nm23-H1基因表达与卵巢癌转移的相关性

背景与目的：转移是卵巢癌治疗失败及患者死亡的首要原因。然而,目前对卵巢癌转移潜能的分子机制知之不多。本研究旨在筛选高频转移卵巢恶性肿瘤细胞,分析卵巢癌高频转移细胞模型中nm23．H1基因表达与肿瘤转移特性的相关性,为系统实验研究和临床实践提供依据。方法：通过反复动物接种和体外培养,观察动物肺转移状况,筛选高频转移细胞株,比较原发肿瘤和转移肿瘤的特征,并应用Northem blot和Westem blot方法测定各类肿瘤细胞nm23 mRNA和蛋白表达水平。结果：8株卵巢恶性肿瘤细胞中,4株有较高转移潜能,多次培养接种可筛选出高频转移细胞亚群。各类细胞nm23mRNA和蛋白表达水平与肿瘤转移特性呈负相关(r=0．96,P=0．0001)。结论：由基因分子水平决定的肿瘤转移趋势在不同肿瘤种类及细胞亚群中有明显差异;卵巢癌中nm23 mRNA和蛋白的表达与其转移能力的降低有密切关系,可作为判定卵巢癌预后的敏感指标。     

Clinical-translational approaches to the Nm23-H1 metastasis suppressor

Nm23-H1 significantly reduces metastasis without effects on primary tumor size and was the first discovered metastasis suppressor gene. At least three mechanisms are thought to contribute to the metastasis-suppressive effect of Nm23-H1: (a) its histidine kinase activity toward ATP-citrate lyase, aldolase C, and the kinase suppressor of ras, with the last inactivating mitogen-activated protein kinase signaling; (b) binding proteins that titer out "free" Nm23-H1 and inhibit its ability to suppress metastasis; and (c) altered gene expression downstream of Nm23-H1, particularly an inverse association with the lysophosphatidic acid receptor endothelial differentiation gene-28 (EDG2). Most metastasis suppressor genes, including Nm23-H1, affect metastatic colonization, which is the outgrowth of tumor cells in distant locations; therefore, they are of high translational interest. A phase II trial is ongoing to test the hypothesis that a compound, high-dose medroxyprogesterone acetate (MPA), used as an unconventional gluocorticoid, will stimulate breast cancer cells to reexpress Nm23-H1 and limit subsequent metastatic colonization.     

P53 is a regulator of the metastasis suppressor gene Nm23-H1

p53, a tumor suppressor gene involved in the G1 cell cycle checkpoint, is also the most frequently mutated gene in human cancer. In addition, p53 modifies the ability of tumor cells to metastasize. The metastasis-associated gene Nm23-H1, which encodes an 18-kDa nucleoside diphosphate kinase, was previously identified in cells with low metastatic potential. Although p53 and Nm23-H1 proteins play an important part in regulating the progression of cancer, any functional relationship between these two proteins is currently unknown. Here we report an association between p53 levels and expression of the Nm23-H1 gene. Our data indicate that wild-type (wt) p53 upregulated the expression of Nm23-H1 at protein and mRNA levels in MCF-7 and J7B cells. This capacity of wt p53 to regulate expression of Nm23-H1 was not only dependent on the endogenous but also the exogenous origin of p53, and could not be reproduced with mutant p53. Subsequently, the invasive ability of MCF-7 and J7B cells was suppressed upon induction of the Nm23-H1 protein by p53. In contrast, increased levels of p53 downregulated the expression of Nm23-H1 at the protein and mRNA levels in RKO and H1299 cells and, as a consequence, increased the invasive ability of both cell types. Thus, our results implicated the differential regulation of Nm23-H1 by p53 in different cell types as an important component in the molecular mechanisms of tumor metastasis.     

The Nm23-H1 metastasis suppressor as a translational target

Nm23 was the first of what has become a field of over 20 known metastasis suppressor genes (MSGs). Since the discovery of Nm23 in 1988, a variety of mechanisms have been attributed to its activity, including a histidine kinase activity, binding of other proteins to regulate metastatic formation, and altered gene expression downstream of Nm23. Here, we will review current efforts to translate the previous work done on this MSG into the clinic, including high-dose medroxyprogesterone acetate (MPA), which has been shown to upregulate Nm23 expression. In addition, we will detail a new potential target downstream of Nm23. LPA1 is one of a group of known cell surface receptors for lysophosphatidic acid (LPA), which has been shown to be inversely correlated with Nm23 expression. A specific LPA1 antagonist could conceivably mimic the effects of Nm23 by downregulating the activity of the LPA1 pathway, which would be of considerable interest for potential clinical use.     

Nm23 and breast cancer metastasis

Summary The majority of breast cancer patients succumb to metastatic disease. We summarize published and recent research concerning thenm23 gene in breast cancer metastasis. In a murine developmental study, nm23 expression increased with the functional differentiation of the mammary gland in nulliparous and pregnant animals. In human breast cancer, five studies have now demonstrated a significant association between reduced nm23 expression, at the RNA or protein levels, and aggressive tumor behavior. Nm23-negative tumor cells have been observed in comedo ductal carcinoma in situ lesions in two independent studies, indicating that decreases innm23 expression begin prior to actual histologically identifiable invasion. Transfection studies, in which humannm23-H1 cDNA was expressed in the metastatic human MDA-MB-435 breast carcinoma cell line, indicate thatnm23-H1 suppresses in vivo metastatic potential by 50-90%. Finally, our data in melanoma and breast carcinoma transfection systems suggest that the biochemical mechanism of nm23 suppressive activity is likely not due to its nucleoside diphosphate kinase activity, association with GAP proteins, or secretion from cells.Supported by an educational grant from CIBA-GEIGY Corp., at the San Antonio Breast Cancer Symposium, Dec. 8, 1992.     

Insights into the biology and prevention of tumor metastasis provided by the Nm23 metastasis suppressor gene

Metastatic disease is the major cause of death among cancer patients. A class of genes, named metastasis suppressors, has been described to specifically regulate the metastatic process. The metastasis suppressor genes are downregulated in the metastatic lesion compared to the primary tumor. In this review, we describe the body of research surrounding the first metastasis suppressor identified, Nm23. Nm23 overexpression in aggressive cancer cell lines reduced their metastatic potential in vivo with no significant reduction in primary tumor size. A complex mechanism of anti-metastatic action is unfolding involving several known Nm23 enzymatic activities (nucleotide diphosphate kinase, histidine kinase, and 3′–5′ exonuclease), protein–protein interactions, and downstream gene regulation properties. Translational approaches involving Nm23 have progressed to the clinic. The upregulation of Nm23 expression by medroxyprogesterone acetate has been tested in a phase II trial. Other approaches with significant preclinical success include gene therapy using traditional or nanoparticle delivery, and cell permeable Nm23 protein. Recently, based on the inverse correlation of Nm23 and LPA1 expression, a LPA1 inhibitor has been shown to both inhibit metastasis and induce metastatic dormancy.     

Downstream targets of Nm23-H1: gene expression profiling of CAL 27 cells using DNA microarray

The human nm23-H1 was discovered as a tumor metastasis suppressor based on its reduced expression in melanoma cell lines with low versus high metastatic potential. It encodes for one of two subunits of the nucleoside-diphosphate kinase. Besides its role in the maintenance of the cells NTP pool, nm23 plays a key role in different cellular processes. The role of nm23-H1 in these processes still has to be elucidated. Our goal was to identify Nm23-H1 downstream targets by subjecting Nm23-H1 overexpressing CAL 27 cells oral squamous cell carcinoma (OSSC) to microarray analysis. The genes with changed expression patterns could be clustered into several groups: transforming growth factor beta (TGFbeta) signaling pathway, cell adhesion, invasion and motility, proteasome machinery, cell-cycle, epithelial structural and related molecules and others. Based on the expression patterns observed we presume that nm23-H1 might have a role in OSSCs, which should be confirmed by future experiments.     

Nm23-H1 suppresses tumor cell motility by down-regulating the lysophosphatidic acid receptor EDG2

Exogenous overexpression of the metastasis suppressor gene Nm23-H1 reduces the metastatic potential of multiple types of cancer cells and suppresses in vitro tumor cell motility and invasion. Mutational analysis of Nm23-H1 revealed that substitution mutants P96S and S120G did not inhibit motility and invasion. To elucidate the molecular mechanism of Nm23-H1 motility suppression, expression microarray analysis of an MDA-MB-435 cancer cell line overexpressing wild-type Nm23-H1 was done and cross-compared with expression profiles from lines expressing the P96S and S120G mutants. Nine genes, MET, PTN, SMO, FZD1, L1CAM, MMP2, NETO2, CTGF, and EDG2, were down-regulated by wild-type but not by mutant Nm23-H1 expression. Reduced expression of these genes coincident with elevated Nm23-H1 expression was observed in human breast tumor cohorts, a panel of breast carcinoma cell lines, and hepatocellular carcinomas from control versus Nm23-M1 knockout mice. The functional significance of the down-regulated genes was assessed by transfection and in vitro motility assays. Only EDG2 overexpression significantly restored motility to Nm23-H1-suppressed cancer cells, enhancing motility by 60-fold in these cells. In addition, silencing EDG2 expression with small interfering RNA reduced the motile phenotype of metastatic breast cancer cells. These data suggest that Nm23-H1 suppresses metastasis, at least in part, through down-regulation of EDG2 expression.     

乳腺癌转移抑制基因1抑制肿瘤转移的机制

乳腺癌转移抑制基因1(BRMS1)具有抑制肿瘤细胞转移的能力,可明显减少转移灶的发生,但不影响肿瘤的生长.其作用机制复杂,与细胞间通讯、磷酸肌醇信号转导以及与转录核因子-KB(NF-KB)的相互作用等有关.故探讨BRMS1抑制肿瘤转移的机制,希望用于肿瘤基因治疗.     

鼻咽癌移植瘤Annexin A3和Nm23-H1蛋白表达与放射敏感相关性的研究

目的:探讨不同放射敏感性鼻咽癌移植瘤中Annexin A3和Nm23-H1蛋白的表达及意义。方法:将不同放射敏感性鼻咽癌细胞CNE-2R和CNE-2分别注射到裸鼠后肢皮下成瘤。成瘤后以X射线分次照射16Gy,计算肿瘤体积及肿瘤倍增时间。采用免疫组化法检测移植瘤中Annexin A3和Nm23-H1蛋白的表达水平。结果:未受照射CNE-2R和CNE-2移植瘤倍增时间分别为4.8和3.9d,受照射CNE-2R和CNE-2移植瘤倍增时间分别为6.2和17.1d。未受照射CNE-2R移植瘤组织Annexin A3蛋白表达明显低于未受照射CNE-2移植瘤组织,MOD值分别为0.035±0.012和0.062±0.009,P=0.000;受照射CNE-2R移植瘤组织明显低于受照射CNE-2移植瘤组织,MOD值分别为0.029±0.007和0.051±0.009,P=0.000。未受照射CNE-2R移植瘤组织Nm23-H1蛋白明显高于未受照射CNE-2移植瘤组织,MOD值分别为0.056±0.007和0.043±0.007,P=0.022;受照射CNE-2R移植瘤组织明显高于受照射CNE-2移植瘤组织,MOD值分别为0.079±0.009和0.046±0.007,P=0.000。结论:CNE-2R移植瘤组织Annexin A3蛋白明显低于CNE-2移植瘤组织,受照射CNE-2R移植瘤组织Nm23-H1蛋白明显高于受照射CNE-2移植瘤组织,与体外实验结论一致,提示与鼻咽癌放射抗拒有关,可作为鼻咽癌放射治疗的新标志。     

Nm23-H1 expression does not predict clinical survival in colorectal cancer patients

The gene Nm23, which encodes for a nucleoside diphosphate kinase, has been defined as a metastasis-suppressor gene because of the inverse correlation between its expression and the metastatic capacity of the tumor cells. For colorectal cancer, however, the findings are equivocal. The aim of our study was to assess, in 160 patients undergoing surgery for colorectal cancer (CRC), the expression of the Nm23-H1 protein and to evaluate its possible associations with traditional clinicopathologic variables, with DNA-ploidy and proliferative activity (S-phase fraction, SPF), and with disease-free and overall survival of patients. Nm23-H1 expressions were evaluated on paraffin-embedded tissue by immunohistochemistry; DNA-ploidy and SPF on frozen tissue by flow-cytometric analysis. The median follow-up time in our study group was 71 months (range 34-115 months). No association was observed between Nm23-H1 protein expression and clinicopathological variables, S-phase fraction and DNA-ploidy. Furthermore, no significant differences were observed in the survival of patients with either moderate or strong Nm23-H1 expression. The major significant predictors for both disease relapse and death were advanced Dukes' stage, DNA aneuploid tumors and high SPF, while lymphohematic invasion was the only independent factor for relapse and non-curative resection for death. Our results indicate that Nm23-H1 activity is tissue-specific and that in CRCs the expression of the protein is not associated with tumor progression and patient prognosis, although further studies are required in order to throw more light on the possible clinical significance of the overexpression of the protein Nm23-H1 in such tumors.     

Nm23-H1和p53基因在乳腺癌细胞增殖及转移评估中的价值研究

目的探讨乳腺癌中nm23-H1、p53基因与肿瘤细胞增殖活性、淋巴结转移之间的关系。方法应用免疫组化技术检测乳腺癌组织中nm23-H1、p53基因蛋白及增殖指标k i-67的表达,结合临床及病理指标,探讨其临床意义。结果乳腺癌组织中nm23-H1、p53蛋白阳性表达率分别为50.8%(61/120)和60.0%(72/120),nm23-H1蛋白的表达与增殖程度(k i-67)和腋窝淋巴结转移有关,k i-67低增殖组nm23-H1阳性表达率为68.0%(49/72),而k i-67高增殖组nm23-H1阳性表达率则为25.0%(12/48),差异有显著性(P<0.05)。无合并淋巴结转移组中nm23-H1阳性表达率为72.2%(39/54),明显高于淋巴结转移组的阳性率33.3%(22/66)(P<0.05)。p53蛋白表达与雌激素受体有关,雌激素受体阳性组p53阳性阳性表达率为55.8%(47/86),阴性组阳性表达率为73.5%(25/34),差异有显著性(P<0.05)。结论nm23-H1和p53基因均与乳腺癌生物学行为有关,检测它们在乳腺癌中的表达可用来评估肿瘤是否具转移和侵袭能力。     

Nm23 gene product expression in invasive breast cancer--immunohistochemical analysis and clinicopathological correlation

The nm23 gene/protein is a putative metastatic suppressor identified a decade ago in a melanoma cell line. A number of laboratory, clinical and pathological studies have been carried out to define its real biological and biochemical function as a step in a complex metastatic process. In our study we examined the significance of nm23 expression in 164 breast cancer patients, aged 35 to 74 years, in comparison to other parameters such as age, menopausal status, histological grade, tumor size, lymph node status, and hormone receptor status. Overall survival (OS) and disease-free survival (DFS) were analyzed. The median follow-up was 84 months. Significant changes in OS were found for tumor size, nodal involvement and histological grade but there was no convincing correlation with nm23 expression. When patients were stratified according to nm23 expression, it was shown that overall survival in nm23 -positive patients was no longer than that in nm23 negative patients. It was also shown that patients who were lymph node negative and older than 50 years had longer OS than nm23 -negative patients. A statistical analysis shows that there is a correlation between axillary node status and nm23 expression (p = 0.018) as well as between patients' ages and nm23 expression (p = 0.043). There was no statistically significant correlation between nm23 expression, lymph node status and their combination on DFS.     

nm23—H1基因在大肠癌中的表达与肝转移及预后的关系

目的：探讨ｎｍ２３－Ｈ１蛋白表达与大肠癌肝转移及预后的关系。方法：对１０１例大肠癌存档石蜡块进行重新切片，采用ｎｍ２３－Ｈ１单克隆抗体进行免疫组化染色（ＬＳＡＢ法）。结果：ｎｍ２３－Ｈ１蛋白表达与年龄、性别、肿瘤大小、部位、组织类型、浆膜侵犯无关；与Ｄｕｋｅｓ分期、淋巴结转移有关；手术时有肝转移组较无肝转移组低，手术后有肝转移复发组较无肝转移复发组低（Ｐ＜００１）。Ｃｏｘ模型分析显示ｎｍ２３－Ｈ１是大肠癌预后的一个保护性指标。结论：ｎｍ２３－Ｈ１基因对大肠癌肝转移和预后具有重要作用。ＬＳＡＢ法检测大肠癌组织中ｎｍ２３－Ｈ１蛋白表达可能是预测大肠癌肝转移及预后的生物学指标之     

转移抑制基因nm23-H1参与肺癌Ras信号传导机理研究

背景与目的nm23-H1基因已被证实为一种肿瘤转移抑制基因,但其作用机理尚不明了,本研究探讨nm23-H1参与肺癌Ras信号传导的机理。方法应用脂质体法将pEGFP-nm23-H1野生型和突变型质粒(WT,H118F,S120G,P96S,S44A)转染人大细胞肺癌细胞株L9981,采用免疫共沉淀与Western blot方法检测野生型和突变型nm23-H1蛋白与Ras支架蛋白KSR的关系。结果在转染了野生型和突变型质粒的五个细胞株中,鼠抗nm23-H1免疫沉淀物在86KDa处可检测到KSR的阳性条带,而各组KSR的量应用单因素方差分析比较无显著性差异(F=0.190,P=0.938)。结论本研究结果表明nm23-H1与KSR存在相互作用,但这种相互作用与nm23-H1有无突变及突变位点无关,nm23-H1可能是通过KSR参与肺癌Ras信号传导的。     

肿瘤转移抑制基因nm23-H1对不同宫颈癌细胞侵袭和增殖的作用

背景与目的：nm23-H1是一种多效性基因,其抑癌作用有一定的组织特异性。本研究目的在于探讨nm23-HI对不同宫颈癌细胞增殖和侵袭的作用。方法：将真核表达载体pcDNA3．1-nm23-HI转染入宫颈癌细胞,Transwell小室法检测转染前后细胞侵袭性改变。M1Tr法绘制生长曲线,比较细胞增殖能力。流式细胞仪检测细胞周期改变。结果：与亲本细胞Caski、SiHa和空载体转染细胞Caski-3．1、SiHa-3．1相比．pcDNA3．1-nm23-H1转染细胞Caski—N和SiHa—N的侵袭力和增殖受到明显抑制（P〈0．05）,G2／M期和S期细胞比例降低（P〈0．05）,而G。／G．期细胞比例明显增加（P〈0．05）。nm23-H1对HeLa细胞增殖、侵袭及细胞周期均无明显影响（P〉0．05）。结论：nm23-H1对不同宫颈癌细胞的增殖和侵袭等细胞表型可产生细胞特异性的抑制作用。