Therapeutic Target In Human Melanoma Research Articles

MITF Expression Predicts Therapeutic Vulnerability to p300 Inhibition in Human Melanoma.

Histone modifications, largely regulated by histone acetyltransferases (HAT) and histone deacetylases, have been recognized as major regulatory mechanisms governing human diseases, including cancer. Despite significant effort and recent advances, the mechanism by which the HAT and transcriptional coactivator p300 mediates tumorigenesis remains unclear. Here, we use a genetic and chemical approach to identify the microphthalmia-associated transcription factor (MITF) as a critical downstream target of p300 driving human melanoma growth. Direct transcriptional control of MITF by p300-dependent histone acetylation within proximal gene regulatory regions was coupled to cellular proliferation, suggesting a significant growth regulatory axis. Further analysis revealed forkhead box M1 (FOXM1) as a key effector of the p300-MITF axis driving cell growth that is selectively activated in human melanomas. Targeted chemical inhibition of p300 acetyltransferase activity using a potent and selective catalytic p300/CBP inhibitor demonstrated significant growth inhibitory effects in melanoma cells expressing high levels of MITF. Collectively, these data confirm the critical role of the p300-MITF-FOXM1 axis in melanoma and support p300 as a promising novel epigenetic therapeutic target in human melanoma. SIGNIFICANCE: These results show that MITF is a major downstream target of p300 in human melanoma whose expression is predictive of melanoma response to small-molecule inhibition of p300 HAT activity.

Interleukin-13 receptor \u03b12 is a novel marker and potential therapeutic target for human melanoma

Malignant melanoma is one of the untreatable cancers in which conventional therapeutic strategies, including chemotherapy, are hardly effective. Therefore, identification of novel therapeutic targets involved in melanoma progression is urgently needed for developing effective therapeutic methods. Overexpression of interleukin-13 receptor α2 (IL13Rα2) is observed in several cancer types including glioma and pancreatic cancer. Although IL13Rα2 is implicated in the progression of various types of cancer, its expression and roles in the malignant melanoma have not yet been elucidated. In the present study, we showed that IL13Rα2 was expressed in approximately 7.5% melanoma patients. While IL13Rα2 expression in human melanoma cells decreased their proliferation in vitro, it promoted in vivo tumour growth and angiogenesis in melanoma xenograft mouse model. We also found that the expression of amphiregulin, a member of the epidermal growth factor (EGF) family, was correlated with IL13Rα2 expression in cultured melanoma cells, xenograft tumour tissues and melanoma clinical samples. Furthermore, expression of amphiregulin promoted tumour growth, implicating causal relationship between the expression of IL13Rα2 and amphiregulin. These results suggest that IL13Rα2 enhances tumorigenicity by inducing angiogenesis in malignant melanoma, and serves as a potential therapeutic target of malignant melanoma.

MiR-767 promoted cell proliferation in human melanoma by suppressing CYLD expression

MicroRNAs (miRNAs) have emerged as critical regulators for cancer development and progression of human melanoma. However, the potential molecular mechanism of miR-767 in human melanoma has not been intensively investigated. In this present study, we confirmed that miR-767 was frequently up-regulated in human melanoma tissues and cell lines. Ectopic expression of miR-767 promoted cell proliferation in human melanoma cell lines A375 and WM35, whereas miR-767-in reversed the function. Bioinformatics analysis revealed that cylindromatosis (CYLD) was hypothesized to be a possible target gene of miR-767, and this was confirmed by luciferase activity assay. Knockdown of CYLD counteracted the proliferation arrest by miR-767-in in melanoma cells A375 and WM35. In conclusion, our study indicated that miR-767 acted as a role of tumor promoter by targeting CYLD in human melanoma, and might serve as a prognostic or therapeutic target for human melanoma.

Abstract 374: KMT2A promotes tumor growth by activating hTERT and CBP signaling and predicts poor prognosis in human melanoma

Abstract Melanoma is an aggressive type of cutaneous malignancy. Although the inhibitors targeting BRAF and/or MEK pathways provide a therapeutic option for non-resectable melanoma driven by BRAF mutation, melanoma, especially metastatic melanoma, has still become one of the most threatening malignancies. Thus, identifying the exact molecular mechanisms involved in melanoma growth and discovering the novel targets for melanoma therapy is urgently needed. In this study, we screened a siRNA library targeting 6024 human genes in human melanoma cells and identified KMT2A as a potential therapeutic target for melanoma. KMT2A was highly expressed in melanoma cell lines and tumor tissues of melanoma patients. Knockdown of KMT2A by siRNA or shRNA significantly inhibited cell viability and colony formation, whereas exogenous expression of KMT2A effectively promoted cell growth in various melanoma cell lines. Further mechanism studies showed that the KMT2A-mediated regulation of melanoma growth was through targeting the hTERT and CBP signaling. Knockdown of KMT2A significantly inhibited hTERT promoter activity and protein expression and attenuated telomerase activity. Overexpression of hTERT rescued the KMT2A knockdown-mediated melanoma cell growth. By contrast, exogenous expression of KMT2A activated hTERT transcription and expression and increased telomerase activity in melanoma cells. Moreover, we found that KMT2A promoted hTERT expression and melanoma cell growth by cooperating with the transcriptional co-activator CBP, which interacted with and acetylated KMT2A. Inhibition of CBP by siRNA or a CBP-specific inhibitor suppressed the acetylation of KMT2A, abrogated the binding of KMT2A on the hTERT promoter and down-regulated hTERT expression, thereby inhibiting the growth of melanoma cells. Conversely, overexpression of CBP increased KMT2A acetylation and the binding to hTERT promoter, resulting in the promotion of hTERT expression and cell growth. The in vivo studies also showed that KMT2A promoted melanoma growth by activating the hTERT signaling in a xenograft tumor mouse model. Furthermore, the analyses for the clinical samples demonstrated that KMT2A expression was positively correlated with hTERT in tumor tissues of melanoma patients, and the high expression of both KMT2A and hTERT was associated with the worse clinical TNM staging and poor prognosis in melanoma patients. Taken together, our results indicate that KMT2A promotes melanoma growth by activating hTERT and CBP signaling in human melanoma. Our study therefore provides new insights into understanding the regulatory mechanism of melanoma growth and suggests that the KMT2A/CBP/hTERT signaling may be a potential therapeutic target for human melanoma. Funding support: This work was supported by the funds from the National Natural Science Foundation of China (81071687, 81272195), the State “973 Program” of China (2014CB542005). Citation Format: Changlin Zhang, Ranran Tang, Kefang Zhang, Wenlin Huang, Wuguo Deng. KMT2A promotes tumor growth by activating hTERT and CBP signaling and predicts poor prognosis in human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 374. doi:10.1158/1538-7445.AM2017-374

Abstract 3597: RPS3 regulates melanoma growth and sensitivity to DNA damage and predicts a poor prognosis by targeting ADT3

Abstract Melanoma, a malignant skin cancer, is resistant to DNA damage-mediated therapy. Discovering and identifying novel therapeutic targets for melanoma is urgently required. In this study, we found that ribosome protein S3 (RPS3) regulated melanoma growth and the sensitivity of melanoma to DNA damage by targeting ADP/ATP translocase 3 (ADT3). Knockdown of RPS3 inhibited cell proliferation and sensitized melanoma cells to DNA damage. RPS3 knockdown also promoted ADT3 translocation to mitochondrial of melanoma cells when exposed to DNA damage. RPS3 interacted with ADT3 in melanoma cells. Knockdown of ADT3 reduced its co-localization with RPS3. RPS3 could not obviously sensitize melanoma cells to DNA damage with ADT3 knockdown. In addition, we found that Lys18 in the death-induce-domain of RPS3 protein played a critical role in the interaction between RPS3 and ADT3. Mutation at Lys18 site could deadlock ADT3 and attenuate cell apoptosis in melanoma cells. Knockdown of RPS3 also inhibited tumor growth in a melanoma mouse model in vivo, but overexpression of the RPS3-18 mutation rescued the growth. Furthermore, we showed that the patients with higher expressed RPS3 had a much shorter median survival, whereas the patients with higher levels of ADT3 had a much longer median survival. Collectively, our results indicate that RPS3 cooperates with ADT3 to regulate the sensitivity of melanoma to DNA damage and suggest that the RPS3/ADT3 pathway is a potential therapeutic target for human melanoma. Grant support: This work was supported by the funds from the National Natural Science Foundation of China (81472178, 81272195) and the State “973 Program” of China (2014CB542005). Citation Format: Yun Tian, Lijun Qin, Wei Guo, Changlin Zhang, Dingbo Shi, Tianze Liu, Wenbing Li, Jingshu Wang, Yixin Li, Ge Qin, Wendan Yu, Xiangsheng Xiao, Tiebang Kang, Wenlin Huang, Wuguo Deng. RPS3 regulates melanoma growth and sensitivity to DNA damage and predicts a poor prognosis by targeting ADT3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3597.

NF-YA promotes invasion and angiogenesis by upregulating EZH2-STAT3 signaling in human melanoma cells.

The process of angiogenesis is essential for tumor development and metastasis. Vascular endothelial growth factor (VEGF), which is overexpressed in most human cancers, has been demonstrated to be a major modulator of angiogenesis. Thus, inhibition of VEGF signaling has the potential for tumor anti-angiogenic therapy. Signal transducer and activator of transcription-3 (STAT3) is a key regulator for angiogenesis by directly binding to the VEGF promoter to upregulate its transcription. Several factors can enhance STAT3 activity to affect angiogenesis. Here, we found that overexpression of nuclear transcription factor-Y alpha (NF-YA) gene could promote cell invasion and angiogenesis accompanying the increase of STAT3 signaling in human melanoma cells. Moreover, the expression and secretion of VEGF was also found to be upregulated by the overexpression of NF-YA gene in melanoma cells. The STAT3 inhibitor was able to attenuate the upregulation of VEGF induced by NF-YA overexpression. Enhancer of zeste homolog2 (EZH2), the catalytic subunit of the Polycomb repressive complex2, enhances STAT3 activity by mediating its lysine methylation. We also showed that NF-YA upregulated the expression of EZH2 and NF-YA‑induced angiogenesis could be inhibited by EZH2 knockdown. Taken together, these findings indicate that overexpression of NF-YA contributes to tumor angiogenesis through EZH2-STAT3 signaling in human melanoma cells, highlighting NF-YA as a potential therapeutic target in human melanoma.

Insulin-like growth factor binding protein 5 (IGFBP5) functions as a tumor suppressor in human melanoma cells.

The insulin-like growth factor binding protein 5 (IGFBP5), which is often dysregulated in human cancers, plays a crucial role in carcinogenesis and cancer development. However, the function and underlying mechanism of IGFBP5 in tumor growth and metastasis has been elusive, particularly in malignant human melanoma. Here, we reported that IGFBP5 acts as an important tumor suppressor in melanoma tumorigenicity and metastasis by a series of experiments including transwell assay, xenograft model, in vivo tumor metastasis experiment, and RNA-Seq. Overexpression of IGFBP5 in A375, a typical human melanoma cell line, inhibited cell malignant behaviors significantly, including in vitro proliferation, anchorage-independent growth, migration and invasion, as well as in vivo tumor growth and pulmonary metastasis. In addition, overexpression of IGFBP5 suppressed epithelial-mesenchymal transition (EMT), and decreased the expression of E-cadherin and the key stem cell markers NANOG, SOX2, OCT4, KLF4, and CD133. Furthermore, IGFBP5 exerts its inhibitory activities by reducing the phosphorylation of IGF1R, ERK1/2, and p38-MAPK kinases and abating the expression of HIF1α and its target genes, VEGF and MMP9. All these findings were confirmed by IGFBP5 knockdown in human melanoma cell line A2058. Taken together, these results shed light on the mechanism of IGFBP5 as a potential tumor-suppressor in melanoma progression, indicating that IGFBP5 might be a novel therapeutic target for human melanoma.

Abstract 4200: Interaction between mPGES-1 and iNOS promotes human melanoma progression

Abstract Melanoma cells in vivo are well known to express varying inflammatory features, which is not limited by somatic mutational subclass. Among the most prevalent of inflammatory markers are iNOS (inducible Nitric Oxide Synthase) generated NO production and COX-2/PGE2, and for which their active pathways have been shown to enhance carcinogenesis and tumor progression, stimulate angiogenesis, support tumor growth, and promote metastasis. COX-2 inhibitors are well studied agents that inhibit tumor growth and prevent tumor development including melanoma, but the focus of research has shifted to develop inhibitors for enzymes, which are downstream of COX-2, especially microsomal PGE2 synthase-1 (mPGES-1), due to the cardiovascular risk associated with the initial COX-2 inhibitory drugs. Here, we demonstrate crosstalk between iNOS/NO and mPGES-1/PGE2 signaling pathways and suggest that mPGES-1 is a novel potential therapeutic target in human melanoma employing a spectrum of well-defined human cell lines, as well as human tissues. Our melanoma stage III survival Tissue MicroArray analysis shows that the expression of mPGES-1 is elevated in 65% of human stage III melanomas and intense expression of mPGES-1 is significantly associated with the short-term survival of patients. More interestingly, about 90 % of patient samples which express mPGES-1 were also positive for iNOS. In addition, treatment of human melanoma cells with NO donors or constitutively-driven expression of iNOS resulted in increased PGE2 production, which was suppressed by mPGES-1 inhibition, indicating that the pathway is active and targetable. Moreover, iNOS was found to interact directly with mPGES-1, and iNOS or NO donors enhance mPGES-1 activity in melanoma cells and in vitro, suggesting that iNOS-mediated NO promotes PGE2 production through mPGES-1 activation. mPGES-1 inhibition by its shRNA or specific inhibitors suppressed human melanoma cell viability and migration. Collectively, these findings suggest that the interaction between iNOS and mPGES-1 is associated with human melanoma progression and could be considered as a novel target for translational application in melanoma growth regulation. Citation Format: Sun-Hee Kim, Yuuri Hashimoto, Suhendan Ekmekcioglu, Elizabeth A. Grimm. Interaction between mPGES-1 and iNOS promotes human melanoma progression. [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 4200. doi:10.1158/1538-7445.AM2014-4200

Prognostic significance of KAI1/CD82 in human melanoma and its role in cell migration and invasion through the regulation of ING4

KAI1/CD82 is a member of the transmembrane 4 superfamily, which was first identified as a metastasis suppressor for prostate cancer. The expression of KAI1 was found to be reduced in many types of cancers, including prostate, breast, ovarian, cervical and endometrial cancer. However, the role of KAI1 in melanoma pathogenesis is not known. In this study, we investigated the expression level of KAI1 in a large set of melanocytic lesions at different stages. We found that the expression of KAI1 is significantly decreased during melanoma progression. In fact, KAI1 expression is drastically reduced in primary melanoma compared with dysplastic nevi (P = 1.8×10(-4)) and further reduced in metastatic melanoma compared with primary melanoma (P = 9.4 × 10(-15)). Furthermore, decreased KAI1 staining is strongly correlated with a worse 5 year and 10 year patient survival. Multivariate Cox regression analysis showed that KAI1 is also an independent prognostic factor for both 5 year and 10 year survival. Moreover, we found that overexpression of KAI1 significantly inhibited melanoma cell migration through suppression of Rho-associated kinase-mediated formation of stress fiber. Our data also suggested that overexpression of KAI1 significantly inhibited melanoma cell invasion by reducing the activity of metalloproteinase-2. In addition, we found that suppression of melanoma cell migration by KAI1 is mediated by another tumor-suppressor protein called inhibitor of growth 4 through the regulation of p65. Taken together, our data suggest that KAI1 may be used as a promising prognostic marker and a possible therapeutic target for human melanoma.

Abstract 2078: Synergistic effects of c-Met and BRAF inhibitors and role of c-Met as a therapeutic target in human melanoma.

Abstract Inhibitors of c-Met and mutated BRAF kinases show limited efficacy due to the development of kinase inhibitor (KI) resistance hence it is important to target a combination of kinases. To study the efficacy of a c-Met and BRAF inhibitor combination, two cell lines were selected: MU, which is positive for the BRAF activating V600E mutation, and MM-RU, which is negative for this mutation. c-Met inhibitor SU11274 resistant cell lines were generated by exposing these parental lines to increasing concentrations of SU11274, until the final IC50 concentrations of resistant cells were 6-8 fold higher than those of parental lines. When c-Met and BRAF kinase inhibitors SU11274 (0.075μM) and vemurafenib (0.15μM) were administered individually and in combination, both drugs were able to significantly (p<0.01) synergize their inhibitory effects in MU parental and SU11274 resistant cells (60% and 58%, respectively) compared to their individual treatments (MUP 41%/5% and MUR 46%/0% vemurafenib/SU11274, respectively). When cells from the RU line were treated with varying concentrations of tivantinib, a c-Met inhibitor currently in clinical trials (0.05μM - 0.3μM) and Vemurafenib (0.025μM - 0.15μM), both parental and resistant cells did not show any synergism in combination. Conversely, MU parental and SU11274 resistant cells again showed synergism (27% and 22% respectively as determined by ANOVA analysis, p<0.01) when treated with 0.15μM vemurafenib and 0.075μM tivantinib in combination. However, minimal inhibition (MUP 9%/4% and MUR 0%/4% vemurafenib/tivantinib) was seen in MU cell lines in response to vemurafenib and tivantinib when given individually. This data suggests that using a vemurafenib and c-Met inhibitor combination therapy on BRAF mutant and c-Met expressing tumors would greatly benefit melanoma patients, even those exhibiting c-Met TKI resistance. To study the effects of an oral/bioavailable c-Met TKI, JNJ, and to determine if c-Met inhibitors could be delivered systemically and targeted to the tumor, the therapeutic effects of JNJ were tested in vivo on MM-RU melanoma cells which produce HGF. Five million MM-RU melanoma cells were injected subcutaneously into the hind flanks of nude mice. When tumors were palpable, they were treated with 20mg/kg JNJ or vehicle (20% Captisol, 0.25% PVP in 0.1N HCL) for 3 weeks. We found that JNJ significantly reduced tumor size by 6 fold (123.77 +/- 56.66mm2 and 16.67 +/-11.19mm2 p<0.03) compared to vehicle. Treatment with JNJ also resulted in 80% +/- 2.35% reduction in blood vessels as seen by CD31 staining and decreased VEGF expression and increased TSP1 expression as seen by immunohistochemistry in MM-RU indicating that inhibition of vessel formation may be one of the mechanisms by which JNJ inhibits tumor growth. Our data indicates that JNJ could be a promising therapeutic option for treating HGF producing melanoma. Citation Format: Deven Etnyre, Manohar Reddy Shambannagari, Neelu Puri. Synergistic effects of c-Met and BRAF inhibitors and role of c-Met as a therapeutic target in human melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2078. doi:10.1158/1538-7445.AM2013-2078

Inhibition of CRM1-Mediated Nucleocytoplasmic Transport: Triggering Human Melanoma Cell Apoptosis by Perturbing Multiple Cellular Pathways

Development of multiple drug resistance mechanisms in melanomas necessitates the identification of new drug targets, which when inhibited could impact multiple cellular pathways, thus circumventing potential resistance. By performing complementary DNA microarray analysis, we identified four key components of the nucleocytoplasmic transport machinery-CRM1, RAN (RAN-GTPase), RANGAP1, and RANBP1-to be overexpressed in human melanoma metastases. Chromosome region maintenance 1 (CRM1) inhibition induced a marked depletion of prosurvival/cytoplasmic extracellular signal-regulated kinase 1/2 (Erk1/2) and p90 ribosomal S6 kinase1 and elicited persistent Erk-signaling hyperactivation. Consistently, CRM1 inhibition inflicted extensive apoptosis in melanoma cells while sparing nontransformed melanocytes and primary lung fibroblasts. Apoptosis required both the intrinsic and extrinsic apoptotic pathways and was associated with a nuclear entrapment and downregulation of the antiapoptotic CRM1 target protein, Survivin. Apoptosis was preceded by a G1 cell-cycle arrest, and even though CRM1 inhibition mediated marked p53 and p21 induction in wild-type p53 melanoma cells, the latter's silencing or inactivation failed to alleviate apoptosis. Notably, CRM1 inhibition induced cell line-specific, G1 to S progression-retarding changes in the expression of multiple cell-cycle regulatory proteins, thus potentially explaining p53 dispensability. We propose CRM1 as a potential therapeutic target in human melanoma, whose inhibition induces loss of prosurvival/cytoplasmic Erk1/2, mediates persistent Erk hyperactivation, and initiates a multitude of cell context-dependent molecular events to trigger G1 arrest followed by massive apoptosis.

Abstract LB-77: Crm1-mediated nucleocytoplasmic transport inhibition perturbs multiple cellular pathways to trigger melanoma cell apoptosis

Abstract Development of multiple resistance mechanisms in melanomas has necessitated the identification of novel drug targets, which when inhibited could impact multiple cellular pathways thus circumventing potential resistance mechanisms. By performing cDNA-microarray analysis, we identified four key components of the nucleocytoplasmic transport machinery, Chromosome Region Maintenance 1 (Crm1), Ran-GTPase, Ran-GTPase Activating Protein 1 (RanGAP1) and Ran Binding Protein 1 (RanBP1) to be overexpressed in human melanoma metastases. Crm1 inhibition, employing the highly specific Crm1 inhibitor Leptomycin B (LMB) induced a dramatic depletion of pro-survival/cytoplasmic Erk1/2 and p90RSK1 and mediated persistent Erk signaling hyperactivation. Consistently, Crm1 inhibition inflicted extensive apoptosis in melanoma cells while sparing immortalized melanocytes and primary lung fibroblasts. Apoptosis required both the intrinsic and extrinsic apoptotic pathways and was associated with a nuclear entrapment and down-regulation of anti-apoptotic Survivin. Apoptosis was preceded by a G1 cell-cycle arrest and even though Crm1 inhibition mediated dramatic p53 and p21 induction exclusively in wild-type p53 melanoma cells, latter's silencing or inactivation failed to alleviate ensuing apoptosis. Notably, Crm1 inhibition elicited cell line specific, G1 to S progression-retarding changes in the expression of multiple cell-cycle regulatory proteins (Cdk2, Cdk4, p27, p16 and p18) thus potentially explaining p53 dispensability. Based on our results, we propose Crm1 as a potential therapeutic target in human melanoma, whose inhibition initiates a multitude of cell context-dependent molecular events to trigger G1-arrest followed by massive apoptosis. 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 LB-77. doi:1538-7445.AM2012-LB-77

Abstract 3418: Regulation of Dicer expression by Sox4 and its role in melanoma cell invasion and patient survival

Abstract Deregulated expression of Sox4 transcription factor has been observed in various types of cancers. We previously reported reduced expression of Sox4 in metastatic melanoma and its role in suppression of cell migration and invasion. We also demonstrated binding of Sox4 to the promoter sequence of Dicer, an RNase involved in maturation of miRNAs. Interestingly, altered expression of Dicer has also been reported in different types of cancers. However, the potential mechanisms which regulate Dicer expression and its potential significance in melanoma are unknown. Here we studied the regulation of Dicer expression by Sox4 and its role in suppression of melanoma invasion. Our data revealed a reduced expression of Dicer mRNA and protein upon Sox4 knockdown. On the other hand, overexpression of Sox4 increased Dicer expression at both mRNA and protein levels. We found that knockdown of Dicer, similar to Sox4 knockdown, enhances the matrigel invasion ability of melanoma cells by at least 2-fold. However, cell growth assays showed that Dicer knockdown does not have a major effect on the growth rate of melanoma cells. In addition, we revealed that overexpression of exogenous Dicer can abrogated the enhanced melanoma cells invasion upon Sox4 knockdown. Furthermore, we examined the expression of Dicer protein in different stages of melanocytic lesions by tissue microarray (TMA) and immunohistochemistry using a construct containing 30 normal nevi, 87 dysplastic nevi, 262 primary and 135 metastatic melanomas. Our data revealed that Dicer expression is inversely correlated with melanoma progression (P<0.0001). Accordingly, the number of samples with positive staining for Dicer reduced from 100% in normal nevi to 85% in dysplastic nevi and 87% in primary melanoma and further reduced to 65% in metastatic melanoma. Expression of Dicer was also negatively correlated with the American Joint Committee on Cancer (AJCC) staging of the melanoma samples (P=0.0004). Strikingly, reduced Dicer expression was correlated with a poorer overall and disease-specific 5-year survival of patients (P=0.015 and 0.0029, respectively). Multivariate Cox regression analysis revealed that reduced Dicer expression is an independent prognostic factor to predict patient outcome (P=0.030). In addition, we analyzed the possible correlation between the expression of Sox4 and Dicer in melanoma TMA samples and found a significant correlation between expression of these markers (P=0.009), further indicating the regulation of Dicer expression by Sox4. Our results pinpoint the regulation of Dicer expression by Sox4 and the critical role of Dicer and probably the miRNA machinery in the suppression of melanoma metastasis. Our data also suggest that Dicer might be a suitable marker for the prognosis of melanoma patients and a potential therapeutic target for human melanoma. 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 3418. doi:1538-7445.AM2012-3418

Prognostic Significance of Sox4 Expression in Human Cutaneous Melanoma and Its Role in Cell Migration and Invasion

The Sox4 transcription factor is involved in various cellular processes, such as embryonic development and differentiation. Deregulated expression of Sox4 in several human cancers has been reported to date, but its role in melanoma is unknown. We explored the role of Sox4 in melanoma pathogenesis in vivo and in vitro. Using tissue microarray, we evaluated Sox4 expression in 180 melanocytic lesions and investigated its role in melanoma cell migration and invasion. Sox4 expression was remarkably reduced in metastatic melanoma compared with dysplastic nevi (P < 0.05) and primary melanoma (P < 0.01). This reduction was correlated with a poorer disease-specific survival of melanoma patients (P = 0.039). Multivariate Cox regression analysis revealed that reduced Sox4 expression is an independent prognostic factor (P = 0.049). Knockdown of Sox4 enhanced melanoma cell invasion, migration, and stress fiber formation. The increased migration and invasion on Sox4 knockdown depends on the presence of nuclear factor (NF)-κB p50 and is abrogated when p50 is knocked down. We further observed inhibition of NF-κB p50 transcription by Sox4, in addition to a reverse pattern of expression of Sox4 and NF-κB p50 in different stages of melanocytic lesions. Our results suggest that Sox4 regulates melanoma cell migration and invasion in an NF-κB p50-dependent manner and may serve as a prognostic marker and potential therapeutic target for human melanoma.

Abstract 2243: Prognostic significance of nuclear Sox4 expression in cutaneous melanoma and its role in cell migration

Abstract Sry-related high-mobility group (Sox) family of genes is consisted of transcription factors involved in various physiological processes including embryonic development, cell fate decision and differentiation. Deregulated expression of Sox4 has been reported in several human cancer types so far; nevertheless the expression and possible role of Sox4 in human melanoma cancer progression is yet to be identified. To investigate the putative role of Sox4 in the development of melanoma and its correlation with patient survival, we examined the expression of Sox4 protein in different stages of melanocytic lesions and its role in melanoma cancer cells proliferation and migration. Using tissue microarray and immunohistochemistry, we evaluated Sox4 protein expression in 43 dysplastic nevi, 89 primary melanomas, and 48 metastatic melanomas. We observed a marked reduction in nuclear Sox4 expression in malignant melanomas compared with dysplastic nevi (Kruskal-Wallis test; p &amp;lt; 0.05) and primary melanomas (Kruskal-Wallis test; p &amp;lt; 0.01). Moreover, the reduced nuclear Sox4 expression was significantly correlated with a poorer disease-specific 5-year survival of patients with primary and metastatic melanoma (Kaplan-Meier test; P = 0.039). Multivariate Cox regression analysis revealed that reduced nuclear Sox4 expression is an independent prognostic factor to predict patient outcome (p = 0.049). We also knocked down the expression of Sox4 in MMRU human melanoma cell line by siRNA and the migratory ability of the cells as well as their proliferation rate were analyzed following Sox4 silencing. Strikingly, the wound healing assay revealed an augmented migration ability of MMRU cells upon siRNA mediated knockdown of Sox4. However, the absence of Sox4 did not affect the rate of melanoma cancer cells proliferation. Our results suggest that Sox4 may be a suitable marker for the prognosis of the melanoma cancer outcome and a potential therapeutic target for human melanoma. 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 2243.

JWA regulates melanoma metastasis by integrin αVβ3 signaling

JWA, a newly identified novel microtubule-associated protein (MAP), was recently demonstrated to be indispensable for the rearrangement of actin cytoskeleton and activation of MAPK cascades induced by arsenic trioxide (As(2)O(3)) and phorbol ester (PMA). JWA depletion blocked the inhibitory effect of As(2)O(3) on HeLa cell migration, but enhanced cell migration after PMA treatment. As cancer cell migration is a hallmark of tumor metastasis and the functional role of JWA in cancer metastasis is not understood, here we show that JWA has an important role in melanoma metastasis. Our data demonstrated that JWA knockdown increased the adhesion and invasion abilities of melanoma cells. Furthermore, JWA knockdown in B16-F10 and A375 melanoma cells significantly promoted the formation and growth of metastatic colonies in vivo. Moreover, in the tumor biopsies from human melanoma patients, JWA expression was significantly decreased in malignant melanoma compared with normal nevi. In addition, we found that JWA knockdown could intensify tumor integrin alpha(V)beta(3) signaling by regulating nuclear factor Sp1. These findings suggest that JWA suppresses melanoma metastasis and may serve a potential therapeutic target for human melanoma.

Role of ING4 in human melanoma cell migration, invasion and patient survival

Inhibitor of growth (ING) 4 has been reported as a tumor suppressor and shown to diminish colony-forming efficiency, induce p53-dependent apoptosis and arrest cell cycle at G(2)-M phase. In this study, we investigated the role of ING4 in human melanoma pathogenesis. Using the tissue microarray technology, we found that ING4 expression is significantly decreased in malignant melanoma compared with dysplastic nevi (P < 0.0001, chi(2) test) and reduced ING4 staining is associated with melanoma thickness, ulceration (P = 0.034 and 0.002, respectively, chi(2) test) as well as poor overall and disease-specific 5-year survival of primary melanoma patients (P = 0.0002 and 0.001, respectively, chi(2) test). Cox regression analysis revealed that reduced ING4 staining is an independent factor for the poor prognosis of patients with primary melanomas. Furthermore, we found that overexpression of ING4 suppressed cell migration by 63% and inhibited the activity of Ras homolog gene family member A (RhoA) small GTPase protein and Rho-associated kinase (ROCK)-mediated formation of stress fiber in melanoma cells. Moreover, our data showed that overexpression of ING4 inhibited melanoma cell invasion by 43% compared with the control (P = 0.006, t-test) and ING4-overexpressing melanoma cells showed significantly reduced activity of matrix metalloproteinase (MMP)-2 and MMP-9. Taken together, this study highlights the importance of ING4 in melanoma pathogenesis and ING4 may serve as a promising prognostic marker and a potential therapeutic target for human melanoma.