Functional Analysis Of One Novel LKB1/STK11Mutant Found In Peutz-jeghers Syndrome

Background and objectivesPeutz-Jeghers Syndrome (PJS, MIM#175200) is an autosomal dominantly-inherited disorder characterized by gastrointestinal hamartomatous polyps, mucocutaneous pigmentation, and an increased risk for development of gastrointestinal (GI) malignancies. The reported estimates of the incidence frequency of PJS vary from1:120000to1:8300. Mutations of the tumor supressor gene LKB1(Liver Kinase B1)/STK11, which lead to impaired protein kinase activity, have been identified as the major cause of various disease including PJS. Until recently, more than200types of mutations in LKB1/STK11gene have been screened to our knowledge in patients with PJ syndrome or sporadic cancers. De novo LKB1mutations are constantly reported, which not only expands mutaion spectrum of LKB1, but also contributes to the research on the relationship between phenotype and genotype in PJS, and provides evidence for genetic counseling, prenatal diagnosis, and follow-up plan. Since its discovery in1998, many researches have focused on identification of its molecular mechanisms. It has been confirmed that LKB1plays an crucial role in tumorigenesis mainly by regulating cell proliferation, apoptosis, cell cycle, cell polarity, chromatin remodeling and energy metabolism, etc. However, the exact pathogenic mechanisms of LKB1are sophisticated, and have not yet been fully elucidated. Different mutations of LKB1may affect specific aspects of cell functions. Therefore in this study, we accumulate clinical information and perform germline mutation screening for PJS patients and their families for further investigation of the possible genotype-phenotype relationship. We also construct a novel LKB1/STK11mutant plasmid to find whether it plays any role in cell growth, apoptosis and its downstream signaling pathway, and therefore further confirm this novel mutation to be the primary cause of this PJS family.Mothods1. PCR primer design12pairs of PCR primers were designed to amplify the STK11gene exons and intron-exon boundaries using the primer sequences in the reported literatures or the software program Primer3.0(http://frodo.wi.mit.edu/primer3A).2. Detection of STK11gene germline mutaion in PJS patientsBlood and(or) polyps of12PJS patients from9families and their healthy family members were collected from Nanfang Hospital, Southern Medical University, Guangzhou. Meanwhile, blood of50normal individuals were also collected as controls from the physical examination center in this hospital. Genomic DNA of PJS probands was extracted from peripheral blood, after DNA amplification and purification of PCR product, we used DNA direct sequencing to detect the germline mutation of STK11gene. Then we aligned mutation sequences investigated with normal DNA sequuences from those50individuals and the healthy family members by HGMD and NCBI database online BLAST alignment, and also using NCBI dbSNP database to rule out the possibility of SNP. To those patients without deteced mutations, we further adopted MLPA assay to detect large-scale genomic deletions/duplications of STK11. MLPA assay results were analyzed using GeneMarker(?) HID STR Human Identity Software.3. Construction of LKB1plasmids.pGCMV-GFP-Vecor empty vector, pGCMV-GFP-LKB1(WT) and pGCMV-GFP-LKB1(G288R) plasmid were synthesized by GenePharma Company in Shanghai, China.4. LKB1expression in different cancer cellsHela、SW1116、DLD1、 HT29、SW620、SW480、LOVO、colo205、HCT116cell lines were maintained in RPMI1640(Gibco) supplemented with10%FBS. The cells were incubated in a humidified incubator at37℃with an atmosphere of5%CO2. Cells were lysed with Trizol or RIPA buffer, and the basic LKB1expression was evaluated by RT-PCR(Reverse transcription polymerase chain reaction) or Western Blot respectively. We chose cell lines in which LKB1was almost not detectable to continue our study.5. LKB1expression in cells transiently transfeced with LKB1plasmids.Hela or SW1116cells seeded in12-well cell culture plate were grown to70%-80%confluence and respectively transfected with3different palsmids by lipofectamin2000according to the instruction of manufacture. The effect of gene overexpression was evaluated by RT-PCR or Western Blot after48hours later.6. Cell proliferation in the transfected cells5*103cells per well seeded in a96-well plate were maintained in100ul RPMI1640(Gibco) supplemented with10%FBS overnight.48hours after transfection, cells proliferation in different transfected groups was measured by CCK-8(cell counting kit-8) according to the manufacturer’s instructions.7. The effect of LKB1plasmid on cell colony forming efficiencyTo examine the effect of LKB1on cell colony forming efficiency,1.5ml of 0.5%agarose in RPMI1640culture medium was allowed to solidify in6-well plates, and were overlaid with1.5ml of0.35%agarose in medium containing4*103cells. Cells were incubated for2weeks at37℃and stained wih0.05%crystal violet. The cell colonies (more than50cells/colony) were counted under the microscope and photograghed.8. The effect of LKB1on cell apoptosisHela or SW1116cells seeded in12-well plate were respectively transfected with palsmids by lipofectamin2000according to the instruction of manufacture when up to70%-80%confluence.48hours after transfection, cells were digested and washed twice with precold PBS. Resuspending cells with1*binding buffer, and adding5ul Annexin V and5ul PI into1*105cells,15min at RT(room temperature), to stain cells. Then performing cell apoptosis analysis by Flow Cytometry.9. The effect of LKB1on LKB1/AMPK/m-TOR signaling pathway and P-AKT expressionEmpty vector or LKB1plasmids transfection were performed when cell confluence was up to70%-80%.48hours after transfection, cells were then starved with medium without FBS. Twelve hours later Western Blot were performed to detect expression of p-AMPK, P70S6K, VEGF, PCNA. On the other hand, protein was also extracted to detect P-AKT expression by Western Blot5minutes after adding50nM insulin into cells transfected.10. Statistical analysisResults obtained were analyzed using SPSS13.0. The data were expressed as Mean±SD with at least3independent expriments. One way ANOVA was used to analyze the quantitive data with significant difference being considered if P values were less than0.05. Results1. Compared against DNA from100normal individuals and the rest of healty family members, seven mutations of STK11gene were found in12patients with PJS from9families, including three point mutaions, one single base insertion and one small deletion by DNA direct sequencing and2large genomic deletions by MLPA assay. We speculated that all of the mutaions found would affect the structure and function of protein. Direct sequencing of the STK11gene showed a G-to-A substitution of nucleotide862in exon6, which altered the wild-type sequence GGG to the mutant sequence AGG at288codon, and resulted in the amino acid substitution from glycine to arginine at residue288within the catalytic kinase domain. A search of the mutational databases for STK11revealed that this mutation was novel and had not been described before.2. RT-PCR and Western Blot showed that LKB1was almost not detectable both at mRNA and protein levels in Hela and SW1116cells, which did not habor this novel mutaion we studied.3. RT-PCR and Western Blot showed that the amount of exogenous mRNA and protein expressed in the cells transfected with the wild-type LKB1/STK11plasmid was similar to that transfected with the novel mutant LKB1/STK11(P>0.05).4. CCK8assay showed that OD450value in both Hela and SW1116cells transfected with wild-type LKBlwas significantly reduced when compared with that in the empty vector group and LKB1mutant transfection group (P<0.01), while there was no significant difference between empty and mutant groups(P>0.05), which means the cell viability was significantly decreased in cells transfected with wildtype plasmid.5. Cell colony assay showed that an over50%reduction in the number of cell colonies expressing wild-type LKB1/STK11was observed in comparison with those expressing mutant LKB1/STK11(p<0.01) and those carrying the empty vector (p<0.01), while there was no significant difference between empty and mutant groups(P>0.05), that is, cell formation efficiency was markedly decreased in wild-type group.6. Flow cytometry to evaluate cell apoptosis showed that a significant increase in the percentage of apoptotic cells (Annexin V-FITC positive) in Hela cells transfected with wildtype (20.78±2.32%for Annexin V-FITC positive;16.46±2.09%for Annexin V-FITC positive and PI negative) and mutant (20.30±2.87;16.67±2.21%) was shown in our study in contrast to that in the empty control (7.67±1.65%;4.43±0.60%)(P<0.01), while the apoptosis rate in wildtype and mutant groups almost had no difference(P>0.05). The same result was obtained in SW1116cells, as assessed by Annexin FITC-V and PI staining, with no significant difference between cells transfected with wild-tye LKB1(21.70±2.07%for Annexin V-FITC positive;16.74±1.63%for Annexin V-FITC positive and PI negative) and mutant(20.96±2.95%;15.62±1.93%); however, an increase in the apoptosis rate was observed when compared with empty vector group(13.97±1.63%;9.46±1.15%)(P<0.01).7. Compared with empty vector or LKB1mutant groups, the expression of P-AMPK(Thr172) was significantly increased (P<0.01), while P-P70S6K(Thr389), VEGF, PCNA, and P-AKT(Ser473) expression was diminished in the Hela or SW1116cells transfected with LKB1(WT)(all P<0.01).Conclusion1. LKB1/STK11mutations are responsible for PJS. Seven mutations found in PJS patients are all pathogenic varients; STK11mutation c.862G>A, that is p.288G>R, which was found in PJS patients is a de novo LKB1germline mutation; 2. This study is the first to use colon adenocarcinoma SW1116cells to analyze the novel LKB1missense mutation; G288R may not affect the LKB1transcription and translation in line with normal expression of LKB1both at mRNA and protein levels;3. G288R reduces cell growth suppression of LKB1, while it may not affect cell apoptosis of LKB1;4. G288R may lead to decreased kinase activity, as assessed by its disability to activate AMPK and inhibition of mTOR activity.Taken togther, alteration of the tumor supressor gene LKB1/STK11was the major cause of PJS. The novel LKB1/STK11missense mutation (G288R) plays an indispensable role in the cell function, and the underlying pathogenic mechanism may be increased cell proliferation mainly due to impaired LKB1kinase activity, which lead to Peutz-Jeghers Syndrome. The novel mutation LKB1(G288R) is responsible for this PJS family.