nm23-H1 Expression Research Articles

NSUN6 Regulates NM23-H1 Expression in an m5C Manner to Affect Epithelial-Mesenchymal Transition in Lung Cancer

Purpose: The expression and regulatory mechanism of NSUN6 in lung cancer are still unclear. Our study explored whether NSUN6 mediates progression of lung cancer by affecting NM23-H1 expression in an m5C-dependent manner. Methods: qRT-PCR, CCK-8, colony formation, transwell, and Western blot analysis were employed to probe the impact of NSUN6 on lung cancer cell proliferation, migration, and epithelial-mesenchymal transition (EMT). RMVar database was utilized to forecast the downstream genes of NSUN6. The mode of interaction between NSUN6 and NM23-H1 was determined by dot blot, luciferase assay, m5C RIP, and cell function assays. The effect of NSUN6 expression on tumor growth was verified in vivo. Results: Expression of NSUN6 was reduced in lung cancer cells, and over-expression of NSUN6 restricted the proliferation of lung cancer cells, migration, and EMT. NSUN6 regulated NM23-H1 expression by modifying the 3′-UTR of NM23-H1 mRNA through m5C and inhibited lung cancer cell proliferation, migration, and EMT. In vivo experiments also showed that over-expression of NSUN6 inhibited the occurrence of lung cancer. Conclusion: NSUN6 regulates NM23-H1 expression in an m5C-dependent manner to affect EMT in lung cancer. Thus, NSUN6 may be considered as a potential therapeutic target for lung cancer.

Heterochiral dipeptide d-phenylalanyl- l-phenylalanine (H-D Phe-L Phe-OH) as a potential inducer of metastatic suppressor NM23H1 in p53 wild-type and mutant cells.

In recent years, significant progress has been made to the use-case of small peptides because of their diversified edifice and hence their versatile application scope in cancer therapy. Here we identify the heterochiral dipeptide H-D Phe-L Phe-OH (F1) as a potent inducer of the metastatic suppressor NM23H1. We divulge the effect of F1 on the major EMT/metastasis-associated genes and the implications on the invasion and migration ability of cancer cells. The anti-invasive potential of F1 was directly correlated with NM23H1 expression. Mechanistically, F1 treatment elevated p53 levels as validated by localization and transcriptional studies. In the NM23H1 knockdown condition, F1 failed to induce any p53 expression/nuclear localization, indicating that the upregulation in p53 expression by F1 is NM23H1 dependent. We also demonstrate how the antimetastatic potential of F1 is primarily mediated through NM23H1 irrespective of the p53 status of the cell. However, both NM23H1 and a functional p53 protein in conjunction govern the apoptotic and cytostatic potential of F1. Coimmunoprecipitation studies unraveled the augmentation of the p53 and NM23H1 interaction in p53 wild-type cells. However, in p53 mutated cells, no such enrichment was evidenced. We employed mouse isogenic cell lines (4T-1 and 4T-1 p53) to determine the in vivo efficacy of F1 (spontaneous and experimental models). Decreased tumor volume in the cohort injected with 4T-1 p53 cells demonstrated that while the antimetastatic potential of F1 was reliant on NM23H1, p53 activation was required for ablation of primary tumor burden. Our findings unravel that F1 treatment induces significant abrogation of the migration, invasion and metastatic potential of both p53 wild-type and p53 deficient cancers mediated through NM23H1.

Prognostic and clinicopathological significance of nm23-H1 expression in non-small cell lung cancer: A meta-analysis.

Background:The relationship between the expression of nm23-H1 and the invasion and prognosis of non-small cell lung cancer (NSCLC) is still controversial. Therefore, we conducted a meta-analysis to determine the prognostic value of nm23-H1 in patients with NSCLC. And to explore the relationship between the expression of nm23-H1 and clinicopathological features in patients with NSCLC.Methods:Literature search in PubMed, EMBASE, Cochrane Library, CNKI, and WanFang database was performed up to June 14, 2021. Studies on the expression and clinical significance of nm23-H1 in NSCLC were included. According to the inclusion and exclusion criteria, 2 researchers independently screened the literatures, extracted the data, and evaluated the quality. Meta-analysis was performed using RevMan 5.4 software (Nordic Cochran Centre, Copenhagen, Denmark).Results:Twenty-five studies met our inclusion criteria and were finally included for the analysis, involving 2198 participants. Our meta-analysis revealed that nm23-H1 expression was associated with tumor differentiation (OR = 0.54, 95% CI: 0.42–0.70, P < .00001), TNM stage (OR = 1.70, 95% CI: 1.23–2.34, P = .001), and lymph node status (OR = 0.26, 95% CI, 0.17–0.39, P < .00001), but have no associate with sex, age, pathological type, and T stages. Additionally, low nm23-H1 expression reduced the 3-year survival rate (OR = 2.74, 95% CI: 1.54–4.86, P = .0006) and 5-year survival rate (OR = 2.78, 95% CI: 1.36–5.69, P = .005).Conclusion:Nm23-H1 can be used as a biomarker to predict tumor invasiveness and evaluate the prognosis of patients with NSCLC.

Nm23-H1 induces apoptosis in primary effusion lymphoma cells via inhibition of NF-κB signaling through interaction with oncogenic latent protein vFLIP K13 of Kaposi's sarcoma-associated herpes virus.

Primary effusion lymphoma (PEL) is an aggressive form of non-Hodgkin lymphoma of B cells caused by Kaposi's Sarcoma-associated Herpes Virus (KSHV). KSHV encoded latent and lytic antigens promote oncogenic transformation and evade apoptosis through the modulation of various host cellular signaling pathways. Nm23-H1 is a known metastatic suppressor whose expression inversely correlates with the metastatic potential of different cancers. Here, we set out to assess the role of Nm23-H1 in PEL development. Flow cytometry and real-time PCR assays were performed for Nm23-H1 expression analysis. Induction of apoptosis was assessed using Western blotting and flow cytometry-based assays in Nm23-H1 overexpressing cells. Co-immunoprecipitation assays, confocal microscopy and imaging flow cytometry were performed to determine Nm23-H1 and vFLIP K13 protein-protein interaction. A PEL cell-induced xenograft model was established in non-obese diabetic/severely combined immunodeficient (NOD/SCID) mice to validate the effect of Nm23-H1 overexpression. We found that Nm23-H1 expression was significantly downregulated both at transcriptional and protein levels in PEL cell lines and that its overexpression triggered mitochondrial-mediated caspase-dependent apoptosis. We revealed Nm23-H1 interacts with the latent protein vFLIP K13 and that Nm23-H1 overexpression leads to inhibition of vFLIP K13 driven nuclear factor-kappa B (NF-κB) signaling with concurrent inhibition of autocrine and paracrine growth factors and downregulation of latent KSHV antigens without induction of active lytic reactivation. We also confirmed the effects of Nm23-H1 overexpression in a PEL cell-induced xenograft model in NOD/SCID mice. Downregulation of Nm23-H1 expression in KSHV-infected PEL cells and its overexpression trigger apoptosis by impairing vFLIP K13-driven NF-κB signaling, suggesting therapeutic implications of Nm23-H1 for primary effusion lymphomas.

Spontaneous Cell Detachment and Reattachment in Cancer Cell Lines: An In Vitro Model of Metastasis and Malignancy.

There is an unmet need for simplified in vitro models of malignancy and metastasis that facilitate fast, affordable and scalable gene and compound analysis. “Adherent” cancer cell lines frequently release “free-floating” cells into suspension that are viable and can reattach. This, in a simplistic way, mimics the metastatic process. We compared the gene expression profiles of naturally co-existing populations of floating and adherent cells in SW620 (colon), C33a (cervix) and HeLa (cervix) cancer cells. We found that 1227, 1367 and 1333 genes were at least 2-fold differentially expressed in the respective cell lines, of which 122 were shared among the three cell lines. As proof of principle, we focused on the anti-metastatic gene NM23-H1, which was downregulated both at the RNA and protein level in the floating cell populations of all three cell lines. Knockdown of NM23-H1 significantly increased the number of floating (and viable) cells, whereas overexpression of NM23-H1 significantly reduced the proportion of floating cells. Other potential regulators of these cellular states were identified through pathway analysis, including hypoxia, mTOR (mechanistic target of rapamycin), cell adhesion and cell polarity signal transduction pathways. Hypoxia, a condition linked to malignancy and metastasis, reduced NM23-H1 expression and significantly increased the number of free-floating cells. Inhibition of mTOR or Rho-associated protein kinase (ROCK) significantly increased cell death specifically in the floating and not the adherent cell population. In conclusion, our study suggests that dynamic subpopulations of free-floating and adherent cells is a useful model to screen and identify genes, drugs and pathways that regulate the process of cancer metastasis, such as cell detachment and anoikis.

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.

NM23-H1 Expression of Head and Neck Squamous Cell Carcinoma in Association With the Response to Irradiation.

A low NM23-H1 expression in head and neck squamous cell carcinoma (HNSCC) was found to be associated with poor clinical outcome. Therefore, we investigated the role of NM23-H1 in the susceptibility of HNSCC cells to irradiation and its clinical significance. An in vitro study was also conducted to validate the results. Furthermore, we used immunohistochemistry to analyze NM23-H1 expression found in specimens of 50 HNSCC patients with cervical metastases receiving postoperative radiotherapy. Low tumor NM23-H1 expression was associated with locoregional recurrence of HNSCC (p=0.040; Hazard ratio=5.62) and poor clinical outcome (p=0.001; Hazard ratio=4.90). To confirm the effect of NM23-H1 on radiation-induced cytotoxicity, we generated several stable clones derived from a human HNSCC cell line (SAS) using knockdown and overexpression of NM23-H1. Knockdown of NM23-H1 decreased the radio-sensitivity of SAS cells, possibly associated with a decrease in the radiation-induced G2/M-phase accumulation and upregulation of cyclin B1. On the contrary, overexpression of NM23-H1 can reverse the aforementioned adverse results. Consequently, we suggest that NM23-H1 expression may be considered as a potential therapeutic treatment option for HNSCC patients.

CTCF and EGR1 suppress breast cancer cell migration through transcriptional control of Nm23-H1

Tumor metastasis remains an obstacle in cancer treatment and is responsible for most cancer-related deaths. Nm23-H1 is one of the first metastasis suppressor proteins discovered with the ability to inhibit metastasis of many cancers including breast, colon, and liver cancer. Although loss of Nm23-H1 is observed in aggressive cancers and correlated with metastatic potential, little is known regarding the mechanisms that regulate its cellular level. Here, we examined the mechanisms that control Nm23-H1 expression in breast cancer cells. Initial studies in aggressive MDA-MB-231 cells (expressing low Nm23-H1) and less invasive MCF-7 cells (expressing high Nm23-H1) revealed that mRNA levels correlated with protein expression, suggesting that transcriptional mechanisms may control Nm23-H1 expression. Truncational analysis of the Nm23-H1 promoter revealed a proximal and minimal promoter that harbor putative binding sites for transcription factors including CTCF and EGR1. CTCF and EGR1 induced Nm23-H1 expression and reduced cell migration of MDA-MB-231 cells. Moreover, CTCF and EGR1 were recruited to the Nm23-H1 promoter in MCF-7 cells and their expression correlated with Nm23-H1 levels. This study indicates that loss of Nm23-H1 in aggressive breast cancer is apparently caused by downregulation of CTCF and EGR1, which potentially drive Nm23-H1 expression to promote a less invasive phenotype.

Anticancer Effects of Valproic Acid via Regulation of Epigenetic Mechanisms in Non-small-cell Lung Cancer A549 Cell Line.

Epigenetic mechanisms are the most important factors contributing to both the development and metastasis of cancer cells. We aimed to scrutinize the role of epigenetic alternations of genes involved in cancer metastasis, including CD44v6 (metastasis indicator) and Nm23-H1 (a novel tumor suppressor), in the A549 lung cancer cell line. The A549 cells were cultured in the DMEM medium. Valproic acid (VPA) was used as a histone deacetylase inhibitor. Caspase-3 activity was assessed by adding DEVD-pNA substrate to the cell lysate. Gene expression was determined by real-time PCR. Finally, protein expression was assessed by western blot. The results showed that VA significantly decreased the expression of the CD44v6 gene and its protein level. This was further accompanied by lower expressions of MMP-2 and MMP-9 genes. On the other hand, the expression of Nm23-H1 and its protein were significantly increased in the cells accompanied by higher activity of caspase-3 (P ˂ 0.05). Our results showed that epigenetic regulation of CD44v6, Nm23-H1, MMP-2, and MMP-9 might be involved in the pathogenesis and metastasis of lung cancer. Therefore, the use of histone deacetylase inhibitors can be effective in the suppression of metastases and the treatment of these tumors.

Mutations at the dimer interface and surface residues of Nm23-H1 metastasis suppressor affect its expression and function.

The Nm23 metastasis suppressor family is involved in a variety of physiological and pathological processes including cell proliferation, differentiation, tumorigenesis, and metastasis. Given that Nm23 proteins may function as hexamers composed of different members of the family, especially Nm23-H1 and H2 isoforms, it is pertinent to assess the importance of interface and surface residues in defining the functional characteristics of Nm23 proteins. Using molecular modeling to identify clusters of residues that may affect dimer formation and isoform specificity, mutants of Nm23-H1 were constructed and assayed for their ability to modulate cell migration. Mutations of dimer interface residues Gly22 and Lys39 affected the expression level of Nm23-H1, without altering the transcript level. The reduced protein expression was not due to increased protein degradation or altered subcellular distribution. Substitution of the surface residues of Nm23-H1 with Nm23-H2-specific Ser131 and/or Lys124/135 affected the electrophoretic mobility of the protein. Moreover, in cell migration assays, several mutants with altered surface residues exhibited impaired ability to suppress the mobility of MDA-MB-231 cells. Collectively, the study suggests that disrupting the dimer interface may affect the expression of Nm23-H1, while the residues at α-helix and β-sheet on the surface of Nm23-H1 may contribute to its metastasis suppressive function.

Biological tumor markers (maspin, CD105, nm23-H1) and disease relapse in laryngeal cancer: cluster analysis.

The aim of this investigation was to see if a panel of biomarkers (maspin, CD105, and nm23-H1) could be used to stratify patients with laryngeal squamous cell carcinoma (LSCC) in homogeneous disease recurrence risk clusters. Cluster analysis was used to classify 89 patients based on their immunohistochemical expression of nm23-H1, CD105, and maspin. Our analysis identified seven homogeneous clusters: the LSCC recurrence rate was lowest in cluster 6 (non-nuclear maspin pattern, nuclear nm23-H1 expression ≥10%, endothelial CD105 expression <6%; P = .009), and highest in cluster 3 (non-nuclear maspin pattern, nuclear nm23-H1 expression <10%, endothelial CD105 expression ≥6%; P <.001). Similar panels of biological variables identified by cluster analysis should be tested in prospective clinical trials to establish whether treating patients identified as being at higher risk of LSCC recurrence more aggressively could significantly improve their recurrence rate and/or disease-specific survival.

Association between Endoglin and nm23-H1 Expression and the Recurrence in Elderly Patients with Laryngeal Squamous Cell Carcinoma

To explore the association between endoglin and nm23-H1 expression and the recurrence in elderly patients with laryngeal squamous cell carcinoma. Patients diagnosed with laryngeal squamous cell carcinoma in The First Affiliated Hospital of Soochow University Suzhou from January 2012 to April 2015 were selected as the subjects, all patients underwent surgical resection and followed up for 5 y, and disease recurrence was chosen as the experimental endpoint. The basic clinical data of all patients were collected, immunohistochemical methods was used to evaluate the expression of endoglin and nm23-H1. According to disease recurrence patients were divided into relapse group and non-relapse group, the differences between the two groups were compared. The independent related factors affecting the relapse of patients were investigated by Logistic regression analysis. 32 patients relapsed with a recurrence rate of 31.37 %. There were significant differences in T stage, clinical stage, and pathological grade between the two groups. The positive expression rate of endoglin in the relapse group was significantly higher than that in the non-relapse group ((6.51±0.64) % vs. (5.63±0.57) %, p<0.001); the positive expression rate of nm23-H1 was significantly lower than that in the non-relapse group ((9.71±0.84) % vs. (10.52±0.78) %). Multivariate logistic regression analysis showed that nm23-H1 and endoglin were independent related factors that affected the recurrence of the disease. Endoglin expression ≥5.91%, nm23-H1 expression <10.27 % are independent risk factors that affect patients’ relapse.

Diffuse Large B Cell Lymphoma (DLBCL)-Released NM23-H1 Promotes Monocyte Survival and Inflammatory Cytokine Release: A Mechanistic Link between the Dual Impacts of NM23-H1 Expression and Reduced Lymphocyte:Monocyte Ratio in DLBCL Prognosis

Elevated circulating levels of NM23-H1 have been associated with poor prognosis in haematological malignancies including AML and non-Hodgkin's lymphomas. In diffuse large B-cell lymphoma (DLBCL), several studies have demonstrated that both elevated circulating plasma levels and intra-tumoural levels of NM23-H1 correlate with poorer prognosis. These observations bring into question whether the mechanistic link of NM23-H1 expression with DLBCL prognosis depends an intrinsic intracellular mechanisms or is mediated by release of NM23-H1. We have shown that DLBCL lymphoma cell lines (Farage, HT, OCI-LY1, OCI-LY3, OCI-LY7, SUDHL4, SUDHL5 SUDHL6, U2932) express both NM23-H1 protein and the highly related protein NM23-H2. We also demonstrated that DLBCL cell lines release significant levels of NM23-H1 into their extracellular environment but not NM23-H2. Release of NM23-H1 was highly variable between cell lines with some releasing very high levels and some releasing much lower levels. Thus, DLBCL cell lines represent appropriate models to investigate the role of high and low levels of NM23-H1 on prognosis. The DLBCL cell lines HT and OCI-LY1 where selected for further study as representative of high and low level releasers of NM23-H1, respectively. CRISPR-Cas9 knockout of NM23-H1 (KO-NM23-H1) in HT and OCI-LY1 DLBCL cells had no impact on cell growth, viability or immuno-phenotype either in normoxic or hypoxic cultures. Thus NM23-H1 appears to not be required intrinsically by DLBCL cell lines. We therefore considered that the link between higher expression and release of NM23-H1 with prognosis is mediated via tumour-host environment interactions. We used an NSG mouse model transplanted subcutaneously with 1x106 CRISPR control (CTRL)-HT, CTRL-OCIL-Y1, KO-NM23-H1-HT or KO-NM23-H1-OCI-LY1 cell lines. We did not observe significant differences in tumour growth between CTRL and KO-NM23-H1 cells in the low NM23-H1 expressing OCI-LY1 DLBCL cell line. However, the high-expressing KO-NM23-H1-HT cells had significantly slower tumour progression and increased host survival when compared to CTRL-HT cells. We interpret to indicate that NM23-H1 release above a certain threshold provides a tumour growth advantage. Reduced lymphocyte:monocyte ratios (LMR) are also associated with poor prognosis in DLBCL. To investigate a potential functional link between NM23-H1 release and monocyte behaviour we first exposed peripheral blood leucocytes to Alexa 647 labelled fluorescent recombinant-NM23-H1 and found that monocytes but not neutrophils, T-cells or B-cells bound NM23-H1. We also observed that monocyte viability and survival were elevated in serum free cultures when supplemented with rNM23-H1, an observation that might indicate that elevated circulating NM23-H1 and reduced LMR in poor prognosis DLBCL may be mechanistically linked. We next co- cultured CTRL-HT or koNM23-H1-HT in a 1:1 ratios with purified monocytes from healthy human donors. Principle component analyses of 27 cytokines simultaneously measured by luminex assays identified that IL-1β, IL-6, IL-8, MIP-1α, MIP-1β and TNF-α were elevated when monocytes were co-cultured with CTRL-HT cells compared to co-culture with koNm23-H1-HT cells. In contrast, IP-10 was found elevated in the co-culture with koNm23-H1-HT cells. These observations indicate potentially important cross talk between the malignant DLBCL cells and innate immune cells of the host. Consistent with this, IL-6 and IL-8 serum levels have been shown to be elevated in pretreatment DLBCL patients compared to control subjects and MIP-1α, IL-6, and IL-8 serum levels have a greater association with DLBCL than follicular lymphoma. In conclusion ours is the first study to investigate potential mechanistic links between the associations of elevated NM23-H1 and poor prognosis in DLBCL and indicate a role for cross-talk between tumour cells and innate immune cells. Our data also indicate that NM23-H1 release from DLBCL cells may be a driving component in driving reduced LMRs that are also associated with poor prognosis. Whether reduced LMR and NM23-H1 release are casually related or not, the possibility that patients with both elevated monocytes and elevated circulating NM23-H1 levels are likely to represent a group with particularly poor prognosis requires urgent investigation. Disclosures Drayson: Abingdon Health: Consultancy, Equity Ownership. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.

Correlation of p16 and nm23-H1 expression levels with incidence and prognosis of soft tissue sarcoma.

The expression levels of p16 and nm23-H1 genes in soft tissue sarcoma (STS) were evaluated to investigate correlation of the expression levels with the incidence and prognosis of STS. Tumor tissues and para-carcinoma normal tissues were collected from 64 STS patients. The messenger ribonucleic acid (mRNA) expression levels of p16 and nm23-H1 in the tissues were detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the protein expression levels of p16 and nm23-H1 in tissues were detected using immunohistochemistry. Spearman's correlation analysis was used for the correlation between expression levels of p16 and nm23-H1 in STS tissues and the correlation between p16 and nm23-H1 mRNA and protein expression. Moreover, the correlation of p16 and nm23-H1 expression levels in tumor tissues with pathological parameters and prognosis of STS patients were analyzed combined with clinical data. Results of RT-qPCR showed that mRNA expression levels of p16 and nm23-H1 in tumor tissues of STS patients were significantly lower than those in para-carcinoma normal tissues (P<0.01). Results of immunohistochemistry showed that the positive expression rates of p16 and nm23-H1 in tumor tissues of STS patients (43.75 and 39.06% respectively) were significantly lower than those in para-carcinoma normal tissues (85.93 and 89.06% respectively). The expression of p16 and nm23-H1 mRNA was positively correlated with protein expression levels. There was a positive correlation between the expression levels of p16 and nm23-H1 in tumor tissues of STS patients. The negative expression of p16 in tumor tissues of STS patients correlated with tumor size, tumor metastasis and clinical staging, and the negative expression of nm23-H1 correlated with tumor metastasis and clinical staging. The overall 5-year survival rate of patients was 54.68%, and the prognosis of patients with positive expression levels of p16 and nm23-H1 was better. Univariate survival analyses revealed that p16 and nm23-H1 were influencing factors of the overall survival rate of STS patients. p16 and nm23-H1 expression in STS is low, and their expression levels are closely related to the pathological parameters and prognosis of STS patients, so they can serve as reference indexes for prognosis estimation of STS.

Nm23-H1 inhibits hypoxia induced epithelial-mesenchymal transition and stemness in non-small cell lung cancer cells.

The Nm23 gene has been acknowledged to play a crucial role in lung cancer metastasis inhibitory cascades controlled by multiple factors. Low expression or allelic deletion of nm23-H1 is strongly linked to widespread metastasis and poor differentiation of non-small cell lung cancer (NSCLC). In this study, nm23-H1 was down regulated in epithelial-mesenchymal transition (EMT) and stemness enhancement under cobalt chloride (CoCl2)-induced hypoxia in NSCLC cells. Moreover, knocking down of nm23-H1 by shRNA apparently promoted hypoxia induced EMT and stemness, which was entirely suppressed via over expression of nm23-H1. Mechanistically, the Wnt/β-catenin signaling pathway was found to participate in the nm23-H1-mediated process. Besides, XAV939 prohibited cell EMT and stemness which could be impaired by knocking down of nm23-H1, while stable transfection of nm23-H1 attenuated hypoxia phonotype induced by lithium chloride (LiCl). Generally, our experiment provided evidence that nm23-H1 can reverse hypoxia induced EMT and stemness through the inhibition of the Wnt/β-catenin pathway, which may furnish a deeper perspective into the better treatment or prognosis for NSCLC.

Nuclear expression of onco-suppressors nm23-H1 and maspin are associated with lower recurrence rate in laryngeal carcinoma.

The main aim of the study was to preliminarily investigate the possibly related role of nuclear onco-suppressors maspin and nm23-H1, a metastasis suppressor, in laryngeal squamous cell carcinoma (LSCC). Maspin expression pattern and nuclear nm23-H1 expression were ascertained in 62 consecutive LSCCs. Recurrence rate was significantly lower in patients with a nuclear maspin pattern of expression; nuclear nm23-H1 expression was significantly lower in patients who experienced disease recurrence. Disease free survival (DFS) was significantly longer in patients with maspin nuclear pattern or with nuclear nm23-H1 expression ≥10%. A significant association was found between nuclear nm23-H1 expression and maspin pattern of expression in LSCC. KNN discriminant analysis considered N status, maspin sub-cellular localization and nuclear nm23-H1 expression. The selected variables' accuracy in terms of relapse was 82%. Positive predictive accuracy was 100%, and negative predictive accuracy 79%. Nuclear nm23-H1 expression and maspin pattern, also in association, show promise as recurrence indicators in LSCC. Further studies are needed to shed more light on the nm23-H1 mechanism of action in LSCC and thus find ways to restore nm23-H1 loss. These preliminary findings suggest that re-activating maspin functions might represent an important goal in the treatment of advanced LSCC.

3181 - Diffuse Large B-Cell Lymphoma Cells Influence Cytokine Secretion by Monocytes via Release of Nm23-H1

High circulating levels of the nucleoside diphosphate kinase (NDPK) Nm23-H1 are associated with worse prognosis in both Acute Myeloid Leukaemia and high grade lymphomas including high grade DLBCL. In DLBCL, the relationship with poorer prognosis is further associated with elevated intracellular Nm23-H1 as revealed by immunohistology; raising the possibility that Nm23-H1 may affect outcome by ether cell intrinsic and/or extrinsic mechanisms. We analysed a panel of DLBCL cell lines (Farage, HT, OCI-LY1, OCI-LY3, OCI-LY7, SUDHL4, SUDHL5 SUDHL6, U2932) for expression and secretion of Nm23-H1 and the closely related NDPK Nm23-H2. Western blotting of cell lysates revealed that all cell lines expressed both Nm23-H1 and Nm23-H2 at equivalence and at similar levels as seen in the AML CD34+ cell line KG1a. Analysis of DLBCL conditioned medium revealed high levels of extracellular Nm23-H1, but little if any Nm23-H2. In contrast, KG1a secreted neither Nm23-H1 nor Nm23-H2. KG1a cells bind and take up exogenous Nm23-H1 however, exposure of DLBCL cell lines to exogenous fluorescent recombinant Nm23-H1 (rNm23-H1) did not result in increased fluorescence by flow cytometry or microscopy suggesting that DLBCL cells do not significantly bind Nm23-H1. Furthermore CRISPR-Cas9 knockout of Nm23-H1 in HT DLBCL cells had no impact on cell growth, viability or immuno-phenotype. Thus it appears that expression and release of Nm23-H1 is not directly necessary for DLBCL cells, suggesting that the impact of Nm23-H1 on prognosis may be mediated by cell extrinsic mechanisms affecting the tumour microenvironment. To test this hypothesis we purified monocytes from normal donors and co-cultured them with wild type (WT) or Nm23-H1 knock-out (KO) HT cells for 48 hours. Of 27 cytokines analysed from the conditioned media IL-1, IL-6, IL-8, MIP-1, and MIP-1 were elevated when monocytes were co-cultured with WT-HT cells compared to co-culture with Nm23-H1 KO-HT cells. Thus, we hypothesise that the association of elevated Nm23-H1 with poor prognosis in DLBCL may be mediated by crosstalk between tumour cells and monocyte/macrophages either in the immediate tumour environment or beyond.

Nm23-H1 is involved in the repair of ionizing radiation-induced DNA double-strand breaks in the A549 lung cancer cell line

BackgroundAlthough originally identified as a putative metastasis suppressor, increasing studies have confirmed a possible role for Nm23-H1 in DNA repair, through the base excision repair and nucleotide excision repair pathways. In this study, we explored whether Nm23-H1 was also involved in double-strand break repair (DSBR).Methods and resultsWe constructed a stable A549-shNm23-H1 cell line with doxycycline-regulated expression of Nm23-H1, and a A549-nNm23-H1 cell line that over expressed a nucleus-localized version of Nm23-H1. Results from both lines confirmed that Nm23-H1 participated in the repair of double-strand breaks induced by X-rays, using Comet and γ-H2AX foci assays. Subsequent studies showed that Nm23-H1 activated the phosphorylation of checkpoint-related proteins including ATM serine/threonine kinase (on S1981), tumor protein p53 (on S15), and checkpoint kinase 2 (Chk2) (on T68). We also detected interactions between Nm23-H1 and the MRE11-RAD50-NBS1 (MRN) complex, as well as Ku80. Moreover, NBS1 and Ku80 levels were comparably higher in Nm23-H1 overexpressing cells than in control cells (t = 14.462, p < 0.001 and t = 5.347, p = 0.006, respectively). As Ku80 is the keystone of the non-homologous end joining (NHEJ) pathway, we speculate that Nm23-H1 promotes DSBR through NHEJ.ConclusionsThe results indicate that Nm23-H1 participates in multiple steps of DSBR.

Nuclear nonmetastatic protein 23-H1 expression and epithelial-mesenchymal transition in laryngeal carcinoma: A pilot investigation.

In epithelial-to-mesenchymal transition, epithelial cells lose their features, acquiring a mesenchymal-like phenotype. Nm23-H1 protein relates to tumor cells' metastatic potential, its low expression in carcinomas often meaning a poor prognosis. This study newly investigated the role of nuclear nm23-H1 in laryngeal squamous cell carcinoma epithelial-to-mesenchymal transition. Immunohistochemical analyses of nuclear nm23-H1, E-cadherin, N-cadherin, Snail, Zinc finger E-box binding homeobox (ZEB)1, and ZEB2 were performed in 33 consecutive patients with laryngeal SCC. Mean nuclear nm23-H1 expression was lower in patients whose disease recurred (P = .0046). Disease-free survival (DFS) was longer for patients whose nuclear nm23-H1 expression was ≥10% (P = .0083). Nuclear nm23-H1 and E-cadherin expressions correlated directly (P = .018). Mean E-cadherin expression was lower in patients whose disease recurred (P = .03). The DFS was shorter in patients with ZEB2 expression ≥5% (P = .006). Nuclear nm23-H1 expression warrants further investigation in laryngeal SCC as a prognostic marker identifying patients at higher risk of recurrence. nm23-H1 targeted treatments may be capable of regulating epithelial-to-mesenchymal transition.

Coexistence of MACC1 and NM23-H1 dysregulation and tumor budding promise early prognostic evidence for recurrence risk of early-stage colon cancer.

The tumor-node-metastasis (TNM) classification, the presence of a mucinous component, and signet ring cells are well-known criteria for identifying patients at a high risk for recurrence and determining the therapeutic approach for early-stage colon cancer (eCC). Nevertheless, recurrence can unexpectedly occur in some eCC cases after surgical resection. The aims of the present study were to evaluate the relation of dysregulated MACC1, c-MET, and NM23-H1 expression with the histopathological features of tumors in recurrence formation in eCC cases. A total of 100 sporadic eCC patients without poor prognosis factors were evaluated in this study. The relationship between the altered expression of MACC1, c-MET, and NM23-H1 and pathological microenvironmental features, including the presence of tumor budding and desmoplasia, were assessed. The primary outcomes, including 5-year overall survival (OS) and disease-free survival (DFS), were also measured. Compared with nonrecurrent patients, the expression level of MACC1 was 8.27-fold higher, and NM23-H1 was 11.36-fold lower in patients with recurrence during the 5-year follow-up (p=0.0345 and p=0.0301, respectively). In addition, the coexistence of high MACC1 and low NM23-H1 expression and tumor budding was associated with short OS (p<0.001). We suggest that the combination of reduced NM23-H1, induced MACC1, and the presence of tumor budding are promising biomarkers for the prediction of recurrence and may aid the stratification of patients with stage II colon cancer for adjuvant chemotherapy.