Activation of Protein Kinase G Is Sufficient to Induce Apoptosis and Inhibit Cell Migration in Colon Cancer Cells

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[CANCER RESEARCH 64, 3966–3973, June 1, 2004] Activation of Protein Kinase G Is Sufficient to Induce Apoptosis and Inhibit Cell Migration in Colon Cancer Cells

Atsuko Deguchi,1 W. Joseph Thompson,3 and I. Bernard Weinstein1,2 1Herbert Irving Comprehensive Cancer Center, 2Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York, and 3OSI Pharmaceuticals, Inc., Farmingdale, New York

ABSTRACT cation channels; it can bind to and activate protein kinase G (PKG); and, under certain conditions, it can bind to and activate protein kinase The activation of protein kinase G (PKG) by cGMP has become of A (8). considerable interest as a novel molecular mechanism for the induction of Two major forms of PKG have been identified in mammalian cells, apoptosis in cancer cells, because sulindac sulfone (exisulind, Aptosyn) and certain derivatives that inhibit cGMP-phosphodiesterases and PKG I and PKG II. In addition, there are two splice variants of PKG thereby increase cellular levels of cGMP appear to induce apoptosis via I, which are designated I␣ and I␤ (9). PKG I is expressed in platelets, this mechanism. However, other effects of these compounds have not been vascular smooth muscle cells, fibroblasts, certain endothelial cells, the excluded, and the precise mechanism by which PKG activation induces lung, the cerebellum, and the heart (10, 11). In endothelial cells, apoptosis has not been elucidated in detail. To directly examine the effects activation of PKG I by cGMP is associated with a reduction in of PKG on cell growth and apoptosis, we generated a series of mutants of thrombin-induced calcium transients and paracellular permeability ␣ ␣ ␣⌬ PKG I : PKG I S65D, a constitutively activated point mutant; PKG I , (12, 13). Direct evidence for functional roles of PKG I in relaxation of a constitutively activated N-terminal truncated mutant; and PKG smooth muscle, inhibition of Ca2ϩ transients, and inhibition of plate- I␣K390R, a dominant-negative point mutant. A similar series of mutants of PKG I␤ were also constructed (Deguchi et al., Mol. Cancer Ther., 1: let adhesion and aggregation was obtained from studies of PKG I 803–809, 2002). The present study demonstrates that when transiently knockout mice (14, 15). Vasodilator-stimulated phosphoprotein expressed in SW480 colon cancer, the constitutively activated mutants of (VASP) is known to be one of the substrates of PKG I, and its PKG I␤, and to a lesser extent PKG I␣, inhibit colony formation and phosphorylation plays a role in inhibiting platelet aggregation and induce apoptosis. We were not able to obtain derivatives of SW480 cells focal adhesion (16). PKG II is expressed mainly in the brush border that stably expressed these constitutively activated mutants, presumably of the intestinal mucosa and in specific regions of the brain and plays because of toxicity. However, derivatives that stably overexpressed wild- a role in transepithelial ClϪ and Naϩ transport in the intestine ␤ type PKG I displayed growth inhibition, whereas derivatives that stably (17, 18). expressed the dominant-negative mutant (KR) of PKG I␤ grew more Expression of PDE 5 has been detected in the normal bladder, rapidly and were more resistant to Aptosyn-induced growth inhibition than vector control cells. Stable overexpression of PKG I␤ was associated colon, pancreas, lung, placenta, prostate, small intestine, and stomach with decreased cellular levels of ␤-catenin and cyclin D1 and increased (19). It is of interest that metastatic breast cancers, colon adenocar- levels of p21CIP1. Reporter assays indicated that activation of PKG I␤ cinoma, bladder squamous carcinoma, and lung cancers often express inhibits the transcriptional activity of the cyclin D1 promoter. We also increased cellular levels of PDE 2 or 5 when compared with adjacent found that transient expression of the constitutively activated mutants of normal tissues (20–25). In addition, the endogenous polypeptide PKG I␤ inhibited cell migration. Taken together, these results indicate guanylyl cyclase activators guanylin and uroguanylin are expressed at ␤ that activation of PKG I is sufficient to inhibit growth and cell migration reduced levels in colon cancer cells (26, 27). Taken together, these and induce apoptosis in human colon cancer cells and that these effects are findings suggest that cGMP-mediated pathways are suppressed in associated with inhibition of the transcription of cyclin D1 and an increase colon cancer cells, presumably to inhibit downstream signaling path- in the expression of p21CIP1. ways related to the activation of PKG. There is also supporting evidence that activation of PKG by cGMP in cardiomyocytes (3), INTRODUCTION pancreatic B cells (4), and cultured smooth muscle cells (28, 29) can cause growth inhibition and apoptosis. In addition, PKG activation cGMP is an important second messenger that mediates the action of negatively regulates interleukin-2 signaling in T cell lines (30). Fur- several hormones, neurotransmitters, and drugs (1) by regulating thermore, recent studies indicate that sulindac sulfone (Aptosyn), a various physiological functions including neurotransmission, platelet metabolite of the nonsteroidal anti-inflammatory drug sulindac, and aggregation, and smooth muscle tone (2). There is increasing evidence two potent derivatives of Aptosyn, OSI-248 and OSI-461, specifically that it can play an important role in cellular proliferation, differenti- inhibit the cGMP-specific PDEs 2 and 5. Evidence has also been ation, and apoptosis (3–5). The intracellular level of cGMP is regu- obtained that the resulting increase in cellular levels of cGMP in lated through a dynamic balance between its rate of synthesis by human colon cancer cells leads to activation of PKG and thereby the guanylyl cyclases and degradation by specific phosphodiesterases induction of apoptosis (31–33). These novel effects of Aptosyn and (PDEs), especially PDEs 2 and 5 (6). cGMP has several intracellular related drugs may explain why these compounds exert anticancer targets. Thus, it can bind to specific PDEs, thereby stimulating or effects in a variety of biological systems even though, in contrast to inhibiting their activities (7); it can bind to and activate cGMP-gated conventional nonsteroidal anti-inflammatory drugs, they do not inhibit cyclooxygenase activity (34). The precise pathway by which PKG Received 12/1/03; revised 2/23/04; accepted 3/3/04. activation leads to apoptosis is not known, although it appears to Grant support: OSI Pharmaceuticals, Inc., Entertainment Industry Foundation- ␤ National Colorectal Cancer Research Alliance (I. Weinstein), the T. J. Martell Foundation involve both a decrease in cellular levels of -catenin and the acti- (I. Weinstein), and the National Foundation for Cancer Research (I. Weinstein). vation of c-Jun NH2-terminal kinase 1 (31, 32, 35, 36). The costs of publication of this article were defrayed in part by the payment of page Cell cycle progression is regulated by the orderly activation and charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. inactivation of a series of cyclins and cyclin-dependent kinases and is Requests for reprints: I. Bernard Weinstein, the Herbert Irving Comprehensive coordinated by both internal and external signals that act at key Cancer Center, College of Physicians and Surgeons, Columbia University, 701 W. 168th St., HHSC-1509, New York, NY 10032-2704. Phone: (212) 305-6921; Fax: (212) 305- checkpoints during cell cycle progression (37). Cyclin D1 is a major 6889; E-mail: [email protected]. positive regulator of the G1-S transition. It acts by binding to and 3966

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. PKG ACTIVATES APOPTOTIC SIGNALING IN COLON CANCER activating cyclin-dependent kinase 4 or cyclin-dependent kinase 6, Determination of Apoptotic Index. SW480 cells were infected with the which then phosphorylates and thereby inactivates the tumor suppres- indicated PKG retrovirus constructs as described above. After 72 h, the cells sor protein pRB (38). These activities are negatively regulated by were harvested and analyzed on a FACSCalibur instrument using CELLQuest p16INK4, p21CIP1 (p21), and p27KIP1 (39). p21CIP1 was first cloned software (Becton Dickinson, Mountain View, CA) with the FL-1 channel to and characterized as a mediator of p53-induced growth arrest (40), but detect GFP. This indicated that the infection efficiency in all samples was about 80%. Therefore, we used the entire cell population to determine the its expression can also be up-regulated by p53-independent mecha- sub-G1 population. The extent of apoptosis was determined using the FL-2 nisms (41). Although the cyclin D1 gene is not amplified in human channel after propidium iodide (Sigma) staining and expressed as a percentage, colon cancer, the expression of cyclin D1 is induced in about 30% of by calculating the number of cells that were in the sub-G1 population divided human adenocarcinoma and also in adenomatous polyps of the colon by the total number of cells examined. For the Annexin V staining assay, the (42, 43). Furthermore, inhibition of cyclin D1 expression with an infected SW480 cells were incubated for 72 h. The harvested cells were then antisense cDNA causes growth inhibition in colon carcinoma cell stained with phycoerythrin-conjugated Annexin V (PharMingen, San Diego, lines (44). Colon cancers also frequently display elevated levels of CA) for 15 min in the dark, according to the manufacturer’s protocol. The ␤-catenin as a result of inactivation of the adenomatous polyposis coli stained cells were then analyzed on a FACSCalibur instrument using molecule or mutations in ␤-catenin (45). This results in increased CELLQuest software (Becton Dickinson). The apoptotic index was expressed binding of ␤-catenin to the transcription element T-cell factor/lymph- as a percentage, by calculating the number of cells that were positive for both phycoerythrin-conjugated Annexin V and GFP divided by the total number of oid enhancer factor-1 and increased transcription of cyclin D1 and GFP-positive cells examined. other genes that enhance growth (46, 47). Therefore, the high expres- Determination of Cell Doubling Time. Cells were plated at a density of sion levels of cyclin D1 in colon cancer are at least in part due to 2 ϫ 104 in 15.6-mm diameter, 24-well dishes. The numbers of cells per well up-regulation of ␤-catenin/T-cell factor transcriptional activity. Re- were counted daily for the next 7 days, using a Coulter counter. The doubling cent studies by Li et al. (48) provide evidence that activated PKG times were measured during the period of exponential growth. directly phosphorylates ␤-catenin in the COOH-terminal region of the Western Blot Analysis. SW480 cells were harvested and then sonicated in molecule, in contrast to GSK3␤, which phosphorylates ␤-catenin at a radioimmunoprecipitation assay (RIPA) buffer, and extracts were examined by Western blot analysis as described previously (33). The lysates were electro- characteristic site in the NH2 terminus, and that this phosphorylation by PKG results in proteolytic degradation of ␤-catenin. phoresed on a 10% polyacrylamide gel and then electophoretically transferred Previous studies implicating PKG in growth inhibition and apopto- to a polyvinylidene difluoride membrane. The membrane was blocked with 5% dry milk and incubated with the indicated antibody. After washing, the mem- sis in colon cancer cells are based on using pharmacological agents brane was incubated with a horseradish peroxidase-conjugated secondary (31–33). In the present study, we obtained more direct evidence by antibody (Amersham Biosciences, Piscataway, NJ). Protein bands were visu- exploring the effects of expressing a series of wild-type (WT), con- alized with the enhanced chemiluminescence Western blotting system (Am- stitutively active, or dominant-negative mutants of PKG, previously ersham Biosciences). Fold expression was determined with NIH Image1.62 developed in our laboratory (33), in the SW480 human colon cancer software. cell line. Luciferase Reporter Assays. SW480 cells were plated at 1 ϫ 105 cells per 6-well 35-mm-diameter plates, and 24 h later, they were transfected with 500 ng of the cyclin D1-luc (51) reporter plasmid, the indicated PKG expression ␤ MATERIALS AND METHODS plasmids, and 10 ng of cytomegalovirus- -galactosidase reporter plasmid (as internal control), using Lipofectin (Invitrogen, Carlsbad, CA). After 18 h of Materials and Cell Culture. The cell-permeable cGMP compound transfection, some of the cells were incubated in fresh growth medium in the 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP) and the anti-PKG I␤ anti- presence or absence of 8-pCPT-cGMP for 24 h before harvesting. Luciferase body were purchased from Calbiochem (San Diego, CA), an anti-␤-catenin activities were normalized to ␤-galactosidase activities, to correct for differ- antibody from Transduction Laboratories (San Diego, CA), a polyclonal anti- ences in transfection efficiency. The relative luciferase activity measured in the body to cyclin D from Upstate USA, Inc. (Charlottesville, VA), an anti-␤-actin vector control cells was assigned the value of 100%. All assays were done in monoclonal antibody from Sigma (St. Louis, MO), an anti-p21CIP1 antibody triplicate. from Santa Cruz Biotechnology (Santa Cruz, CA), and an anti-hemagglutinin Cell Invasion Assays. SW480 cells were infected with the indicated ret- (HA) antibody from Covance (Richmond, CA). Aptosyn and OSI-461 were roviruses of PKG, as described above. After 18 h, the infected cells were provided by OSI Pharmaceuticals, Inc. The construction and characteristics of replated into the upper well of transwell chambers (Matrigel; BD Biosciences, plasmids that encode HA-COOH-terminal tagged WT, constitutively active (⌬ Bedford, MA). After a 24 h incubation with or without 100 ␮M 8-pCPT-cGMP, and SD), and dominant-negative (KR) forms of PKG I␣ and PKG I␤ are the cells on the upper surface of the chamber were removed by scraping with described in our previous publication (33). Similar vector-only plasmids were a cotton swab. Cells that had migrated through the filter were stained with 1% used as controls. SW480 and 293T cells were cultured in DMEM with 10% crystal violet and counted by microscopy. Similar assays were done with cells fetal bovine serum. that stably overexpressed PKG I␣ or I␤ as described in Fig. 4. Colony Formation Assay. SW480 cells were transfected with various Statistical Methods. Results of the experimental studies are reported as constructs of PKG I␣ and PKG I␤ as described previously (33). After 18 h, the mean Ϯ SD. Differences were analyzed by Student’s t test. A value of cells were replated into 100-mm dishes with 1 ϫ 105 cells/dish, and the cells P Ͻ 0.05 was regarded as significant. were cultured in the presence of 1 mg/ml G418 for 3 weeks. The colonies were fixed with formalin, stained with Giemsa, and counted. All assays were done RESULTS in triplicate, and the results are expressed as “% of control,” i.e., the number of colonies obtained compared with the vector control plasmid. Constitutively Activated Mutants of PKG I␣ and PKG I␤ Production and Infection with Retrovirus Constructs. The above- Inhibit Colony Formation in SW480 Cells. To directly examine the described HA-tagged PKG constructs were subcloned into the expression effects of PKG on growth and apoptosis, we constructed a series of vector pMIG upstream of the IRES-enhanced Green Fluorescent Protein (GFP) expression vectors that encode WT-PKG I␣ or the following mutants sequence. pMIG was provided by Dr. Jeremy Luban (Columbia University). of PKG I␣: PKG I␣S65D, a constitutively activated point mutant; 293T cells were cotransfected with pMIG, PMDG, and pCL-Eco as described ␣⌬ previously (49, 50). After 16 h, cells were incubated with fresh DMEM/10% PKG I , a constitutively activated N-terminal truncated mutant; and ␣ fetal bovine serum medium. The retrovirus containing medium was harvested PKG I K390R, a dominant-negative point mutant. A similar series of after another 24 or 48 h. SW480 cells were infected with the indicated expression vectors of PKG I␤ were also constructed, i.e., PKG I␤, retroviruses encoding PKG for 6 h and then incubated in fresh DMEM/10% PKG I␤S80D, PKG I␤⌬, and PKG I␤K405R (33). We transfected fetal bovine serum medium for 72 h. each of these vectors into SW480 cells, the cells were grown in 3967

80% (data not shown). The extent of apoptosis was monitored by

determining the sub-G1 fraction of cells by flow cytometry at 72 h after infection with the various retrovirus constructs (Fig. 2A). We found that when compared with the vector control-infected cells, the PKG I␣,I␣KR, and I␤KR vectors did not induce apoptosis. However, the I␣⌬,I␣SD, I␤, and I␤SD vectors caused about a 1.5-fold increase in apoptosis (P Ͻ 0.05, 0.001, 0.001, and 0.001, respectively). The strongest effect was seen with I␤⌬ vector because it caused about a 2-fold increase in apoptosis (P Ͻ 0.001). Similar effects were seen in a repeat experiment (data not shown). The relatively high apoptosis in the vector control cells is probably due to the infection procedure itself, which involved serum starvation. Significantly greater apoptotic effects were seen with the I␤ WT, ⌬, and SD constructs than the corresponding I␣ constructs. (P Ͻ 0.005, 0.01, and 0.001, respectively). To extend the results obtained with the PKG I␤ constructs, a similar experiment was done, but after infection, the extent of apoptosis was assayed by the Annexin V staining procedure (see “Mate- rials and Methods”). In this study, WT-PKG I␤ caused a small increase in apoptosis (P Ͻ 0.05), but I␤⌬ and I␤SD constructs Fig. 1. Inhibition of colony formation by activated forms of PKG I␣ and I␤ in SW480 cells. The indicated constructs were transfected into SW480 colon cancer cells, and the produced strong increases in apoptosis (P Ͻ 0.001 and 0.01, respec- cells were grown in the presence of the selection agent G418 for 3 weeks. The colonies tively), and again, the I␤KR construct did not induce apoptosis (Fig. were then fixed, stained with Giemsa solution, and counted. The results are representative ␤ of triplicate experiments. Assays were done in triplicate, and the data are plotted as mean 2B). Thus, constitutive activation of PKG I, especially PKG I ,is significant inhibition when compared with the control sufficient to induce apoptosis in SW480 cells. These results are ,ء .(values with SDs (error bars (P Ͻ 0.05). For additional details, see “Materials and Methods.” consistent with the inhibitory effects of activated mutants of PKG I␣ and I␤ on colony formation, described in Fig. 1. selection medium containing G418, and afterward, the colonies were Stable Overexpression of WT-PKG I␤ Inhibits Cell Prolifera- stained with Giemsa and counted (Fig. 1). Transfection with the tion in SW480 Cells. Because the above studies suggested that PKG constitutively activated mutants of PKG I␣ ⌬ and SD and the corre- I␤ appeared to have greater growth inhibitory activity than PKG I␣, sponding constitutively activated mutants of PKG I␤ caused about we examined the effects of PKG I␤ in greater detail by establishing 45% inhibition of colony formation (P Ͻ 0.001). The WT forms of derivatives of SW480 cells that stably overexpress this enzyme. We PKG I␣ and PKG I␤ produced about 30% inhibition, and the KR could not develop derivatives that stably overexpressed the constitu- forms produced about 10% inhibition, but these values were not tively activated mutants PKG I␤⌬ or I␤SD, presumably because of statistically significant. their cytotoxicity. We were however, successful in developing pools Constitutively Activated Mutants of PKG I Induce Apoptosis in of cells that stably overexpressed WT-PKG I␤ or the KR (dominant- SW480 Cells. In view of the above results, it was of interest to negative) mutant of PKG I␤, as demonstrated by Western blot anal- examine whether activation of PKG I is itself sufficient to induce ysis (Fig. 3A). The cells that overexpressed the mutant KR were more apoptosis in SW480 cells. To assure that our PKG I constructs were condensed and more adherent than the vector control cells. Con- expressed with high efficiency, we incorporated the HA-tagged PKGs versely, the cells that overexpressed WT-PKG I␤ were less adherent into the retrovirus vector pMIG. This vector also encodes eGFP so (data not shown). We then compared the growth rates of these two that we could monitor the infection efficiency (see “Materials and types of derivatives with that of a pool of cells derived from SW480 Methods”). Indeed, we found that the infection frequency was about cells that had been transfected with only the control vector. Whereas,

Fig. 2. Constitutively activated mutants of PKG can induce apoptosis in SW480 cells. SW480 cells were infected with retroviral constructs encoding the indicated forms of PKG for 6 h. After 72 h, the cells were analyzed by flow cytometry with FL-1 to detect GFP. More than 80% of the cells were positive for GFP (data not shown). A, the sub-G1 fraction of the entire cell population was determined as described in “Materials and Methods.” The sub-G1 population of noninfected SW480 cells was shown as a negative control (Non). Error significant increases in the apoptotic index when compared with the vector control cells (P Ͻ 0.05). B, cells were infected with the indicated PKG constructs ,ء ;bars indicate SDs as described in A, and after 72 h, the apoptotic index was determined by flow cytometry with phycoerythrin-conjugated Annexin V staining. For additional details, see “Materials and .significant increases compared with the vector control cells (P Ͻ 0.05). These experiments were repeated at least twice and gave similar results ,ء ;Methods.” Error bars indicate SDs 3968

Fig. 3. Growth curves of PKG derivatives of SW480 cells. SW480 cells were transfected with expression vectors encoding wild-type (I␤WT) or dominant-negative (I␤KR) forms of PKG I␤ or a vector control, and the cells were grown in the presence of the selection agent G418 for 3 weeks. We then did Western blot analysis using an anti-HA antibody (A) and growth curves (B)on pools of the PKG I␤WT, PKG I␤KR, and vector control cells. The doubling times during the period of exponential growth for vector control, I␤WT, and I␤KR cells were 21.4 Ϯ 1.0, 29.4 Ϯ 0.4, and 16.8 Ϯ 0.7 h, respectively. P values for differences between the I␤WT and I␤KR and the vector control cells were Ͻ0.0001 and Ͻ0.05, respectively. These experiments were repeated at least twice with similar results.

the exponential doubling time for the vector control cells was luciferase activity (Fig. 5A). To obtain additional evidence that acti- 21.4 Ϯ 1 h, the corresponding values for the WT-PKG I␤ overex- vation of PKG inhibits the transcriptional activity of the cyclin D1 pressor cells and the I␤KR overexpressor cells were 29.4 Ϯ 0.4 and promoter, we examined the effects of treating parental SW480 cells 16.8 Ϯ 0.7 h, respectively (P Ͻ 0.0001 and P Ͻ 0.05, respectively). with two compounds, Aptosyn and OSI-461, that inhibit cGMP PDEs Thus, stable overexpression of PKG I␤ inhibits the proliferation of 2 and 5, thereby activating endogenous PKG (31). It is apparent that SW480 cells. Because the parental SW480 cells express detectable both compounds inhibited luciferase activity in these reporter assays levels of endogenous PKG I␤ (data not shown), the fact that the (Fig. 5B). derivatives that stably express a dominant-negative PKG I␤ grow Effects of PKG I␤ Expression on Sensitivity to Growth Inhibi- more rapidly suggests that endogenous PKG I␤ can itself exert a tion by Aptosyn. It was of interest to determine whether derivatives partial growth inhibitory effect in these cancer cells. that stably overexpress WT-PKG I␤ or the dominant-negative mutant Overexpression of PKG I␤ Inhibits Expression of Cyclin D1 PKG I␤ differ in their sensitivity to growth inhibition by Aptosyn, and ␤-Catenin but Increases Expression of p21CIP1. Because we because as described above, the growth inhibitory and apoptotic found that stable overexpression of PKG I␤ inhibited cell prolifera- effects of this compound appear to be mediated, at least in part, tion, we examined possible effects of PKG I␤ on expression of the cell through the activation of PKG (31–33). Cell viability assays using cycle control proteins cyclin D1 and p21CIP1 and on the signaling SW480 cells carried out with increasing concentrations of Aptosyn ␤ molecule -catenin whose function is often perturbed in colon cancer indicated that with the v#3 control cells, the IC50 for Aptosyn was 300 cells (45). In these studies, we used two clonal derivatives of SW480 cells that stably expressed WT-PKG I␤ (clones I␤#3 and I␤#6), two clonal derivatives that stably overexpressed PKG I␤KR (I␤KR#3 and I␤KR#9), and a vector control clone (v#3). Cell extracts of these clones were obtained from exponentially growing cultures and examined by Western blot analysis. We found that the I␤#3 and I␤#6 cells displayed a marked decrease in the cellular level of cyclin D1 and some decrease in ␤-catenin, when compared with the v#3 cells. The I␤KR#9 cells had higher levels of cyclin D1 than the v#3 cells, but no apparent change in the level of ␤-catenin. There was also a marked increase in the cellular level of p21CIP1 in the I␤#3 and I␤#6 cells when compared with the v#3 cells (Fig. 4). The latter finding is consistent with our finding that treatment of SW480 cell with Apto- syn, OSI-461, or 8-pCPT-cGMP, agents that activate PKG, causes rapid induction of p21CIP1 (data not shown). Induction of p21CIP1 expression by OSI-461 has also been described in chronic lymphocytic leukemia cells (52). Because the SW480 cells that stably overexpress PKG I␤ had decreased levels of the cyclin D1 protein (Fig. 4), we examined whether PKG I␤ affects the transcriptional activity of the promoter region of the cyclin D1 gene by carrying out transient transfection reporter assays using a cyclin D1 promoter-luciferase reporter. Co- Fig. 4. Stable overexpression of PKG I␤ in SW480 cells decreases the expression of ␤ CIP1 transfection of the cells with WT-PKG I␤ partially inhibited luciferase -catenin and cyclin D1 but increases the expression of p21 . The levels of expression of PKG I␤, ␤-catenin, cyclin D1, p21CIP1, and ␤-actin were determined by Western activity, and this was additionally enhanced by treating the transfected blotting with the respective antibodies, in clonal derivatives of SW480 cells. These cells with a cell-permeable activator of PKG 8-pCPT-cGMP (Fig. included: a vector control clone; two clones that overexpress PKG I␤ WT (I␤#3 and ␤⌬ ␤ I␤#6); and two clones that express the dominant-negative mutant of PKG I␤ (I␤KR#3 and 5A), cotransfection with PKG I or PKG I SD caused marked I␤KR#9). Fold expression was analyzed with NIH Image 1.62 software. Similar results inhibition, but cotransfection with PKG I␤KR had no effect, on were obtained in three independent experiments. 3969

Fig. 5. Activation of PKG I␤ inhibits the transcriptional activity of the cyclin D1 promoter in SW480 cells. A, SW480 cells were transfected with the Ϫ1745 cyclin D1 promoter luciferase (luc) reporter and the pCMV-␤-galactosidase reporter and also cotransfected with PKG WT or PKG mutant expression vectors, as indicated. After 18 h of transfection, the cells were incubated in fresh growth medium, in the presence or absence of 250 ␮M 8-pCPT-cGMP (8pCPTcGMP) for 24 h prior, and extracts were assayed for relative luciferase activity. B, SW480 cells were transfected with the Ϫ1745 cyclin D1-luciferase and pCMV-␤-galactosidase reporters. After 18 h, the cells were incubated in fresh growth medium in the presence or absence of 600 ␮M Aptosyn or 10 ␮M OSI-461 for 24 h, and extracts were prepared. The relative luciferase activity measured in the vector control cells was assigned the value of 100%. Error bars indicate SDs of triplicate assays. Similar results were obtained in three independent experiments.

␮M. The I␤#3 clone that stably overexpresses wt-PKG I␤ displayed an VASP plays a role in regulating focal adhesion complexes. Therefore, ␮ Ͼ IC50 of 250 M (P 0.05), but this difference was not statistically it was of interest to examine the effects of our PKG constructs on cell ␮ significant. However, 600 M Aptosyn, which is twice the IC50 for the migration in SW480 cells, employing Matrigel-coated transwell parental SW480 and v#3 cells, caused a significantly greater inhibi- chambers. In one set of studies, SW480 cells were infected with the tion of the I␤#3 cells than that obtained with the v#3 cells (P Ͻ 0.01). above-described series of retroviruses encoding various forms of PKG On the other hand, the PKG I␤KR#9 cells were more resistant to I and then assayed 24 h later for cell migration (Fig. 7A). We found ⌬ ␣ ␤ Aptosyn than the v#3 cells because the IC50 with the former cells was that the WT, , and SD constructs of both PKG I and PKG I , but greater than 600 ␮M (P Ͻ 0.001; Fig. 6). Similar results were obtained not the KR constructs, caused significant inhibition of cell migration. in repeat studies (data not shown). These results provide additional These effects were strongest with the PKG I␤ constructs, especially evidence that PKG plays a role in mediating the growth inhibitory the ⌬ and SD constitutively activated mutants. This inhibition is not effects of Aptosyn and related compounds, although it is possible that simply because of loss of cell viability due to induction of apoptosis, our findings also reflect a more general effect of PKG on cell viability. because although some of these vectors can induce apoptosis at 72 h The possible clinical relevance of these results is mentioned under after infection (see Fig. 2), no increase in apoptosis was seen at 24 h “Discussion.” (data not shown). Nevertheless, it was important to confirm these Activation of PKG Inhibits Cell Migration in SW480 Cells. We effects by also examining the derivatives of SW480 cells that stably previously reported that treatment of SW480 cells with Aptosyn and overexpress PKG I␣ or PKG I␤ and yet remain viable. These studies other compounds that lead to activation of PKG or the overexpression were done with and without the addition of 8-pCPT-cGMP to the of constitutively activated mutants of PKG results in increased phos- medium to enhance the activation of PKG. Fig. 7B indicates that when phorylation of the protein VASP (33). Smolenski et al. (16) found that the vector control derivative was treated with 8-pCPT-cGMP, there activation of PKG in human endothelial cells caused inhibition of cell was significant inhibition of cell migration. Even in the absence of migration through the phosphorylation of VASP, presumably because treatment with this cGMP compound, the PKG I␣#1 clone and the PKG I␤#3 clone displayed significant decreases in cell migration when compared with the untreated vector control cells (P Ͻ 0.001). This inhibition was additionally increased when these cells were treated with the cGMP compound (P Ͻ 0.001). As in our previous studies (Fig. 1; Fig. 2), these effects were greater in the PKG I␤ than in the PKG I␣ cells. Thus, in addition to causing growth inhibition and activation of apoptosis, activation of PKG can inhibit the migration of colon cancer cells. This effect may be mediated, at least in part, through the phosphorylation of VASP, because VASP phosphorylation can cause disruption of focal adhesions (16) and possibly other changes in the cellular cytoskeleton.

DISCUSSION The present studies provide the first direct evidence that activation of PKG is sufficient to induce growth inhibition and apoptosis and also inhibit cell migration in human cancer cells. Our findings are Fig. 6. Effects of expression of PKG I␤ on sensitivity to Aptosyn-induced growth inhibition in SW480 cells. Control v#3 cells, PKGI␤#3 cells that stably overexpress consistent with previous evidence that activation of PKG in vascular WT-PKGI␤ (I␤#3), and PKG I␤KR#9 cells that stably overexpress a dominant-negative smooth muscle cells can have similar effects (28, 29) and that over- ␤ ␤ mutant of PKG I (I KR#9; see Fig. 4) were treated with Aptosyn (Exisulind)atthe expression of a constitutively activated PKG mutant also inhibits indicated concentrations. The numbers of cells were counted after 72 h using a Coulter counter and expressed as the percentage of the respective untreated cells. Error bars growth and cell migration in the porcine vascular system (29). It is of indicate means of triplicate assays. The inhibitory effects of 600 ␮M Aptosyn were interest that in the latter study, vascular surgery decreased the expres- P Ͻ 0.01) and were significantly less in the ,ء) significantly greater in the PKG I␤#3 cells P Ͻ 0.001) than in the v#3 control cells. Similar results were sion of PKG, and this was associated with restenosis. Previous studies ,ءء) PKG I␤KR#9 cells obtained in three independent experiments. indicate that various chemical agents that cause an increase in cellular 3970

Fig. 7. Activation of PKG inhibits cell migration in SW480 cells. A, SW480 cells were infected with the indicated retrovirus constructs of PKG for 6 h. After 24 h, the cells were transferred into Matrigel- coated transwell chambers. After an additional 24 h, cell migration was determined (see “Materials and Methods”), and the results are expressed as the percentage of the vector control. B, migration assays with cells that stably overexpress PKG I␣ or I␤. Cells were seeded into Matrigel-coated transwell chambers. After 6 h, 100 ␮M 8-pCPT-cGMP was added to the medium, as indicated. After 24 h, migration was determined, as described above. Er- assays ,ء ;ror bars indicate SDs of triplicate assays with significant inhibition (P Ͻ 0.05). These experiments were repeated and gave similar results.

levels of cGMP lead to induction of apoptosis in human colon cancer cyclase, caused increased expression of p21CIP1 via a cGMP-depend- cells (31–33, 35), but these findings did not provide direct evidence ent pathway. In addition, we found that treatment of SW480 cells with that activation of PKG was sufficient to produce this effect. Aptosyn or OSI-461, agents that increase cellular levels of cGMP The present study indicates that activation of PKG, specifically (33), causes a rapid increase in the expression of both p21CIP1 mRNA PKG I␤, is also sufficient to cause a decrease in cellular levels of and protein.4 cyclin D1 and an increase in cellular levels of the cell cycle inhibitory The decreased expression of ␤-catenin and cyclin D1 and increased protein p21CIP1 in SW480 colon cancer cells (Fig. 4). Transient expression of p21CIP1 may contribute to the growth inhibitory and transfection reporter assays provide evidence that these effects are due apoptotic effects of PKG activation in SW480 cells. However, the role to inhibition of the transcriptional activity of the cyclin D1 promoter of p21CIP1 in growth inhibition and induction of apoptosis can vary in (Fig. 5). Our results with cyclin D1 differ from those obtained by different cell systems (61). Thus, p21CIP1 null mice display an in- Hanada et al. (53), who found that overexpression of PKG I␤ in crease in tumor incidence (62), and overexpression of p21CIP1 en- murine mesangial cells that were also treated with 8-bromo-cGMP hances growth inhibition and apoptosis in glioma (63) and ovarian inhibited the promoter activity of cyclin E but not cyclin D1. This carcinoma (64) cell lines. On the other hand, up-regulation of p21CIP1 difference may reflect differences between their cell system and ours inhibits transforming growth factor ␤-induced apoptosis in retinal or the fact that they used a reporter plasmid encoding the Ϫ944 to endothelial cells (65). In addition, other effects of PKG activation ϩ139 region of the cyclin D1 promoter, whereas we used a full-length appear to play an important role in the induction of apoptosis by cyclin D1 promoter (Ϫ1745CD1; Ref. 51). Therefore, it is possible Aptosyn and related compounds, including activation of the MEKK1- Ϫ Ϫ that the region of the cyclin D1 promoter between 1745 and 944 SEK1-c-Jun NH2-terminal kinase 1 pathway (36). Furthermore, is required for PKG to exert its inhibitory effect. ␤-Catenin can Shureiqi et al. (66, 67) have recently obtained evidence that increased enhance the transcription of cyclin D1 by binding to the T-cell expression of 15-lipoxygenase-1 can play an important role in the factor/lymphoid enhancer factor-1 site of the cyclin D1 promoter (45, induction of apoptosis in human colon cancer cell lines by Aptosyn, 46). We found that stable overexpression of WT-PKG I␤ decreased and we have recently obtained evidence that PKG activation may also the cellular level of ␤-catenin in SW480 cells (Fig. 4). In addition, play a role in the induction of 15-lipoxygenase-1.5 Thus, the growth there is evidence that ␤-catenin is a substrate of PKG I␤ and that this inhibition and apoptosis caused by activation of PKG is probably phosphorylation can contribute to the degradation of ␤-catenin mediated by a complex series of events. through a GSK3␤-independent pathway (47). This mechanism may It is of interest that SW480 cells that stably express the dominant explain, at least in part, why activation of PKG causes decreased negative (KR) mutant of PKG I␤ were more resistant to the growth expression of cyclin D1, but possible effects on other transcription inhibitory effects of Aptosyn than vector control cells, whereas cells factors may also play a role. that overexpressed WT-PKG I␤ were more sensitive to this drug (Fig. It is known that the expression of p21CIP1 is regulated largely at the 6). These results suggest that the sensitivity of different cancer cells to level of transcription by both p53-dependent and -independent mech- the growth inhibitory effects of this class of compounds may depend, anisms (40). The promoter of the p21CIP1 gene contains two con- at least in part, on their cellular levels of PKG, a finding that may be served p53-binding sites, and at least one of these is required for p53 relevant to the clinical use of these compounds in cancer chemopre- responsiveness after DNA damage (54). In addition, a variety of vention and treatment. We should, however, emphasize that the pres- transcriptional factors that are induced by a number of different signal ent results do not exclude the possibility that the growth inhibitory and pathways can activate p21CIP1 transcription by a p53-independent apoptotic effects of these compounds are also mediated via pathways mechanism, including SP1, SP3, STATs, C/EBP␣, C/EBP␤, and that do not involve the action of PKG. CIP1 Smad3 (55–57). p21 expression is also regulated posttranscrip- Our finding that activation of PKG in SW480 cells inhibited their tionally by both ubiquitin-dependent and -independent proteasome- migration (Fig. 7) may also be of clinical relevance because it sug- mediated degradation (58, 59). Because the SW480 cells that we used gests that Aptosyn and related compounds may exert antitumor effects CIP1 carry a mutant p53, the induction of p21 by PKG appears to be not only by inhibiting cell proliferation but also by inhibiting invasion through a p53-independent pathway. The precise mechanism by and metastasis. We previously reported that activation of PKG in which PKG leads to increased expression of p21CIP1 remains to be determined. Our results are consistent with the finding of Gu et al. 4 Unpublished observations. (60) that treatment of cells with nitric oxide, which activates guanylyl 5 Unpublished observations. 3971

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Atsuko Deguchi, W. Joseph Thompson and I. Bernard Weinstein

Cancer Res 2004;64:3966-3973.

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