MKK4与肿瘤研究现状

MKK4是丝裂原活化蛋白激酶MAPKK家族的一个成员,位于MAPK通路的重要位置,能同时磷酸化并激活下游两条p38和JNK通路,最终活化转录因子,引起一系列复杂的生物学反应。研究发现,MKK4与不同类型肿瘤的发生、发展、恶性转移密切相关,且作用机制复杂。     

MKK4基因启动子区遗传变异与中国南方人群肺癌易感性的相关性研究

目的:探讨丝裂原活化蛋白激酶激酶4(mitogen-activated protein kinase kinase4,MKK4)基因启动子区单核苷酸多态性与中国南方人群肺癌发病风险的关系。方法:采用病例对照研究方法,收集800例肺癌病例和900例正常对照,采用TaqMan技术检测MKK4基因启动子区多态位点rs3826392(-1304T>G)的基因型。应用SAS9.3软件分析其与肺癌易感性的相关性。结果:MKK4基因启动子区-1304T>G基因型在对照组中的频率分布符合Hardy-Weinberg平衡(P=0.149),其在病例组和对照组的分布差异有统计学意义(P=0.001);与携带TT基因型个体相比,携带TG杂合子的个体患肺癌的风险下降25%[校正比值比(oddratio,OR)=0.75,95%可信区间(confidence interval,CI)=0.58～0.97],而携带GG变异纯合子者患肺癌的风险下降45%(校正OR=0.55,95%CI=0.33～0.94);随着变异型等位基因G的个数增加,肺癌发病风险逐步降低(P趋势<0.001)。结论:MKK4基因启动子区-1304T>G基因遗传变异可能降低肺癌发病风险。     

MKK4与肿瘤关系的研究进展

目的:探讨MKK4基因与肿瘤关系的研究进展.方法:以MKK4和肿瘤为关键词,检索1989-2008年PubMed全文数据库和维普资讯-中国科技期刊数据库.纳入标准:1)MKK4基因结构及生物学特性的研究;2)MKK4与肿瘤发生发展关系的研究.粗选有76篇关于MKK4结构特点及其与肿瘤关系研究文章,根据纳入标准,精选45篇文献,最后纳入分析27篇文献.结果:MKK4是MAPK信号转导通路的组成部分,能磷酸化并激活JNK通路和p38通路,将细胞外刺激信号转导至细胞及其核内,引起细胞的增殖、分化、转化及凋亡等生物学反应.MKK4在肿瘤的形成发展过程中具有重要作用,有研究证实MKK4为肿瘤转移抑制基因或抑癌基因,对肿瘤有抑制作用.然而,也有研究证实MKK4为癌基因,有致肿瘤作用.结论:到目前为止,MKK4与肿瘤关系还存在许多争议,需要我们进一步时其进行认识.     

A型激酶锚定蛋白12与肿瘤

A型激酶锚定蛋白12(AKAP12)是一种细胞支架蛋白,能够锚定一些关键信号分子,如蛋白激酶A(PKA)、蛋白激酶C(PKC)和细胞周期素D1(cyclin D1),以及动态调节β2-肾上腺素受体复合物.AKAP12在多条信号转导通路中发挥重要作用.研究发现,AKAP12在前列腺癌、食管癌、胃癌等多种肿瘤中表达下调或缺失,是一个肿瘤抑制基因,同时也是潜在的肿瘤转移抑制基因.     

MKK4基因在喉鳞癌中的表达及其临床意义

目的:研究丝裂原活化蛋白激酶激酶4 (mitogen-activated protein kinase kinase 4,MKK4)在喉鳞状细胞癌组织中的表达及其临床意义.方法:应用RT-PCR和免疫组织化学方法检测42例喉鳞癌、20例喉乳头状瘤及20例声带息肉组织中MKK4 mRNA和蛋白的表达,分析其与喉鳞癌临床病理参数之间的关系.结果:MKK4 mRNA和蛋白表达的阳性率在喉鳞癌、喉乳头状瘤及声带息肉组织中逐渐降低,且差异有统计学意义(P＜0.05).MKK4 mRNA和蛋白的表达随喉鳞癌的浸润深度增加而上升(P＜0.05),在有淋巴结转移组中的表达高于无淋巴结转移组(P＜0.05),在临床Ⅲ～Ⅳ期患者中的表达高于临床Ⅰ～Ⅱ期的患者(P＜0.05).MKK4的表达与患者的年龄、性别、肿瘤发生部位及组织学分级无相关性(P＞0.05).结论:MKK4 mRNA和蛋白在喉鳞癌组织中的表达上调可能与喉鳞癌的发生及浸润转移有关,MKK4可能成为一个潜在的判断喉鳞癌发生与转移的观测指标.     

肿瘤转移抑制基因的功能定位克隆研究

转移是肿瘤的主要恶性表型,严重影响肿瘤患者治疗的疗效和预后。肿瘤转移抑制基因的发现将为肿瘤转移的诊断和干预治疗提供重要的线索。然而,迄今发现的转移抑制基因还不多,国内尚无相关报道。为了促进转移抑制基因的研究与发现,本文对肿瘤转移抑制基因功能定位克隆的基本原理与技术路线(主要包括微细胞介导的染色体转移,序列标签位点的PCR定位,和自发转移动物实验模型)作一综述,并叙述了通过此技术路线发现转移抑制基因(KAI-1,KiSS-1,MKK4,BRMS1)的经过,以及此技术在前列腺癌、黑素瘤和肝癌转移抑制基因的功能定位克隆中的应用情况。     

ras-丝裂原浯化蛋白质激酶通路与肿瘤靶向治疗

ras-丝裂原活化蛋白质激酶通路细胞信号转导网调节细胞的分化、细胞骨架的构建、蛋白质的转运等许多生理过程.近年来在肿瘤治疗中得到了进一步的重视与应用,很多临床研究将重点放在其抑制制剂通路上,如Raf激酶抑制剂、丝裂原活化蛋白激酶(MAPK)激酶(MEK)抑制剂、Src激酶抑制剂通路等.     

MAPK信号转导通路在肿瘤化疗耐药中的作用

丝裂原活化蛋白激酶(MAPK)信号转导通路是调节细胞增殖和凋亡的重要通路.最近研究发现,MAPK信号转导通路可能参与肿瘤化疗耐药,其作用机制可能是调控耐药相关基因的表达,通过对该通路的干预可以提高肿瘤的化疗敏感性,从而逆转化疗耐药.     

pVHL/HIF通路与PI3K/PKB/FOXO通路在肾透明细胞癌发生、发展中的作用

肿瘤抑制基因von Hipple-Lindau(简称VHL基因)的突变失活导致肿瘤细胞中缺氧诱导因子的累积,进而调节其下游靶基因的转录和表达,促进肿瘤内新生血管形成及细胞增殖,从而促进肾透明细胞癌的发生、发展.磷酸肌醇-3激酶/蛋白激酶B通路异常活化,在肾细胞肿瘤的发生、发展、转移中发挥重要作用.上述两通路存在着错综复杂的联系.本文通过分析两通路在肾肿瘤发生,发展中的作用,为探寻肾癌治疗的新靶点提供理论依据.     

MAPK信号传导通路对人炎性乳腺癌细胞系侵袭能力的影响

目的:本研究探讨上皮钙粘蛋白(E-cadherin)基因显性负调控质粒(pcDNA3.1(-)H-2kd-E-cadherin)导入SUM149细胞系后,对丝裂原活化蛋白激酶信号传导系统(mitogenactivatedproteinkinasecascade,MAPK)的相关信号通路及调控机制的改变情况。方法:应用蛋白印迹法,检测了人炎性乳腺癌细胞系SUM149、E-cadherin基因显性负调控突变体高表达的SUM149阳性克隆中MAPK激酶活性的变化。结果:与对照组(未转染及空质粒组)相比,转染后并稳定高表达小鼠H-2kd阳性克隆的细胞中,磷酸化的细胞外信号调节激酶(extracellularsignalregulatedkinase,p-ERK1/2,phospho-P44/42)明显降低;而磷酸化P38激酶无显著变化。结论:在异常高的E-cadherin功能性表达人炎性乳腺癌细胞系SUM149中,下调上皮钙粘着蛋白表达明显抑制其侵袭能力。其可能通过下调丝裂原活化蛋白激酶信号传导系统MAPK中的磷酸化细胞外信号调节激酶(phos-extracellularsignalregulatedkinase)phos-Erk1/2降低基质金属蛋白酶MMP-1、MMP-9等表达,从而明显抑制其侵袭能力。     

Role of mitogen-activated protein kinase kinase 4 in cancer

Mitogen-activated protein (MAP) kinase kinase 4 (MKK4) is a component of stress activated MAP kinase signaling modules. It directly phosphorylates and activates the c-Jun N-terminal kinase (JNK) and p38 families of MAP kinases in response to environmental stress, pro-inflammatory cytokines and developmental cues. MKK4 is ubiquitously expressed and the targeted deletion of the Mkk4 gene in mice results in early embryonic lethality. Further studies in mice have indicated a role for MKK4 in liver formation, the immune system and cardiac hypertrophy. In humans, it is reported that loss of function mutations in the MKK4 gene are found in approximately 5% of tumors from a variety of tissues, suggesting it may have a tumor suppression function. Furthermore, MKK4 has been identified as a suppressor of metastasis of prostate and ovarian cancers. However, the role of MKK4 in cancer development appears complex as other studies support a pro-oncogenic role for MKK4 and JNK. Here we review the biochemical and functional properties of MKK4 and discuss the likely mechanisms by which it may regulate the steps leading to the formation of cancers.     

Mitogen-activated protein kinase kinase 4/stress-activated protein/Erk kinase 1 (MKK4/SEK1), a prostate cancer metastasis suppressor gene encoded by human chromosome 17.

The introduction of a discontinuous approximately 70-cM portion of human chromosome 17 significantly suppresses the metastatic ability of AT6.1 rat prostate cancer cells without affecting tumorigenicity (M. A. Chekmareva et al., Prostate, 33: 271-280, 1997). We have recently demonstrated that AT6.1 cells containing the approximately 70-cM region (AT6.1-17-4 cells) escape from the primary tumor and arrest in the lung but are growth-inhibited unless the metastasis suppressor region is lost (M. A. Chekmareva et al., Cancer Res., 58: 4963-4969, 1998). A series of in vivo studies indicated that the observed growth inhibition was due to the effect of a gene(s) at the metastatic site (M. A. Chekmareva et al., Cancer Res., 58: 4963-4969, 1998). We have now identified the mitogen-activated protein kinase kinase 4/stress-activated protein/Erk kinase 1 (MKK4/SEK1) gene as a candidate metastasis suppressor gene encoded by the approximately 70-cM region. AT6.1 cells were transfected with a MKK4/SEK1 expression construct, and the cells were tested in standard spontaneous metastasis assays. Whereas the metastatic ability of the AT6.1-MKK4/SEK1 cells was significantly reduced as compared with that of transfection controls, the growth rate of the primary tumors was not affected; the average tumor volume at day 29 after injection was approximately 2 cm. Furthermore, histological examination of the lungs of AT6.1-MKK4/SEK1 tumor-bearing animals revealed that the suppression by MKK4/SEK1 is due to an effect at the metastatic site, consistent with the phenotype conferred by the original approximately 70-cM chromosomal region. These studies implicate MKK4/SEK1 as a metastasis suppressor gene encoded by human chromosome 17.     

Suppression of metastatic colonization by the context-dependent activation of the c-Jun NH2-terminal kinase kinases JNKK1/MKK4 and MKK7

Advances in clinical, translational, and basic studies of metastasis have identified molecular changes associated with specific facets of the metastatic process. Studies of metastasis suppressor gene function are providing a critical mechanistic link between signaling cascades and biological outcomes. We have previously identified c-Jun NH2-terminal kinase (JNK) kinase 1/mitogen-activated protein kinase (MAPK) kinase 4 (JNKK1/MKK4) as a prostate cancer metastasis suppressor gene. The JNKK1/MKK4 protein is a dual-specificity kinase that has been shown to phosphorylate and activate the JNK and p38 MAPKs in response to a variety of extracellular stimuli. In this current study, we show that the kinase activity of JNKK1/MKK4 is required for suppression of overt metastases and is sufficient to prolong animal survival in the AT6.1 model of spontaneous metastasis. Ectopic expression of the JNK-specific kinase MKK7 suppresses the formation of overt metastases, whereas the p38-specific kinase MKK6 has no effect. In vivo studies show that both JNKK1/MKK4 and MKK7 suppress the formation of overt metastases by inhibiting the ability of disseminated cells to colonize the lung (secondary site). Finally, we show that JNKK1/MKK4 and MKK7 from disseminated tumor cells are active in the lung but not in the primary tumor, providing a biochemical explanation for why their expression specifically suppressed metastasis while exerting no effect on the primary tumor. Taken together, these studies contribute to a mechanistic understanding of the context-dependent function of metastasis regulatory proteins.     

c-Jun NH2-terminal kinase activating kinase 1/mitogen-activated protein kinase kinase 4-mediated inhibition of SKOV3ip.1 ovarian cancer metastasis involves growth arrest and p21 up-regulation

In many patients without clinical metastases, cancer cells have already escaped from the primary tumor and entered a distant organ. A long-standing question in metastasis research is why some disseminated cancer cells fail to complete steps of metastatic colonization for extended periods of time. Our laboratory identified c-Jun NH(2)-terminal kinase activating kinase 1/mitogen-activated protein kinase kinase 4 (JNKK1/MKK4) as a metastasis suppressor protein in a mouse xenograft model of experimental i.p. ovarian cancer metastasis. In this model, expression of JNKK1/MKK4 via activation of p38 delays formation of >or=1-mm implants and prolongs animal survival. Here, we elucidate the time course of this delay as well as the biological mechanisms underpinning it. Using the Gompertz function to model the net accumulation of experimental omental metastases, we show that MKK4-expressing implants arise, on average, 30 days later than controls. Quantitative real-time PCR shows that MKK4 expression does not have a substantial effect on the number of cancer cells initially adhering to the omentum, and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling analysis shows that there is no increase in apoptosis in these cells. Instead, immunohistochemical quantitation of cell cycle proteins reveals that MKK4-expressing cells fail to proliferate once they reach the omentum and up-regulate p21, a cell cycle inhibitor. Consistent with the time course data, in vitro kinase assays and in vivo passaging of cell lines derived from macroscopic metastases show that the eventual outgrowth of MKK4-expressing cells is not due to a discrete selection event. Rather, the population of MKK4-expressing cells eventually uniformly adapts to the consequences of up-regulated MKK4 signaling.     

Mitogen-activated protein kinase kinase 4 (MKK4) acts as a metastasis suppressor gene in human ovarian carcinoma

Despite improvements in chemotherapy and the recognition that aggressive surgical cytoreduction is beneficial, the majority of patients diagnosed with ovarian cancer will die as a result of metastatic disease. The molecular changes associated with acquisition of metastatic ability in ovarian cancer are poorly understood. We hypothesize that metastasis suppressor gene inactivation or down-regulation plays a role in ovarian cancer progression. Mitogen-activated protein kinase kinase 4 (MKK4), a member of the stress-activated protein kinase signaling cascade, has been identified recently as a metastasis-suppressor gene. An immunohistochemical approach was taken to test the possibility that MKK4 dysregulation occurs during the development of clinical ovarian cancer metastases. MKK4 expression was evaluated in normal and metastatic ovarian tissues. Normal ovarian epithelial cells showed high intensity staining for MKK4, whereas metastatic tissues showed a statistically significant decrease in expression. These results support a role for MKK4 dysregulation in the development of clinical disease. A functional approach was taken to test the ability of MKK4 to suppress metastatic colonization, the process whereby disseminated cancer cells lodge and grow at a secondary site in vivo. The SKOV3ip.1 human ovarian cancer cell line was chosen for these studies because it lacks endogenous MKK4 expression but retains both upstream and downstream components of the signaling cascade of MKK4. Ectopic expression of MKK4 in these cells, when injected into female SCID mice, suppressed the number of overt metastatic implants by nearly 90%. Furthermore, MKK4 expression increased the life span of the animals by 70%. Taken together, these data support a role for MKK4 in the suppression of metastatic colonization in ovarian cancer.     

New paradigms for the function of JNKK1/MKK4 in controlling growth of disseminated cancer cells

Much work has been done in the 20 years since the discovery of the first metastasis suppressor gene to investigate the diverse biochemical functions of the proteins these genes encode. The function of metastasis suppressors cannot be solely predicted from correlative clinical data or in vitro studies. Instead, careful design of in vivo experiments to test broader hypotheses is necessary to pinpoint the mechanism of action of these novel proteins. Our laboratory identified c-Jun NH2-terminal kinase activating kinase 1 (JNKK1)/Mitogen-activated protein kinase (MAPK) kinase 4 (JNKK1/MKK4) as a metastasis suppressor in prostate and ovarian cancer. JNKK1/MKK4 is a stress activated protein kinase (SAPK) involved in a variety of signaling events, ranging from the regulation of hepatoblast survival during mammalian development to metastasis suppression in adult ovarian and prostate cancers. JNKK1/MKK4 function has typically been associated with the c-Jun NH2-terminal kinase (JNK) signaling pathway, particularly in the immune system where JNK plays a role in inflammatory signaling and apoptosis. However, evidence continues to accumulate that JNKK1/MKK4 is also a physiologic activator of p38 under certain conditions, and that activation of p38 arrests cell cycle progression. This review will provide a historical perspective on the role of JNKK1/MKK4 in SAPK signaling, including some recent findings from our own laboratory that shed light on the complicated role for JNKK1/MKK4 in metastatic colonization.     

Mitogen-activated protein kinase kinase 4 metastasis suppressor gene expression is inversely related to histological pattern in advancing human prostatic cancers

We have shown recently (B. A. Yoshida et al., Cancer Res., 59: 5483-5487) that mitogen-activated protein kinase kinase 4 (MKK4) can suppress AT6.1 rat prostate cancer metastases in vivo. Evaluation of the expression of components of the MKK4 signaling cascade showed a loss or down-regulation of expression of MKK4 or c-Jun, a downstream mediator of MKK4, in six of eight human prostate cancer cell lines. Given these findings, we next assessed whether MKK4 dysregulation occurs during the development of clinical prostate cancer. Immunohistochemical studies showed high levels of MKK4 expression in the epithelial but not the stromal compartment of normal prostatic tissues. In neoplastic tissues, a statistically significant, direct, inverse relationship between Gleason pattern and MKK4 was established. These results demonstrate that MKK4 protein is consistently down-regulated during prostate cancer progression and support a role for dysregulation of its signaling cascade in clinical disease. To test the possibility that down-regulation of MKK4 protein is the result of allelic loss, metastatic prostate cancer lesions were examined for loss of heterozygosity (LOH) within the MKK4 locus (D17S969). These studies showed a 31% (5 of 16) LOH of MKK4 that is not associated with coding region mutations, which suggests that the nucleotide sequence of the gene in the remaining allele is infrequently mutated.     

The p38 kinases MKK4 and MKK6 suppress metastatic colonization in human ovarian carcinoma

Despite considerable efforts to improve early detection of ovarian cancer, the majority of women at time of diagnosis will have metastatic disease. Understanding and targeting the molecular underpinnings of metastasis continues to be the principal challenge in the clinical management of ovarian cancer. Whereas the multistep process of metastasis development has been well established in both clinical and experimental models, the molecular factors and signaling pathways involved in successful colonization of a secondary site by disseminated cancer cells are not well defined. We have previously identified mitogen-activated protein kinase (MAPK) kinase 4/c-Jun NH2-terminal kinase (JNK)-activating kinase (MKK4/JNKK1/SEK1, hereafter referred to as MKK4) as a metastasis suppressor protein in ovarian carcinoma. In this study, we elucidate key mechanisms of MKK4-mediated metastasis suppression. Through the use of a kinase-inactive mutant, we show that MKK4 kinase activity is essential for metastasis suppression and prolongation of animal survival. Because MKK4 can activate either of two MAPKs, p38 or JNK, we expressed MKK6 or MKK7, specific activators of these MAPKs, respectively, to delineate which MAPK signaling module was involved in MKK4-mediated metastasis suppression. We observed that MKK6 expression suppressed metastatic colonization whereas MKK7 had no effect. Our finding that MKK4 and MKK6 both suppress metastasis points to the p38 pathway as an important regulatory pathway for metastatic colonization in ovarian cancer.     

MKK7 mediates miR‐493‐dependent suppression of liver metastasis of colon cancer cells

The prognosis of advanced colon cancer patients is profoundly affected by the presence or absence of liver metastasis. miR‐493 functions as a potent suppressor of liver metastasis, and low‐level miR‐493 expression in human primary colon cancer is associated with an elevated incidence of liver metastasis. We previously showed that IGF1R is a target gene of miR‐493, and that the inhibition of IGF1R partly explains how miR‐493 suppresses liver metastasis. However, major functional targets that mediate the antimetastatic activity of miR‐493 remain elusive. Here, we extended our search for target genes and identified MKK7, a mitogen‐activated protein kinase kinase, as a novel target of miR‐493. miR‐493 inhibits MKK7 expression by targeting the binding site at the 3′‐UTR of the mkk7 gene. MKK7 was expressed in six out of seven colon cancer cell lines examined but not in non‐transformed colon epithelial cells, and its expression was required for the activating phosphorylation of JNK. RNA interference‐mediated inhibition of MKK7 resulted in marked suppression of liver metastasis of colon cancer cells. A significant decrease of metastasized cells by the MKK7 knockdown was observed, even at early stages of the metastatic settlement, in accordance with a time course of the miR‐493‐mediated inhibition of the metastasis. Immunohistochemical examination in human primary colon tumors revealed that the occurrence of liver metastasis is associated with elevated levels of MKK7. Thus, MKK7 is a major functional target of miR‐493, and its suppression thwarts liver metastasis of colon cancer cells.     

Inhibition of oncogenic K-ras signaling by aerosolized gene delivery in a mouse model of human lung cancer.

PURPOSE: Transfer of growth-suppressive genes to lung tumors has therapeutic potential, but effective delivery techniques have not been developed. Here, we investigated gene delivery to lung tumors by aerosolization of adenoviral vectors incorporated into calcium phosphate precipitates. EXPERIMENTAL DESIGN: To investigate the efficacy of this delivery method in normal and neoplastic lung, an adenoviral vector expressing beta-galactosidase was administered by jet nebulization to K-ras(LA1) mice, which develop lung adenocarcinomas through activation of a latent allele carrying mutant K-ras(G12D). Furthermore, we investigated whether aerosolized delivery of Ad-MKK4 (KR), an adenoviral vector expressing dominant-negative mutant mitogen-activated protein kinase kinase 4(MKK4), can block Ras-dependent signaling in K-ras(LA1) mice. RESULTS: After a single administration, beta-galactosidase was detected in lung tissue for up to 21 days, and expression was much greater in tumors than in normal lung tissue. MKK4 was activated in the lungs of K-ras(LA1) mice, and aerosolized treatment with Ad-MKK4 (KR) decreased c-Jun-NH(2)-terminal kinase but not extracellular signal- regulated kinase activity, providing evidence that MKK4 was selectively inhibited. CONCLUSIONS: These findings demonstrate a novel approach to targeting oncogenic pathways in lung tumors by aerosolized gene delivery.