Metastasis Suppressor Activity Research Articles

The metastasis suppressor protein NM23-H1 modulates the PI3K-AKT axis through interaction with the p110\u03b1 catalytic subunit

The PI3K pathway is one of the most deregulated pathways in cancer, which is predominantly due to gain of function mutations or altered expression of the PI3KCA gene. This is codified by what is seen for the class I PI3K catalytic subunit p110α, a common feature of many cancers. The metastasis suppressor protein NM23-H1 (NME1), whose ability to suppress the metastasis activities of different tumors has been widely described and was previously reported to alter phosphatidylinositol signaling. Here, we show interaction of NM23-H1 with the p110α subunit and the functional consequence of this interaction. This interaction is predominantly localized at the plasma membrane with some signals seen in the cytoplasmic compartment. Analysis of NM23-H1 levels showed a negative correlation between NM23-H1 expression and Akt phosphorylation, the key marker of PI3K pathway activation. Investigating the functional consequence of this interaction using cell motility and clonogenicity assays showed that expression of NM23-H1 reversed the enhanced migration, invasion, adhesion, and filopodia structure formation in cells expressing the p110α catalytic subunit. A similar trend was seen in anchorage-independent assays. Notably, differential analyses using NM23-H1 mutants which lacked the enzymatic and metastasis suppressor activity, showed no detectable interaction between p110α and the NM23-H1 mutant proteins P96S, H118F, and S120G, as well as no dysregulation of the PI3K-AKT axis.

Resveratrol binds and activates RKIP protein in colorectal cancer

Raf-1 kinase inhibitory protein (RKIP) acts as a tumor cell metastasis suppressor and prognostic indicator for survival in various cancers. Its use is predicted to improve therapy for various malignancies, including colorectal cancer (CRC). RKIP, frequently denoted as phosphatidylethanolamine-binding protein 1, is expressed in all normal mammalian tissues. RKIP functions as an inhibitor of the Raf-1, PI-3K, and MAP kinase (MAPK) pathways. In this study, we found that resveratrol induced the expression of RKIP at protein levels. To elucidate the structural basis of the interaction between resveratrol and RKIP, we performed computational studies that explore the binding affinity and ligand efficacy of resveratrol against RKIP. This study reveals the prognostic significance of RKIP metastasis suppressor activity against CRC and its structural arrangements during drug-target interactions.

Current Status of Raf Kinase Inhibitor Protein (RKIP) in Lung Cancer: Behind RTK Signaling.

Lung cancer is the most deadly neoplasm with the highest incidence in both genders, with non-small cell lung cancer (NSCLC) being the most frequent subtype. Somatic mutations within the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene are key drivers of NSCLC progression, with EGFR inhibitors being particularly beneficial for patients carrying the so-called “EGFR-sensitizing mutations”. However, patients eventually acquire resistance to these EGFR inhibitors, and a better knowledge of other driven and targetable proteins will allow the design of increasingly accurate drugs against patients’ specific molecular aberrations. Raf kinase inhibitory protein (RKIP) is an important modulator of relevant intracellular signaling pathways, including those controlled by EGFR, such as MAPK. It has been reported that it has metastasis suppressor activity and a prognostic role in several solid tumors, including lung cancer. In the present review, the potential use of RKIP in the clinic as a prognostic biomarker and predictor of therapy response in lung cancer is addressed.

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.

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 & 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

B15 - Polymorphism of CDH1 Promoter Is a Predictor of Clinical Outcome in Patients with Metastatic Gastric Cancer Treated with chemotherapy

Background: CDH is a Ca2+-dependent cell adhesion molecule with tumor metastasis suppressor activity. Mutations of CDH1 and its E-cadherin product have been associated with the origin, development, invasion, metastasis, and prognosis of gastric cancer (GC). The present study analyzed CDH1 gene polymorphisms and their impacts on chemotherapeutic event free response (EFS) and survival (OS) in patients with metastatic gastric cancer (MGC) treated with DOC2 (Docetaxel, Oxaliplatin) regimen. The original chemotherapeutic program DOC was developed at the CRO - Cancer Center in Aviano. The phase I study determined the Maximum Tolerated Dose (MTD) and, as per protocol, indicated the level just below the MTD as appropriate for a phase II study. Patients and methods: This is a multi-centre, phase II single arm study of DOC combination in patients with previously untreated surgically unresectable or metastatic gastric cancer. Eligible subjects will be enrolled to receive first-line combination therapy consisting of DOC2 treatment. The foreseen schedule consists of: Docetaxel 30 mg/m2, Oxaliplatin 70 mg/m2, both i.v., on days 1 and 8, associated to Capecitabine 500 mg/m2, p.o., bid, on day 2 to 15, on a three week basis. The study has been designed to determine the activity and safety of the DOC regimen in previously untreated patients. Each patient will be treated for a maximum of 6 cycles until disease progression, unacceptable toxicity, or patient refusal, whichever occur first. However, Capecitabine 1000 mg/m2/die tablets p.o. alone is allowed in patients without tumor progression at the end of chemotherapy (maintenance treatment) for further 4-6 cycles. Up to now the study enrolled 29 out 71 foreseen MGC patients treated with first-line DOC2 chemotherapy with a mean follow-up of 1.2 years (range 0.2 to 3.2). Polymorphisms of the entire CDH1 exons and promoter regions were determined. Results: Among the 37 regions we analyzed, polymorphism in the promoter (P1) was significantly associated with EFS (HR 4.8, 95%CI 1.8-12.7, p = 0.004) and showed an increasing trend of OS (HR 2.8, 95%CI 0.9-8.7). Conclusions: Our result suggest promoter region of CDH1 might be independent predictor of chemotherapeutic clinical outcome.

Abstract 3258: Breast Metastasis Suppressor 1 (BRMS1) phosphorylation appears necessary for metastasis suppressor activity

Abstract Breast cancer is the most common cancer among women worldwide. BRMS1 is a metastasis suppressor that suppresses metastasis without significantly affecting the growth of a primary tumor. BRMS1 contains two coiled-coil motifs important for protein-protein interactions and two putative nuclear localization sequences (NLS - aa198-205 (NLS1) and aa239-245 (NLS2)) and one nuclear export signal which appear to mediate nuclear-cytoplasmic shuttling. Although NLS2 is not necessary for active transport into the nucleus, it is required for metastasis suppression (PMC3563580). A serine immediately upstream of NLS2 (S237) can be phosphorylated by cdk2 (PMC3037622). Therefore, we hypothesized that NLS2 and adjacent amino acids are critical mediators of metastasis suppression. To test this hypothesis, NLS2 (aa231-248) epitope-tagged with HA and GST was transduced into metastatic MDA-MB-231 and -435 breast carcinoma cells expressing green fluorescent protein and stable colonies were selected. BRMS1S237A-expressing MDA-MB-231 cells were no longer metastasis suppressed, suggesting a critical role for phosphorylation of BRMS1 in its metastasis suppressor activity. Studies are underway with full-length BRMS1S237D (phosphomimetic) as well as expression with truncated wild-type NLS2 and mutant (NLS2wt/S237A/S237D) to test whether the BRMS1 phosphorylation or the NLS2 domain itself is critical for metastasis suppressor activity. Support: CA87728, CA134981, P30-CA168524, Natl Fndn Cancer Res, Komen SAC11037, KS Bioscience Auth, NHMRC Citation Format: Lellys M. Contreras, Boris Sarcevic, Keke M. Pounds, Danny R. Welch. Breast Metastasis Suppressor 1 (BRMS1) phosphorylation appears necessary for metastasis suppressor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3258. doi:10.1158/1538-7445.AM2015-3258

Abstract 3253: Metastasis suppression by non-KP54 kisspeptins

Abstract The KISS1 metastasis suppressor blocks metastasis by inhibiting colonization at secondary sites. KISS1 activity requires secretion and extracellular processing by furin into kisspeptins. Kisspeptin-54 (KP54; G68 to F121) binds a G-protein coupled receptor, KISS1R, and the mechanism of metastasis suppression has been assumed to be through the receptor. However, KISS1 is cleaved into kisspeptins outside of the cell, and KISS1R expression is not required for metastasis suppression. Therefore, an alternate hypothesis is that a non-KISS1R binding kisspeptin(s) is responsible for metastasis suppression. To test this hypothesis, we engineered all ten of the possible kisspeptins predicted from known dibasic cleavage sites (M1-Q145; M1-R56; M1-R67; M1-R124; R56-R67; G68-F121; E55-F121; E55-Q145; G68-Q145; E125-Q145) downstream of the KISS1 secretion signal peptide (designated KMP1 to KMP10, respectively) and expressed each individually in B16-F10 melanoma cells. Expression was confirmed by immunoblot and/or RT-QPCR analysis. Following intravenous injection, experimental lung metastases were quantified. KP54 expression suppressed metastasis. Some KMP lacking the KISS1R ligand suppressed metastasis as well as KP54 and full-length KISS1 (p<0.05). Specifically, the N-terminal kisspeptin (M1 - R56) dramatically suppressed metastasis. This suggests that the mechanism of action for metastasis suppression by KISS1 does not necessarily require activation of KISS1R by the peptide ligand but also leaves the possibility for additional receptors. Alternatively, KISS1/KMP interactions with other intracellular proteins may be required for metastasis suppressor activity. Support: CA134981, Natl Fndn Cancer Res, Komen SAC11037, KS Bioscience Auth, P30-CA168524, Steiner Family Fund. Citation Format: Kelsey R. Hampton, Keke M. Pounds, Andrew P. Tremblath, Danny R. Welch. Metastasis suppression by non-KP54 kisspeptins. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3253. doi:10.1158/1538-7445.AM2015-3253

Raf kinase inhibitor protein (RKIP) deficiency decreases latency of tumorigenesis and increases metastasis in a murine genetic model of prostate cancer.

Raf kinase inhibitor protein (RKIP) has been shown to act as a metastasis suppressor gene in multiple models of cancer. Loss of RKIP expression promotes invasion and metastasis in cell transplantation animal models. However, it is unknown if RKIP expression can impact the progression of cancer in an autochthonous model of cancer. The goal of this study was to determine if loss of RKIP expression in a genetic mouse model of prostate cancer (PCa) impacts metastasis. Endogenous RKIP expression was measured in the primary tumors and metastases of transgenic adenocarcinoma of the mouse prostate (TRAMP(+) ) mice. RKIP knockout mice (RKIP(-/-) ) were crossbred with (TRAMP(+) ) mice to create RKIP(-/-) TRAMP(+) mice. Mice were euthanized at 10, 20, and 30 weeks for evaluation of primary and metastatic tumor development. To determine if loss of RKIP alone promotes metastasis, RKIP was knocked down in the low metastatic LNCaP prostate cancer cell line. Endogenous RKIP expression decreased in TRAMP(+) mice as tumors progressed. Primary tumors developed earlier in RKIP(-/-) TRAMP(+) compared to TRAMP(+) mice. At 30 weeks of age, distant metastases were identified only the RKIP(-/-) TRAMP(+) mice. While prostate epithelial cell proliferation rates were higher at 10 and 20 weeks in RKIP(-/-) TRAMP(+) compared to TRAMP(+) mice, by 30 weeks there was no difference. Apoptosis rates in both groups were similar at all timepoints. Decreased RKIP expression did not impact the metastatic rate of LNCaP in an orthotopic PCa model. These results demonstrate that loss of RKIP decreases latency of tumor development and promotes distant metastasis in the TRAMP mouse model in the context of a pro-metastatic background; but loss of RKIP alone is insufficient to promote metastasis. These findings suggest that in addition to its known metastasis suppressor activity, RKIP may promote tumor progression through enhancing tumor initiation. Prostate 75:292-302, 2015. © 2014 Wiley Periodicals, Inc.

SPDEF: a molecular switch for E-cadherin expression that promotes prostate cancer metastasis

SPDEF: a molecular switch for E-cadherin expression that promotes prostate cancer metastasis

TGF-Beta Suppresses VEGFA-Mediated Angiogenesis in Colon Cancer Metastasis

The FET cell line, derived from an early stage colon carcinoma, is non-tumorigenic in athymic nude mice. Engineered FET cells that express TGF-α (FETα) display constitutively active EGFR/ErbB signaling. These cells readily formed xenograft tumors in athymic nude mice. Importantly, FETα cells retained their response to TGF-beta-mediated growth inhibition, and, like the parental FET cells, expression of a dominant negative TGF-beta type II receptor (DNRII) in FETα cells (FETα/DNRII) abrogated responsiveness to TGF-beta-induced growth inhibition and apoptosis under stress conditions in vitro and increased metastatic potential in an orthotopic model in vivo, which indicates metastasis suppressor activity of TGF-beta signaling in this model. Cancer angiogenesis is widely regarded as a key attribute for tumor formation and progression. Here we show that TGF-beta signaling inhibits expression of vascular endothelial growth factor A (VEGFA) and that loss of autocrine TGF-beta in FETα/DNRII cells resulted in increased expression of VEGFA. Regulation of VEGFA expression by TGF-beta is not at the transcriptional level but at the post-transcriptional level. Our results indicate that TGF-beta decreases VEGFA protein stability through ubiquitination and degradation in a PKA- and Smad3-dependent and Smad2-independent pathway. Immunohistochemical (IHC) analyses of orthotopic tumors showed significantly reduced TGF-beta signaling, increased CD31 and VEGFA staining in tumors of FETα/DNRII cells as compared to those of vector control cells. These results indicate that inhibition of TGF-beta signaling increases VEGFA expression and angiogenesis, which could potentially contribute to enhanced metastasis of those cells in vivo. IHC studies performed on human colon adenocarcinoma specimens showed that TGF-beta signaling is inversely correlated with VEGFA expression, indicating that TGF-beta-mediated suppression of VEGFA expression exists in colon cancer patients.

Effect of Inhibition of the Lysophosphatidic Acid Receptor 1 on Metastasis and Metastatic Dormancy in Breast Cancer

Previous studies identified the human nonmetastatic gene 23 (NME1, hereafter Nm23-H1) as the first metastasis suppressor gene. An inverse relationship between Nm23-H1 and expression of lysophosphatidic acid receptor 1 gene (LPAR1, also known as EDG2 or hereafter LPA1) has also been reported. However, the effects of LPA1 inhibition on primary tumor size, metastasis, and metastatic dormancy have not been investigated. The LPA1 inhibitor Debio-0719 or LPA1 short hairpinned RNA (shRNA) was used. Primary tumor size and metastasis were investigated using the 4T1 spontaneous metastasis mouse model and the MDA-MB-231T experimental metastasis mouse model (n = 13 mice per group). Proliferation and p38 intracellular signaling in tumors and cell lines were determined by immunohistochemistry and western blot to investigate the effects of LPA1 inhibition on metastatic dormancy. An analysis of variance-based two-tailed t test was used to determine a statistically significant difference between treatment groups. In the 4T1 spontaneous metastasis mouse model, Debio-0719 inhibited the metastasis of 4T1 cells to the liver (mean = 25.2 liver metastases per histologic section for vehicle-treated mice vs 6.8 for Debio-0719-treated mice, 73.0% reduction, P < .001) and lungs (mean = 6.37 lesions per histologic section for vehicle-treated mice vs 0.73 for Debio-0719-treated mice, 88.5% reduction, P < .001), with no effect on primary tumor size. Similar results were observed using the MDA-MB-231T experimental pulmonary metastasis mouse model. LPA1 shRNA also inhibited metastasis but did not affect primary tumor size. In 4T1 metastases, but not primary tumors, expression of the proliferative markers Ki67 and pErk was reduced by Debio-0719, and phosphorylation of the p38 stress kinase was increased, indicative of metastatic dormancy. The data identify Debio-0719 as a drug candidate with metastasis suppressor activity, inducing dormancy at secondary tumor sites.

NM23 deficiency promotes metastasis in a UV radiation-induced mouse model of human melanoma

Cutaneous malignant melanoma is the most lethal form of skin cancer, with 5-year survival rates of <5 % for patients presenting with metastatic disease. Mechanisms underlying metastatic spread of UVR-induced melanoma are not well understood, in part due to a paucity of animal models that accurately recapitulate the disease in its advanced forms. We have employed a transgenic mouse strain harboring a tandem deletion of the nm23-m1 and nm23-m2 genes to assess the combined contribution of these genes to suppression of melanoma metastasis. Crossing of the nm23-h1/nm23-h2 knockout in hemizygous-null form ([m1m2]+/−) to a transgenic mouse strain (hepatocyte growth factor/scatter factor-overexpressing, or HGF+ strain) vulnerable to poorly-metastatic, UVR-induced melanomas resulted in UVR-induced melanomas with high metastatic potential. Metastasis to draining lymph nodes was seen in almost all cases of back skin melanomas, while aggressive metastasis to lung, thoracic cavity, liver and bone also occurred. Interestingly, no differences were observed in the invasive characteristics of primary melanomas of HGF+ and HGF+ × [m1m2]+/− strains, with both exhibiting invasion into the dermis and subcutis, indicating factors other than simple invasive activity were responsible for metastasis of HGF+ × [m1m2]+/− melanomas. Stable cell lines were established from the primary and metastatic melanoma lesions from these mice, with HGF+ × [m1m2]+/− lines exhibiting increased single cell migration and genomic instability. These studies demonstrate for the first time in vivo a potent metastasis suppressor activity of NM23 in UVR-induced melanoma, and have provided new tools for identifying molecular mechanisms that underlie melanoma metastasis.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-012-9495-z) contains supplementary material, which is available to authorized users.

Raf kinase inhibitor protein (RKIP) in cancer

Raf kinase inhibitory protein (RKIP) was initially identified as phosphatidylethanolamine binding protein in bovine brain. It was later identified as a protein that inhibits Raf kinase activation of MEK. Further exploration has revealed that RKIP modulates several other signaling pathways including NF-κB and G-protein signaling. A gene array screen revealed that RKIP expression was low in a metastatic compared with non-metastatic prostate cancer cell line. Further experiments revealed that RKIP fits the criteria for a metastasis suppressor gene. RKIP expression has been shown to be downregulated in metastatic tissues, compared with non-metastatic tissue in multiple cancers, suggesting that loss of RKIP metastasis suppressor activity is a broad mechanism leading to metastasis. Additionally, loss of RKIP has been shown to impact therapy through conferring radioresistance and chemoresistance. Taken together, these data indicate understanding RKIP's contributions to cancer may lead to important therapeutic strategies to prevent metastasis and promote therapeutic efficacy.

Abstract 3428: Genome-stabilizing functions of NM23 in metastatic melanoma

Abstract The metastasis suppressor protein NM23-H1 exhibits two enzymatic activities in vitro with potential roles in the maintenance of genomic integrity: a nucleoside diphosphate kinase (NDPK) activity that maintains balance in nucleotide pools and a 3′-5′ exonuclease (3′-5′ EXO) activity that could provide proofreading during DNA repair and replication. Studies were conducted in cell culture and in vivo to determine the extent to which NM23-H1 expression contributes to maintenance of genomic integrity, as well as suppression of melanoma initiation and progression. Expression of NM23-H1, and possibly NM23-H2, was shown to be necessary for timely repair of UVR-induced (6-4) photoproducts (6-4PP) in melanoma cell lines, as well as in transgenic mouse-derived melanocytes and embryo fibroblasts, as determined by immunoslot-blot assay. NM23 protein expression also suppressed the incidence of both spontaneous and UVR-induced mutations as determined using the 6-thioguanine resistance colony-forming assay. Point mutations that either selectively or tandemly disrupted the NDPK and 3′-5′ EXO activities of NM23-H1 exhibited compromised DNA repair and mutation-suppressing activities, consistent with contributions of both enzymatic functions. UVR induced rapid (&amp;lt; 5 min) translocation of NM23-H1 to the nucleus, where it colocalized closely in concert with 6-4PPs, strongly suggesting direct participation in the repair of those lesions. Consistent with this proposed function, transgenic mice engineered to be hemizygous-null in expression of both NM23-M1 and NM23-M2 (the mouse homologs of NM23-H1 and NM23-H2, respectively) were rendered susceptible to UVR-induced melanoma and epithelioid follicular cyst formation. Moreover, this NM23-deficient genotype conferred an aggressively metastatic phenotype to the HGF-expressing transgenic mouse model of UVR-inducible melanoma. Metastasis of highly-pigmented cells to draining lymph nodes and the lungs was seen in most cases, with lesions also occasionally seen in liver, brain and bone. Taken together, these results demonstrate a critical role for NM23 proteins in DNA repair and suppressing mutations under basal and genotoxic conditions. Importantly, they highlight for the first time a tumor suppressor function in the context of UVR-induced melanoma in vivo, and suggest a novel metastasis suppressor activity through suppression of metastasis-driving mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3428. doi:1538-7445.AM2012-3428

KAI1 suppresses HIF-1α and VEGF expression by blocking CDCP1-enhanced Src activation in prostate cancer

BackgroundKAI1 was initially identified as a metastasis-suppressor gene in prostate cancer. It is a member of the tetraspan transmembrane superfamily (TM4SF) of membrane glycoproteins. As part of a tetraspanin-enriched microdomain (TEM), KAI1 inhibits tumor metastasis by negative regulation of Src. However, the underlying regulatory mechanism has not yet been fully elucidated. CUB-domain-containing protein 1 (CDCP1), which was previously known as tetraspanin-interacting protein in TEM, promoted metastasis via enhancement of Src activity. To better understand how KAI1 is involved in the negative regulation of Src, we here examined the function of KAI1 in CDCP1-mediated Src kinase activation and the consequences of this process, focusing on HIF-1 α and VEGF expression.MethodsWe used the human prostate cancer cell line PC3 which was devoid of KAI1 expression. Vector-transfected cells (PC3-GFP clone #8) and KAI1-expressing PC3 clones (PC3-KAI1 clone #5 and #6) were picked after stable transfection with KAI1 cDNA and selection in 800 μg/ml G418. Protein levels were assessed by immunoblotting and VEGF reporter gene activity was measured by assaying luciferase activitiy. We followed tumor growth in vivo and immunohistochemistry was performed for detection of HIF-1, CDCP1, and VHL protein level.ResultsWe demonstrated that Hypoxia-inducible factor 1α (HIF-1α) and VEGF expression were significantly inhibited by restoration of KAI1 in PC3 cells. In response to KAI1 expression, CDCP1-enhanced Src activation was down-regulated and the level of von Hippel-Lindau (VHL) protein was significantly increased. In an in vivo xenograft model, KAI1 inhibited the expression of CDCP1 and HIF-1α.ConclusionsThese novel observations may indicate that KAI1 exerts profound metastasis-suppressor activity in the tumor malignancy process via inhibition of CDCP1-mediated Src activation, followed by VHL-induced HIF-1α degradation and, ultimately, decreased VEGF expression.

MiR-31 and its host gene lncRNA LOC554202 are regulated by promoter hypermethylation in triple-negative breast cancer

BackgroundmicroRNAs have been established as powerful regulators of gene expression in normal physiological as well as in pathological conditions, including cancer progression and metastasis. Recent studies have demonstrated a key role of miR-31 in the progression and metastasis of breast cancer. Downregulation of miR-31 enhances several steps of the invasion-metastasis cascade in breast cancer, i.e., local invasion, extravasation and survival in the circulation system, and metastatic colonization of distant sites. miR-31 exerts its metastasis-suppressor activity by targeting a cohort of pro-metastatic genes, including RhoA and WAVE3. The molecular mechanisms that lead to the loss of miR-31 and the activation of its pro-metastatic target genes during these specific steps of the invasion-metastasis cascade are however unknown.ResultsIn the present report, we identify promoter hypermethylation as one of the major mechanisms for silencing miR-31 in breast cancer, and in the triple-negative breast cancer (TNBC) cell lines of basal subtype, in particular. miR-31 maps to the intronic sequence of a novel long non-coding (lnc)RNA, LOC554202 and the regulation of its transcriptional activity is under control of LOC554202. Both miR-31 and the host gene LOC554202 are down-regulated in the TNBC cell lines of basal subtype and over-expressed in the luminal counterparts. Treatment of the TNBC cell lines with either a de-methylating agent alone or in combination with a de-acetylating agent resulted in a significant increase of both miR-31 and its host gene, suggesting an epigenetic mechanism for the silencing of these two genes by promoter hypermethylation. Finally, both methylation-specific PCR and sequencing of bisulfite-converted DNA demonstrated that the LOC554202 promoter-associated CpG island is heavily methylated in the TNBC cell lines and hypomethylated in the luminal subtypes.ConclusionLoss of miR-31 expression in TNBC cell lines is attributed to hypermethylation of its promoter-associated CpG island. Together, our results provide the initial evidence for a mechanism by which miR-31, an important determinant of the invasion metastasis cascade, is regulated in breast cancer.

Abstract A3: miR-31 and its host gene lncRNA LOC554202 are regulated by promoter hypermethylation in triple-negative breast cancer

Abstract Background: microRNAs have been established as powerful regulators of gene expression in normal physiological as well as in pathological conditions, including cancer progression and metastasis. Recent studies have demonstrated a key role of miR-31 in the progression and metastasis of breast cancer. Downregulation of miR-31 enhances several steps of the invasion-metastasis cascade in breast cancer, i.e., local invasion, extravasation and survival in the circulation system, and metastatic colonization of distant sites. miR-31 exerts its metastasis-suppressor activity by targeting a cohort of pro-metastatic genes, including RhoA and WAVE3. The molecular mechanisms that lead to the loss of miR-31 and the activation of its pro-metastatic target genes during these specific steps of the invasion-metastasis cascade are however unknown. Results: In the present report, we identify promoter hypermethylation as one of the major mechanisms for silencing miR-31 in breast cancer, and in the triple-negative breast cancer (TNBC) cell lines of basal subtype, in particular. miR-31 maps to the intronic sequence of a novel long non-coding (lnc)RNA, LOC554202 and the regulation of its transcriptional activity is under control of LOC554202. Both miR-31 and the host gene LOC554202 are down-regulated in the TNBC cell lines of basal subtype and over-expressed in the luminal counterparts. Treatment of the TNBC cell lines with either a de-methylating agent alone or in combination with a de-acetylating agent resulted in a significant increase of both miR-31 and its host gene, suggesting an epigenetic mechanism for the silencing of these two genes by promoter hypermethylation. Finally, both methylation-specific PCR and sequencing of bisulfite-converted DNA demonstrated that the LOC554202 promoter-associated CpG island is heavily methylated in the TNBC cell lines and hypomethylated in the luminal subtypes. Conclusion: Loss of miR-31 expression in TNBC cell lines is attributed to hypermethylation of its promoter-associated CpG island. Together, our results provide the initial evidence for a mechanism by which miR-31, an important determinant of the invasion metastasis cascade, is regulated in breast cancer. Citation Format: Katarzyna Augoff, Brian McCue, Edward F. Plow, Khalid Sossey-Alaoui. miR-31 and its host gene lncRNA LOC554202 are regulated by promoter hypermethylation in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A3.