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The neural crest is a multipotent, highly migratory cell population that gives rise to diverse cell types, including melanocytes. Factors regulating the development of the neural crest and emigration of its cells are likely to influence melanoma metastasis. The transcription factor FOXD3 plays an essential role in premigratory neural crest development and has been implicated in melanoma cell dormancy and response to therapeutics. FOXD3 is downregulated during the migration of the melanocyte lineage from the neural crest, and our previous work supports a role for FOXD3 in suppressing melanoma cell migration and invasion. Alternatively, TWIST1 is known to have promigratory and proinvasive roles in a number of cancers, including melanoma. Using ChIP-seq analysis, TWIST1 was identified as a potential transcriptional target of FOXD3. Mechanistically, FOXD3 directly binds to regions of the TWIST1 gene locus, leading to transcriptional repression of TWIST1 in human mutant BRAF melanoma cells. In addition, depletion of endogenous FOXD3 promotes upregulation of TWIST1 transcripts and protein. Finally, FOXD3 expression leads to a significant decrease in cell migration that can be efficiently reversed by the overexpression of TWIST1. These findings uncover the novel interplay between FOXD3 and TWIST1, which is likely to be important in the melanoma metastatic cascade.Implications: FOXD3 and TWIST1 define distinct subgroups of cells within a heterogeneous tumor. Mol Cancer Res; 12(9); 1314–23. ©2014 AACR.

Supplementary Figures S1-S2. Supplementary Figure S1. 1205Lu cells were transfected with a control non-targeting siRNA or one of two different FOXD3 targeting siRNAs. Lysates and mRNA were harvested four days after siRNA transfection. In addition, control siRNA treated cells were treated for 24 hours with the RAF inhibitor PLX4720 (1 μM) as a positive control for FOXD3 expression. Western blots and quantitative RT-PCR were performed. Columns, average of three independent experiments; bars, SD; *p

Supplementary Figure 1 from Deletion of PTEN Promotes Tumorigenic Signaling, Resistance to Anoikis, and Altered Response to Chemotherapeutic Agents in Human Mammary Epithelial Cells

Supplementary Figure Legends 1-2 from Deletion of PTEN Promotes Tumorigenic Signaling, Resistance to Anoikis, and Altered Response to Chemotherapeutic Agents in Human Mammary Epithelial Cells

Supplementary Figure 2 from Deletion of PTEN Promotes Tumorigenic Signaling, Resistance to Anoikis, and Altered Response to Chemotherapeutic Agents in Human Mammary Epithelial Cells

PDF file - 3777K, TWIST1 expression is not regulated by RSK, STAT3, AKT, or by protein stability in mutant B-RAF cell lines

PDF file - 1510K, TWIST1 is positively regulated by active ERK1/2 signaling in N-RAS mutant melanoma cells

PDF file - 2984K, Oligonucleotide and primer sequences

PDF file - 1651K, Absence of EMT-like protein changes following altered TWIST1 expression in melanoma cells

PDF file - 3308K, High TWIST1 expression allows for melanoma spheroid outgrowth

Many cancers, including breast cancer, harbor loss-of-function mutations in the catalytic domain of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or have reduced PTEN expression through loss of heterozygosity and/or epigenetic silencing mechanisms. However, specific phenotypic effects of PTEN inactivation in human cancer cells remain poorly defined without a direct causal connection between the loss of PTEN function and the development or progression of cancer. To evaluate the biological and clinical relevance of reduced or deleted PTEN expression, a novel in vitro model system was generated using human somatic cell knockout technologies. Targeted homologous recombination allowed for a single and double allelic deletion, which resulted in reduced and deleted PTEN expression, respectively. We determined that heterozygous loss of PTEN in the nontumorigenic human mammary epithelial cell line MCF-10A was sufficient for activation of the phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase pathways, whereas the homozygous absence of PTEN expression led to a further increased activation of both pathways. The deletion of PTEN was able to confer growth factor–independent proliferation, which was confirmed by the resistance of the PTEN−/− MCF-10A cells to small-molecule inhibitors of the epidermal growth factor receptor. However, neither heterozygous nor homozygous loss of PTEN expression was sufficient to promote anchorage-independent growth, but the loss of PTEN did confer apoptotic resistance to cell rounding and matrix detachment. Finally, MCF-10A cells with the reduction or loss of PTEN showed increased susceptibility to the chemotherapeutic drug doxorubicin but not paclitaxel. [Cancer Res 2009;69(21):8275–83]

PDF file - 3105K, TWIST1 is required for melanoma cell migration towards and invasion through collagen type I

PDF file - 11564K, MMP-1 is able to rescue defects in spheroid outgrowth resulting from TWIST1 ablation

Metastatic cancer remains a clinical challenge; however, patients diagnosed prior to metastatic dissemination have a good prognosis. The transcription factor, TWIST1 has been implicated in enhancing the migration and invasion steps within the metastatic cascade, but the range of TWIST1-regulated targets is poorly described. In this study, we performed expression profiling to identify the TWIST1-regulated transcriptome of melanoma cells. Gene ontology pathway analysis revealed that TWIST1 and epithelial to mesenchymal transition (EMT) were inversely correlated with levels of cell adhesion molecule 1 (CADM1). Chromatin immunoprecipitation (ChIP) studies and promoter assays demonstrated that TWIST1 physically interacts with the CADM1 promoter, suggesting TWIST1 directly represses CADM1 levels. Increased expression of CADM1 resulted in significant inhibition of motility and invasiveness of melanoma cells. In addition, elevated CADM1 elicited caspase-independent cell death in non-adherent conditions. Expression array analysis suggests that CADM1 directed non-adherent cell death is associated with loss of mitochondrial membrane potential and subsequent failure of oxidative phosphorylation pathways. Importantly, tissue microarray analysis and clinical data from TCGA indicate that CADM1 expression is inversely associated with melanoma progression and positively correlated with better overall survival in patients. Together, these data suggest that CADM1 exerts tumor suppressive functions in melanoma by reducing invasive potential and may be considered a biomarker for favorable prognosis.

The neural crest is a multipotent, highly migratory cell population that gives rise to diverse cell types, including melanocytes. Factors regulating the development of the neural crest and emigration of its cells are likely to influence melanoma metastasis. The transcription factor FOXD3 plays an essential role in premigratory neural crest development and has been implicated in melanoma cell dormancy and response to therapeutics. FOXD3 is downregulated during the migration of the melanocyte lineage from the neural crest, and our previous work supports a role for FOXD3 in suppressing melanoma cell migration and invasion. Alternatively, TWIST1 is known to have promigratory and proinvasive roles in a number of cancers, including melanoma. Using ChIP-seq analysis, TWIST1 was identified as a potential transcriptional target of FOXD3. Mechanistically, FOXD3 directly binds to regions of the TWIST1 gene locus, leading to transcriptional repression of TWIST1 in human mutant BRAF melanoma cells. In addition, depletion of endogenous FOXD3 promotes upregulation of TWIST1 transcripts and protein. Finally, FOXD3 expression leads to a significant decrease in cell migration that can be efficiently reversed by the overexpression of TWIST1. These findings uncover the novel interplay between FOXD3 and TWIST1, which is likely to be important in the melanoma metastatic cascade. Implications: FOXD3 and TWIST1 define distinct subgroups of cells within a heterogeneous tumor. Mol Cancer Res; 12(9); 1314–23. ©2014 AACR.

Tumor cells often use developmental processes to progress toward advanced disease. The E-box transcription factor TWIST1 is essential to epithelial–mesenchymal transition (EMT) and cell migration in the developing neural crest. In melanoma, which derives from the neural crest cell lineage, enhanced TWIST1 expression has been linked to worse clinical prognosis. However, mechanisms underlying TWIST1 expression and whether aberrant TWIST1 levels promote steps in melanoma progression remain unknown. Here, we report that elevated TWIST1 mRNA/protein expression is dependent on extracellular signal-regulated kinase (ERK)1/2 signaling, which is hyperactive in the majority of melanomas. We show that TWIST1 protein levels are especially high in melanoma cell lines generated from invasive, premetastatic stage tumors. Furthermore, TWIST1 expression is required and sufficient to promote invasion through Matrigel and spheroid outgrowth in three-dimensional dermal-mimetic conditions. Alterations to spheroid outgrowth were not as a result of altered cell death, cell-cycle profile, or paradigm EMT protein changes. Importantly, we identify matrix metalloproteinase-1 (MMP-1) as a novel downstream target of TWIST1. We have determined that TWIST1 acts, in a dose-dependent manner, as a mediator between hyperactive ERK1/2 signaling and regulation of MMP-1 transcription. Together, these studies mechanistically show a previously unrecognized interplay between ERK1/2, TWIST1, and MMP-1 that is likely significant in the progression of melanoma toward metastasis. Cancer Res; 72(24); 6382–92. ©2012 AACR.

Metastatic melanoma is the deadliest form of skin cancer; however, patients diagnosed and treated prior to metastatic dissemination have a good prognosis. The transcription factor, TWIST1 has been implicated in enhancing the migration and invasion of melanoma cells but the range of TWIST1-regulated targets is poorly described. In this study, we performed expression profiling to identify the TWIST1-regulated transcriptome. Gene ontology pathway analysis revealed that TWIST1 and epithelial to mesenchymal transition (EMT) were inversely correlated with levels of cell adhesion molecule 1 (CADM1). Chromatin immunoprecipitation (ChIP) studies and promoter assays demonstrated that TWIST1 physically interacts with the CADM1 promoter, suggesting TWIST1 directly represses CADM1 levels. Modulation of CADM1 resulted in significant effects on the migration and invasion of melanoma cells. In addition, elevated CADM1 elicited cell death in non-adherent conditions, an effect that could not be rescued with a pan-caspase inhibitor. Analyses suggest that CADM1 directed non-adherent cell death is associated with loss of mitochondrial membrane potential and subsequent failure of oxidative phosphorylation pathways. Furthermore, clinical data from TCGA indicates that CADM1 expression is correlated with better overall survival in patients. Together, these data suggest that CADM1 exerts tumor suppressive functions in melanoma by reducing invasive potential and may be a biomarker for improved survival of melanoma patients. Citation Format: Edward J. Hartsough, Michele B. Weiss, Shea A. Heilman, Timothy J. Purwin, Curtis H. Kugel, Sheera R. Rosenbaum, Dan A. Erkes, Manoela Tiago, Inna Chervoneva, Andrew E. Aplin. CADM1 is a TWIST1-regulated suppressor of melanoma invasion and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 105.

Many cancers, including breast cancer, harbor loss-of-function mutations in the catalytic domain of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or have reduced PTEN expression through loss of heterozygosity and/or epigenetic silencing mechanisms. However, specific phenotypic effects of PTEN inactivation in human cancer cells remain poorly defined without a direct causal connection between the loss of PTEN function and the development or progression of cancer. To evaluate the biological and clinical relevance of reduced or deleted PTEN expression, a novel in vitro model system was generated using human somatic cell knockout technologies. Targeted homologous recombination allowed for a single and double allelic deletion, which resulted in reduced and deleted PTEN expression, respectively. We determined that heterozygous loss of PTEN in the nontumorigenic human mammary epithelial cell line MCF-10A was sufficient for activation of the phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase pathways, whereas the homozygous absence of PTEN expression led to a further increased activation of both pathways. The deletion of PTEN was able to confer growth factor–independent proliferation, which was confirmed by the resistance of the PTEN−/− MCF-10A cells to small-molecule inhibitors of the epidermal growth factor receptor. However, neither heterozygous nor homozygous loss of PTEN expression was sufficient to promote anchorage-independent growth, but the loss of PTEN did confer apoptotic resistance to cell rounding and matrix detachment. Finally, MCF-10A cells with the reduction or loss of PTEN showed increased susceptibility to the chemotherapeutic drug doxorubicin but not paclitaxel. [Cancer Res 2009;69(21):8275–83]

The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to “knock in” PIK3CA mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant PIK3CA knockin cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3β phosphorylation. Paradoxically, the GSK3β inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and led to a decrease in the GSK3β target gene CYCLIN D1 . Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant PIK3CA compared with isogenic HCT-116 knockout cells containing only wild-type PIK3CA . Our findings suggest GSK3β is an important effector of mutant PIK3CA , and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations.

Melanoma is the deadliest form of skin cancer; however, with early detection prior to metastatic dissemination, patients generally have a good prognosis. Recently, our lab has implicated the transcription factor TWIST1 in the progression of melanoma towards metastasis. In melanoma cells, we found that RAS-RAF-MEK-ERK (ERK1/2 pathway) signaling increases TWIST1 expression, which promotes invasive properties in the dermal microenvironment at least in part by enhancing levels of the matrix metalloproteinase, MMP1. Other TWIST1 regulated targets are poorly described. In this study, we compared expression profiling data from cells expressing shRNA against TWIST1 or cells overexpressing TWIST1 in order to determine TWIST1 regulated genes. KEGG and GO analysis revealed that TWIST1 is responsible for regulating a number of genes involved in cellular adhesion. We found that TWIST1 levels inversely correlate with levels of cell adhesion molecule 1 (CADM1) (NECL-2, IGSF4, TSLC1, SynCAM). Chromatin immunoprecipitation (ChIP) studies and promoter assays demonstrate that TWIST1 physically interacts with CADM1 promoter and this is associated with reduced CADM1 levels. Additionally, CADM1 expression is inversely associated ERK1/2 signaling and TWIST1 expression. Modulation of cellular CADM1 levels does not seem to affect proliferations rates however, overexpression of CADM1 augments cell-cell interaction, and cell aggregation. Furthermore, exogenous CADM1 inhibited serum directed migration and invasion through matrigel coated boyden chambers, while knockdown of CADM1 was associated with an enhancement in the migratory and invasion properties. Taken together, these data provide evidence that CADM1 is negatively regulated by TWIST1, and may act as a suppressor of melanoma invasion. Citation Format: Edward J. Hartsough, Michele B. Weiss, Curtis H. Kugel, Sheera R. Rosenbaum, Andrew E. Aplin. CADM1 is a TWIST1 regulated suppressor of melanoma invasion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4089. doi:10.1158/1538-7445.AM2015-4089

Melanoma is the deadliest form of skin cancer; however, with early detection prior to metastatic dissemination, patients generally have a good prognosis. Recently, our lab has implicated the transcription factor TWIST1 in the progression of melanoma towards metastasis. In melanoma cells, we found that RAS-RAF-MEK-ERK (ERK1/2 pathway) signaling increases TWIST1 expression, which promotes invasive properties in the dermal microenvironment, at least in part by enhancing levels of the matrix metalloproteinase, MMP1. Other TWIST1-regulated targets are poorly described. In this study, we compared expression-profiling data from cells expressing shRNA against TWIST1 or cells overexpressing TWIST1 in order to identify TWIST1-regulated genes. KEGG and GO analysis revealed that TWIST1 is responsible for regulating a number of genes involved in cellular adhesion. We found that TWIST1 levels inversely correlate with levels of cell adhesion molecule 1 (CADM1, also known as NECL-2, IGSF4, TSLC1, SynCAM). Chromatin immunoprecipitation (ChIP) studies and promoter assays demonstrated that TWIST1 physically interacts with the CADM1 promoter and this is associated with reduced CADM1 levels. Additionally, CADM1 expression is inversely associated with ERK1/2 signaling and TWIST1 expression. Modulation of cellular CADM1 levels did not affect proliferation rates; however, overexpression of CADM1 augmented cell-cell interaction, cell aggregation, and reduced trans-endothelial invasion. Furthermore, exogenous CADM1 inhibited serum-directed migration and invasion through Matrigel-coated Boyden chambers, whereas knockdown of CADM1 was associated with enhanced migratory and invasive properties. Taken together, these data provide evidence that CADM1 is negatively regulated by TWIST1 and may act as a suppressor of melanoma invasion. Citation Format: Edward J. Hartsough, Michele B. Weiss, Sheera R. Rosenbaum, Andrew E. Aplin. CADM1 is a TWIST1 regulated suppressor of melanoma invasion. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A08.

Metastatic melanoma is the deadliest form of skin cancer due to its highly aggressive and chemotherapy-resistant nature. Therefore, determining the factors impacting melanoma advancement toward metastasis is an important and clinically-relevant task. Importantly, RAF/MEK/ERK signaling is hyperactive in the great majority of melanoma cells and is a driver of melanoma growth and invasion. In addition, the TWIST1 protein is up-regulated in melanoma and its expression correlates strongly with poor clinical prognosis. However, the mechanism underlying TWIST1 up-regulation, as well as the role of high TWIST1 expression in traits associated with the progression of melanoma, has yet to be uncovered. In this study, we report that disruption of RAF/MEK/ERK signaling strongly represses TWIST1 at the mRNA and protein level. We further show that TWIST1 protein levels are especially high in B-RAF mutant cells as well as in vertical growth phase melanoma cells, which are those that first begin to invade downward into the deeper dermal layers eventually leading to metastasis. To that effect, depletion of TWIST1 in several invasive melanoma cell lines significantly reduces Matrigel invasion. The inverse phenotype is apparent when TWIST1 is overexpressed in low TWIST1-expressing, non-invasive melanoma cell lines. Additionally, we tested the effect of altered Twist expression in three-dimensional collagen spheroid outgrowth assays, which mimic both tumor architecture and the in vivo collagen-rich dermal layer. We observe significant reduction in spheroid outgrowth when invasive cell lines are depleted for TWIST1 as well as increases in outgrowth when non-invasive cells overexpress TWIST1. Alterations to spheroid outgrowth were not as a result of apoptotic changes or proliferative rate. Importantly, we have found matrix metalloproteinase-1 (MMP-1) to be a downstream effector of TWIST1. Furthermore, TWIST1 acts as a mediator between activated B-RAF signaling and MMP-1 in a dose-dependent and direct manner at the level of transcription. Our findings uncover the novel interplay between B-RAF, TWIST1, and MMP-1 which is likely important in the progression of melanoma towards metastasis. 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 4310. doi:1538-7445.AM2012-4310

Previous studies have indicated that Fn14, the cell surface receptor for the cytokine TWEAK, is over-expressed in multiple human solid tumor types, including brain, breast, and lung cancers, and overexpression can be a negative prognostic indicator. We analyzed a series of melanoma cell lines and tumor tissue microarrays and detected Fn14 expression in ∼60% of the melanoma cell lines, including both B-Raf WT and B-Raf V600E lines. Fn14 expression was elevated in 178/190 (93.6%) of primary melanoma specimens and in 87/150 (58%) of melanoma metastases tested. Fn14 expression was not elevated in normal skin tissues. Initial development and characterization of an immunoconjugate designated ITEM4-rGel targeting Fn14 receptor has been published (Zhou et al., Mol. Cancer Ther. 10:1276 (2011)). We have now developed an Fn14-targeted immunotoxin more suitable for long-term clinical use. Specifically, we generated a humanized, dimeric single-chain version of ITEM-4 and fused this scFv to rGel. The resulting anti-Fn14 immunotoxin, designated hSGZ, bound to Fn14 with a Kd of ∼1.4 nM as determined by Biacore analysis. Confocal immunofluoresence studies showed that hSGZ specifically and rapidly (within 2 hrs) internalized into Fn14-expressing MDA-MB-435 melanoma cells. Cytotoxicity studies showed that hSGZ was highly cytotoxic to a panel of different melanoma cell lines (IC50 ranged from 0.1 pM to 1.1 nM) and was 2.2 to 2.8 ×105 fold more potent than free rGel. Treatment of cells expressing the multidrug resistance protein MDR1 showed no cross-resistance to the fusion construct in vitro. When hSGZ was combined with 5-FU, cisplatin, doxorubicin, etoposide or dacarbazine, we found an additive effect on melanoma cell growth inhibition. Mechanistic studies showed that hSGZ induced melanoma cell death consistent with a necrotic mechanism. Additionally, Fn14-targeted immunotoxins increased Fn14 expression and triggered cell signaling events similar to those induced by the TWEAK ligand. Finally, treatment of mice bearing human melanoma MDA-MB-435 and breast MDA-MB-231 xenografts with either ITEM4-rGel or hSGZ showed significant tumor growth inhibition compared to controls (P< 0.05). Fn14 appears to be an excellent new target for melanoma and the Fn14-targeting construct hSGZ appears to warrant further development as a novel therapeutic agent against Fn14-positive tumors. Melanoma lines appear to be the most sensitive tumor type tested but the reasons for this are unclear. Additional studies are in progress to investigate the biodistribution and pharmacokinetics of hSGZ in tumor-bearing mice. This work was conducted, in part, by the Clayton Foundation for Research (MGR); and supported by NIH grant NS055126 (JAW) and DOD Breast Cancer Concept Award BC086135 (JAW). 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 3866. doi:1538-7445.AM2012-3866

Metastatic melanoma is the deadliest form of skin cancer and a paradigm for highly aggressive, chemotherapy-resistant tumors. Therefore, determining the factors impacting melanoma advancement toward metastasis is an important and clinically-relevant task. The TWIST1 protein is up-regulated in melanoma and its expression correlates strongly with poor clinical prognosis. We tested the role of high TWIST1 expression in traits associated with the progression of melanoma, as well as sought to delineate the mechanism underlying TWIST1 up-regulation. Our data shows that TWIST1 protein levels are higher in melanoma cells, especially invasive lines, compared to normal melanocytes. To evaluate the biological relevance of TWIST1 expression, we generated invasive melanoma cell lines depleted of TWIST1 by shRNA and non-invasive melanoma cell lines which constitutively overexpress TWIST1. First, we utilized these cell lines in standard Matrigel invasion assays through boyden chambers. Depletion of TWIST1 in several invasive melanoma cell lines significantly reduces Matrigel invasion by approximately 40-50%. The inverse phenotype is apparent when TWIST1 is overexpressed in low TWIST1-expressing, non-invasive melanoma cell lines. Additionally, we tested the effect of altered Twist expression in 3D collagen spheroid outgrowth assays, which mimic both tumor architecture and the in vivo collagen-rich dermal layer. We have found significant reduction of spheroid outgrowth when invasive cell lines are depleted for TWIST1 as well as increases in outgrowth when non-invasive cells overexpress TWIST1. Alterations to spheroid outgrowth were not as a result of apoptotic changes or proliferative rate as assessed by live/dead and EdU staining. Importantly, the mechanisms regulating high TWIST1 expression in melanoma are unclear. A comparison between wild-type and mutant B-RAF melanoma cell lines demonstrates that TWIST1 is more highly expressed in mutant B-RAF cells. We have found that disruption of B-RAF/MEK signaling, through siRNA/shRNA knockdown or pharmacological inhibition, strongly represses TWIST1 at the mRNA and protein level. An area of active investigation is the determination of the transcription factor responsible for TWIST1 regulation downstream of mutant B-RAF in melanoma. The data generated from these studies will allow for a greater understanding of the role and regulation of TWIST1 during the progression of melanoma towards metastasis. In addition, it may highlight TWIST1 as an attractive molecule/pathway for novel targeted therapies for melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2334. doi:10.1158/1538-7445.AM2011-2334

Fibroblast growth factor-inducible protein 14 (Fn14), the cell surface receptor for tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is overexpressed in various human solid tumor types and can be a negative prognostic indicator. We detected Fn14 expression in ∼60% of the melanoma cell lines we tested, including both B-Raf WT and B-Raf V600E lines. Tumor tissue microarray analysis indicated that Fn14 expression was low in normal skin, but elevated in 173/190 (92%) of primary melanoma specimens and in 86/150 (58%) of melanoma metastases tested. We generated both a chemical conjugate composed of the recombinant gelonin (rGel) toxin and the anti-Fn14 antibody ITEM-4 (designated ITEM4-rGel) and a humanized, dimeric single-chain antibody of ITEM-4 fused to rGel (designated hSGZ). Both ITEM4-rGel and hSGZ were highly cytotoxic to a panel of different melanoma cell lines. Mechanistic studies showed that both immunotoxins induced melanoma cell necrosis. In addition, these immunotoxins could upregulate the cellular expression of Fn14 and trigger cell-signaling events similar to the Fn14 ligand TWEAK. Finally, treatment of mice bearing human melanoma MDA-MB-435 xenografts with either ITEM4-rGel or hSGZ showed significant tumor growth inhibition compared with controls. We conclude that Fn14 is a therapeutic target in melanoma and the hSGZ construct appears to warrant further development as a therapeutic agent against Fn14-positive melanoma.

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.