Heterogeneity of Melanoma with Stem Cell Properties.

Metastatic melanoma continues to present a significant challenge-with a cure rate of less than 10% and a median survival of 6-9 months. Despite noteworthy advances in the field, the heterogeneity of melanoma tumors, comprised of cell subpopulations expressing a cancer stem cell (CSC) phenotype concomitant with drug resistance markers presents a formidable challenge in the design of current therapies. Particularly vexing is the ability of distinct subpopulations of melanoma cells to resist standard-of-care treatments, resulting in relapse and progression to metastasis. Recent studies have provided new information and insights into the expression and function of CSC markers associated with the aggressive melanoma phenotype, such as the embryonic morphogen Nodal and CD133, together with a drug resistance marker ABCA1. This chapter highlights major findings that demonstrate the promise of targeting Nodal as a viable option to pursue in combination with standard-of-care therapy. In recognizing that aggressive melanoma tumors utilize multiple mechanisms to survive, we must consider a more strategic approach to effectively target heterogeneity, tumor cell plasticity, and functional adaptation and resistance to current therapies-to eliminate relapse, disease progression, and metastasis.

Targeting melanoma with front-line therapy does not abrogate Nodal-expressing tumor cells.

Metastatic melanoma is a highly aggressive skin cancer with a poor prognosis. It is the leading cause of skin cancer deaths with a median overall survival for advanced-stage metastatic disease of <6 months. Despite advances in the field with conventional and targeted therapies, the heterogeneity of melanoma poses the greatest ongoing challenge, ultimately leading to relapse and progression to a more drug-resistant tumor in most patients. Particularly noteworthy are recent findings, indicating that these therapies exert selective pressure on tumors resulting in the activation of pathways associated with cancer stem cells that are unresponsive to current therapy. Our previous studies have shown how Nodal, an embryonic morphogen of the transforming growth factor-beta superfamily, is one of these critical factors that is reactivated in aggressive melanoma and resistant to conventional chemotherapy, such as dacarbazine. In the current study, we sought to determine whether BRAF inhibitor (BRAFi) therapy targeted Nodal-expressing tumor cells in uniquely matched unresectable stage III and IV melanoma patient samples before and after therapy that preceded their eventual death due to disease. The results demonstrate that BRAFi treatment failed to affect Nodal levels in melanoma tissues. Accompanying experiments in soft agar and in nude mice showed the advantage of using combinatorial treatment with BRAFi plus anti-Nodal monoclonal antibody to suppress tumor growth and metastasis. These data provide a promising new approach using front-line therapy combined with targeting a cancer stem cell-associated molecule-producing a more efficacious response than monotherapy.

Melanocytes Affect Nodal Expression and Signaling in Melanoma Cells: A Lesson from Pediatric Large Congenital Melanocytic Nevi.

Expression of Nodal, a Transforming Growth Factor-beta (TGF-ß) related growth factor, is associated with aggressive melanoma. Nodal expression in adult dysplastic nevi may predict the development of aggressive melanoma in some patients. A subset of pediatric patients diagnosed with giant or large congenital melanocytic nevi (LCMN) has shown increased risk for development of melanoma. Here, we investigate whether Nodal expression can help identify the rare cases of LCMN that develop melanoma and shed light on why the majority of these patients do not. Immunohistochemistry (IHC) staining results show varying degree of Nodal expression in pediatric dysplastic nevi and LCMN. Moreover, median scores from Nodal IHC expression analysis were not significantly different between these two groups. Additionally, none of the LCMN patients in this study developed melanoma, regardless of Nodal IHC levels. Co-culture experiments revealed reduced tumor growth and lower levels of Nodal and its signaling molecules P-SMAD2 and P-ERK1/2 when melanoma cells were grown in vivo or in vitro with normal melanocytes. The same was observed in melanoma cells cultured with melanocyte conditioned media containing pigmented melanocyte derived melanosomes (MDM). Since MDM contain molecules capable of inactivating radical oxygen species, to investigate potential anti-oxidant effect of MDM on Nodal expression and signaling in melanoma, melanoma cells were treated with either N-acetyl-l-cysteine (NAC), a component of the anti-oxidant glutathione or synthetic melanin, which in addition to providing pigmentation can also exert free radical scavenging activity. Melanoma cells treated with NAC or synthetic melanin showed reduced levels of Nodal, P-SMAD2 and P-ERK1/2 compared to untreated melanoma cells. Thus, the potential role for Nodal in melanoma development in LCMN is less evident than in adult dysplastic nevi possibly due to melanocyte cross-talk in LCMN capable of offsetting or delaying the pro-melanoma effects of Nodal via anti-oxidant effects of MDM.

Nodal signaling promotes a tumorigenic phenotype in human breast cancer.

The Ras-ERK pathway is deregulated in approximately a third of human cancers, particularly those of epithelial origin. In aggressive, triple-negative, basal-like breast cancers, most tumors display increased MEK and ERK phosphorylation and exhibit a gene expression profile characteristic of Kras or EGFR mutant tumors; however, Ras family genetic mutations are uncommon in triple-negative breast cancer and EGFR mutations account for only a subset of these tumors. Therefore, the upstream events that activate MAPK signaling and promote tumor aggression in triple-negative breast cancers remain poorly defined. We have previously shown that a secreted TGF-ß family signaling ligand, Nodal, is expressed in breast cancer in correlation with disease progression. Here we highlight key findings demonstrating that Nodal is required in aggressive human breast cancer cells to activate ERK signaling and downstream tumorigenic phenotypes both in vitro and in vivo. Experimental knockdown of Nodal signaling downregulates ERK activity, resulting in loss of c-myc, upregulation of p27, G1 cell cycle arrest, increased apoptosis and decreased tumorigenicity. The data suggest that ERK activation by Nodal signaling regulates c-myc and p27 proteins post-translationally and that this cascade is essential for aggressive breast tumor behavior in vivo. As the MAPK pathway is an important target for treating triple-negative breast cancers, upstream Nodal signaling may represent a promising target for breast cancer diagnosis and combined therapies aimed at blocking ERK pathway activation.

Grainyhead-like-2, an epithelial master programmer, promotes interferon induction and suppresses breast cancer recurrence.

Type-I and -III interferons play a central role in immune rejection of pathogens and tumors, thus promoting immunogenicity and suppressing tumor recurrence. Double strand RNA is an important ligand that stimulates tumor immunity via interferon responses. Differentiation of embryonic stem cells to pluripotent epithelial cells activates the interferon response during development, raising the question of whether epithelial vs. mesenchymal gene signatures in cancer potentially regulate the interferon pathway as well. Here, using genomics and signaling approaches, we show that Grainyhead-like-2 (GRHL2), a master programmer of epithelial cell identity, promotes type-I and -III interferon responses to double-strand RNA. GRHL2 enhanced the activation of IRF3 and relA/NF-kB and the expression of IRF1; a functional GRHL2 binding site in the IFNL1 promoter was also identified. Moreover, time to recurrence in breast cancer correlated positively with GRHL2 protein expression, indicating that GRHL2 is a tumor recurrence suppressor, consistent with its enhancement of interferon responses. These observations demonstrate that epithelial cell identity supports interferon responses in the context of cancer.

Tumor cell vasculogenic mimicry: from controversy to therapeutic promise.

In 1999, The American Journal of Pathology published an article entitled "Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry," by Maniotis and colleagues, which ignited a spirited debate for several years and earned distinction as a citation classic. Tumor cell vasculogenic mimicry (VM) refers to the plasticity of aggressive cancer cells forming de novo vascular networks, which thereby contribute to perfusion of rapidly growing tumors, transporting fluid from leaky vessels, and/or connecting with the constitutional endothelial-lined vasculature. The tumor cells capable of VM share a plastic, transendothelial phenotype, which may be induced by hypoxia. Since VM was introduced as a novel paradigm for melanoma tumor perfusion, many studies have contributed new findings illuminating the underlying molecular pathways supporting VM in a variety of tumors, including carcinomas, sarcomas, glioblastomas, astrocytomas, and melanomas. Facilitating the functional plasticity of tumor cell VM are key proteins associated with vascular, stem cell, and hypoxia-related signaling pathways, each deserving serious consideration as potential therapeutic targets and diagnostic indicators of the aggressive, metastatic phenotype.

Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness.

Bidirectional cellular communication is integral to both cancer progression and embryological development. In addition, aggressive tumor cells are phenotypically plastic, sharing many properties with embryonic cells. Owing to the similarities between these two types of cells, the developing zebrafish can be used as a biosensor for tumor-derived signals. Using this system, we show that aggressive melanoma cells secrete Nodal (a potent embryonic morphogen) and consequently can induce ectopic formation of the embryonic axis. We further show that Nodal is present in human metastatic tumors, but not in normal skin, and thus may be involved in melanoma pathogenesis. Inhibition of Nodal signaling reduces melanoma cell invasiveness, colony formation and tumorigenicity. Nodal inhibition also promotes the reversion of melanoma cells toward a melanocytic phenotype. These data suggest that Nodal signaling has a key role in melanoma cell plasticity and tumorigenicity, thereby providing a previously unknown molecular target for regulating tumor progression.

Potential for the embryonic morphogen Nodal as a prognostic and predictive biomarker in breast cancer.

INTRODUCTION: The re-emergence of the tumour growth factor-beta (TGF-beta)-related embryonic morphogen Nodal has recently been reported in several different human cancers. In this study, we examined the expression of Nodal in a series of benign and malignant human breast tissues to determine the clinical significance of this expression and whether Nodal could represent a potential therapeutic target in breast cancer. METHODS: Tissue sections from 431 therapeutically naive patients diagnosed with benign or malignant breast disease were stained for Nodal by immunohistochemistry and analysed in a blinded manner. The degree of Nodal staining was subsequently correlated with available clinical data, such as diagnoses and disease stage. These tissue findings were further explored in breast cancer cell lines MDA-MB-231 and MDA-MB-468 treated with a Nodal blocking antibody to determine biological effects for target validation. RESULTS: A variable degree of Nodal staining was detected in all samples. The intensity of Nodal staining was significantly greater in undifferentiated, advanced stage, invasive breast cancer compared with benign breast disease or early stage breast cancer. Treatment of human breast cancer cells in vitro with Nodal blocking antibody significantly reduced proliferation and colony-forming ability in soft agar, concomitant with increased apoptosis. CONCLUSIONS: These data suggest a potential role for Nodal as a biomarker for disease progression and a promising target for anti-Nodal therapy in breast cancer.

Reactivation of embryonic nodal signaling is associated with tumor progression and promotes the growth of prostate cancer cells.

BACKGROUND: Nodal is a member of the transforming growth factor ß (TGFß) superfamily that directs embryonic patterning and promotes the plasticity and tumorigenicity of tumor cells, but its role in the prostate is unknown. The goal of this study was to characterize the expression and function of Nodal in prostate cancer and determine whether, like other TGFß ligands, it modulates androgen receptor (AR) activity. METHODS: Nodal expression was investigated using immunohistochemistry of tissue microarrays and Western blots of prostate cell lines. The functional role of Nodal was examined using Matrigel and soft agar growth assays. Cross-talk between Nodal and AR signaling was assessed with luciferase reporter assays and expression of endogenous androgen regulated genes. RESULTS: Significantly increased Nodal expression was observed in cancer compared with benign prostate specimens. Nodal was only expressed by DU145 and PC3 cells. All cell lines expressed Nodal's co-receptor, Cripto-1, but lacked Lefty, a critical negative regulator of Nodal signaling. Recombinant human Nodal triggered downstream Smad2 phosphorylation in DU145 and LNCaP cells, and stable transfection of pre-pro-Nodal enhanced the growth of LNCaP cells in Matrigel and soft agar. Finally, Nodal attenuated AR signaling, reducing the activity of a PSA promoter construct in luciferase assays and down-regulating the endogenous expression of androgen regulated genes. CONCLUSIONS: An aberrant Nodal signaling pathway is re-expressed and functionally active in prostate cancer cells.

Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells.

Embryonic stem cells sustain a microenvironment that facilitates a balance of self-renewal and differentiation. Aggressive cancer cells, expressing a multipotent, embryonic cell-like phenotype, engage in a dynamic reciprocity with a microenvironment that promotes plasticity and tumorigenicity. However, the cancer-associated milieu lacks the appropriate regulatory mechanisms to maintain a normal cellular phenotype. Previous work from our laboratory reported that aggressive melanoma and breast carcinoma express the embryonic morphogen Nodal, which is essential for human embryonic stem cell (hESC) pluripotency. Based on the aberrant expression of this embryonic plasticity gene by tumor cells, this current study tested whether these cells could respond to regulatory cues controlling the Nodal signaling pathway, which might be sequestered within the microenvironment of hESCs, resulting in the suppression of the tumorigenic phenotype. Specifically, we discovered that metastatic tumor cells do not express the inhibitor to Nodal, Lefty, allowing them to overexpress this embryonic morphogen in an unregulated manner. However, exposure of the tumor cells to a hESC microenvironment (containing Lefty) leads to a dramatic down-regulation in their Nodal expression concomitant with a reduction in clonogenicity and tumorigenesis accompanied by an increase in apoptosis. Furthermore, this ability to suppress the tumorigenic phenotype is directly associated with the secretion of Lefty, exclusive to hESCs, because it is not detected in other stem cell types, normal cell types, or trophoblasts. The tumor-suppressive effects of the hESC microenvironment, by neutralizing the expression of Nodal in aggressive tumor cells, provide previously unexplored therapeutic modalities for cancer treatment.