The metastasis suppressor NME1 inhibits melanoma cell motility via direct transcriptional induction of the integrin beta-3 gene.

Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility, invasion, and metastasis, but mechanisms underlying these activities are not completely understood. Herein we report a novel mechanism through which NME1 drives formation of large, stable focal adhesions (FAs) in melanoma cells via induction of integrin ß3 (ITGß3), and in one cell line, concomitant suppression of integrin ß1 (ITGß1) transcripts. Forced expression of NME1 resulted in a strong activation of the promoter region (-301 to +13) of the ITGB3 gene. Chromatin immunoprecipitation (ChIP) analysis revealed the transcriptional induction was associated with direct recruitment of NME1 and an increase in the epigenetic activation mark, acetylation of histone 3 on lysine 27 (H3K27Ac) to a 1 kb stretch of 5'-flanking sequence of the ITGB3 gene. Unexpectedly, NME1 did not affect the amount either ITGß1 or ITGß3 proteins were internalized and recycled, processes commonly associated with regulating expression of integrins at the cell surface. The ability of NME1 to suppress motile and invasive phenotypes of melanoma cells was dependent on its induction of ITGß3. Expression of ITGß3 mRNA was associated with increased disease-free survival time in melanoma patients of the TCGA collection, consistent with its potential role as an effector of the metastasis suppressor function of NME1. Together, these data indicate metastasis suppressor activity of NME1 in melanoma is mediated by induction of ITGB3 gene transcription, with NME1-driven enrichment of ITGß3 protein at the cell membrane resulting in attenuated cell motility through the stabilization of large focal adhesions.

Nuclear functions of NME proteins.

The NME family of proteins is composed of 10 isoforms, designated NME1-10, which are diverse in their enzymatic activities and patterns of subcellular localization. Each contains a conserved domain associated with a nucleoside diphosphate kinase (NDPK) function, although not all are catalytically active. Several of the NME isoforms (NME1, NME5, NME7, and NME8) also exhibit a 3'-5' exonuclease activity, suggesting roles in DNA proofreading and repair. NME1 and NME2 have been shown to translocate to the nucleus, although they lack a canonical nuclear localization signal. Binding of NME1 and NME2 to DNA does not appear to be sequence-specific in a strict sense, but instead is directed to single-stranded regions and/or other non-B-form structures. NME1 and NME2 have been identified as potential canonical transcription factors that regulate gene transcription through their DNA-binding activities. Indeed, the NME1 and NME2 isoforms have been shown to regulate gene expression programs in a number of cellular settings, and this regulatory function has been proposed to underlie their well-recognized ability to suppress the metastatic phenotype of cancer cells. Moreover, NME1 and, more recently, NME3, have been implicated in repair of both single- and double-stranded breaks in DNA. This suggests that reduced expression of NME proteins could contribute to the genomic instability that drives cancer progression. Clearly, a better understanding of the nuclear functions of NME1 and possibly other NME isoforms could provide critical insights into mechanisms underlying malignant progression in cancer. Indeed, clinical data indicate that the subcellular localization of NME1 may be an important prognostic marker in some cancers. This review summarizes putative functions of nuclear NME proteins in DNA binding, transcription, and DNA damage repair, and highlights their possible roles in cancer progression.

Identification of a gene expression signature associated with the metastasis suppressor function of NME1: prognostic value in human melanoma.

Although NME1 is well known for its ability to suppress metastasis of melanoma, the molecular mechanisms underlying this activity are not completely understood. Herein, we utilized a bioinformatics approach to systematically identify genes whose expression is correlated with the metastasis suppressor function of NME1. This was accomplished through a search for genes that were regulated by NME1, but not by NME1 variants lacking metastasis suppressor activity. This approach identified a number of novel genes, such as ALDOC, CXCL11, LRP1b, and XAGE1 as well as known targets such as NETO2, which were collectively designated as an NME1-Regulated Metastasis Suppressor Signature (MSS). The MSS was associated with prolonged overall survival in a large cohort of melanoma patients in The Cancer Genome Atlas (TCGA). The median overall survival of melanoma patients with elevated expression of the MSS genes was >5.6 years longer compared with that of patients with lower expression of the MSS genes. These data demonstrate that NMEl represents a powerful tool for identifying genes whose expression is associated with metastasis and survival of melanoma patients, suggesting their potential applications as prognostic markers and therapeutic targets in advanced forms of this lethal cancer.

The HGF/SF Mouse Model of UV-Induced Melanoma as an In Vivo Sensor for Metastasis-Regulating Gene.

Cutaneous malignant melanoma is an aggressive and potentially lethal form of skin cancer, particularly in its advanced and therapy-resistant stages, and the need for novel therapeutics and prognostic tools is acute. Incidence of melanoma has steadily increased over the past few decades, with exposure to the genome-damaging effects of ultraviolet radiation (UVR) well-recognized as a primary cause. A number of genetically-engineered mouse models (GEMMs) have been created that exhibit high incidence of spontaneous and induced forms of melanoma, and a select subset recapitulates its progression to aggressive and metastatic forms. These GEMMs hold considerable promise for providing insights into advanced stages of melanoma, such as potential therapeutic targets and prognostic markers, and as in vivo systems for testing of novel therapies. In this review, we summarize how the HGF/SF transgenic mouse has been used to reveal metastasis-regulating activity of four different genes (CDK4R24C, survivin and NME1/NME2) in the context of UV-induced melanoma. We also discuss how these models can potentially yield new strategies for clinical management of melanoma in its most aggressive forms.

Pigmented Epithelioid Melanocytoma (PEM)/Animal Type Melanoma (ATM): Quest for an Origin. Report of One Unusual Case Indicating Follicular Origin and Another Arising in an Intradermal Nevus.

Pigmented epithelioid melanocytoma (PEM) is a tumor encompassing epithelioid blue nevus of Carney complex (EBN of CNC) and was previously termed animal-type melanoma. Histologically PEMs are heavily pigmented spindled and epithelioid dermal melanocytic tumors with infiltrative borders, however, their origin remains unclear. Stem cells for the epidermis and hair follicle are located in the bulge area of the hair follicle with the potential to differentiate into multiple lineages. Multiple cutaneous carcinomas, including follicular cutaneous squamous cell carcinoma (FSCC), are thought to arise from stem cells in the follicular bulge. We present two cases of PEM/ATM in a 63 year-old male on the scalp with follicular origin and a 72 year-old female on the upper back arising in an intradermal nevus. Biopsy of both cases revealed a proliferation of heavily pigmented dermal nests of melanocytes with atypia. The Case 1 tumor was in continuation with the outer root sheath of the hair follicle in the bulge region. Case 2 arose in an intradermal melanocytic nevus. Rare mitotic figures, including atypical mitotic figures, were identified in both cases. We present two cases of PEM, with histologic evidence suggesting two origins: one from the follicular bulb and one from an intradermal nevus.

Comprehensive molecular profiling of UV-induced metastatic melanoma in Nme1/Nme2-deficient mice reveals novel markers of survival in human patients.

Hepatocyte growth factor-overexpressing mice that harbor a deletion of the Ink4a/p16 locus (HP mice) form melanomas with low metastatic potential in response to UV irradiation. Here we report that these tumors become highly metastatic following hemizygous deletion of the Nme1 and Nme2 metastasis suppressor genes (HPN mice). Whole-genome sequencing of melanomas from HPN mice revealed a striking increase in lung metastatic activity that is associated with missense mutations in eight signature genes (Arhgap35, Atp8b4, Brca1, Ift172, Kif21b, Nckap5, Pcdha2, and Zfp869). RNA-seq analysis of transcriptomes from HP and HPN primary melanomas identified a 32-gene signature (HPN lung metastasis signature) for which decreased expression is strongly associated with lung metastatic potential. Analysis of transcriptome data from The Cancer Genome Atlas revealed expression profiles of these genes that predict improved survival of patients with cutaneous or uveal melanoma. Silencing of three representative HPN lung metastasis signature genes (ARRDC3, NYNRIN, RND3) in human melanoma cells resulted in increased invasive activity, consistent with roles for these genes as mediators of the metastasis suppressor function of NME1 and NME2. In conclusion, our studies have identified a family of genes that mediate suppression of melanoma lung metastasis, and which may serve as prognostic markers and/or therapeutic targets for clinical management of metastatic melanoma.

NME1 Drives Expansion of Melanoma Cells with Enhanced Tumor Growth and Metastatic Properties.

Melanoma is a lethal skin cancer prone to progression and metastasis, and resistant to therapy. Metastasis and therapy resistance of melanoma and other cancers are driven by tumor cell plasticity, largely via acquisition/loss of stem-like characteristics and transitions between epithelial and mesenchymal phenotypes (EMT/MET). NME1 is a metastasis suppressor gene that inhibits metastatic potential when its expression is enforced in melanoma and other cancers. Herein, we have unmasked a novel role for NME1 as a driver of melanoma growth distinct from its canonical function as a metastasis suppressor. NME1 promotes expansion of stem-like melanoma cells that exhibit elevated expression of stem cell markers (e.g., Sox2, Sox10, Oct-4, KLF4, and Ccnb-1), enhanced growth as melanoma spheres in culture, and enhanced tumor growth and lung colonizing activities in vivo. In contrast, NME1 expression did not affect the proliferation of melanoma cell lines in monolayer culture conditions. Silencing of NME1 expression resulted in a dramatic reduction in melanoma sphere size, and impaired tumor growth and metastatic activities of melanoma sphere cells when xenografted in immunocompromised mice. Individual cells within melanoma sphere cultures displayed a wide range of NME1 expression across multiple melanoma cell lines. Cell subpopulations with elevated NME1 expression were fast cycling and displayed enhanced expression of stem cell markers. IMPLICATIONS: Our findings suggest the current model of NME1 as a metastasis-suppressing factor requires refinement, bringing into consideration its heterogeneous expression within melanoma sphere cultures and its novel role in promoting the expansion and tumorigenicity of stem-like cells.