Nme1 and Nme2 genes exert metastasis-suppressor activities in a genetically engineered mouse model of UV-induced melanoma.

NME1 is a metastasis-suppressor gene (MSG), capable of suppressing metastatic activity in cell lines of melanoma, breast carcinoma and other cancer origins without affecting their growth in culture or as primary tumours. Herein, we selectively ablated the tandemly arranged Nme1 and Nme2 genes to assess their individual impacts on metastatic activity in a mouse model (HGF:p16-/-) of ultraviolet radiation (UVR)-induced melanoma. Metastatic activity was strongly enhanced in both genders of Nme1- and Nme2-null mice, with stronger activity in females across all genotypes. The study ascribes MSG activity to Nme2 for the first time in an in vivo model of spontaneous cancer, as well as a novel metastasis-suppressor function to Nme1 in the specific context of UVR-induced melanoma.

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.

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.

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.

Metastasis Suppressor NME1 Directly Activates Transcription of the ALDOC Gene in Melanoma Cells.

BACKGROUND/AIM: NME/NM23 nucleoside diphosphate kinase 1 (NME1) is a metastasis suppressor gene, exhibiting reduced expression in metastatic cancers and the ability to suppress metastatic activity of cancer cells. We previously identified NME1-regulated genes with prognostic value in human melanoma. This study was conducted in melanoma cell lines aiming to elucidate the mechanism through which NME regulates one of these genes, aldolase C (ALDOC). MATERIALS AND METHODS: ALDOC mRNA and protein expression was measured using qRT-PCR and immunoblot analyses. Promoter-luciferase constructs and chromatin immunoprecipitation were employed to measure the impact of NME1 on ALDOC transcription. RESULTS: NME1 enhanced ALDOC transcription, evidenced by increased expression of ALDOC pre-mRNA and activity of an ALDOC promoter-luciferase module. NME1 was detected at the ALDOC promoter, and forced NME1 expression resulted in enhanced occupancy of the promoter by NME1, increased presence of epigenetic activation markers (H3K4me3 and H3K27ac), and recruitment of RNA polymerase II. CONCLUSION: This is the first study to indicate that NME1 induces transcription through its direct binding to the promoter region of a target gene.