ANKHD1 Promotes Cell Proliferation Of Non-Small Cell Lung Cancer By Regulating YAP1 And Its Phosphorylation

The ankyrin repeat motif(ANK)is one of the most common protein motifs found in the protein database.The ankyrin repeat is a 33-amino acid motif present in repeats of12–24 and first identified in yeast and Drosophila.MASK protein consisting of multiple ankyrin repeat and single KH domain was first identified in Drosophila melanogaster through a genetic screen.In humans,an orthologous protein of Drosophila MASK,ANKHD1(Ankyrin Repeat and KH Domain Containing 1),previously named hMASK,was first identified in LNCaP,a prostate cancer cell line,and described by Poulin et.al.ANKHD1 is located on human chromosome 5q31.3 as a single copy and encodes for a protein with multiple ankyrin repeats and as single KH domain(ANKHD1).Ankyrin-repeat-containing proteins regulate multiple cellular functions including transcriptional regulation,cell-cycle regulation,ion channel,cell survival,and cell signaling.In addition,ankyrin repeat proteins also participate in protein–protein interactions via their repeat motifs.ANKHD1 is a protein ubiquitously expressed in normal human tissues and has a varied and high expression in certain cancers.ANKHD1 was predominantly located in the cytoplasm.Some research also reported that ANKHD1 was also lacalised in nuclei.ANKHD1 is located only in the cytoplasm in leukemia and normal hematopoietic cells.But in MM cells,it can be seen both in cytoplasm and nuclei.Due to its direct regulatory effect to p21,it is assumed that ANKHD1 shuttles between cytoplasm and nuclei.ANKHD1 regulates tumor cell proliferation by modulating cell cycle through affecting cell cycle related proteins.The role of ANKHD1 in multiple diseases was studied in the recent decade,mostly in vitro using multiple myeloma,aucute leukemia and prostate cancer cell lines.ANKHD1 was found to play an important role in the activation of YAP1 in the HIPPO pathway in Drosophila and human.In Drosophila S2 cell line,silencing the expression of mask which was essential for tissue development and expression of Yki gene decreased specifically the activity of Yki.In vivo studies revealed that Mask took action by forming complex with Yki.The first ankyrin repeat mortif was detected by probe in the gene expression database of breast cancer patients and it was found that lower MASK1 expression predicted better prognosis.Further investigation utilizing prostate cancer cell lines showed that ANKHD1 regulated cancer cell proliferation and cell cycle progression by means of modulating the expression of CyclinA following activation of YAP1.YAP1 is the nucleus effector of Hippo pathway with gene location of 11q13.It encodes Yes-associated protein(YAP)which is 65kda and consists of 1 to 2 WW domains,a carboxy-terminal PDZ-binding sequence,an amino-terminal proline-rich domain,a14-3-3 protein-binding site,and a SH3-binding motif.Hippo pathway can be activated by various upstream signals including G protein coupled receptor,cell contact,extracellular matrix,cell polarity and cell adhesion molecules or connexins.In human,important Hippo pathway molecules include Yes-associated protein1(YAP1),Large tumor suppressors 1 and 2(LATS1/2),Mammalian STE-20 kinases 1and2(MST1/2)and Msp-one-binder(MOB1).The key pathway molecules are conservative and act as suppressors in cancer development.The activation of Hippo pathway phosphorylates kinase MST1/2,LATS1/2 and YAP1 sequentially,and finally leads to the isolation and degradation of cytoplasm.After inactivation,un-phosphorylated YAP1 is activated and enters nuclei.Then,as a transcriptional activator,YAP1 binds to various transcriptional factors(such as ErbB4,p53,BP-2,RUNX2,TEAD1-4 and p73)and regulates gene expression and transcriptional activity without transcriptional activity per se.Studies show that ANKHD1 is overexpressed in multi-disciplinary cancer patient cells and cell lines.ANKHD1 acts in multiple cell function related pathway in different ways such as transcription,cell cycle and cell survival.To date,there is no study investigating the relation between ANKHD1 expression and prognosis in non small cell lung cancer(NSCLC).The present study aims to reveal whether ANKHD1 modulate the malignant phynotype of NSCLC by altering sequentially YAP expression and Hippo pathway activity.Materials and MethodsPatients and specimens170 cases of tumor specimens including NSCLC tissues and 30 cases of paired non-tumor portions(with>5 cm distance from the primary tumor’s edge)were collected from the First Affiliated Hospital of China Medical University(from 1999 to 2006).All tumor specimens were from the surgical resection without receiving chemotherapy or radiotherapy prior to tumor excision.The selected patients with NSCLC included 94male and 76 female.All specimens were re-evaluated for diagnosis following the criteria for the World Health Organization(WHO)2010 classification.The Cell LinesThe HBE cell line was obtained from the American Type Culture Collection(Manassas,VA,USA).The LK2 cell line was from Japanese Collection of Research Bioresources Cell Bank.The PG-BE1(BE1),PG-LH7(LH7)cell lines was a gift from Dr.Jie Zheng(Department of Pathology,Peking University,Beijing,China).The A549,H1299,BE1,H157,SPC-A-1(SPC),and LTEP-A-2(LTE)cell lines were obtained from Shanghai Cell Bank(Shanghai,China).All cells were cultured in RPMI 1640(Invitrogen,Carlsbad,CA,USA)containing10%fetal calf serum(Invitrogen),100 IU/ml penicillin(Sigma,St.Louis,MO,USA),and 100μg/ml streptomycin(Sigma,St.Louis,MO,USA)supplemented at 37°C in 5%CO2.ImmunohistochemistryAll the tissue blocks were cut into 4-μm slices,deparaffinized in xylene,rehydrated in a graded alcohols,and immunostained with ANKHD1(Abcam,ab199164 1:100).Sections were stained with a S–P system(KIT-9710,Ultrasensitive TM S-P,MaiXin,China).YAP(Cell Signaling Technology,#4912,1:1000/IB),The chromogen used was diaminobenzidine tetrahydrochloride substrate(DAB kit,MaiXin,China),slightly counterstained with hematoxylin,dehydrated and mounted.For the negative controls,the primary antibody was replaced with PBS.Then the slices were incubated with secondary antibody marked by biotin at 37℃for 30 minutes.(Ultrasensitive;MaiXin,Fuzhou,China)。The sections were observed under five random high power fields and 500 tumor cells were counted in each field.The proportion of cells exhibiting ANKHD1 expression was categorized as follows:0=absent,1=1–25%,2=26–50%,3=51–75%,4=≥76%.The staining intensity was categorized as follows:0=negative,1=weak,2=moderate,3=strong.The proportion and intensity scores were then multiplied to obtain a total score.To obtain final statistical results,score less than 2 was considered as negative expression,while scores of 2 or higher were considered as positive expression.The location of ANKHD1 staining was mainly on the plasma.Western BlotCell lines were harvested when in exponential phase.Total protein from cells were extracted in lysis buffer(Pierce,Rockford,IL,USA)and quantified by the Bradford method.Equal amounts of total protein extracts were subjected to 6%SDS-PAGE and then electophoretically transferred to a PVDF membrane(Millipore,Bedford,MA,USA).The membrane was blocked with 5%non-fat milk for 1h and incubated with antibodies against ANKHD1(Abcam,ab117788 1:500)LATS1(Cell Signaling Technology,#9153,1:500);p-YAP(Cell Signaling Technology,#4911,1:500);YAP(Cell Signaling Technology,#4912,1:500);MST1(Cell Signaling Technology,#3682,1:500);CTGF(Santa Cruz Technology,sc-14940,1:100);Cyclin D1(Cell Signaling Technology,#2922,1:500)GAPDH(Sigma#G8795;1:2000);over night at 4°C.The membrane was then incubated with second antibody goat anti-mouse or anti-rabbit IgG at 37℃for2 h.Protein bands were visualized with the ECL and detected using BioImaging Systems(UVP,Upland,CA,USA).Plasmid construction and transfectionThe plasmid of pCMV6-Myc/DDK-ANKHD1(RC221886,Rockville,MD,USA)were purchased from Origene company.siRNA-ANKHD1(Santa Cruz Technology,sc-92073).The above plasmid was transfected into cells by using Lipofectamine 3000Transfection Reagent(Invitrogen,Carlsbad,CA,USA).The protein level was assessed48h or 72 h later by Western blotting.Immunofluorescence stainingThe cells were first fixed in 4%paraformaldehyde in PBS,permeabilized with Triton X-100 then blocked with 1%BSA 1h at room temperature.They were then incubated with antibody ANKHD1(Abcam,ab199164 1:100)YAP(Cell Signaling Technology,#4912,1:100);After washing,the primary antibody was followed by incubation with Goat anti-rabbit/anti-mouse secondary antibody conjugated to rhodamine-labeled.The nuclei were counterstained with DAPI.In the absence of primary antibodies,application of secondary antibodies(negative control)failed to produce any significant staining.Methylthiazoletertrazolium(MTT)assayTwenty-four hours after transfection,the cells were plated in a 96-well plate in medium containing 10%FBS at about 3,000 cells/well,and the quantitation of cell viability was determined by MTT assay.Briefly,20μL of 5 mg/ml MTT(Thiazolyl Blue)solution was added to each well and incubated for 4 h at 37°C,then the media was removed from each well,and the resultant MTT formazan was solubilized in 150μL of DMSO.The results were quantitated spectrophotometrically by using a test wavelength of 490 nm.Migration assaysA 24-well Transwell chamber was used with a pore size of 8μm(Costar)and the inserts were coated with Matrigel(BD Bioscience)in serum-free medium.Forty-eight hours after transfection,100 ul cells were trypsinized and transferred to the upper Matrigel chamber in serum-free medium containing 5×10~5 cells and incubated for 16 h.The lower chamber were filled with 10%BSA in medium,continued to be incubated for48 h.Then the non-invading cells on the upper membrane surface were removed with a cotton tip,and the cells which passed through the filter were fixed with 4%paraformaldehyde and stained with hematoxylin.The number of invaded cells was counted in ten randomly selected high power fields under microscope.Data presented are representative of three individual wells.Colony formation assayForty-eight hours after the transfection,the cells were then planted in 6-cm cell culture dishes(1,000 cells per dish)and incubated for 14 days.Cells were then stained with Giemsa and the number of colonies with more than 50 cells was counted.RNA extraction and Real-time RT-PCRTotal RNA from cells was extracted using RNeasy Plus Mini Kit(Qiagen,Hilden,Germany).1μg RNA was reverse transcripted using the PrimeScriptTM RT Master Mix(TaKaRa,China)according to manufacturer’s instructions.Real-time RT-PCR reaction was 20ul system SYBR Green PCR Master Mix(Applied Biosystems,Foster City,CA)and performed by the ABI7900 system.Real-time PCR system is as follows:50℃2min,95℃10min,95℃40s,40 cycles,and then 60℃60s.GAPDH was used as an internal control,and mRNA values were normalized to GAPDH,All experiments were replicated triplicate.The specific primer sequences are performed as follows:ANKHD1:5-AGACCAATCGGAACACGGCTCT-35-CAGAAGCTGCTTCCATCAAGGG-3YAP1:5-TGTCCCAGATGAACGTCACAGC-35-TGGTGGCTGTTTCACTGGAGCA-3GAPDH:5-GTCTCCTCTGACTTCAACAGCG-35-ACCACCCTGTTGCTGTAGCCAA-3ImmunoprecipitationFor immunoprecipitation,a sufficient amount of antibody was added to 200 ug of protein and gently rotated over night at 4。C.The immunocomplex was captured by adding 25 uL of protein A/G agarose beads(Beyotime,Jiangsu,China)and gently rotated for 3 hours at 4。C.Then,the mixture was centrifuged at 1500×g and 4。C for 5minutes,then the supernatant was discarded.The precipitate was washed three times with ice-cold radioimmunoprecipitation assay buffer,resuspended in sample buffer,and boiled for 5 minutes to dissociate the immunocomplex from the beads.The supernatant was then collected by centrifugation and subjected to Western blot analysis.Xenograft model of tumorigenesisMice were purchased from BeiJing LIHUA company.Experimental groups consisted of 20 4–6 week-old female combined immunodeficiency BALB/c mice,injected with the A549 or SPC after transfection with plasmids.Cells were implanted into the dorsal sub cutis of mice.After 8 weeks,tumors were excised and weighed.All experiments were approved by the Ethics Committee of the China Medical University.Statistical analysisThe statistical software SPSS 19.0(SPSS,Chicago,IL,USA)was used for all analyses.The chi-square test was used to determine the correlation between ANKHD1expression and clinicopathologic factors.The Kaplan–Meier method was used to estimate the probability of patient survival,and differences in the survival of subgroups of patients were compared by using Mantel’s log-rank test.Other results were analyzed using the Student’s t-test.A p value of<0.05 was considered statistical significance.ResultsThe expression of ANKHD1 was up-regulated in non-small cell lung cancerAmong 30 cases of normal tissues,there were 11 cases positively expressed ANKHD1,and the positive expression rate was 36.7%;conversely,19 cases negatively expressed ANKHD1,negative expression rate was 63.3%.Among 170 non-small cell lung cancer cases,ANKHD1 negative expression was found in 53 cases,the negative expression rate of 31.2%,the positive expression of ANKHD1 was found in 117 cases,the positive expression rate was 68.8%,which was significantly higher in lung cancer(p=0.004)Western blot assay was performed to detect the expression of ANKHD1 in non-small cell lung cancer tissues and corresponding adjacent normal tissues.It was found that the expression of ANKHD1 in tumor tissues was significantly higher than that in adjacent normal tissues.The expression of ANKHD1 was correlated with clinicopathological features of non-small cell lung cancerThe expression of ANKHD1 in non-small cell lung cancer tissues was significantly related to p-TNM stage(p=0.019)and lymph node metastasis(p=0.020),but not statistically correlated with age(p=0.615),gender(p=0.740),histological type(p=0.743),the degree of differentiation(p=0.188)and tumor size(p=0.862).Overexpression of ANKHD1 is associated with poor prognosis of patientsSurvival analysis showed that the overall survival time of patients with high expression of ANKHD1 in lung cancer was lower than that of ANKHD1 negative expression(p=0.037),suggesting that ANKHD1 was related to the prognosis of patients.Cox linear regression analysis was used to analyze the risk factors of lung cancer patients.Multivariate analysis revealed that ANKHD1 was an independent prognostic factor in patients with non-small cell lung cancer.(p=0.008,Cox regression model,table 3)ANKHD1 promote the proliferative and invasive abilities of lung cancer cells in vitroThe mRNA and protein levels of ANKHD1 were higher in A549,SPC,H1299,LK2,LTE,BE1,LH7 cell lines than that in human bronchial epithelial cell line(HBE).Therefore,we transfected ANKHD1 plasmid in A549 cell line,which expressed relatively low level of ANKHD1,on the other side,we knocked down the expression of ANKHD1 in SPC,expressing relatively high level of ANKHD1.After 48h,the transfection efficiency were detected by Western blot assay.The colony assay showed that ANKHD1 overexpression could obviously promote colony formation in A549,compared to the control group(control group vs.ANKHD1,140±20 vs.284±28,p<0.05).After knockdown of ANKHD1 in SPC cells,the number of colony was significantly reduced(control group vs.siRNA-ANKHD1,186±41 vs.103±16,p<0.05).Similarly,MTT experiment showed the proliferation rate the A549 cell line which transfected with ANKHD1 plasmid was significantly higher than that control group,In contrast,the proliferation rate decreased in the SPC cell line after ANKHD1 knockdown.The transwell assay results show that the invasive ability the A549 cell line which transfected with ANKHD1 plasmid was significantly enhanced than that control group(control group vs.ANKHD1,43±6 vs.134±5,p<0.05),In contrast,the invasive ability was weakened in the SPC cell line after ANKHD1 knockdown(control group vs.siRNA-ANKHD1,160±10 vs.61±6,p<0.05).Up-regulation of ANKHD1 can promote the formation of transplanted tumor in nude miceThe nude mice experiments showed A549 cells which overexpressed ANKHD1exhibited a more proliferative ability,compared to the control group.The tumor weight was significantly increased(ANKHD1 vs.control group,0.798±0.068g vs 0.330±0.052g,p<0.05);The proliferative ability in the SPC knockdown of ANKHD1 was weakened compared with the control group,the tumor mass decreased significantly(siRNA-ANKHD1 vs.control group,0.712±0.062g vs 0.290±0.067g,p<0.05).ANKHD1 influences Cyclin D1 and CTGF expression,the downstream genes of Hippo pathwayThe expression of Cyclin D1 and CTGF,the Hippo pathway downstream genes,of the A549 cell line was obviously up-regulated after transfection of ANKHD1.In contrast,the expression of CTGF and Cyclin D1 down-regulated in SPC cell line,which was knocked down of ANKHD1 expression.ANKHD1 regulates the activity of Hippo pathway by up-regulating YAP1 expressionImmunohistochemistry results showed that ANKHD1 was positively correlated with the expression of YAP1 in non-small cell lung cancer(p=0.003).ANKHD1 and YAP1 were both located in cytoplasm and nucleus,however,ANKHD1was mainly located in cytoplasm,and YAP1 was mainly localized in the nucleus.Furthermore,the both of them were co localized observed by confocal microscopy.Immunoprecipition showed that ANKHD1 could interact with YAP1,and promote the mRNA and protein expression levels of YAP1.Conclusion1.ANKHD1 was highly expressed in NSCLC.The expression of ANKHD1 was related to pTNM stage,lymph node metastasis status and overall survival.2.ANKHD1 could significantly promote the proliferation and invasion of lung cancer cells.3.ANKHD1 could influence the downstream target gene expression of Hippo pathway.4.ANKHD1 could influence the activities of Hippo pathway and cell proliferation through regulating the expression of YAP1.