Group Ⅱ p21-activated kinases as therapeutic targets in gastrointestinal cancer

P21-activated kinases(PAKs) are central players in various oncogenic signaling pathways. The six PAK family members are classified into group Ⅰ(PAK1-3) and group Ⅱ(PAK4-6). Focus is currently shifting from group Ⅰ PAKs to group Ⅱ PAKs. Group Ⅱ PAKs play important roles in many fundamental cellular processes, some of which have particular significance in the development and progression of cancer. Because of their important functions, group Ⅱ PAKs have become popular potential drug target candidates. However, few group Ⅱ PAKs inhibitors have been reported, and most do not exhibit satisfactory kinase selectivity and "drug-like" properties. Isoform- and kinase-selective PAK inhibitors remain to be developed. This review describes the biological activities of group Ⅱ PAKs, the importance of group Ⅱ PAKs in the development and progression of gastrointestinal cancer, and smallmolecule inhibitors of group Ⅱ PAKs for the treatment of cancer.

The p21-activated kinases in neural cytoskeletal remodeling and related neurological disorders

The serine/threonine p21-activated kinases(PAKs),as main effectors of the Rho GTPases Cdc42 and Rac,represent a group of important molecular switches linking the complex cytoskeletal networks to broad neural activity.PAKs show wide expression in the brain,but they differ in specific cell types,brain regions,and developmental stages.PAKs play an essential and differential role in controlling neural cytoskeletal remodeling and are related to the development and fate of neurons as well as the structural and functional plasticity of dendritic spines.PAK-mediated actin signaling and interacting functional networks represent a common pathway frequently affected in multiple neurodevelopmental and neurodegenerative disorders.Considering specific small-molecule agonists and inhibitors for PAKs have been developed in cancer treatment,comprehensive knowledge about the role of PAKs in neural cytoskeletal remodeling will promote our understanding of the complex mechanisms underlying neurological diseases,which may also represent potential therapeutic targets of these diseases.

p21-activated kinase signalling in pancreatic cancer: New insights into tumour biology and immune modulation

Pancreatic cancer is one of the most aggressive and lethal malignancies worldwide, with a very poor prognosis and a five-year survival rate less than 8%. This dismal outcome is largely due to delayed diagnosis, early distant dissemination and resistance to conventional chemotherapies. Kras mutation is a well-defined hallmark of pancreatic cancer, with over 95% of cases harbouring Kras mutations that give rise to constitutively active forms of Kras. As important down-stream effectors of Kras, p21-activated kinases(PAKs) are involved in regulating cell proliferation, apoptosis, invasion/migration and chemo-resistance. Immunotherapy is now emerging as a promising treatment modality in the era of personalized anti-cancer therapeutics. In this review, basic knowledge of PAK structure and regulation is briefly summarised and the pivotal role of PAKs in Kras-driven pancreatic cancer is highlighted in terms of tumour biology and chemoresistance. Finally, the involvement of PAKs in immune modulation in the tumour microenvironment is discussed and the potential advantages of targeting PAKs are explored.

新型p-21活化激酶4抑制剂的研发进展

目的研究p-21活化激酶4(p21-activated kinase 4,PAK4)抑制剂在肿瘤治疗中的应用,为新型PAK4抑制剂的研发提供参考。方法从母核结构、共晶结构、药物活性、药代动力学、作用机制等方面,对小分子PAK4抑制剂进行论述。结果在多种PAK4抑制剂结构类型中,苯并呋喃类化合物KPT-9274对PAK4的抑制活性显著,对多种癌症具有抗肿瘤作用,是目前唯一处于临床试验阶段的PAK4抑制剂,可以作为今后PAK4抑制剂设计的基础。结论对苯并呋喃类化合物进一步结构优化,有望获得活性更优的PAK4抑制剂用于抗肿瘤研究。

Targeting P21-activated kinase suppresses proliferation and enhances chemosensitivity in T-cell lymphoblastic lymphoma

T-cell lymphoblastic lymphoma(T-LBL)is a highly aggressive non-Hodgkin lymphoma with a poor prognosis.P21-activated kinase(PAK)is a component of the gene expression-based classifier that can predict the prognosis of T-LBL.However,the role of PAK in T-LBL progression and survival remains poorly understood.Herein,we found that the expression of PAK1 was significantly higher in T-LBL cell lines(Jurkat,SUP-T1,and CCRF-CEM)compared to the human T-lymphoid cell line.Moreover,PAK2 mRNA level of 32 relapsed T-LBL patients was significantly higher than that of 37 cases without relapse(P=.012).T-LBL patients with high PAK1 and PAK2 expression had significantly shorter median RFS than those with low PAK1 and PAK2 expression(PAK1,P=.028;PAK2,P=.027;PAK1/2,P=.032).PAK inhibitors,PF3758309(PF)and FRAX597,could suppress the proliferation of T-LBL cells by blocking the G1/S cell cycle phase transition.Besides,PF could enhance the chemosensitivity to doxorubicin in vitro and in vivo.Mechanistically,through western blotting and RNA sequencing,we identified that PF could inhibit the phosphorylation of PAK1/2 and downregulate the expression of cyclin D1,NF-κB and cell adhesion signaling pathways in T-LBL cell lines.These findings suggest that PAK might be associated with T-LBL recurrence and further found that PAK inhibitors could suppress proliferation and enhance chemosensitivity of T-LBL cells treated with doxorubicin.Collectively,our present study underscores the potential therapeutic effect of inhibiting PAK in T-LBL therapy.

蛋白激酶C抑制剂对SACC-83系P_(21)^(ras)、c-myc表达的影响

本实验采用ＰＫＣ抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ作用于ＳＡＣＣ－８３系，观察对Ｐ２１ｒａｓ、ｃ－ｍｙｃ表达的影响．结果表明，Ｓｔａｕｒｏｐｏｒｉｎｅ可明显降低Ｈ－ｒａｓ、ｃ－ｍｙｃ表达程度（ｐ＜０．０１），这提示ＰＫＣ对ｒａｓ、ｃ－ｍｙｃ基因表达具有调控作用，ＰＫＣ抑制剂可能通过调控癌基因的表达来表现抗肿瘤作用的．

Gene polymorphisms of interleukin-28, p21-activated protein kinases 4, and response to interferon-α based therapy in Chinese patients with chronic hepatitis B

Background Peg-lnterferon-a treatment is expensive and associated with considerable adverse effects, selection of patients with the highest probability of response is essential for clinical practice. The objective of this study was to assess the relationship between the gene polymorphisms of interleukin-28 （IL-28）, p21-activated protein kinase 4 （PAK4） and the response to interferon treatment in chronic hepatitis B patients. Methods Two hundred and forty interferon-naive treatment HBeAg seropositive chronic hepatitis B patients were enrolled in the present prospective nested case-control study. Peripheral blood samples were collected, including 92 with favorable response and 148 without response to the interferon treatment. Rs8099917, rs12980602, and rs9676717 SNP was genotyped using matrix assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF MS）. Results IL-28 genotype was not associated with response to interferon treatment （OR for GT/GG vs. TT, 0.881 （95% CI 0.388-2.002）; P=0.762; OR for CT/CC vs. TT, 0.902 （95% CI 0.458-1.778）; P=-0.766）. Rs9676717 in PAK4 genotype was independently associated with the response （OR for CT/CC vs. TT, 0.524 （95% CI 0.310-0.888）; P=0.016）. When adjusting for age, gender, smoking, drinking, levels of hepatitis B virus DNA, and alanine aminotransferase （ALT）, rs9676717 genotype TT appeared to be associated with a higher probability of response for interferon treatment （OR, 0.155 （95% CI 0.034-0.700）; P=0.015）. Conclusion Genotype TTfor rs9676717 in PAK4 gene and no drinking may be predictive of the interferon-a treatment success.

PAK1基因对大肠癌细胞体外侵袭能力的影响

目的研究p21-activated kinase-1(PAK1)基因对大肠癌细胞系体外侵袭能力的影响。方法把重组p21活化蛋白激酶1质粒用脂质体转染大肠癌SW480细胞,同时设立空白对照组和空载体对照组。于转染后48h采用免疫印迹方法检测PAK1的蛋白表达水平,Boyden小室模型检测大肠癌细胞SW480在转染重组PAK1基因质粒后侵袭能力的变化。结果SW480细胞转染p21活化蛋白激酶1重组质粒后,与空白对照和空载体对照相比,PAK1蛋白水平明显增加,细胞的体外侵袭能力增强。结论转染pPAK1重组质粒能够有效上调PAK1基因,增强大肠癌细胞系体外侵袭潜能,提示PAK1基因高表达可能与大肠癌细胞的侵袭和转移等生物学行为相关。

真核绿色荧光蛋白表达载体pEGFP-C1/PAK-1的构建及其在结直肠癌SW480细胞内的表达

目的:构建重组p21-activated kinase-1(PAK1)基因绿色荧光蛋白表达载体pEGFP-C1/PAK1,并转染入结直肠癌细胞SW480中表达.方法:在南方医科大学附属南方医院消化研究所实验室,从人类结直肠癌细胞株SW620细胞提取总RNA,经逆转录聚合酶链式反应获得人PAK1 cDNA片段,经过限制性内切酶进行酶切,T4连接酶进行连接,将目的基因克隆至真核绿色荧光蛋白表达载体pEGFP-C1上,然后转染结直肠癌细胞株SW480,观察其在细胞中表达.结果:重组载体经限制性内切酶酶切鉴定和DNA序列分析验证,显示插入载体的序列与目的基因一致,而且该重组载体能够在SW480细胞中表达.结论:成功构建了真核绿色荧光蛋白表达载体pEGFP-C1/PAK1,为研究PAK1在结直肠癌中的生物学功能奠定了基础.

Activation of Rac1-PI3K/Akt is required for epidermal growth factorinduced PAK1 activation and cell migration in MDA-MB-231 breast cancer cells

Epidermal growth factor （EGF） may increase cell motility, an event implicated in cancer cell invasion and metastasis. However, the underlying mechanisms for EGF-induced cell motility remain elusive. In this study, we found that EGF treatment could activate Ras-related C3 botulinum toxin substrate 1 （Racl）, PI3K/Akt and p21- actived kinase （PAK1） along with cell migration. Ectopic expression of PAK1 K299R, a dominant negative PAK1 mutant, could largely abolish EGF-induced cell migration. Blocking PI3K/Akt signalling with LY294002 or Akt siRNA remarkably inhibited both EGF-induced PAK1 activation and cell migration. Furthermore, expression of dominant-negative Racl （T17N） could largely block EGF-induced PI3K/Akt-PAK1 activation and cell migration. Interestingly, EGF could induce a significant production of ROS, and N-acetyl-L-cysteine, a scavenger of ROS which abolished the EGF-induced ROS generation, cell migration, as well as activation of PI3K/Akt and PAK, but not Racl. Our study demonstrated that EGF-induced cell migration involves a cascade of signalling events, including activation of Racl, generation of ROS and subsequent activation of PI3K/Akt and PAK1.

Molecular determinants of the antitumor effects of trichostatin A in pancreatic cancer cells

AIM:To gain molecular insights into the action of the histone deacetylase inhibitor(HDACI) trichostatin-A(TSA) in pancreatic cancer(PC) cells.METHODS:Three PC cell lines,BxPC-3,AsPC-1 and CAPAN-1,were treated with various concentrations of TSA for def ined periods of time.DNA synthesis was assessed by measuring the incorporation of 5-bromo-2'deoxyuridine.Gene expression at the level of mRNA was quantif ied by real-time polymerase chain reaction.Expression and phosphorylation of proteins was monitored by immunoblotting,applying an infrared imaging technology.To study the role of p38 MAP kinase,the specif ic enzyme inhibitor SB202190 and an inactive control substance,SB202474,were employed.RESULTS:TSA most eff iciently inhibited BrdU incorporation in BxPC-3 cells,while CAPAN-1 cells displayed the lowest and AsPC-1 cells an intermediate sensitivity.The biological response of the cell lines correlated with the increase of histone H3 acetylation after TSA application.In BxPC-3 cells(which are wild-type for KRAS),TSA strongly inhibited phosphorylation of ERK 1/2 and AKT.In contrast,activities of ERK and AKT in AsPC-1 and CAPAN-1 cells(both expressing oncogenic KRAS) were not or were only modestly affected by TSA treatment.In all three cell lines,but most pronounced in BxPC-3 cells,TSA exposure induced an activation of the MAP kinase p38.Inhibition of p38 by SB202190 slightly but signif icantly diminished the antiproliferative effect of TSA in BxPC-3 cells.Interestingly,only BxPC-3 cells responded to TSA treatment by a signif icant increase of the mRNA levels of bax,a pro-apoptotic member of the BCL gene family.Finally,in BxPC-3 and AsPC-1 cells,but not in the cell line CAPAN-1,signif icantly higher levels of the cell cycle inhibitor protein p21Waf1 were observed after TSA application.CONCLUSION:The biological effect of TSA in PC cells correlates with the increase of acetyl-H3,p21Waf1,phospho-p38 and bax levels,and the decrease of phosphoERK 1/2 and phospho-AKT.

MiR-32-5p aggravates intestinal epithelial cell injury in pediatric enteritis induced by Helicobacter pylori

BACKGROUND Pediatric enteritis is one of the infectious diseases in the digestive system that causes a variety of digestive problems,including diarrhea,vomiting,and bellyache in children.Clinically,Helicobacter pylori(H.pylori)infection is one of the common factors to cause pediatric enteritis.It has been demonstrated that aberrant expression of microRNAs(miRNAs)is found in gastrointestinal diseases caused by H.pylori,and we discovered a significant increase of miR-32-5p in H.pylori-related pediatric enteritis.However,the exact role of miR-32-5p in it is still unknown.AIM To investigate the role of aberrant miR-32-5p in pediatric enteritis induced by H.pylori.METHODS MiR-32-5p expression was detected by quantitative real time-polymerase chain reaction.The biological role of miR-32-5p in H.pylori-treated intestinal epithelial cells was evaluated by Cell Counting Kit-8 assay and flow cytometry.The potential target of miR-32-5p was predicted with TargetScanHuman and verified by luciferase assay.The downstream mechanism of miR-32-5p was explored by using molecular biology methods.RESULTS We found that miR-32-5p was overexpressed in serum of H.pylori-induced pediatric enteritis.Further investigation revealed that H.pylori infection promoted the death of intestinal epithelial cells,and increased miR-32-5p expression.Moreover,miR-32-5p mimic further facilitated apoptosis and inflammatory cytokine secretion of intestinal epithelial cells.Further exploration revealed that SMAD family member 6(SMAD6)was the direct target of miR-32-5p,and SMAD6 overexpression partially rescued cell damage induced by H.pylori.The following experiments showed that miR-32-5p/SMAD6 participated in the apoptosis of intestinal epithelial cells induced by transforming growth factor-β-activated kinase 1(TAK1)-p38 activation under H.pylori infection.CONCLUSION Our work uncovered the crucial role of aberrant expression of miR-32-5p in H.pylori–related pediatric enteritis,and suggested that the TAK1-p38 pathway is involved in it.

Ⅰ类PAKs在神经退行性疾病的研究进展

P21活化的蛋白激酶(p21-activated kinases,PAKs)是Rho家族中GTP酶重要效应物,已证实对细胞繁殖和生存有重要意义。研究发现PAKs与神经元树突发育、细胞骨架的形成以及在细胞信号转导等方面起重要作用。本研究对Ⅰ类PAKs在神经退行性疾病中作用研究进展作一综述。

p-21活化激酶-1、Snail在结直肠癌侵袭与转移中的作用

目的观察p-21活化激酶-1（PAK1）、Snail在结直肠癌侵袭与转移中的作用。方法采用荧光原位杂交和免疫组织化学方法分别检测60例结直肠癌患者的正常结直肠黏膜与结直肠癌组织中PAK1和Snail的表达，分析两者在结卣肠癌中表达的意义以及相关性。结果PAK1mRNA、SnailmRNA存结随肠癌中的表达率分别为70．00％（42／60）和73．33％（44／60），均显著高于正常对照组（P〈0．05）；PAK1mRNA在Dukes不同分期中表达差异有统计学意义（x2=6．0708，P〈0．05），在有淋巴结转移的结直肠癌中表达显著高于无淋巴结转移的结直肠癌（x2=5．8764，P〈0．05）；SnailmRNA存Dukes不同分期中表达差异有统计学意义（x2=6．7930，P〈0．05），在有淋巴结转移的结直肠癌中表达显著高于无淋巴结转移的结直肠癌（x2=6．2130，P〈0．05）；PAK1和Snail蛋白在结直肠癌组织巾的表达呈正相关（r=0．31924，P〈0．05）。结论PAK1和Snail的高表达与结直肠癌的侵袭和转移有关；PAK1和Snail在结直肠癌侵袭和转移过程中可能存在相互促进作用。

Antitumor synergism between PAK4 silencing and immunogenic phototherapy of engineered extracellular vesicles

Immunotherapy has revolutionized the landscape of cancer treatment.However,single immunotherapy only works well in a small subset of patients.Combined immunotherapy with antitumor synergism holds considerable potential to boost the therapeutic outcome.Nevertheless,the synergistic,additive or antagonistic antitumor effects of combined immunotherapies have been rarely explored.Herein,we established a novel combined cancer treatment modality by synergizing p21-activated kinase 4(PAK4)silencing with immunogenic phototherapy in engineered extracellular vesicles(EVs)that were fabricated by coating M1 macrophage-derived EVs on the surface of the nano-complex cores assembled with si RNA against PAK4 and a photoactivatable polyethyleneimine.The engineered EVs induced potent PAK4 silencing and robust immunogenic phototherapy,thus contributing to effective antitumor effects in vitro and in vivo.Moreover,the antitumor synergism of the combined treatment was quantitatively determined by the Compu Syn method.The combination index(CI)and isobologram results confirmed that there was an antitumor synergism for the combined treatment.Furthermore,the dose reduction index(DRI)showed favorable dose reduction,revealing lower toxicity and higher biocompatibility of the engineered EVs.Collectively,the study presents a synergistically potentiated cancer treatment modality by combining PAK4 silencing with immunogenic phototherapy in engineered EVs,which is promising for boosting the therapeutic outcome of cancer immunotherapy.

沉默PAK4基因对胶质瘤细胞U251侵袭与迁移能力的影响

目的观察沉默P21活化酶4(p-21 activated kinase 4, PAK4)基因对神经胶质瘤细胞U251侵袭与迁移能力的影响。方法构建稳定沉默PAK4基因的U251细胞系,shRNA-U251(转染空载组作为对照即shNC组);应用免疫荧光实验检测PAK4在胶质瘤细胞系U251中的表达;应用细胞划痕愈合实验与Transwell侵袭实验比较两组细胞的迁移与侵袭能力的变化;通过Western blotting实验检测沉默PAK4基因后U251细胞系中基质金属蛋白酶MMP2与MMP9的表达改变。结果 PAK4蛋白主要表达于胶质瘤细胞系U251细胞核与细胞质; shRNA-U251组细胞划痕愈合率明显降低; shRNA-U251组细胞进入小室下壁的数量明显减少,shRNA-U251组细胞MMP2与MMP9蛋白的表达水平显著下调。结论沉默PAK4基因下调神经胶质瘤细胞U251的迁移与侵袭能力与降低基质金属蛋白酶MMP2、MMP9的表达相关。

Investigation and experimental validation of curcumin-related mechanisms against hepatocellular carcinoma based on network pharmacology

Objective:To determine the potential molecular mechanisms underlying the therapeutic effect of curcumin on hepatocellular carcinoma(HCC)by network pharmacology and experimental in vitro validation.Methods:The predictive targets of curcumin or HCC were collected from several databases.the identified overlapping targets were crossed with Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses using the Database for Annotation,Visualization,and Integrated Discovery(DAVID)platform.Two of the candidate pathways were selected to conduct an experimental verification.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium(MTT)assay was used to determine the effect of curcumin on the viability of Hep G2 and LO2 cells.The apoptosis and autophagy of Hep G2 cells were respectively detected by flow cytometry and transmission electron microscopy.Besides,western blot and real-time polymerase chain reaction(PCR)were employed to verify the p53 apoptotic pathway and adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)autophagy pathway.Hep G2 cells were pretreated with pifithrin-α(PFT-α)and GSK690693 for further investigation.Results:The 167 pathways analyzed by KEGG included apoptosis,autophagy,p53,and AMPK pathways.The GO enrichment analysis demonstrated that curcumin was involved in cellular response to drug,regulation of apoptotic pathway,and so on.The in vitro experiments also confirmed that curcumin can inhibit the growth of Hep G2 cells by promoting the apoptosis of p53 pathway and autophagy through the AMPK pathway.Furthermore,the protein and messenger RNA(m RNA)of the two pathways were downregulated in the inhibitor-pretreated group compared with the experimental group.The damage-regulated autophagy modulator(DRAM)in the PFT-α-pretreated group was downregulated,and p62 in the GSK690693-pretreated group was upregulated.Conclusions:Curcumin can treat HCC through the p53 apoptotic pathway and the AMPK/Unc-51-like kinase 1(ULK1)autophagy pathway,in which the mutual transformation of autophagy and apoptosis may occur through DRAM and p62.

PAK GroupⅠ的异常表达与非小细胞肺癌的恶性程度相关

目的探讨P21活化激酶(p21-activated kinase,PAK)group Ⅰ在非小细胞肺癌中的表达模式和意义。方法应用免疫组化及Western Blot方法,分析检测了肺癌组织中PAK Group Ⅰ的表达情况及其与临床病理因素的关系。结果与正常肺组织相比,PAK Group Ⅰ在肺癌组织中出现明显的胞浆过表达67.31%(105/156),并且其异常表达模式与肺癌组织的组织学类型(P=0.027)、高分期(P=0.007)、淋巴结转移(P=0.035)及患者的不良预后(P<0.001)相关。结论肺癌组织中PAK Group Ⅰ蛋白过表达是非小细胞肺癌的恶性程度的重要标志之一。

PAK1 is a novel cardiac protective signaling molecule

We review here the novel cardiac protective effects of the multifunctional enzyme, p21-aetivated kinase 1 （PAK1）, a member of a serine/threonine protein kinase family. Despite the large body of evidence from studies in noncardiac tissue indicating that PAK1 activity is key in the regulation of a number of cellular functions, the role of PAK1 in the heart has only been revealed over the past few years. In this review, we assemble an overview of the recent findings on PAKI signaling in the heart, particularly its cardiac protective effects. We present a model for PAK1 signaling that provides a mechanism for specifically affecting cardiac cellular processes in which regulation of protein phosphorylation states by protein phosphatase 2A （PP2A） predominates. We discuss the anti-adrenergic and antihypertrophic cardiac protective effects of PAK1, as well as its role in maintaining ventricular Ca2＋ homeostasis and electrophysiological stability under physiological, β-adrenergic and hypertrophic stress conditions.

Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the acti-vation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modifica-tion but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.