Polo-like kinase 1 as a biomarker predicts the prognosis and immunotherapy of breast invasive carcinoma patients

Invasive breast carcinoma(BRCA)is associated with poor prognosis and high risk of mortality.Therefore,it is critical to identify novel biomarkers for the prognostic assessment of BRCA.Methods:The expression data of polo-like kinase 1(PLK1)in BRCA and the corresponding clinical information were extracted from TCGA and GEO databases.PLK1 expression was validated in diverse breast cancer cell lines by quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting.Single sample gene set enrichment analysis(ssGSEA)was performed to evaluate immune infiltration in the BRCA microenvironment,and the random forest(RF)and support vector machine(SVM)algorithms were used to screen for the hub infiltrating cells and calculate the immunophenoscore(IPS).The RF algorithm and COX regression model were applied to calculate survival risk scores based on the PLK1 expression and immune cell infiltration.Finally,a prognostic nomogram was constructed with the risk score and pathological stage,and its clinical potential was evaluated by plotting calibration charts and DCA curves.The application of the nomogram was further validated in an immunotherapy cohort.Results:PLK1 expression was significantly higher in the tumor samples in TCGA-BRCA cohort.Furthermore,PLK1 expression level,age and stage were identified as independent prognostic factors of BRCA.While the IPS was unaffected by PLK1 expression,the TMB and MATH scores were higher in the PLK1-high group,and the TIDE scores were higher for the PLK1-low patients.We also identified 6 immune cell types with high infiltration,along with 11 immune cell types with low infiltration in the PLK1-high tumors.A risk score was devised using PLK1 expression and hub immune cells,which predicted the prognosis of BRCA patients.In addition,a nomogram was constructed based on the risk score and pathological staging,and showed good predictive performance.Conclusions:PLK1 expression and immune cell infiltration can predict post-immunotherapy prognosis of BRCA patients.

Research Progress on Targets and Selective Inhibitors of Polo-like Kinase-1(PLK-1)

In this paper,the biological function of PLK-1,the correlation between PLK-1 and tumors,and the latest research progress on PLK-1 inhibitors under study are reviewed,in order to provide references for the research and development of PLK-1 inhibitors.

RNA干扰技术抑制Polo-like激酶1表达对A549细胞的影响

Polo-like激酶1(Plk1)是参与细胞周期调控的重要分子,已在多种肿瘤中检测到Plk1的高表达,并发现与肿瘤细胞的增殖和预后密切关联.为明确Plk1在肺癌细胞系A549细胞增殖和周期运行中的作用,采用RNA干扰技术,构建能产生siRNA的质粒载体psiRNA-hH1-Plk1并导入A549细胞中.采用RT-PCR检测Plk1mRNA表达的变化,Western印迹检测Plk1、细胞周期蛋白B1、p53蛋白的表达变化,流式细胞术分析细胞周期变化和凋亡;免疫荧光染色检测α微管蛋白的表达.以此观察RNA干扰能否有效抑制Plk1的表达水平,以及抑制后对A549细胞生长的影响.结果表明,psiRNA-hH1-Plk1质粒能特异性地抑制Plk1基因的表达并使其活性下降,细胞周期蛋白B1及p53蛋白的表达水平升高,微管聚集障碍或形成单极的纺锤体,A549细胞增殖减慢,出现G2/M期阻滞并存在细胞凋亡.针对Plk1基因的RNA干扰有望用于肿瘤的基因治疗.

RNA干扰技术抑制Polo-like激酶1表达对A549细胞的影响(英文)

目的:观察RNA干扰技术能否有效抑制非小细胞肺癌细胞株A549细胞中Polo-like激酶1(Plk1)的表达水平,以及抑制后对A549细胞生长的影响。方法:运用脂质体法,以Plk1为靶点,构建能产生siRNA的质粒载体psiRNA-hH1-Plk1并转入A549细胞。RT-PCR检测Plk1 mRNA表达的变化、Western blotting检测Plk1、cyc-linB1、p53蛋白的表达变化、细胞计数分析细胞增殖、流式细胞术分析细胞周期变化和凋亡、免疫荧光染色检测α微管蛋白的表达。结果:psiRNA-hH1-Plk1质粒能特异地抑制Plk1基因的表达并使其活性下降,致使cyclinB1及p53蛋白的表达水平升高,微管聚集障碍或形成单极的纺锤体;A549细胞增殖减慢,出现G2/M期阻滞和凋亡。结论:上述结果提示针对Plk1基因的RNA干扰有望用于肿瘤的基因治疗。

Apoptosis induction with polo-like kinase-1 antisense phosph-orothioate oligodeoxynucleotide of colon cancer cell line SW480

AIM： To investigate the effects of polo-like kinase-1 （PLK1） antisense phosphorothioate oligodeoxynucleotide （ASODN） on apoptosis and cell cycle of human colon cancer cell line SW480. METHODS： After SW480 colon cancer cells were transfected with PLK1 ASODN, Northern and Western blot analyses were used to examine PLK1 gene expression in cancer cells. We studied apoptosis using terminal uridine deoxynucleotidyl nick end labeling. Apoptosis and cell cycle of SW480 cells were examined by fluorescence-activated cell sorter scan. RESULTS： The levels of PLK1 mRNA and protein were greatly inhibited by PLK1 ASODN in SW480 cancer cells transfected with PLK1 ASODN. Apoptosis index （AI） induced PLK1 ASODN in a time- and dose-dependent manner. Results from FLM showed that sub-2N DNA content of transfected cancer cells was significantly increased and arrested at G2/M compared with control groups. CONCLUSION： PLK1 ASODN can induce apoptosis of human colon cancer cell line SW480.

Overexpression of polo-like kinase1 predicts a poor prognosis in hepatocellular carcinoma patients

AIM： To elucidate the role of overexpressed polo-like kinasel （PLK1）in hepatocellular carcinoma （HCC）. METHODS： We prospectively collected clinicopathological, immunohistochemical and semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） data from 135 HCC patients undergoing successful hepatectomy. The correlations between PLK1 mRNA expression and clinicopathologic variables were analyzed by Mann-Whitney U test. Prognostic factors were identified by univariate and multivariate analyses. RESULTS： Immunohistochemical results showed overexpression of PLK1 was mainly found in tumor tissues compared with tumor-free tissue. A similar mRNA result was obtained by semi-quantitative RT-PCR. A total of 111 samples were positive for PLK1 mRNA expression. The positive expression was correlated with venous invasion, tumor nodules and Edmondson grade. Furthermore, 1, 3, 5-year survival rates in the positive expression group were significantly lower than the negative control group. Multivariate analysis showed that positive PLK1 expression was an independent risk factor for HCC. CONCLUSION： PLK1 could be a potential biomarker for diagnosis and therapy for HCC.

Effect of Antisense RNA Targeting Polo-like Kinase 1 on Cell Growth in A549 Lung Cancer Cells

In order to investigate the effect of Polo-like kinase-1 （Plk1） depletion on cell cycle progression and cell growth in lung cancer cells, a recombinant plasmid containing antisense RNA targeting Plk1 （pcDNA3-Plk1） was transfected into A549 cells by lipofectine. RT-PCR and Western-blot were used to detect the Plk1 gene expression. Cell proliferation was evaluated by direct cell counting and bromodeoxyuridine （BrdU） labeling. Cell cycle distribution and apoptosis were examined by flow cytometry, and the inhibition rate （IR） by vinorebline （NVB） was determined by MTF assay. The results showed that after transfection of pcDNA3-Plk1 into A549 cells, the expression levels of Plk1 mRNA and protein were greatly decreased. In pcDNA3-Plk1 transfected groups, abnormal morphological changes of cells and growth inhibition were observed, and the BrdU labeling index was significantly lower than in the control groups （P〈0.05）. Cells in pcDNA3-Plk1 transfected groups were arresed in G2/M phase and apoptosis was detectable 72 h post transfection. IR induced by vinorebline in pcDNA3-Plk1 transfected groups was significantly higher than in other groups. These data suggested that antisense RNA targeting Plk1 could suppress the Plk1 expression, and therefore, significantly inhibit cell proliferation and induce cell cycle arrest and apoptosis. Moreover, it sensitized lung cancer cells to chemotherapy.

Polo-like kinase 1,a new therapeutic target in hepatocellular carcinoma

AIM： To investigate the role of polo-like kinase 1 （PLK1） as a therapeutic target for hepatocellular carcinoma （HCC）. METHODS： PLK1 gene expression was evaluated in HCC tissue and HCC cell lines. Gene knockdown with short-interfering RNA （siRNA） was used to study PLK1 gene and protein expression using real-time reverse transcription polymerase chain reaction （RT-PCR） and Western blotting, and cell proliferation using 3-（4,5-dim ethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2（4-sulf ophenyl）-2H-tetrazolium （MTS） and bromodeoxyuridine （BrdU） assays. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end label- ing （TUNEL） assay, and caspase-inhibition assay. Huh-7 cells were transplanted into nude mice and co-cultured with PLK1 siRNA or control siRNA, and tumor progres- sion was compared with controls. RESULTS： RT-PCR showed that PLK1 was overexpre- ssed 12-fold in tumor samples compared with controls, and also was overexpressed in Huh-7 cells, siRNA against PLK1 showed a reduction in PLK1 gene and protein expression of up to 96% in Huh-7 cells, and a reduction in cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays, respectively. There was a 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this was caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue the apoptotic cells. Immnofluorescence co-localized endonuclease-G to fragmented chromosomes, implicating it in apoptosis. Huh-7 cells transplanted subcutaneously into nude mice showed tumor regression in siPLKl-treated mice, but not in controls. CONCLUSION： Knockdown of PLK1 overexpression in HCC was shown to be a potential therapeutic target, leading to apoptosis through the endonuclease-G path- way.

Dual targeting of Polo-like kinase 1 and baculoviral inhibitor of apoptosis repeat-containing 5 in TP53-mutated hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC),often diagnosed at advanced stages without curative therapies,is the fifth most common malignant cancer and the second leading cause of cancer-related mortality.Polo-like kinase 1(PLK1)is activated in the late G2 phase of the cell cycle and is required for entry to mitosis.Interestingly,PLK1 is overexpressed in many HCC patients and is highly associated with poor clinical outcome.Baculoviral inhibitor of apoptosis repeatcontaining 5(BIRC5)is also highly overexpressed in HCC and plays key roles in this malignancy.AIM To determine the expression patterns of PLK1 and BIRC5,as well as their correlation with p53 mutation status and patient clinical outcome.METHODS The expression patterns of PLK1 and BIRC5,and their correlation with p53 mutation status or patient clinical outcome were analyzed using a TCGA HCC dataset.Cell viability,cell apoptosis,and cell cycle arrest assays were conducted to investigate the efficacy of the PLK1 inhibitors volasertib and GSK461364 and the BIRC5 inhibitor YM155,alone or in combination.The in vivo efficacy of volasertib and YM155,alone or in combination,was assessed in p53-mutated Huh7-derived xenograft models in immune-deficient NSIG mice.RESULTS Our bioinformatics analysis using a TCGA HCC dataset revealed that PLK1 and BIRC5 were overexpressed in the same patient subset and their expression was highly correlated.The overexpression of both PLK1 and BIRC5 was more frequently detected in HCC with p53 mutations.High PLK1 or BIRC5 expression significantly correlated with poor clinical outcome.PLK1 inhibitors(volasertib and GSK461364)or a BIRC5 inhibitor(YM155)selectively targeted Huh7 cells with mutated p53,but not HepG2 cells with wild-type p53.The combination treatment of volasertib and YM155 synergistically inhibited the viability of Huh7 cells via apoptotic pathway.The efficacy of volasertib and YM155,alone or in combination,was validated in vivo in a Huh7-derived xenograft model.CONCLUSION PLK1 and BIRC5 are highly co-expressed in p53-mutated HCC and inhibition of both PLK1 and BIRC5 synergistically compromises the viability of p53-mutated HCC cells in vitro and in vivo.

乳腺癌组织中FOXM1和PLK1的表达及意义

目的探讨叉头框转录因子M1(FOXM1)及Polo样激酶1(PLK1)在乳腺癌组织中的表达及临床意义。方法采用免疫组织化学法检测FOXM1及PLK1蛋白在803例乳腺浸润性导管癌和乳腺癌旁组织中的表达情况及其与乳腺癌组织临床病理特征间的关系。结果 FOXM1及PLK1在乳腺浸润性导管癌组织中的阳性表达率分别为59.78%(480/803)和27.90%(224/803),显著高于其在乳腺癌旁组织中的表达(29.89%,0),差异具有统计学意义(χ~2=145.011,χ~2=260.307,P=0.000)。FOXM1及PLK1蛋白的表达与乳腺癌的组织学分级、淋巴结转移及临床分期相关,而与肿瘤大小无关,且二者表达具有正相关关系(r=0.414,P<0.01)。结论 FOXM1及PLK-1可能协同参与了乳腺癌的发生、发展,并可能成为乳腺癌预后评估的重要指标。

胃癌组织PLK1的表达及其意义

目的:研究Polo样激酶1(Polo-like kinase 1,PLK1)在胃癌中的表达与各临床病理特征、抑癌基因和肿瘤增殖活性的关系,探讨PLK1在胃癌发生发展中的作用以及其临床意义. 方法:运用免疫组织化学的方法检测正常胃组织15例, 非典型增生组织11例,胃癌54例中PLK1,Ki67, P53的表达. 结果:PLK1在正常胃组织中表达均为阴性;11例非典型增生组织有4例弱阳性;胃癌中有48例阳性(88.9%), 其表达与组织分化程度、远处转移和淋巴结转移无关(P>0.05),与浸润深度(x2=6.775,P<0.01)和临床分期(x2=9.009,P<0.01)相关.在胃癌中,P53阳性38例(70.4%),PLK1的表达与P53蛋白积聚相关(x2=6.664, P<0.05).胃癌中Ki67标记指数均值为34.7±13.4%, 范围为10.3-60.1%,PLK1的表达与Ki67呈正相关(r=0.720,P<0.01). 结论:PLK1在胃癌中高表达,与肿瘤的增殖活性和抑癌基因相关,在胃癌发生发展中起重要作用.

丝/苏氨酸蛋白激酶Plk1研究进展

Plk1是一类从酵母到人类都高度保守的丝氨酸/苏氨酸蛋白激酶。Plk1与不同的细胞周期检查点的精密调控有关,从而确保了细胞周期事件按照严格的时间和顺序正常进行。Plk1在增殖活跃的细胞中呈高水平表达,Plk1的高度表达和肿瘤患者的低存活率之间具有显著的统计相关性。Plk1可能是非常有效的抗癌药物设计的靶点。目前已有几种Plk1的小分子抑制剂在体内外的实验中显示出了良好的应用前景。

中心体Plk1在儿童急性白血病中的表达及其意义

目的研究Polo-like kinase1(Plk1)在儿童急性白血病骨髓细胞中的表达水平。方法采用免疫组化法检测Plk1在儿童急性白血病骨髓细胞中的表达水平。结果 30例急性非淋巴细胞白血病(acute non-lymphoblastic Ieukemia,ANLL)及30例急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)儿童患者与19例正常儿童(normal,N)骨髓细胞比较,Plk1表达有显著性差异(χ2=43.947,P<0.01),在ANLL及ALL中表达水平明显高于正常儿童。结论 Plk1在儿童急性白血病中呈异常高表达,可能成为白血病治疗的有效靶点及重要标志物。

ATP非竞争性的Plk1抑制剂研究进展

Polo样激酶1(Plk1)是一种丝/苏氨酸蛋白激酶,在有丝分裂中起着重要作用。目前进入临床的Plk1抑制剂大部分是作用于ATP结合口袋的,此类ATP竞争性抑制剂活性高,但选择性差,易交叉耐药。因此,ATP非竞争性的抑制剂受到关注。本文阐述了ATP非竞争性的Plk1抑制剂的研究进展。

Polo-like kinase 1短发卡RNA重组腺病毒载体构建及其生物学应用

目的构建针对人类Polo-like kinase1（Plk1）基因的短发卡RNA（shRNA）真核表达载体，并进行腺病毒包被。方法设计并合成含有小发夹结构的Plk1 shRNA对应模板序列，退火并与pYr-1．1载体重组质粒；通过LR体外同源重组将pYr-1．1-Plk1—shRNA表达框构建至pAd—DEST腺病毒表达载体上；包装重组腺病毒；感染人类胰腺癌细胞BxPC-3验证干扰效率。实验分3组：对照组（Control）、空载组[rAd-增强绿色荧光蛋白（tAd—EGFP）]、实验组（rAd—shPlk1），实时定量反转录聚合酶链反应（RT-qPCR）和Western blot检测Plk1基因的表达，流式细胞术检测细胞周期的变化及对细胞凋亡的影响。结果腺病毒感染BxPC-3细胞后，RT-qPCR检测Plk1 mRNA表达量：对照组1．047±0．315、空载组1．121±0．199、实验组0．119±0．050；Western blot检测Plk1蛋白表达量：对照组0．760±0．088、空载组0．743±0．101、实验组0．050±0．043，实验组较对照组及空载组Plk1 mRNA和蛋白水平均明显降低（P〈0．01）。流式细胞术检测G2期细胞比例：对照组6．077±0．056、空载组6．533±1．644、实验组33．427±7．774，实验组G：期细胞数比例显著高于空载组和对照组（P〈0．01）。感染24h后实验组的细胞凋亡率明显高于空载组和对照组（P〈0．01）。结论成功构建包被有Plk1 shRNA的腺病毒表达载体，且重组腺病毒载体可以抑制Plk1基因的表达，引起细胞周期G2／M期停滞和促进细胞凋亡。

MCC1019,a selective inhibitor of the Polo-box domain of Polo-like kinase 1 as novel,potent anticancer candidate

Polo-like kinase(PLK1) has been identified as a potential target for cancer treatment.Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain(PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethylbenzofuran-2-carboxylic acid ethyl ester(designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1 PBD was confirmed using fluorescence polarization and microscale thermophoresis.This compound exerted specificity towards PLK1 over PLK2 and PLK3. MCC1019 showed cytotoxic activity in a panel of different cancer cell lines. Mechanistic investigations in A549 lung adenocarcinoma cells revealed that MCC1019 induced cell growth inhibition through inactivation of AKT signaling pathway, it also induced prolonged mitotic arrest—a phenomenon known as mitotic catastrophe, which is followed by immediate cell death via apoptosis and necroptosis. MCC1019 significantly inhibited tumor growth in vivo in a murine lung cancer model without affecting body weight or vital organ size, and reduced the growth of metastatic lesions in the lung. We propose MCC1019 as promising anti-cancer drug candidate.

Effect of antisense RNA targeting polo-like kinase 1 on cell cycle and proliferation in A549 cells

Background Expression of polo-like kinase 1 (Plk1) is elevated in lung cancer and has been proposed as having prognostic value and related to resistance to chemotherapy and radiation. In addition, Plk1 has several functions in mitotic progression. In this study, the authors investigated t he effect of Plk1 depletion on cell cycle progression and proliferation in A549 cells, a lung cancer cell line.Methods A recombinant plasmid containing antisense RNA targeting Plk1 (pcDNA3-Plk1) was transfected into A549 cells. Reverse transcription-polymerase chain reaction and Western blot were used to examine Plk1 gene expression. Cell proliferation was evaluated by direct cell counting and bromodeoxyuridine (BrdU) labelling. Cell cycle and apoptosis were examined by flow cytometry. Expression of α-tubulin was detected by immunofluorescence, and the inhibition rate (IR) by chemotherapeutic agents was determined by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide assay.Results After transfection into A549 cells, pcDNA3-Plk1 reduced Plk1 mRNA by 46.75% for 24 hours and by 61.84% for 48 hours. Plk1 protein was significantly decreased simultaneously (P<0.05). Abnormal morphological changes of cells and growth inhibition were observed in pcDNA3-Plk1 transfected groups. The BrdU labelling index was 25.59% 48 hours after transfection, which was significantly lower than that of the control groups (P<0.05). Forty-eight hours after transfection, there showed absence of microtubule polymerization and spindle abnormalities in staining for α-tubulin. A549 cells showed a strong G 2/M arrest and apoptosis 72 hours post transfection. IR of vinorelbine in pcDNA3-Plk1 transfected groups was significantly higher than that of the other groups (P<0.05, respectively).Conclusions Plk1 depletion interferes with spindle formation, induces cell cycle arrest and apoptosis, and consequently inhibits cell proliferation in A549 cells. Moreover, it sensitizes lung cancer cells to chemotherapy.

Diversity evolution and jump of Polo-like kinase 1 inhibitors

Polo-like kinase 1 （Plkl）, a member of a family of serine/threonine kinases, is an attractive target for the development of anti- cancer drugs because it is involved in the regulation of cell-cycle progression and cytokinesis. This kinase provides two pock- ets for developing Plkl inhibitors： the N-terminal catalytic domain （NCD） and the polo-box domain （PBD）. For both of the two pockets, some natural products were identified as Plkl inhibitors and some synthetic Plkl inhibitors were developed by mimicking ATP and phosphopeptides, natural products binding to NCD and PBD respectively. This article not only reviews the progression of Plkl inhibitors binding to these two pockets, but also discusses diversity evolution and jump in the process of drug development using Plkl inhibitors as examples and how they impact on drug design and pharmacopbore modeling.

Polo-like kinase 1,on the rise from cell cycle regulation to prostate cancer development

Polo-like kinase 1(Plk1),a well-characterized member of serine/threonine kinases Plk family,has been shown to play pivotal roles in mitosis and cytokinesis in eu-karyotic cells.Recent studies suggest that Plk1 not only controls the process of mitosis and cytokinesis,but also,going beyond those previously described functions,plays critical roles in DNA replication and Pten null prostate cancer initiation.In this review,we briefly summarize the functions of Plk1 in mitosis and cytokinesis,and then mainly focus on newly discov-ered functions of Plk1 in DNA replication and in Pten-null prostate cancer initiation.Furthermore,we briefly introduce the architectures of human and mouse pros-tate glands and the possible roles of Plk1 in human prostate cancer development.And finally,the newly chemotherapeutic development of small-molecule Plk1 inhibitors to target Plk1 in cancer treatment and their translational studies are also briefly reviewed.

A combination therapy for KRAS-mutant lung cancer by targeting synthetic lethal partners of mutant KRAS

The KRAS gene is frequently mutated in multiple cancer types,but it fell off the drug discovery radar for many years because of its inherent "undruggable" structure and undefined biological properties.As reported in the paper entitled "Suppression of KRas-mutant cancer through the combined inhibition of KRAS with PLK1 and ROCK" in Nature Communications,we performed a synthetic lethal screening with a combinatorial strategy on a panel of clinical drugs;we found that combined inhibition of polo-like kinase 1 and RhoA/Rho kinase markedly suppressed tumor growth in mice.An increase in the expression of the tumor suppressor P21^(WAF1/CIP1) contributed to the synergistic mechanism of the combination therapy.These findings open a novel avenue for the treatment of KRAS-mutant lung cancer.