Astrocyte elevated gene-1 induces protective autophagy.

Astrocyte-elevated gene-1 (AEG-1) expression increases in multiple cancers and plays a crucial role in oncogenic transformation and angiogenesis, which are essential components in tumor cell development, growth, and progression to metastasis. Moreover, AEG-1 directly contributes to resistance to chemotherapeutic drugs, another important hallmark of aggressive cancers. In the present study, we document that AEG-1 mediates protective autophagy, an important regulator of cancer survival under metabolic stress and resistance to apoptosis, which may underlie its significant cancer-promoting properties. AEG-1 induces noncanonical autophagy involving an increase in expression of ATG5. AEG-1 decreases the ATP/AMP ratio, resulting in diminished cellular metabolism and activation of AMP kinase, which induces AMPK/mammalian target of rapamycin-dependent autophagy. Inhibition of AMPK by siAMPK or compound C decreases expression of ATG5, ultimately attenuating AEG-1-induced autophagy. AEG-1 protects normal cells from serum starvation-induced death through protective autophagy, and inhibition of AEG-1-induced autophagy results in serum starvation-induced cell death. We also show that AEG-1-mediated chemoresistance is because of protective autophagy and inhibition of AEG-1 results in a decrease in protective autophagy and chemosensitization of cancer cells. In summary, the present study reveals a previously unknown aspect of AEG-1 function by identifying it as a potential regulator of protective autophagy, an important feature of AEG-1 that may contribute to its tumor-promoting properties.

Human polynucleotide phosphorylase selectively and preferentially degrades microRNA-221 in human melanoma cells.

MicroRNAs (miRNA), small noncoding RNAs, affect a broad range of biological processes, including tumorigenesis, by targeting gene products that directly regulate cell growth. Human polynucleotide phosphorylase (hPNPase(old-35)), a type I IFN-inducible 3'-5' exoribonuclease, degrades specific mRNAs and small noncoding RNAs. The present study examined the effect of this enzyme on miRNA expression in human melanoma cells. miRNA microarray analysis of human melanoma cells infected with empty adenovirus or with an adenovirus expressing hPNPase(old-35) identified miRNAs differentially and specifically regulated by hPNPase(old-35). One of these, miR-221, a regulator of the cyclin-dependent kinase inhibitor p27(kip1), displayed robust down-regulation with ensuing up-regulation of p27(kip1) by expression of hPNPase(old-35), which also occurred in multiple human melanoma cells upon IFN-beta treatment. Using both in vivo immunoprecipitation followed by Northern blotting and RNA degradation assays, we confirm that mature miR-221 is the target of hPNPase(old-35). Inhibition of hPNPase(old-35) by shRNA or stable overexpression of miR-221 protected melanoma cells from IFN-beta-mediated growth inhibition, accentuating the importance of hPNPase(old-35) induction and miR-221 down-regulation in mediating IFN-beta action. Moreover, we now uncover a mechanism of miRNA regulation involving selective enzymatic degradation. Targeted overexpression of hPNPase(old-35) might provide an effective therapeutic strategy for miR-221-overexpressing and IFN-resistant tumors, such as melanoma.

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.

Astrocyte elevated gene-1 (AEG-1) functions as an oncogene and regulates angiogenesis.

Astrocyte-elevated gene-1 (AEG-1) expression is increased in multiple cancers and plays a central role in Ha-ras-mediated oncogenesis through the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Additionally, overexpression of AEG-1 protects primary and transformed human and rat cells from serum starvation-induced apoptosis through activation of PI3K/Akt signaling. These findings suggest, but do not prove, that AEG-1 may function as an oncogene. We now provide definitive evidence that AEG-1 is indeed a transforming oncogene and show that stable expression of AEG-1 in normal immortal cloned rat embryo fibroblast (CREF) cells induces morphological transformation and enhances invasion and anchorage-independent growth in soft agar, two fundamental biological events associated with cellular transformation. Additionally, AEG-1-expressing CREF clones form aggressive tumors in nude mice. Immunohistochemistry analysis of tumor sections demonstrates that AEG-1-expressing tumors have increased microvessel density throughout the entire tumor sections. Overexpression of AEG-1 increases expression of molecular markers of angiogenesis, including angiopoietin-1, matrix metalloprotease-2, and hypoxia-inducible factor 1-alpha. In vitro angiogenesis studies further demonstrate that AEG-1 promotes tube formation in Matrigel and increases invasion of human umbilical vein endothelial cells via the PI3K/Akt signaling pathway. Tube formation induced by AEG-1 correlates with increased expression of angiogenesis markers, including Tie2 and hypoxia-inducible factor-alpha, and blocking AEG-1-induced Tie2 with Tie2 siRNA significantly inhibits AEG-1-induced tube formation in Matrigel. Overall, our findings demonstrate that aberrant AEG-1 expression plays a dominant positive role in regulating oncogenic transformation and angiogenesis. These findings suggest that AEG-1 may provide a viable target for directly suppressing the cancer phenotype.

Role of excitatory amino acid transporter-2 (EAAT2) and glutamate in neurodegeneration: opportunities for developing novel therapeutics.

Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. Analysis of the 2.5 kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many ß-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a ß-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes through the NF-κB signaling pathway. The NF-κB binding site at -272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.

MDA-7/IL-24 as a cancer therapeutic: from bench to bedside.

The novel cytokine melanoma differentiation associated gene-7 (mda-7) was identified by subtractive hybridization in the mid-1990s as a protein whose expression increased during the induction of terminal differentiation, and that was either not expressed or was present at low levels in tumor cells compared with non-transformed cells. On the basis of conserved structure, chromosomal location and cytokine-like properties, MDA-7, has now been classified as a member of the expanding interleukin (IL)-10 gene family and designated as MDA-7/IL-24. Multiple studies have shown that the expression of MDA-7/IL-24 in a wide variety of tumor cell types, but not in the corresponding equivalent non-transformed cells, causes their growth arrest and ultimately cell death. In addition, MDA-7/IL-24 has been noted to be a radiosensitizing cytokine, which is partly because of the generation of reactive oxygen species and ceramide that cause endoplasmic reticulum stress. Phase I clinical trial data has shown that a recombinant adenovirus expressing MDA-7/IL-24 [Ad.mda-7 (INGN-241)] was safe and had measurable tumoricidal effects in over 40% of patients, which strongly argues that MDA-7/IL-24 may have significant therapeutic value. This review describes what is known about the impact of MDA-7/IL-24 on tumor cell biology and its potential therapeutic applications.

Chemoprevention by perillyl alcohol coupled with viral gene therapy reduces pancreatic cancer pathogenesis.

Pancreatic cancer is one of the deadliest of cancers. Even with aggressive therapy, the 5-year survival rate is <5%, mandating development of more effective treatments. Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) shows potent antitumor activity against most cancers displaying safety with significant clinical efficacy. However, pancreatic cancer cells display inherent resistance to mda-7/IL-24 that is the result of a "protein translational block" of mda-7/IL-24 mRNA in these tumor cells. We now show that a dietary supplement perillyl alcohol (POH) has significant chemopreventive effects for pancreatic cancer and, when coupled with adenovirus-mediated mda-7/IL-24 gene therapy (Ad.mda-7), effectively eliminates s.c. and i.p. xenografts of human pancreatic cancer cells in nude mice, promoting enhanced survival. The combination of POH and Ad.mda-7 efficiently abrogates the mda-7/IL-24 protein translational block, resulting in MDA-7/IL-24 protein production and growth suppression. Of direct translational relevance, clinically achievable concentrations of POH with Ad.mda-7, both of which have been found safe and without toxic effects in human trials, were used. This novel and innovative approach combining a dietary agent and a virally delivered therapeutic cytokine provides a means of both preventing and treating human pancreatic cancer with significant clinical translational potential.

MDA-7 regulates cell growth and radiosensitivity in vitro of primary (non-established) human glioma cells.

We examined the impact of purified bacterially synthesized GST-MDA-7 (IL-24) and ionizing radiation on the proliferation and survival of nonestablished human glioblastoma multiforme (GBM) cells. Glioma cell types expressing mutated PTEN and p53 molecules, activated ERBB1VIII, overexpressing wild type ERBB1 or without receptor overexpression were selected. In MTT assays, GST-MDA-7 caused a dose-dependent reduction in the proliferation of nonestablished glioma cells; however only at higher concentrations did GST-MDA-7 reduce cell viability. The anti-proliferative and cytotoxic effects of GST-MDA-7 were enhanced by radiation in a greater than additive fashion that correlated with JNK1/2/3 activation. The reduction in cell growth and enhancement in cell killing by the combination of GST-MDA-7 and radiation were blocked by an ROS scavenger, N-acetyl cysteine (NAC), a JNK1/2/3 inhibitor SP600125, a pan-caspase inhibitor (zVAD) and by an inhibitor of caspase 9 (LEHD), but not by an inhibitor of caspase 8 (IETD). Low concentrations of either GST-MDA-7 or radiation reduced clonogenic survival, however colony formation ability was significantly further decreased when the two treatments were combined, which was also blocked by inhibition of caspase 9 function. In general agreement with activation of the intrinsic caspase pathway, cell death correlated with reduced BCL-XL expression and with increased levels of the pro-apoptotic proteins BAD and BAX. Inhibition of caspase 9 after combination treatment blunted neither JNK1/2/3 activation nor the enhanced expression of BAD and BAX, but did block caspase 3 cleavage, reduced expression of BCL-XL and inhibition of ERK1/2 activity. In contrast, incubation with NAC blocked JNK1/2/3 activation and cell killing, but not the increases in BAD and BAX expression. These findings argue that after combination treatment JNK1/2/3 activation is a primary pro-apoptotic event and loss of BCL-XL expression and ERK1/2 activity are secondary caspase-dependent processes. This data also argues that GST- MDA-7 induces two parallel pro-apoptotic pathways via ROS-dependent and -independent mechanisms. Infection of primary human astrocytes with a recombinant adenovirus to express MDA-7, Ad.mda-7, but not infection with either Ad.cmv or Ad.mda-7SP- lacking MDA-7 secretion, resulted in the suppression of GBM cell colony formation in soft agar overlay assays, an effect that was enhanced in a greater than additive fashion by radiation. Collectively, our findings demonstrate that MDA-7 reduces proliferation and enhances the radiosensitivity of nonestablished human GBM cells in vitro, and when grown in 3 dimensions, and that sensitization occurs independently of basal EGFR/ERK1/2/AKT activity or the functions of PTEN and p53.

Raf kinase inhibitor RKIP inhibits MDA-9/syntenin-mediated metastasis in melanoma.

Melanoma differentiation associated gene-9 (MDA-9), also known as syntenin, functions as a positive regulator of melanoma progression and metastasis. In contrast, the Raf kinase inhibitor, RKIP, a negative modulator of RAF-stimulated MEKK activation, is strongly downregulated in metastatic melanoma cells. In this study, we explored a hypothesized inverse relationship between MDA-9 and RKIP in melanoma. Tumor array and cell line analyses confirmed an inverse relationship between expression of MDA-9 and RKIP during melanoma progression. We found that MDA-9 transcriptionally downregulated RKIP in support of a suggested cross-talk between these two proteins. Furthermore, MDA-9 and RKIP physically interacted in a manner that correlated with a suppression of FAK and c-Src phosphorylation, crucial steps necessary for MDA-9 to promote FAK/c-Src complex formation and initiate signaling cascades that drive the MDA-9-mediated metastatic phenotype. Finally, ectopic RKIP expression in melanoma cells overrode MDA-9-mediated signaling, inhibiting cell invasion, anchorage-independent growth, and in vivo dissemination of tumor cells. Taken together, these findings establish RKIP as an inhibitor of MDA-9-dependent melanoma metastasis, with potential implications for targeting this process therapeutically.

MDA-9/syntenin: a positive gatekeeper of melanoma metastasis.

Melanoma differentiation associated gene-9 (MDA-9), synonymous with syntenin, is an adapter protein that provides a central role in regulating cell-cell and cell-matrix adhesion. MDA-9/syntenin transduces signals from the cell-surface to the interior through its interaction with a plethora of additional proteins and actively participates in intracellular trafficking and cell-surface targeting, synaptic transmission, and axonal outgrowth. Recent studies demarcate a seminal role of MDA-9/syntenin in cancer metastasis. In the context of melanoma, MDA-9/syntenin functions as a positive regulator of melanoma progression and metastasis through interactions with c-Src and promotes the formation of an active FAK/c-Src signaling complex leading to NF-k B and matrix metalloproteinase (MMP) activation. The present review provides a current perspective of our understanding of the important features of MDA-9/syntenin and its significant role in tumor cell metastasis with special focus on molecular mechanism of action.

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.

Developing an effective gene therapy for prostate cancer: New technologies with potential to translate from the laboratory into the clinic.

Prostate cancer is the second leading cause of cancer-related deaths in men in the U.S. At present, no single or combination therapy has shown efficacy in decreasing disease progression in patients with metastatic disease. A potentially viable approach for treating late-stage prostate cancer is gene therapy. Adenoviruses (Ad) are the most commonly used mode of gene delivery, but progress using this vector has been hampered by concerns over the safety and practicality of viruses including conditionally replicating Ads (CRAds), particularly for intravenous delivery, and the inefficiency of non-viral transfection techniques. Major challenges for effective gene therapy using Ads are the limited infectivity of regular Ad serotype 5 (Ad5) and the inability to specifically deliver the therapeutic directly into diseased tissue without trapping in the liver or elimination by the immune system. The shortcoming in using Ad5 is mostly attributed to a reduction in Coxsackie-adenovirus receptors (CAR) on the surface of cancer cells, which can be mitigated by generating tropism-modified Ads permitting CAR-independent infection of tumor cells. The limitations of systemic gene delivery can now be overcome by using a novel targeted-delivery approach such as ultrasound (US) contrast agents (microbubbles) to deliver effective therapeutic reagents, Ads, or recombinant proteins, combined with ultrasound-targeted microbubble destruction (UTMD), to develop a site-specific therapy in immune competent transgenic mouse models. These unique strategies for enhancing the efficacy of gene therapy provide a direct path to translation from the laboratory into the clinic for developing an effective gene therapy of prostate cancer.

Oncogene AEG-1 promotes glioma-induced neurodegeneration by increasing glutamate excitotoxicity.

Aggressive tumor growth, diffuse tissue invasion, and neurodegeneration are hallmarks of malignant glioma. Although glutamate excitotoxicity is considered to play a key role in glioma-induced neurodegeneration, the mechanism(s) controlling this process is poorly understood. Astrocyte elevated gene-1 (AEG-1) is an oncogene that is overexpressed in several types of human cancers, including more than 90% of brain tumors. In addition, AEG-1 promotes gliomagenesis, particularly in the context of tumor growth and invasion, 2 primary characteristics of glioma. In the present study, we investigated the contribution of AEG-1 to glioma-induced neurodegeneration. Pearson correlation coefficient analysis in normal brain tissues and samples from glioma patients indicated a strong negative correlation between expression of AEG-1 and a primary glutamate transporter of astrocytes EAAT2. Gain- and loss-of-function studies in normal primary human fetal astrocytes and T98G glioblastoma multiforme cells revealed that AEG-1 repressed EAAT2 expression at a transcriptional level by inducing YY1 activity to inhibit CBP function as a coactivator on the EAAT2 promoter. In addition, AEG-1-mediated EAAT2 repression caused a reduction of glutamate uptake by glial cells, resulting in induction of neuronal cell death. These findings were also confirmed in samples from glioma patients showing that AEG-1 expression negatively correlated with NeuN expression. Taken together, our findings suggest that AEG-1 contributes to glioma-induced neurodegeneration, a hallmark of this fatal tumor, through regulation of EAAT2 expression.

Molecular mechanism of chemoresistance by astrocyte elevated gene-1.

Our recent findings show that astrocyte elevated gene-1 (AEG-1) is overexpressed in >90% of human hepatocellular carcinoma (HCC) samples, and AEG-1 plays a central role in regulating development and progression of HCC. In the present study, we elucidate a molecular mechanism of AEG-1-induced chemoresistance, an important characteristic of aggressive cancers. AEG-1 increases the expression of multidrug resistance gene 1 (MDR1) protein, resulting in increased efflux and decreased accumulation of doxorubicin, promoting doxorubicin resistance. Suppression of MDR1 by small interfering RNA or chemical reagents, or inhibition of AEG-1 or a combination of both genes, significantly increases in vitro sensitivity to doxorubicin. In nude mice xenograft studies, a lentivirus expressing AEG-1 short hairpin RNA, in combination with doxorubicin, profoundly inhibited growth of aggressive human HCC cells compared with either agent alone. We document that although AEG-1 does not affect MDR1 gene transcription, it facilitates association of MDR1 mRNA to polysomes, resulting in increased translation, and AEG-1 also inhibits ubiquitination and subsequent proteasome-mediated degradation of MDR1 protein. This study is the first documentation of a unique aspect of AEG-1 function (i.e., translational and posttranslational regulation of proteins). Inhibition of AEG-1 might provide a means of more effectively using chemotherapy to treat HCC, which displays inherent chemoresistance with aggressive pathology.

Astrocyte elevated gene-1: a novel target for human glioma therapy.

Malignant gliomas including glioblastoma multiforme (GBM) and anaplastic astrocytomas are the most common primary brain tumors. Despite multimodal treatment including surgery, chemotherapy, and radiation, median survival for patients with GBMs is only 12 to 15 months. Identifying molecules critical for glioma progression is crucial for devising effective targeted therapy. In the present study, we investigated the potential contribution of astrocyte elevated gene-1 (AEG-1) in gliomagenesis and explored the possibility of AEG-1 as a therapeutic target for malignant glioma. We analyzed the expression levels of AEG-1 in 9 normal brain tissues and 98 brain tumor patient samples by Western blot analysis and immunohistochemistry. AEG-1 expression was significantly elevated in >90% of diverse human brain tumor samples including GBMs and astrocytic tumors, and also in human glioma cell lines compared with normal brain tissues and normal astrocytes. Knockdown of AEG-1 by small interfering RNA inhibited cell viability, cloning efficiency, and invasive ability of U87 human glioma cells and 9L rat gliosarcoma cells. We also found that matrix metalloproteases (MMP-2 and MMP-9) are involved in AEG-1-mediated invasion of glioma cells. In an orthotopic nude mouse brain tumor model using primary human GBM12 tumor cells, AEG-1 small interfering RNA significantly suppressed glioma cell growth in vivo. Taken together, these provocative results indicate that AEG-1 may play a crucial role in the pathogenesis of glioma and that AEG-1 could represent a viable potential target for malignant glioma therapy.

Mechanism of autophagy to apoptosis switch triggered in prostate cancer cells by antitumor cytokine melanoma differentiation-associated gene 7/interleukin-24.

Melanoma differentiation-associated gene 7 (mda-7)/interleukin-24 (IL-24) is a unique member of the IL-10 gene family, which displays a broad range of antitumor properties, including induction of cancer-specific apoptosis. Adenoviral-mediated delivery by Ad.mda-7 invokes an endoplasmic reticulum (ER) stress response that is associated with ceramide production and autophagy in some cancer cells. Here, we report that Ad.mda-7-induced ER stress and ceramide production trigger autophagy in human prostate cancer cells, but not in normal prostate epithelial cells, through a canonical signaling pathway that involves Beclin-1, atg5, and hVps34. Autophagy occurs in cancer cells at early times after Ad.mda-7 infection, but a switch to apoptosis occurs by 48 hours after infection. Inhibiting autophagy with 3-methyladenosine increases Ad.mda-7-induced apoptosis, suggesting that autophagy may be initiated first as a cytoprotective mechanism. Inhibiting apoptosis by overexpression of antiapoptotic proteins Bcl-2 or Bcl-xL increased autophagy after Ad.mda-7 infection. During the apoptotic phase, the MDA-7/IL-24 protein physically interacted with Beclin-1 in a manner that could inhibit Beclin-1 function culminating in apoptosis. Conversely, Ad.mda-7 infection elicited calpain-mediated cleavage of the autophagic protein ATG5 in a manner that could facilitate switch to apoptosis. Our findings reveal novel aspects of the interplay between autophagy and apoptosis in prostate cancer cells that underlie the cytotoxic action of mda-7/IL-24, possibly providing new insights in the development of combinatorial therapies for prostate cancer.

The development of MDA-7/IL-24 as a cancer therapeutic.

The cytokine melanoma differentiation associated gene 7 (mda-7) was identified by subtractive hybridization as a protein whose expression increased during the induction of terminal differentiation, and that was either not expressed or was present at low levels in tumor cells compared to non-transformed cells. Based on conserved structure, chromosomal location and cytokine-like properties, MDA-7, was classified as a member of the interleukin (IL)-10 gene family and designated as MDA-7/IL-24. Multiple studies have demonstrated that expression of MDA-7/IL-24 in a wide variety of tumor cell types, but not in corresponding equivalent non-transformed cells, causes their growth arrest and rapid cell death. In addition, MDA-7/IL-24 has been noted to radiosensitize tumor cells which in part is due to the generation of reactive oxygen species (ROS) and ceramide that cause endoplasmic reticulum stress and suppress protein translation. Phase I clinical trial data has shown that a recombinant adenovirus expressing MDA-7/IL-24 (Ad.mda-7 (INGN-241)) was safe and had measurable tumoricidal effects in over 40% of patients, strongly arguing that MDA-7/IL-24 could have significant therapeutic value. This review describes what is presently known about the impact of MDA-7/IL-24 on tumor cell biology and its potential therapeutic applications.

Mechanism by which Mcl-1 regulates cancer-specific apoptosis triggered by mda-7/IL-24, an IL-10-related cytokine.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), a cytokine belonging to the IL-10 family, selectively induces apoptosis in cancer cells without harming normal cells by promoting an endoplasmic reticulum (ER) stress response. The precise molecular mechanism by which the ER stress response culminates in cell death requires further clarification. The present study shows that in prostate carcinoma cells, the mda-7/IL-24-induced ER stress response causes apoptosis by translational inhibition of the antiapoptotic protein myeloid cell leukemia-1 (Mcl-1). Forced expression of Mcl-1 blocked mda-7/IL-24 lethality, whereas RNA interference or gene knockout of Mcl-1 markedly sensitized transformed cells to mda-7/IL-24. Mcl-1 downregulation by mda-7/IL-24 relieved its association with the proapoptotic protein Bak, causing oligomerization of Bak and leading to cell death. These observations show the profound role of the Bcl-2 protein family member Mcl-1 in regulating cancer-specific apoptosis induced by this cytokine. Thus, our studies provide further insights into the molecular mechanism of ER stress-induced cancer-selective apoptosis by mda-7/IL-24. As Mcl-1 is overexpressed in the majority of prostate cancers, mda-7/IL-24 might provide an effective therapeutic for this disease.

mda-7/IL-24: a unique member of the IL-10 gene family promoting cancer-targeted toxicity.

Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a unique member of the IL-10 gene family that displays nearly ubiquitous cancer-specific toxicity, with no harmful effects toward normal cells or tissues. mda-7/IL-24 was cloned from human melanoma cells by differentiation induction subtraction hybridization (DISH) and promotes endoplasmic reticulum (ER) stress culminating in apoptosis or toxic autophagy in a broad-spectrum of human cancers, when assayed in cell culture, in vivo in human tumor xenograft mouse models and in a Phase I clinical trial in patients with advanced cancers. This therapeutically active cytokine also induces indirect antitumor activity through inhibition of angiogenesis, stimulation of an antitumor immune response, and sensitization of cancer cells to radiation-, chemotherapy- and antibody-induced killing.

Historical perspective and recent insights into our understanding of the molecular and biochemical basis of the antitumor properties of mda-7/IL-24.

A subtraction hybridization approach combined with a differentiation therapy model of human melanoma identified melanoma differentiation associated gene-7 (mda-7) as a gene upregulated during induction of terminal differentiation. Based on conserved structure, chromosomal location and cytokine-like properties, mda-7, has now been classified as a member of the expanding interleukin (IL)-10 gene family and designated as mda-7/IL-24. Extensive in vitro and in vivo human tumor xenograft studies confirm that mda-7/IL-24 induces apoptosis specifically in tumor cells without harming normal cells. Unique properties of mda-7/IL-24 action also include potent "bystander antitumor" activity, an ability to exert anti-angiogenic effects, immune modulating ability and a capacity to enhance the sensitivity of tumor cells to radiotherapy, chemotherapy and monoclonal antibody therapy. Very recent studies from our groups further reveal autocrine regulation and chemoprevention facilitating properties of mda-7/IL-24. Based on these remarkable antitumor attributes, mda-7/IL-24 was evaluated by intratumoral injection with a replication incompetent adenovirus expressing this gene (Ad.mda-7; INGN 241) in a phase I clinical trial in patients with metastatic melanomas and other advanced solid cancers. mda-7/IL-24 was well tolerated with significant clinical activity. This review highlights our current understanding of the molecular and biochemical basis of mda-7/IL-24 antitumor properties and highlights its potential as a viable gene-based therapy for a wide spectrum of primary and advanced cancers.