Human CRKL (obtained from CCSB ORFeome collection) was cloned into pWzl-blast and pLenti6.3-blast-C-terminal V5 epitope tagged vectors. The CRKL mutants (CRKLW160L
, CRKLsilent mutant
) were generated using Quikchange Site-Directed Mutagenesis kit (Stratagene). Human RAP1A, RAP1AQ63E
(provided by Gromoslaw Smolen, Massachusetts General Hospital), RAP1B (Origene) and RAP1BG12V
were cloned into Bam
HI and Bsr
GI sites of pLenti6.3-blast. pLenti6.3-blast-SRC and SRCT351I
have been described (45
). pLenti6.2-blast-LacZ control vector was provided by Guo Wei, Dana-Farber Cancer Institute. pMKO.1-puro-shNF1, pBabe-puro-HRASV12
and myristoylated AKT1 have been described (51
). pLKO.1-puro-shRNA constructs were obtained from The RNAi Consortium. The sequences targeted by CRKL
-specific shRNAs are as follows: shCRKL#1 (TRCN0000006378), 5′-GCTCTGCTCTACCATGTTTAA-3′; shCRKL#2 (TRCN0000006380) 5′-CGTGAAAGTCACAAGGATGAA-3′; shCRKL#3 (TRCC0007470203), 5′-GCCTACTGAGTAGCTTTCATT-3′. A pool of 85 control shRNAs targeting reporter genes (GFP, RFP, Luciferase and LacZ) was used to generate control lentiviruses (Control shRNAs) (30
). The sequences targeted by shGFP or scrambled control shRNA are 5′-ACAACAGCCACAACGTCTATA-3 ′ (TRCN0000072181) and 5′-GTGGACTCTTGAAAGTACTAT-3′, respectively. Other shRNA constructs used are as follows: shARAF#1 (TRCN0000000570), shARAF#2 (TRCN0000000567), shBCAR1#1 (TRCN0000115983), shBCAR#2 (TRCN0000115984), shBRAF#1 (TRCN0000006289), shBRAF#2 (TRCN0000006290), shBRAF#3 (TRCN0000006292), shRAF1#1 (TRCN0000001066), shRAF1#2 (TRCN0000001068), shRAF1#3 (TRCN0000001065), shKRAS#1 (TRCN0000033263), shKRAS#2 (TRCN000003326), shRAP1A#1 (TRCN0000029784), shRAP1A#2 (TRCN0000029787), shRAP1B#1 (TRCN0000029176), shRAP1B#2 (TRCN0000029177), shSOS1#1 (TRCN0000048145), shSOS#2 (TRCN0000048144), shSOS1#3 (TRCN0000048146), shSRC#1 (TRCN0000195339), shSRC#2 (TRCN0000038150), shC3G#1 (TRCN0000048128), shC3G#2 (TRCN0000048129), shC3G#3 (TRCN0000048130) and shC3G#4 (TRCN0000048131).
Cell culture and virus production
HCC515, H1819, HCC1833, H2087 and HCC827 cells were maintained in DMEM (Mediatech) supplemented with 10% fetal bovine serum (FBS, Sigma). HCC1359, H1755, H1437 and H1792 cells were maintained in RPMI1640 (Mediatech) supplemented with 10% FBS. Immortalized human lung airway epithelial (AALE) cells (32
) were maintained in SABM supplemented with SAGM SingleQuots (Lonza). Retroviruses were produced by transfecting 293T packaging cells with pBabe/pWzl/pMKO and pCL-Ampho plasmids (51
). Lentiviruses were produced by transfecting 293T packaging cells with a three-plasmid system (30
). To generate stable cell lines, cells were selected in media containing 2 μg/ml of puromycin for 2 d or 10 μg/ml of blasticidin for 4 d.
Gefitinib, dasatinib and GDC-0941 were purchased from Selleck Chemicals. PHA665752 was purchased from Tocrus Biosciences. LY294002 was purchased from EMD Biosciences.
Cell proliferation assay
Cells were seeded into 96-well plates for 24 h. Six replicates infections were performed for control shRNAs or each gene-specific shRNA in the presence of 4 μg/ml of polybrene for 24 h followed by selection in media containing 2 μg/ml of puromycin. The ATP content was measured at 5 d by using CellTiter-Glo luminescent cell viability assay (Promega). Data represent mean + s.d. of 6 replicate measurements.
For the rescue experiments, H1755 (1.8×105) cells were incubated with lentiviruses expressing LacZ control, CRKL, RAP1, RAP1A63E, RAP1B or RAP1BV12 in 24-well plates in the presence of 4 μg/ml polybrene, and spin-infected at 2,000 rpm for 2 h at 37°C. Cells were then trypsinized and re-plated at a density of 1,500 cells/well of 96-well plates for 24 h.
For measuring IC50, HCC827 cells (1.5×103) expressing CRKL or a control vector were seeded into 96-well plates for 24 h and then incubated with gefitinib for 72 h. The ATP content was measured by using CellTiter-Glo luminescent cell viability assay (Promega). Data represent mean ± s.d. relative to untreated cells for each cell line. For the RNAi experiments, HCC827 cells overexpressing CRKL or a control vector were infected with lentiviruses encoding indicated shRNAs for 24 h, and then selected with 2 μg/ml of puromycin for 2 d. Cells were re-plated into 96-well plates for 24 h before treatment with gefitinib for 72 h.
Luminex immunosandwich assay
) cells were plated onto each well of 6-well plates for 24 h. Triplicate transfections with 3 μg of pLenti-LacZ, CRKL or CRKLW160L
were performed using Fugene 6 (Roche) for 16 h. After the incubation, medium was replaced with fresh medium and cells were cultured for another 32 h before collecting cell lysates. Luminex immunosandwich assay was performed as described (45
Anchorage independent growth assay
Growth in soft agar was determined by plating 5×104
cells in triplicate in 0.4% Noble agar (51
). Colonies greater than 0.2 mm in diameter were counted 4 weeks after plating. Data represent mean + s.d. of 6 replicate measurements from 2 independent experiments.
Tumor xenograft experiments were performed as described (51
). AALE cells expressing indicated constructs were trypsinized and collected in DMEM supplemented with 10% FBS. Cells (8×106
) were resuspended in 400 μl of 1× PBS and mixed with 400μl of Matrigel™
-Basement Membrane Matrix, LDEV-free (BD Biosciences). 200 μl of the cell mixture (containing 2×106
cells) was injected subcutaneously into 6-week-old male BALB/c nude mice (Charles River). Tumor injection sites were monitored for 5 months.
For tumor growth studies, HCC515 cells were infected with lentiviruses expressing Luciferase followed by selection in media containing 10 μg/ml of blasticidin for 5 d. Luciferized HCC515 cells were transduced with lentiviruses encoding pLKO-Tet-On-scrambled control shRNA or pLKO-Tet-On-shCRKL#1 and selected in 2 μg/ml of puromycin to generate stable cell lines. 5×106 cells were resuspended in 200 μl of PBS and injected subcutaneously into 6-week-old male BALB/c nude mice (Charles River). Tumor size was measured by a caliper twice weekly. Non-invasive bioluminescent imaging was performed at 11 and 32 d after implantation. Mice were given a single intraperitoneal injection of a mixture of luciferin (50 mg/kg), ketamine (150 mg/kg) and xylazine (12 mg/kg) in sterile water. After 5 min, mice were placed in a chamber and photons were captured for a time period of 120 s by using IVIS imaging camera (Xenogen). Images were generated by using LIVING IMAGE 2.60.1 software.
Cell lysates were prepared by scraping cells in lysis buffer [50 mM Tris (pH 8), 150 mM NaCl, 1% Nonidet P40, 0.5% Sodium Deoxycholate and 0.1% SDS] containing complete protease inhibitors (Roche) and phosphatase inhibitors (10 mM Sodium Fluoride and 5 mM Sodium Orthovanadate). Protein concentration was measured by using BCA Protein Assay kit (Pierce). An equal amount of protein (30 μg) was separated by NuPAGE Novex Bis-Tris 4–12% gradient gels (Invitrogen) and then transferred onto a polyvinylidene difluoride membrane (Amersham). The membrane was then incubated with primary antibodies for 1 h at room temperature. Phospho-specific antibodies against phospho-S473 AKT (#9271), phospho-Y1068 EGFR (#3777), phospho-Y1289 ERBB3 (#4791), phospho-T202/Y204 ERK1/2 (#4370), phospho-Y118 Paxillin (#2541), phospho-S338 RAF1 (#9427) and phospho-Y416 SRC (#2113), were purchased from Cell Signaling. Antibodies against ERK1/2 (#4695), ARAF (#4432), RAF1 (#9422), Caspase-3 (#9662) and PARP (#9532) were purchased from Cell Signaling Technology. Antibodies against BRAF (sc-5284), phospho-Y504 C3G (sc-12926), C3G (sc-869), CRKL (sc-319), KRAS (sc-30), SOS1 (sc-256) and SRC (sc-19) were from Santa Cruz Biotechnology. Antibodies against phospho-Y249 p130CAS (#558401) and p130CAS (#610272) were from BD Bioscience. Antibodies against p85 (#06-496), RAS (#05-516), RAP1 (#07-916), phospho-MEK (#07-461) and phospho-tyrosine (4G10) were from Millipore. Antibody specific for NF-1 was provided by Karen Cichowski, Brigham and Women’s Hospital. After incubation with the appropriate horseradish peroxidase-linked secondary antibodies (Bio-Rad), signals were visualized by enhanced chemiluminescence plus Western blotting detection reagents (Amersham). Expression of β-actin was also assessed as an internal loading control by using a specific antibody (sc-8432-HRP, Santa Cruz). Intensities of bands were quantified by LabWorks image analysis software (UVP).
Cell lysates were collected by scrapping cells in immunoprecipitation buffer [20 mM Tris (pH 8), 100 mM NaCl, 2 mM EDTA and 0.5% NP-40] containing protease and phosphatase inhibitors. 1 mg of protein extracts was incubated with 2 μg of anti-CRKL antibody (sc-319, Santa Cruz), 5 μg of anti-p85 antibody (#06-496, Millipore) or normal Rabbit IgG (sc-2070, Santa Cruz) for 2 h at 4°C followed by incubation with 30 μl of Protein A/G-PLUS agarose beads (sc-2003, Santa Cruz) for 1 h at 4°C. Beads were collected by centrifugation at 2,000 ×g for 3 min at 4°C and resuspended in 1 ml of immunoprecipitation buffer for washing. After repeated washing and centrifugation for 4 times, beads were boiled for 5 min in 1× NuPAGE LDS sample buffer (Invitrogen).
Pull-down assay for GTP-bound RAS or RAP1 was performed using RAS and RAP1 activation assay kits (Millipore), respectively. For measuring in vitro BRAF kinase activity, 500μg of protein extracts was incubated with 2 μg of anti-BRAF antibody (sc-5284, Santa Cruz) or normal Mouse IgG (sc-2025, Santa Cruz) for 2 h at 4°C followed by incubation with 20 μl of Protein A/G-Plus agarose beads for 1 h at 4°C. After washing with immunoprecipitation buffer for 5 times, beads were incubated with 1.34 μg MEK substrate proteins (#17-359, Millipore) in the presence of 37.5 mM MgCl2 and 250 μM ATP for 30 min at 30°C. Beads were boiled in sample buffer for 5 min and supernatants were resolved for immunoblotting for phospho-MEK1 (#07-461, Millipore).
Real-Time Quantitative Reverse-Transcription PCR
Total RNA was extracted with TRIzol reagent (Invitrogen) and 1 μg of total RNA was used to synthesize the first-strand cDNA using Oligo(dT)20/random hexamer primer cocktails and Superscript III reverse transcriptase (Invitrogen). Quantitative PCR reactions were performed using SYBR green PCR Master Mix (Applied Biosystems). The primer sequences used are as follows: CRKL-primer set 1 (forward: 5′-CTGTCGGTGTCCGAGAACTC-3′; reverse: 5′-ATTGGTGGGCTTGGATACCTG-3′), CRKL-primer set 2 (forward: 5′-AAGCCCACCAATGGGATCTG-3′; reverse: 5′-ACTCCACCACTGTTCTTCAGG-3′) and GAPDH (forward: 5′-CCTGTTCGACAGTCAGCCG; reverse: CGACCAAATCCGTTGACTCC). Triplicate reactions were performed separately on the same cDNA samples by using the ABI 7900HT real time PCR instrument (Applied Biosystems). The mean cycle threshold (Ct) was used for the comparative Ct analysis method (ABI User Bulletin #2).
Quantitative PCR for gene copy number
The standard curve method was used to determine the copy number of CRKL in NSCLC and AALE cells. Genomic DNA was extracted using DNeasy blood and tissue kit (Qiagen). The primer sequences for detecting CRKL were 5′-TTGACAGGCACTGGCTTAGA-3′ and 5′-GGCACTCCACCACTGTTCTT-3′. The primer sequences for detecting LINE-1 were 5′-AAAGCCGCTCAACTACATGG-3′ and 5′-TGCTTTGAATGCGTCCCAGAC-3′. The standard curve was generated by PCR of serially diluted genomic DNA of AALE cells (50, 10, 2, 0.4 and 0.08 ng). Triplicate reactions were performed using 2 ng of genomic DNA extracted from NSCLC cells. The gene copy number was normalized to LINE-1 and normal reference DNA of AALE cells.
Fluorescence in situ hybridization (FISH)
BAC RP11-505B16 clone containing CRKL (Invitrogen) was labeled with digoxigenin (Roche) and BAC RP11-47N6 clone containing 22q13.2 as a reference (Invitrogen) was labeled with biotin using BioPrime labeling mix (Invitrogen). Labeled DNA was precipitated at −80°C for 2 h with glycogen (20 μg/μl), pelleted by centrifugation at 18,000 ×g for 15 min at 4°C, air-dried for 10 min, and resuspended in 50 μl of hybridization buffer (50% deionized formamide, 10% dextran sulfate, 2× SSC).
Slides containing metaphase chromosomes were pretreated with 1:25 Digest-All III (Invitrogen) at 37°C for 6 min, and fixed in 10% buffered formalin for 1 min. Slides were dehydrated for 2 min each in 70%, 90% and 100% ethanol. Probes were prepared by mixing 2 μl of each labeled probe, 1 μl Cot-1 DNA (1 mg/ml; Invitrogen), and 11 μl of hybridization buffer. Probes were applied to air-dried slides and covered with coverslips. Slides were incubated at 72°C for 5 min to denature probes. Hybridization was performed for 18 h at 37°C in a dark humid chamber. After hybridization, slides were washed in 0.5× SSC at 72°C for 5 min and rinsed in PBS containing 0.025% Tween-20 at room temperature. Slides were blocked with CAS-Block containing 10% normal goat serum (Invitrogen) and incubated with FITC-anti-digoxigenin (Roche) and Alexa Fluor 594 Streptavidin (Invitrogen). Slides were washed in PBS containing 0.025% Tween-20 and counterstained with DAPI (Invitrogen). Images were captured using Zeiss Axio Observer Z1 microscope and AxioVision imaging software (Zeiss).
Tumor specimens from erlotinib-treated patients were obtained from Dana-Farber Cancer Institute, Brigham and Women’s Hospital and Massachusetts General Hospital. All samples were analyzed under IRB-approved protocols. The presence of EGFR
mutations in each specimen was confirmed as described (54
). FISH analysis of tumor specimens using CRKL
-specific probe (BAC RP11-505B16) and chromosome 22 telomere-specific probe (Abbott Molecular) were performed as described (54