Local and systemic inhibition of lung tumor growth after nanoparticle-mediated mda-7/IL-24 gene delivery

The human melanoma differentiation associated gene-7 (mda-7), also known as interleukin-24 (IL-24), is a novel gene with tumor suppressor, antiangiogenic, and cytokine properties. In vitro adenovirus-mediated gene transfer of the human mda-7/IL-24 gene (Ad-mda-7) results in ubiquitous growth suppression of human cancer cells with minimal toxicity to normal cells. Intratumoral administration of Ad-mda-7 to lung tumor xenografts results in growth suppression via induction of apoptosis and antiangiogenic mechanisms. Although these results are encouraging, one limitation of this approach is that its locoregional clinical application-systemic delivery of adenoviruses for treatment of disseminated cancer is not feasible at the present time. An alternative approach that is suitable for systemic application is non-viral gene delivery. We recently demonstrated that DOTAP:cholesterol (DOTAP:Chol) nanoparticles effectively deliver tumor suppressor genes to primary and disseminated lung tumors. In the present study, therefore, we evaluated nanoparticle-mediated delivery of the human mda-7/IL-24 gene to primary and disseminated lung tumors in vivo. We demonstrate that DOTAP:Chol efficiently delivers the mda-7/IL-24 gene to human lung tumor xenografts, resulting in suppression of tumor growth. Growth-inhibitory effects were observed in both primary (P=0.001) and metastatic lung tumors (P=0.02). Furthermore, tumor vascularization was reduced in mda-7/IL-24-treated tumors. Finally, growth was also inhibited in murine syngenic tumors treated with DOTAP:Chol-mda-7 nanoparticles (P=0.01). This is the first report demonstrating (1) systemic therapeutic effects of mda-7/IL-24 in lung cancer, and (2) antitumor effects of human mda-7 in syngeneic cancer models. Our findings are important for the development of mda-7/IL-24 treatments for primary and disseminated cancers.     

mda-7/IL-24 differentially regulates soluble and nuclear clusterin in prostate cancer

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), a unique member of the IL-10 gene family, displays a broad range of antitumor properties including cancer-specific induction of apoptosis, inhibition of tumor angiogenesis, and modulation of anti-tumor immune responses. Here, we identify clusterin (CLU) as a MDA-7/IL-24 interacting protein in DU-145 cells and investigate the role of MDA-7/IL-24 in regulating CLU expression and mediating the antitumor properties of mda-7/IL-24 in prostate cancer. Ad.mda-7 decreased expression of soluble CLU (sCLU) and increased expression of nuclear CLU (nCLU). In the initial phase of Ad.mda-7 infection sCLU expression increased and CLU interacted with MDA-7/IL-24 producing a cytoprotective effect. Infection of stable clones of DU-145 prostate cancer cells expressing sCLU with Ad.mda-7 resulted in generation of nCLU that correlated with decreased cell viability and increased apoptosis. In the presence of mda-7/IL-24, sCLU-DU-145 cells displayed G(2)/M phase arrest followed by apoptosis. Similarly, Ad.mda-7 infection decreased cell migration by altering cytoskeleton in sCLU-DU-145 cells. Ad.mda-7-treated sCLU-DU-145 cells displayed a significant reduction in tumor growth in mouse xenograft models and reduced angiogenesis when compared to the vector control group. Tumor tissue lysates demonstrated enhanced nCLU generated from sCLU with increased apoptosis in the presence of MDA-7/IL-24. Our findings reveal novel aspects relative to the role of sCLU/nCLU in regulating the anticancer properties of MDA-7/IL-24 that may be exploited for developing enhanced therapies for prostate cancer.     

Ionizing radiation enhances adenoviral vector expressing mda-7/IL-24-mediated apoptosis in human ovarian cancer

Ovarian cancer is the fifth most common cause of cancer-related death in women. Current interventional approaches, including debulking surgery, chemotherapy, and/or radiation have proven minimally effective in preventing the recurrence and/or mortality associated with this malignancy. Subtraction hybridization applied to terminally differentiating human melanoma cells identified melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), whose unique properties include the ability to selectively induce growth suppression, apoptosis, and radiosensitization in diverse cancer cells, without causing any harmful effects in normal cells. Previously, it has been shown that adenovirus-mediated mda-7/IL-24 therapy (Ad.mda-7) induces apoptosis in ovarian cancer cells, however, the apoptosis induction was relatively low. We now document that apoptosis can be enhanced by treating ovarian cancer cells with ionizing radiation (IR) in combination with Ad.mda-7. Additionally, we demonstrate that mda-7/IL-24 gene delivery, under the control of a minimal promoter region of progression elevated gene-3 (PEG-3), which functions selectively in diverse cancer cells with minimal activity in normal cells, displays a selective radiosensitization effect in ovarian cancer cells. The present studies support the use of IR in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in ovarian cancer, particularly in the context of tumors displaying resistance to radiation therapy.     

IL-24选择性诱导肿瘤细胞凋亡机制

白细胞介素24/MDA-7基因是在黑色素瘤分化过程中被分离得到的,具有抑制黑色素瘤增生和促进其终末分化的能力。IL-24作为细胞因子不仅具有免疫调节功能,而且能选择性诱导多种肿瘤细胞凋亡。这种抑制作用不依赖p53、Rb和p16等抑癌基因,对正常细胞没有影响。白细胞介素24具有显著的选择性抗肿瘤特性,成为肿瘤治疗的研究新热点。     

Enhanced delivery of mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) in combination with the Apogossypol derivative BI-97C1 (Sabutoclax) improves therapeutic efficacy in low CAR colorectal cancer cells

Adenovirus (Ad)-based gene therapy represents a potentially viable strategy for treating colorectal cancer. The infectivity of serotype 5 adenovirus (Ad.5), routinely used as a transgene delivery vector, is dependent on Coxsackie-adenovirus receptors (CAR). CAR expression is downregulated in many cancers thus preventing optimum therapeutic efficiency of Ad.5-based therapies. To overcome the low CAR problem, a serotype chimerism approach was used to generate a recombinant Ad (Ad.5/3) that is capable of infecting cancer cells via Ad.3 receptors in a CAR-independent manner. We evaluated the improved transgene delivery and efficacy of Ad.5/3 recombinant virus expressing melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an effective wide-spectrum cancer-selective therapeutic. In low CAR human colorectal cancer cells RKO, wild-type Ad.5 virus expressing mda-7/IL-24 (Ad.5-mda-7) failed to infect efficiently resulting in lack of expression of MDA-7/IL-24 or induction of apoptosis. However, a recombinant Ad.5/3 virus expressing mda-7/IL-24 (Ad.5/3-mda-7) efficiently infected RKO cells resulting in higher MDA-7/IL-24 expression and inhibition of cell growth both in vitro and in nude mice xenograft models. Addition of the novel Bcl-2 family pharmacological inhibitor Apogossypol derivative BI-97C1 (Sabutoclax) significantly augmented the efficacy of Ad.5/3-mda-7. A combination regimen of suboptimal doses of Ad.5/3-mda-7 and BI-97C1 profoundly enhanced cytotoxicity in RKO cells both in vitro and in vivo. Considering the fact that Ad.5-mda-7 has demonstrated significant objective responses in a Phase I clinical trial for advanced solid tumors, Ad.5/3-mda-7 alone or in combination with BI-97C1 would be predicted to exert significantly improved therapeutic efficacy in colorectal cancer patients.     

MDA7/IL-24 is an anti-viral factor that inhibits influenza virus replication

Melanoma differentiation associated gene-7 (mda-7)/interleukin- 24 (IL-24) is a secreted cytokine, which plays an essential role in tumor suppression. Although its role as a multifunctional protein affecting broad types of cancers is well described, functions of IL-24 in host defense against virus infection are yet to be determined. In this study, we explored the anti-viral effect of recombinant IL-24 treatment during influenza infection. Infection of human lung adenocarcinoma cells (A549) with the influenza A virus up-regulated IL-24 mRNA and protein expression in a time-dependent manner. Pre-treatment of A549 cells with recombinant IL-24 protein effectively suppressed viral plaque formation. Furthermore, IL-24 treatment of A549 cells reduced viral non-structural protein 1 (NS1) synthesis, whereas IL-24 knockdown resulted in increased viral replication. Interestingly, IL-24 treatment following influenza A virus infection led to up-regulation of interferon (IFN)-induced antiviral signaling. Taken together, our results suggest that IL-24 exerts a potent suppressive effect on influenza viral replication and can be used in the treatment of influenza infection.     

人mda-7／IL-24的抗肿瘤作用机制

mda-7／IL-24是20世纪90年代中期发现的一个新基因。由于mda-7／IL-24与人IL-10家族具有相当的同源性,后来HUGO基因命名委员会将之重新命名为IL-24,并将其归类到IL-10家族。近年研究表明,采用复制缺陷的腺病毒表达载体Ad．mda-7使其在肿瘤细胞异位表达,引起多种肿瘤细胞的生长抑制。尽管mda．7／IL-24肿瘤靶向性的作用机制还不是很清楚,但大量的实验结果表明该基因作为一个有效的肿瘤治疗基因,能够区分正常细胞和肿瘤细胞、诱导各种不同肿瘤细胞凋亡、启动抗肿瘤“旁观者效应”、增强肿瘤细胞对射线敏感性、抑制动物模型体内移植瘤的生长和血管新生以及具有调节免疫应答能力。     

Mda-7/IL-24 induces apoptosis of diverse cancer cell lines through JAK/STAT-independent pathways

Experimental evidence documents that the MDA-7/IL-24 protein (an IL-10 family cytokine) binds to IL-20 and IL-22 receptor complexes resulting in the activation of JAK/STAT signaling pathways. Recent published reports utilizing human blood derived primary lymphocytes have provided additional confirmatory evidence relating to the cytokine properties of this molecule. A notable attribute of mda-7/IL-24 is its cancer cell-specific apoptosis inducing capacity, which currently remains incompletely understood. Treatment with distinctive tyrosine kinase inhibitors (Genistein and AG18) or a JAK-selective inhibitor (AG490) did not prevent Ad.mda-7 induced apoptosis in diverse cell lines. In addition, there is no apparent correlation between patterns of expression of IL-20R1, IL-20R2, and IL-22R mRNA and susceptibility to Ad.mda-7 in different cell lines. Furthermore, Ad.mda-7 is able to induce killing in STAT/JAK deficient cells. In contrast, treatment with the p38(MAPK) selective inhibitor SB203580, partially inhibited apoptosis induced by Ad.mda-7 in different cell lines. These results demonstrate for the first time that signaling events leading to susceptibility to Ad.mda-7 induced apoptosis, might be tyrosine kinase independent and can thus be distinguished from its cytokine function related properties mediated by the IL-20/IL-22 receptor complexes that require JAK/STAT kinase activity.     

Autophagy switches to apoptosis in prostate cancer cells infected with melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24)

MDA-7/IL-24 has noteworthy potential as an anticancer therapeutic because of its diversity of antitumor properties, its lack of toxicity toward normal cells and tissues, and its safety and efficacy as evidenced in a phase I clinical trial. In a recent study, we document that Ad.mda-7-induced ER stress and ceramide production leads to early autophagy that subsequently switches to apoptosis in human prostate cancer cells. During the apoptotic phase, the MDA-7/IL-24 protein physically interacts with Beclin 1 and this interaction might inhibit Beclin 1 function culminating in apoptosis. Conversely, Ad.mda-7 infection leads to calpain-mediated cleavage of the Atg5 protein that might also facilitate a biochemical switch from autophagy to apoptosis. Our recent paper reveals novel aspects of the interplay between autophagy and apoptosis that underlie the cytotoxic action of MDA-7/IL-24 in prostate cancer cells. These new insights into MDA-7/IL-24 action provide intriguing leads for developing innovative combinatorial approaches for prostate cancer therapy.     

mda-7 (IL-24): signaling and functional roles

One hallmark of neoplasia is abnormal differentiation. Induction of differentiation, by chemical or biological methods, provides a possible therapeutic intervention. "Differentiation therapy" is well documented in several model systems. These include melanoma, in which treatment with interferon-beta and the protein kinase C activator mezerein induces irreversible growth arrest and terminal differentiation culminating in programmed cell death. Subtraction hybridization between terminally differentiated and untreated melanoma cells identified melanoma differentiation-associated gene-7 (mda-7), which is selectively induced during the process of melanoma terminal differentiation. Since its identification seven years ago, mda-7 has been the object of intense focus because of its unique biological properties. Firstly, mda-7 is a secreted protein having cytokine-like properties and belonging to the IL-10 cytokine family. Based on this consideration, mda-7 was renamed IL-24. Secondly if delivered by means of an adenoviral vector, mda-7 induces selective apoptosis in cancer cells of diverse origin, while sparing their normal cellular counterparts. As such, mda-7 has become a novel tool for cancer gene therapy and is currently undergoing phase II clinical trials to determine its clinical efficacy in patients. The present review examines the biological properties of mda-7 and the signaling pathways that contribute to its unique cancer-specific apoptosis-inducing properties.     

Autophagy switches to apoptosis in prostate cancer cells infected with melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24)

MDA-7/IL-24 has noteworthy potential as an anticancer therapeutic because of its diversity of antitumor properties, its lack of toxicity toward normal cells and tissues, and its safety and efficacy as evidenced in a phase I clinical trial. In a recent study, we document that Ad.mda-7-induced ER stress and ceramide production leads to early autophagy that subsequently switches to apoptosis in human prostate cancer cells. During the apoptotic phase, the MDA-7/IL-24 protein physically interacts with Beclin 1 and this interaction might inhibit Beclin 1 function culminating in apoptosis. Conversely, Ad.mda-7 infection leads to calpain-mediated cleavage of the Atg5 protein that might also facilitate a biochemical switch from autophagy to apoptosis. Our recent paper reveals novel aspects of the interplay between autophagy and apoptosis that underlie the cytotoxic action of MDA-7/IL-24 in prostate cancer cells. These new insights into MDA-7/IL-24 action provide intriguing leads for developing innovative combinatorial approaches for prostate cancer therapy.     

MDA-7/IL-24: novel cancer growth suppressing and apoptosis inducing cytokine

The melanoma differentiation-associated gene-7 (mda-7) was cloned by subtraction hybridization as a molecule whose expression is elevated in terminally differentiated human melanoma cells. Current information based on structural and sequence homology, has led to the recognition of MDA-7 as an IL-10 family cytokine member and its renaming as IL-24. Northern blot analysis revealed mda-7/IL-24 expression in human tissues associated with the immune system such as spleen, thymus, peripheral blood leukocytes and normal melanocytes. The MDA-7/IL-24 mouse counterpart, FISP, appears to be a Th2-specific protein and the rat counterpart, C49A/MOB-5, is associated with wound healing and is also induced as a consequence of ras-transformation. A notable property of MDA-7/IL-24 is its ability to induce apoptosis in a large spectrum of human cancer derived cell lines, in mouse xenografts and upon intratumoral injection in human tumors (phase I clinical trials). Various aspects of this intriguing molecule including its cytokine and anti-tumoral effects are described and discussed.     

Combinatorial treatment of non-small-cell lung cancers with gefitinib and Ad.mda-7 enhances apoptosis-induction and reverses resistance to a single therapy

Activation of the epidermal growth factor receptor (EGFR) contributes to the pathogenesis of non-small-cell lung carcinomas (NSCLC) and gefitinib, a selective reversible EGFR inhibitor, is effective in treating patients with NSCLC. However, clinical resistance to gefitinib is a frequent occurrence highlighting the need for improved therapeutic strategies. Melanoma differentiation associated gene-7 (mda-7)/Interleukin-24 (IL-24) (mda-7/IL-24) displays cancer-selective apoptosis induction when delivered via a replication-incompetent adenovirus (Ad.mda-7). In this study, the effect of Ad.mda-7 infection, either alone or in combination with gefitinib, was analyzed in a panel of NSCLC cell lines carrying wild-type EGFR (H-460 and H-2030) or mutant EGFR (H-1650 and H-1975). While H-2030 and H-1650 cells were sensitive, H-460 and H-1975 cells were resistance to growth inhibition by Ad.mda-7, which was reversed by the combination of Ad.mda-7 and gefitinib. This combination increased MDA-7/IL-24 and downstream effector double-stranded RNA-activated protein kinase (PKR) protein expression, promoting apoptosis induction of NSCLC cells. Inhibition of PKR significantly inhibited apoptosis induction by Ad.mda-7 when administered alone but not when used in combination with gefitinib. The combination treatment also augmented inhibition of EGFR signaling. Our findings indicate that a combinatorial treatment with Ad.mda-7 and gefitinib may provide benefit in the treatment of NSCLC, especially in patients displaying resistance to clinically used EGFR inhibitors.     

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 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.     

Targeted combinatorial therapy of non-small cell lung carcinoma using a GST-fusion protein of full-length or truncated MDA-7/IL-24 with Tarceva

Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), a cytokine belonging to the IL-10 family, displays cancer-specific apoptosis-inducing properties when delivered by a replication-incompetent adenovirus (Ad.mda-7) or as a GST-tagged recombinant protein (GST-MDA-7). Previous studies demonstrated that an adenovirus expressing M4, a truncated version of MDA-7/IL-24 containing amino acid residues 104-206, also induced similar cancer-specific apoptosis. We generated recombinant GST-M4 proteins and examined the potency of GST-MDA-7 and GST-M4 on a panel of epidermal growth factor receptor (EGFR) wild type and mutant non-small cell lung carcinoma (NSCLC) cells either as a single agent or in combination with a reversible EGFR inhibitor, Tarceva. The combination of either GST-MDA-7 or GST-M4 ( approximately 0.1 microM) and Tarceva (10 microM), at sub-optimal apoptosis-inducing concentrations synergistically enhanced growth inhibition and apoptosis induction over that observed with either agent alone. The combination treatment also augmented inhibition of EGFR signaling, analyzed by phosphorylation of EGFR and its downstream effectors AKT and ERK1/2, over that with single-agent therapy. Tarceva enhanced GST-MDA-7 and GST-M4 toxicity in cells expressing mutated EGFR proteins that are resistant to the inhibitory effects of Tarceva. In total, these data suggest that combined treatment of NSCLC cells with an EGFR inhibitor can augment the efficacy of GST-MDA-7 and GST-M4 and that the EGFR inhibitor Tarceva may mediate this combinatorial effect by inhibiting multiple tyrosine kinases in addition to the EGFR. This approach highlights a potential new combinatorial strategy, which may prove beneficial for NSCLC patients with acquired resistance to EGFR inhibitors.     

The protein product of the tumor suppressor gene,melanoma differentiation-associated gene 7, exhibits immunostimulatory activity and is designated IL-24

The melanoma differentiation-associated gene 7 (mda-7) has been studied primarily in the context of its tumor suppressor activity. Although mda-7 has been designated as IL-24 based on its gene location in the IL-10 locus and its mRNA expression in leukocytes, no functional evidence supporting this cytokine designation exists. To further characterize MDA-7/IL-24 expression patterns in the human immune system, MDA-7/IL-24 protein levels were examined in human PBMC. MDA-7/IL-24 was detected in PHA- and LPS-stimulated whole PBMC lysate by Western blot and in PHA-activated CD56 and CD19 subsets by immunohistochemistry. The biological function of MDA-7/IL-24, secreted from Ad-MDA7-transfected HEK 293 cells, was assessed by examining the effect of MDA-7/IL-24 on the cytokine secretion profile of PBMC. Within 48 h MDA-7/IL-24 induced secretion of high levels of IL-6, TNF-alpha, and IFN-gamma and low levels of IL-1beta, IL-12, and GM-CSF from human PBMC as measured by ELISA. The MDA-7/IL-24-mediated induction of these Th1-type cytokines was inhibited by the addition of IL-10 to the PBMC cultures, suggesting that these two related protein family members may provide antagonistic functions. Therefore, because human blood leukocytes can be stimulated to produce MDA-7/IL-24, as well as respond to MDA-7/IL-24 by expressing secondary cytokines, MDA-7/IL-24 has the expression profile and major functional attributes that justify its designation as an IL.     

PERK-dependent regulation of MDA-7/IL-24-induced autophagy in primary human glioma cells

Melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The studies by Yacoub et al. (Mol Cancer Ther 2008; 7:314-29) further defines the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that were dependent on activation of JNK1-3 with subsequent activation of BAX and the induction of mitochondrial dysfunction. Activation of JNK1-3 was dependent upon protein kinase R-like endoplasmic reticulum kinase (PERK) and GST-MDA-7 lethality was suppressed in PERK(-/-) cells. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methyladenine, expression of HSP70 or of BiP/GRP78, or by knockdown of ATG5 or Beclin 1 expression, but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin 1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data demonstrate that GST-MDA-7 induces an ER stress response that, via the induction of autophagy, is causal in the activation of pro-apoptotic pathways that converge on the mitochondrion and ultimately culminate in decreased glioma cell survival.     

Splice variants of mda-7/IL-24 differentially affect survival and induce apoptosis in U2OS cells

Interleukin-24 (mda-7/IL-24) is a cytokine in the IL-10 family that has received a great deal of attention for its properties as a tumor suppressor and as a potential treatment for cancer. In this study, we have identified and characterized five alternatively spliced isoforms of this gene. Several, but not all of these isoforms induce apoptosis in the osteosarcoma cell line U2OS, while none affect the survival of the non-cancerous NOK cell line. One of these isoforms, lacking three exons and encoding the N-terminal end of the mda-7/IL-24 protein sequence, caused levels of apoptosis that were higher than those caused by the full-length mda-7/IL-24 variant. Additionally, we found that the ratio of isoform expression can be modified by the splice factor SRp55. This regulation suggests that alternative splicing of mda-7/IL-24 is under tight control in the cell, and can be modified under various cellular conditions, such as DNA damage. In addition to providing new insights into the function of an important tumor suppressor gene, these findings may also point toward new avenues for cancer treatment.