In the present study, we used CCLS to identify differentially expressed genes in mouse lung adenomas. The CCLS method allows one to define differentially expressed genes, based on competitive hybridization between normal lung RNA and RNA derived from lung adenomas, in a nonselective manner and allows one to readily clone differentially expressed genes. There are many advantages in the use of CCLS to determine gene expression changes in cancer. For example, expression differences for both known and novel genes can be detected. Because the cDNA library is not normalized to ensure approximately equal representation of polyA+ RNA sequences, detection frequencies of differentially expressed genes can be determined, indicating the relative frequency of mRNA expression in the normal lung tissue. Additionally, in-depth sampling of gene expression changes for more than 100,000 clones is possible. Although CCLS is laborious and time-consuming, screening data are extensive and allow for the further characterization and functional analysis of unknown genes and examination of the potential roles of known genes in lung tumorigenesis. Some disadvantages of CCLS also exist. The use of CCLS methodology that employs a normal mouse lung cDNA library yields a number of implications. First, if a gene that is found in tumor tissue were not expressed at all in the normal lung, it would not be detected in this study. Secondly, genes that are expressed at very low levels in normal lung were probably missed using this method. Thirdly, because the cDNA library is not normalized, clones associated with significantly overexpressed genes may be repeatedly selected. Thus, there were hundreds of independent clones identified that proved by sequencing to be either surfactants or CC10.
Sixty-five genes were found to be differentially expressed in lung adenomas when compared to normal lung. Nineteen genes were underexpressed and 46 were overexpressed. Seven clones do not match any of the known genes in the NCBI sequence database, whereas 58 had high homology to known genes. For most of the genes, the changes were highly reproducible; thus, 37 of 49 genes displaying overexpression in tumors demonstrated such overexpression in at least two or three of the adenomas. Moreover, 24 of 37 genes were overexpressed at least three-fold and 12 of 37 at least five-fold. Similarly, 10 of 19 underexpressed genes were underexpressed in at least two or three of the adenomas and 6 of 19 were underexpressed at least three-fold in adenomas. Although some of the genes appear to be mechanistically more relevant to the cancer process or are more obvious candidate targets for therapy (see Discussion below), any of the defined genes may be candidate markers for early detection of lesions and as potential endpoint biomarkers. Known genes found to be differentially expressed in lung adenomas including 45S pre-rRNA, pancotin, α-globin, β-globin, fibrinogen A α-chain, paroxanase, cysteinyl-tRNA synthetase, homolog of D. melanogaster flightless I gene, von Ebner minor salivary gland protein, and TNFα-stimulated ABC protein. Although the role of these genes in mouse lung tumorigenesis is still unknown, they are candidate biomarkers for lung tumorigenesis and potential targets for chemoprevention studies. Many of the differentially expressed genes were detected reproducibly and were highly altered in tumors versus normal parenchyma including JAK-1, zinc finger 96, α-1 protease inhibitor, and homolog of D. melanogaster flightless I gene.
Three particularly intriguing overexpressed genes code for the kinases: ERK-1, JAK-1
, and CHUK
. All three genes are overexpressed in at least 67% of adenomas, and JAK-1
levels are overexpressed almost five-fold in adenomas. Members of the various kinase families are particularly appealing for chemotherapy studies using mouse lung tumor model because small molecule inhibitors have been developed against this family of enzymes. ERK-1
belongs to the MAK kinase family and is a component of signaling pathways that influences cellular proliferation and differentiation, while JAK-1
is a member of the intracellular tyrosine kinase family (Janus kinases), and activation of JAKs is the initial step in cytokine signaling. Studies have shown that ERK activation increased 15-fold, whereas ERK expression levels were only 1.3-fold higher in prostate cancer [19
contains a serine-threonine kinase catalytic domain and may be targeted to a helix-loop-helix and/or a leucine zipper transcription factor [20
links kinase cascades to NF-κB activation [21
RasGAP was downregulated and both ERK-1
were upregulated in mouse lung tumors. RasGAP, which is downregulated in adenomas, is a ubiquitous 120-kDa protein that hydrolyzes GTP bound to p21Ras [22,23
]. Underexpression of RasGAP, which should increase the levels of Ras proteins in the activated state, and overexpression of ERK-1
appear to be crucial to Ras-RasGAP cycling-Raf-1-MAPK kinase signal transduction in mouse lung tumor development. Studies show that α-adaptin interacts with GHR and mediates endocytosis of GHR [24
] upon hormone stimulation. The interaction of Shc with α-adaptin is also involved in receptor endocytosis [25
]. Our data showed that α-adaptin was overexpressed in 80% of mouse lung adenomas (five of six).
NFAT1, CA150, CHUK
, and zinc finger protein 96
were overexpressed in 50%, 67%, 83%, and 100% of lung tumors, respectively. These four genes play crucial roles in signal transduction and gene expression. NFAT1
orients the two subunits of AP-1, c-Jun, and c-Fos on DNA through direct protein-protein interaction to regulate transcription [26,27
]. Evidence suggests that CA150, a nuclear protein associated with the human RNA polymerase II holoenzyme, plays a role in the regulation of cellular transcriptional processes [28
]. The functions of CA150 in the mouse have not as yet been reported. CHUK
contains a serine-threonine kinase catalytic domain and may be targeted to helix-loop-helix and/or leucine zipper transcription factors [20
links kinase cascades to NF-κB activation [21
]. The zinc finger motif is generally present in most transcription factors that regulate gene expression. Overexpression of NFAT1, CA150, CHUK
, and the zinc finger protein 96
in mouse lung tumor cells is likely to facilitate DNA transcription upon growth stimulation during tumor development.
The result showing a decrease in cyr61 appears to contradict the explanation that it is a factor that will stimulate tumor growth. Cyr61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible early gene, which acts as an extracellular, matrix-associated signaling molecule promoting the adhesion of endothelial cells through interaction with integrin αV
]. Studies suggest that cyr61 is an angiogenic inducer that promotes tumor growth and vascularization through integrin αV
-dependent pathways [32
]. We found that cyr61 was underexpressed in five of six lung tumors.
Three genes that encode metabolizing enzymes were differentially expressed in mouse lung tumors. CA IV and ALDH II were downregulated in 50% and 83% of tumors, respectively. CYP2C40 was overexpressed in 67% of tumors. CA IV is a glycoprotein associated with cell membranes in lung and kidney [33,34
]. Altered expression of various CA isozymes has been observed in a variety of tumor types. CA, an NADPH-dependent enzyme, has many functions: elimination of CO2
and metabolites, pH regulation, and participation in membrane transport events during active cell growth [35
]. ALDH II is a member of the ALDH family and plays a role in ethanol detoxification [36
]. Similar to other p450 enzymes, CYP2C40 plays an important role in bioactivation and detoxification of certain hepatoxins.
In this study, sulfated glycoprotein-2 (clusterin) was overexpressed in more than 67% of lung tumors. Clusterin is a widely expressed, well-conserved, secreted glycoprotein that inhibits apoptosis. Secreted proteins such as clusterin become particularly attractive candidate proteins as biomarkers of cancer in serum. Recent studies indicate that overexpression of clusterin confers cellular protection against heat shock and oxidative stress [37
] and exogenous clusterin reduces the sensitivity of cells to TNF [38
MHC class I, immunoglobulin, and complement components are involved in immune surveillance [39
]. In this study, both the MHC class I heavy-chain precursors, H-2D(k) and H-2K(k), are upregulated in tumors. Alterations in expression of these genes may reflect differences in the numbers of lymphoid cells observed in tumors as contrasted with normal lung parenchyma. Complement C3 is also upregulated, whereas 12A1 immunoglobulin heavy chain is downregulated.
A number of genes that are commonly expressed in normal lung parenchyma were overexpressed or underexpressed in lung adenomas. We have also shown that surfactant-associated proteins (SPs) A, B, and C are upregulated in three of six (50%), six of six (100%), and six of six (100%), whereas CC10 was downregulated in all lung adenomas examined. These results suggest that most of the lung adenoma cells were derived from Type II cells instead of Clara cells. Alternatively, altered expression of these genes may have functional implications. For example, CC10 may function to bind to calcium, proteins, or other ligands and may be an important immunomodulatory and anti-inflammatory protein [40,41
]. Overexpression of CC10 cDNA in the NSCLC cell line A549 markedly reduces its invasiveness. CC10-transfected cell lines also exhibit decreased adhesiveness to fibronectin [42
]. These results support the conclusion that loss of CC10 may contribute to carcinogenesis. SP-A, SP-B, and SP-C, are known to be required for optimal surfactant function [43
]. The functional significance of these proteins is unknown. Studies have shown that SP mRNA are present in all lung tumors, with SP-A, SP-B, and SP-C being coexpressed in 10 of 12 (83%) adenomas and four of five (80%) carcinomas [44
There is a possibility that some of the differentially expressed genes are due to differences in the density of mouse Type II cells or Clara cells in normal lungs versus and in lung adenomas with respect to amounts of relative to stromal and other contaminating cells. We have not been successful in isolating pure Type II cells or Clara cells from normal surrounding lungs of the animals bearing adenomas. Furthermore, numerous steps and treatments required for the current methodology available for isolating these cells would not make the isolated cells suitable control cells for lung adenomas that did not undergo such a process for gene expression profiling studies.
As was mentioned in the Introduction
, one of the strengths of the CCLS technique is its ability to allow the identification and cloning of unknown or minimally described genes. Thus, in addition to the better known genes described above, an additional seven unknown genes were described as being overexpressed in the majority of lung adenomas. In fact, most of these genes were overexpressed at least three-fold. Two of the genes were further characterized by extensive sequencing and comparison of both mouse and human cDNA: a hypothetical protein (FLJ11240) and a GEF homologue (). FLJ11240 hypothetical protein was found to have partial homology with a peptidase, E1–E2 ATPase, His kinase, and Ppx/GppA phosphatase when searching the Protein-BLAST database (NCBI, NIH). The GEF
gene was found to be highly homologous (>98%) to mouse neuronal GEF. GEFs have been shown to play important roles in the Ras signaling pathway, which is frequently activated by the binding of Ras to Raf protein kinases, Type I phosphatidylinositol-3 (PI3) kinases, or Ral-specific guanine nucleotide exchange factors (RalGEFs) [13
]. RalGEFs interact with Ras to form the GTP-bound state of the Ral family GTPases, leading to enhanced transcription of c-fos, cyclin D1, and genes containing the TATA-binding protein promoter [14–17
]. Recently, activation of the RalGEF pathway has been shown to promote tumor metastasis [18
]. This result suggests that overexpression of the neuronal GEF
gene is associated with lung carcinogenesis in mice. These results show the strength of this approach for identifying unknown or minimally characterized genes with altered expression.
Finally, several genes differentially expressed, observed in this study using the CCLS method, are also found to be differentially expressed in mouse lung tumors through immunohistochemistry. For example, Mason et al. [45
] reported increased expression of SP-A and SP-C in mouse lung adenomas and the lack of expression of CC10 in mouse lung adenomas regardless of morphology (solid or papillary) using immunohistochemistry. Another report by Ramakrishna et al. [46
] found that expression of Erk1/2 was increased in mouse lung tumors using immunoblotting method. These reports provide further confirmation of our results using a different methodology.
The genes identified in this study can be employed in a variety of ways: 1) for use as early detection markers for lung lesions in the A/J model; 2) to compare the gene expression changes observed in the A/J model compared with human adenocarcinomas; 3) for basic understanding of the cancer process; 4) to help define potential molecular targets, which can be tested in this highly reproducible lung tumor model; and 5) to serve as potential modulatable biomarkers, which can be employed in screening for potential agents or in determining the efficacy of those agents.