| Lung cancer and colon cancer have high incidence rates of malignant tumours in China,and the high morbidity and mortality of lung cancer are reasons why it ranks first amongst all malignant tumours.On the basis of different pathological features,lung cancer can be divided into non-small cell lung cancer(NSCLC)and small cell lung cancer.NSCLC accounts for 80%of the total number of lung cance.Although treatment methods have greatly improved,the overall prognosis of lung cancer remains poor,and the 5-year survival rate of patients with NSCLC is about 15%.Molecular targeted therapy is a new approach for cancer treatment.Unlike traditional chemotherapy,these drugs target specific molecules to prevent cancer cell growth or target normal cells that have been "hijacked" by the tumour in its attempts to grow.Therefore,targeted drugs have fewer side effects than other cytotoxic drugs used for cancers.The most common gene mutation in lung cancer is epidermal growth factor receptor(EGFR)mutation,which accounts for about 10-35%of lung cancer patients.Several lung cancer molecule-targeted drugs,such as gefitinib or erlotinib that target epidermal growth factor receptor(EGFR),are especially effective in the treatment of patients with NSCLC who harbour activating EGFR mutations and the median survival was 52.2 months.Another TKI agent that targets tyrosine kinase anaplastic lymphoma kinase(ALK)is also effective for ALK rearrangement in patients with NSCLC.Nevertheless,in lung cancer,ALK rearrangement only account for 3%-7%of the total lung cancer.The other major gene mutation is KRAS mutation,which accounts for about 15-25%of patients with lung cancer.However,the drugs targeted for KRAS mutation fail to achieve effective therapeutic effect.Therefore,the development of new targeted drugs is imperative.Protein kinase,including tyrosine kinases and serine/threonine kinases,is a kind of enzyme that catalyses protein phosphorylation.P21 activated kinases(PAKs)are a kind of evolutionarily conservative serine/threonine kinase,which are activated by RAS,Rac1 and Cdc42.PAKs are involved in cell activities such as cell migration,cytoskeletal rearrangement,cell apoptosis and survival,gene transcriptional regulation and cell transformation.As the pivot of signal transduction networks of cancer cells,PAK has vital functions in tumour formation,development and metastasis.It has been reported that PAK developed lung cancer through the RAS/PAKI/Crk pathway.In RAS mutation colon cancer cells,PAKs is activated by GTPase binding to RAS;thus,blocking PAK significantly inhibits tumour cell proliferation.PAKs may be a substitute for targeting Ras in lung cancers with Ras mutation.However,whether PAK1 plays a role in KRAS-mutated cancers must be explored,and the role of PAK1 may differ in various types of cancers.PAKs are divided into two subgroups depending on their structural characteristics:group Ⅰ includes PAK1,PAK2 and PAK3,and group Ⅱ includes PAK4,PAK5 and PAK6.PAK1 and PAK4 may play important roles in tumour development.Our previous study showed that both PAK1 and PAK4 are highly expressed in patients with colon cancer;overexpression of PAK1 is associated with lymph node metastasis,and PAK4 is associated with colon cancer serosal invasion.These findings indicated that PAK1 is correlated with patients,prognosis[Part Ⅳ section],so PAK1 may be suitable as a prognostic marker.Overexpression of PAK4 has been reported to be associated with NSCLC invasion and metastasis.However,reports about PAK1 and NSCLC remain limited.This study mainly included four parts.It aimed to(1)determine PAK1 expression in NSCLC tissues and its correlation with KRAS mutation,pERK and pAKT;(2)conduct bioinformatics analysis of the relationship between PAK1 and the prognosis of NSCLC;(3)analyse the effects of targeted inhibition of PAK1 on the proliferation and metastasis of NSCLC cells;and(4)study the expression of PAK1 and PAK4 in colon cancer tissues and their relationship with prognosis.Part I PAK1 expression in NSCLC tissues and its correlation with RAS mutation via the ERK and AKT signalling pathwaysObjective:This study aimed to investigate the expression of PAK1 in NSCLC tissues and determine its correlation between PAK1 and KRAS mutation and ERK and AKT signalling.Methods:This study involved a total of 110 cases of NSCLC,Tumour tissue and its adjacent normal lung tissue were obtained through surgery.PAK1 mRNA level was detected by qPCR,whereas PAK1,pERK and pAKT protein expression were detected by immunohistochemistry.The relationship between PAK1 expression and prognosis of patients with lung cancer was analysed.Survival analysis was performed by Kaplan-Meier analysis and log-rank test.KRAS mutation was detected in patients with lung adenocarcinoma,and the correlation between KRAS mutation and PAK1,as well as between PAK1 and pAKT and pERK protein,was analysed.Results:1.The relative expression of PAK1 mRNA in lung cancer tissues was significantly higher than that in normal tissues(0.60±1.12 vs 0.31±0.69,P<0.05).PAK1 mRNA was significantly higher in the low differentiation group than in the high differentiation group(0.93 ± 1.12 vs 0.31 ± 0.66,P<0.05).PAK1 expression was higher in patients with positive lymph nodes than in patients with negative lymph nodes(0.81 ± 0.57 vs 0.33 ±0.23,P<0.05).The expression of PAK1 increased gradually with the clinical stage in carcinoma tissues,and the difference was statistically significant(0.35± 0.25 for stage I,0.45± 0.46 for stage II and 0.85 ± 0.63 for stage III + IV,P = 0.013).2.PAK1 protein expression with PAK1,pERK and pAKT in NSCLC was detected.In NSCLC,PAK1 was highly expressed in 62.7%(69/110)of all patients with lung cancer,64.1%(41/64)in lung adenocarcinoma and 57.8%(22/38)in lung squamous cell carcinoma.PAK1 was significantly correlated with tumour size(P = 0.028),clinical stage(P<0.001)and lymph node metastasis(P<0.001).The positive expression rate of pERK was 40.9%in NSCLC,whereas that of pAKT was 43.6%.Both pERK and pAKT protein expression were correlated with low differentiation(P = 0.031 for pERK;P = 0.032 for pAKT)and lymph node metastasis(P = 0.007 for pERK;P = 0.014 for pAKT).3.Survival analysis showed that the total survival time of patients with low PAK1 expression was significantly longer than that of patients with high expression(1858± 58 vs 1338±71 days,P<0.001).Multiple COX analysis showed that high PAK1 expression was an independent prognostic factor for poor prognosis(HR = 4.265,P = 0.01 1).4.KRAS mutation assay demonstrated that KRAS mutation was present in 23.4%(15/64)of patients with lung adenocarcinoma.However,the expression of PAK1 was not significantly correlated with KRAS mutation.In the KRAS mutation group,PAK1 was significantly positively correlated with pERK(r = 0.722,P = 0.002)and pAKT(r = 0.739,P = 0.002).In the KRAS wild group,PAK1 was also significantly positively correlated with pERK(r = 0.344,P = 0.016)and pAKT(r = 0.466,P = 0.011).Conclusions:1.The PAK1 gene was highly expressed in NSCLC and indicated poor prognosis of NSCLC.2.PAK1 expression was not correlated with KRAS mutation in NSCLC.However,PAK1was significantly correlated with the ERK and AKT signalling pathways in both KRAS mutant and KRAS wild-type NSCLC.Part II Bioinformatics analysis of PAK1 expression and prognosis of NSCLCObjective:This study aimed to analyse PAK1 expression in NSCLC and its prognostic value by using bioinformatics data.Methods:Using online information databases,RNA-seq gene expression profile data of lung adenocarcinoma(LUAD)and lung squamous cell carcinoma(LUSC)were downloaded from the public TCGA database at http://tega-data.nci.nih.gov/tega and GEO database at https://www.ncbi.nlm.nih.gov/,respectively.The PAK1 mRNA level data were selected and downloaded with the clinical data of patients with lung cancer.Gene expression data and gene mutation data of cancer cell lines were downloaded from the website https://portals.broadinstitute.org/ccle.Correlations of PAK1 gene expression and KRAS mutation were analysed.Results:1.The TCGA database included 585 cases of LUAD and 504 cases of LUSC.In LUAD,PAK1 mRNA high expression was significantly correlated with increased T stage(P =0.046)and N stage(P = 0.043).The total survival time of the PAK1 low-expression group was significantly longer than that of the PAK1 high expression group(3060± 336 vs 2010± 398 days,P = 0.002).In LUSC,PAK1 mRNA was significantly higher in male patients than in female patients(P = 0.036),and the total.survival time of the PAK1 low expression group was also longer than that of the PAK1 high expression group.(2086±138 vs 1831±212 days,P = 0.853);the difference was not statistically significant.In the TCGA database,there were 191 lung adenocarcinoma samples with both RNA expression information and gene mutation information.The analysis showed that PAK1 gene expression in the KRAS mutation group and KRAS wild-type group was 11.95± 0.89 and 11.93± 0.77(P;0.888),respectively.PAK1 expression was not significantly correlated with KRAS mutation(r =0.019,P = 0.797).2.After conditional selection,PAK1 expression was analysed in 11 group data with 1123 patients with lung cancer in the GEO database.Five groups were tested for PAK1 gene expression by Affymetrix Human Genome U133A chip.The other four groups were tested by Illumina HumanRef-8 v3.0 expression bead chip.The results showed that PAK1 expression in tumour tissues was significantly higher than that in normal lung tissues(6.49± 1.00 vs 5.31± 0.44[P<0.01]and 6.52±1.06 vs 4.62± 0.35.[P<0.01],respectively.Analysis of 480 samples with both PAK1 expression and clinical survival data showed that the total survival time of patients in the PAK1 low expression group was significantly longer than that in the high expression group(2265± 210 days vs 1886± 212 days,P=0.032),and the difference was statistically significant.In addition,clinical staging was an important factor influencing survival(P<0.001).3.The Broad-Novartis Cancer Cell Line Encyclopedia(https://portals.broadinstitute.org/ccle/)included 1457 kinds of tumour cell lines,with a total of 136 strains of the NSCLC line.A total of 129 cell lines with both transcriptome gene expression and gene mutation test data were analysed.Correlation analysis showed that PAK1 expression was not correlated with KRAS mutant in lung cancer cells.Conclusions:1.Bioinformatics analysis showed that PAK1 was highly expressed in NSCLC tissues and correlated with poor prognosis of NSCLC.2.Bioinformatics analysis showed that PAK1 expression was not correlated with KRAS mutation in NSCLC.Part Ⅲ Effect and mechanism of targeting PAK1 to inhibit the proliferation and invasion of NSCLCObjective:This study aimed to explore the effects and mechanism of the down-regulation of PAK1 on the growth,proliferation,invasion and metastasis of NSCLC cell.This study also explored whether PAK1 is sensitive to KRAS mutation signals and plays a role through the MEK/ERK and PI3K/AKT signalling pathways.Methods:1.The expression of PAK1 mRNA and protein in a panel of NSCLC cells(A549,H460,H520,H1975,HCC827 and H1299)with different RAS mutation status was detected by qPCR and Western blot.2.The PAK1 gene interference sequence was designed and synthesised.Lentiviral vectors were used to transfect A549 cells(KRAS mutant)and H520 cells(KRAS wild-type),and the lentiviral PAK1 gene interference cell strain was constructed.A small-molecule PAK1 inhibitor IPA-3 was applied to the above mentioned A549,H460,H520,H1975,HCC827 and H1299 cells.3.Cell viability and survival were detected by MTT and colony formation.Cell apoptosis and cell cycle were analysed by flow cytometry.Cell migration and invasion were investigated by cell scratch assay and Transwell assay.The gene chip was used to detect the downstream gene expression regulated by PAK1.Western blot was conducted to detect the expression of downstream proteins of PAK1.4.MEK/ERK inhibitors and PI3K/AKT inhibitors were applied alone or in combination to the above cells to investigate whether PAK1 affects the proliferation and invasion of lung cancer cells by regulating the ERK and AKT signalling pathways.5.To establish a A549 xenograft tumour model in nude mice,the effects of PAK1 gene inhibition on the growth and proliferation of A549 lung.cancer cells,as well as ERK and AKT,were detected in vivo.Results:1.PAK1 mRNA and protein expression were observed in lung cancer cell lines with different RAS mutations.No significant difference in PAK1 expression level was found in RAS mutant and RAS wild-type lung cancer cells.2.Two interference sequences of PAK1 were transfected into A549 and H520 cells by lentivirus vector.Transfection with sh#1 and sh#2sequence in PAK1 mRNA of A549 and H520 cells decreased by about 91%and 89%and 58.7%and 60.7%,respectively.Western blot demonstrated that PAK1 protein of A549 and H520 cells decreased by 80.8%and 79.2%with sh#1 and 68.7%and 88.7%with sh#2.3.MTT assay showed that the cell growth inhibition rate gradually increased with the increase in IPA-3 concentration,thereby presenting a dose-time effect in all the lung cancer cells.IPA-3(2.5-10 μmol/L)showed significant differences in cell growth inhibition compared with the control cells(P<0.05).Cell counting showed that the growth of A549 and H520 cells with PAK1 sh#1 and sh#2 was significantly lower than that of the control group(P<0.05).In addition,colony formation rates of A549 and H520 cells with IPA-3 treatment and PAK1 silencing were significantly lower than that of the control group(P<0.05).4.Different concentrations of IPA-3(0,1,2.5 and 5 μmol/L)were applied to 549,H520,H460 and H1975 cells,respectively,and the apoptosis rate gradually increased in each drug concentration group and presented a dose-time effect.Caspase-3 activity of A549 and H520 cells interfered by PAK1 gene was higher than that of the control group.Apoptosis-related proteins PARP and caspase-3 expression increased in the IPA-3 treatment and PAK1 interference groups.5.Wound healing assay showed that the migration ability of PAK1 interfering A549 and H520 cells and IPA-3 treatment of A549,H520 and H1975 cells significantly decreased(P<0.05).Transwell assay showed that PAK1 gene silencing and IPA-3 treatment with A549,H520,H460 and H1975 cells were significantly reduced(P<0.01).6.The results of cDNA microarray revealed that the reduction in PAK1 expression in A549 cells up-regulated 391 genes and down-regulated 533 genes.The down-regulated genes were mainly involved in the interaction of cytokines and their receptors,TNF signalling pathway,NF-KB signalling pathway,FoxO signalling pathway and MAPK signalling pathway.The up-regulated genes were mainly involved in peroxidase,propionic acid metabolism and cytoskeletal control signalling pathway.The expression levels of TNFAIP3,NGFR,EDA2R and TNFRSF25 in the-TNF signalling pathway were verified by Taqman PCR.Western blot showed that IPA-3 could reduce the expression of pPAK1 and alter MEK/ERK and AKT signalling.7.Specific MEK/ERK inhibitor(U0126)and PI3K/AKT inhibitor(LY294002)were applied to the above cells.MTT results showed that LY294002 was significantly inhibited,whereas U0126 slightly inhibited cell proliferation of all lung cancer cells.The combination of U0126 and LY294002 significantly inhibited the proliferation of these cells.PAK1 gene interference(A549 and H520)and IPA-3 treatment(A549,H520,H460 and H1975)had the same effect on cell proliferation as the control cells treated with U0126 and LY294002.Transwell assay showed that both U0126 and LY294002 could inhibit cell invasion to some extent.The effect of PAK1 interference(A549 and H520)cells and IPA-3 treatment(A549,H520,H460 and H1975)on the inhibition of cell invasion was consistent with that of the combined treatment with U0126 and LY294002 in the control cells.In addition,the expression of pERK and pAKT decreased by 20%-50%with U0126 and LY294002,respectively.Combination of LY294002 and U0126 inhibited pERK and pAKT to their levels in PAK1 interference or IPA-3-treated cells.These results showed that PAK1 mediated the proliferation and invasion of lung cancer cells by regulating the phosphorylation of the ERK and AKT pathways.8.Nude mouse xenograft assay demonstrated that inhibition of PAK1 by IPA-3 and PAK1 interference could reduce the tumour growth rate and decrease tumour volume.This effect was accompanied with a decrease in pPAK1,pERK and pAKT expression.Conclusions:1.Down-regulation of PAK1 reduced cell growth,proliferation and invasion of NSCLC cells and induced apoptosis.2.PAK1 mediated proliferation and invasion of lung cancer cells by regulating the ERK and AKT pathways.Part IV Correlation of PAK1 and PAK4 expression and colorectal cancer prognosisObjective:This study aimed to explore the relationship between PAK1 and PAK4 and clinicopathologic features of colorectal cancer,especially cancer metastasis and survival.Methods:Human tissue samples were obtained from surgical specimens of 186 patients with colorectal cancer.TaqMan real-time polymerase chain reaction and immunohistochemistry were conducted to detect the mRNA and protein expression of PAK1 and PAK4.Differences and/or correlations between groups were calculated using Student’s t-test andχ2 test.Kaplan-Meier analysis and log-rank test were employed for survival analysis.Results:1.The relative expression levels of PAK1 and PAK4 genes in colorectal carcinoma tissues were 0.454± 0.153 and 0.603± 0.208,respectively,which were significantly higher than those in normal tissues(0.179 ± 0.063 and 0.212 ± 0.071,respectively,P<0.01).PAK1 high mRNA expression was associated with lymph node metastasis(P<0.05),whereas PAK4 high mRNA.expression was associated with infiltration into the serous layer(P<0.05).2.Immunohistochemical results showed that PAK1 and PAK4 protein expression levels were higher in colon cancer tissues than in normal tissues.PAK1 and PAK4 protein expression levels were positively correlated with mRNA expression levels.The expression level of PAK1 and PAK4 increased gradually with the clinical stages(P<0.01).PAK1 expression was higher in patients with positive lymph nodes,and PAK4 expression was higher in patients with infiltration into the serous layer(P<0.05).3.The PAK1 overexpression group had a higher recurrence/metastasis rate than the PAK1 low expression group(28.9%vs 11.6%,P<0.05).Follow up with stage Ⅲ and IV patients showed that the median progression-free survival time of the high PAK1 expression group was significantly shorter than that of the PAK1 low expression group(28.8 month vs 43.7 months,P<0.05).Conclusions:1.PAK1 and PAK4 expression levels were associated with colorectal cancer lymph node metastasis and serous layer infiltration.2.PAK1 can be used as a prognostic marker for patients with colorectal cancer. |
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