Strategy For Treatment Of Melanoma Research Articles

The role of stress and beta-adrenergic system in melanoma: current knowledge and possible therapeutic options.

The aim of the present review was to discuss recent findings on the role of beta-adrenergic system in melanoma, in order to provide information on the biological responses elicited by its activation and its potential application for melanoma treatment. A literature search was performed, and evidences regarding the involvement of stress and beta-adrenergic system in cancer and melanoma were found and discussed. Our search pointed out that beta-adrenergic system is a key regulator of important biological processes involved in the onset and progression of some solid tumors. In the last decade, functional beta-adrenoceptors have been also identified on melanoma cells, as well as on their microenvironment cells. Similarly to other common cancers too, the activation of such adrenoceptors by catecholamines, usually released under stress conditions, has been found to trigger pro-tumorigenic pathways contributing to cell proliferation and motility, immune system regulation, apoptosis, epithelial-mesenchymal transition, invasion and neoangiogenesis. The biological evidences we found clarify and sustain the clinical evidences reporting the involvement of chronic stress in melanoma onset and progression. In such scenario, it is conceivable that a therapeutic approach targeting beta-adrenergic system could constitute a novel and promising strategy for melanoma treatment.

Combined inhibition of Hsp90 and heme oxygenase-1 induces apoptosis and endoplasmic reticulum stress in melanoma

Heat shock proteins are ubiquitous molecular chaperones involved in post-translational folding, stability, activation and maturation of many proteins that are essential mediators of signal transduction and cell cycle progression. Heat shock protein 90 (Hsp90) has recently emerged as an attractive therapeutic target in cancer treatment since it may act as a key regulator of various oncogene products and cell-signaling molecules. Heme oxygenase-1 (HO-1; also known as Hsp32) is an inducible enzyme participating in heme degradation and involved in oxidative stress resistance. Recent studies indicate that HO-1 activation may play a role in tumor development and progression. In the present study we investigated the chemotherapic effects of combining an Hsp90 inhibitor (NMS E973) and an HO-1 inhibitor (SnMP) on A375 melanoma cells. NMS E973 treatment was able to reduce cell viability and induce endoplasmic reticulum (ER) stress (i.e. Ire1α, ERO1, PDI, BIP and CHOP). Interestingly, no significant effect was observed in reactive oxygen species (ROS) formation. Finally, NMS E973 treatment resulted in a significant HO-1 overexpression, which in turn serves as a possible chemoresistance molecular mechanism. Interestingly, the combination of NMS E973 and SnMP produced an increase of ROS and reduced cell viability compared to NMS E973 treatment alone. The inhibitors combination exhibited higher ER stress, apoptosis as evidenced by bifunctional apoptosis regulator (BFAR) mRNA expression and lower phosphorylation of Akt when compared to NMS E973 alone. In conclusion, these data suggest that HO-1 inhibition potentiates NMS E973 toxicity and may be exploited as a strategy for melanoma treatment.

Comparative gene-expression profiling of CD133(+) and CD133(-) D10 melanoma cells.

The present study aimed to compare the gene-expression profiling of CD133(+) and CD133(-) D10 cells. Cancer stem cell-like properties and gene-expression profiling of CD133(+) D10 cells versus CD133(-) cells were evaluated. The CD133(+) D10 cells showed significantly higher clonogenic and spheroid forming potential, also higher expression of stemness genes NANOG and OCT4A compared with the CD133(-) cells. Gene-expression profiling of CD133(+) versus CD133(-) D10 cells revealed that 130 genes including ABC transporter superfamily (ABCC1, ABCG2 and ABCC6) were upregulated, while 61 genes including apoptosis modifying genes (CASP8 and TNFRSF4) were downregulated. We conclude that many genes involved in drug resistance and tumor aggressiveness are upregulated in CD133(+) D10 cells and targeting them might be an efficient strategy for treatment of melanoma.

Ursolic acid and resveratrol synergize with chloroquine to reduce melanoma cell viability.

Malignant melanoma is associated with a 5-year survival rate of less than 20% once metastasized. Malignant melanoma cells exhibit increased levels of autophagy, a process of intracellular digestion that allows cells to survive various stresses including chemotherapies, resulting in reduced patient survival. Autophagy can be inhibited by chemicals like chloroquine (CQ), which prevents fusion of autophagosomes to lysosomes, resulting in autophagosome accumulation in most systems. Here, we describe how tested CQ to see whether it could sensitize B16F10 metastatic mouse melanoma cells to the anticancer activities of the natural compounds ursolic acid (UA) and resveratrol (RES). CQ with UA or RES strongly and synergistically reduced the viability of B16F10 mouse melanoma and A375 human melanoma cells. Surprisingly, flow cytometry of acridine orange-stained cells showed that UA or RES in combination with CQ significantly reduced autophagosome levels. Western blotting analysis revealed that CQ plus UA or RES paradoxically increased LC3II, indicative of autophagosome accumulation. In addition, CQ plus RES synergistically decreased the levels of both autophagy initiator beclin-1 and autophagy supporter p62. These results indicate that CQ with UA or RES strongly and synergistically reduces the viability of B16F10 and A375 melanoma cells. However, studies on B16F10 cells have shown that the synergistic effect was not mediated by inhibition of autophagy induced by UA or RES. These compounds are well-tolerated in humans, and CQ has shown promise as an adjuvant therapy. These combinations may be valuable treatment strategies for melanoma.

Combining a BCL2 Inhibitor with the Retinoid Derivative Fenretinide Targets Melanoma Cells Including Melanoma Initiating Cells

Investigations from multiple laboratories support the existence of melanoma initiating cells (MICs) that potentially contribute to melanoma's drug resistance. ABT-737, a small molecule BCL-2/BCL-XL/BCL-W inhibitor, is promising in cancer treatments, but not very effective against melanoma, with the anti-apoptotic protein MCL-1 as the main contributor to resistance. The synthetic retinoid fenretinide (4-HPR) has shown promise for treating breast cancers. Here, we tested whether the combination of ABT-737 with 4-HPR is effective in killing both the bulk of melanoma cells and MICs. The combination synergistically decreased cell viability and caused cell death in multiple melanoma cells lines (carrying either BRAF or NRAS mutations), but not in normal melanocytes. The combination increased the NOXA expression and caspase-dependent MCL-1 degradation. Knocking-down NOXA protected cells from combination-induced apoptosis, implicating the role of NOXA in the drug synergy. The combination treatment also disrupted primary spheres (a functional assay for MICs) and decreased the percentage of ALDHhigh cells (a marker of MICs) in melanoma cell lines. Moreover, the combination inhibited the self-renewal capacity of MICs, measured by secondary sphere forming assays. In vivo, the combination inhibited tumor growth. Thus, this combination is a promising treatment strategy for melanoma, regardless of mutation status of BRAF or NRAS.

Universes collide: combining immunotherapy with targeted therapy for cancer.

There have been significant advances in the past several years with regard to targeted therapy and immunotherapy for cancer. This is highlighted in melanoma, where treatment with targeted therapy (against the BRAF oncoprotein) results in responses in the majority of patients, although the duration of response is limited. In contrast, treatment with immunotherapy results in a lower response rate, but one that tends to be more durable. Insights about mechanisms of response and potential synergy between these treatment strategies for melanoma are a focus of this review, with opportunities to extend these insights to the treatment of other cancers. Two major advances in melanoma have occurred concurrently and involve treatment with targeted therapy and immune checkpoint blockade. However, each of these approaches has limitations with regard to overall response rates or duration of response. To address this, investigators have proposed combining these strategies, and this concept is being tested empirically in clinical trials. There is a scientific rationale supporting the combination of targeted therapy and immunotherapy, and these concepts are discussed herein.

67-kDa Laminin Receptor-dependent Protein Phosphatase 2A (PP2A) Activation Elicits Melanoma-specific Antitumor Activity Overcoming Drug Resistance

The Ras/Raf/MEK/ERK pathway has been identified as a major, druggable regulator of melanoma. Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, resulting in constitutive melanoma hyperproliferation. A selective BRAF inhibitor showed remarkable clinical activity in patients with mutated BRAF. Unfortunately, most patients acquire resistance to the BRAF inhibitor, highlighting the urgent need for new melanoma treatment strategies. Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) inhibits cell proliferation independently of BRAF inhibitor sensitivity, suggesting that increased understanding of the anti-melanoma activity of EGCG may provide a novel therapeutic target. Here, by performing functional genetic screening, we identified protein phosphatase 2A (PP2A) as a critical factor in the suppression of melanoma cell proliferation. We demonstrated that tumor-overexpressed 67-kDa laminin receptor (67LR) activates PP2A through adenylate cyclase/cAMP pathway eliciting inhibitions of oncoproteins and activation of tumor suppressor Merlin. Activating 67LR/PP2A pathway leading to melanoma-specific mTOR inhibition shows strong synergy with the BRAF inhibitor PLX4720 in the drug-resistant melanoma. Moreover, SET, a potent inhibitor of PP2A, is overexpressed on malignant melanoma. Silencing of SET enhances 67LR/PP2A signaling. Collectively, activation of 67LR/PP2A signaling may thus be a novel rational strategy for melanoma-specific treatment.

Abstract 5149: EZH2 as a novel therapeutic target in melanoma treatment

Abstract Histone modifications are increasingly being recognized as an important epigenetic mechanism that governs chromatin structure and gene expression. EZH2 is the catalytic subunit of the polycomb repesssive complex 2 (PRC2), responsible for tri-methylation of lysine 27 residues on type 3 histones (H3K27Me3) that leads to gene silencing. EZH2 is found to be over-expressed in many different types of cancers, where it is postulated to abnormally repress tumor suppressor genes. Activating mutations described within the catalytic SET domain of EZH2 at codon 646 confer its oncogenic activity, as identified in approximately 3% of melanomas. We sought to assess whether direct inhibition of EZH2's methyltransferase activity is a suitable strategy for melanoma treatment. This was tested using a small molecule inhibitor known as GSK126, a potent, highly selective drug that competes with S-adenosyl-methionine substrate. We found that both mutant and non-mutant EZH2 melanoma cell lines over-express EZH2 compared to primary cells, via western blot and that levels of H3K27 methylation were reduced following drug treatment. GSK126 significantly decreased proliferation and reduced the percentage of cells in the G1 phase of the cell cycle, most likely attributed to a G2 arrest. Flow cytometery also revealed an increased sub-G1 population, indicative of drug induced apoptosis. This was confirmed by Annexin-V staining. Growth inhibition by GSK126 was particularly effective in melanoma cells harboring EZH2 activating mutations and had little effect on primary cells that displayed low levels of EZH2. Expression arrays and chromatin immunoprecipitation are being used to determine which PCR2 target genes are being de-repressed and to decipher the mechanisms of GSK126 growth inhibition. Collectively our initial data indicate that EZH2 represents a promising gene target for therapeutic intervention in melanoma in vitro and that in vivo studies are warranted. Citation Format: Jessamy Tiffen, Dilini Gunatilake, Stuart Gallagher, Kavitha Gowrishankar, Peter Hersey. EZH2 as a novel therapeutic target in melanoma treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5149. doi:10.1158/1538-7445.AM2014-5149

Proteasomal Degradation of Mcl-1 by Maritoclax Induces Apoptosis and Enhances the Efficacy of ABT-737 in Melanoma Cells

Background and purposeMetastatic melanoma remains one of the most invasive and highly drug resistant cancers. The over expression of anti-apoptotic protein Mcl-1 has been associated with inferior survival, poor prognosis and chemoresistance of malignant melanoma. A BH3 mimetic, ABT-737, has demonstrated efficacy in several forms of cancers. However, the efficacy of ABT-737 depends on Mcl-1. Because the over expression of Mcl-1 is frequently observed in melanoma, specifically targeting of Mcl-1 may overcome the resistance of ABT-737. In this study, we investigated the effects of Maritoclax, a novel Mcl-1-selective inhibitor, alone and in combination with ABT-737, on the survival of human melanoma cells.Experimental approachFor cell viability assessment we performed MTT assay. Apoptosis was determined using western blot and flow cytometric analysis.Key resultsThe treatment of Maritoclax reduced the cell viability of melanoma cells with an IC50 of between 2.2–5.0 µM. Further, treatment of melanoma cells with Maritoclax showed significant decrease in Mcl-1 expression. We found that Maritoclax was able to induce apoptosis in melanoma cells in a caspase-dependent manner. Moreover, Maritoclax induced Mcl-1 degradation via the proteasome system, which was associated with its pro-apoptotic activity. We also found that Maritoclax treatment increased mitochondrial translocation of Bim and Bmf. Importantly, Maritoclax markedly enhanced the efficacy of ABT-737 against melanoma cells in both two- and three-dimensional spheroids.Conclusions and implicationsTaken together, these results suggest that targeting of Mcl-1 by Maritoclax may represent a new therapeutic strategy for melanoma treatment that warrants further investigation as a single therapy or in combination with other agents such as Bcl-2 inhibitors.

Sticky siRNAs targeting survivin and cyclin B1 exert an antitumoral effect on melanoma subcutaneous xenografts and lung metastases

BackgroundMelanoma represents one of the most aggressive and therapeutically challenging malignancies as it often gives rise to metastases and develops resistance to classical chemotherapeutic agents. Although diverse therapies have been generated, no major improvement of the patient prognosis has been noticed. One promising alternative to the conventional therapeutic approaches currently available is the inactivation of proteins essential for survival and/or progression of melanomas by means of RNA interference. Survivin and cyclin B1, both involved in cell survival and proliferation and frequently deregulated in human cancers, are good candidate target genes for siRNA mediated therapeutics.MethodsWe used our newly developed sticky siRNA-based technology delivered with linear polyethyleneimine (PEI) to inhibit the expression of survivin and cyclin B1 both in vitro and in vivo, and addressed the effect of this inhibition on B16-F10 murine melanoma tumor development.ResultsWe confirm that survivin and cyclin B1 downregulation through a RNA interference mechanism induces a blockage of the cell cycle as well as impaired proliferation of B16-F10 cells in vitro. Most importantly, PEI-mediated systemic delivery of sticky siRNAs against survivin and cyclin B1 efficiently blocks growth of established subcutaneaous B16-F10 tumors as well as formation and dissemination of melanoma lung metastases. In addition, we highlight that inhibition of survivin expression increases the effect of doxorubicin on lung B16-F10 metastasis growth inhibition.ConclusionPEI-mediated delivery of sticky siRNAs targeting genes involved in tumor progression such as survivin and cyclin B1, either alone or in combination with chemotherapeutic drugs, represents a promising strategy for melanoma treatment.

Gene electrotransfer of siRNAs against CD146 inhibits migration and invasion of human malignant melanoma cells SK-MEL28

Targeting molecules involved in tumor invasion may be useful in future strategies for melanoma treatment aiming to reduce the progression of the disease and prevention of metastatic spread. During melanoma progression to metastatic disease, a significant overexpression of melanoma cell adhesion molecule CD146 occurs. It has been correlated with tumor progression and metastatic potential. Various approaches for targeting CD146 in melanoma cells have been exploited and CD146 has been shown to be a promising target for antitumor therapy. In our study, a new approach of gene electrotransfer (GET) of small interfering RNA (siRNA) against CD146 was evaluated in human malignant melanoma cells. We demonstrated for the first time that downregulation of CD146 mRNA after GET is more significant than after lipid-mediated transfer. Furthermore, reduced cell migration and invasion of melanoma cells was observed after GET of therapeutic siRNAs. GET of therapeutic siRNAs also reduced cell survival, but had no effect on cell proliferation. These findings suggest that targeting CD146 expression by GET of siRNAs against CD146 is effective for reducing the metastatic potential of melanoma cells in vitro. CD146 is therefore a potential target for the development of adjuvant therapies for metastatic melanoma.

Rapamycin induces autophagy in the melanoma cell line M14 via regulation of the expression levels of Bcl-2 and Bax

Cancer therapy with rapamycin has been successfully implemented for kidney cancer, glioblastoma and prostate cancer. However, there are few studies concerning the effects of rapamycin on the treatment of human melanoma. In this study, we investigated whether rapamycin may be a promising strategy for the effective treatment of melanoma and explored the possible mechanism for this by culturing M14 cells in vitro and treating with rapamycin at three concentrations (10, 50 or 100 nmol/l). MDC and LC3B staining, western blot analysis, flow cytometry and transmission electron microscopy were employed. We revealed that rapamycin induced autophagy and inhibited the proliferation of M14 cells in a concentration-dependent manner, Furthermore, western blot analysis revealed an upregulated expression of Bcl-2 and downregulated expression of Bax in M14 cells. In conclusion, rapamycin induced autophagy and inhibited the growth of M14 cells. The mechanism may involve regulation of the expression of Bcl-2 family proteins. Rapamycin appears to be a promising strategy for the effective treatment of melanoma.

An Ester Extract of Cochinchina Momordica Seeds Induces Differentiation of Melanoma B16 F1 Cells via MAPKs Signaling

Cochinchina momordica seeds (CMS) have been widely used due to antitumor activity by Mongolian tribes of China. However, the details of the underlying mechanisms remain unknown. In the present study, we found that an EtOAc (ethyl ester) extract of CMS (CMSEE) induced differentiation and caused growth inhibition of melanoma B16 F1 cells. CMSEE at the concentration of 5-200 μg/ml exhibited strongest anti-proliferative effects on B16 F1 cells among other CMS fractions (water or petroleum ether). Moreover, CMSEE induced melanoma B16 F1 cell differentiation, characterized by dendrite-like outgrowth, increasing melanogenesis production, as well as enhancing tyrosinase activity. Western blot analysis showed that sustained phosphorylation of p38 MAP accompanied by decrease in ERK1/2 and JNK dephosphorylation were involved in CMSEE-induced B16 F1 cell differentiation. Notably, 6 compounds that were isolated and identified may be responsible for inducing differentiation of CMSEE. These results indicated that CMSEE contributes to the differentiation of B16 F1 cells through modulating MAPKs activity, which may throw some light on the development of potentially therapeutic strategies for melanoma treatment.

Abstract 2823: PHA-739358, a pan-Aurora kinase inhibitor, induces apoptosis and inhibits invasion in melanoma cell lines

Abstract Cutaneous melanoma (CM) is the most lethal form of skin cancer and is highly invasive and markedly resistant to conventional therapy. The incidence of malignant melanoma has been increasing worldwide for the past three decades, however, the standard care for patients with metastatic melanoma has not changed significantly, therefore, new strategies for treatment of metastatic melanoma are urgently needed. Significant insights have recently been gained into the molecular events underpinning the development of melanoma. Aurora kinases play an essential role as key mitotic regulators, controlling chromosome alignment, segregation, centrosomal maturation, mitotic spindle formation, and cytokinesis during mitosis, and are frequently overexpressed in various human cancers including that of CM making them ideal targets for anti-cancer therapy. Several small molecule inhibitors of the aurora kinases have been developed and tested in vivo and in vitro, and some of them have shown promising clinical efficacy in a number of early phaseI/II clinical trials. Among those, one of the most advanced clinical compounds currently is PHA-739358 (PHA). The goal of this study is to investigate the anti-proliferative and anti-invasive features of PHA in three melanoma cell lines including WM3211, Lu1205, and SK-Mel28. The results showed a time- and dose- dependent growth inhibition in all the cell lines tested with low μM IC50s upon PHA treatment, and significant induction of caspase activity was observed as well. FACScan cell cycle analysis showed significant induction of G2/M population of cells in all three cell lines and increased polyploidy proportion of cells in WM3211 cell line. For the first time, we showed that PHA inhibits melanoma cell invasion in a dose- and time- dependent manor using a Matrigel Invasion Assay. More interestingly, addition of PHA enhanced Temozolomide (TMZ)-induced caspase activation in melanoma cell lines indicating a potential combination therapeutic strategy for the treatment of melanoma. Thus, targeting aurora kianses with PHA is a promising therapeutic strategy administered alone or in combination with TMZ for patients with advanced stages of CM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2823. doi:1538-7445.AM2012-2823

Elevated expression of Rad18 regulates melanoma cell proliferation

The E3 ligase Rad18 is a key regulator for the lesion bypass pathway, which plays an important role in genomic stability. However, the status of Rad18 expression in melanoma is not known. Using melanoma tissue microarray (TMA), we showed that nuclear Rad18 expression was upregulated in primary and metastatic melanoma compared to dysplastic nevi. Rad18 expression was significantly reduced in sun-exposed sites compared to the sun-protected sites. Strong Rad18 expression correlated with worse 5-year patient survival and was an independent prognostic factor for melanoma found in the sun-protected sites. Furthermore, we showed that melanoma cell proliferation and the expression of pAkt and cyclin D1 were reduced upon Rad18 knockdown. We, for the first time, showed that Rad18 is significantly increased in melanoma and predicts the poor outcome for melanoma in the sun-protected sites. Rad18 is involved in the regulation of melanoma cell proliferation, which can be exploited in designing new strategy for melanoma treatment.

Paying “Particle” Attention to Novel Melanoma Treatment Strategies

Malignant melanoma remains the deadliest form of skin cancer because of its highly aggressive nature and the lack of effective treatments. Recent investigations into alternative treatment strategies have highlighted the exciting potential of nanoparticles to increase melanoma cell delivery and the efficacy of small interfering RNAs (siRNAs) and pharmacological inhibitors. In this issue, Chen et al. report a new liposomal nanoparticle for c-Myc siRNA delivery, noting it to be highly effective in reducing c-Myc expression and inhibiting melanoma tumor growth in mouse models. This preclinical study underscores the importance of investigating nanoparticle treatment options for chemoresistant melanomas.

Simultaneous Inhibition of MEK and CDK4 Leads to Potent Apoptosis in Human Melanoma Cells

ABSTRACT Deregulation of RAS-RAF-MEK-ERK and p16INK4A-cycylin D:CDK4/6-RB pathways is important for melanoma development. Chemotherapeutic agents targeting both pathways were developed but results of clinical studies with monotherapies were disappointing. We examined the effect of co-targeting both pathways with MEK inhibitor PD98059 and CDK4 inhibitor 219476 on human melanoma cells lines, and found that combinatorial treatment dramatically increased apoptosis compared to the single agent treatment. The apoptosis was associated with downregulation of BCL2, BCL2L1, BIRC5, and upregulation of BIM. Our results indicate that simultaneously targeting ERK and RB pathways is a promising strategy for melanoma treatment and should encourage further in-depth investigations.

Abstract LB-232: MEK2 signaling pathway alone is sufficient but not necessary for melanoma cell proliferation

Abstract The Raf-MEK-ERK signaling pathway plays a critical role in survival of many types of cancers, including melanoma. Targeting this pathway by using MEK inhibitors is a rational therapeutic strategy for melanoma treatment. Despite their success in pre-clinical studies, MEK inhibitors have failed to obtain satisfactory responses in clinical cancer trials. An improved understanding of MEK signaling in melanoma survival will facilitate the design of more effective therapeutics. There are two isoforms of MEK; MEK1 and MEK2. Although they are thought to be functionally redundant, recent studies have suggested that these two signaling molecules have non-overlapping cellular functions. To test this we developed a novel experimental system to allow signaling through individual MEK pathway in human melanoma SK-MEL-28 cells. In this system, multiple MEK/MKK pathways are inhibited by anthrax lethal toxin (LeTx), a Bacillus anthracis-secreted toxin which specifically and proteolytically inhibits members of MEK/MKK proteins. MEK1 or MEK2 signaling is rescued by expressing a cleavage-resistant MEK mutant (MEK1cr or MEK2cr) able to actively signal in the presence of LeTx. Using this approach, we determined that MEK2 signaling, and not MEK1, is sufficient for melanoma cell proliferation and anchorage-independent growth. To explain this we hypothesized that MEK1 and MEK2 signaling differentially regulate downstream effectors to control cell survival. To test this and to identify the non-overlapping effectors downstream to MEK1 and MEK2, we compared gene expression profiles using Agilent 60-mer Whole Human Genome Microarrays. We found that global suppression of MKK signaling by LeTx treatment significantly altered expression of 20% of represented genes. These mainly include genes involved in survival pathways such as the pathways of DNA replication, E2F transcription factors, rRNA processing, and cell proliferation, as well as oncogenic pathways such as Myc and Ras. Of these genes, approximately half were rescued by expression of one of MEK1cr or MEK2cr. Whereas MEK2cr had a more pronounced effect on expression and predominantly rescued signaling associated with cell proliferation, MEK1cr rescued signaling normally associated with epithelial to mesenchymal transition. Our findings demonstrate that MEK1 and MEK2 have non-redundant cellular functions and differentially regulate downstream effectors in SK-MEL-28 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-232.

Abstract 1175: PCR array-based gene expression profiling identifies CD70 and CD74 as molecular markers and potential therapeutic targets for human melanoma

Abstract Melanoma is one of the most prevalent cancers. While early detection and surgical excision can cure melanoma, patients with advanced disseminated disease have a very poor prognosis due to its extremely aggressive growth characteristics and high resistance to chemotherapy and radiation. Improvement of our understanding of the mechanisms that lead to progression of metastatic melanoma, as well as development of novel therapeutic strategies are urgently required for patients. In our study, we applied PCR arrays and bioinformatics analysis to evaluate gene expression of metastatic melanoma cells and normal skin cells. We used the Human Inflammatory Cytokine and Receptors RT2 Profiler™ PCR Array to analyze the differential expression of genes in metastatic melanoma cell lines (A375) and primary normal epidermal melanocytes (NHEM), which are the precursor cell of melanoma. We found the 43 genes were highly expressed in A375 which were upregulated by more than 20 fold in comparison to NHEM. We next selected the top-ten differentially expressed genes (CD70, IL-1β, MGLL, SERPINA3, MDK, IL13RA2, CD74, VEGFA, CXCL1, and IL8), whose expression levels in A375 cells were > 250-fold higher than in the primary normal NHEM, for further validation. The results from our RT-PCR, immunoblot, immunocytochemistry (ICC) and tissue microarray (TMA) analysis showed that CD70, SERPINA3, IL13RA2 and CD74 genes were highly expressed at mRNA and protein levels in various melanoma cell lines and metastatic melanoma tumor tissues. A much lower level of expression of each gene was detected in normal human skin cell lines and normal skin tissues. These results thus demonstrated that the four genes identified by our PCR array are likely of molecular markers and should be pursued as therapeutic targets for melanoma treatment. To further verify their potentials as potential melanoma therapeutic targets, we also evaluated the effects of siRNAs targeting CD70 and CD74 genes and the monoclonal antibodies against CD70 and CD74 proteins for regulation of melanoma growth. Treatment with CD70 or CD74-specific siRNAs and also monoclonal antibodies dramatically inhibited cell growth in various melanoma cell lines, indicating that CD70 and CD74 are also attractive molecular targets in the treatment of human metastatic melanoma. Our findings have important implications in understanding the metastatic process of melanoma and discovering novel therapeutic strategies for melanoma treatment. The molecular markers and therapeutic targets developed in this study will have a direct and immediate translational application. If successful, our study will represent a significant advance in the diagnosis and treatment of melanoma. (This study was partially supported by the grant from the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and the Melanoma SPORE grant from the NCI, P50, CA093459). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1175.

Simultaneous Inhibition of MEK and CDK4 Leads to Potent Apoptosis in Human Melanoma Cells

ABSTRACTDeregulation of RAS-RAF-MEK-ERK and p16INK4A-cycylin D:CDK4/6-RB pathways is important for melanoma development. Chemotherapeutic agents targeting both pathways were developed but results of clinical studies with monotherapies were disappointing. We examined the effect of cotargeting both pathways with MEK inhibitor PD98059 and CDK4 inhibitor 219476 on human melanoma cells lines, and found that combinatorial treatment dramatically increased apoptosis compared to the single agent treatment. The apoptosis was associated with downregulation of BCL2, BCL2L1, BIRC5, and upregulation of BIM. Our results indicate that simultaneously targeting ERK and RB pathways is a promising strategy for melanoma treatment and should encourage further in-depth investigations.