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1.  The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer 
Background
Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer.
Methods and Results
A genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide.
Conclusion
These results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.
doi:10.1186/1479-5876-10-97
PMCID: PMC3388458  PMID: 22591714
CRKL; Gastric cancer; Cell proliferation; Overexpression; Copy number amplification
2.  Amplification of CRKL induces transformation and EGFR inhibitor resistance in human non small cell lung cancers 
Cancer discovery  2011;1(7):608-625.
We previously identified a region of recurrent amplification on chromosome 22q11.21 in a subset of primary lung adenocarcinomas. Here we show that CRKL, encoding for an adaptor protein, is amplified and overexpressed in non-small cell lung cancer (NSCLC) cells that harbor 22q11.21 amplifications. Overexpression of CRKL in immortalized human airway epithelial cells promoted anchorage independent growth and tumorigenicity. Oncogenic CRKL activates SOS1-RAS-RAF-ERK and SRC-C3G-RAP1 pathways. Suppression of CRKL in NSCLC cells that harbor CRKL amplifications induced cell death. Overexpression of CRKL in EGFR mutant cells induces resistance to gefitinib by activating ERK and AKT signaling. We identified CRKL amplification in an EGFR inhibitor treated lung adenocarcinoma that was not present prior to treatment. These observations show that CRKL overexpression induces cell transformation, credential CRKL as a therapeutic target for a subset of NSCLC that harbor CRKL amplifications and implicate CRKL as an additional mechanism of resistance to EGFR-directed therapy.
doi:10.1158/2159-8290.CD-11-0046
PMCID: PMC3353720  PMID: 22586683
non small cell lung cancer; CRKL; cell transformation; RAP1; oncogene
3.  Genomic Profiling Identifies GATA6 as a Candidate Oncogene Amplified in Pancreatobiliary Cancer 
PLoS Genetics  2008;4(5):e1000081.
Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46%) primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies.
Author Summary
Pancreatic cancer is a devastating disease, having among the lowest survival rates of any cancer. A better understanding of the molecular basis of pancreatic cancer may lead to improved rationale therapies. We report here the discovery of amplification (i.e. extra copies) of the GATA6 gene in many human pancreatic cancers. GATA6 is a regulator of gene expression and functions in the development of the normal pancreas. Our findings indicate that its amplification and aberrant overexpression contribute to pancreatic cancer development. GATA6 joins a growing list of cancer genes with key roles in normal human development but pathogenic roles in cancer when aberrantly expressed. Our discovery of GATA6 amplification provides a new foothold into understanding the pathogenic mechanisms underlying pancreatic cancer, and suggests new strategies for therapy by targeting GATA6 or the genes it regulates.
doi:10.1371/journal.pgen.1000081
PMCID: PMC2413204  PMID: 18535672
4.  Structural Requirements for Function of the Crkl Adapter Protein in Fibroblasts and Hematopoietic Cells 
Molecular and Cellular Biology  1998;18(9):5082-5090.
Crkl is an adapter protein and phosphotyrosine-containing substrate implicated in transformation by the bcr-abl oncogene and in signaling by cytokines. When phosphorylated, Crkl binds through its Src homology 2 (SH2) domain to other tyrosine phosphoproteins such as paxillin and Cbl. Overexpression of Crkl in fibroblasts induces transformation. Here we examine the role of Crkl in hematopoietic cells and find that overexpression of Crkl confers a signal leading to increased adhesion to fibronectin. In both fibroblasts and hematopoietic cells, individual mutations or deletions of each SH2 and SH3 domain abrogated transformation and adhesion, respectively, indicating that interactions with other proteins such as Cbl and paxillin (SH2 domain) and Abl, Sos, and C3G (N-terminal SH3 domain) are essential for biological activity. In vivo and in vitro tryptic phosphopeptide mapping studies show that Crkl is phosphorylated on multiple tyrosine residues when overexpressed or when activated by Bcr-Abl. Mutation at tyrosine 207, a residue conserved in c-Crk, abrogates all in vivo tyrosine phosphorylation of Crkl. Despite this loss of phosphotyrosine, mutation at this site enhanced Crkl function as measured by complex formation with SH2 binding proteins, signal transduction to Jun Kinase, and fibroblast transformation. These observations implicate Crkl in cellular adhesion and demonstrate that Y207 functions as a negative regulatory site.
PMCID: PMC109093  PMID: 9710592
5.  Differential expression of CRKL and AXL genes in lung adenocarcinoma subtypes according to the epidermal growth factor receptor and anaplastic lymphoma kinase gene status 
Biomedical Reports  2014;2(4):481-489.
Non-small-cell lung cancer (NSCLC) is the most common cause of cancer-related mortality. Adenocarcinoma (AC) is the predominant histological type of NSCLC; however, AC consists of several subtypes. It has not yet been determined whether there is a correlation of CRKL and AXL expression with epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) gene status in lung AC. We assayed exons 18 through 21 of the EGFR gene by direct sequencing; ALK rearrangement and the expression of CRKL and AXL were detected by immunostaining. A total of 212 cases of AC were included in this study, diagnosed using the novel classification system established by the International Association for the Study of Lung Cancer, the American Thoracic Society and the European Respiratory Society in 2011, including 69 acinar ACs, 17 lepidic predominant ACs (LPAs), 63 papillary, 14 mucinous, 17 micropapillary and 32 solid ACs. Of the 212 cases, 101 harbored EGFR mutations. The most common subtypes carrying delK745-S753 were papillary and acinar ACs. ALK rearrangement was found in 23 cases (11%) of lung ACs. Acinar and solid ACs were the most frequent subtypes with ALK aberrance, particularly in acinar ACs with cribriform structure (4/5 cases, 80%). The expression of CRKL was significantly different among the AC subtypes (P=0.01), with the highest and lowest expression levels of CRKL protein in papillary ACs and LPAs, respectively (P<0.05). AXL expression was also significantly different among the AC subtypes (P=0.002) and was correlated with lymph node infiltration in acinar ACs. ACs with EGFR mutations exhibited high levels of AXL protein expression compared to those without mutations (P<0.001). Acinar AC with cribriform structure is a distinct subtype that frequently harbors ALK rearrangement. The activation of AXL may be one of the factors contributing to the invasion of acinar and micropapillary ACs.
doi:10.3892/br.2014.261
PMCID: PMC4051477  PMID: 24944792
lung adenocarcinoma; epidermal growth factor receptor gene mutation; anaplastic lymphoma kinase gene rearrangement; CRKL; AXL
6.  Characterization of fibroblast growth factor receptor 2 overexpression in the human breast cancer cell line SUM-52PE 
Breast Cancer Research : BCR  2000;2(4):311-320.
The fibroblast growth factor receptor (FGFR)2 gene has been shown to be amplified in 5-10% of breast cancer patients. A breast cancer cell line developed in our laboratory, SUM-52PE, was shown to have a 12-fold amplification of the FGFR2 gene, and FGFR2 message was found to be overexpressed 40-fold in SUM-52PE cells as compared with normal human mammary epithelial (HME) cells. Both human breast cancer (HBC) cell lines and HME cells expressed two FGFR2 isoforms, whereas SUM-52PE cells overexpressed those two isoforms, as well as several unique FGFR2 polypeptides. SUM-52PE cells expressed exclusively FGFR2-IIIb isoforms, which are high-affinity receptors for fibroblast growth factor (FGF)-1 and FGF-7. Differences were identified in the expression of the extracellular Ig-like domains, acid box and carboxyl termini, and several variants not previously reported were isolated from these cells.
Introduction:
The FGFR family of receptor tyrosine kinases includes four members, all of which are highly alternatively spliced and glycosylated. For FGFR2, alternative splicing of the second half of the third Ig-like domain, involving exons IIIb and IIIc, is a mutually exclusive choice that affects ligand binding specificity and affinity [1,2,3]. It appears that the second half of the third Ig-like domain can dictate high affinity for FGF-2 or keratinocyte growth factor (KGF), whereas affinity for FGF-1 appears to remain the same [3]. Alternative splicing of the carboxyl terminus has been shown to involve at least two different exons that can produce at least three different variants. The C1-type and C2-type carboxyl termini are encoded by the same exon, and have two different splice acceptor sites, whereas the C3-type carboxyl terminus is encoded by a separate exon [4]. The biologic significance of the C1 carboxyl terminus, as compared with the shorter C3 variant found primarily in tumorigenic samples, has been studied in NIH3T3 transfection assays, in which C3 variants were able to produce three times more transformed foci in soft agar than C1 variants (both IIIb), whereas full length FGFR2 and FGFR1 (both IIIc variants) showed no transforming activity [4].
Previous studies [5,6] have found amplification and overexpression of FGFR2 in 5-10% of primary breast cancer specimens. A recent study [7] done using a tissue array consisting of 372 primary breast cancer specimens found a 5% incidence of FGFR2 amplification. To our knowledge, none of the HBC cell lines studied thus far have an FGFR2 gene amplification, although overexpression of FGFR2 message and protein has been documented for some breast cancer cell lines [6,8,9].
SUM-52PE is a breast cancer cell line previously isolated in our laboratory that grows under serum-free and epidermal growth factor-free conditions, has high levels of tyrosine-phosphorylated membrane proteins, and has the capacity to invade and grow under anchorage-independent conditions [10,11,12]. This cell line exhibits all of the important hallmarks of transformed, highly malignant cells. Therefore, SUM-52PE was used as a model to study the diversity of FGFR2 expression in a breast cancer cell line that has true amplification and overexpression of FGFR2.
Objectives:
This study was conducted to examine the degree of FGFR2 amplification and overexpression in the breast cancer cell line SUM-52PE. Subsequent sequencing and characterization of individual FGFR2 variants cloned from the SUM-52PE cell line was completed to determine the complexity of FGFR2 alternative splicing in the context of a highly metastatic breast cancer cell line.
Methods:
Southern, Northern and Western blot analyses were done in order to determine the degree of FGFR2 amplification and overexpression in the breast cancer cell line SUM-52PE. Individual FGFR2 variants were cloned out of SUM-52PE using FGFR2-specific primers in a reverse transcription (RT) polymerase chain reaction (PCR). FGFR2 cDNAs were characterized by restriction fragment analysis, sequencing and transient transfection into 293 cells to examine the protein expression of each FGFR2 clone.
Results:
The results of the Southern blot showed that there was a 12-fold amplification of FGFR2 in the SUM-52PE cell line. Northern blot analysis of SUM-52PE showed FGFR2 transcripts to be highly overexpressed compared with other breast cancer cell lines and normal HME cells. Several overexpressed bands of approximately 6.3, 5.0, 4.0, and 2.8kb were observed in SUM-52PE cells. The most prominent band, at 2.8kb, was so abundant that it was difficult to discern other individual bands clearly. Western blot analysis showed that both normal HME and HBC cells expressed two FGFR2 variants of 95 and 135kDa. The SUM-52PE cell line greatly overexpressed not only these two polypeptides, as compared with HME and HBC cells, but also overexpressed two unique variants of FGFR2 - 85 and 109kDa polypeptides - as well as several smaller polypeptides in the 46-53kDa range. The antibody used in Western blot analysis only recognizes FGFR2 isoforms that express the C1 carboxyl termini, therefore greatly underestimating the actual number of different FGFR2 variants that are overexpressed in this cell line.
PCR was performed to determine the proportion of C1/C2 variants as compared with C3 variants in the SUM-52PE cell line. Results of this analysis indicated the presence of all three types of variants in this cell line, although the C1/C2 variants were predominant as compared with the C3 variants in SUM-52PE.
Four different FGFR2-C1 clones were isolated and sequenced from SUM-52PE cells, which differed in their signal sequence, first Ig-like loop, and acid box. Two FGFR2-C2 clones were isolated from the SUM-52PE cell line, which were identical to each other except for the variable expression of the number of Ig-like domains (two or three). Three C3 clones were isolated and sequenced, two of which have not previously been described in the literature. Clone C3-#3 contained two Ig-like domains, but no acid box. C3-#5 was missing the first two Ig-like domains and the acid box, but did contain the third Ig-like domain.
Discussion:
There is an extensive amount of evidence implicating erbB-2, a gene that is overexpressed in approximately 30% of breast cancer cases, as a breast cancer gene [13]. The identification of other breast oncogenes that function in the remaining 70% of cases is an ongoing challenge, as is establishing a causal role for such oncogenes in HME cell transformation.
FGFR1 and FGFR2, previously established oncogenes, were found to be amplified within large amplicons on 8p11 and 10q26, respectively, in the breast cancer cell line SUM-52PE [14]. Previous studies have shown that the FGFR2 gene is amplified in about 5-10% of breast cancer cases.
Our results showed that SUM-52PE cells overexpressed many alternatively spliced isoforms of FGFR2 at both the transcript and protein level as compared with normal HME cells. The variability in FGFR2 isoform expression is complex and involves exon IIIb/c, which encodes the second half of the third Ig-like loop; variations in the carboxyl terminal end of the receptor, involving the C1/C2 or C3 domains; and variable expression of the Ig-like loops and acid box in the extracellular portion of the receptor. The characterization of three unique FGFR2 isoforms that were cloned from SUM-52PE may build on the findings of others concerning the transforming potential of FGFR2 variants [4]. In particular, because it has been demonstrated that expression of C3-IIIb variants may have more transforming activity than C1-IIIb variants, differences between the three C3 clones we have isolated may provide information regarding the influence of particular structural domains on transforming potential.
Ongoing studies are aimed at characterizing the transforming ability of FGFR2 isoforms obtained from SUM-52PE cells by transducing these genes into normal HME cells. By overexpressing FGFR2 isoforms in a physiologically relevant system, we hope to determine the isoform(s) that acts in a dominant way in the process of cell transformation, and to determine whether different regions present in individual clones drive specific phenotypes associated with transformation.
PMCID: PMC13919  PMID: 11056689
alternative splicing; breast cancer; fibroblast growth factor receptor; receptor tyrosine kinase; SUM-52PE
7.  SOX2 Is an Oncogene Activated by Recurrent 3q26.3 Amplifications in Human Lung Squamous Cell Carcinomas 
PLoS ONE  2010;5(1):e8960.
Squamous cell carcinoma (SCC) of the lung is a frequent and aggressive cancer type. Gene amplifications, a known activating mechanism of oncogenes, target the 3q26-qter region as one of the most frequently gained/amplified genomic sites in SCC of various types. Here, we used array comparative genomic hybridization to delineate the consensus region of 3q26.3 amplifications in lung SCC. Recurrent amplifications occur in 20% of lung SCC (136 tumors in total) and map to a core region of 2 Mb (Megabases) that encompasses SOX2, a transcription factor gene. Intense SOX2 immunostaining is frequent in nuclei of lung SCC, indicating potential active transcriptional regulation by SOX2. Analyses of the transcriptome of lung SCC, SOX2-overexpressing lung epithelial cells and embryonic stem cells (ESCs) reveal that SOX2 contributes to activate ESC-like phenotypes and provide clues pertaining to the deregulated genes involved in the malignant phenotype. In cell culture experiments, overexpression of SOX2 stimulates cellular migration and anchorage-independent growth while SOX2 knockdown impairs cell growth. Finally, SOX2 over-expression in non-tumorigenic human lung bronchial epithelial cells is tumorigenic in immunocompromised mice. These results indicate that the SOX2 transcription factor, a major regulator of stem cell function, is also an oncogene and a driver gene for the recurrent 3q26.33 amplifications in lung SCC.
doi:10.1371/journal.pone.0008960
PMCID: PMC2813300  PMID: 20126410
8.  Loss-of-function screen in rhabdomyosarcoma identifies CRKL-YES as a critical signal for tumor growth 
Oncogene  2013;32(47):5429-5438.
To identify novel signaling pathways necessary for rhabdomyosarcoma (RMS) survival, we performed a loss-of-function screen using an inducible small hairpin RNA (shRNA) library in an alveolar and an embryonal RMS cell line. This screen identified CRKL expression as necessary for growth of alveolar RMS and embryonal RMS both in vitro and in vivo. We also found that CRKL was uniformly highly expressed in both RMS cell lines and tumor tissue. As CRKL is a member of the CRK adapter protein family that contains an SH2 and two SH3 domains and is involved in signal transduction from multiple tyrosine kinase receptors, we evaluated CRKL interaction with multiple tyrosine kinase receptor signaling pathways in RMS cells. While we saw no interaction of CRKL with IGFIR, MET or PI3KAKT/mTOR pathways, we determined that CRKL signaling was associated with SRC family kinase (SFK) signaling, specifically with YES kinase. Inhibition of SFK signaling with dasatinib or another SFK inhibitor, sarcatinib, suppressed RMS cell growth in vitro and in vivo. These data identify CRKL as a novel critical component of RMS growth. This study also demonstrates the use of functional screening to identify a potentially novel therapeutic target and treatment approach for these highly aggressive pediatric cancers.
doi:10.1038/onc.2012.590
PMCID: PMC3898328  PMID: 23318429
rhabdomyosarcoma; CRKL; SRC; YES; dasatinib
9.  CRKL as a lung cancer oncogene and mediator of acquired resistance to EGFR inhibitors: is it all that it is cracked up to be? 
Cancer discovery  2011;1(7):560-561.
Summary
Cheung et al show that amplified CRKL can function as a driver oncogene in lung adenocarcinoma, activating both RAS and RAP1 to induce MAPK signaling. In addition, they show that CRKL amplification may be another mechanism for primary or acquired resistance to EGFR kinase inhibitors.
doi:10.1158/2159-8290.CD-11-0295
PMCID: PMC3247017  PMID: 22211192
10.  Amplification and overexpression of the ID4 gene at 6p22.3 in bladder cancer 
Molecular Cancer  2005;4:16.
Background
Amplifications at 6p22.3 are prevalent in advanced stage bladder cancer (TCC). Previous studies have identified SOX4, CDKAL, and E2F3 as targets of this amplification and therefore potential oncogenes, but the more telomeric DEK gene too has been reported as overexpressed and amplified. We have therefore investigated whether the intermediate region harboring the oncogene candidate ID4 is also part of the amplicon.
Results
Expression of E2F3, DEK, and ID4 was investigated by real-time RT-PCR in 28 TCC compared to 6 normal bladder tissues and in 15 TCC cell lines compared to cultured normal urothelial cells. Expression of E2F3 as well as DEK increased on average in tumor vs. normal tissues (3-fold and 2.5-fold, resp.), but only the increase for E2F3 was statistically significant (p = 0.039). ID4 overexpression was observed in selected specimens. Each of the three genes was overexpressed in several cell lines, up to 150-fold (ID4), 30-fold (E2F3), and 9-fold (DEK), but these increases were not correlated to each other. Instead, moderate (DEK) to excellent (ID4) correlations were observed with copy number increases of microsatellites near each gene. Microsatellite copy number increases were highly heterogeneous across the investigated several Mb region revealing at least three subregions of amplification.
Conclusion
Extending previous reports, our data indicate that the 6p22.3 amplicon in TCC is highly heterogeneous and targets several genes in a variable fashion. Among these, expression of E2F3 and DEK appear to be generally increased in TCC, with additional increases caused by amplifications. In contrast, over-expression of ID4, which is normally predominantly expressed in testes and brain, appears to depend more strictly on gene amplification. Accordingly, the effect of amplifications at 6p22.3 in bladder cancer is expected to be non-uniform, thereby contributing to the highly variable biological and clinical behavior of advanced stage tumors. ID4 is a potential oncogene in a small subset of bladder cancers.
doi:10.1186/1476-4598-4-16
PMCID: PMC1131921  PMID: 15876350
11.  DNA amplification is a ubiquitous mechanism of oncogene activation in lung and other cancers 
Oncogene  2008;27(33):4615-4624.
Chromosomal translocation is the best-characterized genetic mechanism for oncogene activation. However, there are documented examples of activation by alternate mechanisms, for example gene dosage increase, though its prevalence is unclear. Here, we answered the fundamental question of the contribution of DNA amplification as a molecular mechanism driving oncogenesis. Comparing 104 cancer lines representing diverse tissue origins identified genes residing in amplification ‘hotspots’ and discovered an unexpected frequency of genes activated by this mechanism. The 3431 amplicons identified represent ~10 per hematological and ~36 per epithelial cancer genome. Many recurrently amplified oncogenes were previously known to be activated only by disease-specific translocations. The 135 hotspots identified contain 538 unique genes and are enriched for proliferation, apoptosis and linage-dependency genes, reflecting functions advantageous to tumor growth. Integrating gene dosage with expression data validated the downstream impact of the novel amplification events in both cell lines and clinical samples. For example, multiple downstream components of the EGFR-family-signaling pathway, including CDK5, AKT1 and SHC1, are overexpressed as a direct result of gene amplification in lung cancer. Our findings suggest that amplification is far more common a mechanism of oncogene activation than previously believed and that specific regions of the genome are hotspots of amplification.
doi:10.1038/onc.2008.98
PMCID: PMC2849646  PMID: 18391978
gene amplification; array CGH; gene expression; integrative analysis; lung cancer; EGFR signaling
12.  Crk adaptor proteins act as key signaling integrators for breast tumorigenesis 
Introduction
CT10 regulator of kinase (Crk) adaptor proteins (CrkI, CrkII and CrkL) play a role in integrating signals for migration and invasion of highly malignant breast cancer cell lines. This has important implications, as elevated CrkI/II protein levels were observed in a small cohort of breast cancer patients, which identified a potential role for Crk proteins in breast cancer progression. Numerous in vitro studies identified a role for Crk proteins in cell motility, but little is known about how Crk proteins contribute to breast cancer progression in vivo.
Methods
The clinical significance of Crk proteins in human breast cancer was assessed by analyzing published breast cancer datasets using a gene expression signature that was generated following CrkII over-expression and by examining Crk protein expression in tissue microarrays of breast tumors (n = 254). Stable knockdown of Crk (CrkI/CrkII/CrkL) proteins was accomplished using a short hairpin RNA (shRNA)-mediated approach in two basal breast cancer cell lines, MDA-231 1833TR and SUM1315, where the former have a high affinity to form bone metastases. Both in vitro assays (cell migration, invasion, soft agar growth) and in vivo experiments (intra-cardiac, tibial and mammary fat pad injections) were performed to assess the functional significance of Crk proteins in breast cancer.
Results
A gene signature derived following CrkII over-expression correlated significantly with basal breast cancers and with high grade and poor outcome in general. Moreover, elevated Crk immunostaining on tissue microarrays revealed a significant association with highly proliferative tumors within the basal subtype. RNAi-mediated knockdown of all three Crk proteins in metastatic basal breast cancer cells established a continued requirement for Crk in cell migration and invasion in vitro and metastatic growth in vivo. Furthermore, Crk ablation suppressed anchorage independent growth and in vivo orthotopic tumor growth. This was associated with diminished cell proliferation and was rescued by expression of non-shRNA targeted CrkI/II. Perturbations in tumor progression correlated with altered integrin signaling, including decreased cell spreading, diminished p130Cas phosphorylation, and Cdc42 activation.
Conclusions
These data highlight the physiological importance of Crk proteins in regulating growth of aggressive basal breast cancer cells and identify Crk-dependent signaling networks as promising therapeutic targets.
doi:10.1186/bcr3183
PMCID: PMC3446336  PMID: 22569336
13.  Models of Crk Adaptor Proteins in Cancer 
Genes & Cancer  2012;3(5-6):341-352.
The Crk family of adaptor proteins (CrkI, CrkII, and CrkL), originally discovered as the oncogene fusion product, v-Crk, of the CT10 chicken retrovirus, lacks catalytic activity but engages with multiple signaling pathways through their SH2 and SH3 domains. Crk proteins link upstream tyrosine kinase and integrin-dependent signals to downstream effectors, acting as adaptors in diverse signaling pathways and cellular processes. Crk proteins are now recognized to play a role in the malignancy of many human cancers, stimulating renewed interest in their mechanism of action in cancer progression. The contribution of Crk signaling to malignancy has been predominantly studied in fibroblasts and in hematopoietic models and more recently in epithelial models. A mechanistic understanding of Crk proteins in cancer progression in vivo is still poorly understood in part due to the highly pleiotropic nature of Crk signaling. Recent advances in the structural organization of Crk domains, new roles in kinase regulation, and increased knowledge of the mechanisms and frequency of Crk overexpression in human cancers have provided an incentive for further study in in vivo models. An understanding of the mechanisms through which Crk proteins act as oncogenic drivers could have important implications in therapeutic targeting.
doi:10.1177/1947601912459951
PMCID: PMC3513787  PMID: 23226572
Crk adaptor proteins; signal transduction; regulation; mouse models
14.  BCL-XL Mediates the Strong Selective Advantage of a 20q11.21 Amplification Commonly Found in Human Embryonic Stem Cell Cultures 
Stem Cell Reports  2013;1(5):379-386.
Summary
Human embryonic stem cells (hESCs) regularly acquire nonrandom genomic aberrations during culture, raising concerns about their safe therapeutic application. The International Stem Cell Initiative identified a copy number variant (CNV) amplification of chromosome 20q11.21 in 25% of hESC lines displaying a normal karyotype. By comparing four cell lines paired for the presence or absence of this CNV, we show that those containing this amplicon have higher population doubling rates, attributable to enhanced cell survival through resistance to apoptosis. Of the three genes encoded within the minimal amplicon and expressed in hESCs, only overexpression of BCL2L1 (BCL-XL isoform) provides control cells with growth characteristics similar to those of CNV-containing cells, whereas inhibition of BCL-XL suppresses the growth advantage of CNV cells, establishing BCL2L1 as a driver mutation. Amplification of the 20q11.21 region is also detectable in human embryonal carcinoma cell lines and some teratocarcinomas, linking this mutation with malignant transformation.
Graphical Abstract
Highlights
•The presence of the 20q11.21 CNV protects hESCs against apoptosis•20q11.21 CNV cells have increased levels of antiapoptotic BCL-XL, driving selection•hECCs and primary embryonal carcinoma samples also display the 20q11.21 CNV•20q11.21 CNV could be a feature of neoplastic progression
Avery and colleagues report that BCL2L1 (gene product BCL-XL) is the driver mutation for the copy number variant (CNV) amplification of chromosome 20q11.21 (present in 25% of normal-karyotype hESC lines). CNV cells exhibit enhanced cell survival through BCL-XL-associated resistance to apoptosis and rapidly outcompete nonmutant cells. Routine screening for this mutation should be considered for hESCs to be used in therapy.
doi:10.1016/j.stemcr.2013.10.005
PMCID: PMC3841249  PMID: 24286026
15.  Crk Adapter Proteins Promote an Epithelial–Mesenchymal-like Transition and Are Required for HGF-mediated Cell Spreading and Breakdown of Epithelial Adherens Junctions 
Molecular Biology of the Cell  2002;13(5):1449-1461.
Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor (HGF), promotes an epithelial–mesenchymal transition and cell dispersal. However, little is known about the HGF-dependent signals that regulate these events. HGF stimulation of epithelial cell colonies leads to the enhanced recruitment of the CrkII and CrkL adapter proteins to Met-dependent signaling complexes. We provide evidence that signals involving CrkII and CrkL are required for the breakdown of adherens junctions, the spreading of epithelial colonies, and the formation of lamellipodia in response to HGF. The overexpression of a CrkI SH3 domain mutant blocks these HGF-dependent events. In addition, the overexpression of CrkII or CrkL promotes lamellipodia formation, loss of adherens junctions, cell spreading, and dispersal of colonies of breast cancer epithelial cells in the absence of HGF. Stable lines of epithelial cells overexpressing CrkII show enhanced activation of Rac1 and Rap1. The Crk-dependent breakdown of adherens junctions and cell spreading is inhibited by the expression of a dominant negative mutant of Rac1 but not Rap1. These findings provide evidence that Crk adapter proteins play a critical role in the breakdown of adherens junctions and the spreading of sheets of epithelial cells.
doi:10.1091/mbc.01-10-0477
PMCID: PMC111118  PMID: 12006644
16.  Identification of Druggable Cancer Driver Genes Amplified across TCGA Datasets 
PLoS ONE  2014;9(5):e98293.
The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 16 cancer subtypes and identified 486 genes that were amplified in two or more datasets. The list was narrowed to 75 cancer-associated genes with potential “druggable” properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 42 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 42 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapters GRB2 and GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts.
doi:10.1371/journal.pone.0098293
PMCID: PMC4038530  PMID: 24874471
17.  A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth 
BMC Cancer  2009;9:198.
Background
Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells.
Methods
DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively.
Results
DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity.
Conclusion
Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified KRAS as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of KRAS amplification and overexpression.
doi:10.1186/1471-2407-9-198
PMCID: PMC2717977  PMID: 19545448
18.  Identification and functional analysis of 9p24 amplified genes in human breast cancer 
Oncogene  2011;31(3):10.1038/onc.2011.227.
Previously, our group identified a novel amplicon at chromosome 9p24 in human esophageal and breast cancers, and cloned the novel gene, GASC1 (gene amplified in squamous cell carcinoma 1, also known as JMJD2C/KDM4C), from this amplicon. GASC1 is a histone demethylase involved in the deregulation of histone methylation in cancer cells. In the current study, we aimed to comprehensively characterize the genes in the 9p24 amplicon in human breast cancer. We performed extensive genomic analyses on a panel of cancer cell lines and narrowed the shortest region of overlap to approximately 2 Mb. Based on statistical analysis of copy number increase and overexpression, the 9p24 amplicon contains six candidate oncogenes. Among these, four genes (GASC1 UHRF2, KIAA1432 and C9orf123) are overexpressed only in the context of gene amplification while two genes (ERMP1 and IL33) are overexpressed independent of the copy number increase. We then focused our studies on the UHRF2 gene, which has a potential involvement in both DNA methylation and histone modification. Knocking down UHRF2 expression inhibited the growth of breast cancer cells specifically with 9p24 amplification. Conversely, ectopic overexpression of UHRF2 in non-tumorigenic MCF10A cells promoted cell proliferation. Furthermore, we demonstrated that UHRF2 has the ability to suppress the expression of key cell-cycle inhibitors, such as p16INK4a, p21Waf1/Cip1 and p27Kip1. Taken together, our studies support the notion that the 9p24 amplicon contains multiple oncogenes that may integrate genetic and epigenetic codes and have important roles in human tumorigenesis.
doi:10.1038/onc.2011.227
PMCID: PMC3886828  PMID: 21666724
chromosome 9p24; GASC1; UHRF2; gene amplification
19.  A chimeric receptor/oncogene that can be regulated by a ligand in vitro and in vivo. 
Journal of Clinical Investigation  1997;100(7):1708-1715.
The BCR/ABL oncogene encodes an activated tyrosine kinase that causes human chronic myelogenous leukemia. The mechanism of transformation, however, is complex and not well understood. One of the important contributions of BCR to transformation is believed to be dimerization or oligomerization of ABL, thereby activating ABL tyrosine kinase activity. We reasoned that if ABL was dimerized through other mechanisms, activation of the tyrosine kinase activity should also result, and the activated kinase may also be transforming. Erythropoietin is known to activate its receptor by causing dimerization, and therefore a synthetic oncogene was created by linking the extracytoplasmic and transmembrane domains of the EPO receptor with c-ABL. This chimeric receptor was stably expressed in Ba/F3 cells and, in the absence of EPO, had no detectable biological effect on the cells. EPO, however, induced a rapid, dose-dependent activation of ABL tyrosine kinase activity and phosphorylation of several cellular proteins. The major target proteins have been identified, and are very similar to the known substrates of BCR/ABL, including Shc, CBL, CRKL, and several proteins in the cytoskeleton. EPO treatment also resulted in biological effects that were remarkably similar to those of BCR/ABL, including improved viability, altered integrin function, and a weak mitogenic signal. The biological effects were in part dose-dependent, in that low EPO concentrations enhanced viability but did not cause proliferation. At high EPO doses, kinase activation was maximal, and a mitogenic effect was also revealed. In nude mice, Ba/F3 cells expressing this chimeric receptor did not cause detectable disease without administration of pharmacologic doses of EPO. If EPO was given intraperitoneally 5 days a week, however, a dose-dependent lethal leukemia resulted. This ligand-regulatable oncogene mimics some of the biological effects of BCR/ABL, and analysis of ABL mutants in this system will be useful to dissect the signaling pathways that cause CML.
PMCID: PMC508353  PMID: 9312168
20.  Identification of CrkL-SH3 binding proteins from embryonic murine brain: Implications for Reelin signaling during brain development 
Journal of proteome research  2011;10(10):4453-4462.
The Crk and Crk-like (CrkL) adaptor proteins play important roles in numerous signaling pathways, bridging tyrosine kinase substrates to downstream signaling effectors by virtue of their phosphotyrosine-binding SH2 domains and their effector-binding SH3 domains. Critical to understanding the diverse roles of Crk/CrkL is the identification of tissue- and signal-specific tyrosine phosphorylated substrates to which they are recruited and the tissue-specific effector proteins they chaperone into signaling complexes. Crk and CrkL are known biochemically and genetically to be essential mediators of Reelin/Disabled-1 (Dab1) signaling which governs proper mammalian brain development. Multimeric Reelin clusters its receptors as well as the receptor-bound intracellular scaffolding protein Dab1. Clustering induces Fyn/Src-dependent Dab1 tyrosine phosphorylation which recruits Crk/CrkL and SH3-bound effectors. Previously 21 Crk/CrkL-SH3 binding proteins were identified from diverse cell types. We present here the proteomic identification of 101 CrkL-SH3 binding proteins from embryonic murine brain. The identified proteins are enriched in the Crk/CrkL-SH3 binding motif and signaling activities regulating cell adhesion and motility. These results suggest Reelin-induced Dab1 tyrosine phosphorylation may generate a multi-faceted signaling scaffold containing a rich array of Crk/CrkL-SH3 binding effectors and may explain a growing diversity of cellular activities suggested to be influenced by Reelin/Dab1 signaling.
doi:10.1021/pr200229a
PMCID: PMC3189288  PMID: 21879738
Crk-like (CrkL); Crk; Src Homology 2 (SH2); Src Homology 3 (SH3); Reelin; Disabled-1 (Dab1); Proteomics; mass spectrometry; neuronal migration; brain development
21.  A BCR-ABL Mutant Lacking Direct Binding Sites for the GRB2, CBL and CRKL Adapter Proteins Fails to Induce Leukemia in Mice 
PLoS ONE  2009;4(10):e7439.
The BCR-ABL tyrosine kinase is the defining feature of chronic myeloid leukemia (CML) and its kinase activity is required for induction of this disease. Current thinking holds that BCR-ABL forms a multi-protein complex that incorporates several substrates and adaptor proteins and is stabilized by multiple direct and indirect interactions. Signaling output from this highly redundant network leads to cellular transformation. Proteins known to be associated with BCR-ABL in this complex include: GRB2, c-CBL, p62DOK, and CRKL. These proteins in turn, link BCR-ABL to various signaling pathways indicated in cellular transformation. In this study we show that a triple mutant of BCR-ABL with mutations of the direct binding sites for GRB2, CBL, p62DOK and CRKL, is defective for transformation of primary hematopoietic cells in vitro and in a murine CML model, while it retains the capacity to induce IL-3 independence in 32D cells. Compared to BCR-ABL, the triple mutant's ability to activate the MAP kinase and PI3-kinase pathways is severely compromised, while STAT5 phosphorylation is maintained, suggesting that the former are crucial for the transformation of primary cells, but dispensable for transformation of factor dependent cell lines. Our data suggest that inhibition of BCR-ABL-induced leukemia by disrupting protein interactions could be possible, but would require blocking of multiple sites.
doi:10.1371/journal.pone.0007439
PMCID: PMC2757918  PMID: 19823681
22.  Crkl Deficiency Disrupts Fgf8 Signaling in a Mouse Model of 22q11 Deletion Syndromes 
Developmental cell  2006;10(1):71-80.
Summary
Deletions on chromosome 22q11.21 disrupt pharyngeal and cardiac development and cause DiGeorge and related human syndromes. CRKL (CRK-Like) lies within 22q11.21, and Crkl-/- mice have phenotypic features of 22q11 deletion (del22q11) syndromes. While human FGF8 does not localize to 22q11, deficiency of Fgf8 also generates many features of del22q11 syndrome in mice. Since Fgf8 signals via receptor-type tyrosine kinases, and Crk family adaptor proteins transduce intracellular signals downstream of tyrosine kinases, we investigated whether Crkl mediates Fgf8 signaling. In addition to discovering genetic interactions between Crkl and Fgf8 during morphogenesis of structures affected in del22q11 syndrome, we found that Fgf8 induces tyrosine phosphorylation of FgfRs 1 and 2 and their binding to Crkl. Crkl is required for normal cellular responses to Fgf8, including survival and migration, Erk activation, and target gene expression. These findings provide mechanistic insight into disrupted intercellular interactions in the pathogenesis of malformations seen in del22q11 syndrome.
doi:10.1016/j.devcel.2005.12.003
PMCID: PMC1780033  PMID: 16399079
23.  Allele-Specific Amplification in Cancer Revealed by SNP Array Analysis 
PLoS Computational Biology  2005;1(6):e65.
Amplification, deletion, and loss of heterozygosity of genomic DNA are hallmarks of cancer. In recent years a variety of studies have emerged measuring total chromosomal copy number at increasingly high resolution. Similarly, loss-of-heterozygosity events have been finely mapped using high-throughput genotyping technologies. We have developed a probe-level allele-specific quantitation procedure that extracts both copy number and allelotype information from single nucleotide polymorphism (SNP) array data to arrive at allele-specific copy number across the genome. Our approach applies an expectation-maximization algorithm to a model derived from a novel classification of SNP array probes. This method is the first to our knowledge that is able to (a) determine the generalized genotype of aberrant samples at each SNP site (e.g., CCCCT at an amplified site), and (b) infer the copy number of each parental chromosome across the genome. With this method, we are able to determine not just where amplifications and deletions occur, but also the haplotype of the region being amplified or deleted. The merit of our model and general approach is demonstrated by very precise genotyping of normal samples, and our allele-specific copy number inferences are validated using PCR experiments. Applying our method to a collection of lung cancer samples, we are able to conclude that amplification is essentially monoallelic, as would be expected under the mechanisms currently believed responsible for gene amplification. This suggests that a specific parental chromosome may be targeted for amplification, whether because of germ line or somatic variation. An R software package containing the methods described in this paper is freely available at http://genome.dfci.harvard.edu/~tlaframb/PLASQ.
Synopsis
Human cancer is driven by the acquisition of genomic alterations. These alterations include amplifications and deletions of portions of one or both chromosomes in the cell. The localization of such copy number changes is an important pursuit in cancer genomics research because amplifications frequently harbor cancer-causing oncogenes, while deleted regions often contain tumor-suppressor genes. In this paper the authors present an expectation-maximization-based procedure that, when applied to data from single nucleotide polymorphism arrays, estimates not only total copy number at high resolution across the genome, but also the contribution of each parental chromosome to copy number. Applying this approach to data from over 100 lung cancer samples the authors find that, in essentially all cases, amplification is monoallelic. That is, only one of the two parental chromosomes contributes to the copy number elevation in each amplified region. This phenomenon makes possible the identification of haplotypes, or patterns of single nucleotide polymorphism alleles, that may serve as markers for the tumor-inducing genetic variants being targeted.
doi:10.1371/journal.pcbi.0010065
PMCID: PMC1289392  PMID: 16322765
24.  Domain organization differences explain Bcr-Abl’s preference for CrkL over CrkII 
Nature chemical biology  2012;8(6):590-596.
CrkL is a key signaling protein that mediates the leukemogenic activity of Bcr-Abl. CrkL is thought to adopt a structure that is similar to that of its CrkII homolog. The two proteins share high sequence identity and indistinguishable ligand binding preferences; yet they have distinct physiological roles. Here we show that the structures of CrkL and phosphorylated CrkL are drastically different than the corresponding structures of CrkII. As a result, the binding activities of the SH2 and SH3 domains in the two proteins are regulated in a distinct manner and to a different extent. The different structural architecture of CrkL and CrkII may account for their distinct functional roles. The data show that CrkL forms a constitutive complex with Abl thus explaining the strong preference of Bcr-Abl for CrkL. The results also highlight how the structural organization of the modular domains in adaptor proteins can control signaling outcome.
doi:10.1038/nchembio.954
PMCID: PMC3423979  PMID: 22581121
25.  Overexpression of OLC1, Cigarette Smoke, and Human Lung Tumorigenesis 
Background
Exposure to cigarette smoke is a major risk factor for lung cancer, but how it induces cancer is unclear. The overexpressed in lung cancer 1 (OLC1) gene is one of 50 candidate lung cancer genes identified by suppression subtractive hybridization as having higher expression in squamous cell carcinoma (SCC) than normal lung epithelia.
Methods
We used immunohistochemistry (IHC) to measure OLC1 protein levels in primary lung cancer samples from 559 patients and used fluorescence in situ hybridization to measure OLC1 copy number in primary SCC samples from 23 patients. We compared OLC1 protein expression in SCC samples of 371 patients with and without a smoking history using the Pearson χ2 test. We assayed OLC1 protein levels by immunoblotting in H1299 human lung cancer cells, immortalized human bronchial epithelial cells, and primary cultured normal human bronchial epithelial cells that were treated with cigarette smoke condensate. We assayed tumor formation in athymic mice using NIH3T3 mouse fibroblast cells transfected with OLC1 (eight mice) and analyzed apoptosis and colony formation of H1299 and H520 lung cancer cells transfected with scrambled (negative) or OLC1 small interfering RNAs (siRNAs) (s1).
Results
OLC1 protein was overexpressed in 387 of 464 (83.4%) of primary lung cancers, as detected by IHC, and OLC1 was amplified in 14 of 23 (60%) of SCC samples. OLC1 protein overexpression was more common in SCC patients with a smoking history than those without (77.1% vs 45.8%, P < .001). In addition, cigarette smoke condensate increased OLC1 protein levels in H1299 cells, immortalized human bronchial epithelial cells, and primary cultured normal human bronchial epithelial cells. Overexpression of OLC1 induced tumor formation in athymic mice (control vs OLC1, 0% vs 100%). Knockdown of OLC1 increased apoptosis (mean percentage of apoptotic H1299 cells, s1 vs negative: 30.3% vs 6.4%, difference = 23.9%, 95% confidence interval [CI] = 19.1% to 28.5%, P = .002; mean percentage of apoptotic H520 cells, s1 vs negative: 21.6% vs 4.9%, difference = 16.7%, 95% CI = 10.6% to 22.8%, P = .007) and decreased colony formation (mean no. of colonies of H1299 cells transfected with siRNAs, negative vs s1: 84 vs 4, difference = 80, 95% CI = 71 to 88, P < .001; mean no. of colonies of H520 cells transfected with siRNAs, negative vs s1: 103 vs 24, difference = 79, 95% CI = 40 to 116, P = .005).
Conclusions
OLC1 is a candidate oncogene in lung cancer whose expression may be regulated by exposure to cigarette smoke.
doi:10.1093/jnci/djn379
PMCID: PMC3299210  PMID: 19001599

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