Functional Analysis Of Novel STK11 Mutations Found In Peutz-Jeghers Syndrome

BackgroundPeutz-Jeghers Syndrome?PJS,MIM#175200?is all 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 from 1:120 000 to 1:8300.Multiple gastrointestinal polyps of the PJS patients often caused intussusception,intestinal obstruction and gastrointestinal bleeding in adolescence.PJS causes not only higher cancer risk in digestive tract,but there is also an age-dependent increased risk for development of extra-intestinal malignancies compared to the general population.There is no intervention to prevent the occurrence of PJS polyps development or their malignant transformation.Therefore,exploration of PJS related genes,pathways and the mechanisms of PJS polyp development and malignant transformation has great significance.STK11 has been implicated as an important regulator of cell proliferation and apoptosis,which may require the tumor suppressor function of this kinase and/or its catalytic activity.Moreover,STK11 has been associated with the control of cell growth via multiple signaling pathways.The mammalian target of rapamycin?mTOR?is one of these major downstream pathways that can be regulated by STK11.The ribosomal protein S6 kinase?S6K1?is the first mTOR substrate and has been an extensively studied effector of mammalian target of rapamycin complex 1?mTORC1?.Furthermore,S6K1 activation requires mTORC1-mediated phosphorylation in vitro,which acts to improve S6K1 activity towards its S6 substrate.Signals from this activation are involved in a variety of cellular functions,including the regulation of protein translation,cell growth,angiogenesis,and metabolism.Thus,STK11 mutations that inactivate its endogenous activity can negatively regulate mTORCl signaling,resulting in phosphorylation and activation of its downstream targets.In turn,this can de-represses protein synthesis,ultimately promoting cell growth and tumorigenesis.Take together,we directly interrogated STK11 genetic mutations from PJS patients in 11 unrelated Chinese families,8?72.7%,8/11?different mutations were found,including six point mutations and two large deletions.Of these,three were novel and two of them were missense mutations?p.Asp30Asn and p.Leu290Pro?.We also detected these two novel missense mutations could disrupt the function of tumor suppressor STK11 gene.This disruption led to phosphorylation of S6K1 and S6 to promote protein synthesis and cell proliferation.These findings made sure the pathogenicity of the two novel missense mutations,and really enlarged the STK11 gene mutation spectrum.Understanding the dysregulation of STK11 functioning could better know the molecular mechanisms implicated in PJS pathogenesis and even carcinogenesis,potentially providing appropriate targets for future clinical therapies.Materials and Methods1.Patient RecruitmentPeripheral blood samples of 21 PJS patients from 6 PJS families and 5 sporadic PJS cases were collected during the year of 2012 to September 2015 in Nanfang Hospital.The diagnostic criteria for PJS was determined according to our previous reports.Endoscopic or surgical polypectomy,including histopathologic examination?reviewed by at least two pathologists?,was performed in all patients.Three PJS patients were performed surgeries because of benign or malignant tumors in other organs including breast and cervix.The family cancer history was collected by the patients' medical records.All participating PJS patients and their family members gave informed consent for this investigation and the principles outlined in the Declaration of Helsinki were followed.The project was approved for human study by the Medical Ethics Committee,Nanfang Hospital of Southern Medical University.2.PCR primers designGenomic DNA was extracted from peripheral blood using the commercially available QiAamp DNA Blood Midi Kit?Qiagen,Hilden,Germany?.PCR primers were designed using the software program Primer 3.0?http://frodo.wi.mit.edu/primer3/?and sought to amplify both the STK11 gene exons and intron-exon boundaries?GenBank NM 000455.4?.3.Detection of STK11 gene germline mutations by Sanger sequencingSanger sequencing was performed for each index case in the 6 familial and 5 sporadic cases as previously describedo[16].Specific details regarding the exact primer sequences and PCR conditions are available upon request.In familial cases,identified mutations were further tested in all available family members to confirm segregation of the mutation with the disease.All variants identified were screened against the dbSNP database to rule out the possibility of these variants representing polymorphisms.4.Detection of STK11 gene germline mutations by MLPA assayMLPA assay was performed for large intragenic deletions using the MLPA test kit?SALSA P101-B1 STK11;MRCHolland,Amsterdam,the Netherlands?as previously described.Deletion screening was performed according to the manufacturer's instructions and the results were analyzed using GeneMarker???HID STR Human Identity Software?Softgenetics LLC,State College,PA,USA?.Values of 0.85-1.15 indicate normal results?presence of two copies?,and values of 0.35-0.65 or 1.35-1.65 indicate a deletion or duplication,respectively.All identified deletions were confirmed in a second independent reaction and confirmed to segregate within the relevant family members.5.Cell lines and cultureOur research group has previously maintained the following cell lines in our laboratory:human colon cancer cell lines?SW1116,HT29,SW620,SW480,Lovo,Caco2,and Hctl 16?,human cervical carcinoma cell line?HeLa?,normal human gastric epithelial cell line?GES-1?,and human kidney cell line?293T?.These cell lines were cultured in DMEM supplemented with 10%fetal bovine serum and maintained in a humidified atmosphere of 5%CO2 at 37?.6.Cell lines transfectionThe sequences of all STK11 gene mutant plasmids were confirmed through genetic sequencing.Of these vectors,pGCMV-GFP-V ecor?empty vector?,pGCMV-GFP-STK11?WT?,pGCMV-GFP-STK11?p.Leu290Pro?and pGCMV-GFP-STAK11?p.Asp30Asn?were synthesized by Gene Pharma Company?Shanghai,China?.Cells seeded in tissue culture plates were grown to 70-80%confluences and transfected with the aforementioned vectors using lipofectamin 3000?Invitrogen,Carlsbad,CA,USA?according to the manufacturer's instructions.At 24-48 h post-transfection,gene overexpression was evaluated via qRT-PCR and protein expression by Western blot.Stable transfections were performed after 48 h transfection,and the transfected cells were grown in a final concentration of 400 mg/ml antibiotic G418?Sigma,St.Louis,MO,USA?for 4-8 weeks to allow for positive selection.7.Quantitative Real-time PCR?qRT-PCR?Total RNA was extracted using Trizol?Invitrogen,Carlsbad,CA,USA?and cDNA was synthesized by oligo dT primed reverse transcription?Promega,San Luis Obispo,CA,USA?from 1?g of total RNA.Quantitative Real-time PCR?qRT-PCR?was performed using All-in-OneTM qPCR Mix?Applied GeneCopoeia Inc.USA?on a LightCycler 480 System.Primer sequences for STK11 qPCR were as follows:Forward,5'-AGGGCCGTCAAGATCCTCAA-3' and reverse,5'-GCATGCCACACACGCAGTA-3'.The housekeeping gene GAPDH was used as an internal control for later normalization.The relative expression of STK11 was calculated according to the 2-??Ct method.8.ImmunohistochemistryImmunohistochemistry was performed as previously described.Sections were deparaffinized and incubated with a p-S6?Ser240/244?primary antibody?Cell Signaling Technology,Inc.,USA,1:400 dilution?.A known p-S6 positive CRC was used as a positive control for p-S6 staining.p-S6 staining was scored as positive when more than 10%of the cells showed nuclear expression.9.Western blottingTotal protein was extracted using RIPA lysis buffer with protease and phosphatase inhibitors?1:100,Roche,Nutley,NJ,USA?.Total protein was quantified using the BCA method with commercially available reagents?Thermo Scientific,Waltham,MA,USA?.Total protein?25 ?g?was resolved using 10%SDS-PAGE gel electrophoresis?Bio-Rad,Philadelphia,PA,USA?and transferred to polyvinylidene difluoride?PVDF?membranes?Millipore,Bedford,MA?.Membranes were incubated with various primary antibodies in specific concentrations according to the manufacturer's instructions.Primary antibodies against STK1l?1:1000 dilution?,S6K1?1:1000 dilution?,S6?1:1000 dilution?,p-S6K1?Thr389??1:1000 dilution?,p-S6?Ser240/244??1:1000 dilution?and HRP?horseradish peroxidase?-conjugated goat anti-rabbit IgG?1:2000 dilution?were obtained from CST?Cell Signaling Technology,Inc.,USA?.Mouse polyclonal anti-GAPDH antibody?1:3000 dilution?and HRP-conjugated goat anti-mouse IgG?1:5000 dilution?were purchased from Santa Cruz?Santa Cruz,CA,USA?.GAPDH was used as the loading control.Protein expression was detected using enhanced chemiluminescence?Thermo Scientific,Waltham,MA,USA?.10.Cell biology experiments?1?Cell Counting Kit-8 assay,To determine the effect of novel STKll mutants on cell proliferation,transfected cells were measured using a commercially available Cell Counting Kit-8?Dojindo,Tokyo,Japan?following the kit assay protocols.?2?colony formation assay,Proliferation was also assessed by a colony forming assay,whereby transfected cells were counted and seeded into 6-well plates at a density of 1000 cells per well.Two weeks after seeding,the numbers of colonies containing more than 50 cells were stained with crystal violet and counted.And the relative cell colony numbers were evaluated at the ratio of the last visible colonies to the initialization.?3?Cell-cycle analysis,The cell-cycle distribution was analyzed by propidium iodide?PI?staining and flow cytometry.?4?Flow cytometry,Cell apoptosis was quantitated using the Annexin V-APC/7-AAD apoptosis kit?Sigma,St.Louis,MO,USA?as detailed in the manufacturer's instructions.Transfected cells were collected at 48 h after incubation with the aforementioned STK11 plasmids.Cells were washed with cold PBS and stained with annexin V-APC and 7-ADD,mixed gently,and incubated on ice for 15 min in the dark.Apoptotic cells?acquired 10,000 cells/test?were quantified by flow cytometer?BD Biosciences,USA?.?5?Cell migration assay,The cells were resuspended in serum-free RPMI-1640 medium.Then the cells were added to each transwell chamber with 10%fetal bovine serum.After cultured for 24h,the cells fixed and stained under the microscope.?6?Cell invasion assay,Matrigel was added into the Transwell chamber uniformly after thawing?50ul?in 37? foster 12h.The remaining operation was the same cell migration assay.11.Statistical analysisAll statistical analyses were performed using SPSS v17.0 software?SPSS Inc,Chicago,IL?.The data are shown as mean±SD unless otherwise noted.Comparisons among multiple groups were determined using one-way ANOVA.A value of P<0.05 was considered statistically significant.Results1.STK11 mutations and genomic deletionsWe identified six different germline point mutations in 6 out of 11?52.4%?index cases.Of these,four were found in familial?4 out of 6,66.7%?and two in sporadic cases?2 out of 5,40%?.We detected four different truncation mutations and two missense mutations.Three mutations?3 out of 6,50%?were associated with cancer in either index patients or in relatives with PJS.We next used MLPA to determine the presence or absence of exonic rearrangements in the five PJS probands in whomno mutations were identified by Sanger sequencing.Two large deletions were detected,with one being associated with breast cancer in the patients' relative.Both deletions were predicted to affect the kinase domain of the protein.2.Spectrum of two novel STK11 missense mutationsIn summery,the total mutation detection rate in our index cases was 72.7%?8 out of 11?when applied direct genomic sequencing together with the MLPA assay;this indicated that genetic alteration of STK11 predisposing to PJS that increased risk for the development of PJS-associated cancers.To our knowledge,of these mutations,one novel truncation mutation?c.143₁44insA?and two novel missense mutations?c.88G>A and c.869T>C?were found.and then two novel missense mutations were predicted to be pathogenic?PolyPhen2:http://genetics.bwh.harvard.edu/pph2/?.3.Two novel STK11 missense mutations induced p-S6 expression in PJS patients' tissueTo investigate whether two novel missense mutations had effects on the phosphorylation of mTOR pathway key downstream target gene S6;Immunohistochemistry?IHC?was performed to detect p-S6?Ser240/244?expression in individuals with or without STK11 mutations.Positive p-S6 was observed in nucleus of epithelial cells of colon hamartomas obtained from the PJS patient ?:2 with the mutations c.869T>C?p.Leu290Pro?and in the breast intraductal papillary tumor from the patient ?:2 with c.88G>A?p.Asp30Asn?,respectively.By contrast,both colorectal mucosal deriving from the relative 111:3 and the breast hyperplasia glandular epithelial from the relative 11:2 stained negative for p-S6 protein,both of which companied without STK11 mutation.These findings suggest that the two novel missense mutations may disrupt the protein function of STK11 serine/threonine kinase inducing S6 phosphorylation.4.Expression of novel STK11 mutation in HeLa and SW1116 cellsWe firstly used both qRT-PCR and Western blot to detect endogenous expression levels of STK11 in human cell lines.As shown,STK11 expression levels were nearly undetectable in HeLa and SW1116 cells when compared with all other tested cell lines.We then focused on HeLa and SW1116 cells for our further studies,beginning with an evaluation of the effects of novel STK11 mutations.We transfected STK11 plasmids including empty vector,wild-type vector,p.Leu290Pro vector,and p.Asp30Asn vector into both cell lines and examined the STK11 expression by both qRT-PCR and Western blot analysis.As shown in Fig.3B and C,the expression of transfected STK11 novel mutants as well as wild-type vector were all significantly increased in HeLa and SW1116 cells when compared to empty control vector.However,there was no obvious difference between the wild-type vector and novel mutants.Taken together,these results suggest that the novel mutants have negligible effects on STK11 transcription and translation.5.Two novel STK11 missense mutations promote cell proliferation.To further determine whether the novel missense STK11 mutations could effectively suppress cell growth and proliferation,we performed CCK-8 and colony-forming assays.As shown in,both HeLa and SW1116 cells transfected with wild-type STK11 vectors had a marked reduction in cell growth?versus the mutants?.Comparatively,the colony-forming efficiencies of HeLa and SW1116 cells transfected with either of the STK11 mutants?p.Leu290Pro or p.Asp30Asn?were both significantly increased?p<0.01?when compared with the empty vector control.These results suggest that STK11 gene is a tumor-suppressor gene in two cell lines,the two novel mutations abrogated cellular growth suppression function of STK11 gene,contributing to tumorigenesis.Actually,we also investigated the effect of the two novel missense STK11 mutations on cell apoptosis.When compared to empty vector control,wide-type STK11 promoted apoptosis,while the two novel STK11 missense mutations had no significant effect.This findings indicate that STK11 gene play apotosis promotion roles in HeLa and SW1116 cells respectively.6.Two novel STK11 missense mutations induce phosphorylation of S6K1 and S6 in cell lines.We then sought to determined whether two novel STK11 missense mutations affect gene function resulting in disregulation of its downstream mTOR-mediated pathway.Phosphorylation of S6K1 at Thr389 and S6 at Ser240/244--both of which are mTORC1 substrates--were measured using Western blot.As shown in Fig.,compared with the empty vector control,there were elevated expression levels of p-S6K1?Thr389?and p-S6?Ser240/244?in both HeLa and SW1116 cell lines transfected with the two novel missense STK11 mutants.Contrastingly,cells transfected with wild-type STK11 vectors had a marked decrease in levels of both p-S6K1 and p-S6.These results demonstrated that novel STK11 mutants disrupted their endogenous protein kinase activity that switching on mTORC1 pathway signals amplification,yielding a dysregulation of STK11-S6K1 axis.In turn,we hypothesized that this disruption leads to abnormal de-repression of protein synthesis and promotion of cell growth.Conclusion1.STK11 mutation in PJS family probability is 72.7%,the family was 80%,and sporadic family about 33.3%,suggests the presence of a new mutation in or low penetrance,explaining the PJS with genetic heterogeneity.2.Three new STK11 gene mutations?c.143₁44insa,c.869 t>c and c.88 g>A?were found,which were pathogenic mutations and enrich the STK11 gene mutation spectrum.3.STK11 missense mutation c.869 t>c and c.88 g>A were both no missing amount of protein expression,suggesting the mutations may not affect STK11 gene transcription and translation process.4.STK11 missense mutation c.869 t>c and c.88 g>A both led to the decrease of the encoding protein kinase activity or missing,protein function damage and promote cell proliferation,accelerate the process of cell cycle,and had no obvious effect on cell apoptosis,enhance the ability of cell migration and invasion;At the same time reduces the STK11 of p-AMPK activation ability;The abnormal activation of mTOR pathways,improve the level of downstream target genes P70S6K and phosphorylation of 4 ebpl.5.mTOR signaling pathway is downstream of STK11 may be associated with the onset of PJS and malignant polyp,the key target genes P70S6K and 4 ebpl of mTOR pathway become PJS clinical cure or the possibility of targeted drugs.