mda-9/Syntenin regulates the metastatic phenotype in human melanoma cells by activating nuclear factor-kappaB

mda-9/Syntenin is a scaffolding PDZ domain-containing protein overexpressed in multiple human cancers that functions as a positive regulator of melanoma metastasis. Using a normal immortal human melanocyte cell line and weakly and highly metastatic human melanoma cell lines, we presently show that mda-9/syntenin initiates a signaling cascade that activates nuclear factor-kappaB (NF-kappaB) in human melanoma cells. As a consequence of elevated mda-9/syntenin expression, tumor cell growth and motility, fundamental components of tumor cell invasion and metastatic spread of melanoma cells, are enhanced through focal adhesion kinase (FAK)-induced and p38 mitogen-activated protein kinase (MAPK)-induced activation of NF-kappaB. Inhibiting mda-9/syntenin, using an adenovirus expressing antisense mda-9/syntenin, NF-kappaB, using an adenovirus expressing a mutant super-repressor of IkappaBalpha, or FAK, and using a dominant-negative mutant of FAK (FRNK), blocks melanoma cell migration, anchorage-independent growth, and invasion. Downstream signaling changes mediated by mda-9/syntenin, which include activation of FAK, p38 MAPK, and NF-kappaB, promote induction of membrane-type matrix metalloproteinase-1 that then activates pro-MMP-2-promoting migration and extracellular matrix invasion of melanoma cells. These results highlight the importance of mda-9/syntenin as a key component of melanoma metastasis providing a rational molecular target for potentially intervening in the metastatic process.     

mda-9/Syntenin: more than just a simple adapter protein when it comes to cancer metastasis

Cancer is a progressive disease that, in many instances, if untreated, can culminate in metastatic spread of primary tumor cells to distant sites in the body. Metastasis frequently confers virulence and therapy resistance to cancer cells, and defining the molecular events that control metastasis will be mandatory to develop rational, targeted therapies for effective intervention, prevention of recurrence, and the "holy grail" of engendering a cure. Adapter proteins are physiologically pertinent molecules that, through interactions with key regulatory proteins via specific conserved domains, control important cellular events. Melanoma differentiation associated gene-9 (mda-9), also known as syntenin, is a PDZ domain-containing adapter protein that is involved in organization of protein complexes in the plasma membranes, regulation of B-cell development, intracellular trafficking and cell-surface targeting, synaptic transmission, and axonal outgrowth. Recent studies now define a seminal role for mda-9/syntenin in cancer metastasis. The present review provides a current perspective of our understanding of this important aspect of mda-9/syntenin, suggesting that this gene and its encoded protein and interacting protein partners may provide viable targets for intervening in the final and invariably the most lethal stage of cancer progression, namely, cancer metastasis.     

Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas.

The melanoma differentiation associated gene-7 (mda-7) has a potential inhibitory role in melanoma progression, although the mechanisms underlying this effect are still unknown. mda-7 mRNA has been found to be present at higher levels in cultured normal melanocytes compared with metastatic melanoma cell lines. Furthermore, levels of mda-7 message have shown an inverse correlation with melanoma progression in human tumor samples, suggesting that mda-7 may be a novel tumor suppressor gene. We have designed this study to investigate MDA-7 protein expression in different stages of melanoma progression and to examine its antiproliferative effects in vitro. Our data demonstrate that MDA-7 protein can be found in normal melanocytes and early stage melanomas. It is also observed in smooth muscle cells in the skin. However, in keeping with a possible role as a tumor suppressor, MDA-7 expression is decreased in more advanced melanomas, with nearly undetectable levels in metastatic disease. We also investigated antitumor effects of overexpressed MDA-7 on human melanoma cells in vitro. Our results demonstrate that Ad-mda-7 induces apoptosis and G2/M cell cycle arrest in melanoma cells, but not in normal human melanocytes.     

mda-7/IL-24, novel anticancer cytokine: focus on bystander antitumor, radiosensitization and antiangiogenic properties and overview of the phase I clinical experience (Review)

Subtraction hybridization applied to a 'differentiation therapy' model of cancer employing human melanoma cells resulted in the cloning of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24). Initial studies confirm an inverse correlation between mda-7 expression and melanoma development and progression. Forced expression of mda-7 by means of a plasmid or via a replication incompetent adenovirus (Ad.mda-7) promotes growth suppression and induces apoptosis in a broad array of human cancers. In contrast, mda-7 does not induce growth suppressive or toxic effects in normal cells. Based on structure (containing an IL-10 signature motif), secretion by cells (including subsets of T-cells) and location on chromosome 1q (in an area containing IL-10-family genes), mda-7 has now been renamed mda-7/IL-24. Studies by several laboratories have uncovered many of mda-7/IL-24's unique properties, including cancer-specific apoptosis-induction, cell cycle regulation, an ability to inhibit angiogenesis, potent 'bystander antitumor activity' and a capacity to enhance the sensitivity of tumor cells to radiation, chemotherapy and monoclonal antibody therapy. Moreover, based on its profound cancer tropism, substantiated by in vivo human xenograft studies in nude mice, mda-7/IL-24 (administered as Ad.mda-7) was evaluated in a phase I clinical trial in patients with melanomas and solid cancers. These studies document that mda-7/IL-24 is well tolerated and demonstrates evidence of significant clinical activity. In these contexts, mda-7/IL-24 represents a unique cytokine gene with potential for therapy of human cancers. The present review focuses on three unique properties of mda-7/IL-24, namely its potent 'bystander antitumor activity', ability to sensitize tumor cells to radiation, and its antiangiogenesis properties. Additionally, an overview of the phase I clinical trial is provided. These studies affirm that mda-7/IL-24 has promise for the management of diverse cancers.     

Tumor cell intrinsic Toll-like receptor 4 signaling promotes melanoma progression and metastatic dissemination

Most melanoma-associated deaths result from the early development of metastasis. Toll-like receptor 4 (TLR4) expression on nontumor cells is well known to contribute to tumor development and metastatic progression. The role of TLR4 expression on tumor cells however is less well understood. Here we describe TLR4 as a driver of tumor progression and metastatic spread of melanoma cells by employing a transplantable mouse melanoma model. HCmel12 melanoma cells lacking functional TLR4 showed increased sensitivity to tumor necrosis factor α induced cell killing in vitro compared to cells with intact TLR4. Interestingly, TLR4 knockout melanoma cells also showed impaired migratory capacity in vitro and a significantly reduced ability to metastasize to the lungs after subcutaneous transplantation in vivo. Finally, we demonstrate that activation of TLR4 also promotes migration in a subset of human melanoma cell lines. Our work describes TLR4 as an important mediator of melanoma migration and metastasis and provides a rationale for therapeutic inhibition of TLR4 in melanoma.     

Acquired and innate resistance to the cancer-specific apoptosis-inducing cytokine, mda-7/IL-24: not insurmountable therapeutic problems

Despite significant progress in early cancer detection and aggressive therapies, effective treatments for metastatic disease frequently fall short of producing the desired effect of engendering a 'cure.' This can be attributed in part to inherent and acquired resistance of primary and evolving tumor cells to conventional therapeutic approaches. Agents that can interfere with critical aberrant cell signaling and survival pathways in tumor cells while displaying minimal or preferably no toxicity to normal cells represent potentially powerful tools for cancer therapy. A recently identified cancer gene therapeutic is melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) that has the unique ability of inducing apoptosis in diverse cancer cells without harming normal cells or tissues. As a secreted cytokine mda-7/IL-24 also exerts anti-angiogenic, radiosensitizing and immunomodulatory effects and the therapeutic benefits of an adenovirus expressing mda-7/IL-24 (Ad.mda-7) in human xenograft animal models has been successfully recapitulated in a Phase I clinical trial in patients with advanced cancers. However, as with most treatment modalities, particular subsets of tumor cells might be inherently resistant to mda-7/IL-24, as observed in pancreatic and specific colorectal cancer cells, or they might acquire resistance because of repeated exposure to this cytokine. Pataer et al. have developed Ad.mda-7-resistant lung cancer cells in vitro and demonstrated that the combination of Ad.mda-7 with bcl-2 siRNA or 17AAG could overcome this resistance. Indeed, previous studies have also demonstrated that combinatorial approaches employing Ad.mda-7 and diverse other modalities, such as chemo- or radiotherapy, small molecule inhibitors and monoclonal antibodies, can either augment the therapeutic effect of Ad.mda-7 or overcome resistance to this gene. In these contexts, should resistance to monotherapy with mda-7/IL-24 occur, combinatorial therapies with this cytokine might provide a viable path for potentially curing patients of primary and metastatic cancer.     

MDA-9/syntenin is a key regulator of glioma pathogenesis

Background

The extraordinary invasiveness of human glioblastoma multiforme (GBM) contributes to treatment failure and the grim prognosis of patients diagnosed with this tumor. Consequently, it is imperative to define further the cellular mechanisms that control GBM invasion and identify promising novel therapeutic targets. Melanoma differentiation associated gene–9 (MDA-9/syntenin) is a highly conserved PDZ domain–containing scaffolding protein that promotes invasion and metastasis in vitro and in vivo in human melanoma models. To determine whether MDA-9/syntenin is a relevant target in GBM, we investigated its expression in tumor samples and involvement in GBM invasion and angiogenesis.

Materials

We assessed MDA-9/syntenin levels in available databases, patient tumor samples, and human-derived cell lines. Through gain-of-function and loss-of-function studies, we analyzed changes in invasion, angiogenesis, and signaling in vitro. We used orthotopic xenografts with GBM6 cells to demonstrate the role of MDA-9/syntenin in GBM pathogenesis in vivo.

Results

MDA-9/syntenin expression in high-grade astrocytomas is significantly higher than normal tissue counterparts. Forced overexpression of MDA-9/syntenin enhanced Matrigel invasion, while knockdown inhibited invasion, migration, and anchorage-independent growth in soft agar. Moreover, overexpression of MDA-9/syntenin increased activation of c-Src, p38 mitogen-activated protein kinase, and nuclear factor kappa-B, leading to elevated expression of matrix metalloproteinase 2 and secretion of interleukin-8 with corresponding changes observed upon knockdown. GBM6 cells that stably express small hairpin RNA for MDA-9/syntenin formed smaller tumors and had a less invasive phenotype in vivo.

Conclusions

Our findings indicate that MDA-9/syntenin is a novel and important mediator of invasion in GBM and a key regulator of pathogenesis, and we identify it as a potential target for anti-invasive treatment in human astrocytoma.     

Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo

Overexpression of the melanoma differentiation associated gene-7 (mda-7) in vitro results in suppression of lung cancer cell proliferation. However, the ability of MDA-7 to suppress lung cancer in vivo has not been previously demonstrated. In this study, we investigated the possibility of inducing overexpression of the mda-7 gene in human non-small cell lung carcinoma cells in vivo and its effects on tumor growth. Adenovirus-mediated overexpression of MDA-7 in p53-wild-type A549 and p53-null H1299 subcutaneous tumors resulted in significant tumor growth inhibition through induction of apoptosis. In addition, decreased CD31/PECAM expression and upregulation of APO2/TRAIL were observed in tumors expressing MDA-7. In vivo studies correlated well with in vitro inhibition of lung tumor cell proliferation and endothelial cell differentiation mediated by Ad-mda7. These data demonstrate that Ad-mda7 functions as a multi-modality anti-cancer agent, possessing both, pro-apoptotic and anti-angiogenic properties. We demonstrate for the first time the potential therapeutic effects of Ad-mda7 in human lung cancer.     

Mda-9/syntenin promotes human brain glioma migration through focal adhesion kinase (FAK)-JNK and FAK-AKT signaling

Invasion is usually recognized as the main reason for the high recurrence and death rates of glioma and restricts the efficacy of surgery and other therapies. Therefore, we aimed to investigate the mechanism involved in promotion effects of mda-9/syntenin on human glioma cell migration. The wound healing method was used to test the migration ability of human glioma cells CHG-5 and CHG-hS, stably overexpressing mda-9/syntenin. Western blotting was performed to determine the expression and phosphorylation of focal adhesion kinase (FAK) and JNK in CHG-5 and CHG-hS cells. The migration ability of CHG-hS cells was significantly higher than that of CHG-5 cells in fibronectin (FN)-coated culture plates. Phosphorylation of FAK on tyrosine 397, 576, and 925 sites was increased with time elapsed in CHG-hS cells. However, phosphorylated FAK on the tyrosine 861 site was not changed. Phosphorylated Src, JNK and Akt levels in CHG-hS cells were also significantly upregulated. Phosphorylation of JNK and Akt were abolished by the specific inhibitors SP600125 and LY294002, respectively. and the migration ability of CHG-hS cells was decreased, indicating that the JNK and PI3K/Akt pathways play important roles in regulating mda-9/syntenin-induced human brain glioma migration. Our results indicate Mda- 9/syntenin overexpression could activate FAK-JNK and FAK-Akt signaling and then enhance the migration capacity of human brain glioma cells.     

Syntenin is overexpressed and promotes cell migration in metastatic human breast and gastric cancer cell lines

Two human breast cancer cell lines of differing invasive and metastatic potential, MDA-MB-435 and MCF7, were examined using subtractive suppression hybridization in a search for any genes associated with metastasis. Of the 17 cDNAs identified as being differentially expressed genes, it was determined that syntenin was overexpressed in metastatic MDA-MB-435 cells. Expression analysis showed that the expression level of syntenin was well correlated with invasive and metastatic potential in various human breast and gastric cancer cell lines. Moreover, gastric tumor tissues exhibited a much higher syntenin mRNA expression than their normal counterparts. Syntenin-transfected MCF7 cells migrated more actively, and showed an increased invasion rate relative to vector-transfectants or parental MCF7 in vitro, without evidencing any effect on the adhesion to fibronectin, type I collagen and laminin. Similarly, the forced expression of syntenin to human gastric cancer cell line Az521 increased its migratory and invasive potential in vitro. Syntenin-expressing MCF7 cells were associated with the appearance of numerous cell surface extensions and with pseudopodia formation on collagen I, suggesting that syntenin may be involved in the signaling cascade to actin-reorganization. Mutation study suggested that PDZ2 domain of syntenin could be an essential role in its stimulatory effect on the cell migration. This is the first demonstration that syntenin, a PDZ motif-containing protein, can be overexpressed during the metastatic progression of human breast and gastric cancer cells and that it can function as a metastasis-inducing gene.     

Is mda-7/IL-24 a "magic bullet" for cancer?

The "holy grail" of cancer therapy is to identify and exploit genetic elements and signal transduction pathways capable of selectively destroying tumor cells without eliciting harmful effects in normal cells or tissues. To achieve this objective, subtraction hybridization was combined with a "differentiation therapy" model of cancer in which human melanoma cells were induced to revert to a more "normal" state, growth arrest irreversibly, and terminally differentiate by treatment with fibroblast IFN and mezerein. This strategy permitted the cloning of a variety of genes involved in regulating important physiologic processes, including cell cycle, response to cytokines and viruses, tumorigenesis and metastasis, cancer growth control, apoptosis, and senescence. A specific gene, melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), displaying cancer-specific apoptosis-inducing properties isolated using this scheme has now come into the limelight as a new gene therapy for divergent cancers. Although the mechanism of cancer cell selectivity of mda-7/IL-24 remains to be delineated, numerous attributes enable this gene as an effective therapy for cancer, including an ability to discriminate between normal and cancer cells, induce apoptosis in diverse tumor cells, promote "bystander" antitumor effects, inhibit tumor growth and angiogenesis in animal models, synergize with radiation, and modulate immune responses. These unique features combined with successful transition into the clinic instill confidence that mda-7/IL-24, as a single or more likely as part of a combinatorial approach, may provide profound therapeutic benefit for cancer patients.     

The cancer growth suppressing gene mda-7 induces apoptosis selectively in human melanoma cells

Human melanoma cells growth arrest irreversibly, lose tumorigenic potential and terminally differentiate after treatment with a combination of fibroblast interferon (IFN-beta) and the protein kinase C activator mezerein (MEZ). Applying subtraction hybridization to this model differentiation system permitted cloning of melanoma differentiation associated gene-7, mda-7. Expression of mda-7 inversely correlates with melanoma development and progression, with elevated expression in normal melanocytes and nevi and increasingly reduced expression in radial growth phase, vertical growth phase and metastatic melanoma. When expressed by means of a replication incompetent adenovirus (Ad.mda-7) growth of melanoma, but not normal early passage or immortal human melanocytes, is dramatically suppressed and cells undergo programmed cell death (apoptosis). Infection of metastatic melanoma cells with Ad.mda-7 results in an increase in cells in the G(2)/M phase of the cell cycle and changes in the ratio of pro-apoptotic (BAX, BAK) to anti-apoptotic (BCL-2, BCL-XL) proteins. Ad.mda-7 infection results in a temporal increase in mda-7 mRNA and intracellular MDA-7 protein in most of the melanocyte/melanoma cell lines and secretion of MDA-7 protein is readily detected following Ad.mda-7 infection of both melanocytes and melanoma cells. The present studies document a differential response of melanocytes versus melanoma cells to ectopic expression of mda-7 and support future applications of mda-7 for the gene-based therapy of metastatic melanoma.     

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.     

Elevated expression of syntenin in breast cancer is correlated with lymph node metastasis and poor patient survival

Introduction

Syntenin is a scaffolding-PDZ domain-containing protein. Although it is reported that syntenin is associated with melanoma growth and metastasis, the possible role of syntenin in breast cancer has not been well elucidated. The present study investigated the expression and function of syntenin in breast cancer.

Methods

Real-time polymerase chain reaction (PCR) and Western blots were used to determine the mRNA and protein expression of syntenin. With a combination of overexpression and RNA interference, the effect of syntenin on migration, invasion, and ERK1/2 activation was examined in breast cancer cell lines. The effect of syntenin in vivo was assessed with an orthotropic xenograft tumor model in BALB/c nu/nu mice. In addition, the expression level of syntenin in clinical breast cancer tissues was evaluated with immunohistochemistry. The Kaplan-Meier survival curve was used to evaluate patient survival, and the Cox proportional hazards model was used for multivariate analysis.

Results

Our study showed that syntenin expression was upregulated in high-metastasis breast cancer cell lines and breast cancer tissues. Overexpression of syntenin in breast cancer cells promoted cell migration and invasion in vitro. Moreover, overexpression of syntenin promoted breast tumor growth and lung metastasis in vivo. We further showed that activation of integrin β1 and ERK1/2 was required for syntenin-mediated migration and invasion of breast cancer cells. The correlation between syntenin expression and tumor size (P = 0.011), lymph node status (P = 0.001), and recurrence (P = 0.002) was statistically significant. More important, syntenin expression in primary tumors was significantly related to patients'' overall survival (OS; P = 0.023) and disease-free survival (DFS; P = 0.001). Its status was an independent prognostic factor of OS (P = 0.049) and DFS (P = 0.002) in our cohort of patients.

Conclusions

These results suggest that syntenin plays a significant role in breast cancer progression, and it warrants further investigation as a candidate molecular marker of breast cancer metastasis and a potential therapeutic target.     

High level of messenger RNA for BRMS1 in primary breast carcinomas is associated with poor prognosis

BRMS1 is regarded as a metastasis suppressor gene for its ability to reduce metastatic potential of human and murine breast cancer cells as well as human melanoma cells. However, BRMS1 association to human tumor progression is not clearly understood. In the present study we analyzed BRMS1 mRNA expression in tumor progression and its potential prognostic value for breast carcinoma. BRMS1 mRNA expression level was quantified by real-time PCR in 47 tumoral, in 14 peritumoral and in 15 metastatic microdissected cellular populations from 47 breast cancer patients with 10-year follow up. We found BRMS1 expression to be higher in carcinoma cells than in matching normal epithelial cell populations in 10 out of 14 cases (p = 0.0005), while lymph-nodal carcinoma cells showed lower BRMS1 expression in 9 out of 15 cases (p = 0.001). Using both in vivo (human mammary breast carcinomas) and in vitro systems (breast cancer cell lines) we were able to demonstrate that BRMS1 overexpression was not a bias effect induced by cell proliferation rate. BRMS1 expression levels did not correlate with standard breast cancer prognostic factors but BRMS1 higher expression was associated with patient shorter disease-free and overall survival. Our findings are apparently inconsistent with the concept of BRMS1 as a metastasis suppressor gene. One possible explanation is that epithelial cells increase their BRMS1 expression as a compensatory response to tumor formation or metastasis progression, which is elevated in proportion to tumor aggressiveness, whereas those cells of the primary tumor that cannot upregulate BRMS1 escape to form metastasis.     

Unique aspects of mda-7/IL-24 antitumor bystander activity: establishing a role for secretion of MDA-7/IL-24 protein by normal cells

Melanoma differentiation associated gene-7 (mda-7) was cloned using subtraction hybridization from terminally differentiated human melanoma cells. Based on structural and functional properties, mda-7 is now recognized as interleukin-24 (IL-24), a new member of the expanding IL-10 gene family. Unique properties of mda-7/IL-24 include its ability to selectively induce growth suppression, apoptosis and radiosensitization in diverse human cancer cells, without causing similar effects in normal cells. The utility of mda-7/IL-24, administered by means of a replication-incompetent adenovirus, as a gene therapy for cancer has recently received validation in patients, highlighting an important phenomenon initially observed in pancreatic tumor cells, namely a 'potent bystander apoptosis-inducing effect' in adjacent tumor cells not initially receiving this gene product. We presently investigated the contribution of mda-7/IL-24 secreted by normal cells in mediating this 'bystander effect', and document that normal cells induced to produce mda-7/IL-24 following infection with recombinant adenoviruses expressing this cytokine secrete mda-7/IL-24, which modifies the anchorage-independent growth, invasiveness, survival and sensitivity to radiation of cancer cells that contain functional IL-20/IL-22 receptors, but not in cancer cells that lack a complete set of receptors. Moreover, the combination of secreted mda-7/IL-24 and radiation engenders a 'bystander antitumor effect' not only in inherently mda-7/IL-24 or radiation-sensitive cancer cells, but also in tumor cells overexpressing the antiapoptotic proteins bcl-2 or bcl-x(L) and displaying resistance to either treatment alone. The present studies provide definitive evidence that secreted mda-7/IL-24 from normal cells can induce direct antitumor and radiation-enhancing effects that are dependent on the presence of canonical receptors for this cytokine on tumor cells. Moreover, we now describe a novel means of enhancing mda-7/IL-24's therapeutic potential by targeting normal cells to produce and release this cancer-specific apoptosis-inducing cytokine, a strategy that could be employed as an innovative way of using this unique gene product for treating metastatic disease.     

Loss of MDA-7 expression with progression of melanoma.

PURPOSE: Ectopic transfer of the melanoma differentiation-associated gene-7 (mda-7) has been shown in vitro to suppress growth and induce apoptosis in a variety of human tumor cell lines; similar effects are not elicited in normal cells. Thus, the mda-7 gene seems to function as a novel tumor suppressor, and there is interest in the potential of mda-7 gene transfer as cancer therapy. The objective of this study was to determine if MDA-7 protein is lost during primary melanoma progression from superficial to invasive stages and from localized to metastatic tumor. As a secondary objective, we analyzed MDA-7 protein expression in primary melanomas for correlation with predictors of outcome and with survival. MATERIALS AND METHODS: MDA-7 protein expression was evaluated by immunohistochemistry in 41 primary melanomas and 41 metastases, including 24 paired samples. Each sample was scored for the percentage of positive cells and the overall intensity of immunolabeling. RESULTS: Significant decreases in MDA-7 immunostaining, reflected in both number and intensity scores, were observed when comparing the intraepidermal and superficially invasive portions with the deeply invasive portions of primary tumors. Significant differences were also observed when comparing primary tumors to paired metastases. CONCLUSION: Downregulation of MDA-7 expression in primary melanomas facilitates progression to invasive and metastatic stages. These data support the development of Ad-mda7 as gene therapy for advanced melanoma.     

Prognostic significance of BRMS1 expression in human melanoma and its role in tumor angiogenesis

Breast cancer metastasis suppressor 1 (BRMS1) has been reported to suppress metastasis without significantly affecting tumorigenicity in breast cancer and ovarian cancer. To investigate the role of BRMS1 in human melanoma progression and prognosis, we established tissue microarray and BRMS1 expression was evaluated by immunohistochemistry in 41 dysplastic nevi, 90 primary melanomas and 47 melanoma metastases. We found that BRMS1 expression was significantly decreased in metastatic melanoma compared with primary melanoma or dysplastic nevi (P=0.021 and 0.001, respectively, χ(2) test). In addition, reduced BRMS1 staining was significantly correlated with American Joint Committee on Cancer stages (P=0.011, χ(2) test), but not associated with tumor thickness, tumor ulceration and other clinicopathological parameters. Furthermore, BRMS1 expression was significantly correlated with disease-specific 5-year survival of melanoma patients (P=0.007, log-rank test). Multivariate Cox regression analysis revealed that BRMS1 staining was an independent prognostic factor for melanoma patients (relative risk=0.51; confidence interval=0.29-0.91; P=0.022). Moreover, we demonstrated that BRMS1 overexpression inhibited endothelial cell growth and tube formation ability by suppressing NF-κB activity and IL-6 expression in vitro. We also showed that knockdown of BRMS1 increased IL-6 expression and promoted endothelial cell growth and tube formation. In addition, our data revealed that the BRMS1-mediated IL-6 expression is dependent on NF-κB. Strikingly, our in vivo studies using nude mice confirmed that BRMS1 inhibited blood vessel formation and the recruitment of CD31-positive cells in matrigel plugs. Taken together, BRMS1 expression was decreased in metastatic melanomas, which resulted in deficient suppression of angiogenesis and contributed to melanoma progression. BRMS1 may serve an important prognostic marker and therapeutic target for melanoma patients.     

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.     

Melanoma differentiation associated gene-7, mda-7/IL-24, selectively induces growth suppression,apoptosis and radiosensitization in malignant gliomas in a p53-independent manner

Malignant gliomas are extremely aggressive cancers currently lacking effective treatment modalities. Gene therapy represents a promising approach for this disease. A requisite component for improving gene-based therapies of brain cancer includes tumor suppressor genes that exhibit cancer constrained inhibitory activity. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7) as a gene associated with melanoma cell growth, differentiation and progression. Ectopic expression of mda-7 by means of a replication-incompetent adenovirus (Ad), Ad.mda-7, induces growth suppression and apoptosis selectively in diverse human cancers, without producing any apparent harmful effect in normal cells. We presently demonstrate that Ad.mda-7 induces growth inhibition and apoptosis in malignant human gliomas expressing both mutant and wild-type p53, and these effects correlate with an elevation in expression of members of the growth arrest and DNA damage (GADD) gene family. In contrast, infection with a recombinant Ad expressing wild-type p53, Ad.wtp53, specifically affects mutant p53 expressing gliomas. When tested in early passage normal and immortal human fetal astrocytes, growth inhibition resulting from infection with Ad.mda-7 or Ad.wtp53 is significantly less than in malignant gliomas and no toxicity is evident in these normal cells. Moreover, infection of gliomas with Ad.mda-7 or treatment with purified GST-MDA-7 protein sensitizes both wild-type and mutant p53 expressing tumor cells to the growth inhibitory and antisurvival effects of ionizing radiation, and this response correlates with increased expression of specific members of the GADD gene family. Since heterogeneity in p53 expression is common in evolving gliomas, the present findings suggest that Ad.mda-7 may, in many instances, prove more beneficial for the gene-based therapy of malignant gliomas than administration of wild-type p53.