Promoter Methylation of the CADM 1 and 4 . 1 B Genes Occurs Independently of the EGFR or the KRAS 2 Mutation in Non-Small Cell Lung Cancer

Objective: Targeting mutated EGFR by EGFR-tyrosine kinase inhibitors (EGFR-TKI) is a potent approach to a subset of non-small cell lung cancer (NSCLC). However, the response to EGFR-TKI varies in individual cases even among tumors carrying the same EGFR mutation, suggesting the involvement of modifying factors. To characterize possible modifiers, we examined mutation state of the EGFR and the KRAS genes in Japanese NSCLC and compared them with the methylation state of lung tumor suppressors, the CADM1 and 4.1B, whose products have potentials to modify the functions of EGFR or KRAS. Materials and methods: A total of 103 Japanese NSCLC and 11 NSCLC cell lines were examined. Genomic DNA of exons 18 21 of the EGFR and exons 1 and 2 of the KRAS were amplified by polymerase chain reaction (PCR), followed by single-strand conformation polymorphism analysis and direct sequencing. Methylation status of gene promoters in NSCLC cells were examined by methylation-specific PCR. Results: Mutations of the EGFR and KRAS were detected mutually exclusively in 27 and 11 out of 103 NSCLC cases, respectively. EGFR mutations were observed exclusively in adenocarcinoma (27 of 69, 41%) and preferentially in tumors from female and non-smokers (p < 0.00001). Eight (30%) and 12 (44%) of 27 tumors carrying mutated EGFR and 4 (36%) and 8 (73%) of 11 tumors carrying mutated KRAS showed methylation of the CADM1 and 4.1B, respectively. EGFR-mutated tumors with methylation of either CADM1 or 4.1B showed more malignant features than those with unmethylated CADM1 and 4.1B (p < 0.05). ConCorresponding author.


Introduction
Lung cancer is a leading cause of cancer death in developed countries, including USA and Japan, and about 80% -85% of them are histologically classified as non-small cell lung cancer (NSCLC) [1].NSCLC develops and progresses with multiple genetic and epigenetic alterations.Mutations of the EGFR and the KRAS oncogenes and fusion of the EML4-ALK gene are representative driver mutations occurred in the early stage of NSCLC.On the other hand, mutations of the tumor suppressor TP53, RB1and CDKN2A genes, as well as hypermethylation of the tumor suppressor CADM1 and 4.1B genes, occur in the late stage of tumorigenesis and play important roles in malignant progression, including invasion and metastasis of NSCLC [2].It is important, therefore, to know whether these genetic and epigenetic alterations accumulate in the same cancer cells and participate synergistically in the malignant progression of tumors or some of the alterations occur mutually exclusively and play roles in independent pathway of lung tumorigenesis.
Recently, several molecular targeting drugs have been developed and clinically used for the treatment of NSCLC, especially for lung adenocarcinoma.EGFR-tyrosine kinase inhibitors (EGFR-TKI), including Gefitinib and Erlotinib, are established for the treatment of lung adenocarcinomas carrying the specific mutations of the EGFR gene [3].ALK-tyrosine kinase inhibitor, Crizotinib, is also effective to a subset of lung adenocarcinoma expressing the fusion gene, EML4-ALK [4].On the other hand, Cetuximab, a monoclonal antibody against KRAS protein, is widely used for the treatment of a subset of colorectal cancer with wild-type KRAS gene [5], although treatment by Cetuximab or other RAS-targeting drugs has not been established in NSCLC over-expressing KRAS.Targeting to such specific driver oncogenes is getting a major approach to develop new molecular targeting drugs against cancer.However, the responses of tumors to the relevant molecular targeting drugs show variation in individual cases even among tumors expressing the same specific markers, suggesting that additional factors would modulate the responsiveness to the relevant drugs [6].
We have previously identified a tumor suppressor gene, the CADM1/TSLC1, on chromosome 11q23, which encodes a transmembrane glycoprotein that is involved in adhesion in various epithelial cells [7].We have subsequently identified 4.1B as a direct binding protein of CADM1 in its cytoplasmic domain [8].4.1B is an actin-binding protein which transmits the signals of cell adhesion to the cytoskeletal reorganization [9].We have previously reported that the promoter of the CADM1 and 4.1B genes are methylated in 45 (44%) and 59 (57%) of 103 NSCLCs, respectively, both of which provide indicators of poor prognosis of lung adenocarcinoma [10] [11].Promoter methylation as well as loss of expression of CADM1 and 4.1B is also observed in various cancers [12]- [14], suggesting that CADM1-4.1B is an important tumor suppressor cascade of NSCLC [15]- [17].
Interestingly, it has been reported that CADM1 protein associates with a member of EGFR family proteins, ERBB3 [18], suggesting that CADM1 might possibly modify EGFR pathway.It is also noteworthy that loss of CADM1 expression was specifically reported in an invasive model of murine lung adenocarcinoma in the mutated KRAS knock-in mice, suggesting that loss of CADM1 might enhance the malignant features of tumor cells caused by mutated KRAS [19].However, correlation of mutated EGFR or KRAS and inactivation of CADM1 or 4.1B in NSCLC have not been reported yet.
Here, we examined the mutation state of the EGFR and KRAS genes in a series of 103 NSCLC tumors as well as 11 NSCLC cell lines and compared to our previous results of the methylation state of the CADM1 and 4.1B genes.Then, we analyzed the clinico-pathological features of the tumors carrying these genetic and epigenetic alterations to investigate whether methylation of CADM1 or 4.1B could serve as modifiers of the malignant phenotype of NSCLC.

Surgical Materials and Cell Lines
A total of 103 Japanese subjects (74 men and 29 women; median age: 67 years old) provided informed, written consent in accordance with the ethics board of the University of Tokyo (ID: 20-36-0918) and were included in this study.All subjects were surgically resected and histologically diagnosed as having NSCLC at the National Cancer Research Center Hospital, Japan.After surgical removal, all samples were immediately frozen and stored at −135˚C.NSCLC cell lines, Calu-3, NCI-H441, NCI-H522, SK-LU-1, and NCI-H596 were obtained from the American Type Culture Collection; ABC-1, RERF-LC-MS, RERF-LC-OK and VMRC-LCD were from the Health Science Research Resources Bank, Japan; and A549 and PC-14 cells were from RIKEN Cell Bank, Japan.These cells were cultured according to the supplier's recommendation.

Single-Strand Conformational Polymorphism (SSCP) Analysis
Genomic DNA was extracted using the AllPrep Mini Kit (Qiagen, Madison, WI, USA).For SSCP analysis, the DNA fragments corresponding to exons 18 -21 of the EGFR and exons 1 and 2 of the KRAS as well as their flanking sequences were amplified by PCR using KOD FX polymerase (TOYOBO, Osaka, Japan) and a specific pair of primers [20].Primers used for PCR were shown in Table A1, where one of the primers in each pair was end-labeled with Texas Red.The PCR product was diluted 10 times with a loading buffer (90% deionized formamide, 0.01% New Fuchsin and 10 mM EDTA), heat-denatured for 3 min at 95˚C, cooled on ice for 3 min and then loaded onto the gel (0.5xMDE Gel Solution; BMS, Rockland, ME).Electrophoresis was carried out for 120 min at 20˚C using SF5200 (Hitachi Electronics Engineering, Tokyo, Japan) with cooling systems.The results were analyzed using a DNA Fragment Analyzed (Hitachi Electronics Engineering).

Sequencing Analysis
PCR products were separated by electrophoresis on agarose gel, and fragments were cut from the gel and purified by Wizard SV Gel and PCR Clean-Up System (Promega.Madison, WI, USA).Sequencing was conducted by reactions with Applied Biosystems' BigDye 3.1 kit on a 3130xl DNA sequencer.Sequence results were analyzed using Lasergene (DNASTAR, Madison, WI, USA), and statistical analysis was done using SPSS software.The significance level was set at p < 0.05.

Methylation-Specific PCR
Genomic DNA was extracted from cells using a Wizard SV Genomic DNA Purification System (Promega) according to the manufacturer's instructions.For methylation analysis, 500 ng of genomic DNA was subjected to bisulfite conversion using a MethylCode Bisulfite Conversion Kit (Invitrogen, Carlsbad, CA, USA).Then, the bisulfate-converted DNA (50 ng) was subjected to PCR to amplify methylated and unmethylated DNA of cancer related genes, including RASSF1A, CDKN2A, APC, CDH1, GATA-4 and GATA-5, by Platinum Taq DNA Polymerase (Invitrogen).Pairs of primers specific to methylated and unmethylated DNA were as listed in Table A1 [21]- [25].Methylation state of the CADM1 and 4.1B genes in 11 cells were analyzed by bisulfite SSCP as described previously [10] [11].

Statistical Analysis
Fisher exact test were used for categorical variables.Stat View 5.0 (SAS Institute, Cary, NC) was used for the analysis.Differences with P value of less than 0.05 were considered significant.

Mutation of the EGFR and the KRAS Genes in Japanese NSCLC
SSCP analysis followed by sequencing analysis of exons 18 -21 and their flanking regions of the EGFR gene idenified 27 mutations (26%) out of 103 HNSCC cases (Figure 1 and Figure 2, Table 1).These include 14 inframe deletions of 15 nucleotides corresponding to codon 746 to codon 750, nine T to G transversions at codon 858 (leucine to arginine), and four G to T transversions at codon 719 (glycine to cysteine), all of which are  known mutations of the EGFR.On the other hand, SSCP and sequencing analyses of exons 1 and 2 and their flanking regions of the KRAS gene identified 11 mutations (11%) out of 103 NSCLC cases, all of which were known activating mutations of the KRAS gene (Figure 1 and Figure 2, Table 2).

Clinico-Pathological Features of NSCLC with the EGFR or the KRAS Mutations
It is widely accepted that the EGFR mutation is preferentially observed in female cases of adenocarcinoma without smoking background, whereas the KRAS mutation is frequently detected in NSCLC with heavy smoking background.The present study confirmed these characteristics (Table 1 and Table 2).The EGFR mutation is  observed exclusively in adenocarcinoma (27/69; 39%) but not in 26 squamous cell carcinoma, 2 adenosquamous carcinoma, or 6 large cell neuroendocrine carcinoma (LCNEC) (p < 0.0001).The incidence of the EGFR mutation is also significantly higher in female cases (17/29; 59%) than that in male cases (10/74; 14%) (p < 0.00001).
In addition, the incidence of the EGFR mutation is significantly higher in never smokers (16 of 26; 62%) in comparison with that in ex-smokers (6 of 26; 23%) or in current smokers (5 of 51; 10%) (p < 0.00001).Moreover, mutation of the EGFR and the KRAS gene was also detected mutually exclusively as previously reported.
On the other hand, the incidence of the KRAS mutation do not show significant difference in male or female cases (10 of 74; 14% and 1 of 29; 3%, respectively) or in current smokers (7 of 51; 14%) in comparison with that in in ex-smokers (3 of 26; 12%) or in never smokers (1 of 26; 4%) (Table 2).In contrast to the EGFR mutation, the KRAS mutation is detected in considerable portions of various histological subtypes of NSCLC, including adenocarcinoma (6 of 69; 9%), squamous cell carcinoma (2 of 26; 8%), adenosquamo cell carcinoma (1 of 2; 50%) and large cell neuroendocrine carcinoma (2 of 6; 33%).When clinico-pathological features of the EGFR-mutated tumors were examined, the CADM1 or 4.1B methylation appeared to be preferentially observed in tumors with moderately differentiated histology or with stages II or III in comparison with tumors with well differentiated histology or those with stage I, although the difference was not statistically significant.Since CADM1 and 4.1B act in the same cascade of tumor suppression [8] [16], this cascade would be disrupted when either the CADM1 or 4.1B is methylated, whereas this cascade would be intact only when both the CADM1 and 4.1B are unmethylated.When we divide 27 EGFR-mutated tumors into two groups from this point of view, methylation of either the CADM1 or 4.1B was more frequently observed in moderately differentiated tumors (12 of 15, 80%) than in well differentiated tumors (4 of 12; 33%) (p < 0.05) or in tumors with stages II and III (10 of 12; 87%) than in stage I tumors (6 of 15; 40%) (p < 0.05) (Table 3).

Mutation of the EGFR or the KRAS
Similarly, the KRAS gene mutation was observed in 4 of 45 (9%) NSCLC tumors with the CADM1 methylation, while in 7 of 58 (12%) tumors without the CADM1 methylation.The KRAS mutation was also observed in 8 of 59 (14%) NSCLC tumors with the 4.1B methylation and in 3 of 44 (7%) tumors without the 4.1B methylation (Table 2).The differences in the incidence are not statistically significant, suggesting that the mutation status of the KRAS gene is independent of the methylation status of the CADM1 or 4.1B genes in Japanese NSCLC.Furthermore, histological differentiation and pathological stages of the KRAS-mutated tumors in terms of the methylation status of the CADM1 or 4.1B did not show any significant difference (Table A2).
Finally, we have examined the mutation of the EGFR and the KRAS genes in 11 NSCLC cell lines and found 1 EFGR and 3 KRAS mutations, respectively, whereas methylation of the CADM1 and 4.1B were detected in 4 and 7 cell lines, respectively.The patterns of alteration in individual cells suggest that the EGFR and KRAS mutations and the CADM1 and 4.1B methylation are also independent events in NSCLC cell lines (Table 4).To know the overall tendency of gene methylation in each cell line, methylation state of 6 additional gene promoters were analyzed by methylation-specific PCR.These include RASSF1A, CDKN2A, APC, CDH1, GATA-4 and GATA-5 genes.As shown in Table 4, NCI-H441 and ABC-1 cells showed total 6 methylated genes, whereas NCI-H522, Calu-3, PC-14, SKLU-1 and VMRC-LCD cells showed only 2 methylated genes.

Discussion
Molecular targeting therapies in association with specific genetic markers provide promising approaches to the treatment of NSCLC.However, the effectiveness of or resistance to EGFR-TKI inhibitors to lung adenocarci- noma shows diversity in individual tumors even if they are carrying the specific EGFR gene mutation.One of the possible molecular mechanisms of this variation would be additional factors that modulate the function of EGFR and its signaling in tumor cells.Thus, it would be important to characterize such modifiers for generating more effective personalized therapy.Expression profiling of these tumors would provide valuable information of possible genetic and epigenetics modifiers [26].Alternatively, a candidate gene approach to examine the expression of specific molecules showing functional cross-talk with EGFR would be also useful.It was reported that inactivation of the CDKN2A gene could serve as a poor prognostic factor in NSCLC [27].
In the present study, we examined the mutation status of the EGFR gene in 103 NSCLC and compared the results with the methylation status of the CADM1 and/or 4.1B.CADM1 was chosen because CADM1 was shown to interact with ERBB3, a member of EGFR family proteins, and partly suppresses ERBB3 phosphorylation by recruiting a phosphatase PTPN13 [18].Since ERBB3 forms a dimer with EGFR, CADM1 could modulate EGFR signaling.As 4.1B is a binding protein of CADM1 and acts in the downstream of CADM1 cascade, we also compared the EGFR mutation with methylation status of the 4.1B.As widely accepted, we have confirmed that 27 EGFR-mutated tumors are exclusively adenocarcinoma (p < 0.00001) and developed in female (p < 0.00001) and non-smokers (p < 0.00001).We have also confirmed that the EGFR mutation and the KRAS mutation are mutually exclusive (Table 1).
The original finding in the present study is independent occurrence of the mutation of a driver oncogene, EGFR, and methylation of the CADM1 and 4.1B, tumor suppressor genes involved in tumor progression in 103 primary tumors and 11 cell lines of NSCLC.This would be reasonable considering that CADM1-4.1Bcascade transmits the signals of cell adhesion to the development and maintenance of epithelia-like cell structure through reorganizing the cytoskeleton, which is not directly related to growth factor signaling triggered by EGFR [8] [28].Thus, 27 EGFR-mutated tumors can be divided into 16 tumors with methylation of either the CADM1 or 4.1B genes showing disrupted CADM1-4.1Bcascade, and 11 tumors with unmethylated CADM1 and 4.1B genes showing intact CADM1-4.1Bcascade.Another important finding of this study is that the disruption of CADM1-4.1Bcascade by methylation of either CADM1 or 4.1B genes are associated with more malignant phenotype in histological differentiation (p < 0.05) and pathological stages (p < 0.05) even in EGFR-mutated tumors (Table 3).In addition to pathological characterization, further studies for estimating clinical outcome of the patients with these two groups of tumors, including responses to the TKI-therapy and possible recurrence, would be required to answer the question whether CADM1 and 4.1B act as epigenetic modifiers in the treatment of TKI.
As to the methylation in cancer, it should be noted that some gene promoters are often methylated not only by causative gene silencing but also by passenger mechanisms due to the methylator phenotype of cancer cells [29].In fact, among 8 gene promoters examined, 6 genes (75%) were methylated in 2 cell lines, whereas only 2 genes (25%) were methylated in 5 cell lines, including PC-14 cells, suggesting that methylator phenotype could be involved at least in a subset of NSCLC cell lines.In PC-14 and Calu-3 cells, however, the CADM1 and 4.1B were the only genes methylated among 8 genes analyzed, suggesting that silencing of the CADM1 and 4.1B by promoter methylation would confer some biological advantages to these cancer cells.Since PC-14 carried the EGFR mutation, restoration of CADM1 and/or 4.1B expression might modulate the response of PC-14 cells to EGFR-TKI, although PC-14 is essentially sensitive to EGFR-TKI.
Mutation of the KRAS gene is another common alteration of driver oncogenes in various tumors, including NSCLC and HNSCC [5] [20] [30].To understand the molecular mechanisms of NSCLC development and progression, Jackson et al. established an elegant mouse model, in which Cre-recombinase was infected into the murine airway epithelial cells through retroviral gene transfer and the mutated KRAS knocked-in gene was activated in the airway epithelial cells to develop lung adenocarcinoma [31] [32].Subsequent expression profile analysis identified the CADM1 as one of 18 genes whose expression was significantly down-regulated in the aggressive tumors showing chromosome instability [18].These finding suggest that loss of CADM1 expression synergistically enhances the malignant phenotype of NSCLCs carrying the KRAS mutation.Our study demonstrated that the mutation status of the KRAS and the methylation status of the CADM1 or 4.1B were independent phenomena.In fact, among 11 KRAS-mutated tumors, 9 tumors showed methylation of either CADM1 or 4.1B, whereas 2 tumors showed both unmethyalted CADM1 and the 4.1B (Table A2).However, differences in the histological differentiation and pathological stages of these two groups showed borderline or no statistical significance in this study.Further studies to check the status of CADM1-4.1Bcascade in the KRAS-mutated tumors and their clinico-pathological features in larger cohort would be helpful to understand the pathobiological characterstics and clinical outcome of the KRAS-mutated NSCLC.

Conclusion
This is the first report to demonstrate that the methylation status of the CADM1 or the 4.1B gene is independent of the mutation status of the EGFR or the KRAS gene in NSCLC.Moreover, among the EGFR-mutated lung adenocarcinoma, methylation of both or either of the CADM1 or 4.1B was observed in tumors showing less differentiated histology and more advanced pathological stages.Since CADM1 and 4.1B proteins have potentials to modify the function of EGFR and KRAS signaling, methylation status of the CADM1 and/or 4.1B in tumors could provide useful information not only for deep understanding of the pathological features but also for predicting the response or recurrence of lung adenocarcinoma to the EGFR-TKI therapy.A2

Figure 1 .
Figure 1.Single-strand conformation polymorphism (SSCP) analysis of exon 18 (A) and exon 19 (B) of the EGFR and exon 1 of the KRAS genes.T1-T12 are DNAs from NSCLC, while N4, N8, N12 are DNAs from non-cancerous lung tissues from the same patients.T3, T7 and T10 showed shift of the mobility caused by nucleotide substitutions indicated by red arrow.Primer pairs used for amplification of exons 18 -21 of the EGFR gene and exons 1 and 2 of the KRAS gene are listed in Table A1.Red arrows indicate bands with mobility shift caused by higher structural changes due to nucleotide substitutions or deletions.

Figure 2 .
Figure 2. Sequencing analysis of the EGFR and the KRAS genes.A-C.A G to T transversion of codon 719 (Glycine (GGC) to Cysteine (TGC)) in exon 18 (A), an in-frame deletion of 15-bp from codon 746 to codon 750 in exon 19 (B) and T to G transversion of codon 858 (CTG (Leucine) to CGG (Arginine) (C) of the EGFR gene are shown.(D) and (E) A G to T transversion of codon 12 (GGT (Glycine) to TGT (Cysteine)) in exon 1 (D) and a G to T transversion of codon 61 (CAA (Glutamine) to CTA (Leucine)) in exon 2 (E) of the KRAS gene are shown.

Table 1 .
EGFR mutation and clinicopathological characteristics of primary NSCLCs.

Table
KRAS mutation and clinicopathological characteristics of primary NSCLCs.

Genes Is Independent of the Promoter Methylation of the CADM1 or 4.1B Genes in Japanese NSCLC
Next, mutation state of the EGFR and KRAS gene obtained in this study was compared with the methylation state of the CADM1 or 4.1B gene promoters that we have previously reported in the same series of NSCLC tumors[10] [11].As shown in Table1, the EGFR mutation was observed in 8 of 45 (18%) NSCLC tumors with hypermethylation of the CADM1 and in 19 of 58 (33%) tumors without hypermehylation of the CADM1.Similarly, the EGFR mutation was observed in 12 of 59 (20%) NSCLC tumors with hypermethylation of the 4.1B and in 15 of 44 (34%) tumors without hypermehylation of the 4.1B.These incidences do not show any significant difference, suggesting that the CADM1 or the 4.1B methylation is an independent event of the EGFR mutation.

Table
Methylation status of the CADM1 and the 4.1B and clinico-pathological charactersitcs of EGFR-mutated lung adenocarcinoma.
NS: not significant; * W/D vs M/D, ** I vs II + III.

Table 4 .
Mutation status of the EGFR and the KRAS genes and methylation status of the CADM1, 4.1B and other genes in human NCLCL cell lines.

.
Methylation status of the CADM1 and the 4.1B and pathological charactersitcs of KRAS-mutated lung adenocarcinoma.
NS: not significant.