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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 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.

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.

Reduced NME1 expression in melanoma cell lines, mouse models of melanoma, and melanoma specimens in human patients is associated with increased metastatic activity. Herein, we investigate the role of NME1 in repair of double-stranded breaks (DSBs) and choice of double-strand break repair (DSBR) pathways in melanoma cells. Using chromatin immunoprecipitation, NME1 was shown to be recruited rapidly and directly to DSBs generated by the homing endonuclease I-PpoI. NME1 was recruited to DSBs within 30 min, in concert with recruitment of ataxia-telangiectasia mutated (ATM) protein, an early step in DSBR complex formation, as well as loss of histone 2B. NME1 was detected up to 5 kb from the break site after DSB induction, suggesting a role in extending chromatin reorganization away from the repair site. shRNA-mediated silencing of NME1 expression led to increases in the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways of double-strand break repair (DSBR), and reduction in the low fidelity, alternative-NHEJ (A-NHEJ) pathway. These findings suggest low expression of NME1 drives DSBR towards higher fidelity pathways, conferring enhanced genomic stability necessary for rapid and error-free proliferation in invasive and metastatic cells. The novel mechanism highlighted in the current study appears likely to impact metastatic potential and therapy-resistance in advanced melanoma and other cancers.

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.

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.

Knowledge of protein-protein interactions in the plant cell is invaluable for furthering our understanding of the functions of these proteins. Many of the methods available for the study of these interactions, such as yeast two-hybrid and co-immunoprecipitation assays, rely on in vitro techniques. Here we describe the use of bimolecular fluorescence complementation for the study of protein-protein interactions in vivo, using simple techniques and accessible materials.

Knowledge of protein-protein interactions in the plant cell is invaluable for furthering our understanding of the functions of these proteins. Many of the methods available for the study of these interactions, such as yeast two-hybrid and co-immunoprecipitation assays, rely on in vitro techniques. Here we describe the use of bimolecular fluorescence complementation for the study of protein-protein interactions in vivo, using simple techniques and accessible materials.

NME1 is a potent suppressor of metastasis, and reduced expression has been associated with aggressive melanoma. The nucleoside diphosphate kinase and 3’-5’ exonuclease (3’-5’ EXO) activities of NME1 protein both contribute to its metastasis suppressor activity. 3’-5’ exonucleases are important for proofreading during DNA repair and synthesis, suggesting a potential role for NME1 in maintenance of genomic stability in melanoma. Although impairment of DNA repair activity has been implicated in melanoma risk, its contribution to melanoma initiation and progression is not well understood. We have shown previously that both enzymatic activities of NME1 are critical for repair of UV-induced DNA damage via nucleotide excision repair. In the current study, chromatin immunoprecipitation was used to demonstrate recruitment of NME1 to DNA double-strand breaks (DSBs), representing the first evidence of direct recruitment of NME1 to sites of DNA damage. Induction of DSBs with gamma irradiation (γ-IR) or bleomycin promoted rapid physical association of NME1 with γH2AX, ATM, NBS1 and RAD50. In addition, treatment of melanoma cells with γ-IR triggered a physical association of NME1 with XRCC4, an effector of the non-homologous end-joining (NHEJ) pathway. We are currently investigating whether knockout of NME1 induces a switch from NHEJ to the homologous recombination (HR) pathway, a slower but higher fidelity form of DSB repair, using plasmid repair assays. We are also using plasmid rescue to study the role of NME1 on the fidelity of NHEJ repair. Overall, our study indicates for the first time direct participation of NME1 in a molecular mechanism of DSB repair in both normal and transformed cells. In addition, these data suggest a novel mechanism of metastasis suppressor function for NME1, with loss of NME expression in tumor cells conferring genetic instability and enhanced metastatic potential. Citation Format: Gemma S. Puts, Stuart G. Jarrett, Devin Snyder, Richard Vincent, Ying Wang, Katie Leonard, Ben Portney, Feyruz Rassool, Michal Zalzman, David M. Kaetzel. The metastasis suppressor NME1 is recruited to DNA double strand breaks [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 4846. doi:10.1158/1538-7445.AM2017-4846