Supplementary Table S1 from Mitotic Arrest, Apoptosis, and Sensitization to Chemotherapy of Melanomas by Methionine Deprivation Stress

O(6)-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, reverses mutagenic and cytotoxic effects of O(6)-alkylguanine in DNA induced by chemotherapeutic N-alkyl N-nitrosourea and procarbazine type drugs by dealkylating the adduct. MGMT expression is down-regulated by wild-type p53 (WTp53) in human tumor cells. Here we report that p53 sequesters the Sp1 transcription factor to prevent its binding to the cognate cis elements in the MGMT promoter and thus inhibits MGMT expression. Sp1 overexpression abrogated the inhibitory effect of p53 on the MGMT promoter activity in a dose-dependent manner. Stable interaction of Sp1 with WTp53 was observed in HCT116 cells. Moreover, WTp53 overexpression reduced the binding of the nuclear extract to the Sp1 consensus sequence, even though recombinant p53 alone did not bind to the same sequence. Taken together, these results suggest that sequestration of Sp1 could be one of the mechanisms by which p53 negatively regulates MGMT expression, thus enhancing sensitivity of tumor cells to O(6)-alkylguanine generating drugs.

O6-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, reverses mutagenic and cytotoxic effects of O6-alkylguanine in DNA induced by chemotherapeutic N-alkyl N-nitrosourea and procarbazine type drugs by dealkylating the adduct. MGMT expression is down-regulated by wild-type p53 (WTp53) in human tumor cells. Here we report that p53 sequesters the Sp1 transcription factor to prevent its binding to the cognate cis elements in the MGMT promoter and thus inhibits MGMT expression. Sp1 overexpression abrogated the inhibitory effect of p53 on the MGMT promoter activity in a dosedependent manner. Stable interaction of Sp1 with WTp53 was observed in HCT116 cells. Moreover, WTp53 overexpression reduced the binding of the nuclear extract to the Sp1 consensus sequence, even though recombinant p53 alone did not bind to the same sequence. Taken together, these results suggest that sequestration of Sp1 could be one of the mechanisms by which p53 negatively regulates MGMT expression, thus enhancing sensitivity of tumor cells to O6alkylguanine generating drugs. Alkylation of the O6-position of guanine in DNA, induced by methylating (temozolomide, procarbazine) and chloroethylating [carmustine (BCNU); nimustine, (ACNU)], is considered to be the most mutagenic and carcinogenic lesion (1, 2). Alkylating agents such as nitrogen mustards, procarbazine and nitrosoureas are among the most widely used chemotherapeutic agents. Mutagenic and cytotoxic adducts are removed from the O6 position of guanine by O6 methylguanine-DNA methyltransferase (MGMT) (3-6). MGMT is a ubiquitous DNA repair protein that acts in a stoichiometric reaction in which an alkyl group attached to the O6 position of guanine is transferred to a specific cysteine residue in the protein’s active site [reviewed in (7, 8)]. This in situ dealkylation restores the original guanine in DNA while inactivating the protein MGMT (9). The MGMT level varies widely in both normal and tumor tissues and in tumor cell lines. Approximately 20% of in vitro transformed tumor cell lines have no detectable MGMT and are highly sensitive to methylating and chloroethylating agents; these are called Mer–/Mex– (10-14). Several studies showed that human MGMT down-regulation in human Mer–/Mex– cells was linked to the presence of methylated CpGs in the promoter region, while other studies indicated that regulation of MGMT expression was complex (15-18). Increased MGMT expression was observed in rat hepatoma cells after treatment with alkylating agents or UV light (19). One recent study showed that treatment with cysteine prodrugs and herbal antioxidant such as curcumin also increased the MGMT level in many cancer cells (20). Tumor cells with high levels of MGMT are known to have high resistance to BCNU, and depletion of MGMT activity by pseudosubstrates (such as O6-benzylguanine, O6-BG) reverses this resistance (4, 21, 22). Hence the level of MGMT expression could be a key determinant in tumor cell resistance to alkylating agents. The MGMT promoter, like that of many housekeeping genes, does not have any TATA or CAAT boxes, but contain six putative Sp1-binding sequences, two glucocorticoid responseelements (GRE-1), and two activator protein-1 (AP-1) binding sequences (23-27). The involvement of Sp1, AP-1, GRE-1 in MGMT expression has been previously documented (26, 27). Interestingly, in spite of the lack of p53-binding sites in the MGMT promoter, several studies have explored the impact of 3741 *Present Address: Department of Investigational Cancer Therapeutics, The University of Texas, M.D. Anderson Cancer Center, Mail Unit 455, Houston, TX 77030, U.S.A. Correspondence to: Kishor K. Bhakat, Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 6.136 Medical Research building, Route 1079. Galveston, TX 77555, U.S.A. Tel: +1 4097721779, Fax: +1 4097478608. e-mail: kkbhakat@utmb.edu

Melanoma is the most malignant of skin cancers, highly resistant to chemotherapy and radiotherapy. Temozolomide, a promising new derivative of dacarbazine, is currently being tested for treatment of metastatic melanoma. Resistance to alkylating agents such as temozolomide correlates with increased expression of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Interleukin-24 (IL-24; mda-7) is a tumor suppressor cytokine that selectively inhibits tumor cell growth by inducing apoptosis and cell cycle arrest in melanoma cell lines and solid tumors. This tumor-selective activity has been observed in multiple preclinical animal models and in clinical trials. In this study, we analyzed the ability of Ad-IL-24 and its protein product, IL-24, to overcome temozolomide resistance in human melanoma cells. We have shown that Ad-IL-24 via exogenous IL-24 protein induces combinatorial synergy of temozolomide-induced cell killing in temozolomide-resistant melanoma cells by inhibition of MGMT. Neutralizing antibodies against IL-24 or its receptors significantly blocked the apoptotic activity of IL-24 + MGMT treatment. We show that accumulation of functional p53 is essential for IL-24-induced down-regulation of MGMT. Using either MGMT small interfering RNA, p53 small interfering RNA, or a p53 dominant-negative mutant to block MGMT protein expression resulted in increased sensitization to temozolomide. However, MGMT blockade in combination with IL-24 + temozolomide resulted in loss of combinatorial synergy, indicating that MGMT expression is required for the reversal of temozolomide resistance in melanoma cells. This study shows that IL-24 can play a significant role in overcoming temozolomide resistance and that the clinical efficacy of temozolomide may be improved by using a biochemotherapy combination with IL-24. [Mol Cancer Ther 2008;7(12):3842–51]

Conventional cancer treatments include cytotoxic chemotherapies and radiotherapy, which result in significant collateral toxicities. The goal for future cancer treatments is to leverage improved understanding of cancer biology mechanisms and thereby develop targeted drugs that display exquisite tumor selectivity and avoid iatrogenic damage. In this review, we discuss the potential of tumor suppressor genes for development of cancer-selective drugs using the tumor suppressor p53 as an archetype.

PURPOSE:Activation of the double-stranded RNA-activated protein kinase (PKR) leads to the induction of various pathways including the down-regulation of translation through phosphorylation of the eukaryotic translation initiation factor 2alpha (eIF-2alpha). There have been no reports to date about the role of PKR in radiation sensitivity.EXPERIMENTAL DESIGN:A clonogenic survival assay was used to investigate the sensitivity of PKR mouse embryo fibroblasts (MEF) to radiation therapy. 2-Aminopurine (2-AP), a chemical inhibitor of PKR, was used to inhibit PKR activation. Nuclear factor-kappaB (NF-kappaB) activation was assessed by electrophoretic mobility shift assay (EMSA). Expression of PKR and downstream targets was examined by Western blot analysis and immunofluorescence.RESULTS:Ionizing radiation leads to dose- and time-dependent increases in PKR expression and function that contributes to increased cellular radiation resistance as shown by clonogenic survival and terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) apoptosis assays. Specific inhibition of PKR with the chemical inhibitor 2-AP restores radiation sensitivity. Plasmid transfection of the PKR wild-type (wt) gene into PKR(-/-) MEFs leads to increased radiation resistance. The protective effect of PKR to radiation may be mediated in part through NF-kappaB and Akt because both NF-kappaB and Akt are activated after ionizing radiation in PKR+/+ but not PKR-/- cells.CONCLUSIONS:We suggest a novel role for PKR as a mediator of radiation resistance modulated in part through the protective effects of NF-kappaB and Akt activation. The modification of PKR activity may be a novel strategy in the future to overcome radiation resistance.

The melanoma differentiation-associated gene-7 (mda-7/IL-24) is a unique member of the interleukin 10 (IL-10) family of cytokines, with ubiquitous tumor cell pro-apoptotic activity. Recent data have shown that IL-24 is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and as a potent anti-angiogenic molecule. In this study, we analyzed the activity of Ad-mda7 and its protein product, secreted IL-24, against human breast cancer cells. We show that Ad-mda7 transduction of human breast cancer cells results in G(2)/M phase cell cycle arrest and apoptotic cell death, which correlates with secretion of IL-24 protein. Neutralizing antibody against IL-24 significantly inhibited Ad-mda7 cytotoxicity. IL-24 and IL-10 both engage their cognate receptors on breast cancer cells resulting in phosphorylation and activation of STAT3, however, IL-10 receptor binding failed to induce cell killing, indicating that tumor cell killing by IL-24 is independent of STAT3 phosphorylation. Treatment with exogenous IL-24 induced apoptosis in breast cancer cells and this effect was abolished by addition of anti-IL-24 antibody or anti-IL-20R1, indicating that bystander cell killing is mediated via IL-24 binding to the IL-20R1/IL-20R2 heterodimeric receptor complex. Co-administration of the related cytokine IL-10 inhibited killing mediated by IL-24 and concomitantly inhibited IL-24 mediated up-regulation of the tumor suppressor proteins, p53 and p27(Kip1). In summary, we have defined a tumor-selective cytotoxic bystander role for secreted IL-24 protein and identified a novel receptor-mediated death pathway in breast cancer cells, wherein the related cytokines IL-24 and IL-10 exhibit antagonistic activity.

Adenovirus-mediated mda-7 (Ad-mda7) gene transfer has been shown to induce apoptosis in various human cancer cells while sparing normal cells. Vitamin E succinate (VES) is also known to exhibit antitumor activity against a number of human cancer cell lines. We hypothesized that a combination of the two agents would produce an enhanced antitumor effect in MDAH2774 human ovarian cancer cells. Treatment of MDAH2774 cells with Ad-mda7 plus VES resulted in enhanced antitumor activity that involved the activation of two apoptotic pathways. Activation of the extrinsic pathway was demonstrated by increased cell-surface Fas expression and cleavage of Bid and caspase-8. Activation of the intrinsic pathway was demonstrated by disruption of mitochondrial potential; and activation of downstream capase-9 and caspase-3 via cytochrome C release. In contrast, the combination of Ad-mda7 plus VES did not show any antitumor activity against normal fibroblasts, indicating selective tumor cell killing. Our in vitro results provide a basis for further preclinical testing of Ad-mda7 plus VES as a potential cancer treatment strategy.

The protein encoded by the melanoma differentiation-association gene 7 (MDA-7/IL-24) is a novel interleukin (IL)-10 family cytokine with unique tumor-specific apoptotic and antiangiogenic properties that make it especially attractive for use in cancer gene therapy applications. Mda-7 gene transfer with a replication incompetent adenoviral vector (Ad-mda7) induces apoptosis in a tumor specific manner, an effect that is independent of the status of other tumor suppressor genes, such as p53, Rb, or pl6INK4. In addition to its direct cytotoxic effects, Ad-mda7 transduction causes secretion of a processed, glycosylated form of MDA-7 protein. MDA-7 is a novel interleukin (IL-24) with unique apoptotic functions. Studies on the secreted MDA-7/IL-24 protein have shown that it can act as a pro-Thl cytokine, and induces secretion of interferon-gamma, tumor necrosis factor-α, IL-6, IL-12, and granulocyte macrophage colony-stimulating factor in human peripheral blood mononuclear cells. Additional studies in syngeneic mice indicate that MDA-7 can function as an immune adjuvant and enhance immune reactivity against tumors. More recently, our group demonstrated that MDA-7 protein functions as a potent antiangiogenic factor in vitro and in vivo that is 50-fold more active than angiostatin or endostatin. In phase I clinical trials intratumoral delivery of Ad-mda7 showed tumor regression in patients with advanced carcinomas who failed conventional therapies. This chapter provides a comprehensive perspective on MDA-7/IL-24 research, highlighting its proapoptotic, antimetastatic, and antiangiogenic properties. The combination of these potent effector mechanisms makes mda-7/IL-24 a promising and novel approach for the treatment of cancer.

The early discoveries elucidating p53 function were based on cell culture experiments. Most of our fundamental knowledge of the role of p53 in cell signaling, stress response, cell cycle control, and apoptosis are a result of these in vitro studies (Giaccia and Kastan, 1998; Ko and Prives, 1996; Levine, 1997; Vogelstein et al., 2000). However, a greater depth of understanding was facilitated by the advent first of transgenic mouse methodologies and then by embryonic stem (ES) cell-based genetic manipulations. The sequencing of the mouse genome (www.ensembl.org and www.myscience.appliedbiosystems.com) has greatly simplified and accelerated the generation of null alleles. Methods have been developed to generate single nucleotide substitutions in the germline of mice, and importantly, to generate somatic mutations in genes to study somatic inactivation as occurs in most human cancers. The availability of whole genome analysis at the RNA expression level (arrays) and at the genomic level (array CGH) provides another level of analysis that is sure to provide insights into the molecular changes that lead to the initiation, progression, and maintenance of the tumor phenotype.

The melanoma differentiation-associated gene-7 (mda-7) is a member of the interleukin-10 cytokine family and a novel tumor suppressor gene. Adenoviral-mediated mda-7 (Ad-mda7) gene transfer has tumor-specific growth inhibitory and proapoptotic effects in a broad spectrum of cancer cells. In breast cancer cells, adenoviral-induced mda-7 expression triggers antiproliferative effects by downregulation of survival signals, such as Bcl-2 and Akt. The anti-human epidermal growth factor receptor-2 (Her-2) monoclonal antibody, Trastuzumab (Herceptin), increases the sensitivity of Her-2/neu-overexpressing breast cancer cells to chemotherapeutic agents and radiotherapy. In this study, we evaluate the effects of treatment with Ad-mda7 and Herceptin combination therapy in a panel of Her-2/neu-overexpressing cell lines, and in established tumors in nude mice. Compared to individual treatments, the combination of Ad-mda7 and Herceptin elicits supra-additive antitumor activity in Her-2/neu-overexpressing tumor cell lines: increased cell death, cell cycle block and apoptosis. The Ad-mda7 and Herceptin interaction was shown to be synergistic by isobologram analysis. Ad-mda7 does not alter cell surface Her-2/neu levels, but the combination of Ad-mda7+Herceptin results in increased expression of cell surface E-cadherin with concomitant translocation of β-catenin from the nucleus to the cell membrane. In vivo, the combination of Ad-mda7 and Herceptin showed significantly increased antitumor activity (P<0.003) against Her-2/neu-overexpressing tumors. These data suggest that the combination of Ad-mda7 with Herceptin may be a novel therapy for breast cancer patients whose tumors overexpress Her-2/neu. The observed synergistic effect may improve treatment options for otherwise poorly responsive, Her-2-positive, breast cancer patients.

Abstract Methionine deprivation stress (MDS) eliminates mitotic activity in melanoma cells regardless of stage, grade, or TP53 status, whereas it has a negligible effect on normal skin fibroblasts. In most cases, apoptosis accounts for the elimination of up to 90% of tumor cells from the culture within 72 hours after MDS, leaving a scattered population of multinucleated resistant cells. Loss of mitosis in tumor cells is associated with marked reduction of cyclin-dependent kinase (CDK) 1 transcription and/or loss of its active form (CDK1-P-Thr161), which is coincident with up-regulation of CDKN1A, CDKN1B, and CDKN1C (p21, p27, and p57). Expression of the proapoptotic LITAF, IFNGR, EREG, TNFSF/TNFRSF10 and TNFRSF12, FAS, and RNASEL is primarily up-regulated/induced in cells destined to undergo apoptosis. Loss of Aurora kinase B and BIRC5, which are required for histone H3 phosphorylation, is associated with the accumulation of surviving multinucleated cells. Nevertheless, noncycling survivors of MDS are sensitized to temozolomide, carmustin, and cisplatin to a much greater extent than normal skin fibroblasts possibly because of the suppression of MGMT/TOP1/POLB, MGMT/RAD52/RAD54, and cMET/RADD52, respectively. Sensitivity to these and additional genotoxic agents and radiation may also be acquired due to loss of cMET/OGG1, reduced glutathione reductase levels, and a G2-phase block that is a crucial step in the damage response associated with enhancement of drug toxicity. Although the genes controlling mitotic arrest and/or apoptosis in response to low extracellular methionine levels are unknown, it is likely that such control is exerted via the induction/up-regulation of tumor suppressors/growth inhibitor genes, such as TGFB, PTEN, GAS1, EGR3, BTG3, MDA7, and the proteoglycans (LUM, BGN, and DCN), as well as the down-regulation/loss of function of prosurvival genes, such as NFκB, MYC, and ERBB2. Although MDS targets several common genes in tumors, mutational variability among melanomas may decide which metabolic and signal transduction pathways will be activated or shutdown. (Mol Cancer Res 2006;4(8):575–89)

Our previous studies demonstrated that adenovirus-mediated overexpression of melanoma differentiation-associated gene-7 (Ad-mda7) leads to rapid induction of double-stranded RNA-dependent protein kinase (PKR) and activation of its downstream targets, resulting in apoptosis induction in human lung cancer cells. Here, we report that Ad-mda7 and the benzoquinone ansamycin geldanamycin (GA) interact in a highly synergistic manner to induce cell death in human lung cancer cells. Co-administration of Ad-mda7 and GA did not modify expression of MDA-7, and was not associated with further PKR induction and activation; instead the enhanced cytotoxicity of this combination was associated with inactivation of AKT by GA. By surface staining using anti-E-cadherin monoclonal antibody and flow cytometry, we found that treatment with the combination of Ad-mda7 and GA increased E-cadherin levels in these cancer cells. Ad-mda7 and GA cotreatment also inhibited lung cancer cell motility by increasing the β -catenin/E-cadherin association. Moreover, combination of GA derivative 17-allyl-amino, 17-demethoxygeldanamycin (17AAG), with Ad-mda7 resulted in enhancement of cell death in A549 and H460 human lung cancer cells.

712We have previously demonstrated that adenoviral mediated overexpression of mda-7 (Ad-mda7) leads to a rapid induction and activation of PKR, which correlates with apoptosis in A549 and H1299 human lung cancer cells. To our surprise, we found that Ad-mda7 induced activation of not only PKR, but also AKT in these cells. Western analysis revealed that Ad-mda7-mediated activation of AKT is induced by phosphorylation at the S437 site, in A549 and H1299 human lung cancer cells in a time- and dose-dependent manner. Inhibition of phospho-AKT by geldanamycin (GA) enhances Ad-mda7-mediated cell killing in these cells. Additionally, PKR siRNA 48 h treatment of these cells induces a dramatic down-regulation of PKR and phospho-AKT protein levels. To determine whether PKR is required for activation of AKT, we used Western blot analysis to evaluate the effects of PKR, phospho-PKR, AKT, and …

B52 The p53 signaling pathway integrates diverse environmental stimuli and serves as the nodal point for regulation of cell proliferation or cell death via apoptosis. This pathway is disrupted or dysfunctional in >50% of human tumors. In squamous cell carcinoma of the head and neck (SCCHN), overexpression of p53 protein (p53 + ) is associated with poor patient outcome and poor tumor response to therapy. With the goal of evaluating p53 pathway markers, we have used immunohistochemical (IHC) analysis to evaluate tumor samples from 64 individuals with SCCHN. Overexpression of p53 protein (p53 + ) was found in 47/64 (73.4%) samples examined, as defined by ≥20% of cells positive for nuclear staining using the monoclonal DO7 antibody. Additional p53 pathway proteins were also examined, including HDM2, Bcl-2, p14 ARF , and survivin. Utilizing ≥20% of cells positive as criteria for expression, 47.6% (30/63) of tumors analyzed expressed HDM2, 27.0% (17/63) Bcl-2, 21.9% (14/64) p14 ARF , and 74.6% (44/59) survivin. A subset of the individuals with recurrent SCCHN was treated in Phase II clinical trials using p53 gene therapy (Advexin; Adenovirus-p53). In order to determine whether any of these proteins could potentially be used as a predictive biomarker for p53 gene therapy efficacy, analysis was carried out on the subset of patients treated with Advexin for whom clinical outcome information was available (n=18). Of the markers evaluated, only p53 protein overexpression had a statistically significant correlation with locoregional tumor disease control (CR, PR, or SD, all of >3 months duration) and increased median survival following Advexin treatment. 72.7% of patients with p53 + tumors demonstrated locoregional disease control, compared to only 14% of patients with p53 - tumors (p=0.05). In addition, the median survival of patients with tumors overexpressing p53 protein (p53 + ) was 11.0 months (95% CI 6.5-16.0), compared to only 3.0 months (95% CI 1.5-3.5) in patients whose tumors were p53 - (p

The melanoma differentiation-associated gene-7 (mda-7/IL-24) is a unique member of the interleukin 10 (IL-10) family of cytokines, with ubiquitous tumor cell pro-apoptotic activity. Recent data have shown that IL-24 is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and as a potent anti-angiogenic molecule. In this study, we analyzed the activity of Ad-mda7 and its protein product, secreted human IL-24, against human breast cancer cells. We show that Ad-mda7 transduction of breast cancer cells results in G2/M phase cell-cycle arrest and killing, which correlates with secretion of IL-24 protein. Neutralizing antibody against IL-24 significantly inhibited Ad-mda7 killing. IL-24 and IL-10 both engage their cognate receptors on breast cancer cells resulting in phosphorylation and activation of STAT3, however, IL-10 receptor binding failed to induce cell killing, indicating that tumor cell killing by IL-24 is independent of STAT3 phosphorylation. Exogenous L-24 killed breast cancer cells by induction of apoptosis and this effect was abolished by addition of anti-IL-24 antibody or anti-IL-20R1, indicating that bystander cell killing is mediated via IL-24 binding to the IL-20R1/IL-20R2 heterodimeric receptor complex. Furthermore, IL-10 treatment inhibits killing mediated by IL-24. In summary, we have defined a tumor-selective cytotoxic bystander role for secreted IL-24 protein and identified a novel receptor- mediated death pathway in breast cancer cells, wherein the related cytokines IL-24 and IL-10 exhibit antagonistic activity. Sunil Chada, Minghong Zheng, and Dora Bocangel are Introgen employees.