Caveolin-1 Correlates With Sensitivity To EGFR-TKIs In Human Lung Adenocarcinoma With EGFR Mutation

Lung cancer is the leading cause of cancer death worldwide.Approximately 85% of lung cancers are grouped as NSCLC,and lung adenocarcinoma is the most common subtype of NSCLC.Unfortunately,in spite of advances in early detection of cancer,the majority of patients are diagnosed with advanced-stage disease resulting in poor prognosis.EGFR is overexpressed in 40% to 80% of NSCLC.Homodimerization or heterodimerization and autophosphorylation of EGFR result in the activation of MAPK and PI3 K signaling pathways that mediate cell proliferation,survival and migration.EGFR-TKIs block the activity of EGFR by competitive binding to the intracellular ATP-binding domain,thereafter,inhibit the tumor growth.Moreover,studies of Paez and Lynch confirmed early that specific types of cancer cell mutations in the EGFR gene correlate with clinical response to EGFR-TKIs.The effective population for EGFR-TKIs treatment was NSCLC patients with EGFR gene exon 19 deletion mutation(delE746-A750),exon 21 point mutation(L858R)or exon 18 point mutation(G719S).Thus,it has been recommended as the first line therapy for NSCLC patients with EGFR mutations.Unfortunately,there is a significant individual difference in the degree of benefit to EGFR-TKIs in patients.It was reported that approximately 25% of patients with EGFR mutations have primary resistance to EGFR-TKIs.Moreover,the clinical efficacy of patients who initially benefit from EGFR-TKIs is not consistent,and the majority of patients would acquire resistance eventually.Although studies of EGFR mutation itself(T790M),EGFR pathway-related molecule(c-MET amplification),and other pathways have been partially demonstrated to be associated with EGFR-TKIs resistance,it is still insufficient to explain the difference in efficacy in lung adenocarcinoma patients with EGFR mutations.As a principal structural component of caveolae,a 21-24 kDa integral membrane protein,Cav-1 regulates many signal transduction processes via a scaffolding domain which has the ability to engage in complex interactions with a variety of signaling proteins(EGFR,ErbB2,H-Ras,MEK,etc.)in the caveolae and to affect their functions.Some studies have shown that Cav-1plays an important role in the development of many tumors.Our previous experiments have also shown that Cav-1 could promote the proliferation,migration and invasion of lung adenocarcinoma cells by up-regulating phosphorylation of EGFR and activity MAPK and PI3 K pathways.In addition,Cav-1 closely correlates with the drug resistance in cancer cells.High expression levels of Cav-1 and high surface density of caveolae have been identified in a number of drug resistance cancer cell lines.However,whether Cav-1 contributes in modulating EGFR-TKIs sensitivity to human lung adenocarcinoma is unknown.In this study,we investigated the role of Cav-1 in sensitivity to EGFR-TKIs in tumor tissues from advanced lung adenocarcinoma patients,and verified in human lung adenocarcinoma PC-9 cells in vitro and in vivo and provided a potential therapeutic target for reversing EGFR-TKIs resistance in human lung adenocarcinoma.Part one Caveolin-1 correlates with the effect of gefitinib in advanced human lung adenocarcinoma with EGFR mutationObjective: To investigate the expression of Cav-1 in EGFR-mutation advanced lung adenocarcinoma and its correlation with the efficacy of gefitinib.Methods:1 Clinical specimens20 specimens were collected from patients with advanced(stage IIIB or IV)lung adenocarcinoma harboring EGFR mutations(deletion mutation in exon 19)who undergoing first-line EGFR-TKIs treatment(gefitinib,250mg/d)until disease progression in the Bethune International Peace Hospital from July 2011 to July 2014.All samples were CT-guided lung adenocarcinomatissues,adequate tissue samples for histological examination,and graded according to TNM stage in the AJCC 7th staging system by clinical pathologists.The index of short-term clinical efficacy which was evaluated using Spiral CT while following RECIST 1.1 criteria every 6 weeks of gefitinib treatment includes complete response(CR),partial response(PR),stable disease(SD)and progressive disease(PD).Progression free survival(PFS)and overall survival(OS)indicate long-term outcomes.2 ImmunohistochemistryImmunostaining was performed on 5-?m formalin-fixed,paraffin-embedded tissue sections incubated with antibodies against Cav-1and Ki-67.The assessment of immunohistochemistry(IHC)signals was performed by two experienced pathologists in a blinded manner.Semi-quantitative scoring for Cav-1 was performed using the H-score method.The staining intensity was scored as 0 – 3(0 = none,1 = weak,2 = moderate,3 = strong),and the proportion of the tumor staining for that intensity was recorded from 0 to 100.A final H-score was derived as(% of cells stained at intensity category 1 × 1)+(% of cells stained at intensity category 2 × 2)+(%of cells stained at intensity category 3 × 3).The percentage of nuclear Ki-67 immunoreactivity was calculated by determining the average expression of positive Ki-67 in 500 nuclei.Results:1 Expression of Cav-1 in human lung adenocarcinomaCav-1 was positively expressed both on the cell membrane and in the cytoplasm,but the positive expression of Ki-67 in cell nucleus.Based on the scoring by two independent pathologists,there were significant positive correlations between the immunoreactivity for Cav-1 and Ki-67.2 High expression of Cav-1 correlates with poor clinical response of gefitinib and worse PFS of patientsThe relationship between different clinical parameters and Cav-1expression in univariate analysis.The level of Cav-1 expression had no difference in gender and other clinical parameters,except for the clinicalresponse to gefitinib,PFS and OS.After gefitinib treatment,the total response rate was 60% in all patients,and H-score value of Cav-1 in(CR + PR)group was lower than that in(SD + PD)group(P<0.05).The median PFS of all patients was 8 months(95%CI: 6.91-9.09)and the median OS was 16.5months(95%CI: 13.58-19.42).Shorter PFS and OS groups showed higher H-score value of Cav-1(P<0.05).Gather the cases based on the median H-score value of Cav-1,patients with high Cav-1 expression in their tumors had a markedly poor disease response than that with low Cav-1 content(P<0.05).High Cav-1 expression group had a shorter PFS compared with low Cav-1 group(P<0.05).However,there was no difference on OS between two groups(P>0.05).Part two Effects of knockdown of Caveolin-1 on sensitivity of EGFR-TKIs in lung adenocarcinoma cells with EGFRmutationObjective: To investigate the effects of knockdown of Cav-1 on sensitivity of EGFR-TKIs in lung adenocarcinoma cells with EGFR mutation.Methods:1 TransfectionCav-1 shRNA plasmid and Control shRNA plasmid were obtained from Santa Cruz.1 mg of shRNA plasmids were transfected into PC-9 cell lines using Plasmid Transfection Medium.Stably transfected clones(PC-9/KD and PC-9/Ctrl)were selected and expanded after incubation in Puromycin containing selection median(2 ?g/ml).2 Proliferation Inhibition assayCells were seeded into 96-well plates,allowed to adhere overnight,then EGFR-TKIs(gefitinib or erlotinib)were added at a final concentration of0.0125–3.2 ?M.After incubation for 48 hours,20 m l MTS was added,and continue to incubate for 2 hours,then,the optical density(OD)values were read at 490 nm using a microplate reader.Half-maximal inhibitory concentration(IC50)was defined as the drug concentration at which cell growth was inhibited by 50%.Subsequently,proliferation inhibition rate atdifferent intervals(24,48 and 72 hours)was tested using the lower IC50 of EGFR-TKIs incubated for 48 hours in each pair of stably transfected cell lines.Each sample was plated in triplicate and three independent experiments were performed.3 Colony formation assayClonogenic assay was measured cell proliferation in a cell contact independent way.Cells were seeded at a low density 300 cells/35 mm dish,and incubated in the presence or absence of EGFR-TKIs.After cultured for8-12 days,cell colonies were stained with hematoxylin and counted.4 Migration assayIn brief,cells were seeded in 24-well plates,allowed to adhere and form a confluent monolayer,created a wound with a pipet tip and then incubated in medium with or without EGFR-TKIs.Images were acquired at the specified point-in-time.5 Invasion assayDiluted matrigel was added to the center of each upper chamber of transwell plates.After 600 ?l of medium with 10% FBS was added to the lower chamber,cells in medium without FBS supplemented with or no EGFR-TKIs were placed in the upper chamber.After incubation for 24 hours,invasive cells were stained with H&E,then photographed and counted under a microscope at 200× magnification in five fields.6 Western blottingRIPA lysis buffer,50 mM Tris(p H 7.4),150 mM NaCl,1% NP-40,0.5%sodium deoxycholate,was used to cleave cells.Cell lysates were separated on10% SDS-PAGE and transferred onto polyvinylidene difluoride(PVDF)membranes which then were blocked with 5% nonfat milk or 3% bovine serum albumin at room temperature for 2 hours,incubated with primary antibodies at 4 °C overnight and followed by incubation with secondary antibodies at room temperature for 1 hour.The immunoreactive proteins were visualized using enhanced chemiluminescence(ECL)detection reagents.The lysate proteins were incubated with EGFR antibody or Cav-1antibody and non-immune IgG overnight at 4 °C followed by protein G-conjugated agarose for 2 hours at 4 °C.Immune complexes were pelleted by centrifugation at 500 g for 1 min,washed twice with lysis buffer and resuspended in suitable sample buffer.Subsequently,cell extracts and immune complexes were separated by 10% SDS-PAGE for detection of Cav-1 or EGFR by western blot as described.8 Laser scanning confocalCells were grown on glass coverslips,fixed with 4% paraformaldehyde for 20 min,permeabilized by 0.1% Triton X-100 for 5 min,blocked in 8%BSA for 1 hour,and then incubated with EGFR(1:200)and Cav-1(1:200)antibodies overnight at 4 °C and Alexa Flour 488-conjugated goat anti-mouse IgG(1:200)and 555-conjugated goat anti-rabbit Ig G(1:200)for 1 hour at room temperature in the dark.The cell nuclei were stained by DAPI for 5 min.After extensive PBS washes,cells were examined with a laser scanning confocal microscope.More than 100 cells were inspected per experiment,and the results displayed the typical cells.9 In vivo experimentsCells were subcutaneously injected into the posterior flank of BALB/c nude mice of 5-week-old individually.And the tumor volume was measured weekly using the following equation: Volume = 1/2 × length × width2.Once the tumor size reached 40 mm3,mice were randomly divided into 2 groups:gefitinib group(6 mg/kg)or no drug group(equal volume of ddH2O).All mice were sacrificed after 4 weeks of intragastric administration.Tumor tissues were weighted to calculate the tumor inhibition rate.Then all tumor tissues were analyzed by western blot and IHC as mentioned previously.Results:1 Construction of stable transfected cell linesThe expression level of Cav-1 was dramatically downregulated in PC-9/KD cells compared with PC-9/Ctrl cells(P<0.05).7 ImmunoprecipitationEGFR-TKIsCell viability of stably transfected cells treated with the indicated doses of EGFR-TKIs(gefitinib or erlotinib)for 48 hours was assessed with MTS assay.Whether gefitinib or erlotinib,the inhibition rate and IC50 of PC-9/KD cells was lower than that of PC-9/Ctrl cells(P<0.05).Inhibition rate was tested at different intervals(24,48 and 72 hours)using the lower IC50 of 48 hours in each pair of stably transfected cell lines(P<0.05).The inhibition was dose and time dependent manner.Our results confirmed that knockdown of Cav-1 increased the sensitivity to EGFR-TKIs.3 Knockdown of Cav-1 inhibits the proliferation,migration and invasion of PC-9 cells and enhances the inhibitory effect of EGFR-TKIs on cell biological behaviorThe clone formation and colony forming efficiency of PC-9/KD cells was significantly lower and the inhibitory effect of EGFR-TKIs on the formation of the clones was obviously increased as compared with PC-9/Ctrl cells(P<0.05).Wound healing assay showed that PC-9/KD cells exhibited a prolonged healing time and an increased ability of EGFR-TKIs to inhibit migration as compared with PC-9/Ctrl cells(P<0.05);Subsequently,transwell invasion assay similarly indicates a reduction in the number of invasive cells and an increased ability of EGFR-TKIs to inhibit invasion(P<0.05).All of the above experiments confirmed that knockdown of Cav-1inhibited the proliferation,migration and invasion of PC-9 cells and enhanced the inhibitory effect of EGFR-TKIs on biological behavior.4 Knockdown of Cav-1 enhances EGFR-TKIs sensitivity to PC-9 cells by regulating EGFR phosphorylationThe phosphorylation levels of EGFR,Akt and Erk in PC-9/KD cells were significantly lower than that in PC-9/Ctrl cells and the inhibitory effect of EGFR-TKIs on signal proteins was significantly enhanced(P<0.05).We confirmed that Cav-1 functionally regulate the activity of EGFR in PC-9 cells due to direct interaction which would require physical contact in the same region.IP assay showed that proteins of Cav-1 and EGFR could be2 Knockdown of Cav-1 enhances the sensitivity of PC-9 cells toco-immunoprecipitated from cell lysates.Confocal microscopy also found that Cav-1 co-localizes with EGFR.The yellow fluorescent points indicated co-localization of Cav-1(red)and EGFR(green)in the same domain.In summary,these results suggest that knockdown of Cav-1 dramatically inhibited cell growth and enhanced EGFR-TKIs sensitivity by down-regulating phosphorylation of EGFR and inhibition of MAPK and PI3 K pathways.5 Knockdown of Cav-1 inhibits tumor growthThe tumor volumes of PC-9/KD group were significantly less than those of PC-9/Ctrl group(P<0.05),whether or not treated with gefitinib.A significant lighter tumor weight was observed in PC-9/KD group than that of PC-9/Ctrl group(P<0.05).6 Knockdown of Cav-1 increases gefitinib sensitivity in vivoAfter 2 weeks of gefitinib administration,the tumor growth of PC-9/KD group was inhibited evidently and that of PC-9/Ctrl group got basically unchanged.The tumor inhibition rate of PC-9/KD group were significantly greater than those of PC-9/Ctrl group(P<0.05).Western blot analysis was performed with tumor tissue of each nude mouse and the results were consistent with those of in vitro.IHC assays showed that xenograft tumors from PC-9/KD cells exhibited lower Cav-1 and Ki-67 expression as compared with respective control.Moreover,the level of Ki-67 in nude mice treated with gefitinib was significantly lower than that of no treated with gefitinib.A significant positive correlation was detected between the immunoreactivity for Cav-1 and Ki-67 in nude mice no treated with gefitinib.Part three Effects of overexpression of Caveolin-1 on sensitivity of EGFR-TKIs in lung adenocarcinoma cells with EGFR mutationObjective: To investigate the effects of overexpression of Cav-1 on sensitivity of EGFR-TKIs in lung adenocarcinoma cells with EGFR mutation. PC-9 cells were transfected either with pcDNA3.1/Cav-1 plasmid or pcDNA3.1 empty plasmid,and selected by G418(1000 ?g/ml),resulting in PC-9/Cav-1 and PC-9/pcDNA3.1 stably transfected clones.2 The rest of experimental methods are the same as part two.Results:1 Construction of stable transfected cell linesThe expression level of Cav-1 was significantly upregulated in PC-9/Cav-1 cells compared with PC-9/pcDNA3.1 cells(P<0.05).2 Overexpression of Cav-1 inhibits the sensitivity of PC-9 cells to EGFR-TKIsCell viability of stably transfected cells treated with the indicated doses of EGFR-TKIs for 48 hours was assessed with MTS assay.Whether gefitinib or erlotinib,the inhibition rate and IC50 of PC-9/Cav-1 cells was higher than that of PC-9/pcDNA3.1 cells(P<0.05).Inhibition rate was tested at different intervals(24,48 and 72 hours)using the lower IC50 of 48 hours in each pair of stably transfected cell lines(P<0.05).The inhibition was dose and time dependent manner.Our results confirmed that overexpression of Cav-1increase the tolerance of PC-9 cells to EGFR-TKIs.3 Overexpression of Cav-1 promotes the proliferation,migration and invasion of PC-9 cells and weakens the inhibitory effect of EGFR-TKIs on cell biological behaviorThe clone formation and colony forming efficiency of PC-9/Cav-1 cells was higher than PC-9/pcDNA3.1 cells and the inhibitory effect of EGFR-TKIs was obviously decreased(P<0.05).Wound healing assay showed that PC-9/Cav-1 cells exhibited a shorter healing time and an decreased ability of EGFR-TKIs to promote migration as compared with PC-9/pcDNA3.1cells(P<0.05);Subsequently,transwell invasion assay similarly indicates an increasing in the number of invasive cells and an decreased ability of EGFR-TKIs to inhibit invasion(P<0.05).All of the above experiments confirmed that overexpression of Cav-1 promoted cell proliferation,migration and invasion and weakened the inhibitory effect of EGFR-TKIs.Methods:1 Transfection4 Overexpression of Cav-1 weakens EGFR-TKIs sensitivity to PC-9 cells by regulating EGFR phosphorylationThe phosphorylation levels of EGFR,Akt and Erk in PC-9/Cav-1 cells were obviously higher than that in PC-9/pcDNA3.1 cells and the inhibitory effect of was reduced(P<0.05).IP assay showed that proteins of Cav-1 and EGFR could be co-immunoprecipitated from cell lysates.Confocal microscopy also found that Cav-1 co-localizes with EGFR.The yellow fluorescent points indicated co-localization of Cav-1(red)and EGFR(green)in the same domain.In summary,these results suggest that Cav-1up-regulation significantly promoted cell growth and decreased EGFR-TKIs sensitivity.5 Overexpression of Cav-1 promotes tumor growthThe tumor volumes of PC-9/Cav-1 group were obviously greater than those of PC-9/pcDNA3.1 group(P<0.05),whether or not treated with gefitinib.Tumor weight of PC-9/Cav-1 group was significantly higher than that of PC-9/pc DNA3.1 group(P<0.05).6 Overexpression of Cav-1 decreases gefitinib sensitivity to PC-9 cells in vivoAfter 2 weeks of gefitinib administration,the tumor size of PC-9/Cav-1group had been in a state of slow growth and that of PC-9/pcDNA3.1 group tended to be unchanged.The tumor inhibition rate of PC-9/Cav-1 group were obviously less than those of PC-9/pcDNA3.1 group(P<0.05).Western blot analysis was performed with tumor tissue of each nude mouse and the results were consistent with those of in vitro.IHC assays showed that xenograft tumors from PC-9/Cav-1 cells exhibited higher Cav-1 and Ki-67 expression as compared with respective controls.Moreover,the level of Ki-67 in nude mice treated with gefitinib was significantly lower than that of no treated with gefitinib.A significant positive correlation was detected between the immunoreactivity for Cav-1 and Ki-67 in nude mice no treated with gefitinib.Conclusion:1 The expression level of Cav-1 was negatively correlated with thecurative effect of EGFR-TKIs in lung adenocarcinoma.2 Knockdown of Cav-1 could inhibit cell proliferation,migration and invasion and enhance EGFR-TKIs sensitivity to PC-9 cells.3 Overexpression of Cav-1 could promote cell proliferation,migration and invasion and decrease EGFR-TKIs sensitivity to PC-9 cells.4 Cav-1 could promote cell growth and decrease EGFR-TKIs sensitivity to lung adenocarcinoma cells by up-regulating phosphorylation of EGFR and activating downstream signal transduction of MAPK and PI3 K pathways.