Matrix Metalloproteinases 9 and 10 Inhibit Protein Kinase C–Potentiated, P53-Mediated Apoptosis

Research Article

Matrix Metalloproteinases 9 and 10 Inhibit Protein Kinase C–Potentiated, p53-Mediated Apoptosis

Eric Meyer, Jean-Yves Vollmer, Raymonde Bovey, and Ivan Stamenkovic

Division of Experimental Pathology, Institut Universitaire de Pathologie, Universite´ de Lausanne, Lausanne, Switzerland

Abstract or cell death through modification of the expression level of several target genes. p53 target genes include, among many others, Bax (6), p53, a major sensor of DNA damage, is a transcription factor WAF1 that, depending on its phosphorylation status, regulates the Mdm2 (7), Apaf1 (8), Fas (9), p21 ,andDR5 (10). The cell cycle, DNA repair, or apoptosis. The protein kinase C consequences of the induction of these target genes are a function (PKC) family of isozymes is also implicated in cell cycle and of the cellular context. programmed cell death (PCD) control and has recently been Recently, a new mode of action has been proposed for the shown to influence p53 function. Using three human colon proapoptotic activity of p53 (11). According to this view, p53 may adenocarcinoma cell lines SW480, EB-1, and HCT116 that function in a transcription-independent manner and may induce either lack p53 function and were engineered to express cell death by modifying mitochondrial membrane permeability, inducible wild-type p53 (wt p53), or that constitutively express thereby activating the intrinsic apoptotic pathway. Regulation of wt p53, we show that phorbol ester–mediated PKC activation p53 activity occurs at several levels, including post-translational potentiates p53-induced PCD. Despite the effectiveness of modification (phosphorylation and acetylation), induction of the PKC/p53 synergy in inducing SW480 tumor cell death, Mdm2 protein, which in turn induces ubiquitin-mediated p53 however, a fraction of the cells invariably survive. To address degradation (12), and intracellular localization. To act as a the putative mechanisms that underlie resistance to PKC/p53- transcription factor, p53 must translocate from the cytosol to the induced cell death, we generated a phorbol 12-myristate 13- nucleus. In contrast, its transcription-independent activity acetate/p53–resistant SW480 subline and compared the gene requires its presence at the mitochondrial surface where it can expression profile of resistant and parental cells by DNA associate with the mitochondrial regulators of apoptosis Bax and microarray analysis. The results of these experiments show Bcl-XL (13). Localization is regulated in part by Mdm2, which that PKC/p53-resistant cells express a higher level of several sequesters p53 and forces its translocation back toward the matrix metalloproteinases (MMP), including MMP-9, MMP-10, cytosol, augmenting its availability for mitochondrial association and MMP-12, and corresponding real-time PCR assays (14, 15). indicate that p53 is a negative regulator of MMP-9 gene Recent evidence suggests that protein kinase C (PKC) isozymes expression. Using MMP inhibitors and MMP-specific small are involved in the regulation of the p53 DNA-binding activity by interfering RNA, we show that MMP function confers directly phosphorylating p53 on the Ser378 residue (16). It has also protection from PKC/p53-induced apoptosis and identify the been proposed that PKCy regulates p53 expression at the protective MMPs as MMP-9 and MMP-10. Taken together, transcriptional level and contributes to the accumulation of the these observations provide evidence that MMPs are implicat- p53 protein (17). PKC family members are serine/threonine ed in tumor cell resistance to the synergistic proapoptotic protein kinases that are divided into three subfamilies depending effect of PKC and p53. (Cancer Res 2005; 65(10): 4261-72) on their structure and mode of activation. The classic PKC subfamily includes the a, hI, hII, and g isotypes, which are Introduction activated by diacylglycerol and phosphatidylserine in a calcium- Resistance to apoptosis is a hallmark of cancer. Although dependent manner. The atypical PKCs, which constitute the E numerous molecular events can enable cells to survive in the face second subfamily, include the ~ and isotypes that are activated of proapoptotic stimuli, the full repertoire of mechanisms used by by phosphatidylserine alone. Finally, the novel PKC subfamily is y q D cancer cells to resist programmed cell death has yet to be composed of the , , , and u, isotypes, which are activated by established. In the physiologic setting, the transcription factor p53 diacylglycerol and phosphatidylserine in a calcium-independent plays a major role in regulating cell survival, in addition to several manner. These kinases play a role in multiple cellular functions, other cellular processes that include cell cycle checkpoint control one of them being apoptosis. However, the precise role played by (1), DNA repair and senescence (2, 3). It is therefore not surprising each of the different isotypes in programmed cell death (PCD) y that p53 is among the most commonly affected molecules in remains to be fully elucidated. For example, the novel PKC, PKC , cancer, >50% of malignant human tumors bearing mutations in the is considered proapoptotic and is cleaved by caspase-3 (18, 19), TP53 gene. p53 is activated in response to a multitude of signals, one of the effector caspases whose activation is common to including oncogene activation, hypoxia (4), and DNA damage (5), several apoptotic pathways. The resulting cleaved fragment is and when required, induces a cell cycle checkpoint blockade and/ proapoptotic and initiates an apoptotic amplification loop by targeting its own activator, caspase-3. On the other hand, the full- size PKCy can mediate apoptosis at the mitochondrial level h h Note: E. Meyer and J-Y. Vollmer contributed equally to this work. (20, 21). In contrast, the classic PKCs, PKCa,PKC I, and PKC II Requests for reprints: Ivan Stamenkovic, Institute of Pathology, Centre are believed to exert antiapoptotic activity. PKCa, for example, is Hospitalier Universitaire Vaudois, Rue du Bugnon 25, 1011 Lausanne, Switzerland. Phone: 121-314-7136; Fax: 121-314-7110; E-mail: [email protected]. able to phosphorylate and stabilize the antiapoptotic molecule I2005 American Association for Cancer Research. Bcl-2 (22). Several other reports show data suggesting that classic www.aacrjournals.org 4261 Cancer Res 2005; 65: (10). May 15, 2005

PKCs may display proapoptotic activity. The overall picture cDNA Array Analysis emerging from these observations is that even if the role played Total RNA was isolated from SMF1 or S1R6 cells after 8 hours of DMSO by PKC family members in apoptosis differs from isotype to or PMA/tetracycline stimulation using the RNeasy RNA extraction kit isotype, the consequences of their activation are most likely (QIAGEN, Basel, Switzerland) according to the manufacturer’s recommendations. The isolated RNA was linear-amplified using the Arcturus dependent on the molecular and cellular context and the type of (Mountain View, CA) riboamp RNA amplification kit. For each experiment, apoptotic stimulus. 6 Ag of sample 1 mRNA were labeled with dCTP-Cy3 and 6 Ag of sample 2 In the present study, we sought to determine the effect of PKC mRNA were labeled with dCTP-Cy5. Labeled samples were pooled and activity on p53-induced PCD and the physiologic mechanisms that hybridized in duplicate to human cDNA microarray (Hu10K) chips from underlie resistance to p53-mediated apoptosis in tumor cells. To NCCR/ISREC containing 11,552 spotted elements corresponding to 9,500 this end, we engineered the SW480 human colon adenocarcinoma unique human cDNAs (for an accurate description see http://intranet. cell line, which contains an endogenous mutant p53 (23), to isrec.ch/microarrays/nccr_isrec.html). Hybridization was done in hybrid- j overexpress wild-type p53 (wt p53) under the regulation of a ization chambers (Corning, Baar, Switzerland) in a 64 C water bath for tetracycline (Tet-ON) conditional system. Using this model system 16 hours. After several careful washing steps, the chips were scanned in a Perkin-Elmer/GSI Lumonics ScanArray 4000 scanner and scanned slide as well as additional colon carcinoma cell lines that express images were converted to a tagged image file format. Spot intensity was inducible exogenous or constitutive endogenous p53, we show that analyzed with the ScanAlyze software (http://rana.lbl.gov/EisenSoftware. PKC activation and p53 expression function in synergy to induce htm) and further primary data analysis was done using com.braju.sma apoptosis in colon carcinoma cells and that resistance to the routines in the R statistical package (http://www.maths.lth.se/help/R/ and combined proapoptotic effect of these molecules is conferred, at http://www.r-project.org/). We used the nonlinear Scaled print-tip normal- least in part, by matrix metalloproteinase 9 (MMP-9) and MMP-10 ization method (25). Quality control of slide hybridization was done using expression. variables described on the corresponding web site: http://intranet.isrec.ch/ microarrays/nccr_isrec.html. When comparing SMF1-DMSO– and SMF1-PMA/tetracycline–treated Materials and Methods cells, we considered spots with an average ratio value superior to 1.85 to correspond to positively regulated genes; a ratio of 1.00 was used for Cell Culture and Reagents comparisons between PMA/tetracycline-treated SMF1 and S1R6 cells. The The SW480 parental human colon adenocarcinoma cell line was average ratio was determined for each set of experiments as the experiment obtained from European Collection of Animal Cell Cultures (Porton Down, background intensity plus two SDs. United Kingdom) and the HCT p53+/+, HCT p53/, HCT Bax+/ control, and HCT Bax/ cells were a gift from Bert Vogelstein (The Johns Hopkins Real-time PCR University Medical Institutions, Baltimore, MD). The p53-inducible EB-1 Total RNA was isolated with the RNeasy extraction kit (QIAGEN) and colon adenocarcinoma cell line was a gift from Phil Shaw (Department cDNAs were produced by reverse transcription with the Moloney murine of Pathology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzer- leukemia virus Reverse Transcriptase RNase H minus, point mutant land; ref. 24). Cell stocks were maintained in DMEM (Life Technologies, (Promega, Wallisellen, Switzerland) and Random primers (Promega). Real- Basel, Switzerland) supplemented with penicillin, streptomycin, and 10% time PCR was done in an Applied Biosystems 7900HT Sequence Detection fetal bovine serum (FBS, Life Technologies). Tetracyclin (Calbiochem, System using the ABI Taqman Master mix chemistry. The sets of primers Schwalbach, Germany) and ZnCl (Sigma Co., Buchs, Switzerland) were and Taqman probes were as follows: cyclophilin A, ABI human PPIA 2 endogenous control [VIC/minor groove binder (MGB) Taqman probe]; large dissolved in water, whereas 5-fluorouracil (5-FU) and phorbol 12-myristate ribosomal protein, ABI human RLPO endogenous control (VIC/MGB 13-acetate (PMA, both from Sigma) were dissolved in DMSO. z-VAD-fmk Taqman probe); MMP-9 (26): forward primer 5V-CCTGGAGACCTGAGAAC- (BIOMOL Res. Lab., Butler Pike, PA) and 1,10-phenanthroline monohydrate CAATCT-3V, reverse primer 5V-TGCCACCCGAGTGTAACCA-3V,FAM/ (Sigma) were dissolved in DMSO. The metalloproteinase inhibitor GM6001 TAMRA-Taqman probe 5V-CAGCTGGCAGAGGAATACCTGTACCGCT-3V; and its negative control (Calbiochem) were dissolved in DMSO, as were the MMP-10, ABI Assay on Demand Ref. Hs00233987_m1 (FAM/MGB Taqman PKC inhibitors rottlerin/mallotoxin, bisindolylmaleimide I, and Go¨6976 probe); MMP-12, ABI Assay on Demand Ref. Hs00159178_m1 (FAM/MGB (Calbiochem). Taqman probe). Tetracycline-Regulated Expression System Construction The resulting data were analyzed with the comparative Ct method for The human wt p53 cDNA sequence was subcloned as a 1,200-bp BamHI- relative gene expression quantification. EcoRI fragment into the pcDNA4/TO/myc-His A vector (Invitrogen, Basel, Cell Death Analysis Switzerland). The resulting vector (p4TO-hwtp53) allowed expression of wt Propidium iodide sub-G peak quantification (Nicoletti stain). p53 under the control of a tetracycline operator 2 sequence–containing 1 Following stimulation, dead cell–containing supernatants were collected cytomegalovirus promoter. The SW480 cell line was first stably transfected and pooled with trypsinized living cells. Samples were then centrifuged and with the pcDNA6/TR vector (Invitrogen), containing sequences encoding washed with PBS. After a second centrifugation step, cells were permeabilized the tetracycline repressor protein, using LipofectAMINE 2000 (Invitrogen) with 70% ethanol, washed in PBS, and treated for 15 minutes at 37jC with according to the manufacturer’s recommendations. The bulk population RNase A (Sigma, 1 mg/mL in PBS) and for 15 additional minutes at room was then stably transfected with the p4TO-hwtp53 plasmid. After selection temperature with propidium iodide (PI, Sigma, 100 Ag/mL in PBS). Samples with Zeocin and Blasticidin (Invitrogen), a monoclonal population was where then analyzed on a FACS analyzer (Becton Dickinson, San Jose, CA). isolated and named SMF1. The percentage of specific cell death was calculated using the following Isolation of a PKC/p53-Induced Apoptosis-Resistant Cell formula: Population SMF1 cells were treated with tetracycline and PMA (2 and 0.16 Amol/L, ððS CÞ=ð100 CÞÞ Â 100 ¼ %specific cell death; where respectively) for 24 hours. Resistant cells were rinsed with PBS and cultured S ¼ sample cell death and C ¼ control sample cell death: in DMEM/10% FBS for 2 weeks. The resulting population was subjected to additional rounds of tetracycline + PMA stimulation and selection. After the Annexin V staining. The Annexin V-Biotin Apoptosis Detection Kit from fifth round of selection, surviving cells were tested for p53/PKC resistance Oncogene Research Products (Lucerne, Switzerland) was used according to and preservation of tetracycline-inducible wt p53 expression. The resulting the manufacturer’s protocol. Briefly, dead and living cells were pooled and SMF1-derived cell line was named S1R6. washed once with PBS. The Annexin V-Biotin conjugate was added to the

Cancer Res 2005; 65: (10). May 15, 2005 4262 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. MMP-9 and MMP-10 Inhibit p53/PKC-Induced Apoptosis sample in binding buffer [10 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, GGAAACAUUAUAUCACCUAtt-3V and antisense sequence 5V-UAGGUGAU-

2.5 mmol/L CaCl2, 1 mmol/L MgCl2, 4% bovine serum albumin] and AUAAUGUUUCCtc-3V. incubated at room temperature in the dark for 15 minutes, centrifuged, and All siRNAs were synthesized by Ambion, Inc. (Huntingdon, United resuspended in 0.5 mL binding buffer supplemented with 15 AL Kingdom). streptavidin-FITC (eBioscience, San Diego, CA; 15 Ag/mL) and 10 ALPI (Sigma, 1 Ag/mL). Cells were kept at 4jC in the dark and analyzed Gelatin Zymography immediately by flow cytometry. After cell stimulation, the serum-free supernatant was concentrated with Terminal deoxynucleotidyl transferase–mediated nick-end labeling centricon column with a 30-kDa cutoff value (Millipore, Volketswil, Switzerland). The concentrated solution was mixed with Laemmli sample staining. Terminal deoxynucleotidyl transferase–mediated nick-end label- h ing (TUNEL) assays were done using the In situ Cell Death Detection Kit, buffer, without -mercaptoethanol, and loaded onto a gelatin (1 mg/mL) AP from Roche Diagnostics (Rotkreuz, Switzerland), according to the containing 10% SDS-PAGE gel. Following the completion of electrophoresis, the gel was washed in Triton X-100 solution and the proteases were manufacturer’s instructions. Briefly, cells were grown on glass chamber activated overnight at 37jC in 5 mmol/L CaCl and 50 mmol/L Tris (pH 8). slides. Following stimulation, the slides were washed in PBS, air-dried, and 2 fixed in 4% paraformaldehyde in PBS (pH 7,4). After a second washing The gel was then stained with Coomassie blue. step, cells were permeabilized in 0.1% Triton X-100 with 0.1% sodium citrate. Samples were labeled by adding fluorescein-dUTP conjugate and Results terminal deoxynucleotidyl transferase and incubating for 1 hour at 37jC in the dark. After several washes in PBS, the cells were examined under a To address the mechanisms that underlie tumor cell resistance to fluorescence microscope. The signal was converted using an AP- apoptosis and to elucidate the effect of PKC activation on p53- conjugated anti-fluorescein antibody. Phosphatase activity was detected induced proapoptotic signaling in human cancer cells, we selected by immersion of the slides in AP substrate–containing staining solution the SW480 cell line established from a primary human colon [nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate mix in 0.1 adenocarcinoma. These cells express a p53 protein containing two mol/L Tris (pH 9,5), 0.05 mol/L MgCl2, and 0.1 mol/L NaCl] for 45 minutes distinct mutations: G273A (ref. 23; converting an arginine to a in the dark. The reaction was stopped by addition of double distilled histidine residue) and C309T (converting a proline to a serine residue; water. ref. 27), which result in p53 inactivation. To study the effect of wt p53 Mitochondrial transmembrane potential (#W ) analysis. Briefly, m overexpression in SW480 cells, we applied a tetracycline-regulated wt DMSO- or PMA/tetracycline-stimulated cells (5 Â 105 cells per sample) were washed twice with cold PBS containing 1% serum. For each sample, two p53 expression system (28). PKC activation was achieved by tubes were prepared, one containing cells stained with the carbocyanine dye exogenous administration of the phorbol ester PMA, which is a 3,3V-dihexyloxacarbocyanine iodide (Molecular Probes, Eugene, OR) and the major activator of PKCs as well as of other signaling pathways, other containing unstained cells that served as negative controls for including mitogen-activated protein kinases and nuclear factor nh. fluorescence quantification. After 15 minutes of incubation at 37jC, the SMF1 cells overexpress wild-type human p53 under the samples were washed in PBS containing 1% serum and resuspended in a control of tetracycline (Tet-ON system) and initiate a cell final volume of 200 AL PBS/1% FBS. Samples were kept at 4jC in the dark death programme accelerated by phorbol 12-myristate 13- and analyzed immediately by flow cytometry. acetate stimulation. SMF1 cells were generated by genetically Crystal violet staining. DMSO-, PMA-, 5-FU-, or 5-FU/PMA-treated cells modifying SW480 cells to stably express the tetracycline repressor were washed with PBS. Cells were stained with 0.5% Crystal violet solution protein allowing conditional expression of wt p53. Expression of (25% methanol) for 15 minutes. After two washes with distilled water, cells exogenous p53 was achieved by addition of tetracycline to the were lysed in 1% SDS and absorbance at 620 nm was measured. All 620-nm culture medium (Fig. 1A). DMSO and PMA stimulation (Fig. 1A, absorbance values were expressed as a percentage of values obtained from lanes 1 and 3, respectively) had no effect on endogenous p53 lysates of DMSO-treated cells, giving the percentage of cell viability. Cell death rate was calculated by subtracting this percentage from 1. expression in SMF1 cell lysates, whereas lysates from SMF1 cells stimulated with either tetracycline alone or tetracycline and PMA Western Blotting show expression of both the endogenous and the wild-type SDS-PAGE protein electrophoresis and Western blotting were done exogenous protein (Fig. 1A, lanes 2 and 4, respectively). Twenty- according to standard procedures. For immunodetection of the human p53 four hours following induction of expression, p53-dependent cell protein, we used the DO-1 mouse monoclonal IgG2a antibody (Santa Cruz Biotechnology, Santa Cruz, CA). For caspase-3/Yama/Apopain/CPP32, we death was almost negligible (mean 1%, Fig. 1B). Stimulation with used the Ab-1 mouse monoclonal IgG1 antibody (Oncogene Research PMA alone was not accompanied by significant induction of cell Products), which recognizes both the zymogen and the cleaved form of the death, but costimulation with tetracycline and PMA resulted in 73% caspase-3. For human h-actin, we used the AC-74 mouse monoclonal IgG2a cell death at 24 hours (Fig. 1B). Observations by light microscopy antibody (Sigma). The secondary antibody was a horseradish peroxidase– indicated the presence of detectable cell death 12 hours following conjugated antimouse IgG and the signal was revealed using the Enhanced combined p53 induction and PMA stimulation in SMF1 cells (data Chemiluminescence PLUS substrate solutions (Amersham Biosciences., not shown). Thus, PMA treatment sensitized SMF1 cells to p53- Otelfingen, Switzerland). dependent cell death. The notion that this effect was p53 dependent mRNA Interference and not related to some property of the tetracycline molecule itself For specific gene silencing, we used the small interfering RNA (siRNA) is supported by the observation that PMA-induced sensitization did technology. Cells grown to 30% to 40% confluence in 24-well culture plates not occur in tetracycline-treated parental SW480 cells that do not were transfected with siRNA solution consisting of oligofectamine reagent express wt p53 (Fig. 1B). In addition, tetracycline stimulation of and the Opti-MEM (both from Invitrogen). Following transfection, cells SW480 cells stably transfected with tetracycline repressor and the were maintained in DMEM/10% FCS without antibiotics for 48 hours. We p53-independent gene PLSCR1 did not result in a higher ratio of cell used the following siRNA: control, sense sequence 5V-AAAGGAAACUG- GAAAAAUGtt-3Vand antisense sequence 5V-CAUUUUUCCAGUUUCCUUUtt-3V; death than in the parental SW480 cells (data not shown). anti–MMP-9, sense sequence 5V-CAUCACCUAUUGGAUCCAAtt-3V and To ensure that the observed sensitization of p53-mediated cell antisensesequence5V-UUGGAUCCAAUAGGUGAUGtt-3V; anti–MMP-10, death by PMA was not a cell type–specific event, we tested the sense sequence 5V-GGAGGACUCCAACAAGGAUtt-3Vand antisense sequence effect of p53 induction in the presence and absence of PMA in the 5V-AUCCUUGUUGGAGUCCUCCtc-3V; anti–MMP-12, sense sequence 5V- EB-1 colon carcinoma cell line (24) engineered to express wt p53 www.aacrjournals.org 4263 Cancer Res 2005; 65: (10). May 15, 2005

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Cancer Research gene under the control of the metallothionein MT-1 promoter. costimulation occurs in a more physiologic context, we stimulated Unlike SMF1 cells, which contain mutated p53, EB-1 cells lack HCT116 colon carcinoma cells, which constitutively express wt p53 +/+ / / endogenous p53 expression. Treatment of EB-1 cells with ZnCl2 (HCT ), and their p53 derivatives (HCT )withthe led to a robust induction of p53 (Fig. 1C) accompanied by 20% anticancer drug 5-FU, a known p53 inducer. Treatment of HCT+/+ cell death at 24 hours, whereas costimulation with ZnCl2 and cells with 5-FU augmented endogenous p53 expression and PMA resulted in 40% cell death at the same time point (Fig. 1C). induced 34% cell death at 24 hours (Fig. 1D). Consistent with the To determine whether the observed effect of p53/PMA observations using SMF1 and EB-1 cells, 5-FU–induced cell death was potentiated by costimulation with PMA (Fig. 1D). 5-FU cytotoxicity was due, in part, to p53-independent events, as 5-FU treatment of HCT p53/ cells resulted in 15% to 19% cell death (Fig. 1D). Importantly, however, PMA costimulation did not enhance 5-FU–mediated death of HCT/ cells (Fig. 1D). Cell death observed in phorbol 12-myristate 13-acetate/ tetracycline stimulated cells shows features of apoptosis, including DNA cleavage, phosphatidylserine externalization, and mitochondrial transmembrane potential #Wm alteration. Differentiation between apoptotic and necrotic cell death is based on several morphologic and functional criteria. We therefore applied a series of approaches to determine the type of cell death that was induced by the combination of p53 and PMA. Analysis of genomic DNA from dying cells revealed a DNA ladder (data not shown) which is a hallmark of apoptosis. p53/ PMA costimulation was observed to induce several other apoptosis-specific molecular and cellular features. Thus, TUNEL staining (Fig. 2A) showed the presence of nick-containing DNA in the PMA/tetracycline-stimulated cells, which occurs in most forms of apoptosis as a result of endonuclease activation. Annexin V-biotin conjugate reactivity revealed externalization of phosphatidylserine (29, 30), another characteristic feature of programmed cell death (Fig. 2B). Similar phosphatidyl serine externalization was observed in EB-1 and HCT+/+ cells in response to ZnCl2 and 5-FU stimulation, respectively (data not shown). Finally, using a mitochondrial membrane carbocyanine dye (Fig. 2C), we could show that p53/PMA altered the mitochondrial transmembrane potential DWm (31, 32), in SMF1 cells, suggesting that activation of the intrinsic mitochondrial apoptotic pathway may contribute to cell death induced by the combined effect of p53 and PMA. Selection of S1R6 cells that are partially resistant to p53/ phorbol 12-myristate 13-acetate costimulation induced apoptosis. Because all three cell types displayed a similar response to p53/PMA costimulation, we selected SMF1 cells for subsequent experiments. Although most of the SMF1 cells treated with tetracycline and PMA underwent apoptosis, a small fraction (<10%) survived. To address the mechanisms that underlie tumor cell resistance to the synergistic cell death inducing effect of PMA and p53, we subjected the surviving cells to several rounds of

Figure 1. Tetracycline-inducible p53 expression in SMF1 cells and PMA potentiation of p53-induced cell death. A, Western blot analysis of SMF1 cell lysates using anti-human p53 and anti-human h-actin antibodies. SMF1 cells were stimulated with DMSO (lane 1), DMSO + 2 Amol/L tetracycline (lane 2), 0.16 Amol/L PMA (lane 3) or PMA + tetracyclin (lane 4) for 24 hours. B and C, sub-G1 apoptotic DNA quantification by flow cytometry. B, SMF1 cells (black bars) and SW480 parental cells (white columns) were stimulated with DMSO (D), PMA (P), tetracycline (T), or PMA + tetracycline (PT) for 24 hours. C, EB-1 cells were stimulated with DMSO (D), PMA (P), 100 Amol/L ZnCl2 (Z), or 100 Amol/L ZnCl2 + PMA (Z+P) for 24 hours. Inset, Western blot analysis of p53 induction using the anti-p53 monoclonal antibody DO-1. D, crystal violet staining: HCT 116 p53+/+ (black columns) or HCT 116 p53/ (white columns) were stimulated with DMSO (D), PMA (P), 50 Ag/mL 5-FU, or 50 Ag/mL 5-FU + PMA (5-FU + P) for 24 hours. Inset, Western blot analysis of p53 induction in HCT+/+ and HCT/ cells using the anti-p53 monoclonal antibody DO-1. Columns, mean of experiments done in triplicate; bars, SD.

Cancer Res 2005; 65: (10). May 15, 2005 4264 www.aacrjournals.org

Figure 2. S1R6 cells display augmented resistance to PMA/tetracycline-induced apoptosis. A, TUNEL staining of SMF1 and S1R6 cells treated with DMSO or 0.16 Amol/L PMA + 2 Amol/L tetracycline (PT) for 24 hours. B, Annexin V staining of phosphatidylserine externalization. SMF1 (black column) and S1R6 (white column) cells were stimulated with DMSO (D) or PMA + tetracycline (P+T) for 24 hours. Columns, mean of triplicate experiments; bars, SD. C, mitochondrial transmembrane potential analysis by flow cytometry. SMF1 and S1R6 cells were treated with DMSO or PMA + tetracycline (PT) for 20 hours. Solid arrows, peaks corresponding to cells with normal mitochondria; hatched arrows, peaks corresponding to cells with damaged mitochondria. D, sub-G1 apoptotic DNA quantification by flow cytometry. SMF1 cells (n) or S1R6 cells (.) were stimulated with PMA + tetracycline for 24 or 48 hours. Points, mean of three different experiments; bars, SD. tetracycline/PMA treatment and selection. The resulting S1R6 cell Phorbol 12-myristate 13-acetate/tetracycline–induced apo- line displayed markedly different sensitivity to PMA/p53 compared ptosis is caspase dependent. Most of the apoptotic signaling with the parental SMF1 cells (Fig. 2). Thus, in the TUNEL assay pathways described thus far are mediated by the caspase family of (Fig. 2A), PMA/tetracycline stimulation induced only weak staining cysteine-proteases (33). The activated forms of these enzymes in S1R6 cells, whereas the majority of the SMF1 cells were strongly cleave diverse targets that are important for several key cellular positive (DMSO treated SMF1 and S1R6 cells, used as controls, functions, including DNA repair and cell structure maintenance, were TUNEL negative). Similarly, phosphatidylserine externaliza- and are also able to activate other caspases. We therefore tion in S1R6 was weaker than in SMF1 (18% compared with 45%, addressed their implication in PMA/p53-induced apoptosis. Fig. 2B). In addition, whereas there was little relevant mitochon- Caspase-3 (Apopain/YAMA/CPP32), a member of the effector drial transmembrane potential difference between DMSO- and caspase subfamily, was observed to be activated by PMA/p53, as PMA/tetracycline-treated S1R6 cells (DMSO: 14% and PMA/ shown by the cleavage of the 32-kDa zymogen (Fig. 3). The active tetracycline: 16%), a major difference was observed in SMF1 cells 17-kDa fragment was detectable only in the PMA/tetracycline- (DMSO: 9% and PMA/tetracycline: 49%, Fig. 2C). A cell death time stimulated SMF1 cell lysates after 24 hours (Fig. 3A). Cleavage was course (Fig. 2D) indicates that at 24 and 48 hours, the cell death not detected in SMF1 cell lysates at 8 hours or in S1R6 cell lysates observed in S1R6 represents 21% and 46%, respectively, of that at any time. The 116-kDa form of poly(ADP-ribose) polymerase induced in SMF1 cells. (PARP), a nuclear protein implicated in DNA repair and known to www.aacrjournals.org 4265 Cancer Res 2005; 65: (10). May 15, 2005

Figure 3. PMA/tetracycline-dependent apoptosis is mediated by caspase-3. A, Western blot analysis of SMF1 and S1R6 cell lysates using antihuman caspase-3 antibody. SMF1 and S1R6 cells were stimulated for 8 or 24 hours with DMSO (D) or 0.16 Amol/L PMA + 2 Amol/L tetracycline (PT). Representative of two independent experiments. B, sub-G1 apoptotic DNA quantification by flow cytometry. SMF1 cells (white columns) and S1R6 cells (black columns) were stimulated for 15 hours with DMSO or 0.16 Amol/L PMA + 2 Amol/L tetracycline. z-VAD-fmk was added 3 hours before stimulation at a concentration of 100 Amol/L. Representative of three independent experiments. C, crystal violet staining: HCT 116 Bax+/ (black columns) or HCT 116 Bax/ (white columns) cells were stimulated with DMSO (D), PMA (P), 50 Ag/mL 5-FU, or 50 Ag/mL 5-FU + PMA (5-FU + P) for 24 hours. Columns, mean of experiments performed in triplicate; bars, SD. be a target of at least caspase-3 and caspase-7 was also found to be cell lines. These observations suggest that the potentiation of p53- cleaved in SMF1 cells after PMA + tetracycline stimulation (data induced cell death by PMA in SW480 cells is mediated by PKCs of not shown). Finally, using the generic caspase inhibitor, z-VAD-fmk the classic subfamily and antagonized by PKCs of the novel (34), which inactivates most members of the family, we observed subfamily. complete inhibition of PMA/tetracycline-induced apoptosis in S1R6 S1R6 expresses higher levels of matrix metalloproteinases cells but incomplete inhibition in SMF1 cells (Fig. 3B). after phorbol 12-myristate 13-acetate/tetracycline stimula- Because activation of Bax, which leads to the release of tion. To address the mechanisms responsible for the observed cytochrome c from mitochondria and caspase activation, is a key resistance of S1R6 cells to PKC/p53-induced apoptosis, we did a mechanism underlying p53-mediated cell death, we tested the microarray gene expression analysis of SMF1 and S1R6 cells. effect of 5-FU in the presence and absence of PMA on HCT 116 First, we compared gene expression in DMSO- and PMA/ Bax/ cells. Cell death induced by 5-FU and 5-FU/PMA was lower tetracycline-stimulated SMF1 cells. Among 474 genes that in HCT Bax/ cells than in HCT Bax+/ counterparts (Fig. 3C). displayed a >3-fold induction in cells treated with PMA/ The observed cell death in HCT Bax/ cells most likely cor- tetracycline, were several p53 target genes, PKC-related genes, responds to p53-independent cytotoxicity of 5-FU consistent with and genes implicated in apoptosis (data not shown). P53 target the notion that Bax is implicated in apoptosis induced by p53 and genes included PIG11, CDKN1A (encoding p21), and MDM2. The p53/PMA. gene encoding p53 itself, TP53, was up-regulated, and as Phorbol 12-myristate 13-acetate sensitization to p53- expected, genes encoding PKC family members, including PRKCA induced apoptosis is protein kinase C mediated. Because the and PRKCZ, and genes encoding proapoptotic proteins, phorbol ester PMA is known to activate several members of the including BIK, CASP10, and APAF1 were also induced by PMA/ PKC family in addition to other signaling pathways, we attempted p53 (data not shown). to identify the PKC family members implicated in the observed A second round of microarray analysis was done to compare gene PMA/p53-induced apoptosis using specific PKCs inhibitors, expression in SMF1- versus S1R6-PKC/p53–stimulated cells. Ninety- including rottlerin (35), a novel PKC subfamily inhibitor, Go¨6976 seven genes were found to display a >2 fold induction in S1R6 cells (36, 37), a specific inhibitor of the classic PKCs a and h, and bis- (Table 1 and data not shown). At least two groups of genes of indolylmaleimide I (38), a broad classic PKC inhibitor. When potential interest were up-regulated. The first includes genes pretreated with either classic PKC inhibitor, both SMF1 and S1R6 encoding proteins involved in cell adhesion and cell-cell commu- cells displayed a marked reduction in cell death following PMA/ nication. The second includes genes encoding matrix metal- tetracycline stimulation (Fig. 4). In contrast, the novel PKC loproteinases (MMP), and more specifically, MMP-9, MMP-10, and inhibitor rottlerin enhanced PMA/tetracycline sensitivity of both MMP-12. Because of their implication in the regulation of cell

Cancer Res 2005; 65: (10). May 15, 2005 4266 www.aacrjournals.org

phosphatidylserine externalization in a similar way (data not shown). Matrix metalloproteinase 9– and matrix metalloproteinase 10–specific small interfering RNAs sensitize S1R6 cells towards phorbol 12-myristate 13-acetate/tetracycline–induced cell death. To identify the MMPs that confer protection to S1R6 cells from PKC/p53-induced cell death, we did siRNA- transient transfection experiments (41) to specifically silence MMP- 9, MMP-10, and MMP-12 transcripts. Each siRNA induced a reduction of the corresponding mRNA expression as detected by real-time PCR analysis (Fig. 5B-D). MMP-9 and MMP-10 gene silencing was observed to be followed by corresponding cell sensitization to PMA/tetracycline-induced cell death (Fig. 5B and C). MMP-9– and MMP-10–specific siRNAs induced 257% and 161% sensitization, respectively. In contrast, MMP-12 siRNA failed to modify S1R6 cell sensitivity to PKC/p53-induced apoptosis (Fig. 5D). Thus, consistent with the results obtained using chemical MMP inhibitors, MMP-9 and MMp-10 expression may play a protective role in colon cancer against PKC/p53-induced apoptosis. This notion is supported by the observation that sensitization of EB-1 cells, which express MMP-9, to ZnCl2/PMA-mediated apoptosis could also be achieved by MMP-9–specific siRNA, with a 30% increase in cell death (data not shown). By contrast, HCT+/+ cells, which are negative for MMP-9 expression, as assessed by gelatin zymography, displayed no sensitization to p53-mediated cell death following transfection with MMP-9 siRNA (data not shown). p53 down-regulates expression of matrix metalloproteinase 9 at the transcriptional level. p53 is known to induce the expression of several MMPs, but its effect on MMP-9 has not been fully explored. To determine the effect of p53 on MMP-9 Figure 4. PMA sensitization to p53-induced apoptosis is PKC mediated. Sub-G1 apoptotic DNA quantification by flow cytometry. A, SMF1 cells were stimulated expression, we quantified the mRNA expression of the gene for 24 hours with DMSO or 0.16 Amol/L PMA + 2 Amol/L tetracycline. PKC encoding MMP-9 by real-time PCR in response to p53 induction. inhibitors bisindolylmaleimide I (1 Amol/L), Go¨6976 (1 Amol/L), or rottlerin (25 Amol/L) were added 3 hours before stimulation (white columns, no Surprisingly, p53 was observed to be a negative regulator of the pretreatment with PKC inhibitors; black columns, pretreatment with Go¨6976; dotted columns, pretreatment with bisindolylmaleimide I; hatched columns, pretreatment with rottlerin). B, S1R6 cells were treated as in (A). Representative of three independent experiments. Table 1. S1R6 cells display augmented MMP expression compared with SMF1 cells survival, we chose to address the functional role played by the Cluster Overexpression Gene Description MMPs in response to PMA/p53 stimulation. Matrix metalloproteinase activity plays a role in protein MMPs 4 MMP-9 MMP-9 kinase C/p53-induced apoptosis. To test the potential role of (gelatinase B) MMP-mediated proteolysis in colon carcinoma cell response to 4 MMP-10 MMP-10 PMA/p53 signals, we first blocked MMP proteolytic activity using (stromelysin 2) two broad spectrum MMP inhibitors: the metal chelating agent 3 MMP-12 MMP-12 o-phenanthroline (PHE), a molecule known to interfere with the (macrophage MMP-zinc ion fixation (39), and GM6001 (40), a hydroxamic acid elastase) isomer known to be a potent collagenase inhibitor. Figure 5A Cell adhesion 8 CLDN7 Claudin 7 illustrates the effect of pretreatment of SMF1 or S1R6 cells with 5 CDH1 E-cadherin 4 OCLN Occludin these drugs. PMA/tetracycline-induced apoptosis was augmented 2 CLDN1 Claudin1 in both cell lines following PHE compared with DMSO treatment 2 CLDN11 Claudin 11 (64% and 36% for SMF1 and 53% and 24% for S1R6, respectively). Similarly, treatment with GM6001 increased the fraction of cells undergoing apoptosis in response to PMA/ NOTE: SMF1 and S1R6 cells were treated for 8 h with 0.16 Amol/L tetracycline to 75% and 35% among SMF1 and S1R6 cells, PMA + 2 Amol/L tetracyclin, and mRNA was extracted and amplified respectively. Thus, MMP activity seems to provide the tumor for microarray analysis. Clusters of genes that were upregulated in cells with partial protection against apoptosis induced by p53/ PMA/tetracyclin-stimulated S1R6 cells compared with PMA/tetracy- PKC costimulation. To make sure that these effects were not clin-treated SMF1 cells. The fold increase was rounded off to the specific to the DNA degradation marker of apoptosis, we did nearest whole number. Annexin V binding analysis and found that both inhibitors affect www.aacrjournals.org 4267 Cancer Res 2005; 65: (10). May 15, 2005

Figure 5. PKC/p53-induced apoptosis is inhibited by MMP inhibitors. A, sub-G1 apoptotic DNA quantification by flow cytometry. SMF1 and S1R6 cells were stimulated for 24 hours with 0.16 Amol/L PMA + 2 Amol/L tetracycline. No inhibitors (white columns) or MMP inhibitors, 100 Amol/L PHE (black columns), 100 Amol/L GM (dotted columns), and 100 Amol/L GM+ (hatched columns) were added 3 hours before stimulation. Representative of three independent experiments. B, sub-G1 apoptotic DNA quantification by flow cytometry (% specific cell death) as a function of MMP expression. S1R6 cells were transfected with control siRNA (V) or MMP-9–specific siRNA (M) and 48 hours later, stimulated with 0.16 Amol/L PMA + 2 Amol/L tetracycline for 24 hours. Expression was determined by quantification of MMP-9 mRNA by real-time PCR 48 hours post-transfection in S1R6 cells (relative expression with respect to control siRNA transfected cells). C and D, experiment was performed as in (B) but using MMP-10 siRNA (C) and MMP-12 siRNA (D). Representative of three independent experiments.

MMP-9 gene. Figure 6A shows relative quantification using the One of the principal observations of the present work is that cyclophilin A mRNA as endogenous control. As expected, PMA PMA treatment of three different colon carcinoma cell lines induced MMP-9 (15.6-fold), but the addition of tetracycline expressing inducible exogenous or constitutive endogenous p53 together with PMA reduced this induction to 6.07-fold. It is accelerates and enhances cell death induced by p53 expression important to note that the mRNA was extracted from PMA + alone. Importantly, cell death triggered as a result of 5-FU– tetracycline stimulated SMF1 cells before the onset of cell death. induced endogenous p53 expression, which reflects a more To eliminate the possibility of an effect due to intrinsic properties physiologic situation than tetracycline- or ZnCl2-mediated of the tetracycline molecule (independent of p53 induction), induction of exogenously introduced p53, was augmented in we did the same experiment in the SW480 parental cells. We the presence of PMA. observed a similar induction by PMA, but the addition of Several criteria support the notion that the observed cell death tetracycline failed to down-regulate MMP-9 (data no shown). was apoptotic. First, phosphatidylserine, normally present in the These observations suggest that p53 is a negative regulator of inner layer of the cytoplasmic membrane, was externalized in all MMP-9 mRNA expression. Accordingly, MMP-9 protein levels, as three cell types. Second, SMF1 cells were positive for TUNEL assessed by gelatin zymography, were decreased by p53 (Fig. 6B). staining. Third, chromatolysis and DNA fragmentation occurred in a nonstochastic manner. Fourth, caspases were involved in this Discussion process as shown by the cleavage of PARP and caspase-3. However, Inactivation of p53-dependent cell death by mutation or z-VAD-fmk was not able to completely abrogate SMF1 cell death deletion of the TP53 gene is one of the key events in even at a high concentration (100 Amol/L), suggesting that the cell carcinogenesis. Nevertheless, many malignant tumors retain p53 death we observed is predominantly but not exclusively caspase expression and function, and an understanding of the mecha- dependent. Interestingly, mitochondria were also affected as shown nisms that potentiate p53-mediated tumor cell death as well as by the modification of the mitochondrial transmembrane potential, those that tumor cells deploy to survive in the face of p53 suggesting that the intrinsic mitochondrial apoptotic pathway is signals may have important implications in cancer treatment. also involved here. As might be expected, the use of HCT116 cells

Cancer Res 2005; 65: (10). May 15, 2005 4268 www.aacrjournals.org

both of these subfamilies are implicated in the potentiation of p53- mediated cell death. Consistent with this notion, both classic PKC and novel PKC inhibitors affected p53-dependent apoptosis but in opposite directions. Thus, bis-indolylmaleimide I, which inhibits PKCa,PKChI,PKChII, PJCg,PKCy, and PKCq isozymes, was the strongest inhibitor of PMA-dependent cell death potentiation, with Go¨6976, which inhibits PKCa and PKChI isozymes, displaying a lesser degree of inhibition. Based on these findings, it would seem that the observed PMA-associated cell death enhancement was mediated by the PKCa,PKChI,PKChII,PKCg, and PKCq isozymes. In contrast, PKCy and PKCu seemed to have an antiapoptotic effect in p53-stimulated SW480 cells. PKC targets independent of the p53 may contribute to the observed synergistic proapoptotic effect of PKC activation and p53 induction (Fig. 7). The function of PKCy, which is both a substrate and an activator of caspase-3, has been suggested to be linked to the mitochondrial release of cytochrome c during apoptosis. Consistent with these observations, the phorbol ester 12-O- Figure 6. p53 is a negative regulator of MMP-9 mRNA and protein expression. tetradecanoylphorbol-13-acetate induces the release of cytochrome A, relative quantification of MMP-9 mRNA expression by real-time PCR. SMF1 c and activation of caspase-3. Our observation that inhibition cells were stimulated with DMSO (D), 0.16 Amol/L PMA (P), 2 Amol/L of PKCy by rottlerin sensitized colon carcinoma cells to p53 in tetracycline (T), or PMA + tetracycline (PT) for 24 hours. The 2DDCT value represents MMP-9 expression relative to that in DMSO treated cells. B, MMP-9 our in vitro system may therefore be somewhat surprising and activity as illustrated by gelatin zymography. SMF1 cells were treated as would seem to contradict the evidence suggesting that PKCy is indicated for 24 hours. Columns, mean of experiments performed in triplicate; bars, SD. proapoptotic. However, rottlerin has been suggested to inhibit the intrinsic cell death pathway but to potentiate extrinsic cell death pathways (49). Thus, depending on the type of proapoptotic with homozygous deletion of Bax showed that Bax is involved in stimulus, rottlerin may be expected to exert a stimulatory or p53/PMA-mediated apoptosis. inhibitory effect. In the case of our model system, the observed The mechanism that underlies PMA-mediated potentiation of effect of rottlerin is consistent with the possibility that even if there p53-induced cell death is consistent with PKC involvement. was activation of the intrinsic pathway, as suggested by changes Phorbol esters activate the PKC family whose members in the mitochondrial transmembrane potential, the extrinsic pro- phosphorylate and thereby regulate the activity of numerous apoptotic pathway was likely to be dominant. cellular substrates, possibly including p53 itself. p53 activity is Selection of SMF1 cells that were resistant to PMA/p53-induced regulated, at least in part, by phosphorylation at several levels. cell death (termed S1R6), allowed the identification of potential First, p53 stability is increased by the activity of several kinases, mechanisms that underlie the resistance. S1R6 cells were observed including ATM, ATR, Chk1, and Chk2 (42), which directly to retain tetracycline-regulated p53 expression, such that their phosphorylate p53 and/or its negative regulator MDM2 (43). resistance could not be attributed to loss of p53 induction. Second, by phosphorylating sites at its NH2 terminus, kinases can However, comparison of the gene expression profiles of S1R6 and inactivate a nuclear export signal and thereby regulate the cellular SMF1 cells showed up-regulation of a relatively small number of localization of p53 (44). Third, the activity itself of p53 is prone to genes among which two groups seemed of immediate interest: regulation by kinases. Thus, phosphorylation of p53 has been MMPs and cell adhesion molecules. shown to control both its DNA binding ability and its Members of the MMP family, also called matrixins, are cysteine transcriptional activity. In addition, p53 can be phosphorylated proteases with zinc ion–dependent proteolytic activity (reviewed in by the c-Jun NH2-terminal protein kinases, known to be activated refs. 50–52). Thus far, >20 MMPs have been identified in mammals by PMA-induced PKC activity (45), and by the CAK (46) complex and are currently grouped according to structural features (51). The (CDK7, cyclin H, and p36MAT1). Changes in p53 phosphorylation majority of MMPs are secreted into the extracellular matrix (ECM), may therefore provide at least one mechanism to explain the a subset being membrane bound by virtue of a transmembrane or PKC-mediated sensitization of p53-dependent cell death observed PI-linked domain (51). All are produced as inactive zymogens and in this study. are activated by proteolysis. MMPs were originally described as Members of the PKC family have been shown to interact with being primarily involved in the proteolysis of ECM components p53 and modulate p53-induced apoptosis and cell growth arrest. and therefore believed to primarily mediate tissue remodeling. The classic PKCs are able to induce p53 translocation to the However, it is becoming increasingly clear that these enzymes play nucleus and the G1 cell cycle control checkpoint (47) and PKCy has a critical role in a broad range of physiologic and pathologic been shown to induce an accumulation of p53 (17). Moreover, p53 processes. Thus, MMPs can cleave and regulate the activity of a contains a PKC binding site in the central oligomerization domain host of growth factors, chemokines, and cytokines as well as cell and can be directly phosphorylated by PKCs at Ser378 and by PKCa surface adhesion receptors and proteoglycans involved in intercel- and PKC~ at Ser371 (48). In addition, a recent study has shown lular communication and cell migration that are directly implicat- activation by cyclin-dependent kinase 2/cyclin A and PKC of the ed in tumor progression and metastasis (50–52). p53-specific DNA sequence binding ability (16). Functional analysis in the present study showed that Because all novel PKCs and classic PKCs contain a phorbol ester expression of MMP-9, and to a lesser extent that of MMP-10 binding motif, it would seem reasonable to expect that either or but not MMP-12, confer partial resistance to PMA/p53-mediated www.aacrjournals.org 4269 Cancer Res 2005; 65: (10). May 15, 2005

Figure 7. Schematic representation of the effect of PKC isoforms and MMP-9 on p53-mediated cell death. PKCs directly affect p53 phosphorylation, localization, and expression at the protein level. Classic PKCs such as the a and h isoforms synergize with p53 in inducing apoptosis, by directly influencing p53 activity and/or promoting non–p53-mediated proapoptotic events, whereas PKCy has an antagonistic effect. In addition to inducing intracellular proapoptotic pathways, p53 inhibits transcription of MMP-9, which in its active form, promotes cell survival by proteolytically activating and/or inhibiting extracellular survival and proapoptotic factors, respectively. These factors may function in an autocrine and/or paracrine manner. cell death. A potential issue in our approach is the use of production and relative availability of proteolytically released and tetracycline to induce p53, because tetracycline and its derivative activated death-inducing and/or growth and survival factors. doxycyline have been shown to inhibit MMP activity and MMP-2 There are several potential mechanisms whereby MMP-9 may gene expression. However, inhibition occurs with an IC50 of 30 to promote tumor cell resistance to proapoptotic stimuli. MMP-9 300 Amol/L, whereas we used a concentration of 2 Amol/L, well can release and activate transforming growth factor h (TGF-h; ref. below the reported inhibitory levels. Accordingly, gelatin 50), cleave several chemokines and activate other MMPs including zymography assays showed no difference between lysate and MMP-1 (49–51). TGF-h can promote survival in transformed cells, supernatant MMP activity from cells treated or not with as can the chemokine IL-8, whose function is potentiated by tetracycline. The presence of tetracycline therefore seems to MMP-9–mediated proteolysis (50–52). Activation of MMP-1 leads have no effect on the activity of MMP-9 in our system at the to the release of IGF-I from ECM-sequestered IGF binding concentration used. proteins (50–52). However, the full range of MMP substrates is far Similar to PKC, MMP activity may have both proapoptotic and from elucidated, and it is quite possible that proteolytic antiapoptotic effects. Proapoptotic effects can be due to the activation or inactivation of other regulators of cell survival proteolytic degradation of ECM proteins, such as laminin, that may be implicated in the observed antiapoptotic effect of MMP-9 serve as ligands of the integrin class of cell surface adhesion expression. High expression of MMP-9 is often associated with receptors (50–52). In the absence of ligand, specific subclasses of poor prognosis in cancer patients and, in at least some tumor integrins can no longer trigger signals necessary for normal types, correlates with increased angiogenesis and metastatic epithelial and endothelial cell survival, resulting in a form of proclivity (50–52). apoptosis known as anoikis. Conversely, ECM degradation can Interestingly, regulation of MMP expression is reported to be result in the release and augmented bioactivity of potent survival subjected, at least in part, to p53 control. Thus, p53 has been factors, such as insulin-like growth factor I (IGF-I) that may shown to induce transcriptional activation of the MMP-2 (53) gene directly promote tumor cell resistance to proapoptotic signals and promoter and to attenuate the production of MMP-1 (54) and favor tumor growth. MMP-mediated cleavage of the integral MMP-13 (55). Consistent with earlier work by others (53), we have membrane ligand of the death-inducing receptor Fas (FasL) can found that MMP-2 expression is enhanced by p53 (data not shown). result in increased or reduced apoptosis depending on the In the present study, we observed that p53 represses the human physiologic context (50–52). The proapoptotic or antiapoptotic MMP-9 gene, which in and of itself may contribute to p53-induced effect of any given MMP may therefore depend on the local cellular cell death. It is also interesting to note that the two MMPs (MMP-2

Cancer Res 2005; 65: (10). May 15, 2005 4270 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. MMP-9 and MMP-10 Inhibit p53/PKC-Induced Apoptosis and MMP-9) that form the gelatinase subfamily of MMPs, based on totic PKC/p53 signals. Understanding of the precise mechanism of structural similarities and shared substrate specificity (50–52), are PKC/p53 synergy and identification of the relevant PKC targets may differentially regulated by p53. help develop therapeutic strategies that, in combination with selective The second group of genes of potential interest found to be MMP inhibitors, could augment the effectiveness of tumor cell overexpressed in S1R6 cells encode proteins involved in cell-cell elimination. communication and/or adhesion. Several are components of tight junctions (zonula occludens) and contribute to the epithelial Acknowledgments barrier function (claudin and occludin family of proteins), Received 8/16/2004; revised 12/15/2004; accepted 1/4/2005. whereas E-cadherin is part of the zonula adherens junctions that Grant support: National Center for Competence in Research Molecular Oncology, generate signals sensed by p53 (56). Although further work will be Swiss National Scientific Foundation grant 31-65090.01, and Oncosuisse grant 1267-08- required to address their role, it is conceivable that elevated 2002. The costs of publication of this article were defrayed in part by the payment of page expression of these proteins contributes to resistance to PKC/p53- charges. This article must therefore be hereby marked advertisement in accordance dependent apoptosis. with 18 U.S.C. Section 1734 solely to indicate this fact. Taken together, our observations provide evidence that PKCs are We thank Josiane Wyniger and Otto Hagenbu¨chle for providing the cDNA microarrays, Bert Vogelstein for the HCT cell lines, Phil Shaw for EB-1 cells, Richard able to potentiate p53-induced apoptosis and that expression of Iggo for the wt human p53 cDNA, and Nathalie Mayran for her advice regarding siRNA MMP-9 and MMP-10 confers partial protection against the proapop- experiments.

References tion by down-regulation of protein kinase Cy. J Biol 31. Quillet-Mary A, Jaffrezou JP, Mansat V, Bordier C, Chem 2004;279:9970–7. Naval J, Laurent G. Implication of mitochondrial 1. Schwartz D, Rotter V. p53-dependent cell cycle 18. Koriyama H, Kouchi Z, Umeda T, et al. Proteolytic hydrogen peroxide generation in ceramide-induced control: response to genotoxic stress. Semin Cancer activation of protein kinase C y and epsilon by caspase- apoptosis. J Biol Chem 1997;272:21388–95. Biol 1998;8:325–36. 3 in U937 cells during chemotherapeutic agent-induced 32. Zamzami N, Marchetti P, Castedo M, et al. Sequential 2. Hofseth LJ, Hussain SP, Harris CC. p53: 25 years after apoptosis. Cell Signal 1999;11:831–8. reduction of mitochondrial transmembrane potential its discovery. Trends Pharmacol Sci 2004;25:177–81. 19. Leverrier S, Vallentin A, Joubert D. Positive feedback and generation of reactive oxygen species in early 3. Oren M. Decision making by p53: life, death and of protein kinase C proteolytic activation during programmed cell death. J Exp Med 1995;182:367–77. cancer. Cell Death Differ 2003;10:431–42. apoptosis. Biochem J 2002;368:905–13. 33. Nunez G, Benedict MA, Hu Y, Inohara N. Caspases: 4. Koumenis C, Alarcon R, Hammond E, et al. Regulation 20. Li L, Lorenzo PS, Bogi K, Blumberg PM, Yuspa SH. the proteases of the apoptotic pathway. Oncogene of p53 by hypoxia: dissociation of transcriptional Protein kinase C y targets mitochondria, alters 1998;17:3237–45. repression and apoptosis from p53-dependent trans- mitochondrial membrane potential, and induces apo- 34. Slee EA, Zhu H, Chow SC, MacFarlane M, Nicholson activation. Mol Cell Biol 2001;21:1297–310. ptosis in normal and neoplastic keratinocytes when DW, Cohen GM. Benzyloxycarbonyl-Val-Ala-Asp (OMe) 5. Selivanova G, Wiman KG. p53: a cell cycle regulator overexpressed by an adenoviral vector. Mol Cell Biol fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by activated by DNA damage. Adv Cancer Res 1995;66:143–80. 1999;19:8547–58. blocking the processing of CPP32. Biochem J 1996;315: 6. Miyashita T, Reed JC. Tumor suppressor p53 is a direct 21. Brodie C, Blumberg PM. Regulation of cell apoptosis 21–4. transcriptional activator of the human bax gene. Cell by protein kinase C y. Apoptosis 2003;8:19–27. 35. Gschwendt M, Muller HJ, Kielbassa K, et al. Rottlerin, 1995;80:293–9. 22. Ruvolo PP, Deng X, Carr BK, May WS. A functional a novel protein kinase inhibitor. Biochem Biophys Res 7. Juven T, Barak Y, Zauberman A, George DL, Oren M. role for mitochondrial protein kinase Ca in Bcl2 Commun 1994;199:93–8. Wild type p53 can mediate sequence-specific trans- phosphorylation and suppression of apoptosis. J Biol 36. Qatsha KA, Rudolph C, Marme D, Schachtele C, May activation of an internal promoter within the mdm2 Chem 1998;273:25436–42. WS. Go 6976, a selective inhibitor of protein kinase C, gene. Oncogene 1993;8:3411–6. 23. Rodrigues NR, Rowan A, Smith ME, et al. p53 is a potent antagonist of human immunodeficiency 8. Moroni MC, Hickman ES, Denchi EL, et al. Apaf-1 is a mutations in colorectal cancer. Proc Natl Acad Sci virus 1 induction from latent/low-level-producing transcriptional target for E2F and p53. Nat Cell Biol U S A 1990;19:7555–9. reservoir cells in vitro. Proc Natl Acad Sci U S A 2001;3:552–8. 24. Shaw P, Bovey R, Tardy S, Sahli R, Sordat B, Costa J. 1993;90:4674–8. 9. Owen-Schaub LB, Zhang W, Cusack JC, et al. Wild-type Induction of apoptosis by wild-type p53 in a human 37. Martiny-Baron G, Kazanietz MG, Mischak H, et al. human p53 and a temperature-sensitive mutant induce colon tumor-derived cell line. Proc Natl Acad Sci U S A Selective inhibition of protein kinase C isozymes by Fas/APO-1 expression. Mol Cell Biol 1995;15:3032–40. 1992;89:4495–9. the indolocarbazole Go 6976. J Biol Chem 1993;268: 10. Takimoto R, El-Deiry WS. Wild-type p53 trans- 25. The R engine was used with the SMA library 9194–7. activates the KILLER/DR5 gene through an intronic (Statistic for MicroArray, Speed’s laboratory, University 38. Toullec D, Pianetti P, Coste H, et al. The bisindo- sequence-specific DNA-binding site. Oncogene 2000;19: of Berkeley, CA) and the BRAJU library (Bengtsson H., lylmaleimide GF 109203X is a potent and selective 1735–43. University of Lund, Sweden). inhibitor of protein kinase C. J Biol Chem 1991;266: 11. Leu JI, Dumont P, Hafey M, Murphy ME, George DL. 26. Sanceau J, Truchet S, Bauvois B. Matrix metal- 15771–81. Mitochondrial p53 activates Bak and causes disrup- loproteinase-9 silencing by RNA interference triggers 39. Uhm JH, Dooley NP, Villemure JG, Yong VW. Glioma tion of a Bak-Mcl1 complex. Nat Cell Biol 2004;6: the migratory-adhesive switch in Ewing’s sarcoma cells. invasion in vitro: regulation by matrix metalloprotease- 443–50. J Biol Chem 2003;278:36537–46. 2 and protein kinase C. Clin Exp Metastasis 1996;14: 12. Michael D, Oren M. The p53-Mdm2 module and the 27. Abarzua P, LoSardo JE, Gubler ML, Neri A. Microin- 421–33. ubiquitin system. Semin Cancer Biol 2003;13:49–58. jection of monoclonal antibody PAb421 into human 40. Grobelny D, Poncz L, Galardy RE. Inhibition of 13. Chipuk JE, Kuwana T, Bouchier-Hayes L, et al. Direct SW480 colorectal carcinoma cells restores the tran- human skin fibroblast collagenase, thermolysin, and activation of Bax by p53 mediates mitochondrial scription activation function to mutant p53. Cancer Res Pseudomonas aeruginosa elastase by peptide hydroxa- membrane permeabilization and apoptosis. Science 2004; 1995;55:3490–4. mic acids. Biochemistry 1992;31:7152–4. 303:1010–4. 28. Gossen M, Bujard H. Tight control of gene 41. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, 14. Momand J, Zambetti GP, Olson DC, George D, Levine expression in mammalian cells by tetracycline- Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs AJ. The mdm-2 oncogene product forms a complex with responsive promoters. Proc Natl Acad Sci U S A 1992; mediate RNA interference in cultured mammalian cells. the p53 protein and inhibits p53-mediated trans- 89:5547–51. Nature 2001;411:494–8. activation. Cell 1992;69:1237–45. 29. Martin SJ, Reutelingsperger CP, McGahon AJ, et al. 42. Caspari T. How to activate p53. Curr Biol 2000; 15. Geyer RK, Yu ZK, Maki CG. The MDM2 RING-finger Early redistribution of plasma membrane phosphati- 10:R315–7. domain is required to promote p53 nuclear export. Nat dylserine is a general feature of apoptosis regardless 43. Khosravi R, Maya R, Gottlieb T, Oren M, Shiloh Y, Cell Biol 2000;2:569–73. of the initiating stimulus: inhibition by overexpres- Shkedy D. Rapid ATM-dependent phosphorylation of 16. Pospisilova S, Brazda V, Kucharikova K, et al. sion of Bcl-2 and Abl. J Exp Med 1995;182:1545–56. MDM2 precedes p53 accumulation in response to Activation of the DNA-binding ability of latent p53 30. Boersma AW, Nooter K, Oostrum RG, Stoter G. DNA damage. Proc Natl Acad Sci U S A 1999;96: protein by protein kinase C is abolished by protein Quantification of apoptotic cells with fluorescein 14973–7. kinase CK2. Biochem J 2004;378:939–47. isothiocyanate-labeled Annexin V in Chinese hamster 44. Zhang Y, Xiong Y. A p53 amino-terminal nuclear 17. Abbas T, White D, Hui L, Yoshida K, Foster DA, ovary cell cultures treated with cisplatin. Cytometry export signal inhibited by DNA damage-induced Bargonetti J. Inhibition of human p53 basal transcrip- 1996;24:123–30. phosphorylation. Science 2001;292:1910–5.

www.aacrjournals.org 4271 Cancer Res 2005; 65: (10). May 15, 2005

45. Hu MC, Qiu WR, Wang YP. JNK1, JNK2 and JNK3 are 49. Basu A, Miura A. Differential regulation of extrinsic metalloproteinase 2) promoter. Mol Cell Biol 1997;17: p53 N-terminal serine 34 kinases. Oncogene 1997;15: and intrinsic cell death pathways by protein kinase C. 6330–8. 2277–87. Int J Mol Med 2002;10:541–5. 54. Sun Y, Sun Y, Wenger L, Rutter JL, Brinckerhoff CE, 46. Ko LJ, Shieh SY, Chen X, et al. p53 is phosphorylated 50. Stamenkovic I. Extracellular matrix remodelling: the Cheung HS. p53 down-regulates human matrix metal- by CDK7-cyclin H in a p36MAT1-dependent manner. role of matrix metalloproteinases. J Pathol 2003;200: loproteinase-1 (collagenase-1) gene expression. J Biol Mol Cell Biol 1997;17:7220–9. 448–64. Chem 1999;274:11535–40. 47. Scotto C, Delphin C, Deloulme JC, Baudier J. 51. Egelblad M, Werb Z. New functions for the matrix 55. Ala-aho R, Grenman R, Seth P, Kahari VM. Adenoviral Concerted regulation of wild-type p53 nuclear accumu- metalloproteinases in cancer progression. Nat Rev delivery of p53 gene suppresses expression of collage- lation and activation by S100B and calcium-dependent Cancer 2002;2:161–74. nase-3 (MMP-13) in squamous carcinoma cells. Onco- protein kinase C. Mol Cell Biol 1999;19:7168–80. 52. McCawley LJ, Matrisian LM. Matrix metalloprotei- gene 2002;21:1187–95. 48. Youmell M, Park SJ, Basu S, Price BD. Regulation of nases: they’re not just for matrix anymore! Curr Opin 56. Yamaguchi M, Hirose F, Inoue YH, et al. Genetic the p53 protein by protein kinase C a and protein Cell Biol 2001;13:534–40. link between p53 and genes required for formation kinase C ~. Biochem Biophys Res Commun 1998;245: 53. Bian J, Sun Y. Transcriptional activation by p53 of the of the zonula adherens junction. Cancer Sci 2004;95: 514–8. human type IV collagenase (gelatinase A or matrix 436–41.

Cancer Res 2005; 65: (10). May 15, 2005 4272 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Matrix Metalloproteinases 9 and 10 Inhibit Protein Kinase C− Potentiated, p53-Mediated Apoptosis

Eric Meyer, Jean-Yves Vollmer, Raymonde Bovey, et al.

Cancer Res 2005;65:4261-4272.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/65/10/4261

Cited articles This article cites 53 articles, 22 of which you can access for free at: http://cancerres.aacrjournals.org/content/65/10/4261.full#ref-list-1

Citing articles This article has been cited by 6 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/65/10/4261.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/65/10/4261. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.