Metastasis Suppressor Function Research Articles

CD44 and RHAMM Are Microenvironmental Sensors with Dual Metastasis Promoter and Suppressor Functions.

The progression of primary tumors to metastases remains a significant roadblock to the treatment of most cancers. Emerging evidence has identified genes that specifically affect metastasis and are potential therapeutic targets for managing tumor progression. However, these genes can have dual tumor promoter and suppressor functions that are contextual in manifestation, and that complicate their development as targeted therapies. CD44 and RHAMM/HMMR are examples of multifunctional proteins that can either promote or suppress metastases, as demonstrated in experimental models. These two proteins can be viewed as microenvironmental sensors and this minireview addresses the known mechanistic underpinnings that may determine their metastasis suppressor versus promoter functions. Leveraging this mechanistic knowledge for CD44, RHAMM, and other multifunctional proteins is predicted to improve the precision of therapeutic targeting to achieve more effective management of metastasis.

OBSCN restoration via OBSCN-AS1 long-noncoding RNA CRISPR-targeting suppresses metastasis in triple-negative breast cancer

Mounting evidence implicates the giant, cytoskeletal protein obscurin (720 to 870 kDa), encoded by the OBSCN gene, in the predisposition and development of breast cancer. Accordingly, prior work has shown that the sole loss of OBSCN from normal breast epithelial cells increases survival and chemoresistance, induces cytoskeletal alterations, enhances cell migration and invasion, and promotes metastasis in the presence of oncogenic KRAS. Consistent with these observations, analysis of Kaplan-Meier Plotter datasets reveals that low OBSCN levels correlate with significantly reduced overall and relapse-free survival in breast cancer patients. Despite the compelling evidence implicating OBSCN loss in breast tumorigenesis and progression, its regulation remains elusive, limiting any efforts to restore its expression, a major challenge given its molecular complexity and gigantic size (~170 kb). Herein, we show that OBSCN-Antisense RNA 1 (OBSCN-AS1), a novel nuclear long-noncoding RNA (lncRNA) gene originating from the minus strand of OBSCN, and OBSCN display positively correlated expression and are downregulated in breast cancer biopsies. OBSCN-AS1 regulates OBSCN expression through chromatin remodeling involving H3 lysine 4 trimethylation enrichment, associated with open chromatin conformation, and RNA polymerase II recruitment. CRISPR-activation of OBSCN-AS1 in triple-negative breast cancer cells effectively and specifically restores OBSCN expression and markedly suppresses cell migration, invasion, and dissemination from three-dimensional spheroids invitro and metastasis invivo. Collectively, these results reveal the previously unknown regulation of OBSCN by an antisense lncRNA and the metastasis suppressor function of the OBSCN-AS1/OBSCN gene pair, which may be used as prognostic biomarkers and/or therapeutic targets for metastatic breast cancer.

Knock-out of mitochondrial nucleoside diphosphate kinase NME4 reveals surprising tissue-distribution and remodeled cell signaling, suggestive of multiple functions.

The NME protein family (also called nucleoside diphosphate kinases, NDPK or NM23) comprises ten isoforms that are mostly multi-functional and localize to different cellular compartments. Three of them are found in mitochondria: NME3, NME4 and NME6. NME4 binds to the inner membrane, largely facing the intermembrane space, where it locally regenerates GTP and is involved in cardiolipin intermembrane transfer. Loss-of-function mutations in different human cell lines identified NME4 as a metastasis suppressor. However, few is known on NME4 physiology in animals in vivo. Here we characterize a germ line KO mouse model of NME4. While NME4 immunodetection is hampered by the lack of isoform-specific antibodies, comparison of NME levels between wild-type and KO animals allowed a first profiling of NME4 expression in various organs and tissues. Surprisingly, high levels of NME4 were found in uterus, testis, spleen, bladder, and kidney, while NME4 levels in mitochondria-rich organs like liver and heart were low. No compensatory upregulation of mitochondrial NME3 and NME6 was observed. NME4 KO animals occurred to have less mitochondrial mass (as expressed by protein levels, e.g. ATP synthase) in different organs, and a decreased ATP/ADP ratio in liver. Thus, although this organ has few NME4, depletion of these low quantities is sufficient to impact mitochondria. Finally, an unbiased large-scale kinome analysis in liver revealed that lack of NME4 alters cellular signaling cascades. Modified cell signaling was already implicated in the loss of the metastasis suppressor function of NME4. In summary, our data suggest different functions of NME4, with low levels critical for mitochondria and cell signaling in liver and heart, and high levels as in reproductive organs necessary for putative additional functions.

A germline SNP in BRMS1 predisposes patients with lung adenocarcinoma to metastasis and can be ameliorated by targeting c-fos.

About 50% of patients with early-stage, surgically resected lung cancer will develop distant metastasis. There remains an unmet need to identify patients likely to develop recurrence and to design innovative therapies to decrease this risk. Two primary isoforms of BRMS1, v1 and v2, are present in humans. Using next-generation sequencing of BRMS1 on matched human noncancerous lung tissue and non-small cell lung cancer (NSCLC) specimens, we identified single-nucleotide polymorphism (SNP) rs1052566 that results in an A273V mutation of BRMS1v2. This SNP is homozygous (BRMS1v2A273V/A273V) in 8% of the population and correlates with aggressive biology in lung adenocarcinoma (LUAD). Mechanistically, we show that BRMS1v2 A273V abolishes the metastasis suppressor function of BRMS1v2 and promotes robust cell invasion and metastases by activation of c-fos-mediated gene-specific transcriptional regulation. BRMS1v2 A273V increases cell invasion in vitro and increases metastases in both tail-vein injection xenografts and LUAD patient-derived organoid (PDO) intracardiac injection metastasis in vivo models. Moreover, we show that BRMS1v2 A273V fails to interact with nuclear Src, thereby activating intratumoral c-fos in vitro. Higher c-fos results in up-regulation of CEACAM6, which drives metastases in vitro and in vivo. Using both xenograft and PDO metastasis models, we repurposed T5224 for treatment, a c-fos pharmacologic inhibitor investigated in clinical trials for arthritis, and observed suppression of metastases in BRMS1v2A273V/A273V LUAD in mice. Collectively, we elucidate the mechanism of BRMS1v2A273V/A273V-induced metastases and offer a putative therapeutic strategy for patients with LUAD who have this germline alteration.

Abstract 2439: Targeting c-fos to suppress metastasis inBRMS1v2germline mutantlung adenocarcinoma

Abstract Introduction: Approximately 50% of patients with early-stage, surgically resected lung cancer develop distant metastasis. There remains an unmet need to identify patients likely to develop recurrence, and for innovative therapies to decrease this risk. Methods: The prevalence of single nucleotide polymorphisms (SNPs) rs105266, which causes a missense mutation at codon 273 (Ala to Val) of the metastasis suppressor BRMS1 isoform 2 (BRMS1v2), was assessed using the TCGA cohorts of lung adenocarcinoma (LUAD) and breast cancer, and validated in our LUAD cohort. Next, to explore the function of BRMS1v2 and the impact of BRMS1v2 A273V in LUAD, 3 distinct experimental systems were employed, including 1) Two LUAD cells stably expressing ectopic V5-epitope BRMS1v2 wild type (WT), or A273V mutant, by a retroviral system, 2) LUAD homozygous BRMS1v2A273V/A273V isogenic cells generated via a CRISPR knock-in approach, and 3) patient-derived LUAD organoids (PDO) with homozygous BRMS1v2A273V/A273V. The metastatic capabilities of LUAD cells and PDOs were evaluated by in vitro invasion chamber assays and two in vivo metastasis models: tail-vein injection and intracardiac injection. To interrogate the molecular mechanism of action, RNA-seq. and loss and gain of function experiments using genetic engineering and pharmacological inhibitors were performed. Results: The homozygous rs1052566 (BRMS1v2A273V/A273V) is present in 8% of individuals of European ancestry and 28% of South Asians. Importantly, BRMS1v2A273V/A273V is associated with an increased risk of metastases and a worse progression-free survival in patients with early-stage disease in the TCGA LUAD cohort (N=278). We mechanistically elucidated that BRMS1v2 A273V abolishes the metastasis suppressor function of BRMS1v2, promoting invasion and metastases. BRMS1v2 A273V fails to interact with nuclear Src, thereby activating intratumoral c-fos. Higher c-fos results in upregulation of CEACAM6, which drives metastases. Using a patient-derived organoid metastasis model, we repurposed a c-fos pharmacologic inhibitor investigated in clinical trials for arthritis and observed suppression of metastases in BRMS1v2A273V/A273V LUAD. Conclusion: Our data suggest that individuals harboring homozygotic BRMS1v2A273V/A273V have a predisposition for developing metastatic LUAD. Moreover, following external validation, BRMS1v2A273V/A273V could potentially serve as a biomarker to predict the development of metastatic disease in early-stage LUAD. Using a PDO metastasis model we show that pharmacologic targeting of c-fos in BRMS1v2A273V/A273V LUAD results in significantly less metastases in vivo. This offers the first pre-clinical evidence of a targeted therapeutic strategy in LUAD patients with this germline alteration. Citation Format: Yuan Liu, David Jones. Targeting c-fos to suppress metastasis inBRMS1v2germline mutantlung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2439.

The Prognostic Relevance of PMCA4 Expression in Melanoma: Gender Specificity and Implications for Immune Checkpoint Inhibition.

PMCA4 is a critical regulator of Ca2+ homeostasis in mammalian cells. While its biological and prognostic relevance in several cancer types has already been demonstrated, only preclinical investigations suggested a metastasis suppressor function in melanoma. Therefore, we studied the expression pattern of PMCA4 in human skin, nevus, as well as in primary and metastatic melanoma using immunohistochemistry. Furthermore, we analyzed the prognostic power of PMCA4 mRNA levels in cutaneous melanoma both at the non-metastatic stage as well as after PD-1 blockade in advanced disease. PMCA4 localizes to the plasma membrane in a differentiation dependent manner in human skin and mucosa, while nevus cells showed no plasma membrane staining. In contrast, primary cutaneous, choroidal and conjunctival melanoma cells showed specific plasma membrane localization of PMCA4 with a wide range of intensities. Analyzing the TCGA cohort, PMCA4 mRNA levels showed a gender specific prognostic impact in stage I–III melanoma. Female patients with high transcript levels had a significantly longer progression-free survival. Melanoma cell specific PMCA4 protein expression is associated with anaplasticity in melanoma lung metastasis but had no impact on survival after lung metastasectomy. Importantly, high PMCA4 transcript levels derived from RNA-seq of cutaneous melanoma are associated with significantly longer overall survival after PD-1 blockade. In summary, we demonstrated that human melanoma cells express PMCA4 and PMCA4 transcript levels carry prognostic information in a gender specific manner.

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.

Emerging Molecular Connections between NM23 Proteins, Telomeres and Telomere-Associated Factors: Implications in Cancer Metastasis and Ageing.

The metastasis suppressor function of NM23 proteins is widely understood. Multiple enzymatic activities of NM23 proteins have also been identified. However, relatively less known interesting aspects are being revealed from recent developments that corroborate the telomeric interactions of NM23 proteins. Telomeres are known to regulate essential physiological events such as metastasis, ageing, and cellular differentiation via inter-connected signalling pathways. Here, we review the literature on the association of NM23 proteins with telomeres or telomere-related factors, and discuss the potential implications of emerging telomeric functions of NM23 proteins. Further understanding of these aspects might be instrumental in better understanding the metastasis suppressor functions of NM23 proteins.

Tektin4 loss promotes triple-negative breast cancer metastasis through HDAC6-mediated tubulin deacetylation and increases sensitivity to HDAC6 inhibitor.

Progression of triple-negative breast cancer (TNBC) constitutes a major unresolved clinical challenge, and effective targeted therapies are lacking. Because microtubule dynamics play pivotal roles in breast cancer metastasis, we performed RNA sequencing on 245 samples from TNBC patients to characterize the landscape of microtubule-associated proteins (MAPs). Here, our transcriptome analyses revealed that low expression of one MAP, tektin4, indicated poor patient outcomes. Tektin4 loss led to a marked increase in TNBC migration, invasion, and metastasis and a decrease in microtubule stability. Mechanistically, we identified a novel microtubule-associated complex containing tektin4 and histone deacetylase 6 (HDAC6). Tektin4 loss increased the interaction between HDAC6 and α-tubulin, thus decreasing microtubule stability through HDAC6-mediated tubulin deacetylation. Significantly, we found that tektin4 loss sensitized TNBC cells, xenograft models, and patient-derived organoid models to the HDAC6-selective inhibitor ACY1215. Furthermore, tektin4 expression levels were positively correlated with microtubule stability levels in clinical samples. Together, our findings uncover a metastasis suppressor function of tektin4 and support clinical development of HDAC6 inhibition as a new therapeutic strategy for tektin4-deficient TNBC patients.

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.

TCF21 Promotes Luminal-Like Differentiation and Suppresses Metastasis in Bladder Cancer.

Little is known regarding the subclone evolution process in advanced bladder cancer, particularly with respect to the genomic alterations that lead to the development of metastatic lesions. In this project, we identify gene expression signatures associated with metastatic bladder cancer through mRNA expression profiling of RNA isolated from 33 primary bladder cancer and corresponding lymph node (LN) metastasis samples. Gene expression profiling (GEP) was performed on RNA isolated using the Illumina DASL platform. We identified the developmental transcription factor TCF21 as being significantly higher in primary bladder cancer compared with LN metastasis samples. To elucidate its function in bladder cancer, loss- and gain-of-function experiments were conducted in bladder cancer cell lines with high and low expression of TCF21, respectively. We also performed GEP in bladder cancer cell lines following TCF21 overexpression. We identified 2,390 genes differentially expressed in primary bladder cancer and corresponding LN metastasis pairs at an FDR cutoff of 0.1 and a fold change of 1. Among those significantly altered, expression of TCF21 was higher in the primary tumor compared with LN metastasis. We validated this finding with qPCR and IHC on patient samples. Moreover, TCF21 expression was higher in luminal cell lines and knockdown of TCF21 increased invasion, tumor cell dissemination, and metastasis. In contrast, overexpression of TCF21 in highly metastatic basal bladder cancer cell lines decreased their invasive and metastatic potential. IMPLICATIONS: TCF21 is differentially overexpressed in primary bladder cancer compared with matched LN metastasis, with in vitro and in vivo studies demonstrating a metastasis suppressor function of this transcription factor.

Chromatin remodeling factor ARID2 suppresses hepatocellular carcinoma metastasis via DNMT1-Snail axis

Recurrence and metastasis remain the major obstacles to successful treatment of hepatocellular carcinoma (HCC). Chromatin remodeling factor ARID2 is commonly mutated in HCC, indicating its important role in cancer development. However, its role in HCC metastasis is largely elusive. In this study, we find that ARID2 expression is significantly decreased in metastatic HCC tissues, showing negative correlation with pathological grade, organ metastasis and positive association with survival of HCC patients. ARID2 inhibits migration and invasion of HCC cells in vitro and metastasis in vivo. Moreover, ARID2 knockout promotes pulmonary metastasis in different HCC mouse models. Mechanistic study reveals that ARID2 represses epithelial-mesenchymal transition (EMT) of HCC cells by recruiting DNMT1 to Snail promoter, which increases promoter methylation and inhibits Snail transcription. In addition, we discover that ARID2 mutants with disrupted C2H2 domain lose the metastasis suppressor function, exhibiting a positive association with HCC metastasis and poor prognosis. In conclusion, our study reveals the metastasis suppressor role as well as the underlying mechanism of ARID2 in HCC and provides a potential therapeutic target for ARID2-deficient HCC.

Tumor metastasis suppressor functions of Ets transcription factor through integrin β3-mediated signaling pathway.

Prostate-derived Ets transcription factor (PDEF) is a member of the Ets transcription factor family originally identified in prostate tissue. PDEF functions like a tumor metastasis suppressor. Although the underlying mechanism is not very clear. In this study, a significantly higher expression level of integrin β3 was observed in aggressive prostate cancer (PCa) cells; PC3, that lack the expression of PDEF, was confirmed by quantitative real-time polymerase chain reaction and the immunoblot analysis. Dual immunofluorescence studies have confirmed the lower expression of PDEF and higher expression level of integrin β3 in PCa cells compared with non-tumorigenic prostate epithelial RWPE-1 cells. Then, it was attempted to identify integrin β3-mediated downstream signaling pathways that modulate actin cytoskeleton remodeling in PCa cells. Inhibition of PDEF by RNA interference (PDEFKD ) in DU145 cells confirmed by transcript and western blot analysis, exhibited significantly higher expression level of integrin β3 and its downstream signaling molecules talin and paxillin, associated with promoting migration and invasion of cells. Given the involvement of integrin-mediated invasion of cells, PDEFKD DU145 cells were treated with Echistatin, a potent integrin β3-specific antagonist and found that the cells significantly reduced the transcript and protein expression levels of talin and paxillin; and reduced the invasion of cells. Overall, the cytoskeleton remodeling by integrin β3, modulated by PDEF, may represent a novel molecular link with cell adhesion and migration leading to the suppression of metastasis in PCa cells.

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 β3 (ITGβ3), and in one cell line, concomitant suppression of integrin β1 (ITGβ1) 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 ITGβ1 or ITGβ3 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 ITGβ3. Expression of ITGβ3 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 ITGβ3 protein at the cell membrane resulting in attenuated cell motility through the stabilization of large focal adhesions.

VAMP8, a vesicle-SNARE required for RAB37-mediated exocytosis, possesses a tumor metastasis suppressor function

We previously identified a metastasis suppressor RAB37 small GTPase that regulated exocytosis of tissue inhibitor of metalloproteinases 1 (TIMP1) to suppress lung cancer metastasis. Here, we show that vesicle-associated membrane protein 8 (VAMP8), a v-SNARE (vesicle soluble N-ethylmaleimide-sensitive factor activating protein receptor), interacts with RAB37 and drives the secretion of TIMP1 to inhibit tumor metastases. Confocal and total internal reflection fluorescence microscopic images demonstrated that VAMP8 co-localized with RAB37 and facilitated trafficking of RAB37-TIMP1 vesicles. Reconstitution experiments using tail-vein injection and lung-to-lung metastasis in mice showed that VAMP8 was essential for RAB37-regulated vesicle trafficking of TIMP1 to suppress cancer metastasis. Lung cancer patients with low VAMP8 showed distant metastasis, poor overall survival and progression-free survival. Importantly, multivariate Cox regression analysis indicated that patients with low VAMP8/low RAB37 expression profile showed significantly high risk of death (hazard ratio = 3.42, P < 0.001) even after adjusting for tumor metastasis parameter. Our findings reveal that VAMP8 is a novel v-SNARE crucial for RAB37-mediated exocytic transport of TIMP1 to suppress lung tumor metastasis. VAMP8 possesses a tumor metastasis suppressor function with a prognostic value in lung cancer.

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.

Abstract 4845: Induction of aldolase C expression by NME1 may mediate metastasis suppressor activity in malignant melanoma

Abstract Although the metastasis suppressor activity of NME1 has been demonstrated in a number of settings of human cancer, the underlying molecular mechanisms are not well understood. NME1 has two known enzymatic activities, nucleoside diphosphate kinase (NDPK) and a 3’ to 5’ exonuclease. To determine the relative contribution either of these activities has on the metastasis suppressor function of NME1, our lab previously generated site directed mutants to abrogate one or both of the enzymatic activities (Ma et al., 2004). These studies showed that disrupting the 3’ to 5’ exonuclease activity with the point substitutions glutamate5-to-alanine (E5A) and lysine12-to-glutamine (K12Q) also significantly compromised the metastasis suppressor function of NME1 (Zhang et al., 2011). To identify genes that are regulated by NME1 and potentially modulate its metastasis suppressor activity, we compared the gene expression profile induced by forced expression of wild-type NME to those obtained with the E5A and K12Q mutants. Using the human Exon 1.0 Affymetrix microarray , we identified 13 genes whose expression was modulated by wild-type NME1 expression, but not by the suppressor-deficient mutants, which were designated as metastasis suppressor signature (MSS) genes. We are currently refining the list to genes that have the highest clinical relevance through observing expression profiles of the MSS genes in clinical datasets. Preliminary results of unsupervised hierarchal clustering of GEO melanoma dataset GSE8401 showed that expression of the MSS gene, aldolase C (ALDOC), was significantly lower in a subset of metastatic melanoma lesions compared to primary melanomas. Western blot and qPCR analysis confirm that NME1 induces expression of ALDOC in both WM793 and M14 melanoma cell lines, and that induction of ALDOC is dose-responsive to NME1 expression as observed in the WM1158 metastatic melanoma cell line. Further, NME1 induction of ALDOC is specific compared to the ALDOA and ALDOB isoforms. Recent studies have indicated aldolase isoforms can modulate motile and invasive phenotypes of cancer cells (Li et al., 2015 &amp; Hu et al., 2016), suggesting regulation of ALDOC expression may represent a key mechanism underlying the metastasis suppressor function of NME1. Current work is focused on measuring the impact of NME1-induced expression of ALDOC on the metastatic potential of melanoma cells in culture and in vivo. Citation Format: Nidhi Pamidimukkala, Katie Leonard, Joseph McCorkle, Qingbei Zhang, Anup Mahurkar, Amol Shetty, David Kaetzel. Induction of aldolase C expression by NME1 may mediate metastasis suppressor activity in malignant melanoma [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 4845. doi:10.1158/1538-7445.AM2017-4845

Abstract 4846: The metastasis suppressor NME1 is recruited to DNA double strand breaks

Abstract 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

Abstract 4840: SNP rs1052566 (Ala273Val) in BRMS1 variant2 promotes metastasis in lung adenocarcinoma

Abstract Background: Metastatic lung cancer kills over 180,000 people in the United States annually. Family history is one risk factor for lung adenocarcinoma (LUAD), suggesting a putative genetic predisposition to the development of LUAD. Single nucleotide polymorphisms (SNPs) are the most common genetic variations in the human genome and major determinants of variations in disease susceptibility, prognosis, response to medication, and toxicity. Recent studies indicate that SNPs play important roles in the progression of LUAD. We have previously shown that breast cancer metastasis suppressor-1 (BRMS1) plays a critical role in inhibiting the progression of lung cancer. We observed that SNP rs1052566 homo-variant in breast cancer metastasis suppressor-1 (BRMS1) variant 2 (v2) causes an A273V mutation. However, the role of this SNP in the progression of lung cancer remains to be determined. Methods: We utilized next-generation DNA sequencing and Taqman SNP assays to detect the prevalence of rs1052566 in 40 surgically resected LUAD specimens. Next, we generated a retroviral expressional system (pBabe puro) encoding V5-tagged BRMS1v2 wild-type (WT) and A273V mutant and created NSCLC H1299 and A549 cell lines that stably express V5-tagged BRMS1v2 WT, A273V, or vector control. The metastatic capabilities of these stable cells were evaluated by invasion chamber assays in vitro and a tail-vein injection metastasis model in vivo. To explore the mechanism(s) through which BRMS1v2 A273V promotes metastasis, we performed RT profiler PCR assay. Results: We determined that SNP rs1052566 is present in 45% of LUAD patients. Of these, the homo-variant occurs in 7.5% of patients (3 of 40). Importantly, BRMS1v2 A273V significantly promotes metastasis of NSCLC cells in vitro and in vivo. Additionally, in our mouse model, A273V was associated with decreased survival, compared with BRMS1v2 WT or control. On RT profiler PCR array, 7 genes, including L1CAM, were found to be significantly differentially regulated by BRMS1v2 WT and A273V. Confirmatory RT-PCR and immunoblots proved that BRMS1v2 A273V increases both the transcript and protein levels of L1CAM. Furthermore, we found that either c-Jun or c-Fos promotes the transcription of L1CAM. However, BRMS1v2 A273V specifically enhances c-Fos-, but not c-Jun-, induced L1CAM transcription. Conclusion: We observed that BRMS1v2 A273V abolishes the metastasis suppressor function of BRMS1 and promotes metastasis. Up-regulation of c-Fos-induced L1CAM transcription is a putative mechanism of BRMS1v2 A273V-induced metastasis. Our data suggest that SNP rs1052566, especially its homo-variant, is associated with an increased risk of metastasis in LUAD. Note: This abstract was not presented at the meeting. Citation Format: Neel P. Chudgar, Yuan Liu, Joseph Montecalvo, Marty W. Mayo, Prasad S. Adusumilli, David R. Jones. SNP rs1052566 (Ala273Val) in BRMS1 variant2 promotes metastasis in lung adenocarcinoma [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 4840. doi:10.1158/1538-7445.AM2017-4840

RD3 loss dictates high-risk aggressive neuroblastoma and poor clinical outcomes.

Clinical outcomes for high-risk neuroblastoma patients remains poor, with only 40-50% 5-Year overall survival (OS) and <10% long-term survival. The ongoing acquisition of genetic/molecular rearrangements in undifferentiated neural crest cells may endorse neuroblastoma progression. This study recognized the loss of Retinal Degeneration protein 3, RD3 in aggressive neuroblastoma, and identified its influence in better clinical outcomes and defined its novel metastasis suppressor function. The results showed ubiquitous expression of RD3 in healthy tissues, complete-loss and significant TNM-stage association of RD3 in clinical samples. RD3-loss was intrinsically associated with reduced OS, abridged relapse-free survival, aggressive stage etc., in neuroblastoma patient cohorts. RD3 was transcriptionally and translationally regulated in metastatic site-derived aggressive (MSDAC) cells (regardless of CSC status) ex vivo and in tumor manifolds from metastatic sites in reproducible aggressive disease models in vivo. Re-expressing RD3 in MSDACs reverted their metastatic potential both in vitro and in vivo. Conversely muting RD3 in neuroblastoma cells not only heightened invasion/migration but also dictated aggressive disease with metastasis. These results demonstrate the loss of RD3 in high-risk neuroblastoma, its novel, thus-far unrecognized metastasis suppressor function and further imply that RD3-loss may directly relate to tumor aggressiveness and poor clinical outcomes.