Skip Navigation


Human Molecular Genetics Advance Access originally published online on December 22, 2004
Human Molecular Genetics 2005 14(4):475-482; doi:10.1093/hmg/ddi043
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
14/4/475    most recent
ddi043v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (12)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Garnis, C.
Right arrow Articles by Lam, W. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Garnis, C.
Right arrow Articles by Lam, W. L.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics, Vol. 14, No. 4 © Oxford University Press 2005; all rights reserved

Involvement of multiple developmental genes on chromosome 1p in lung tumorigenesis

Cathie Garnis*, Jennifer Campbell{dagger}, Jonathan J. Davies{dagger}, Calum MacAulay, Stephen Lam and Wan L. Lam

British Columbia Cancer Research Centre, Vancouver, BC V5Z 1L3, Canada

Received October 4, 2004; Revised December 8, 2004; Accepted December 15, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Lung cancer is the leading cause of cancer death in North America. Despite advances in lung cancer treatment, the overall 5 year survival rate for those diagnosed with the disease is bleak presumably due to the late stage of diagnosis. Owing to the difficulty of early detection, preneoplastic specimens are rare. However, studying both preinvasive and invasive stages of disease is necessary to fully understand lung cancer progression. Aberration of chromosome arm 1p is common in lung and other cancers. In this study, we used a genomic array with complete tiling coverage of 1p to profile preinvasive and invasive squamous non-small cell lung carcinoma samples. With this technology, multiple novel submegabase alterations were identified. Three of the 1p alterations harbored genes belonging to gene families known to be involved in cancer development through either the Wnt or the Notch developmental pathways. Our finding of a 0.4 Mb amplified region at 1p36.12 containing WNT4 in preinvasive lung cancer, coupled with the identification of three additional alterations in invasive tumors that also contain genes related to the Notch and Wnt pathways, strongly suggests an intricate role of these pathways in early and late stages of lung cancer development. Furthermore, ectopic expression of DVL1, LRP8 and Notch2 in malignant lung tissue validates the biological impact of these genetic alterations. Importantly, this implication of pathways known only to be activated in fetal lung development lends support to the proposed model of lung cancer ontology whereby tumors arise from dysregulated pleuripotent stem cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Aberration of the short arm of chromosome 1 (1p) is a common event in lung and many types of cancer (e.g. colorectal, gastric, neuroblastoma, liver, pancreatic and breast cancer) (1). Cytogenetic studies have shown that 1p harbors structural rearrangements in non-small cell lung cancer (NSCLC) (2), whereas microsatellite analysis demonstrates frequent loss of heterozygosity (LOH) at 1p36 in all major lung cancer subtypes (3). Moreover, the alignment of LOH data revealed multiple regions of alteration on 1p (3–6). Although there appears to be a high frequency of LOH across the entire chromosome arm, three large but distinct regions (1p36, 1p32 and 1p12) have been reported, suggesting the presence of more than one oncogene/tumor suppressor gene on the short arm of chromosome 1. Notably, there are several genes associated with the Wnt and Notch developmental pathways mapping to chromosome 1p.

Pathways involved in development may play a role in tumorigenesis. Recent focus on the Wnt pathway in colorectal cancer has prompted gene expression analysis of pathway components in other cancer types including breast, prostate and lung (7–9). Wnt ligands bind to their target membrane receptors, frizzled (FZD) and low-density lipoprotein receptor-related proteins (LRPs), and interfere with the multi-protein APC/ß-catenin destruction complex, resulting in the downstream activation of gene transcription by ß-catenin. While the complex role of ß-catenin in cell proliferation and cell adhesion has been the main focus of many mechanistic studies, it is becoming increasingly evident that upstream components such as WNT, FZD, SFRP, LRP and DVL genes are also involved in cancer (9–19). Recent reports of DVL3 and WNT2 overexpression in NSCLC suggest a potential role of this pathway in cell proliferation (17,20). However, WNT7a silencing, which leads to ß-catenin-mediated loss of E-Cadherin expression and subsequently impacts cell migration, suggests that the Wnt pathway plays a role in the later stages of lung cancer (including metastasis) (21). In addition, the Notch developmental pathway, which interacts with Wnt components, has also been implicated in cancer. For example, NOTCH3 translocation and overexpression has been observed in lung cancer (22). Interestingly, aberrant Wnt pathway signaling is an early event found in ~90% of colorectal cancers (23). This possibility has not been explored in lung cancer.

In this study, we investigated 1p alterations in early stage development of lung cancer. Hitherto, genomic analysis of early stage lesions has been limited presumably owing to the difficulty in obtaining preinvasive lesions and the minute size of such material, which yield limited DNA sufficient only to support locus-specific analysis but far too impractical for large-scale gene discovery. In this work, we combine the use of fluorescent bronchoscopy to capture preinvasive lesions and a bacterial artificial chromosome (BAC) genomic array to comprehensively analyze the 1p arm in microdissected preinvasive bronchial carcinoma in situ (CIS) and invasive squamous cell carcinoma (SqCC) specimens. The BAC clones of the 1p specific array span the chromosome arm in a tiling fashion, allowing focused characterization of genetic alterations in order to identify candidate genes that may be causal to the development of lung cancer.

As mentioned earlier, the role of developmental pathways has become increasingly appreciated in cancer etiology. Remarkably, candidate genes within three of the five novel regions of genetic alteration defined in this study are associated with the Notch and Wnt developmental pathways.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Detecting genetic alterations by 1p array CGH
Tumor DNA samples isolated from microdissected lung cancer cells were analyzed for regional copy number changes using a BAC array spanning the chromosome 1p arm at tiling resolution. In order to define gains and losses, we first established the baseline of technical variation by performing normal versus normal reference DNA comparisons. Clones with aberrant signal intensity were removed from the analysis. To ensure the detection of single copy alterations, normal male and female DNA samples were differentially labeled and co-hybridized to the 1p array, which contains control spots of X chromosome loci as a measure of detection sensitivity. Figure 1 shows the 1p profiles from two SqCC samples. Multiple segmental copy number changes were evident across the chromosome arm. Owing to the redundancy and the high degree of overlap of clones, small regions of genetic alterations were readily detected. While such small alterations may have gone undetected by marker-based techniques and conventional chromosomal CGH, the tiling nature of the array enables the definition of breakpoints flanking an altered region. In our example, we magnified three regions to illustrate simultaneous localization of multiple breakpoints (Fig. 1). One of the regions located at 1p12 contains Notch2.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 1. Detection of copy number gains and losses on chromosome 1p. Profiles of two lung tumor samples (T7734 and T8611) as viewed by SeeGH software. Each black dot represents a single BAC clone. Purple line represents log 2 signal intensity ratio of 0, green line is log 2 signal intensity ratio of –1 and red line represents +1. Far right is a magnified picture showing a breakpoint (top), a 1.59 Mb amplification (middle) and the amplified region containing Notch2 (bottom).

 
Recurrent amplification in preinvasive stage
Genetic alterations critical to lung cancer are likely to be present in multiple patients. Twenty lung SqCCs (Eight CIS and 12 tumors) were microdissected and the DNA analyzed. Although there are sporadic alterations on 1p in the CIS samples, only one region was deemed to be recurrent under our stringent criteria (half of the CIS samples had to contain the amplification). This recurrent alteration was identified by the alignment of 1p profiles, generated from a two clone moving average across the entire chromosome arm (Fig. 2). Amplification at 1p36.12 was observed in six of the eight CIS samples defining the ~0.2 Mb minimal region of alteration between BAC clones RP11-489K12 and RP11-415K20. This region contains two known genes, interestingly, one of which is WNT4, a member of the wingless-type MMTV integration site family (region A1, Table 1). WNT4 has been shown to be abnormally expressed in human breast cancer cell lines (24). The involvement of the Wnt pathway in cancer is best known through adenomatous polyposis coli (APC) mutations in colorectal cancer (23). In the context of lung cancer, this pathway has been implicated in NSCLC, owing to the overexpression of dishevelled homolog 3 (DVL3) (17). Although the amplification was observed in six of the eight samples, the small sample size precludes the generalization of frequency in squamous cell lung cancer. However, to our knowledge, this study is the first to suggest early involvement of the Wnt pathway in preinvasive lung lesions.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 2. Amplification of WNT4 region in CIS specimens. Representative CIS copy number profiles. Each BAC clone is represented by a black line segment. The moving average for a two clone window is plotted. Shading indicates the ±0.15 threshold range (see Materials and Methods). Wnt4 resides within this minimal region (bottom panel).

 

View this table:
[in this window]
[in a new window]
  		Table 1. Summary of regions of recurrent amplification on 1p
 
Recurrent regions of amplification in squamous lung carcinoma
In contrast to the CIS samples, all of the tumors analyzed contained both segmental amplifications and deletions. Figure 3 is a summary of the segmental copy number changes in the tumor samples by colorimetric representation. Examination of 1p at a continuous resolution of overlapping BAC DNA segments has allowed for the identification of novel small recurrent alterations. Recurrent regions of alteration were observed in cytobands 1p36, 1p33–35, 1p32 and 1p11 (regions A–E in Fig. 3). Regions A and B are wide (>20 Mb), while regions C, D and E are localized to 0.13, 0.32 and 0.05 Mb, respectively, as revealed by the alignment of array CGH profiles (Table 1). Regions B–E were detected at very low frequencies in the CIS samples (Table 1).



View larger version (66K):
[in this window]
[in a new window]
  		Figure 3. Colorimetric representation of 1p array tumor data. Visualization of the 1p arm as viewed by Java TreeView. Each row corresponds to a BAC clone and each column represents a tumor sample. Red indicates a gain in copy number, whereas green, black and grey indicate a loss in copy number, no change in copy number and uninformative data, respectively. Regions of frequent alteration (A–E) are denoted by a vertical light blue line. Genes of interest have been mapped to the 1p heat map on the left. The close-up view of regions C and D is shown to the right with the genes of interest represented by dark blue lines. Note that the lines simply indicate which BACs the genes reside in and do not imply that the genes overlap.

 
Region A at 1p36, observed in five of the 12 samples, is the largest and most prominent region of alteration, from BAC clone RP11-158F2 to RP11-277F18. This 21 Mb region encompasses the 0.4 Mb A1 region observed in the CIS samples. Previous searches for tumor suppressor genes on chromosome 1p in NSCLC have largely focused on this frequently altered region. However, definitions of the minimal region on the basis of LOH studies have yielded conflicting results, raising the possibility of more than one candidate gene at 1p36 (25). Many of the genes in region A could potentially be related to tumor development. TP73, located at 1p36.33, has been implicated as a possible tumor suppressor gene owing to its homology to TP53. However, TP73 mutations are infrequently detected in lung cancer (26). In addition to TP73, there are over 250 other known and predicted genes in this region, of which several could potentially be linked to cancer. They include WNT4, whose amplification was observed in most of the CIS samples (discussed earlier); EGFL3, which resembles epidermal growth factor receptor (EGFR) in structure and DVL1, which shares sequence identity with DVL3, whose overexpression is critical to Wnt-signaling activation and cell growth in NSCLC (17,27). The expression profiling of these three genes in NSCLC is described subsequently. Region B at 1p33–1p35.1 is observed in five of the 12 tumors (Table 1). This large 15 Mb region is also gene rich. Interestingly, in a sixth tumor a much smaller (0.79 Mb) amplification within the 15 Mb was observed containing the L-MYC gene. L-MYC is thought to be activated late in lung cancer, although the mechanism by which MYC regulates tumorigenecity is not yet established (28–31). This is consistent with our observation that the L-MYC region is amplified in only one of the eight CIS samples. The 0.13 Mb region C at 1p32.3 is present in seven of the 12 tumors (Table 1; Fig. 4). LRP8 is the only gene mapping within this region. Intriguingly, LRP8 belongs to the same low-density LPR family as LRP5 and LRP6, which are known co-receptors for Wnt ligands (32). The 0.32 Mb region D is at 1p32.1. Amplification is observed in eight of the 12 tumor samples but contains no known genes. Interestingly, region D is only 90 kb centromeric to the JUN oncogene, but RT–PCR assay of paired normal and tumor samples did not detect elevated JUN expression levels (discussed later). However, five ORFs are located in the amplified region according to the Genescan track at UCSC (http://genome.ucsc.edu/), and its recurrence suggests an importance in lung cancer development. Region E is the smallest of the recurrent alterations, spanning only 0.05 Mb between BACs RP11-641F2 and RP11-754O9 at 1p11 (Table 1; Fig. 3). This region near the centromere is amplified in nine of the 12 tumors and contains a single Refseq gene NOTCH2 (Drosophila Notch homologue 2). Amplification of this region has been recently reported in small cell lung cancer cell lines (33).



View larger version (34K):
[in this window]
[in a new window]
  		Figure 4. Expression analysis of candidate genes. The expression levels of DVL1, LRP8 and NOTCH2 were assessed by RT–PCR analysis of paired microdissected normal and tumor frozen samples. (A) DVL1 reverse transcription product amplified from cDNA of the matched pairs resolved by polyacrylamide gel electrophoresis and stained with SYBR green. Below is a bar graph of expression levels of DVL1 relative to that of ß-actin. White bars are normal samples and black bars are tumor samples. (B) LRP8 reverse transcription product. Below is a bar graph of expression levels of LRP8 relative to that of ß-actin. (C) NOTCH2 reverse transcription product. Below is a bar graph of expression levels of NOTCH2 relative to that of ß-actin.

 
Interestingly, nearly all of the altered regions identified on chromosome 1p harbored genes belonging to gene families known to be involved in cancer development through either the Wnt or the Notch developmental pathway. Coupling our early observation of an amplified region containing WNT4 in CIS and the identification of amplified regions containing additional genes related to these developmental pathways in the tumors prompted further evaluation of these genes at the expression level.

Expression of candidate genes in the Wnt and Notch pathways
We investigated the expression pattern of WNT4, DVL1 and EGFL3 (region A), LRP8 (region C), JUN (region D) and NOTCH2 (region E). As a control, NOTCH2 expression levels were compared with those of the neighboring ADAM30 and REGIV. We examined the expression levels of these genes in 13 microdissected matched normal and tumor SqCC cases by RT–PCR (Fig. 4). The expression analysis revealed significant overexpression in tumors of Notch and Wnt pathway gene family members DVL1 (P=0.003), LRP8 (P=0.008) and NOTCH2 (P=0.029). EGFL3, JUN, ADAM30 and REGIV showed no significant expression changes. WNT4 levels were too low to allow for the detection of differential expression. However, though expression levels of WNT4 are not high in tumors, this does not preclude the importance of WNT4 in early development as the genetic evidence suggested. Further investigation of the role of WNT4 at the expression level in preinvasive lesions—albeit challenging to obtain—will be necessary for validation.

Pair-wise comparison of DVL1 expression data showed a large difference between five of the 11 (Tumor 1, 3, 5, 10, 11) matched tumors and normal samples (Fig. 4A). This is in contrast to Uematsu et al. (17), who report only the overexpression of DVL3 and not DVL1 and DVL2 in NSCLC; however, their sample set was small, comprising only four squamous and four adenocarcinomas. The Drosophila dishevelled gene (Dsh) encodes a cytoplasmic phosphoprotein that regulates cell proliferation, acting as a transducer protein for developmental processes that include segmentation and neuroblast specification (34). It has been reported that Dsh is required for the function of the Wingless gene product, Wg, a segment polarity gene homologous to the mammalian proto-oncogene WNT1 (35–37) (Fig. 5). Human DVL1 shares ~64% amino acid identity with DVL3 and therefore may serve similar functions (27).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 5. Schematic diagram highlighting the interaction of the Wnt, Notch and reelin pathways. FZD and its co-receptor low-density LRP bind to the extracellular wingless ligand, thereby phosphorylating the intracellular disheveled (DVL) homologue which inhibits GSK-3ß leading to the stabilization of free ß-catenin. Stable ß-catenin translocates to the nucleus and acts as a transcription factor. Activation of NOTCH by an extracellular ligand such as delta (DLL) results in processing via presenilin (PSEN) and subsequent cleavage of the NOTCH intracellular domain (NICD) which translocates to the nucleus and acts as a transcription factor.

 
Expression analysis of LRP8 was striking, with overexpression observed in 11 of the 13 samples (Fig. 4B). The LRP family encodes membrane proteins involved in receptor-mediated endocytosis of many ligands and signal transduction pathways (32). Although LRPs appear to have multiple functions (38), LRP5, 6 and 12 have been linked to carcinogenesis (12,39–41). LRP5 and LRP6 are known to be co-receptors for Wnt ligands in Drosophila, Xenopus and mice (32). The role of LRP5 and 6 in Wnt signaling in humans is observed, but the mechanism remains unclear (42). It has been suggested that LRP5 is a possible marker for progression in high-grade osteosarcoma (39). In addition, LRP12 has recently been shown to be amplified and overexpressed in oral SqCC (12). LRP8, or ApoER, is a lipophilic protein that mediates high-affinity binding of APOE-containing lipoproteins, one of which is reelin, to the very low-density lipoprotein (VLDL) receptor. Binding of reelin to the receptor results in the phosphorylation of Dab1; this activates an unknown chain of events that ultimately suppresses the activity of glycogen synthase kinase (GSK) 3ß. Interestingly, the Wnt signaling pathway also functions through the regulation of GSK3ß (43,44). The amplification and overexpression of LRP8 in squamous cell lung carcinoma reported here combined with previous functional reports of other LRP family members suggests that LRP8 may play an oncogenic role in lung cancer.

In addition to alterations affecting the Wnt developmental pathway, we also observe alterations in the Notch developmental pathway. Not only is NOTCH2 frequently amplified in lung tumors (discussed earlier) but expression is also higher in the tumor samples assayed (Fig. 4C). The Notch signaling pathway affects a wide variety of cellular processes, including the maintenance of stem cells, specification of cell fate, differentiation, proliferation and apoptosis (45). Although little is known about the role of NOTCH2 in cancer, its involvement is not surprising as NOTCH1 has been repeatedly implicated in disease (45). Furthermore, a NOTCH3 translocation has been previously observed in NSCLC (22). The Notch and Wnt developmental pathways interact through a number of mechanisms. Depending on the mechanism of action, these pathways can act either synergistically or antagonistically (46–52).

Of the 13 tumor normal pairs, we were able to obtain DNA for five of the samples (case number 3, 5, 8, 10, 13 in Fig. 4) for the purpose of genomic profiling. Notably, there was concordance between amplification and overexpression for these three genes. All amplifications correspond to overexpression.

In summary, this study provided evidence for the genetic basis of Wnt and Notch involvement in both the early and the late stages of tumorigenesis. Furthermore, previous description of Wnt7a involvement in cell adhesion suggests its role in lung cancer metastasis (21). Through the comparison of preinvasive and invasive lung carcinomas, we have identified novel genetic alterations which contain overexpressed genes related to both the developmental pathways (Fig. 5). These pathways are important for normal lung development (53–55); however, the ectopic expression of genes within these pathways reported here may lead to abnormal cellular activity. Importantly, our implication of developmental pathways in lung cancer supports the proposed model of lung cancer ontology whereby tumors arise from dysregulated pleuripotent stem cells (56). Also, by identifying five novel regions on chromosome 1p in tumors as well as a recurrent alteration in CIS samples, this study demonstrates the value of genetically profiling early preinvasive lesions and the use of a comprehensive tiling set approach to array CGH. Extension of this approach to the analysis of entire tumor genomes, for example, using the newly developed whole genome submegabase resolution tiling set array (57), will provide more complete understanding of lung cancer progression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Lung tissue samples
The detection and capture of minute precancerous and cancerous bronchial lesions has been facilitated by the development of fluorescence bronchoscopy technology (58). The isolation of CIS specimens was guided by the use of the LIFE-Lung device (59). Formalin-fixed, paraffin-embedded CIS tissue blocks were retrieved from the cancer imaging collection at the British Columbia Cancer Agency and diagnoses were confirmed by a lung pathologist. In addition, formalin-fixed and frozen lung tumor specimens (for DNA profiling and gene expression analysis, respectively) were provided by Dr James Hogg, St Paul's Hospital, Vancouver, BC. Tumor cells were microdissected from tissue sections and DNA was isolated as previously described (60).

BAC array synthesis
Construction of a 1p chromosome arm specific BAC array for CGH analysis has been described by Henderson et al. (33). Clone lists with map position of individual BACs are available publicly at WWW.bccrc.ca/cg/ArrayCGH_Group.html. The procedures used for BAC DNA isolation, HindIII fingerprint clone verification and linker-mediated PCR amplification have been described previously (61). All clones were spotted in triplicate using a VersArray ChipWriter Pro (BioRad) with Stealth Micro Spotting Pins (Telechem/ArrayIT SMP2.5) as previously described. Quality control was applied to each batch of arrays synthesized to ensure reproducibility and robust detection of single copy gains and losses (33,62). While this 1p array containing 642 unique BACs was constructed to accommodate the profiling of minute CIS samples, in order to identify progressive changes on 1p during tumorigenesis, CIS profiles were compared with tumor profiles created from the 1280 clones that mapped to 1p on the SMRT array (57).

Probe labeling and hybridization
Probe labeling was previously described (61). Briefly, Cyanine3 and Cyanine5 dCTPs were used to label the reference and test DNA, respectively (100 ng each for CIS samples and 200 ng each for tumors). The DNA probes were pooled, denatured and annealed in a solution containing 100 µg of human Cot-1 DNA (Invitrogen, Mississauga, Ontario) in 25 µl of DIG Easy hybridization solution (Roche, Quebec), 50 µg of sheared herring sperm DNA (Sigma-Aldrich, Oakville, Ontario) and 250 µg of yeast tRNA (Calbiochem, CA, USA). The probe mixture was applied to the slide surface and hybridized for 36 h at 42°C. Arrays were washed five times with 0.1xSSC, 0.1% SDS for 5 min each at room temperature, rinsed five times with 0.1xSSC and then dried by centrifugation.

Array CGH imaging and analysis
Hybridized arrays were imaged using a charge-coupled device-based imaging system and analyzed using the SoftWorx array analysis program (ArrayWorx eAuto, API, Issaquah, WA, USA). Data for each dye channel were normalized by applying a scale factor to each channel, which placed the median signal ratio to 1. The average signal ratios and standard deviations for each triplicate spot set were calculated as previously described (62) and displayed as a plot of the normalized Cyanine5/Cyanine3 log 2 signal ratio versus relative tiling path position using Excel for the 1p array and versus basepair position using SeeGH software for the SMRT array (63). A log 2 signal ratio of 0 at a spot represents equivalent copy number between the sample and the reference DNA. Normal versus normal hybridizations on the 1p array and SMRT array were performed to establish the technical variation throughout the arrays. We found that the standard deviations for all clones were 0.074 and 0.086 for the 1p array and SMRT array, respectively. As previously described by Veltman et al. (64), we selected thresholds of 0.15 for the 1p array and 0.18 for the SMRT array, which is slightly more conservative than two times the standard deviation for a normal versus normal hybridization. In addition, alterations had to have more than one consecutive BAC with copy number change to be scored as a region of alteration. For a region to be considered as altered, the local average had to be greater than or equal to 0.15 on the 1p array and 0.18 on the SMRT array. To identify regions of recurrent alteration, the data from each sample were aligned and visualized as heat maps of the copy number profiles using Java TreeView (http://jtreeview.sourceforge.net).

Gene expression analysis
Semi-quantitative analysis of gene expression was performed by RT–PCR. RNA was extracted from frozen tissue sections using Trizol reagent (Invitrogen, Burlington, Ontario). cDNA was synthesized using the Superscript II RNase H reverse transcriptase system (Invitrogen). Gene expression levels were determined by gene-specific PCR, using ß-actin levels for normalization between samples. Gene-specific primers were used to assay the quantity of DVL1 (5'-CCCAACCCCTTGTGTCTGGT-3', 5'-GGTGGGACAGATGACAGGGT-3'), EGFL3 (5'-AGTTGGACTGAGGACAGGTG-3', 5'-AGCCTGCAGACCTGAATCAG-3'), JUN (5'-AACCGCATCGCTGCCTCCAA-3', 5'-CAACTGCTGCGTTAGCATGA-3'), NOTCH2 (5'-GCCAATCGAGACATCACAGA-3', 5'-CCCACAGATGACAGGTGAGA-3'), WNT4 (5'-ACATGCAACAAGACGTCCAA-3', 5'-TCATCGGCACGTGTGCAACT-3'), LRP8 (5'-ATGTTGCGGAAAGGTAACCA-3', 5'-TGAGTAAGGTACTGTGCCAT-3'), REG4 (5'-GAACAAGCACTGTGTCGAGA-3', 5'-ATGAGCAGATTTAGCCAGGC-3') and ADAM30 (5'-GGGTGTTTGCAACAACAGAA-3', 5'-TGGACACAACCCAAATTGAC-3'). PCR cycle conditions were as follows: one cycle of 95°C, 1 min; 30–35 cycles of 95°C, 30 s; annealing at temperature optimal for primers used, 30 s; 72°C, 1 min; and a 10 min extension at 72°C. Annealing temperatures were as follows: 55°C for ß-actin, ADAM30 and NOTCH2; 56°C for EGFL3; 59°C for DVL1; 53°C for WNT4; 50°C for JUN and LRP8 and 55°C for REG4. PCR products were resolved by polyacrylamide gel electrophoresis, imaged by SYBR green staining (Roche, Laval, Quebec) on a Storm phosphoimager and quantified using ImageQuant software (Molecular Dynamics, Piscataway, NJ). Because these genes were hypothesized to be overexpressed owing to DNA amplification, a one-tailed Wilcoxon sign-rank test was used to determine whether overexpression of these genes was significant in a set of matched tumor and normal samples.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Drs James Hogg and Kim Lonergan for providing tumor specimens and cDNA samples, Dr Jean LeRiche for histopathology evaluation, Chris Dawe and Arek Siwoski for microdissection and Bradley P. Coe, Spencer K. Watson and Chad Malloff for array analysis and synthesis. This work was supported by funds from the National Cancer Institute of Canada, Canadian Institute of Health Research, Genome British Columbia/Genome Canada and scholarships to C.G. and J.J.D. from the Michael Smith Foundation of Health Research and the National Science Engineering Research Council.


   FOOTNOTES
 
* To whom correspondence should be addressed at: BC Cancer Research Centre, 601 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada. Tel: +1 6048776149; Fax: +1 60487760002421; Email: cgarnis{at}bccrc.ca

{dagger} These authors contributed equally. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Han, S., Semba, S., Abe, T., Makino, N., Furukawa, T., Fukushige, S., Takahashi, H., Sakurada, A., Sato, M., Shiiba, K. et al. (1999) Infrequent somatic mutations of the p73 gene in various human cancers. Eur. J. Surg. Oncol., 25, 194–198.[CrossRef][ISI][Medline]

  2. Testa, J.R., Siegfried, J.M., Liu, Z., Hunt, J.D., Feder, M.M., Litwin, S., Zhou, J.Y., Taguchi, T. and Keller, S.M. (1994) Cytogenetic analysis of 63 non-small cell lung carcinomas: recurrent chromosome alterations amid frequent and widespread genomic upheaval. Genes Chromosomes Cancer, 11, 178–194.[ISI][Medline]

  3. Nomoto, S., Haruki, N., Tatematsu, Y., Konishi, H., Mitsudomi, T. and Takahashi, T. (2000) Frequent allelic imbalance suggests involvement of a tumor suppressor gene at 1p36 in the pathogenesis of human lung cancers. Genes Chromosomes Cancer, 28, 342–346.[CrossRef][ISI][Medline]

  4. Girard, L., Zochbauer-Muller, S., Virmani, A.K., Gazdar, A.F. and Minna, J.D. (2000) Genome-wide allelotyping of lung cancer identifies new regions of allelic loss, differences between small cell lung cancer and non-small cell lung cancer, and loci clustering. Cancer Res., 60, 4894–4906.[Abstract/Free Full Text]

  5. Ragnarsson, G., Eiriksdottir, G., Johannsdottir, J.T., Jonasson, J.G., Egilsson, V. and Ingvarsson, S. (1999) Loss of heterozygosity at chromosome 1p in different solid human tumours: association with survival. Br. J. Cancer, 79, 1468–1474.[CrossRef][ISI][Medline]

  6. Gasparian, A.V., Laktionov, K.K., Belialova, M.S., Pirogova, N.A., Tatosyan, A.G. and Zborovskaya, I.B. (1998) Allelic imbalance and instability of microsatellite loci on chromosome 1p in human non-small-cell lung cancer. Br. J. Cancer, 77, 1604–1611.[ISI][Medline]

  7. Taketo, M.M. (2004) Shutting down Wnt signal-activated cancer. Nat. Genet., 36, 320–322.[CrossRef][ISI][Medline]

  8. Wissmann, C., Wild, P.J., Kaiser, S., Roepcke, S., Stoehr, R., Woenckhaus, M., Kristiansen, G., Hsieh, J.C., Hofstaedter, F., Hartmann, A. et al. (2003) WIF1, a component of the Wnt pathway, is down-regulated in prostate, breast, lung, and bladder cancer. J. Pathol., 201, 204–212.[CrossRef][ISI][Medline]

  9. Lee, A.Y., He, B., You, L., Dadfarmay, S., Xu, Z., Mazieres, J., Mikami, I., McCormick, F. and Jablons, D.M. (2004) Expression of the secreted frizzled-related protein gene family is downregulated in human mesothelioma. Oncogene, 23, 6672–6676.[CrossRef][ISI][Medline]

  10. Brennan, K., Gonzalez-Sancho, J.M., Castelo-Soccio, L.A., Howe, L.R. and Brown, A.M. (2004) Truncated mutants of the putative Wnt receptor LRP6/Arrow can stabilize beta-catenin independently of Frizzled proteins. Oncogene, 23, 4873–4884.[CrossRef][ISI][Medline]

  11. Tamai, K., Semenov, M., Kato, Y., Spokony, R., Liu, C., Katsuyama, Y., Hess, F., Saint-Jeannet, J.P. and He, X. (2000) LDL-receptor-related proteins in Wnt signal transduction. Nature, 407, 530–535.[CrossRef][Medline]

  12. Garnis, C., Coe, B.P., Zhang, L., Rosin, M.P. and Lam, W.L. (2004) Overexpression of LRP12, a gene contained within an 8q22 amplicon identified by high-resolution array CGH analysis of oral squamous cell carcinomas. Oncogene, 23, 2582–2586.[CrossRef][ISI][Medline]

  13. Uthoff, S.M., Eichenberger, M.R., McAuliffe, T.L., Hamilton, C.J. and Galandiuk, S. (2001) Wingless-type frizzled protein receptor signaling and its putative role in human colon cancer. Mol. Carcinog., 31, 56–62.[CrossRef][ISI][Medline]

  14. Lu, D., Zhao, Y., Tawatao, R., Cottam, H.B., Sen, M., Leoni, L.M., Kipps, T.J., Corr, M. and Carson, D.A. (2004) Activation of the Wnt signaling pathway in chronic lymphocytic leukemia. Proc. Natl Acad. Sci. USA, 101, 3118–3123.[Abstract/Free Full Text]

  15. Suzuki, H., Watkins, D.N., Jair, K.W., Schuebel, K.E., Markowitz, S.D., Dong Chen, W., Pretlow, T.P., Yang, B., Akiyama, Y., Van Engeland, M. et al. (2004) Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer. Nat. Genet., 36, 417–422.[CrossRef][ISI][Medline]

  16. Caldwell, G.M., Jones, C., Gensberg, K., Jan, S., Hardy, R.G., Byrd, P., Chughtai, S., Wallis, Y., Matthews, G.M. and Morton, D.G. (2004) The Wnt antagonist sFRP1 in colorectal tumorigenesis. Cancer Res., 64, 883–888.[Abstract/Free Full Text]

  17. Uematsu, K., He, B., You, L., Xu, Z., McCormick, F. and Jablons, D.M. (2003) Activation of the Wnt pathway in nonsmall cell lung cancer: evidence of dishevelled overexpression. Oncogene, 22, 7218–7221.[CrossRef][ISI][Medline]

  18. Uematsu, K., Kanazawa, S., You, L., He, B., Xu, Z., Li, K., Peterlin, B.M., McCormick, F. and Jablons, D.M. (2003) Wnt pathway activation in mesothelioma: evidence of dishevelled overexpression and transcriptional activity of beta-catenin. Cancer Res., 63, 4547–4551.[Abstract/Free Full Text]

  19. Okino, K., Nagai, H., Hatta, M., Nagahata, T., Yoneyama, K., Ohta, Y., Jin, E., Kawanami, O., Araki, T. and Emi, M. (2003) Up-regulation and overproduction of DVL-1, the human counterpart of the Drosophila dishevelled gene, in cervical squamous cell carcinoma. Oncol. Rep., 10, 1219–1223.[ISI][Medline]

  20. You, L., He, B., Xu, Z., Uematsu, K., Mazieres, J., Mikami, I., Reguart, N., Moody, T.W., Kitajewski, J., McCormick, F. et al. (2004) Inhibition of Wnt-2-mediated signaling induces programmed cell death in non-small-cell lung cancer cells. Oncogene, 23, 6170–6174.[CrossRef][ISI][Medline]

  21. Ohira, T., Gemmill, R.M., Ferguson, K., Kusy, S., Roche, J., Brambilla, E., Zeng, C., Baron, A., Bemis, L., Erickson, P. et al. (2003) WNT7a induces E-cadherin in lung cancer cells. Proc. Natl Acad. Sci. USA, 100, 10429–10434.[Abstract/Free Full Text]

  22. Dang, T.P., Gazdar, A.F., Virmani, A.K., Sepetavec, T., Hande, K.R., Minna, J.D., Roberts, J.R. and Carbone, D.P. (2000) Chromosome 19 translocation, overexpression of Notch3, and human lung cancer. J. Natl Cancer Inst., 92, 1355–1357.[Free Full Text]

  23. Fodde, R. (2002) The APC gene in colorectal cancer. Eur. J. Cancer, 38, 867–871.[CrossRef][ISI][Medline]

  24. Huguet, E.L., McMahon, J.A., McMahon, A.P., Bicknell, R. and Harris, A.L. (1994) Differential expression of human Wnt genes 2, 3, 4, and 7B in human breast cell lines and normal and disease states of human breast tissue. Cancer Res., 54, 2615–2621.[Abstract/Free Full Text]

  25. Nomoto, S., Haruki, N., Kondo, M., Konishi, H. and Takahashi, T. (1998) Search for mutations and examination of allelic expression imbalance of the p73 gene at 1p36.33 in human lung cancers. Cancer Res., 58, 1380–1383.[Abstract/Free Full Text]

  26. Stiewe, T. and Putzer, B.M. (2002) Role of p73 in malignancy: tumor suppressor or oncogene? Cell Death Differ., 9, 237–245.[CrossRef][ISI][Medline]

  27. Pizzuti, A., Novelli, G., Mari, A., Ratti, A., Colosimo, A., Amati, F., Penso, D., Sangiuolo, F., Calabrese, G., Palka, G. et al. (1996) Human homologue sequences to the Drosophila dishevelled segment-polarity gene are deleted in the DiGeorge syndrome. Am. J. Hum. Genet., 58, 722–729.[ISI][Medline]

  28. Nau, M.M., Brooks, B.J., Battey, J., Sausville, E., Gazdar, A.F., Kirsch, I.R., McBride, O.W., Bertness, V., Hollis, G.F. and Minna, J.D. (1985) L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer. Nature, 318, 69–73.[CrossRef][Medline]

  29. Kaye, F., Battey, J., Nau, M., Brooks, B., Seifter, E., De Greve, J., Birrer, M., Sausville, E. and Minna, J. (1988) Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing. Mol. Cell. Biol., 8, 186–195.[Abstract/Free Full Text]

  30. Johnson, B.E., Russell, E., Simmons, A.M., Phelps, R., Steinberg, S.M., Ihde, D.C. and Gazdar, A.F. (1996) MYC family DNA amplification in 126 tumor cell lines from patients with small cell lung cancer. J. Cell. Biochem. Suppl., 24, 210–217.[Medline]

  31. Zajac-Kaye, M. (2001) Myc oncogene: a key component in cell cycle regulation and its implication for lung cancer. Lung Cancer, 34 (Suppl. 2), S43–S46.[Medline]

  32. Schneider, W.J. and Nimpf, J. (2003) LDL receptor relatives at the crossroad of endocytosis and signaling. Cell. Mol. Life Sci., 60, 892–903.[CrossRef][ISI][Medline]

  33. Henderson, L.J., Coe, B.P., Lee, E., Girard, L., Gazdar, A.F., Minna, J., Lam, S., MacAulay, C. and Lam, W.L. (2004) Genomic and gene expression profiling of minute alterations of chromosome arm 1p in small cell lung carcinoma cells. B. J. Cancer, in press.

  34. Klingensmith, J. and Nusse, R. (1994) Signaling by wingless in Drosophila. Dev. Biol., 166, 396–414.[CrossRef][ISI][Medline]

  35. Dickinson, M.E. and McMahon, A.P. (1992) The role of Wnt genes in vertebrate development. Curr. Opin. Genet. Dev., 2, 562–566.[CrossRef][Medline]

  36. Rijsewijk, F., Schuermann, M., Wagenaar, E., Parren, P., Weigel, D. and Nusse, R. (1987) The Drosophila homolog of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless. Cell, 50, 649–657.[CrossRef][ISI][Medline]

  37. Nusse, R. and Varmus, H.E. (1992) Wnt genes. Cell, 69, 1073–1087.[CrossRef][ISI][Medline]

  38. Herz, J. and Strickland, D.K. (2001) LRP: a multifunctional scavenger and signaling receptor. J. Clin. Invest., 108, 779–784.[CrossRef][ISI][Medline]

  39. Hoang, B.H., Kubo, T., Healey, J.H., Sowers, R., Mazza, B., Yang, R., Huvos, A.G., Meyers, P.A. and Gorlick, R. (2004) Expression of LDL receptor-related protein 5 (LRP5) as a novel marker for disease progression in high-grade osteosarcoma. Int. J. Cancer, 109, 106–111.[CrossRef][ISI][Medline]

  40. Qiang, Y.W., Endo, Y., Rubin, J.S. and Rudikoff, S. (2003) Wnt signaling in B-cell neoplasia. Oncogene, 22, 1536–1545.[CrossRef][ISI][Medline]

  41. Rodriguez, S., Jafer, O., Goker, H., Summersgill, B.M., Zafarana, G., Gillis, A.J., van Gurp, R.J., Oosterhuis, J.W., Lu, Y.J., Huddart, R. et al. (2003) Expression profile of genes from 12p in testicular germ cell tumors of adolescents and adults associated with i(12p) and amplification at 12p11.2–p12.1. Oncogene, 22, 1880–1891.[CrossRef][ISI][Medline]

  42. He, X., Semenov, M., Tamai, K. and Zeng, X. (2004) LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way. Development, 131, 1663–1677.[Abstract/Free Full Text]

  43. Herz, J. and Bock, H.H. (2002) Lipoprotein receptors in the nervous system. Annu. Rev. Biochem., 71, 405–434.[CrossRef][ISI][Medline]

  44. Ohkubo, N., Lee, Y.D., Morishima, A., Terashima, T., Kikkawa, S., Tohyama, M., Sakanaka, M., Tanaka, J., Maeda, N., Vitek, M.P. et al. (2003) Apolipoprotein E and Reelin ligands modulate tau phosphorylation through an apolipoprotein E receptor/disabled-1/glycogen synthase kinase-3beta cascade. FASEB J., 17, 295–297.[Abstract/Free Full Text]

  45. Radtke, F. and Raj, K. (2003) The role of Notch in tumorigenesis: oncogene or tumour suppressor? Nat. Rev. Cancer, 3, 756–767.[CrossRef][ISI][Medline]

  46. Axelrod, J.D., Matsuno, K., Artavanis-Tsakonas, S. and Perrimon, N. (1996) Interaction between Wingless and Notch signaling pathways mediated by dishevelled. Science, 271, 1826–1832.[Abstract]

  47. Wesley, C.S. (1999) Notch and wingless regulate expression of cuticle patterning genes. Mol. Cell. Biol., 19, 5743–5758.[Abstract/Free Full Text]

  48. Soriano, S., Kang, D.E., Fu, M., Pestell, R., Chevallier, N., Zheng, H. and Koo, E.H. (2001) Presenilin 1 negatively regulates beta-catenin/T cell factor/lymphoid enhancer factor-1 signaling independently of beta-amyloid precursor protein and notch processing. J. Cell Biol., 152, 785–794.[Abstract/Free Full Text]

  49. Saura, C.A., Tomita, T., Soriano, S., Takahashi, M., Leem, J.Y., Honda, T., Koo, E.H., Iwatsubo, T. and Thinakaran, G. (2000) The nonconserved hydrophilic loop domain of presenilin (PS) is not required for PS endoproteolysis or enhanced abeta 42 production mediated by familial early onset Alzheimer's disease-linked PS variants. J. Biol. Chem., 275, 17136–17142.[Abstract/Free Full Text]

  50. De Strooper, B. and Annaert, W. (2001) Where Notch and Wnt signaling meet. The presenilin hub. J. Cell. Biol., 152, F17–F20.[Abstract/Free Full Text]

  51. Noll, E., Medina, M., Hartley, D., Zhou, J., Perrimon, N. and Kosik, K.S. (2000) Presenilin affects arm/beta-catenin localization and function in Drosophila. Dev. Biol., 227, 450–464.[CrossRef][ISI][Medline]

  52. Brown, M.S., Ye, J., Rawson, R.B. and Goldstein, J.L. (2000) Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell, 100, 391–398.[CrossRef][ISI][Medline]

  53. Collins, B.J., Kleeberger, W. and Ball, D.W. (2004) Notch and lung development and lung cancer. Semin. Cancer Biol., 14, 357–364.[CrossRef][ISI][Medline]

  54. Okubo, T. and Hogan, B.L. (2004) Hyperactive Wnt signalling changes the developmental potential of embryonic lung ectoderm. J. Biol., 3, 11.[CrossRef][Medline]

  55. Tebar, M., Destree, O., de Vree, W.J. and Ten Have-Opbroek, A.A. (2001) Expression of Tcf/Lef and sFrp and localization of beta-catenin in the developing mouse lung. Mech. Dev., 109, 437–440.[CrossRef][ISI][Medline]

  56. Reya, T., Morrison, S.J., Clarke, M.F. and Weissman, I.L. (2001) Stem cells, cancer, and cancer stem cells. Nature, 414, 105–111.[CrossRef][Medline]

  57. Ishkanian, A.S., Malloff, C.A., Watson, S.K., DeLeeuw, R.J., Chi, B., Coe, B.P., Snijders, A., Albertson, D.G., Pinkel, D., Marra, M.A. et al. (2004) A tiling resolution DNA microarray with complete coverage of the human genome. Nat. Genet., 36, 299–303.[CrossRef][ISI][Medline]

  58. McWilliams, A., MacAulay, C., Gazdar, A.F. and Lam, S. (2002) Innovative molecular and imaging approaches for the detection of lung cancer and its precursor lesions. Oncogene, 21, 6949–6959.[CrossRef][ISI][Medline]

  59. Lam, S., MacAulay, C., leRiche, J.C. and Palcic, B. (2000) Detection and localization of early lung cancer by fluorescence bronchoscopy. Cancer, 89, 2468–2473.[CrossRef][ISI][Medline]

  60. Siwoski, A., Ishkanian, A., Garnis, C., Zhang, L., Rosin, M. and Lam, W.L. (2002) An efficient method for the assessment of DNA quality of archival microdissected specimens. Mod. Pathol., 15, 889–892.[CrossRef][ISI]

  61. Garnis, C., Baldwin, C., Zhang, L., Rosin, M.P. and Lam, W.L. (2003) Use of complete coverage array comparative genomic hybridization to define copy number alterations on chromosome 3p in oral squamous cell carcinomas. Cancer Res., 63, 8582–8585.[Abstract/Free Full Text]

  62. Garnis, C., Coe, B.P., Ishkanian, A., Zhang, L., Rosin, M.P. and Lam, W.L. (2004) Novel regions of amplification on 8q distinct from the MYC locus and frequently altered in oral dysplasia and cancer. Genes Chromosomes Cancer, 39, 93–98.[CrossRef][ISI][Medline]

  63. Chi, B., DeLeeuw, R.J., Coe, B.P., MacAulay, C. and Lam, W.L. (2004) SeeGH—a software tool for visualization of whole genome array comparative genomic hybridization data. BMC Bioinformatics, 5, 13.[CrossRef][Medline]

  64. Veltman, J.A., Fridlyand, J., Pejavar, S., Olshen, A.B., Korkola, J.E., DeVries, S., Carroll, P., Kuo, W.L., Pinkel, D., Albertson, D. et al. (2003) Array-based comparative genomic hybridization for genome-wide screening of DNA copy number in bladder tumors. Cancer Res., 63, 2872–2880.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
A. J. Ross, L. A. Dailey, L. E. Brighton, and R. B. Devlin
Transcriptional Profiling of Mucociliary Differentiation in Human Airway Epithelial Cells
Am. J. Respir. Cell Mol. Biol., August 1, 2007; 37(2): 169 - 185.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
M. J. Hardman, A. Waite, L. Zeef, M. Burow, T. Nakayama, and G. S. Ashcroft
Macrophage Migration Inhibitory Factor: A Central Regulator of Wound Healing
Am. J. Pathol., December 1, 2005; 167(6): 1561 - 1574.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
14/4/475    most recent
ddi043v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (12)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Garnis, C.
Right arrow Articles by Lam, W. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Garnis, C.
Right arrow Articles by Lam, W. L.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?