Dora Bocangel

Human interleukin 24 (MDA-7/IL-24) protein kills breast cancer cells via the IL-20 receptor and is antagonized by IL-10

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 G2/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 p27Kip1. 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.

mda-7 gene transfer sensitizes breast carcinoma cells to chemotherapy, biologic therapies and radiotherapy: correlation with expression of bcl-2 family members.

Current therapies used in the treatment of breast cancer are limited by systemic toxicity, rapid drug metabolism and intrinsic and acquired drug resistance. We have previously shown that adenoviral-mediated transfer of the melanoma differentiation-associated gene-7 (mda-7) elicits growth inhibition and apoptosis in various tumor types. Here, we evaluate the effects of Ad-mda7, alone and in combination with other therapies, against a panel of nine breast tumor cell lines and their normal counterparts; we report selective Ad-mda7-mediated p53-independent growth inhibition, G2/M cell cycle arrest, and apoptosis. In vivo, Ad-mda7 induced p53-independent tumor growth inhibition (P<0.004) in multiple xenograft models. We then evaluated the combination of Ad-mda7 with agents commonly used to treat breast cancer: radiotherapy (XRT), Tamoxifen, Taxotere, Adriamycin, and Herceptin. These agents exhibit diverse modes of action, including formation of bulky adducts, inhibition of DNA replication (Adriamycin, XRT), damage to microtubules (Taxotere), nonsteroidal estrogen antagonists (Tamoxifen), or Her2/neu receptor blockade (Herceptin). Treated with conventional anticancer drugs or radiation, MDA-7-expressing cells display additive or synergistic cytotoxicity and apoptosis that correlates with decreased BCL-2 expression and BAX upregulation. In vivo, animals that received Ad-mda7 and XRT underwent significant reduction of tumor growth (P<0.002). This is the first report of the synergistic effects of Ad-mda7 combined with chemotherapy or radiotherapy on human breast carcinoma cells.

Interleukin-24 overcomes temozolomide resistance and enhances cell death by down-regulation of O6-methylguanine-DNA methyltransferase in human melanoma cells

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]

Combinatorial synergy induced by adenoviral-mediated mda-7 and Herceptin in Her-2+ breast cancer cells

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.

The Double-Stranded RNA-Activated Protein Kinase Mediates Radiation Resistance in Mouse Embryo Fibroblasts through Nuclear Factor κB and Akt Activation

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 2α (eIF-2α). 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-κB (NF-κB) 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-κB and Akt because both NF-κB 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-κB and Akt activation. The modification of PKR activity may be a novel strategy in the future to overcome radiation resistance.

Enhancement of adenoviral MDA-7-mediated cell killing in human lung cancer cells by geldanamycin and its 17-allyl- amino-17-demethoxy analogue.

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.

287. mda-7: A Remedy for Oncogene Addiction in Breast Cancer?

The concept of |[ldquo]|oncogene addiction|[rdquo]| has been proposed to support the rationale for development of molecularly targeted therapies. It is clear that acquisition of genetic mutations results in dysregulation of oncogenic signaling pathways in tumor cells, and recent studies have indicated that persistence of these dysregulated oncogenes is essential for maintaining the tumorigenic phenotype. We evaluated the role of mda-7 gene transfer in oncogene-addicted breast cancer cells. Current therapies used in the treatment of breast cancer are limited by systemic toxicity, rapid drug metabolism and intrinsic and acquired drug resistance. We previously showed that adenoviral mediated transfer of the melanoma differentiation-associated gene-7 (mda-7) elicits growth inhibition and apoptosis in various tumor types. Here, we evaluate the effects of Ad-mda7, alone and in combination with other therapies, against a panel of nine breast tumor cell lines and their normal counterparts; we report tumor-selective p53-independent growth inhibition, G2/M cell cycle arrest, and apoptosis induction by Ad-mda7. In vivo, Ad-mda7 induced p53-independent tumor growth inhibition (p<0.004) in multiple xenograft models. We then evaluated the combination of Ad-mda7 with agents commonly used to treat breast cancer: radiotherapy (XRT), Tamoxifen, Taxotere, Adriamycin, and Herceptin. These agents exhibit diverse modes of action, including formation of bulky adducts, inhibition of DNA replication (Adriamycin, XRT), damage to microtubules (Taxotere), non-steroidal estrogen antagonists (Tamoxifen), or Her2/neu receptor blockade (Herceptin). Treated with conventional anti-cancer drugs or radiation, MDA-7-expressing cells display additive or synergistic cytotoxicity and apoptosis that correlates with decreased BCL-2 expression and BAX up-regulation. In vivo, animals that received Ad-mda7 and XRT underwent significant reduction of tumor growth (p<0.002). This is the first report of the synergistic effects of Ad-mda7 combined with chemotherapy or radiotherapy on human breast carcinoma cells, and suggests that mda-7 gene transfer can overcome the oncogene addiction observed in breast cancer.