Detecting Tumor Metastases: The Road to Therapy Starts Here.

Metastasis is the complex process by which primary tumor cells migrate and establish secondary tumors in an adjacent or distant location in the body. Early detection of metastatic disease and effective therapeutic options for targeting these detected metastases remain impediments to effectively treating patients with advanced cancers. If metastatic lesions are identified early, patients might maximally benefit from effective early therapeutic interventions. Further, monitoring patients whose primary tumors are effectively treated for potential metastatic disease onset is also highly valuable. Finally, patients with metastatic disease can be monitored for efficacy of specific therapeutic interventions through effective metastatic detection techniques. Thus, being able to detect and visualize metastatic lesions is key and provides potential to greatly improve overall patient outcomes. In order to achieve these objectives, researchers have endeavored to mechanistically define the steps involved in the metastatic process as well as ways to effectively detect metastatic progression. We presently overview various preclinical and clinical in vitro and in vivo assays developed to more efficiently detect tumor metastases, which provides the foundation for developing more effective therapies for this invariably fatal component of the cancerous process.

AEG-1/MTDH/LYRIC: A Promiscuous Protein Partner Critical in Cancer, Obesity, and CNS Diseases.

Since its original discovery in 2002, AEG-1/MTDH/LYRIC has emerged as a primary regulator of several diseases including cancer, inflammatory diseases, and neurodegenerative diseases. AEG-1/MTDH/LYRIC has emerged as a key contributory molecule in almost every aspect of cancer progression, including uncontrolled cell growth, evasion of apoptosis, increased cell migration and invasion, angiogenesis, chemoresistance, and metastasis. Additionally, recent studies highlight a seminal role of AEG-1/MTDH/LYRIC in neurodegenerative diseases and obesity. By interacting with multiple protein partners, AEG-1/MTDH/LYRIC plays multifaceted roles in the pathogenesis of a wide variety of diseases. This review discusses the current state of understanding of AEG-1/MTDH/LYRIC regulation and function in cancer and other diseases with a focus on its association/interaction with several pivotal protein partners.

Evolving Strategies for Therapeutically Targeting Cancer Stem Cells.

Cancer is a multifactor and multistep process that is affected intrinsically by the genetic and epigenetic makeup of tumor cells and extrinsically by the host microenvironment and immune system. A key component of cancer involves a unique subpopulation of highly malignant cancerous cells referred to as cancer stem cells (CSCs). CSCs are positioned at the apex of the tumor hierarchy with an ability to both self-renew and also generate non-CSC/differentiated progeny, which contribute to the majority of the tumor mass. CSCs undergo functional changes and show plasticity that is stimulated by specific microenvironmental cues and interactions in the tumor niche, which contribute to the complexity and heterogeneity of the CSC population. The prognostic value of CSCs in the clinic is evident since there are many examples in which CSCs serve as markers for poor patient prognosis. CSCs are innately resistant to many standard therapies and they display anoikis resistance, immune evasion, tumor dormancy, and field cancerization, which may result in metastasis and relapse. Many academic laboratories and biotechnology companies are currently focusing on strategies that target CSCs. Combination therapies, epigenetic modifiers, stemness inhibitors, CSC surface marker-based therapies, and immunotherapy-based CSC-targeting drugs are currently undergoing clinical trials. Potential new targets/strategies in CSC-targeted therapy include MDA-9/Syntenin (SDCBP), Patched (PTCH), epigenetic targets, noncoding RNAs, and differentiation induction. Defining ways of targeting and destroying CSCs holds potential to impact significantly on cancer therapy, including prevention of metastasis and cancer recurrence.

BI-69A11 enhances susceptibility of colon cancer cells to mda-7/IL-24-induced growth inhibition by targeting Akt.

BACKGROUND: Akt and its downstream signalling pathways contribute to the aetiology and progression of colorectal carcinoma (CRC). Targeting the Akt pathway is an attractive strategy but few chemotherapeutic drugs have been used to treat CRC with only limited success. BI-69A11, a small molecule inhibitor of Akt, efficiently inhibits growth in melanoma cells. Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 promotes cancer-selective apoptosis when delivered by a tropism-modified replication incompetent adenovirus (Ad.5/3-mda-7). However, Ad.5/3-mda-7 displays diminished antitumour efficacy in several CRC cell lines, which correlates with the expression of K-RAS. METHODS: The individual and combinatorial effect of BI-69A11 and Ad.5/3-mda-7 in vitro was studied by cell viability, cell cycle, apoptosis and invasion assays in HT29 and HCT116 cells containing wild type or mutant K-ras, respectively. In vivo HT29 tumour xenografts were used to test the efficacy of the combination treatment. RESULTS: BI-69A11 inhibited growth and induced apoptosis in CRC. However, combinatorial treatment was more effective compared with single treatment. This combination showed profound antitumour and anti angiogenic effects in vitro and in vivo by downregulating Akt activity. CONCLUSIONS: BI-69A11 enhances the antitumour efficacy of Ad.5/3-mda-7 on CRC overexpressing K-RAS by inducing apoptosis and regulating Akt activity thereby warranting further evaluation in treating CRC.

Chemoprevention gene therapy (CGT) of pancreatic cancer using perillyl alcohol and a novel chimeric serotype cancer terminator virus.

Conditionally replication competent adenoviruses (Ads) that selectively replicate in cancer cells and simultaneously express a therapeutic cytokine, such as melanoma differentiation associated gene- 7/Interleukin-24 (mda-7/IL-24), a Cancer Terminator Virus (CTV-M7), hold potential for treating human cancers. To enhance the efficacy of the CTV-M7, we generated a chimeric Ad.5 and Ad.3 modified fiber bipartite CTV (Ad.5/3-CTV-M7) that can infect tumor cells in a Coxsackie Adenovirus receptor (CAR) independent manner, while retaining high infectivity in cancer cells containing high CAR. Although mda-7/IL-24 displays broad-spectrum anticancer properties, pancreatic ductal adenocarcinoma (PDAC) cells display an intrinsic resistance to mda-7/IL-24-mediated killing due to an mda-7/IL-24 mRNA translational block. However, using a chemoprevention gene therapy (CGT) approach with perillyl alcohol (POH) and a replication incompetent Ad to deliver mda-7/IL-24 (Ad.mda-7) there is enhanced conversion of mda-7/IL-24 mRNA into protein resulting in pancreatic cancer cell death in vitro and in vivo in nude mice containing human PDAC xenografts. This combination synergistically induces mda-7/IL-24-mediated cancer-specific apoptosis by inhibiting anti-apoptotic Bcl-xL and Bcl-2 protein expression and inducing an endoplasmic reticulum (ER) stress response through induction of BiP/GRP-78, which is most evident in chimeric-modified non-replicating Ad.5/3- mda-7- and CTV-M7-infected PDAC cells. Moreover, Ad.5/3-CTV-M7 in combination with POH sensitizes therapy-resistant MIA PaCa-2 cell lines over-expressing either Bcl-2 or Bcl-xL to mda-7/IL-24-mediated apoptosis. Ad.5/3-CTV-M7 plus POH also exerts a significant antitumor 'bystander' effect in vivo suppressing both primary and distant site tumor growth, confirming therapeutic utility of Ad.5/3-CTV-M7 plus POH in PDAC treatment, where all other current treatment strategies in clinical settings show minimal efficacy.

Chemoprevention gene therapy (CGT): novel combinatorial approach for preventing and treating pancreatic cancer.

Pancreatic cancer remains one of the deadliest of all cancers despite aggressive surgical treatment combined with adjuvant radiotherapy and chemotherapy. Chemoresistance and radioresistance are the principal causes of failure of pancreatic cancer patients to respond to therapy. Conditionally replication competent adenovirus (CRCA)-based cancer gene therapy is an innovative strategy for treating cancers displaying inherent resistance to treatment. Limitations of current adenovirus (Ad)-based gene therapies for malignant tumors include lack of cancer-specificity, and effective and targeted delivery. To remedy this situation, CRCAs have been designed that express E1A, necessary for Ad replication, under the control of a cancer-specific progression elevated gene-3 promoter (PEG-Prom) with concomitant expression of an immunomodulatory cytokine, such as mda-7/IL-24 or interferon-γ (IFN-γ), under the control of a ubiquitous and strong cytomegalovirus promoter (CMV-Prom) from the E3 region. These bipartite CRCAs, when armed with a transgene, are called cancer terminator viruses (CTVs), i.e., Ad.PEG-E1A-CMV-mda-7 (CTV-M7) and Ad.PEG-E1A-CMV-IFN-γ (CTV-γ), because of their universal effectiveness in cancer treatment irrespective of p53/pRb/p16 or other genetic alterations in tumor cells. In addition to their selective oncolytic effects in tumor cells, the potent 'bystander antitumor' properties of MDA-7/IL-24 and IFN-γ embody the CTVs with expanded treatment properties for both primary and distant cancers. Pancreatic cancer cells display a "translational block" of mda-7/IL-24 mRNA, limiting production of MDA-7/IL-24 protein and cancer-specific apoptosis. Specific chemopreventive agents abrogate this "translational block" resulting in pancreatic cancer-specific killing. This novel chemoprevention gene therapy (CGT) strategy holds promise for both prevention and treatment of pancreatic cancers where all other strategies have proven ineffective.

GADD45 proteins: central players in tumorigenesis.

The Growth Arrest and DNA Damage-inducible 45 (GADD45) proteins have been implicated in regulation of many cellular functions including DNA repair, cell cycle control, senescence and genotoxic stress. However, the pro-apoptotic activities have also positioned GADD45 as an essential player in oncogenesis. Emerging functional evidence implies that GADD45 proteins serve as tumor suppressors in response to diverse stimuli, connecting multiple cell signaling modules. Defects in the GADD45 pathway can be related to the initiation and progression of malignancies. Moreover, induction of GADD45 expression is an essential step for mediating anti-cancer activity of multiple chemotherapeutic drugs and the absence of GADD45 might abrogate their effects in cancer cells. In this review, we present a comprehensive discussion of the functions of GADD45 proteins, linking their regulation to effectors of cell cycle arrest, DNA repair and apoptosis. The ramifications regarding their roles as essential and central players in tumor growth suppression are also examined. We also extensively review recent literature to clarify how different chemotherapeutic drugs induce GADD45 gene expression and how its up-regulation and interaction with different molecular partners may benefit cancer chemotherapy and facilitate novel drug discovery.

IL-6 promotes prostate tumorigenesis and progression through autocrine cross-activation of IGF-IR.

As an established mediator of inflammation, interleukin-6 (IL-6) is implicated to facilitate prostate cancer progression to androgen independence through transactivation of the androgen receptor. However, whether IL-6 has a causative role in de novo prostate tumorigenesis was never investigated. We now provide the first evidence that IL-6 can induce tumorigenic conversion and further progression to an invasive phenotype of non-tumorigenic benign prostate epithelial cells. Moreover, we find that paracrine IL-6 stimulates the autocrine IL-6 loop and autocrine activation of insulin-like type I growth factor receptor (IGF-IR) to confer the tumorigenic property and also that activation of signal transducer and activator of transcription 3 (STAT3) is critical in these processes. Inhibition of STAT3 activation or IGF-IR signaling suppresses IL-6-mediated malignant conversion and the associated invasive phenotype. Inhibition of STAT3 activation suppresses IL-6-induced upregulation of IGF-IR and its ligands, namely IGF-I and IGF-II. These findings indicate that IL-6 signaling cooperates with IGF-IR signaling in the prostate microenvironment to promote prostate tumorigenesis and progression to aggressiveness. Our findings suggest that STAT3 and IGF-IR may represent potential effective targets for prevention or treatment of prostate cancer.

Human polynucleotide phosphorylase (hPNPase(old-35)): an evolutionary conserved gene with an expanding repertoire of RNA degradation functions.

Human polynucleotide phosphorylase (hPNPase(old-35)) is an evolutionary conserved RNA-processing enzyme with expanding roles in regulating cellular physiology. hPNPase(old-35) was cloned using an innovative 'overlapping pathway screening' strategy designed to identify genes coordinately regulated during the processes of cellular differentiation and senescence. Although hPNPase(old-35) structurally and biochemically resembles PNPase of other species, overexpression and inhibition studies reveal that hPNPase(old-35) has evolved to serve more specialized and diversified functions in humans. Targeting specific mRNA or non-coding small microRNA, hPNPase(old-35) modulates gene expression that in turn has a pivotal role in regulating normal physiological and pathological processes. In these contexts, targeted overexpression of hPNPase(old-35) represents a novel strategy to selectively downregulate RNA expression and consequently intervene in a variety of pathophysiological conditions.

Inhibition of AP-1 by SARI negatively regulates transformation progression mediated by CCN1.

Enhanced expression of the CCN family of secretory integrin-binding proteins correlates with many essential components of the cancerous state, including tumor cell adhesion, proliferation, invasion and migration. Consequently, CCN1 expression is elevated in various cancers, including breast cancer, and its expression directly correlates with poor patient prognosis. Using subtraction-hybridization, combined with induction of cancer cell terminal differentiation, we cloned SARI (suppressor of activator protein (AP)-1, regulated by interferon (IFN)), an IFN-beta-inducible, potent tumor suppressor gene that exerts cancer-selective growth inhibitory effects. Forced expression of SARI using an adenovirus (Ad.SARI) inhibits AP-1 function and downregulates CCN1 expression in multiple cancer lineages, resulting in a profound inhibition in anchorage-independent cell growth and tumor cell invasion. Overexpression of SARI reduces CCN1-promoter activity through inhibition of AP-1 binding. Accordingly, SARI selectively blocks expression of the transformed state in rat embryo fibroblast cells that stably overexpress c-Jun. These results illustrate that SARI inhibits AP-1 transactivating factor binding to the cis-element of the CCN1 promoter, possibly through its interaction with c-Jun. Overall, SARI can directly inhibit CCN1-induced transformation by inhibiting the transcription of CCN1, as well as indirectly by inhibiting the expression of c-Jun (and hence blocking AP-1 activity). In these contexts, transformed cells 'addicted' to AP-1 activity are rendered susceptible to SARI-mediated inhibition of expression of the transformed phenotype.

Src kinase activation is mandatory for MDA-9/syntenin-mediated activation of nuclear factor-kappaB.

The scaffolding postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain-containing protein melanoma differentiation associated gene-9 (MDA-9)/syntenin is a tandem PDZ protein overexpressed in human melanoma, and breast and gastric cancer cells. MDA-9/syntenin affects cancer cell motility and invasion through distinct biochemical and signaling pathways, including focal adhesion kinase and p38 mitogen-activated protein kinase (MAPK), resulting in activation of the nuclear factor (NF)-kappaB pathway. MDA-9/syntenin also promotes melanoma metastasis by activating c-Src, but how c-Src regulates NF-kappaB activation is unclear. Using a human melanoma model, we document that MDA-9/syntenin-c-Src interactions are positive regulators of NF-kappaB activation. Inhibition of c-Src by PP2 treatment, by blocking c-Src or mda-9/syntenin expression with small interfering RNA, or in c-Src (-/-) knockout cell lines, reduces NF-kappaB activation following overexpression of mda-9/syntenin or c-Src. Deletion or point mutations of the PDZ binding motif preventing MDA-9/syntenin association with c-Src reveals that both PDZ domains, with PDZ2 being the dominant module, are required for activating downstream signaling pathways, including p38 MAPK and NF-kappaB. We also document that MDA-9/syntenin-c-Src complexes functionally cooperate with NF-kappaB to promote anchorage-independent growth, motility and invasion of melanoma cells. These findings underscore PDZ domains of MDA-9/syntenin as promising potential therapeutic targets for intervening in a decisive component of cancer progression, namely, metastatic tumor spread.

Enhanced delivery of mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) improves therapeutic efficacy in low CAR prostate cancer cells.

Gene therapy is being examined as a potential strategy for treating prostate cancer. Serotype 5 adenovirus (Ad.5) is routinely used as a vector for transgene delivery. However, the infectivity of Ad.5 is dependent on Coxsackie-adenovirus receptors (CARs); many tumor types show a reduction in this receptor in vivo, thereby limiting therapeutic gene transduction. Serotype chimerism is one approach to circumvent CAR deficiency; this strategy is used to generate an Ad.5/3-recombinant Ad that infects cancer cells through Ad.3 receptors in a CAR-independent manner. In this report, the enhanced transgene delivery and efficacy of Ad.5/3-recombinant virus was evaluated using an effective wide-spectrum anticancer therapeutic melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24). Our data show that in low CAR human prostate cancer cells (PC-3), a recombinant Ad.5/3 virus delivering mda-7/IL-24 (Ad.5/3-mda-7) is more efficacious than an Ad.5 virus encoding mda-7/IL-24 (Ad.5-mda-7) in infecting tumor cells, expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis, inhibiting in vivo tumor growth and exerting an antitumor 'bystander' effect in a nude mouse xenograft model. Considering the fact that Ad.5-mda-7 has shown significant objective responses in a phase I clinical trial for solid tumors, Ad.5/3-mda-7 is predicted to exert enhanced therapeutic benefit in patients with prostate cancer.

Astrocyte elevated gene-1 contributes to the pathogenesis of neuroblastoma.

Neuroblastoma, derived from neural crest progenitor cells, is the most common extracranial solid tumor of childhood. Astrocyte elevated gene-1 (AEG-1) is a primary mediator of tumor progression and metastasis in several human cancers. In this study, we investigated the potential contribution of AEG-1 in human neuroblastoma pathogenesis. AEG-1 expression was significantly elevated in neuroblastoma patient-derived samples and neuroblastoma cell lines as compared with normal peripheral nerve tissues, normal astrocytes and immortalized melanocytes. Knockdown of AEG-1 by small interfering RNA reduced the tumorigenic properties of highly aggressive neuroblastoma cells. Conversely, over-expression of AEG-1 enhanced proliferation and expression of the transformed state in less aggressive neuroblastoma cells through activation of the phosphatidylinositol 3-kinase-Akt-signaling pathway and stabilization of MYCN. These provocative results indicate that AEG-1 may play a crucial role in the pathogenesis of neuroblastoma and could represent a potential target for therapeutic intervention.

Characterization of infectivity of knob-modified adenoviral vectors in glioma.

Malignant glioma continues to be a major target for gene therapy and virotherapy due to its aggressive growth and the current lack of effective treatment. However, these approaches have been hampered by inefficient infection of glioma cells by viral vectors,particularly vectors derived from serotype 5 adenoviruses (Ad5). This results from limited cell surface expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on tumor cells. To circumvent this problem, Ad fiber pseudotyping,the genetic replacement of either the entire fiber or fiber knob domain with its structural counterpart from another human Ad serotype that recognizes a cellular receptor other than CAR, has been shown to enhance Ad infectivity in a variety of tumor types,including human glioma. Here, we have extended the paradigm of genetic pseudotyping to include fiber domains from non-human or"xenotype" Ads for infectivity enhancement of human glioma cell populations. In this study, we evaluated the gene transfer efficiency of a panel of Ad vectors which express one of five different "xenotype"fiber knob domains, including those derived from murine,ovine, porcine and canine species, in both human glioma cell lines as well as primary glioma tumor cells from patients. Adenovirus vectors displaying either canine Ad or porcine Ad fiber elements had the highest gene transfer to both glioma cell lines and primary tumor cells. The correlation between the viral infectivity of modified adenovirus vectors and expression of human CAR and CD46(an adenovirus type B receptor) on the surfaces of tumor cells was also analyzed. Taken together, human adenovirus vectors modified with "xenotype" fiber elements could be excellent candidates to target human glioma.

A cancer terminator virus eradicates both primary and distant human melanomas.

The prognosis and response to conventional therapies of malignant melanoma inversely correlate with disease progression. With increasing thickness, melanomas acquire metastatic potential and become inherently resistant to radiotherapy and chemotherapy. These harsh realities mandate the design of improved therapeutic modalities, especially those targeting metastases. To develop an approach to effectively treat this aggressive disease, we constructed a conditionally replication-competent adenovirus in which expression of the adenoviral E1A gene, necessary for replication, is driven by the cancer-specific promoter of progression-elevated gene-3 (PEG-3) and which simultaneously expresses mda-7/IL-24 in the E3 region of the adenovirus (Ad.PEG-E1A-mda-7), a cancer terminator virus (CTV). This CTV produces large quantities of MDA-7/IL-24 protein as a function of adenovirus replication uniquely in cancer cells. Infection of Ad.PEG-E1A-mda-7 (CTV) in normal human immortal melanocytes and human melanoma cells demonstrates cancer cell-selective adenoviral replication, mda-7/IL-24 expression, growth inhibition and apoptosis induction. Injecting Ad.PEG-E1A-mda-7 CTV into xenografts derived from MeWo human metastatic melanoma cells in athymic nude mice completely eliminated not only primary treated tumors but also distant non-treated tumors (established in the opposite flank), thereby implementing a cure. These provocative findings advocate potential therapeutic applications of this novel virus for treating patients with advanced melanomas with metastases.

Astrocyte elevated gene-1 activates cell survival pathways through PI3K-Akt signaling.

Astrocyte elevated gene-1 (AEG-1) displays oncogenic properties. Its expression is elevated in diverse neoplastic states and it cooperates with Ha-ras to promote cellular transformation. Overexpression of AEG-1 augments invasion and anchorage-independent growth of transformed cells, while AEG-1 siRNA inhibits Ha-ras-mediated colony formation, supporting a potential functional role in tumorigenesis. Additionally, oncogenic Ha-ras induces AEG-1 expression through the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. In the present study, we investigated whether AEG-1 could induce serum-independent cell growth, another property of oncogenes. Overexpression of AEG-1 inhibited serum starvation-induced apoptosis through activation of PI3K-Akt signaling, one of the effector pathways induced by activated Ras. AEG-1 also affected the phosphorylation state of Akt substrates that are implicated in apoptosis suppression, including glycogen synthase kinase 3beta, c-Myc, murine double minute 2, p53, p21/mda-6 and Bad. Additionally, AEG-1 blocked the activity of serum starvation-induced caspases. Taken together, these observations provide evidence that AEG-1 is an oncogene cooperating with Ha-ras as well as functioning as a downstream target gene of Ha-ras and may perform a central role in Ha-ras-mediated carcinogenesis. Activation of survival pathways may be one mechanism by which AEG-1 exerts its oncogenic properties.

Targeting inhibition of K-ras enhances Ad.mda-7-induced growth suppression and apoptosis in mutant K-ras colorectal cancer cells.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a cancer-specific, growth-suppressing and apoptosis-inducing gene with broad-spectrum antitumor activity. However, when administered by means of a replication-incompetent adenovirus, Ad.mda-7, several colorectal carcinoma cell lines are resistant to its antiproliferative and antisurvival effects. We have presently endeavored to determine if K-ras mutations, present in approximately 40-50% of colorectal cancers and which may mediate resistance to chemotherapy and radiotherapy, represent a predisposing genetic factor mitigating reduced sensitivity to Ad.mda-7. To suppress ras expression, three structurally different replication-incompetent adenoviral vectors were engineered that express (1) an intracellular, neutralizing single-chain antibody (scAb) to p21 ras (Ad.K-ras scAb), (2) an antisense (AS) K-ras gene (Ad.K-ras AS) or (3) both mda-7/IL-24 and a K-ras AS gene in a single bipartite virus (Ad.m7.KAS). Simultaneous inhibition of K-ras and expression of mda-7/IL-24 enhanced killing of colorectal carcinoma cells with mutated K-ras, but not with wild-type K-ras. The extent of killing depended on the degree of K-ras downregulation, with Ad.K-ras AS being generally more efficient than Ad.K-ras scAb in combination with Ad.mda-7. These findings support an effective dual-combinatorial approach for the therapy of colorectal cancers that employs a unique cancer-specific suppressor gene (mda-7/IL-24) with targeted inhibition of oncogene (ras) expression.

Activation of Ras/Raf protects cells from melanoma differentiation-associated gene-5-induced apoptosis.

Melanoma differentiation-associated gene-5 (mda-5) was the first molecule identified in nature whose encoded protein embodied the unique structural combination of an N-terminal caspase recruitment domain and a C-terminal DExD/H RNA helicase domain. As suggested by its structure, cumulative evidences documented that ectopic expression of mda-5 leads to growth inhibition and/or apoptosis in various cell lines. However, the signaling pathways involved in mda-5-mediated killing have not been elucidated. In this study, we utilized either genetically modified cloned rat embryo fibroblast cells overexpressing different functionally and structurally distinct oncogenes or human pancreatic and colorectal carcinoma cells containing mutant active ras to resolve the role of the Ras/Raf signaling pathway in mda-5-mediated growth inhibition/apoptosis induction. Rodent and human tumor cells containing constitutively activated Raf/Raf/MEK/ERK pathways were resistant to mda-5-induced killing and this protection was antagonized by intervening in this signal transduction cascade either by directly inhibiting ras activity using an antisense strategy or by targeting ras-downstream factors, such as MEK1/2, with the pharmacological inhibitor PD98059. The present findings provide a further example of potential cross-talk between growth-inhibitory and growth-promoting pathways in which the ultimate balance of these factors defines cellular homeostasis, leading to survival or induction of programmed cell death.

Defining the mechanism by which IFN-beta dowregulates c-myc expression in human melanoma cells: pivotal role for human polynucleotide phosphorylase (hPNPaseold-35).

Type I interferons (IFN-alpha/-beta) are capable of suppressing c-myc mRNA expression by modulating post-transcriptional processing. However, the molecular mechanism of this phenomenon is poorly understood. We previously established that human polynucleotide phosphorylase (hPNPase(old-35)), a type I IFN-inducible 3',5' exoribonuclease involved in mRNA degradation, induces G1 cell cycle arrest and eventually apoptosis by specifically degrading c-myc mRNA. We now demonstrate a close association between IFN-beta-induced hPNPase(old-35) upregulation and c-myc downregulation in human melanoma cells. Employing stable melanoma cell clones expressing hPNPase(old-35) small inhibitory RNA, we demonstrate that hPNPase(old-35) is a key molecule coupled with IFN-beta-mediated downregulation of c-myc mRNA. Inhibition of hPNPase(old-35) or overexpression of c-myc protects melanoma cells from IFN-beta-mediated growth inhibition, emphasizing the importance of hPNPase(old-35) upregulation and consequent c-myc downregulation in IFN-beta-induced growth inhibition and apoptosis induction. In these contexts, targeted overexpression of hPNPase(old-35) might be a novel therapeutic strategy for c-myc-overexpressing and IFN-resistant tumors, such as melanomas.

Ionizing radiation enhances therapeutic activity of mda-7/IL-24: overcoming radiation- and mda-7/IL-24-resistance in prostate cancer cells overexpressing the antiapoptotic proteins bcl-xL or bcl-2.

Subtraction hybridization applied to terminally differentiating human melanoma cells identified mda-7/IL-24, a cytokine belonging to the IL-10 gene superfamily. Adenoviral-mediated delivery of mda-7/IL-24 (Ad.mda-7) provokes apoptosis selectively in a wide spectrum of cancers in vitro in cell culture, in vivo in human tumor xenograft animal models and in patients with advanced carcinomas and melanomas. In human prostate cancer cells, a role for mitochondrial dysfunction and induction of reactive oxygen species in the apoptotic process has been established. Ectopic overexpression of bcl-xL and bcl-2 prevents these changes including apoptosis induction in prostate tumor cells by Ad.mda-7. We now document that this resistance to apoptosis can be reversed by treating bcl-2 family overexpressing prostate tumor cells with ionizing radiation in combination with Ad.mda-7 or purified GST-MDA-7 protein. Additionally, radiation augments apoptosis induction by mda-7/IL-24 in parental and neomycin-resistant prostate tumor cells. Radiosensitization to mda-7/IL-24 is dependent on JNK signaling, as treatment with the JNK 1/2/3 inhibitor SP600125 abolishes this effect. Considering that elevated expression of bcl-xL and bcl-2 are frequent events in prostate cancer development and progression, the present studies support the use of ionizing radiation in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in prostate cancer, particularly in the context of tumors displaying resistance to radiation therapy owing to bcl-2 family member overexpression.