Genomic and functional analysis identifies CRKL as an oncogene amplified in lung cancer


DNA amplifications, leading to the overexpression of oncogenes, are a cardinal feature of lung cancer and directly contribute to its pathogenesis. To uncover such novel alterations, we performed an array-based comparative genomic hybridization survey of 128 non-small-cell lung cancer cell lines and tumors. Prominent among our findings, we identified recurrent high-level amplification at cytoband 22q11.21 in 3% of lung cancer specimens, with another 11% of specimens exhibiting low-level gain spanning that locus. The 22q11.21 amplicon core contained eight named genes, only four of which were overexpressed (by transcript profiling) when amplified. Among these, CRKL encodes an adapter protein functioning in signal transduction, best known as a substrate of the BCR-ABL kinase in chronic myelogenous leukemia. RNA-interference-mediated knockdown of CRKL in lung cancer cell lines with (but not without) amplification led to significantly decreased cell proliferation, cell-cycle progression, cell survival, and cell motility and invasion. In addition, overexpression of CRKL in immortalized human bronchial epithelial cells led to enhanced growth factor-independent cell growth. Our findings indicate that amplification and resultant overexpression of CRKL contribute to diverse oncogenic phenotypes in lung cancer, with implications for targeted therapy, and highlight a role of adapter proteins as primary genetic drivers of tumorigenesis.


Lung cancer is the leading cause of cancer death in the United States, accounting for almost 30% of all cancer-related mortality (Jemal et al., 2008). Nearly 80% of lung cancers diagnosed are non-small-cell lung cancers (NSCLCs), which are classified into three main histological subtypes: adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Despite the advancement of surgical, cytotoxic and radiological treatment options over the years, lung cancer therapy remains largely ineffective from a clinical standpoint, as evidenced by a low 5-year survival rate (<15%), and underscores the aggressive nature of the disease.

Much effort has been directed toward elucidating the pathogenetic alterations underlying the initiation and progression of NSCLC, with the hope of developing novel therapeutics to selectively target those alterations in vivo. Indeed, recent application of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors has been moderately successful in the treatment of NSCLCs harboring activating point mutations of EGFR (Lynch et al., 2004; Paez et al., 2004). It is likely that other genetic alterations in NSCLC await discovery, and once characterized might provide useful targets for therapy.

Genomic DNA amplifications are a frequent class of aberrations in NSCLC, where increased gene dosage leads to overexpression of key cancer genes. Genomic profiling studies of NSCLC, using cDNA (Tonon et al., 2005; Kwei et al., 2008), BAC (Garnis et al., 2006), oligonucleotide (Tonon et al., 2005; Kendall et al., 2007) and single nucleotide polymorphism (Zhao et al., 2005; Weir et al., 2007) arrays have revealed focal amplicons harboring known oncogenes, such as KRAS (12p12.1), EGFR (7p12.2), ERBB2 (17q12), MET (7q31.2), MYC (8q24.1), CDK4 (12q14.1) and CCND1 (11q13.2), and have led to the recent discovery of TITF1 (14q13) as a lineage-dependent oncogenic transcription factor amplified in lung cancer (Kendall et al., 2007; Weir et al., 2007; Kwei et al., 2008). For other recurrent amplicons, the driver oncogene(s) have not yet been identified, and mapping such loci provides a starting point for cancer gene discovery and characterization. Here, from a cDNA microarray-based genomic profiling analysis of 128 lung cancer specimens, we identify amplification of CRKL (22q11) as a recurrent genetic event promoting cell proliferation, survival and invasion in lung cancer.


Recurrent 22q11 amplicon in NSCLC spans CRKL

To identify recurrent DNA amplifications pinpointing novel oncogenes in NSCLC, we analyzed cDNA array comparative genomic hybridization (CGH) data generated on 52 NSCLC cell lines and 76 NSCLC tumors, the latter comprising 36 adenocarcinomas (including 2 metastases) and 40 squamous cell carcinomas (with 1 metastasis), totaling 128 samples. One of the most frequently amplified loci not associated with a known oncogene occurred at cytoband 22q11.21 (Figure 1a), where high-level amplification (fluorescence ratios >3, corresponding to >5-fold amplification; Pollack et al., 1999) was found in 4 of 128 samples analyzed (3%), with low-level gain spanning 22q11.21 present in an additional 14 of 128 samples (11%). There was no significant difference in the frequency of 22q11.21 gain/amplification between adenocarcinoma and other histologies (both for cell lines and for tumors), nor in the NSCLC lines was there a significant association between 22q11.21 gain/amplification and mutation of KRAS, EGFR or TP53 (data not shown). Conversely, we observed significant (false discovery rate <0.01) association of 22q11.21 gain with concomitant gains elsewhere in the genome, namely at 9q34.3, 11q13.2–q13.3 (CCND1), 15q24.1 and 21q22.3 (Supplementary Table 2), suggesting possible cooperative interactions.

Figure 1

Recurrent 22q11 amplicon in non-small-cell lung cancer (NSCLC) spans CRKL. (a) Frequency plot of cytobands harboring high-level DNA amplification in NSCLC cell lines. Cytobands consisting known oncogenes in lung cancer are indicated, with 22q11.21 highlighted in red. (b) Heat map representation of array comparative genomic hybridization (CGH) profiles of NSCLC cell lines and tumors representing a segment of 22q11.21. Each column represents a different sample (histologies indicated, M=metastasis) and each row represents a different gene ordered by chromosome position. Red indicates positive tumor/normal array CGH ratios (scale shown), and samples called gained (by cghFLasso) or highly amplified at 22q11.21 are marked below by closed black or red circles. Genes residing within the smallest common region of gain (amplicon core) are indicated; those with increased expression when amplified (see Figure 2a) are highlighted in red. AIFM3 (asterisked) was not present on the array but resides where shown. (c) fluorescence in situ hybridization (FISH) validation of CRKL amplification in NSCLC cell lines HCC515 and H1819. DNA amplification is indicated by increased CRKL (green) to telomere-22q (red) signals or signal clusters.

The smallest region of recurrent amplification (or amplicon core) (Figure 1b), spanned approximately 170 kb within 22q11.21, and contained eight known RefSeq genes, which included SNAP29 (synaptosomal-associated protein 29), CRKL (v-crk avian sarcoma virus CT10 oncogene homologue-like), AIFM3 (apoptosis-inducing factor, mitochondrion-associated 3), LZTR1 (leucine-zipper-like transcriptional regulator 1), THAP7 (THAP domain-containing protein 7), FLJ39582 (hypothetical protein LOC439931), P2RXL1 (purinergic receptor P2X-like 1, orphan receptor) and SLC7A4 (solute carrier family 7 (cationic amino-acid transporter, y+ system), member 4). Expression profiling, performed in parallel with array CGH, revealed that only four genes (SNAP29, CRKL, LZTR1 and THAP7) were overexpressed when amplified (P<0.05, Student's t-test) (Figure 2a), thus effectively narrowing the list of candidate ‘driver’ oncogenes. One of these genes, CRKL, encodes an Src homology 2 and 3 (SH2/SH3) domain-containing adapter protein that shares homology with the CRK proto-oncogene (ten Hoeve et al., 1993). Best known as a substrate of the BCR-ABL oncogenic kinase in chronic myelogenous leukemia (Sattler and Salgia, 1998), a role of CRKL in other cancer types remains largely unexplored. We therefore sought to characterize a possible role of CRKL amplification in lung cancer.

Figure 2

CRKL is overexpressed when amplified. (a) mRNA transcript levels (centered log2 ratios, measured by microarray) of genes residing within the 22q11.21 amplicon core, plotted separately for specimens with and without 22q11.21 amplification. Box plots show 25th, 50th (median) and 75th percentiles of expression for samples; genes whose expression is significantly (Student's t-test, P<0.05) elevated with DNA amplification are indicated (*). (b) Western blot analysis of CRKL protein in representative non-small-cell lung cancer (NSCLC) cell lines confirming overexpression in NSCLC cell lines with (compared to without) 22q11.21 amplification. Total and phosphorylated (active) CRKL (pY207) levels are shown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as a loading control. Note, CRKL amplification appears better correlated with p-CRKL than total CRKL (see Discussion section).

CRKL amplification promotes cell proliferation and survival

To assess the functional significance of CRKL amplification and overexpression in NSCLC, we used small-interfering RNAs (siRNAs) directed against CRKL in two cell lines, HCC515 and H1819, for which CRKL amplification had been validated by fluorescence in situ hybridization (Figure 1c), and with increased levels of total and phosphorylated (activated) CRKL protein (Figure 2b). Transfection of two different siRNAs targeting distinct sequences within CRKL each led to decreased levels of total and p-CRKL proteins (Figure 3a), and significantly decreased cell proliferation (measured using the WST-1 assay) compared with a negative control siRNA targeting an irrelevant gene, green fluorescent protein (GFP; Figure 3b). In contrast, knockdown of CRKL in H157, a lung cancer cell line without CRKL amplification and with comparatively less expression of CRKL, led to a more subtle effect on cell proliferation, supporting the specificity of CRKL targeting.

Figure 3

CRKL amplification contributes to cell proliferation and survival. (a) Confirmation of siRNA-mediated knockdown of CRKL protein by western blot analysis. Two different siRNA constructs targeting CRKL reduce total and phosphorylated protein levels in CRKL-amplified cell lines (HCC515 and H1819) and a cell line without amplification (H157), compared with a negative control siRNA targeting an irrelevant gene, green fluorescent protein (GFP). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as a loading control. (b) siRNA-mediated knockdown of CRKL results in decreased cell proliferation, compared to control siRNA, as measured by WST-1 assay in CRKL-amplified cells (HCC515 and H1819), with less pronounced effects in a nonamplified line (H157) expressing lower levels of CRKL (*P<0.05, Student's t-test, CRKL siRNA compared to control). (c) Knockdown of CRKL reduces cell-cycle progression as measured 72 h after transfection by 5-bromo-2-deoxyuridine (BrdU) incorporation, indicated by a decrease in S-phase fraction with G1 block compared to control siRNA (*P<0.05, Student's t-test). Representative flow cytometry plots are also shown. (d) Knockdown of CRKL leads to increased apoptosis levels 72 h after transfection, as quantified by flow cytometry-based Annexin V staining, compared to control siRNA (*P<0.05, Student's t-test). Representative flow cytometry plots are also shown; L, live; A, apoptotic; D, dead.

The observed decrease in cell proliferation might be attributable to decreased cell-cycle progression, increased apoptosis or both. To distinguish these possibilities, we assayed cell-cycle progression by measuring 5-bromo-2-deoxyuridine (BrdU) incorporation, and apoptosis by Annexin V staining. Targeted knockdown of CRKL in the amplified lines HCC515 and H1819 resulted in decreased cell-cycle progression, as evidenced by a significant decrease in S-phase fraction with G1 block (Figure 3c) compared with control siRNA. CRKL knockdown in amplified cell lines also led to increased apoptosis, evidenced by the higher fraction of Annexin V-positive cells with siRNA targeting CRKL versus control (though reaching significance only for HCC515) (Figure 3d).

CRKL amplification promotes cell migration and invasion

Earlier studies have implicated a role for CRKL in epithelial cell migration and invasion, with relevance to metastatic potential (Feller, 2001). We therefore sought to evaluate a possible role of CRKL amplification in cell migration/invasion in lung cancer. Targeted knockdown of CRKL in amplified NSCLC cell lines HCC515 and H1819 led to a significant inhibition of both cell migration (Figure 4a) and invasion (Figure 4b), compared with control siRNA. Furthermore, no such effect was observed in the lung cancer cell line H157 without amplification (Figures 4a and b), revealing a specific connection between CRKL amplification and cell migration and invasion. Co-transfection of a siRNA-resistant CRKL cDNA (containing silent mutations in the siRNA target site) largely rescued CRKL/p-CRKL levels and invasiveness of H1819 cells (Figure 4c), further confirming siRNA targeting specificity.

Figure 4

CRKL amplification potentiates cell motility and invasion. siRNA-mediated knockdown of CRKL, compared to control siRNA, results in significantly decreased cell counts for (a) migration and (b) invasion in cell lines with CRKL amplification (HCC515 and H1819) (*P<0.05, Student's t-test). No significant effects are seen in H157, a cell line without CRKL amplification. Representative microphotographs depicting cell migration and invasion (CRKL siRNA compared to control) are shown. (c) Rescue of siRNA knockdown confirms targeting specificity. H1819 cells were co-transfected with siRNAs and cDNA expression vectors as indicated, and invasion was assayed. pCMV6-CRKLR contains silent mutations within the siRNA#1 target site, creating an siRNA-resistant transcript (see Materials and methods section). Western blot (Figure) confirms knockdown and rescue of CRKL and p-CRKL levels; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as loading control.

CRKL overexpression promotes growth factor independence

To complement the RNAi knockdown studies, we sought to determine whether CRKL overexpression might promote oncogenic phenotypes in nontumorigenic lung epithelial cells. HBEC3 (Ramirez et al., 2004) is a human bronchial epithelial cell line immortalized by hTERT and Cdk4 (the latter bypassing p16-associated growth arrest), and provides a useful model for assessing the contribution of lung cancer genes (Sato et al., 2006). By retroviral transduction, we engineered HBEC3 cells stably overexpressing CRKL, though CRKL levels did not approach those observed in CRKL-amplified NSCLC lines (Figure 5a). Nonetheless, overexpression of CRKL in HBEC3 cells significantly enhanced growth factor (EGF)-independent cell growth (Figures 5b and c). However, CRKL overexpression in HBEC3 was not sufficient to promote cell invasion or anchorage-independent soft agar colony growth (data not shown).

Figure 5

CRKL overexpression promotes growth-factor-independent proliferation. (a) Western blot confirmation of CRKL and p-CRKL overexpression in stably transduced HBEC3 cells. H1819 and HCC515 lines provide a comparison for CRKL-amplified levels. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as a loading control. (b) CRKL overexpression leads to enhanced growth factor (EGF)-independent cell growth, measured by WST assay. (c) Time course confirmation of CRKL-promoted EGF-independent cell growth (*P<0.05; **P<0.01, Student's t-test; pMSCV-CRKL compared to pMSCV control).


By genomic profiling, we have identified a focal and recurrent amplicon at cytoband 22q11.21 in NSCLC cell lines and tumor samples. Within the amplicon core, CRKL is one of only four genes that are overexpressed when amplified. Interestingly, amplification appears even better correlated with p-CRKL levels than with total CRKL protein (Figure 2b), suggesting that amplification occurs within a genetic or cellular context appropriate for CRKL signaling. RNA interference studies in NSCLC cell lines with 22q11.21 amplification show a role and functional dependency on CRKL amplification for tumor cell proliferation, survival and motility/invasion. Overexpression studies in nontumorigenic HBEC3 cells also reveal a function of CRKL in growth-factor-independent proliferation, a classic oncogenic phenotype and, similar to that previously reported for K-RASV12 expression and p53 knockdown (Sato et al., 2006). However, in contrast to p53 (knockdown) and K-RASV12 (Sato et al., 2006), CRKL alone (at least at the expression levels achieved) appears insufficient to enhance anchorage-independent growth of HBEC3 cells. Further studies may reveal possible cooperative effects with other lung cancer genes.

Other recently published genomic profiling studies using various microarray platforms have also reported amplifications spanning 22q11.21 in NSCLC. Using submegabase resolution tiling BAC arrays, Garnis et al. (2006) identified high-level amplification at 22q11.21 in 2 of 28 NSCLC cell lines analyzed, both of which were verified in our study. Using 115K single nucleotide polymorphism arrays, Zhao et al. (2005) described 22q11.21 amplification in a panel of lung cancer cell lines (including HCC515, HCC1359 and H1819) and primary lung tumors. They were able to localize the amplicon to a 1 Mb region in 22q11.21, and suggested CRKL and PIK4CA (catalytic subunit of phosphatidylinositol 4-kinase-α) (centromeric to CRKL, and in our data set mapping outside the amplicon core), as possible driver genes. Interestingly, they went on to exclude CRKL as the likely driver because its protein levels were not increased in cell lines with amplification (their data not shown), a finding clearly discordant with ours. Most recently, Weir et al. (2007) used 250K single nucleotide polymorphism arrays to survey 371 lung adenocarcinomas, reporting high-level amplification at 22q11.21 in 2.4% of tumors, a frequency consistent with our findings. However, from the specimens surveyed they were only able to narrow down the amplicon to an 1 Mb interval consisting about 15 genes. Our own studies define an 140 Kb amplicon core, including four genes that are overexpressed when amplified. Though our functional studies implicate CRKL as the driver, we cannot exclude the possibility that one or more of the other three genes (SNAP29, LZTR1 and THAP7) contributes, though we note that their known functions do not relate in obvious ways to carcinogenesis. Of interest, very recently, Luo et al. (2008) identified CRKL through a short-hairpin RNA screen as essential for cell proliferation in a subset of NSCLC cell lines studied.

Our findings define a role of CRKL amplification in NSCLC pathogenesis. CRKL (Crk-Like) (ten Hoeve et al., 1993) is a member of the human Crk adapter protein family, which also includes two alternatively spliced isoforms (CRKI/II) of CRK, the cellular homologue of the avian retroviral v-crk oncogene (Feller, 2001). CRKL contains SH2 and SH3 domains that mediate protein–protein interactions connecting tyrosine-phosphorylated upstream signaling components (for example, p130CAS, paxillin, CBL, GAB1) to downstream effectors (for example, C3G, DOCK180), regulating diverse cellular processes like cell adhesion, migration and immune cell responses (Feller, 2001). Early studies identified CRKL as a key substrate and effector of the BCR-ABL oncogenic tyrosine kinase in chronic myelogenous leukemia (ten Hoeve et al., 1994; Senechal et al., 1996; Hemmeryckx et al., 2001). CRKL overexpression was also shown to activate Ras and JUN kinase (JNK) signaling pathways, and to transform Rat-1 (Senechal et al., 1996), though not NIH-3T3 (de Jong et al., 1997), rodent fibroblasts.

In epithelial cells, CRKL has been shown to potentiate hepatocyte growth factor (scatter factor)-induced cell motility through protein complexes connecting the MET receptor tyrosine kinase to downstream activation of effector proteins like Rap1 and Rac (Furge et al., 2000; Feller, 2001). The connection to MET is particularly intriguing given that MET is activated by mutation or amplification in some lung cancers (Ma et al., 2003; Zhao et al., 2005). In our NSCLC cell line panel, amplification of MET and CRKL was mutually exclusive (data not shown), consistent with their function in the same pathway (with CRKL downstream of MET). However, in preliminary studies, whereas knockdown of CRKL in an NSCLC cell line with MET amplification (H1648) reduced cell proliferation and survival, knockdown of MET in that same cell line reduced proliferation/survival (consistent with a recent report (Lutterbach et al., 2007)) but not phospho-CRKL levels (unpublished findings). Further studies are required to clarify the connection between MET and CRKL in NSCLCs with MET amplification.

Although we report here the amplification of CRKL in lung cancer, CRKL may have a pathogenic role in other epithelial cancers as well. Recently, Singer et al. (2006) described overexpression of phospho-CRKL, measured by immunohistochemistry, in breast, ovarian and colon cancer, as well as lung cancer, in comparison with the corresponding normal tissues. Our own array CGH studies have identified amplifications spanning 22q11.21 in breast and pancreatic cancers (Bashyam et al., 2005; Bergamaschi et al., 2006). Further studies are needed to establish a functional role of CRKL amplification/overexpression in these cancers. We also note that the related adapter CRK (at 17p13.3), although occasionally found within broad low-level gains in lung cancer, is not focally amplified as we have found CRKL.

A finding of particular interest from our study is that the amplification of an adapter protein, functioning solely to assemble other proteins, promotes strong and pleiotropic oncogenic phenotypes. Prototypic oncogenic amplifications include tyrosine kinases (ERBB2, EGFR, MET), ras proteins (KRAS), cell-cycle modulators (CCND1, CDK4) and transcription factors (MYC). Although overexpression of SH2/SH3 adapter proteins such as GRB2 (Daly et al., 1994), GRB7 (Stein et al., 1994) and GAB2 (Daly et al., 2002) has been implicated in oncogenesis, and v-Crk itself was discovered as an avian oncogene, our findings now place amplification of an adapter protein gene (CRKL) as a primary genetic event driving human cancer.

Our findings also underscore the potential of novel therapeutics targeting adapter protein interactions, and targeting CRKL in lung cancer in particular. Indeed, a small molecule inhibitor disrupting interactions of the GRB2 SH2 domain has been described (Gay et al., 1999), and more recently a peptide inhibitor selectively targeting the CRKL SH3 domain, disrupting its interaction with BCR-ABL (Kardinal et al., 2000). In summary, our combined genomic and functional analysis defines a novel role of CRKL amplification in lung carcinogenesis, potentiating cell proliferation, growth-factor independence, survival and migration/invasion, highlighting the oncogenic role of adapter proteins and suggesting a new point for therapeutic intervention.

Materials and methods

Lung cancer cell lines and tumors

‘NCI-H’ series NSCLC cell lines, established at the National Cancer Institute, and ‘HCC’ series cell lines, established at the Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center (Dallas, TX, USA), together totaling 52 cell lines, were obtained from the latter's tissue culture repository (most lines are currently available through the American Type Culture Collection, Manassas, VA, USA). For functional studies, we cultured cells in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Hyclone; Fisher Scientific, Pittsburgh, PA, USA). HBEC3 cells (Ramirez et al., 2004), obtained from the same repository, were grown in K-SFM medium (Invitrogen) containing 50 μg/ml bovine pituitary extract (Invitrogen) with 5 ng/ml EGF (Invitrogen). A total of 76 freshly frozen lung tumors were banked at the University Hospital Charité, Berlin, Germany, with institutional review board approval. Specimens were verified by hematoxylin and eosin staining to contain at least 70% tumor cells.

Genomic and gene expression profiling

cDNA microarray-based genomic profiling by CGH, and mRNA transcript profiling, of 128 lung cancer cell lines and tumors was described in a preliminary report of our study, focused on the identification of TITF1 amplification (Kwei et al., 2008). cDNA microarrays contained 39 632 human cDNAs, representing 22 279 mapped human genes and 4230 additional mapped expressed sequence tags. The complete microarray data sets are available at Stanford Microarray Database ( and at the Gene Expression Omnibus (accession GSE9995).

Microarray data analysis

Background-subtracted fluorescence ratios were normalized by mean-centering genes for each array. For array CGH analysis, we selected only those genes whose Cy3 reference-channel fluorescence signal intensity was at least 40% above background in at least 50% of samples. Map positions for arrayed cDNA clones were assigned using the NCBI genome assembly (Build 36), accessed through the UCSC genome database (Kent et al., 2002). We used the method cghFLasso (R package) to identify DNA gains and losses (Tibshirani and Wang, 2008). High-level DNA amplifications were defined as contiguous regions called by cghFLasso where at least 50% of genes show fluorescence ratios 3. To detect associations between DNA copy number alterations at distinct loci, we computed a Pearson's correlation between the mean copy number of a given cytoband and that of all other cytobands. Statistically significant correlations were determined by randomly permuting cytoband labels and recalculating correlations 100 times; a false discovery rate of 1% was used to establish a significance threshold. For expression profiling, fluorescence ratios were normalized for each array, and then well-measured genes (fluorescence intensities for the Cy5 or Cy3 channel at least 50% above background) were subsequently mean-centered (that is, reported for each gene relative to the mean ratio across all samples).

Fluorescence in situ hybridization

Probe labeling and fluorescence in situ hybridization were performed using Vysis (Downers Grove, IL, USA; now Abott Molecular) reagents according to the manufacturer's protocols. A locus-specific BAC probe targeting CRKL at 22q11.21 (RP11-1058B20) (BACPAC Resources Center, Oakland, CA, USA) was labeled with Spectrum green-dUTP, and co-hybridized with Spectrum orange-dUTP-labeled chromosome 22 telomeric probe (TelVysion 22q; Vysis). Chromosomal locations of labeled BAC probes were validated on metaphase slides prepared from normal donors. Slides were counterstained with 46-diamidino-2-phenyl indole, and imaged using an Olympus BX51 fluorescence microscope with Applied Imaging (San Jose, CA, USA) CytoVision 3.0 software.

siRNA transfections

Two different siRNAs targeting CRKL, along with a negative control siRNA targeting an irrelevant gene, GFP, were obtained from Qiagen, Valencia, CA, USA. Complete siRNA sequences are provided (Supplementary Table 1). Cell lines were maintained at 37 °C in complete media of RPMI 1640 with 10% FBS before transfection. For transfection, 80 000–175 000 cells were seeded per six-well plate, and transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. Cells were transfected with a final concentration of 50 nM siRNA for 6 h, and subsequently replaced with complete growth media.

Plasmid constructs

A full-length human CRKL cDNA expression vector, pCMV6/XL4-CRKL, and the parent vector, pCMV6/XL4, were purchased from OriGene (Rockville, MD, USA). A CRKL siRNA#1-resistant CRKL cDNA was engineered using the QuikChange XL II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), with the following mutational primers: 5′-IndexTermGGTTGGTGACATCGTGAAGGTGACCCGGATGAACATTAATGGCCAGTGGGAAG-3′ (degenerate, mutated bases denoted by bold) and 5′-IndexTermCTTCCCACTGGCCATTAATGTTCATCCGGGTCACCTTCACGATGTCACCAACC-3′. To generate HBEC3 cells stably overexpressing CRKL, we created a retroviral expression vector by PCR-amplifying full-length CRKL cDNA and subcloning the product into the HpaI and XhoI restriction sites of the vector pMSCV-Hyg (Clontech, Mountain View, CA, USA).

Viral transduction and stable selection

293T cells were transfected with either pMSCV-Hyg or pMSCV-Hyg-CRKL together with pVpack-VSVG and pVpack-GP vectors (Stratagene) to generate replication-defective retrovirus. Viral supernatants were collected 48 h after transfection, and used to infect HBEC3 cells, which were subsequently treated with 10 μg/ml hygromycin B (Invitrogen) for 14 days for stable selection.

Western blot analysis

Cells were lysed in 1 × RIPA Lysis buffer (Upstate/Chemicon, San Francisco, CA, USA) supplemented with 1 × Complete Protease Inhibitor (Roche, Indianapolis, IN, USA), 0.1 mM sodium orthovanadate, 1 mM sodium fluoride and 1 mM phenylmethylsulfonyl fluoride, and protein was quantified using the BCA assay (Pierce, Rockford, IL, USA). For western blot analysis, 20–30 μg of protein lysate was electrophoresed on a 4–15% Criterion Tris–HCl polyacrylamide gradient gel (Bio-Rad, Hercules, CA, USA) and transferred overnight to a PVDF membrane (Bio-Rad). After blocking in TBS-T buffer (20 mM Tris–HCl (pH 7.4), 0.15 M NaCl, 0.1% Tween 20) with 5% dry milk for 45 min, blots were incubated with primary antibody overnight (for phospho specificity) at 4 °C or 90 min (for native) at room temperature. After sequential washing steps, blots were incubated with horseradish-peroxidase-conjugated secondary antibody for 45 min at room temperature in TBS-T buffer. The following antibodies were used: phospho-CRKL Y207 (1:500; catalogue no. 3181; Cell Signaling, Danvers, MA, USA), anti-CRKL rabbit polyclonal antibody (1:1000; catalogue no. sc-319; Santa Cruz Biotechnology, Santa Cruz, CA, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, for loading control; Santa Cruz Biotechnology) and horseradish-peroxidase-conjugated anti-rabbit IgG (1:20 000; Pierce).

Proliferation assay

Cell proliferation was quantified by WST-1 assay (Roche), a colorimetric assay based on the metabolic cleavage of the tetrazolium salt WST-1 in viable cells, according to the manufacturer's protocol. WST-1 reagent (100 μl) was added to 1 ml of culture volume in six-well plates and incubated at 37 °C for 30 min. Absorbance was then measured at 450 nm with reference to 650 nm using a SpectraMax 190 plate reader (Molecular Devices, Sunnyvale, CA, USA). Transfections were performed in triplicate and average (±1 s.d.) OD reported.

Cell-cycle analysis

Cell-cycle distribution analysis was performed by flow cytometry using the BrdU-FITC Flow kit (BD Biosciences, San Jose, CA, USA) per the manufacturer's instructions. Cells were incubated with 10 μM BrdU at 37 °C for 5 h, then fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences). Cellular DNA was treated with DNase at 37 °C for 1 h to expose incorporated BrdU, then cells were stained with anti-BrdU fluorescein isothiocyanate antibody to quantify incorporated BrdU and 7-aminoactinomycin D to quantify total DNA content. A total of 15 000 events were scored by FACSCalibur (BD Biosciences) flow cytometer and analyzed using CellQuest software (BD Biosciences). Transfections were performed in triplicate and average (±1 s.d.) cell-cycle fractions were reported.

Apoptosis assay

Apoptosis levels were assayed by Annexin V staining, quantified by flow cytometry using the Vybrant Apoptosis Assay kit 2 (Invitrogen) per the manufacturer's recommendations. Briefly, floating and trypsinized adherent cells were pooled and resuspended in 200 μl 1 × Annexin binding buffer. One microliter Alexa Fluor 488 Annexin V and 1 μl of 100 μg/ml propidium iodide solution were added and cells were incubated for 15 min at room temperature. Cells were then resuspended in equal volume of 1 × Annexin binding buffer and analyzed immediately by flow cytometry. A total of 15 000 events were scored by FACSCalibur and analyzed using CellQuest software. Transfections were performed in triplicate, and average (±1 s.d.) percent apoptosis was reported.

Invasion and migration assays

Invasion and migration assays were carried out using BD Biocoat (BD Biosciences) modified Boyden chambers and control inserts with polyethylene membrane, respectively. For invasion assays, precoated filters (8 μm pore size, Matrigel 100 μg/cm2) were rehydrated with 500 μl of complete growth media (RPMI 1640, 10% FBS), then 1 to 5 × 104 cells resuspended in RPMI 1640 media with 2% FBS were seeded into the upper chamber. Following incubation for 16–72 h at 37 °C, cells were fixed in 10% buffered formalin and then stained with crystal violet. Migration assays were performed similarly to invasion assays, with the exception of using control inserts (with/without Matrigel coating) and using fewer cells (1 to 2.5 × 104 cells) and shorter time points (16–48 h). Noninvaded (or migrated) cells in the upper membrane were removed by swabbing, and the amount of invasion (or migration) was quantified by counting stained cells on the underside of the membrane. Assays were performed in triplicate and the average (±1 s.d.) cell count was reported.

Conflict of interest

The authors declare no conflict of interest.

Accession codes




  1. Bashyam MD, Bair R, Kim YH, Wang P, Hernandez-Boussard T, Karikari CA et al. (2005). Array-based comparative genomic hybridization identifies localized DNA amplifications and homozygous deletions in pancreatic cancer. Neoplasia 7: 556–562.

  2. Bergamaschi A, Kim YH, Wang P, Sorlie T, Hernandez-Boussard T, Lonning PE et al. (2006). Distinct patterns of DNA copy number alteration are associated with different clinicopathological features and gene-expression subtypes of breast cancer. Genes Chromosomes Cancer 45: 1033–1040.

  3. Daly RJ, Binder MD, Sutherland RL . (1994). Overexpression of the Grb2 gene in human breast cancer cell lines. Oncogene 9: 2723–2727.

  4. Daly RJ, Gu H, Parmar J, Malaney S, Lyons RJ, Kairouz R et al. (2002). The docking protein Gab2 is overexpressed and estrogen regulated in human breast cancer. Oncogene 21: 5175–5181.

  5. de Jong R, ten Hoeve J, Heisterkamp N, Groffen J . (1997). Tyrosine 207 in CRKL is the BCR/ABL phosphorylation site. Oncogene 14: 507–513.

  6. Feller SM . (2001). Crk family adaptors-signalling complex formation and biological roles. Oncogene 20: 6348–6371.

  7. Furge KA, Zhang YW, Vande Woude GF . (2000). Met receptor tyrosine kinase: enhanced signaling through adapter proteins. Oncogene 19: 5582–5589.

  8. Garnis C, Lockwood WW, Vucic E, Ge Y, Girard L, Minna JD et al. (2006). High resolution analysis of non-small cell lung cancer cell lines by whole genome tiling path array CGH. Int J Cancer 118: 1556–1564.

  9. Gay B, Suarez S, Caravatti G, Furet P, Meyer T, Schoepfer J . (1999). Selective GRB2 SH2 inhibitors as anti-ras therapy. Int J Cancer 83: 235–241.

  10. Hemmeryckx B, van Wijk A, Reichert A, Kaartinen V, de Jong R, Pattengale PK et al. (2001). Crkl enhances leukemogenesis in BCR/ABL P190 transgenic mice. Cancer Res 61: 1398–1405.

  11. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T et al. (2008). Cancer statistics, 2008. CA Cancer J Clin 58: 71–96.

  12. Kardinal C, Konkol B, Schulz A, Posern G, Lin H, Adermann K et al. (2000). Cell-penetrating SH3 domain blocker peptides inhibit proliferation of primary blast cells from CML patients. FASEB J 14: 1529–1538.

  13. Kendall J, Liu Q, Bakleh A, Krasnitz A, Nguyen KC, Lakshmi B et al. (2007). Oncogenic cooperation and coamplification of developmental transcription factor genes in lung cancer. Proc Natl Acad Sci USA 104: 16663–16668.

  14. Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM et al. (2002). The human genome browser at UCSC. Genome Res 12: 996–1006.

  15. Kwei KA, Kim YH, Girard L, Kao J, Pacyna-Gengelbach M, Salari K et al. (2008). Genomic profiling identifies TITF1 as a lineage-specific oncogene amplified in lung cancer. Oncogene 27: 3635–3640.

  16. Luo B, Cheung HW, Subramanian A, Sharifnia T, Okamoto M, Yang X et al. (2008). Highly parallel identification of essential genes in cancer cells. Proc Natl Acad Sci USA 105: 20380–20385.

  17. Lutterbach B, Zeng Q, Davis LJ, Hatch H, Hang G, Kohl NE et al. (2007). Lung cancer cell lines harboring MET gene amplification are dependent on Met for growth and survival. Cancer Res 67: 2081–2088.

  18. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW et al. (2004). Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350: 2129–2139.

  19. Ma PC, Kijima T, Maulik G, Fox EA, Sattler M, Griffin JD et al. (2003). c-MET mutational analysis in small cell lung cancer: novel juxtamembrane domain mutations regulating cytoskeletal functions. Cancer Res 63: 6272–6281.

  20. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S et al. (2004). EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304: 1497–1500.

  21. Pollack JR, Perou CM, Alizadeh AA, Eisen MB, Pergamenschikov A, Williams CF et al. (1999). Genome-wide analysis of DNA copy-number changes using cDNA microarrays. Nat Genet 23: 41–46.

  22. Ramirez RD, Sheridan S, Girard L, Sato M, Kim Y, Pollack J et al. (2004). Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res 64: 9027–9034.

  23. Sato M, Vaughan MB, Girard L, Peyton M, Lee W, Shames DS et al. (2006). Multiple oncogenic changes (K-RAS(V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells. Cancer Res 66: 2116–2128.

  24. Sattler M, Salgia R . (1998). Role of the adapter protein CRKL in signal transduction of normal hematopoietic and BCR/ABL-transformed cells. Leukemia 12: 637–644.

  25. Senechal K, Halpern J, Sawyers CL . (1996). The CRKL adaptor protein transforms fibroblasts and functions in transformation by the BCR-ABL oncogene. J Biol Chem 271: 23255–23261.

  26. Singer CF, Hudelist G, Lamm W, Mueller R, Handl C, Kubista E et al. (2006). Active (p)CrkL is overexpressed in human malignancies: potential role as a surrogate parameter for therapeutic tyrosine kinase inhibition. Oncol Rep 15: 353–359.

  27. Stein D, Wu J, Fuqua SA, Roonprapunt C, Yajnik V, D'Eustachio P et al. (1994). The SH2 domain protein GRB-7 is co-amplified, overexpressed and in a tight complex with HER2 in breast cancer. EMBO J 13: 1331–1340.

  28. ten Hoeve J, Kaartinen V, Fioretos T, Haataja L, Voncken JW, Heisterkamp N et al. (1994). Cellular interactions of CRKL, and SH2-SH3 adaptor protein. Cancer Res 54: 2563–2567.

  29. ten Hoeve J, Morris C, Heisterkamp N, Groffen J . (1993). Isolation and chromosomal localization of CRKL, a human crk-like gene. Oncogene 8: 2469–2474.

  30. Tibshirani R, Wang P . (2008). Spatial smoothing and hot spot detection for CGH data using the fused lasso. Biostatistics 9: 18–29.

  31. Tonon G, Wong KK, Maulik G, Brennan C, Feng B, Zhang Y et al. (2005). High-resolution genomic profiles of human lung cancer. Proc Natl Acad Sci USA 102: 9625–9630.

  32. Weir BA, Woo MS, Getz G, Perner S, Ding L, Beroukhim R et al. (2007). Characterizing the cancer genome in lung adenocarcinoma. Nature 450: 893–898.

  33. Zhao X, Weir BA, LaFramboise T, Lin M, Beroukhim R, Garraway L et al. (2005). Homozygous deletions and chromosome amplifications in human lung carcinomas revealed by single nucleotide polymorphism array analysis. Cancer Res 65: 5561–5570.

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We thank the SFGF for fabrication of microarrays and the SMD for database support. We thank Eon Rios for assistance with flow cytometry analysis. We also thank the members of the Pollack lab for helpful discussion. This work was supported in part by grants from the NIH: R01 CA97139 (JRP), SPORE P50CA70907 (JDM), EDRN U01CA084971 (AFG); DOD VITAL (JDM); Longenbaugh Foundations (JDM); TRDRP (17FT-0062; KAK) and the Deutsche Krebshilfe: 108003 (IP). KS was supported by the Medical Scientist Training Program and is a Paul & Daisy Soros fellow.

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Correspondence to J R Pollack.

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Kim, Y., Kwei, K., Girard, L. et al. Genomic and functional analysis identifies CRKL as an oncogene amplified in lung cancer. Oncogene 29, 1421–1430 (2010).

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  • CRKL
  • lung cancer
  • DNA amplification
  • genomic profiling
  • adapter protein

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