| Part ⅠEndothelial PAS domain-containing protein 1 confers TKIresistance by mediating EGFR and MET pathways in non-small cell lung cancer cellsLung cancer has become the urban population of malignant tumor 1 bit to the cause of death of malignant tumors. Non-small cell lung cancer, including common squamous cell carcinoma, adenocarcinoma and large cell carcinoma, etc., compared with small cell carcinoma of the cancer cells divide more slowly, spread relatively late. Non-small cell lung cancer accounts for about 80% of all lung cancer, of which about 75% of the patients has been found in the middle-late, 5-year survival rate is very low. In recent years, with the development of pharmacogenomics, significantly enhance,drug research about non-small cell lung cancer becoming popular. one hand, which to better play to the effect of chemotherapy play a guiding role; On the other hand, more and more targeted drugs have been developed, which has played a lot of encouraging results in cancer treatment. We made a lot of resistance about the targeted therapy of lung cancer, transfer factor, durg resistance,etc.,Now for the first generation of EGFR- TKI targeted therapy drugs, such as gefitinib and erlotinb, for patients with non-small cell lung cancer treated first, the median PFS can reach 10 months, but followed by resistance function to be solved. So some new protein gene receptor related research is hot. To explore if there are other factors mediating EGFR-MET interaction, we carried out a genome-wide search for HIF-1α homologs. To assess whether EPAS1 is directly involved in EGFR signaling pathway, we co-expressed HA-tagged EPAS1（HA-EPAS1） and wild-type EGFR（Myc-EGFR） in HCC827 cells and performed co-immunoprecipitation assay using anti-HA as bait. To rule out the possibility whether this selective interaction between EPAS1 and T790MEGFR was a cell line specific effect, we did the same expression and co-immunoprecipitation assay in another NSCLC cell line A549.Next we investigated whether the interaction between EPAS1 and T790 M EGFR was a direct binding or not, through protein crosslinking assay using dithio-bismaleimidoethane（DTME） as the crosslinker. HCC827 cells expressing HA-EPAS1 were also transfected with either wild-type or T790 M EGFR and protein lysates were subjected to immunoprecipitation with HA antibody after the crosslinking. To test whether the mechanism for EPAS1 binding to T790 M EGFR to up-regulate MET is more likely to operate independently of the extracellular ligand binding domain we constructed a truncated version of T790 M EGFR, where amino acids 2 to 620 coding for the ligand binding domain was deleted from its open reading frame, namely ΔN-T790 M.To assess whether interaction of EPAS1 to T790 M EGFR has physiological functions in NSCLC, we performed MTS proliferation and growth assay in HCC827 cells co-expressing EPAS1 and T790 M EGFR, by treating them with increasing concentrations of either gefitinib or erlotinib. To validate this result we performed colony formation assay to assess the survival of NSCLC cells co-expressing T790 M EGFR and EPAS1 with TKI treatment. In order to rule out the possibility that TKI-resistance we observed above could still be explained as a direct result of EPAS1 expression independent of MET activation, we knocked down endogenous MET expression by RNAi and repeated the MTS proliferation and growth assay.Methods: Cell culture NSCLC HCC827 and A549 cells were obtained from the American Type Culture Collection（Manassas） and passaged for less than 6 months after receipt for completion of the studies. Cells were maintained in DMEM containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 m M L-glutamine at 37°C in 5% CO2. Plasmids EPAS1 c DNA coding region was cloned into the p CMV-HA vector withan HA-tag in the N-terminal. The human EGFR c DNA coding region and the ligand binding domain（amino acid 2-620） deletion mutant EGFR were cloned into the pc DNA3.1 vector with a Myc-tag in the N-terminal. Immunofluorescence HCC827 cells transfected with HA-tagged EPAS1 were grown on cell culture wells and fixed in 4% formaldehyde for 30 min, followed by permeabilization using 0.2% Triton X-100 for 5 min. After fixation and permeabilization, cells were labeled with blocking buffer（5% BSA） with mouse monoclonal HA-probe as primary antibody and Alexa Fluor 488 goat anti-mouse as secondary antibody for 30 min at room temperature. DAPI was used to visualize DNA. Cells were washed three times with PBS after each antibody labeling step. Coverslips were mounted using Pro Long Gold Antifade reagent. Immunoprecipitation and immunoblotting Protein lysates were extracted using RIPA buffer（50 m M Tris-HCl, p H 7.4, 150 m M Na Cl, 1 m M EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitors）. Lysates were centrifuged at 12,000 rpm/min for 10 min at 4°C. The supernatants were rotated for 2 hours at 4°C with Protein A Sepharose beads coupled to anti-HA or anti-Myc antibodies. After incubation samples were thoroughly washed three times with lysis buffers and then twice with a final wash buffer（50 m M Tris, p H 7.5, and 50 m M Na Cl）. Proteins were eluted by adding SDS-PAGE sample buffer followed by boiling at 98°C for 5 min and were analyzed by SDS-PAGE and transferred to PVDF membrane using wet transfer unit. The membrane was then immersed in PBST（PBS, 0.1% Tween-20） with 5% nonfat milk for 2h and then incubated with appropriate primary antibody overnight at 4°C. After washes with PBST, blots were incubated with HRP-conjugated secondary antibody for 2h, washed again with PBST, and visualized using Luminata Forte Western HRP Substrate. Protein crosslinking assay HCC827 cells co-transfected with EPAS1 with either wild-type orT790M EGFR were collected by centrifugation at 800 g for 10 min at 4°C and resuspended in PBS buffer. Crosslinker was dissolved in DMSO immediately prior to use, and added to the cell suspension at a final concentration of 0.1 m M, followed by incubation for 2 hours at 4°C. Protein lysates were extracted using RIPA buffer, and subjected to immunoprecipitation. Proteins were eluted by adding SDS-PAGE sample buffer with or without DTT and analyzed by SDS-PAGE, followed by western blot analysis. RNA interference（RNAi） methods and Quantitative real-time PCR For RNAi knock-down experiments, lentiviral sh RNA constructs V2LHS113750 and V3LHS318637 targeting EPAS1, and V2LHS76544 and V3LHS642482 targeting MET, as well as GIPZ Non-silencing Lentiviral sh RNA Control were purchased from GE Dharmacon. Cells were transduced with the packaged virus and subsequently maintained with 1 μg/ml puromycin for stable knockdown. To assess the efficiency of RNAi assay, total RNA was isolated from cells and 1 μg of total RNA was reverse-transcribed with Easy Script Reverse Transcriptase[M-MLV,RNase H-] Kit（Trans Gen Biotech） as recommended by the manufacturer. GAPDH m RNA levels were measured for normalization and all data were presented as relative expression. Cell proliferation, growth and survival assays Growth and inhibition of growth were assessed by 3-（4, 5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-te trazolium（MTT） assay according to previously described methods. The cells were treated with TKIs for 72 hours, and the number of cells was scored as percentage relative to mock treatment. Colony formation assay was performed according to previously described methods. Each concentration of drug was performed five times. All data were presented as mean ± SEM. Statistical analysis All data were presented as mean ± SEM. Two-tailed student’s t-test was used to establish significant differences between groups. Data were determined to be statistically different when P < 0.05.Results:EPAS1（NCBI Reference Sequence: NP001421.2） was returned as the top hit with 48% amino acid sequence identity. When expressed in NSCLC cell line HCC827, EPAS1 localized mainly inside the nucleus, with lesser distribution throughout the cytoplasm, suggesting besides the role of transcription factors, it performs other cellular functions.The EGFR variant（Myc-T790M）, which contains the threonine 790 to methionine mutation responsible for adapted resistance in TKI therapies treating NSCLC, was pulled down together with EPAS1.This specific interaction between EPAS1 and T790 M EGFR was validated to be specific, since Myc-T790 M was only detected in the pull-down in the presence of HA-EPAS1 and vice versa. EPAS1 only interacted with T790 M but not wild-type EGFR in A549 cells, meaning the binding between these two proteins is a bona fide interaction across different cell lines. T790 M but not wild-type EGFR was pull-down together with HA-EPAS1. EPAS1 and T790 M EGFR synergistic up-regulation of MET indeed reflected a general mechanism in NSCLC cells. EPAS1 and T790 M EGFR interaction up-regulates MET pathway, through mechanism independent of EGF ligand binding. Both sh RNAs were able to degrade EPAS1 expression significantly compared with non-silencing control. Interaction with TKI-resistant EGFR to amplify MET signaling is an intrinsic function of EPAS1 protein. Conclusion: 1 EPAS1 is a HIF-1α homolog and directly interacts with T790 M EGFR in NSCLC cells. 2 EPAS1 and T790 M EGFR interaction up-regulates MET pathway independent of EGF ligand binding. 3 EPAS1 mediates T790 M EGFR and MET pathways to confer TKI-resistance in NSCLC cells.Part Ⅱ Micro RNA-200 a targets EGFR and c-Met to inhibit migration, invasion and gefitinib resistance in non-small cell lung cancerObjects: In order to investigate the role of mi R-200 a in human NSCLC, we next examined mi R-200 a expression in several NSCLC cancer cell lines（H3255, H1975 and HCC827） and normal lung cells（MRC-5 and CCD-19Lu） using RT-PCR.To confirm EGFR and c-Met are bona fide targets of mi R-200 a, we performed luciferase reporter assay, using sequences from original 3’-UTR on EGFR and c-Met m RNA as well as their mutated versions. We next transfected mi R-200 a into NSCLC cell lines and analyzed both m RNA and proteins levels of EGFR and c-Met. Next using antibodies against EGFR and c-Met, we detect whether their levels were consistently down-regulated in mi R-200 a transfected NSCLC cells.To test whether mi R-200 a can reduced the groth through its targets EGFR and c-Met, we performed wound-healing assay and cell invasion assay in both mi R-200 a and mi R-control transfected NSCLC cell lines. Moreover, MTS assay Brd U incorporation assay were performed to assess the cell viability and proliferation, respectively. We next investigated how different NSCLC cell lines transfected with mi R-200 a responded to gefitinib treatment by exposing them to increasing concentrations of the drug in cell proliferation and growth assay.Methods: Cell culture The human normal lung cell lines MRC-5 and CCD-19 Lu, and human lung cancer cell lines H3255, H1975 and HCC827 were obtained from the American Type Culture Collection（ATCC）.H3255 has the L858 R EGFR allele, H1975 has L858R/T790 M EGFR allele, and HCC827 harbors an in-frame EGFR E746-A750 deletion. Cells were cultured according to ATCC instructions and passaged for less than 6 months after receipt for completion of the studies. All cell lines were cultured as monolayers in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin-neomycin, in a humidified incubator at 37°C in a 5% CO2 atmosphere. Mi RNA transfection Hsa-mi R-200 a mir Vana mi RNA mimic, mature mi RNA sequence 5’-UAACACUGUCUGGUAACGAUGU-3’, was purchased from Applied Biosystems along with mir Vana mi RNA Mimic Negative Control, andtransfected into cell lines according to manufacturer’s instructions. 10 μM mi RNA mimic and 10 μl Lipofectamine 2000 each in 500μl Opti-MEM were mixed and incubated at room temperature for 20 min. The mixture was then added to cells pre-seeded in 6-well plates at 60% confluency, followed by 2-day incubation at 37°C. RNA extraction and quantitative Real time PCR（q RT-PCR） Total m RNA was extracted with Trizol according to the manufacturer’s instructions. The expression level of mi R-200 a was quantified using mi RNA-specific Taq Man Pri-mi RNA Assays and normalized by RNU48 Control mi RNA Assay. To quantify EGFR and c-Met m RNA levels, 1 μg of total RNA was reverse-transcribed with Easy Script Reverse Transcriptase as recommended by the manufacturer. 2 μl from a total of 20 μl c DNA was used for RT-PCR. GAPDH m RNA levels were measured for normalization. Luciferase reporter assay The putative mi R-200 a binding sites at the 3’-UTR of EGFR and c-Met were cloned downstream of a CMV promoter-driven firefly luciferase cassette in a p MIR-REPORT vector Mutant forms of the luciferase constructs were also generated using standard PCR-based overlap-extension protocols. For luciferase reporter assay, HCC827 cells（80000） were plated in a 24-well plate and then co-transfected with hsa-mi R-200 a or mi R-control, either wild-type or mutant luciferase constructs, and p RL-TK, using Lipofectamine2000 according to manufacturer’s instruction. Cells were collected 48 h after transfection and analyzed using the Dual-Luciferase reporter Assay System. Protein isolation and western blot Protein lysates were extracted using RIPA buffer（50 m M Tris-HCl, p H 7.4, 150 m M Na Cl, 1 m M EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitors）. Lysates were centrifuged at 12,000 rpm/min for 10 min at 4°C. Proteins were analyzed by SDS-PAGE and transferred to PVDF membrane using wet transfer unit. The membrane was then immersed in PBST（PBS, 0.1% Tween-20） with 5% nonfat milk for 2h and then incubated with anti-EGFR or anti-c-Met) overnight at 4°C. Afterwashes with blocking buffer, blots were incubated with HRP-conjugated secondary antibody for 20 min, washed again with blocking buffer, and visualized using Luminata Forte Western HRP Substrate. Wound-healing assay 1×105 cells transfected with mir Vana mi RNA Mimic Negative Control #1 and hsa-mi R-200 a were seeded into six-well plate after transfection. A linear wound was carefully made by a 10 μl sterile pipette tip across the confluent cell monolayer, and the cell debris was removed by washing with fresh media. The migrated distance of the growing edge on wounded monolayers was measured at 24 h after being wounded. Cell invasion assay 1×105 cells transfected with mir Vana mi RNA Mimic Negative Control #1 and hsa-mi R-200 a were placed into the upper chamber per well with the Matrigel-coated membrane, which was diluted with serum-free culture medium. The lower compartment was filled with 500 μl of medium containing 10% fetal bovine serum as a chemo-attractant. The cells were incubated at 37 °C in a 5% CO2 humidified incubator for 24 h. The cells were counted under six random microscopic fields for each well, using NIH Image J software. Cell proliferation and growth assay Growth and inhibition of growth in the absence or presence of tyrosine kinase inhibitor gefitinib were assessed by MTT assay and Brd U incorporation assay according to previously described methods. The cells were treated with gefitinib at indicated concentrations for 72 hours, and the number of cells was scored as percentage relative to mock treatment. Data are shown as mean ± SEM of five independent experiments. Statistical analysis All data were presented as mean ± SEM. Two-tailed student’s t-test was used to establish significant differences between groups. Data were determined to be statistically different when P < 0.05.Results:mi R-200 a expression in H3255 was less than 20% of MRC-5. H1975 and HCC827 were slightly higher, but still only 33% and 25% compared to MRC-5. These results clearly indicated that compared with normal lung cells, mi R-200 a levels were significantly down-regulated in NSCLC cell lines. As a result, EGFR-wt luciferase activity was dramatically reduced by co-transfection of mi R-200 a, to less than 40% of mi R-control transfected levels. In contrast, luciferase activity of EGFR-mt construct was approximately 90% of the control under the same conditions. These results clearly indicated EGFR 3’-UTR was indeed direct target of mi R-200 a. The same experiment was performed for c-Met-wt and-mt luciferase constructs, also indicating that mi R-200 a directly targets the 3’-UTR of c-Met m RNA. H3255, H1975 and HCC827 cells transfected with control mi RNA migrated average 281 μm, 326 μm and 252 μm respectively. In contrast, when these cells were transfected with mi R-200 a, the distance migrated was greatly decreased to 134 μm, 168 μm and 83 μm for H3255, H1975 and HCC827 cells, showing at least 47% reduction to control. As expected, NSCLC cells transfected with mi R-200 a also exhibited severe defect in cell invasion assay compared to control The results indicated that, upon mi R-200 a transfection, the viability and proliferation of all three NSCLC cell lines were significantly repressed, compared to mi R-control transfected experiments. Compared with mi R-control transfection, EC50 of H3255 dropped to 0.008 μM, indicating mi R-200 a increased the sensitivity of H3255 to gefitinib. H1975 exhibited the biggest reduction in gefitinib resistance, with EC50 dropping from over 10 μM in control experiment to below 0.004 μM after mi R-200 a transfection. TKI-resistance of HCC827 remained the same between mi R-200 a and mi R-control transfected groups. Taken together, these data strongly indicated mi R-200 a was able to increase response to gefitinib in TKI-resistant NSCLC cell lines. Conclusion: 1 Mi R-200 a expression is down-regulated in NSCLC cell lines; 2 Mi R-200 a directly targets and down-regulates EGFR and c-Met levelsin NSCLC cell lines; 3 Mi R-200 a inhibits migration and invasion of NSCLC cells; 4 Mi R-200 a increases sensitivity to gefitinib in TKI-resistant NSCLC cell lines... |
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