oxidative the growth and survival of cancers. stress and associated apoptosis and (b) promoted mitogen- GSTP1 monomers are also promiscuous ligandins, binding to activated protein kinase-extracellular signal-regulated kinase nonenzymatic substrates, such as steroids, bile acid, and c-Jun NH2-terminal kinase (JNK; refs. 20–22). In this context of binding kinase/extracellular signal-regulated kinase–mediated G1-S cell cycle progression. In vivo,GSTP1wascriticalfor to the stress signaling protein JNK, there is emerging evidence that engraftment and growth of HCT116 tumor xenografts. These GSTP1 may affect the proliferation of cells. GSTP1-null mouse studies directly show that GSTP1 promotes clonogenic embryonic fibroblasts proliferated faster and had higher JNK survival and proliferation in HCT116 human colon cancer levels than their wild-type counterparts, which would suggest cells. (Cancer Res 2005; 65(20): 9485-94) that GSTP1 inhibits proliferation (23, 24). Forced overexpression of GSTP1 in NIH 3T3 fibroblasts increased JNK and extracellular signal-regulated kinase (ERK) phosphorylation and protected Introduction against hydrogen peroxide (H2O2)–induced cell death, but the Glutathione S-transferases (GST) are phase II detoxification direct effect of GSTP1 on cell proliferation was not reported (25). enzymes that catalyze the conjugation of electrophilic compounds Specific inhibition of GSTP1 by the compound TLK199 enhanced with reduced glutathione. Based on their biochemical properties, myeloproliferation; however, HL60 leukemia cells treated with the cytosolic GSTs are divided into a, A, N, k, j, u, and ~ sub- noncytotoxic doses of TLK199 had no alterations in growth, albeit classes. GST k1 (hereafter called GSTP1) is frequently overex- increased basal activities of ERK and JNK (23). Thus, the direct role pressed in many cancers, including tumors of the brain, breast, of GSTP1 overexpression in cancer cells remains speculative. ovary, esophagus, stomach, pancreas, colon, skin, kidney, lung, bile Studies in mice have shown that GSTP1 has a protective role ducts, and lymphatic and hematopoietic systems (1–6). In contrast, against tumor formation. GSTP1-null mice have increased myelo- loss of GSTP1 expression by promoter hypermethylation is a signa- proliferation and are at increased risk for carcinogen-induced skin ture of prostate cancer (7). As such, there has been considerable papillomas (26). Mice deficient for GSTP1 in a wild-type p53 clinical interest in GSTP1 as a tumor marker and as a therapeutic background developed more lung adenomas (27). Mouse embry- target (8, 9). onic fibroblasts from GSTP1-null mice exhibit increased prolifer- Colorectal cancer is the second leading cause of cancer deaths ation rates compared with their wild-type counterparts and are in the United States. Colorectal cancer develops through multiple protected against acetaminophen-induced apoptosis (23, 24, 26). steps, with the sequential acquisition of genetic alterations in key Thus, the data in mice would suggest that GSTP1 is protective tumor suppressors and oncogenes (10). GSTP1 is overexpressed in against tumors. In this article, we report the consequences of genetic disruption of GSTP1 in the human colon cancer cell line HCT116 by targeted Requests for reprints: Long H. Dang and Duyen T. Dang, Department of Internal Medicine, University of Michigan, MSRB I, Room 6514, 1150 West Medical Center homologous recombination. We find that GSTP1 directly mediates Drive, Ann Arbor, MI 48109-0682. Phone: 734-647-2964; Fax: 734-763-2535; E-mail: clonogenic survival and proliferation under growth-limiting con- [email protected]. I2005 American Association for Cancer Research. ditions. Consistent with our in vitro observations, we find that doi:10.1158/0008-5472.CAN-05-1930 GSTP1 promotes in vivo tumor engraftment and growth. www.aacrjournals.org 9485 Cancer Res 2005; 65: (20). October 15, 2005

Materials and Methods densities. For cell proliferation studies, cells were harvested after 4 and 8 days and stained with trypan blue, and viable cells were counted on a Tissue culture. HCT116, RKO, SW480, LOVO, and HT29 cells were tf hemacytometer. Doubling time was calculated as N = N 2 , where N is the acquired from the American Type Culture Collection (Manassas, VA) and t 0 t number of cells at 8 days, N is the number of cells initially, t is time (days), cultured in recommended medium supplemented with 10% fetal bovine 0 and f is the frequency of cell cycles per unit time (1/day; ref. 40). For serum (FBS) and 1% penicillin/streptomycin (complete medium). For calculations of apoptotic index, cells were harvested at 4 and 8 days and growth-limiting experimental conditions, cells were seeded at densities of stained with Hoescht 33258, and the number of cells with pyknotic nuclei Â 3 Â 3 Â 3 Â 3 2 7.5 10 , 2.0 10 , 1.0 10 , or 0.5 10 per cm and cultured in medium and intact nuclei was counted as described previously (41). Apoptotic index supplemented with 10% or 1% FBS and 1% penicillin/streptomycin (28, 29). was calculated as the number of pyknotic nuclei divided by the total Real-time reverse transcription-PCR analyses. Total RNA extracted number of cells counted per Â100 power field. For clonogenic survival from normal human colon and colon cancer tissues of three different assays, colonies were allowed to grow undisturbed for 2 weeks and stained patients were acquired from Clontech (Palo Alto, CA), and total RNA from with crystal violet. cell lines was extracted. All total RNA was treated with DNase I and single- Flow cytometry. For cell cycle analyses, cells were harvested, permeated stranded cDNA was generated using Moloney murine leukemia virus with 70% ice-cold ethanol, and stained with 50 Ag/mL propidium iodide in reverse transcriptase (Bio-Rad, Hercules, CA). Real-time PCR reactions PBS containing 0.2% Tween 20 and 2.5 Ag/mL RNase. DNA contents of were done in triplicate on reverse transcription–derived cDNA using SYBR 10,000 cells were measured on a FACSCalibur cytometer (Becton Dickinson, Green Supermix (Bio-Rad). Crossing point at which fluorescence increases Franklin Lakes, NJ) and data were analyzed using Modfit Lt software (Verity appreciably above the background fluorescence was determined. Primers Software House, Topsham, ME). V V used were GSTP1 (BC010915) 5-CCTCACCCTGTACCAGTCC-3 (forward) Oxidative stress assays. Cellular release of H O was measured using V V 2 2 and 5-GATGTATTTGCAGCGGAGGT-3 (reverse) and glyceraldehyde-3- the Amplex Red reagent (Molecular Probes, Carlsbad, CA). In the presence V V phosphate dehydrogenase (GAPDH)5-AAAGGGCCCTGACAACTCTT-3 of HRP, the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) (forward) and 5V-GGTGGTCCAGGGGTCTTACT-3V(reverse). PCR products reacts with H2O2 to produce highly fluorescent resorufin, which can be met three criteria to be included in the study: (a) the signal from the measured at 560 nm. Equal numbers of cells were harvested and reverse transcription–derived cDNA was at least 100-fold greater than that incubated with HRP and Amplex red reagent, and absorbance was read of control reactions done without reverse transcription, (b) PCR products every 5 minutes for 1 hour. At each time point, Vmax was calculated, from the reactions with reverse transcription had to be the expected size which represented the rate of H2O2 production. Fold increase in H2O2 on gel electrophoresis, and (c) melt curve analyses were consistent with production was calculated by dividing the Vmax after 30 minutes of specificity of PCR. Relative expression of GSTP1 to GAPDH was calculated incubation by the V after 5 minutes of incubation. The lipid À[crossing point GSTP1 À crossing point GAPDH] max using the formula: ratio = 2 (30). peroxidation product malondialdehyde was measured using the thiobar- Strategy for disruption of the human GSTP1 gene. The endogenous bituric acid–reactive substances (TBARS) assay. Malondialdehyde forms a GSTP1 locus, adeno-associated virus (AAV) knockout construct, and 1:2 adduct with thiobarbituric acid, which can be measured at 532 nm. resulting targeted locus are shown in Fig. 1B. The targeting strategy is as Cells were harvested and protein concentrations were equalized. Whole described previously (31–34). The AAV method of transgene insertion has homogenates were incubated with thiobarbituric acid and centrifuged, been shown to result primarily in single-site insertions by Southern blotting and absorbance of the supernatant was read (Oxitech, Buffalo, NY). (35). The AAV technology has proven useful in the generation of somatic Results were plotted against a standard curve of known malondialdehyde cell knockouts (31–34, 36). Somatic cell knockouts, in turn, have provided concentrations. For analyses of intracellular 8-oxoguanosine, cells were added insights into gene function in cancer (31, 36–39). Exon 2 of GSTP1 harvested, fixed, and stained with the FITC-conjugated antibody against was targeted for disruption with an AAV cassette containing the Neo the 8-oxoguanine moiety of 8-oxoguanosine (Biotrin, Dublin, Ireland). The resistance gene under the constitutive control of a SV40 promoter flanked fluorescence intensity in 10,000 cells was measured on a FACSCalibur by left and right homology arms f1 kb long. Successful disruption resulted cytometer. The percentage of cells with 8-oxoguanosine was calculated as in a 100-bp insertion and translation stop codons in all three reading the difference in fluorescence of 8-oxoguanine-FITC–stained and un- frames. Cells exhibiting neomycin resistance were screened with locus- stained cells. The activities of all types of superoxide dismutases (SOD) specific PCR. Once the first allele was successfully targeted, the Neo were measured using the OxyScan SOD-525 Assay (Oxis Research, resistance gene was excised using Cre recombinase (Microbix Biosystems, Portland, OR). Cells were harvested, disrupted by several freeze-thaw Inc., Toronto, Ontario, Canada). The same targeting vector was then used to cycles, clarified by centrifugation, and assayed for SOD activity. SOD target the second allele. For locus-specific PCR to confirm homologous mediates an increase in the rate of auto-oxidation of 5,6,6a,11b- integration of the targeting vector, genomic DNA was amplified using tetrahydro-3,9,10-trihydroxybenzo[c]fluorene to yield a chromophore, primers specific for exon 2 of GSTP1. Loss of GSTP1 was confirmed by which can be measured at 525 nm. Results were plotted against a Western blot analyses. standard curve of known SOD concentrations. Western blot analyses. Whole-cell extracts were prepared from various Treatment with mitogen-activated protein kinase-extracellular cell lines with radioimmunoprecipitation assay lysis buffer containing signal-regulated kinase kinase inhibitor. Cells were treated with 10 protease inhibitors (Roche, Indianapolis, IN). Proteins (50 Ag) were Amol/L of the MEK inhibitor U0126 (Cell Signaling Technology). U0126 is a separated by electrophoresis, transferred to nitrocellulose membranes, highly selective inhibitor of MEK1 and MEK2 (42). The concentration of 10 probed with primary and horseradish peroxidase (HRP)–coupled secondary Amol/L is consistent with manufacturer recommendations as well as with antibodies, and visualized by chemiluminescence reagent (Perkin-Elmer, previous studies in HCT116 cells (43). Cells were observed during Norton, OH). At the end of the experiment, membranes were stripped and incubation and harvested after 4 days for cell cycle analyses. reprobed for a-tubulin to confirm equal loading. Antibodies were obtained In vivo tumorigenesis. GSTP1+/+ and GSTP1À/À cells were grown in from Oxford Biomedical Research, Inc. (Oxford, MI; rabbit anti-human complete medium and harvested for in vivo studies as described GSTP1), Cell Signaling Technology [Beverly, MA; cleaved caspase-7, cleaved previously (44, 45). Either 7.5 Â 106 or 1.0 Â 106 cells were implanted s.c. poly(ADP-ribose) polymerase (PARP), caspase-7, PARP, phospho–stress- into the flanks of 6-week-old female athymic nu/nu mice (Charles River activated protein kinase (SAPK)/JNK (Thr183/Tyr185), SAPK/JNK, phospho– Labs, Wilmington, MA). Tumor sizes in two dimensions were measured mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) 1/2 (Ser221), with calipers, and volumes were calculated with the formula: (L Â W2) phospho–ERK p42/p44 (Thr202/Tyr204), MEK1/2, ERK p42/p44, anti-rabbit Â 0.5, where L is length and W is width. Mice were euthanized once HRP, and anti-mouse HRP], and Sigma (St. Louis, MO; a-tubulin). Antibody overwhelmed by tumor burden as defined by animal care guidelines. dilutions were as recommended by the manufacturer. Mice were housed in barrier environments, with food and water provided Measurement of cell proliferation, apoptotic index, and clonogenic ad libitum as approved by the University of Michigan Animal Care and survival. Cells were trypsinized, counted, and plated at various seeding Use Committee.

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Statistics. In vitro experiments were done in triplicate and repeated primary colon cancer tissues, and colorectal cancer cell lines by twice. In vivo experiments were done with n = 10 in each group and real-time reverse transcription-PCR (RT-PCR) analyses (Fig. 1A). F repeated once. Results are expressed as averages SD of all experiments. Consistent with the literature, GSTP1 is highly expressed in colon Statistical analyses of data were done using Student’s paired t test and Ps< cancer. 0.05 were considered significant. Targeted disruption of GSTP1 in HCT116 cells. Somatic cell knockout technology was used to generate clones of HCT116 Results human colon cancer cells without GSTP1. We chose HCT116 cells GSTP1 is highly expressed in colorectal cancer. We examined because they express GSTP1, are near diploid, and have been the expression of GSTP1 in normal human colonic epithelia, shown to be stable in culture (31–34). We targeted exon 2 of GSTP1

Figure 1. Expression and disruption of GSTP1 in colon cancer. A, expression of GSTP1 relative to GAPDH by real-time RT-PCR in normal colon, colon cancer, and colon cancer cell lines. n = 9 for each column. *, P < 0.01, Student’s paired t test comparing colon cancer tissues or cell lines to normal colon tissues. B, disruption of GSTP1. The endogenous GSTP1 locus, AAV knockout construct, and resulting targeted locus are shown. Numbered boxes, exons; triangles, loxP sites. ITR, inverted terminal repeats; HA, homology arm; P, SV40 promoter; Neo, neomycin resistance gene; pA, polyA tail. C, confirmation of disruption of GSTP1 by locus-specific PCR. Primers P1 and P2, which amplify exon 2, are shown on the endogenous and targeted GSTP1 locus diagrams. Lane 2, the endogenous locus contains a f200-bp fragment; lanes 3 and 4 after successful targeting of the first allele and treatment with Cre recombinase, the amplification product is a larger, f300-bp fragment. With successful targeting of the second allele, the amplification product is a f1,650-bp fragment encompassing the Neo gene. D, confirmation of loss of GSTP1 by Western blot analysis with rabbit anti-human GSTP1 and mouse anti-human a-tubulin (for loading). www.aacrjournals.org 9487 Cancer Res 2005; 65: (20). October 15, 2005

Â Table 1. Cell doubling time in GSTP1+/+ and GSTP1À/À clones significantly increased to 39 to 41 hours when seeded at 2 3 2 z 3 2 cells 10 cells/cm and to 121 hours when seeded at 1 Â 10 cells/cm . Moreover, at the lowest seeding density of 0.5 Â 103 cells/cm2, Cell line Seeding density Doubling GSTP1À/À cells never grew to double in number. GSTP1+/À (cells/cm2), Â103 time (h) heterozygote cells behaved similarly to GSTP1+/+ cells (data not shown). Our findings are best illustrated with clonogenic survival GSTP1+/+ 7.5 29 F 4 assays, which again show the significant decreases in colony 2.0 24 F 2 formation in GSTP1À/À cells compared with GSTP1+/+ cells 1.0 28 F 8 (Fig. 2A). Strikingly, at the two lowest seeding densities, GSTP1À/À 0.5 40 F 3 colonies did not form. F GSTP1À/À clone 1 7.5 31 5 Morphologically, GSTP1À/À cells in growth-limiting conditions F 2.0 39 8* seemed to undergo marked cell death, with sporadic live cells that 1.0 121 F 20* did not form colonies. To confirm morphologic apoptosis in the 0.5 >196* GSTP1À/À clone 2 7.5 34 F 4 cells, we stained for pyknotic nuclei and calculated apoptotic 2.0 41 F 7* indexes. We found no significant differences in apoptotic index in 3 2 1.0 >196* cells seeded at densities of 7.5 Â 10 cells/cm (Fig. 2B). However, 3 2 0.5 >196* starting with a lower seeding density of 2.0 Â 10 cells/cm , there was a 2-fold increase in the apoptotic index in GSTP1À/À cells compared with GSTP1+/+ cells, particularly after 8 days in culture. 3 3 3 3 NOTE: Cells were plated at 7.5 Â 10 , 2.0 Â 10 ,1Â 10 , or 0.5 Â 10 Â 3 2 2 Furthermore, at the lowest seeding density of 0.5 10 cells/cm , per cm and counted after 4 and 8 days in culture at 1% FBS, and cell there was an up to 10-fold increase in the apoptotic index in doubling time was calculated. n = 9 for each value. GSTP1À/À cells compared with GSTP1+/+ cells. *P < 0.01, Student’s paired t test comparing GSTP1À/À to GSTP1+/+ cells in the same conditions. Together, our findings show that GSTP1 is required for cell survival and proliferation in growth-limiting conditions. Notably, the differences in survival and growth at low-density seeding were not evident when the cells were cultured in 10% serum (data for disruption as outlined in Fig. 1B. Homozygous disruption of not shown). Furthermore, when the cells were cultured at high GSTP1 was identified by genomic PCR (Fig. 1C). Loss of GSTP1 densities, there were no differences in their proliferation in low- protein in the knockouts was confirmed by Western blot analysis serum conditions (data not shown). Thus, the protective effects of using GSTP1 antibody (Fig. 1D). The resulting cells (hereafter called GSTP1 may be replaced by mitogens and nutrients in serum or GSTP1À/À cells) are similar to their parental HCT116 (GSTP1+/+) alternatively by autocrine and/or paracrine factors in high-density cells, with the exception of ablated GSTP1 expression. To control seeding (53, 54). for clonal variation with passage and random vector integration, Effect of GSTP1 on apoptosis and G1-S progression. To begin parental GSTP1+/+ cells were passed in parallel with knockout to understand the mechanisms by which GSTP1-mediated cell cells, and one independent clone of GSTP1+/À heterozygous cells survival and growth, we analyzed cell cycle variables. As our studies as well as two independent clones of GSTP1À/À cells were selected thus far have noted GSTP1-dependent differences in conditions of for further analyses. The two GSTP1À/À clones were isolated from both low seeding density and low serum, we attempted to different plates and were absolutely distinct. The same clones were distinguish the contribution from these two culture conditions. used for all ensuing studies. On the one hand, serum deprivation induces growth arrest (55, 56). GSTP1 is required for growth in growth-limiting conditions. On the other hand, low seeding density increases apoptosis and The GSTP1+/+, GSTP1+/À, and GSTP1À/À cells were found to be decreases cell colonization (47, 57). To assess the effects of low indistinguishable with respect to morphology and growth rates serum alone, we examined cells at a seeding density of 7.5 Â 103 under standard culture conditions (data not shown). To rigorously cells/cm2 in 10% or 1% serum supplementation. To assess the compare growth and survival, we examined the cells under combined effects of both low serum and low seeding density, we low-density seeding and low-serum supplementation conditions. compared cells grown in 1% serum at seeding densities of 7.5 Â 103 Low-density seeding and low-serum supplementation are growth- or 2.0 Â 103 cells/cm2. limiting conditions and thus stringently reflect the clonogenic and We found that in both low serum alone and combined with low proliferative potential of cells (28, 29, 46–50). Certainly, one of the seeding density, GSTP1+/+ cells exhibited small increases in hallmarks of cancer is their continued growth in growth-limiting apoptosis, with the sub-G1 population increasing from 2% to 5% conditions, aptly termed oncogenic resistance to growth-limiting to 7% of the total population of cells (Fig. 2C). Interestingly, serum conditions (51). deprivation did not induce G1-S arrest in GSTP1+/+ cells, with the To quantitate cell proliferation under growth-limiting condi- G1-S ratio remaining equivalent in both low serum alone and tions, cells were seeded at various densities in low serum and combined with low seeding density. These findings again show that counted, and doubling time was calculated (Table 1). Basal GSTP1+/+ cells have escaped normal growth control mechanisms. doubling time for GSTP1+/+ cells was f29 hours, which was Our findings in GSTP1À/À cells were more dramatic. When consistent with previous reports (52). Basal doubling time for exposed to low serum, GSTP1À/À cells underwent G1-S arrest as f GSTP1À/À cells was 31 to 34 hours, which was not significantly evidenced by at least 2-fold increases in the G1-S ratio (0.8-2.3 in different from their GSTP1+/+ counterparts. When seeding density clone 1 and 1.1-2.5 in clone 2; Fig. 2C). In addition, the percentage 3 3 3 2 f f was decreased from 7.5 Â 10 to 2 Â 10 or 1 Â 10 cells/cm , the of cells in the sub-G1 phase increased from 4% to 12%, doubling time in GSTP1+/+ cells remained roughly the same at 24 indicating a significant amount of apoptosis. Furthermore, when to 28 hours. In contrast, the doubling time in both GSTP1À/À cultured in low serum combined with low seeding density, the

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Figure 2. Clonogenic survival, apoptosis, and cell cycle analyses in GSTP1+/+ and GSTP1À/À cells. A, clonogenic survival assays. Cells were seeded at the noted densities in 1% serum, incubated for 2 weeks, and stained with crystal violet. B, apoptotic index as calculated by the number of pyknotic nuclei divided by the total number of cells. Cells were seeded at the noted densities in 1% serum and examined by Hoescht 33258 staining after 4 and 8 days in culture. n =30 for each column. *, P < 0.01, Student’s paired t test comparing GSTP1À/À to GSTP1+/+ cells in the same conditions. C, cell cycle analyses. Cells were seeded and cultured at the noted densities and serum supplementation for 4 days, harvested, and analyzed for DNA content. Columns, average percentage of cells in G1,S,G2-M, or sub-G1 phases. Numbers above the brackets, G1-S ratio (average percent of cells in G1 divided by average percent of cells in S phase). n =3.*,P < 0.01, Student’s paired t test comparing GSTP1À/À with GSTP1+/+ cells in the same conditions. www.aacrjournals.org 9489 Cancer Res 2005; 65: (20). October 15, 2005

percentage of GSTP1À/À cells in the sub-G1 population increased GSTP1+/+ cells (Fig. 3E). Altogether, these experiments show that another 2-fold to f23% in both clones. Altogether, our findings GSTP1 is critical for cytoprotection against oxidative stress and show that GSTP1 protects from cell cycle arrest under serum associated apoptosis under low-serum and low-density seeding deprivation and apoptosis under low seeding density conditions. To conditions. our knowledge, these are the first data to link GSTP1 to cancer GSTP1 is required for mitogen-activated protein kinase- cells’ resistance to growth-limiting conditions and again point to extracellular signal-regulated kinase kinase/extracellular the critical role of GSTP1 in the clonogenic survival and signal-regulated kinase signaling in growth-limiting condi- proliferation of HCT116 cancer cells. tions. Although our studies have shown that GSTP1 is critical for Effect of GSTP1 on cellular oxidative stress. As GSTP1 is protection against oxidative stress and associated apoptosis under important for maintaining cellular redox status (58–60), and low-serum and low-density seeding, it remains unclear how GSTP1 increased cellular oxidative stress has been associated with mediates continued cell cycle progression under low-serum apoptosis under low seeding densities (53, 57), we examined the conditions. We next examined JNK and ERK phosphorylation effect of GSTP1 on cellular oxidative stress under growth-limiting in GSTP1+/+ and GSTP1À/À cells. The GSTP1 monomer is a conditions. We used four independent assays as indicators of direct endogenous inhibitor of the stress-activated kinase JNK cellular oxidative stress: cellular release of H2O2, production of the in nonstressed fibroblasts (22). Furthermore, forced expression lipid peroxidation end-product malondialdehyde, the presence of of GSTP1 has been associated with altered regulation of ERK the oxidized DNA product 8-oxoguanosine, and total SOD activity. kinase (25). H2O2 is a ROS generated from the breakdown of superoxide Under the least stressful culture conditions, phospho–SAPK/JNK anions (61). Malondialdehyde is an aldehyde by-product of lipid was increased in GSTP1À/À cells compared with GSTP1+/+ cells peroxidation, a major mechanism of ROS-mediated cellular injury (Fig. 4A, column 1). These data support previous findings that (61). ROS can also generate nucleic acids adducts, for example, GSTP1 inhibits JNK phosphorylation in nonstressed cells (22). the hydroxylation of 2V-deoxyguanosine to form 8-oxoguanosine However, under the more stressful growth-limiting conditions, (61). 8-Oxoguanosine is biologically significant, as it can induce phospho–SAPK/JNK was equivalent in GSTP1À/À cells compared G:C to T:A transversions in DNA replication. Thus, lipid with GSTP1+/+ cells (Fig. 4A, columns 2 and 3). These data support peroxidation and DNA adduct products reflect oxidative stress previous findings that cellular stress induces GSTP1 dimerization and damage to the cell. SODs are metalloenzymes that catalyze and abolishes the inhibitory interaction between GSTP1 monomers the dismutation of superoxide anions into oxygen and H2O2 and and JNK (22). Altogether, our data are consistent with the thus are an indicator of cellular oxidative stress and antioxidant published concepts that (a) GSTP1 inhibition of JNK phosphory- capacity (62). lation occurs primarily in nonstressed cells and (b) phosphoryla- We found that serum deprivation alone did not alter oxidative tion of JNK becomes largely independent of GSTP1 expression in stress in both cell lines. Cellular production of H2O2 in both stressed cells (22, 63). Thus, in HCT116 cells, altered JNK cell lines was not altered with serum deprivation (Fig. 3A). To phosphorylation is unlikely a mechanism by which GSTP1 determine cellular damage from oxidative stress, we measured mediates continued cell cycle progression under growth-limiting levels of the lipid peroxidation product malondialdehyde. We found conditions. comparable basal levels of malondialdehyde in GSTP1À/À and In contrast, under growth-limiting conditions, GSTP1À/À cells GSTP1+/+ cells, with no significant changes after treatment with exhibited significant decreases in phospho-ERK compared with serum deprivation (Fig. 3B). In the same manner, there were no GSTP1+/+ cells (Fig. 4B). ERK is a component of the MAPK pathway significant changes in 8-oxoguanosine after treatment with serum that controls the growth and survival of tumors (64). In fact, signals deprivation (Fig. 3C). Interestingly, basal SOD activity was up to from a multitude of growth factors, cytokines, and proto-oncogenes 40 units/mg higher in GSTP1À/À compared with GSTP1+/+ cells converge on the G protein RAS, which activates the serine/threonine (Fig. 3D). With serum deprivation, both cell lines had similar kinase RAF, which activates the MAPK kinase MEK, which in turn increases in SOD activity (by f5 units/mg protein in both cell activates ERK. The MEK-ERK cascade is critical in tumor growth and lines). These findings would suggest that basal SOD antioxidant progression (65). As ERK is phosphorylated by MEK, we next activity is up-regulated in GSTP1À/À cells, but the response to examined phosphorylation of MEK1 and MEK2 in our model system. serum starvation is quantitatively similar in GSTP1À/À compared We found significantly decreased phospho-MEK1/2 in GSTP1À/À with GSTP1+/+ cell lines. Altogether, our results would suggest that cells compared with GSTP1+/+ cells (Fig. 4B). Together, these data loss of GSTP1 did not significantly alter cellular oxidative stress suggest that GSTP1 is important for MEK-ERK survival signaling under serum deprivation. As such, although cleavage of the apo- under growth-limiting conditions. Although it is not surprising that ptosis mediators PARP and caspase-7 were increased with serum activation of the MEK-ERK cascade is important for tumorigenesis, deprivation, the increases were similar between GSTP1+/+ and the association of GSTP1 to MEK-ERK phosphorylation and GSTP1À/À cells (Fig. 3E). The finding of increased basal levels clonogenic survival is novel. of SOD in GSTP1À/À cells suggests a potential compensatory To determine whether GSTP1-dependent augmentation of MEK mechanism when GSTP1 is ablated. phosphorylation was functionally contributing to the growth and In contrast, loss of GSTP1 resulted in marked increases in survival of HCT116 cancer cells, we treated GSTP1+/+ and oxidative stress when the cells were cultured at low seeding density GSTP1À/À cells with the MEK inhibitor U0126. We reasoned that combined with low-serum conditions. The combined treatments if the decreases in MEK-ERK phosphorylation in GSTP1À/À cells induced significant increases in H2O2 production, malondialde- functionally impaired their growth in growth-limiting conditions, hyde, 8-oxoguanosine, and SOD activity in GSTP1À/À cells then (a) treatment of parental GSTP1+/+ cells with MEK inhibitors compared with GSTP1+/+ cells (Fig. 3A-D, last columns). This was would render them more sensitive to G1-S arrest and apoptosis associated with marked cleavage of the apoptosis mediators under growth-limiting conditions and (b) GSTP1À/À cells caspase-7 and PARP in GSTP1À/À cells compared with would be more sensitive to the effects of MEK inhibition. Indeed,

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Figure 3. Oxidative stress analyses in GSTP1+/+ and GSTP1À/À cells. Cells were seeded and harvested after 4 days in culture for respective assays. A, Amplex red assay to determine the cellular release of H2O2. Columns, average fold increase in H2O2 production rate: V max at 30 minutes divided by V max at 5 minutes. B, TBARS assay to show malondialdehyde lipid peroxidation products. Columns, average amount of TBARS detected (nmol/mg). C, 8-oxoguanine-FITC binding protein fluorescence to show the presence of 8-oxoguanosine DNA adducts. Columns, average percent of 8-oxoguanine-FITC–stained cells that were fluorescent minus the percent of unstained cells that were fluorescent. D, SOD activity in cells. Columns, average SOD activity (units/mg protein). E, Western blots for cleaved PARP and cleaved caspase-7. Cell type, serum supplementation, and density seeding are as labeled. For 3A-3D, n = 9 for each column, with the exception of (C), where n = 3 for each columns. *, P < 0.01, Student’s paired t test comparing GSTP1À/À to GSTP1+/+ cells in the same conditions.

f GSTP1+/+ cells treated with U0126 underwent significant G1-S in sub-G1 dramatically increased to 10% when treated with arrest under serum deprivation, with a 3-fold increase in the ratio U0126 and serum deprivation. Thus, MEK signaling is important of cells in the G1 phase compared with the S phase (Fig. 4C). Under for the survival of cells under serum deprivation. However, this the same conditions, GSTP1À/À cells were significantly more aspect of MEK signaling did not seem to be dependent on GSTP1, sensitive to the effects of U0126, developing an 8-fold increase in as GSTP1À/À cells were no more sensitive to apoptosis than the ratio of cells in the G1 phase compared with S phase. Thus, GSTP1+/+ cells. We were not able to address the effects of U0126 in GSTP1-dependent MEK activation contributes to oncogenic G1-S conditions of low serum combined with low seeding density, as the progression in growth-limiting conditions. cells were not viable under these conditions (data not shown). Treatment with U0126 also induced significant apoptosis Altogether, these data point to a role for GSTP1 in mediating MEK/ (Fig. 4D). In both cell lines, the percentage of the cell population ERK–dependent cell cycle progression. www.aacrjournals.org 9491 Cancer Res 2005; 65: (20). October 15, 2005

GSTP1 promotes in vivo tumorigenesis. Thus far, our in vitro reasoned that the presence or absence of GSTP1 would be critical data have shown that, under growth-limiting conditions, GSTP1 in low seeding conditions. We found that all of the GSTP1+/+ cells promotes continued HCT116 oncogenic cell survival and growth by engrafted and grew as xenografts over 35 days (Fig. 5B). In contrast, (a) protecting cells from oxidative stress and associated apoptosis most the GSTP1À/À cells did not engraft. Specifically, 100% of and (b) promoting MEK/ERK mitogenic signaling for continued G1- GSTP1À/À clone 1 and 60% of GSTP1À/À clone 2 did not engraft S progression. These cell culture–based findings prompted us to test after 77 days. These results are consistent with our in vitro data and the effects of GSTP1 in vivo. show that GSTP1 is critical for HCT116 tumor engraftment and We proceeded to implant the GSTP1+/+ cells and the two early growth. GSTP1À/À clones used in the preceding studies into the flanks of athymic nude mice. When high numbers of tumor cells (7.5 Â 106) were implanted, all of the tumors engrafted. However, loss of GSTP1 Discussion significantly hindered xenograft growth (Fig. 5A). After 21 days, Our results directly show that GSTP1 is critical for the GSTP1+/+ xenografts attained volumes that were f3-fold that of clonogenic survival and proliferation of HCT116 human colon both clones of GSTP1À/À xenografts. Interestingly, loss of GSTP1 cancer cells. Specifically, GSTP1 promotes HCT116 tumor engraft- seems to preferentially affect the lag phase rather than the log phase ment and growth by (a) protecting against oxidative stress and of tumor growth. These in vivo data are reminiscent of our in vitro associated apoptosis and (b) augmenting MEK/ERK mitogenic findings, in which no differences were noted between GSTP1À/À signaling in growth-limiting conditions. Thus, GSTP1 mediates and GSTP1+/+ cells at higher seeding density. Thus, the presence of oncogenic survival and growth under physiologically stressful GSTP1 may be particularly critical in early HCT116 xenograft conditions. growth. Surprisingly, we found that GSTP1 was not critical for HCT116 We next implanted low numbers (1 Â 106) of tumor cells into the cancer cell survival and growth in standard culture conditions. flanks of athymic nude mice. Based on our in vitro data, we Furthermore, in mice xenograft studies, once a tumor mass had

Figure 4. JNK and MEK/ERK phosphorylation in GSTP1+/+ and GSTP1À/À cells. A, Western blots for phospho–SAPK/JNK and total SAPK/JNK. Cells were seeded at noted densities and serum supplementation and harvested after 4 days in culture, and Western blot analyses were done on whole-cell extracts. B, Western blots for phospho-ERK, phospho-MEK1/2, total ERK, and total MEK1/2. Cells were seeded at noted densities and serum supplementation and harvested after 4 days in culture, and Western blot analyses were done on whole-cell extracts. C, G1-S arrest after treatment with the MEK inhibitor U0126. Cells were seeded at 7.5 Â 103 cells/cm2, treated with 10% or 1% serum with or without 10 Amol/L U0126 for 4 days, and harvested. Cell cycle analyses were done, G1-S ratios were calculated, and averages were graphed. D, apoptosis after treatment with U0126. Cells were seeded in triplicate at 7.5 Â 103 cells/cm2, treated with 10% or 1% serum with or without 10 Amol/L U0126 for 4 days, and harvested. Cell cycle analyses were done and the average percentage of cells in sub-G1 phase is depicted. n = 3 for each column. *, P < 0.01, Student’s paired t test comparing values from U0126 treatment to no U0126 treatment.

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Figure 5. In vivo tumorigenesis in GSTP1+/+ and GSTP1À/À xenografts. A, xenograft growth after implantation of 7.5 Â 106 cells s.c. B, xenograft growth after implantation of 1.0 Â 106 cells s.c. After 35 days, the remaining graph line represents 100% of GSTP1À/À clone 1 and 60% of GSTP1À/À clone 2 that did not engraft. n = 20 for each symbol. *, P < 0.01, paired Student’s t test comparing GSTP1+/+ to both clones of GSTP1À/À xenografts at the same time point.

formed, the presence or absence of GSTP1 did not significantly Certainly, further investigations are warranted and ongoing. As we alter its growth curve. We can speculate that GSTP1 overexpression were seeking to determine the effects of GSTP1 on tumorigenicity, we is sustained in a HCT116 tumor mass because it continues to have yet to determine the effects of GSTP1 on drug response and support proliferation and detoxification; however, these functions metastasis in this experimental model. Furthermore, this model could in a larger tumor mass may be redundant. be used to characterize the molecular interactions of GSTP1 with Clonal cell survival and expansion are reminiscent of small, signaling proteins and for drug discovery efforts. Nevertheless, these indolent growths in the clinical setting, such as those found are the first data to conclusively show that GSTP1 promotes clonogenic in precancerous lesions, remain postoperatively, become drug survival and proliferation in HCT116 human colon cancer cells. resistant, or metastasize to a new site (66). It is notable that GSTP1 overexpression has been associated with these stages in Acknowledgments colorectal cancer (6, 12, 67). Our data, integrated with the Received 6/2/2005; revised 7/21/2005; accepted 8/3/2005. literature, would suggest that GSTP1 may contribute to clono- Grant support: University of Michigan Comprehensive Cancer Center Pilot Grant genic survival in these ‘‘low-tumor burden’’ stages. It is notable (L.H. Dang), NIH grant K08DK59970, American Gastroenterological Association that GSTP1 inhibitors, such as ethacrynic acid and TLK199, have Research Scholar Award, Glaxo Institute for Digestive Health Basic Research Award, and Michigan Gastrointestinal Peptide Research Center Pilot Grant (D.T. Dang). been shown to modulate tumor drug resistance (16). Based on The costs of publication of this article were defrayed in part by the payment of page our findings, we may postulate that an additional setting for a charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. GSTP1 inhibitor would be low-tumor burden settings. These We thank Bert Vogelstein and Bill Nelson for helpful suggestions on the article, approaches would have to be extensively explored first, as Stephanie Knock for assistance with mice tumor measurements, the University of hypermethylation of the GSTP1 promoter and loss of GSTP1 Michigan Comprehensive Cancer Center Flow Cytometry Core for assistance with fluorescence-activated cell sorting analyses, and the University of Michigan Juvenile expression have been reported in prostate cancer, among other Diabetes Research Foundation’s Oxidative Stress Core for assistance with oxidative cancers (7, 68). stress measurements.

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Duyen T. Dang, Fang Chen, Manu Kohli, et al.

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