siRNA沉默LKB1基因激活Hedgehog信号通路及对人乳腺癌裸鼠移植瘤模型生长的实验研究

目的探讨应用小分子干扰RNA(small interfering RNA,siRNA)沉默抑癌基因LKB1对人乳腺癌细胞MDA-MB-231中Hedgehog信号通路相关因子的表达及人乳腺癌裸鼠移植瘤模型的肿瘤生长的影响。方法构建LKB1基因siRNA质粒LKB1-siRNA;建立LKB1表达抑制的MDA-MB-435细胞模型;裸鼠乳晕皮下接种,建立人乳腺癌裸鼠移植瘤动物模型;成瘤后,观察肿瘤体积变化、裸鼠生存时间;并用Western印迹法检测瘤组织中LKB1和Hedgehog信号通路中信号肽Shh、Sufu、膜受体Ptch、Smo、转录因子Gli1、Hip蛋白表达的变化。结果 LKB1-siRNA质粒组裸鼠的肿瘤体积明显增长(P<0.05);肿瘤内LKB1基因表达水平明显下降,而Hedgehog信号通路相关因子Shh、Gli1、Ptch、Smo的表达升高,Hedgehog信号通路抑制因子Sufu、Hip表达下降。结论 LKB1基因siRNA能够明显抑制人乳腺癌裸鼠移植模型的LKB1基因的表达,上调Hedgehog信号通路相关因子的表达,促进肿瘤生长。LKB1基因和Hedgehog信号通路在乳腺癌细胞中呈现负相关表达。     

LKB1基因与Hedgehog信号通路对乳腺癌MDA-MB-231细胞抑制机制的探讨

目的:探讨乳腺癌细胞中LKB1基因与Hedgehog信号转导途径的关系.方法:以乳腺癌MDA-MB-231细胞为研究对象,LKB1基因转染或干扰处理后,反转录-聚合酶链反应和蛋白质印迹法分别检测Hedgehog信号通路中Shh、Sufu、Ptch、Smo、Gli 1、Hip mRNA和蛋白表达的变化.建立LKBI 基因表达抑制的细胞移植瘤模型,验证体外实验结果并进行对比分析.结果:Hedgehog 信号通路激动因子Ptch的mRNA和蛋白的表达在各组细胞中差异无统计学意义.而激动因子Shh、Gli1和Smo的mRNA和蛋白的表达在MDA-MB-231正常细胞及其空质粒细胞、siRNA无关序列细胞中差异无统计学意义;在LKB1转染细胞中降低(F=29.56,P＜0.05),在LKBI转染siRNA细胞中升高(F=32.15,P＜0.05).而抑制因子Sufu、Hip的mRNA和蛋白的表达正好相反,在LKB1转染细胞中升高,在LKB1转染siRNA细胞中降低,F=30.25,P＜0.05.移植瘤检测Shh、Sufu、Smo、Gli 1、Hip蛋白表达与体外实验相似,F=8.40,P＜0.01.而且发现LKB1转染siRNA细胞组裸鼠肿瘤平均体积显著大于对照组(F=4.68,P＜0.05),生存时间显著短于对照组,F=3.50,P＜0.01.LKB1基因与Hedgehog信号通路相关因子表达呈负相关,r=0.232,P＜0.05.结论:LKB1基因与Hedgehog信号通路相关因子表达在乳腺癌细胞中呈负相关,LKB1基因可能通过Hedgehog信号通路抑制乳腺癌细胞的发生,两者之间的相互作用对乳腺癌的诊治可能有重要影响.     

乳腺癌中Hedgehog信号通路的表达及其与LKB1基因相关性的研究

目的：探讨Hedgehog（Hh）信号通路主要蛋白在乳腺癌中的表达及其与LKB1基因的相关性,以及与临床病理特征、预后的关系。方法：采用免疫组化方法检测75例人乳腺癌标本中Hh信号通路主要蛋白（Shh、Smo、Sufu、Gli1、Ptch）及LKB1基因的表达,分析二者相关性,并分析Hh信号通路的表达与临床病理特征及预后的关系。结果：在75例乳腺癌样本中,阳性表达Shh、Gli1、Smo、Ptch、Sufu、LKB1分别为69（92.0%）、58（77.3%）、65（86.7%）、50（66.7%）、34（45.3%）、33（44.0%）,而在其相邻的正常乳腺上皮不表达或低表达。Hh信号通路与LKB1的表达呈负相关;Hh信号通路的表达情况与患者年龄、淋巴结转移情况、分子分型具有一定的相关性,且高表达的患者预后较差。结论：Hh信号通路在乳腺癌中过度激活,与临床病理特征及预后相关,并与LKB1基因的表达呈负相关,两者之间的相互作用对乳腺癌的诊治及预后可能有重要影响。     

Sonic Hedgehog信号通路与乳腺癌抑癌基因LKB1的相互影响

目的：研究环靶明（cyclopamine）抑制Sonic Hedgehog（SHH）信号通路后,转染LKB1基因的乳癌细胞的凋亡、周期及信号通路相关基因表达的改变。方法：抑癌基因LKB1转染人乳腺癌细胞MDA-MB-231,分MDA-MB-231组（231组）和转染LKB1基因的MDA-MB-231（LKB1组）两组,每组分别采用0,5×10-6mol/L,10×10-6mol/L,20×10-6mol/L 4种浓度的环靶明处理细胞,各小组细胞分别采用流式细胞仪检测细胞凋亡和周期,用RT-PCR法和Western blot法检测Sonic Hedgehog信号通路相关基因Shh、Smo、Ptch、Sufu、Hip及LKB1的mRNA和蛋白表达水平。结果 ：在LKB1组和231组中,各小组细胞的凋亡率变化与Sonic Hedgehog相关基因Shh、Smo表达变化一致,随环靶明浓度增大凋亡率增加、基因表达受抑制,在环靶明浓度达10×10-6mol/L时变化最明显,且LKB1组较231组的变化更为明显。在231组中,各小组细胞周期变化与Sonic Hedgehog相关抑制基因Sufu、Hip表达变化一致。而在LKB1组中,各小组细胞周期与抑制基因Sufu、Hip表达变化均无明显差异。在231组中,各小组抑癌基因LKB1的表达随药物浓度增加渐增强。Ptch表达在两组均无明显变化。结论 ：抑癌基因LKB1可协同Sonic Hedgehog信号通路抑制剂环靶明促进乳腺癌细胞凋亡,调控细胞周期,推测其机制可能是通过如上信号通路相关基因表达变化而实现。信号通路抑制剂环靶明可提高乳腺癌细胞抑癌基因LKB1的表达,推测此也是信号通路抑制剂降低癌细胞活性机制之一。     

Hedgehog信号通路通过协同抑制Cyclin D1增强Let-7a对三阴性乳腺癌肿瘤细胞的抑制作用

目的:研究Hedgehog信号通路在Let-7a抑制三阴性乳腺癌肿瘤进展过程中的作用。方法:应用MTT实验及流式实验检测Hedgehog通路对Let-7a所引起的乳腺癌细胞凋亡的影响。以Western blot实验,luc-assay实验验证Hedgehog信号通路所引起的和Let-7a有关的下游信号通路的变化。免疫荧光实验检测Hedgehog信号通路抑制剂环巴胺引起的Let-7a对乳腺癌干细胞表型改变的影响,免疫组化实验检测Hedgehog通路激活的和Let-7a有关的下游分子在不同分期的乳腺癌组织中的表达。结果:抑制Hedgehog信号通路可以抑制MDA-MB-231细胞和BT-20细胞的增殖,并且有助于增敏Let-7对MDA-MB-231和BT-20细胞的相关作用。Let-7a对TNBC细胞中Cyclin D1水平的抑制作用虽然不是十分显著,但Hedgehog通路抑制剂可以明显增强其相关作用,我们通过Western blot,luc-assay和免疫荧光实验对这一作用进行了验证。此外,研究结果还显示环巴胺可以抑制MDA-MB-231和BT-20细胞中的ALDH1(+)亚群细胞,并可以加强Let-7a对TNBC细胞自我更新能力的抑制作用。结论:Hedgehog通路的抑制剂可以增强Let-7 miRNA在三阴性乳腺癌中所引起的肿瘤抑制作用。     

Hedgehog信号通路与肿瘤的关系研究进展

Hedgehog信号通路在胚胎发育中起着重要的作用,控制着细胞的"命运"和增殖[1].1980年,Nusslein-Vollhard和Wieschaus在研究果蝇的基因突变时发现了hedgehog基因和patched基因.近几年人们逐渐发现许多疾病都与这个信号通路有关.当这个信号通路异常激活时,会引起肿瘤的发生和发展.     

Hedgehog信号通路GLI1与SuFu在口腔鳞癌中的研究进展

Hedgehog（Hh）信号通路在胚胎发育、细胞增殖和分化中起着重要的调节作用，此通路异常激活可能会导致全身恶性肿瘤（肺癌、胃癌）的发生，尤其是口腔鳞癌的发生，因此Hh信号通路与口腔鳞癌密切相关。Hh信号通路主要有分泌型糖蛋白配体 Hedgehog（SHH）配体，跨膜蛋白受体Ptched（PTCH）受体，跨膜蛋白Smoothened（SMO），核转录因子GLI蛋白、Fu抑制子（SuFu）及下游目的基因组成。其中GLI1作为Hh信号通路的正向调控基因，起着激活Hh信号通路的作用。而SuFu作为Hh信号通路负向调控基因，起着抑制Hh信号通路的作用。本文就Hh信号通路在口腔鳞癌中信号传导机制及其相关基因GLI1、SuFu的研究进展进行综述，为未来口腔鳞癌的治疗、诊断和预防提供依据。     

Hedgehog信号通路相关蛋白Gli1在乳腺癌中的表达及其意义

目的 探讨Hedgehog信号通路转录因子Gli1在乳腺癌组织中的表达及其临床意义。方法 应用免疫组织化学染色检测Hedgehog信号通路相关蛋白Gli1在102例乳腺浸润性导管癌及30例癌旁正常组织中的表达,并分析其表达与临床病理特征的关系。结果 Gli1蛋白在乳腺癌组织中的阳性表达率为75.5％（77／102）,高于癌旁正常组织的26.7％（8／30）,两者差异有统计学意义（P＜0.05）。Gli1蛋白的表达与腋窝淋巴结转移、雌激素受体（ER）表达有关（P＜0.05）,而与患者的年龄、绝经状况、肿瘤直径、病理分期、组织学分级、孕激素受体（PR）及人表皮生长因子受体-2（HER-2）表达状况均无关（P＞0.05）。结论 Hedgehog信号通路相关蛋白Gli1在乳腺癌组织中存在高表达,其一定程度上对ER表达起到负向调节作用。     

乳腺癌组织中Hedgehog相关蛋白的表达及其临床意义

目的 探讨Hedgehog相关蛋白在乳腺癌中的表达及其临床意义。方法 从334例乳腺癌 组织中制备组织块。免疫组织化学法检测六种Hedgehog信号蛋白（Shh、ptch、Smo、Gli-1、 Gli-2和Gli-3）的表达。结果 Hedgehog信号蛋白表达与一些预后因素显著相关,包括淋巴结转 移与Shh（P=0.001）、Gli-3 (P<0.001)、Ptch(P=0.025)的相关性、分期与Shh和Ptch表达（P分别 为0.007和0.037）的相关性,核分级与Smo(P=0.004)和Gli-3（P=0.026）的相关性,组织学分级与 Smo(P=0.007)的相关性。Shh、Ptch、Smo、Gli-1、Gli-2和Gli-3表达在表型之间差异有统计学意义 （P分别为<0.0001、<0.0001、0.004、0.039、0.031和0.003）。Gli-2表达与较差的整体生存结果相关 （P=0.012）。结论 Hedgehog通路表达激活与乳腺癌的侵袭性相关,可能是乳腺癌的一个有效的治 疗靶点。Gli-2可作为乳腺癌的一个预后指标。     

LKB1基因在不同基因亚型乳腺癌中表达的意义

目的:检测LKB1基因在不同基因亚型乳腺癌中的表达,并探讨其在乳腺癌中表达的意义.方法: 应用组织芯片及免疫组织化学法对180例乳腺浸润性导管癌、18例导管原位癌及20例正常乳腺组织中LKB1基因、 ER 、PR、HER-2、CK56、和P53 的表达进行检测;根据最近提出的乳腺癌分子分型进行分组,对LKB1基因在不同基因亚型中的表达及与其他临床病理特征的关系进行统计学分析. 结果: LKB1基因在乳腺导管原位癌的阳性率为66.11%(11/18),浸润性导管癌的阳性率为36.67%(66/180),两组间差异有统计学意义(P<0.05);在浸润性导管癌中,LKB1基因在淋巴结转移组阳性率为47.61%(40/84),无转移组阳性率为83.33%(80/96),两组间差异具有统计学意义(P<0.01);在不同基因亚型中中LKB1基因的表达差异具有统计学意义(P<0.05);其中Luminal A组与HER-2+组及三阴性组两两比较差异具有统计学意义(P<0.05).结论: LKB1基因的在乳腺浸润性导管癌中的表达明显低于导管原位癌,与淋巴结转移呈负相关,且在生物学行为更为恶性的HER-2+组和三阴性组具有更低的表达水平,提示LKB1基因对乳腺癌的浸润与转移过程有抑制作用.     

Evaluation of liver kinase B1 downstream signaling expression in various breast cancers and relapse free survival after systemic chemotherapy treatment

LKB1-signaling has prominent roles in cancer development and metastasis. This report evaluates LKB1-signaling pathway gene expression associations with patient survival in overall breast cancer, specific subtypes, as well as pre- and post-chemotherapy. Subtypes analyzed were based on intrinsic molecular subtyping and traditional biomarker classifications. Intrinsic molecular subtypes included were Luminal-A, Luminal-B, HER2-enriched, and Basal-like. The biomarker subtypes assessed were Estrogen-Receptor Positive (ER+) and Negative (ER-), Wild-Type TP53 (WT-TP53) & Mutant-TP53, and Triple-Negative Breast Cancer (TNBC). Additionally, comparisons were made between these subtypes and breast cancer overall, and analyses between LKB1 signaling to patient survival before and after chemotherapy were made. We used the Kaplan-Meier Online Tool (KM Plotter) to correlate the relationship between mRNA expression of known LKB1 scaffolding proteins (CAB39 and LYK5), and downstream signaling targets (AMPK, MARK1, MARK2, MARK3, MARK4, NUAK1, NUAK2, PAK1, SIK1, SIK2, BRSK1, BRSK2, SNRK, and QSK), and patient survival across each subtype and treatment group. Our findings provide evidence that LKB1-signaling is associated with improved survival in overall breast cancer. Stratification into breast cancer subtypes show a more complicated relationship; NUAK2, for example, is correlated with improved survival in ER- but is worse in ER+ breast cancer. In evaluating the association of LKB1-signaling pathway expression with relapse free survival of varying breast cancer tumors exposed to chemotherapy or treatment-naive tumors, our data provides baseline knowledge for understanding the pathway dynamics that affect survival and therefore are linked to pathology. This establishes a foundation for studying LKB1 targets with the goal of identifying druggable targets.     

LKB1 when associated with methylatedERα is a marker of bad prognosis in breast cancer

Although the presence of nuclear estrogen receptor is widely used to guide breast cancer therapy, less attention has been paid to the receptor cytoplasmic signaling. Recently, we have shown that this pathway is operative in vivo and is activated in aggressive tumors representing a new potential target for breast cancer therapy. Here, we identified LKB1 as a partner of ERα and we explored its potential role in estrogen nongenomic signaling. The associations between LKB1 expression and the actors of this pathway, namely the methylated form of ERα (metERα), Src and PI3K, have been analyzed both in cultured cells and in 154 primary breast tumor samples. We found that LKB1 is a component of the cytoplasmic signaling complex in breast cell lines as well as in primary breast tumors. Moreover, an inverse correlation between the localization of LKB1 in nuclear and cytoplasmic compartments is observed. Importantly, high expression of cytoplasmic LKB1 is an independent marker of poor prognosis, associated with reduced overall survival (OS) and disease free survival (DFS). Conversely, the presence of nuclear LKB1 associates with increased OS and DFS. In conclusion, our results highlight that LKB1 expression in breast cancer appears to have opposite effects depending on its subcellular localization and may be used as a new prognostic biomarker.     

The tumor suppressor gene LKB1 is associated with prognosis in human breast carcinoma.

PURPOSE: LKB1 (also called STK11) is a recently identified tumor suppressor gene in which its mutation can lead to Peutz-Jeghers syndrome, characterized by gastrointestinal polyps and cancers of different organ systems. Weak expression of this gene does occur at a certain frequency in sporadic breast cancer. This indicates that LKB1 gene may relate to the tumorigenesis of breast cancer. EXPERIMENTAL DESIGN: To investigate the function of the LKB1 gene in sporadic breast cancer, we reintroduced LKB1 into breast cancer cell lines which lack the LKB1 gene. Also, we examined the LKB1 protein expression in human breast cancer samples. RESULTS: We found that reintroducing LKB1 into breast cancer cell lines suppresses cell growth by G(1) cell cycle block. The LKB1-mediated G(1) cell cycle arrest is caused by up-regulation of the expression of p21(WAF1/CIP1) in breast cancer MDA-MB-435 cells. We also demonstrated that low LKB1 protein expression correlates with higher histological grade (P = 0.013), larger tumor size (P = 0.001), progesterone receptor status (P = 0.048), and presence of lymph node metastasis (P = 0.003). Furthermore, LKB1 low expression was associated with a higher relapse rate (P = 0.002) and a worse OS (P = 0.008). CONCLUSIONS: LKB1 plays a role in tumor suppressor function in human breast cancer. LKB1 expression may be a useful prognostic marker in human breast cancer.     

LKB1 mutations and their correlation with LKB1 and Rheb expression in bladder cancer

Although there are extensive studies on the genetics of bladder cancer, several questions remain unanswered. One of the pathways which are altered in bladder cancer is the mTOR signaling pathway. In the present study, we analyzed the expression of Rheb gene and genetic alterations in the LKB1 gene which are the key components of mTOR pathway. Nine exons of the LKB1 gene were analyzed by direct sequencing in 51 bladder cancer patients. To investigate the expression of Rheb and LKB1, real‐time quantitative RT‐PCR was performed in bladder tumor and normal bladder tissue samples. We did not observed a statistically significant difference in Rheb or LKB1 expression between the tumor and normal tissue samples. We detected a novel missense mutation creating stop codon in a high percent of the tumor samples. Five different single nucleotide substitutions were also observed in the introns. Our results indicate that LKB1 gene may play a role in the progression of bladder cancer. © 2012 Wiley Periodicals, Inc.     

The tumor suppressor LKB1 antagonizes WNT signaling pathway through modulating GSK3β activity in cell growth of esophageal carcinoma

The tumor suppressor LKB1 gene encodes a serine–threonine kinase that regulates cell proliferation and polarity. Inactivation of LKB1 by mutations in LKB1 or loss of its expression is highly correlated with lung, ovarian, and pancreatic cancers, and WNT/β-catenin pathway is also known to be involved in many human malignancies. However, the relationship between LKB1 and WNT signaling pathway in esophageal carcinoma remains unknown. The expression of LKB1 in 62 cases of esophageal cancer patients was determined by quantitative real-time PCR. It was found that LKB1 mRNA level was significantly lower than the adjacent normal epithelium and that the LKB1 downregulation was correlating with TNM stages. Moreover, the expression of WNT target genes such as Cyclin D1, C-MYC, MMP2, and FZD2 was significantly upregulated in esophageal cancer tissues. LKB1 overexpression in TE10 cells inhibited TOPFlash luciferase reporter activity and WNT target gene expression even in the presence of WNT3A. Conversely, LKB1 knockdown enhanced WNT signaling activity in esophageal cancer cells. It was also found that LKB1 antagonized WNT signaling pathway through interaction with GSK3β to downregulate β-catenin expression level. Functional investigation revealed that LKB1 suppressed the promotion effects of WNT3A on the cell growth of TE10 cells. The LKB1 functions in regulating cell growth and WNT target genes expression were impaired by GSK3β inhibition, suggesting that LKB1 antagonized WNT-induced cell proliferation through enhancement of GSK3β activity. Together, the interaction between LKB1 and GSK3β upregulates GSK3β activity to suppress WNT-induced cell proliferation in esophageal carcinoma cells. Loss of LKB1 expression may result in the deregulation of WNT/β-catenin pathway to promote malignant progression of esophageal cancer.