Malignant Progression Of Non-small Cell Lung Cancer Research Articles

Ginsenoside Rh2 shifts tumor metabolism from aerobic glycolysis to oxidative phosphorylation through regulating the HIF1-α/PDK4 axis in non-small cell lung cancer

BackgroundGinsenoside Rh2 (G-Rh2), a steroidal compound extracted from roots of ginseng, has been extensively studied in tumor therapy. However, its specific regulatory mechanism in non-small cell lung cancer (NSCLC) is not well understood. Pyruvate dehydrogenase kinase 4 (PDK4), a central regulator of cellular energy metabolism, is highly expressed in various malignant tumors. We investigated the impact of G-Rh2 on the malignant progression of NSCLC and how it regulated PDK4 to influence tumor aerobic glycolysis and mitochondrial function.MethodWe examined the inhibitory effect of G-Rh2 on NSCLC through I proliferation assay, migration assay and flow cytometry in vitro. Subsequently, we verified the ability of G-Rh2 to inhibit tumor growth and metastasis by constructing subcutaneous tumor and metastasis models in nude mice. Proteomics analysis was conducted to analyze the action pathways of G-Rh2. Additionally, we assessed glycolysis and mitochondrial function using seahorse, PET-CT, Western blot, and RT-qPCR.ResultTreatment with G-Rh2 significantly inhibited tumor proliferation and migration ability both in vitro and in vivo. Furthermore, G-Rh2 inhibited the tumor’s aerobic glycolytic capacity, including glucose uptake and lactate production, through the HIF1-α/PDK4 pathway. Overexpression of PDK4 demonstrated that G-Rh2 targeted the inhibition of PDK4 expression, thereby restoring mitochondrial function, promoting reactive oxygen species (ROS) accumulation, and inducing apoptosis. When combined with sodium dichloroacetate, a PDK inhibitor, it complemented the inhibitory capacity of PDKs, acting synergistically as a detoxifier.ConclusionG-Rh2 could target and down-regulate the expression of HIF-1α, resulting in decreased expression of glycolytic enzymes and inhibition of aerobic glycolysis in tumors. Additionally, by directly targeting mitochondrial PDK, it elevated mitochondrial oxidative phosphorylation and enhanced ROS accumulation, thereby promoting tumor cells to undergo normal apoptotic processes.

DIRAS3 enhances RNF19B-mediated RAC1 ubiquitination and degradation in non-small-cell lung cancer cells

Distant metastasis remains the leading cause of high mortality in patients with non-small-cell lung cancer (NSCLC). DIRAS3 is a candidate tumor suppressor protein that is decreased in various tumors. However, the regulatory mechanism of DIRAS3 on metastasis of NSCLC remains unclear. Here, we found that DIRAS3 suppressed the migration of NSCLC cells. Besides, DIRAS3 stimulated the polyubiquitination of RAC1 and suppressed its protein expression. Furthermore, RNF19B, a member of the RBR E3 ubiquitin ligase family, was observed to be the E3 ligase involved in the DIRAS3-induced polyubiquitination of RAC1. DIRAS3 could promote the binding of RAC1 and RNF19B, thus enhancing thedegradation of RAC1 by the ubiquitin-proteasome pathway. Finally, the DIRAS3-RNF19B-RAC1 axis was confirmed to be associated with the malignant progression of NSCLC. These findings may be beneficial for developing potential prognostic markers of NSCLC and may provide an effective treatment strategy.

HIF‑1α inhibits ferroptosis and promotes malignant progression in non‑small cell lung cancer by activating the Hippo‑YAP signalling pathway.

Ferroptosis and hypoxia-inducible factor 1α (HIF-1α) have critical roles in human tumors. The aim of the present study was to investigate the associations between ferroptosis, HIF-1α and cell growth in non-small cell lung cancer (NSCLC) cells. The lung cancer cell lines SW900 and A549 were evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the expression of HIF-1α. Cell Counting Kit-8, flow cytometry and Transwell migration assays were used to measure cell viability, apoptosis and invasion, respectively. The production of reactive oxygen species (ROS) and levels of malondialdehyde (MDA), glutathione (GSH) and ferrous ion (Fe2+) were determined using detection kits. The expression levels of glutathione peroxidase 4 (GPX4) and Yes-associated protein 1 (YAP1) were detected using RT-qPCR and western blotting. The results showed that the expression of HIF-1α was significantly upregulated in NSCLC cells compared with normal human bronchial epithelial cells. Small interfering RNA specific to HIF-1α (si-HIF-1α) significantly decreased the proliferation and invasion of NSCLC cells and increased their apoptosis. si-HIF-1α also increased the levels of ROS, MDA and Fe2+ but decreased GSH and GPX4 levels in A549 cells. Additionally, si-HIF-1α increased phosphorylated (p-)YAP1 levels, suppressed GPX4 and YAP1 expression, and attenuated the YAP1 overexpression-induced changes in YAP1, p-YAP1 and GPX4 levels and cell viability. The ferroptosis antagonist ferrostatin-1 partially attenuated the effects of si-HIF-1α on the NSCLC cells, while the ferroptosis agonist erastin further inhibited NSCLC growth by blocking HIF-1α expression. In conclusion, the silencing of HIF-1α induces ferroptosis by suppressing Hippo-YAP pathway activation in NSCLC cells. The present study provides novel insights into the malignant progression of NSCLC and suggests that HIF-1α is an effective target for the treatment of NSCLC.

Knockdown of growth differentiation factor-15 inhibited nonsmall cell lung cancer through inactivating PTEN/PI3K/AKT signaling pathway.

Non-small cell lung cancer (NSCLC) is characterized by high morbidity and mortality in the world. Growth and differentiation factor 15 (GDF15) has been proved to play an important role in regulating tumor progression. However, the influence of GDF15 on NSCLC remains unclear. We aimed to investigate the regulatory role of GDF15 in NSCLC. The correlation between GDF15 expression and prognosis, stage of NSCLC was examined with bioinformatics method. The cell proliferation was detected with CCK8 and EdU staining. Wound healing, Transwell, flow cytometry assays were used to measure cell migration, invasion, and apoptosis, respectively. High expression of GDF15 is correlated with worse survival and malignant progression of NSCLC. Knockdown of GDF15 restrained the proliferation, invasion, migration, but accelerated apoptosis of lung cancer cells through regulating PTEN/PI3K/AKT signaling pathway. sh-GDF15 suppressed epithelial mesenchymal transition (EMT) process and promoted the chemotherapy sensitivity of lung cancer cells. GDF15 plays an important role in NSCLC progression. GDF15 mediated PTEN/PI3K/AKT signaling pathway might be the potential therapeutic targets for the prevention and treatment of GDF15.

CircDLG1 promotes malignant development of non-small cell lung cancer through regulation of the miR-630/CENPF axis.

Circular RNAs (circRNAs) have been reported to be crucial modulatory molecules in the etiology of non-small cell lung cancer (NSCLC). This study aimed to probe the precise role and mechanism of circRNA discs large MAGUK scaffold protein1 (circDLG1) in the malignant progression of NSCLC. The abundances of circDLG1, miR-630, and centromere proteinF (CENPF) mRNAs were gauged by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was tested in 3‑(4, 5‑dimethylthiazol-2-yl)-2, 5‑diphenyltetrazolium bromide (MTT) assay and 5‑ethynyl-2'-deoxyuridine (EdU)-incorporation assay. Cell apoptosis was analyzed by flow cytometry. Cell migration and invasion were assessed by transwell assay. Western blot was exploited to examine the levels of all proteins. The interaction between miR-630 and circDLG1 or CENPF was verified by dual-luciferase reporter, RNA pull-down, and/or RNA immunoprecipitation assays. Tumor xenograft assay and immunohistochemistry (IHC) were executed for the role of circDLG1 in tumor growth in vivo. CircDLG1 and CENPF were highly expressed in NSCLC, while miR-630 was downregulated. CircDLG1 silencing repressed proliferation, migration, and invasion, and expedited apoptosis of NSCLC cells in vitro. Mechanistically, circDLG1 deficiency modulated NSCLC cell malignant development through interacting with miR-630. Furthermore, CENPF was targeted by miR-630, and circDLG1 could positively control CENPF expression through acting as an miR-630 sponge. Furthermore, CENPF overexpression reversed the repressive impacts of circDLG1 inhibition in the malignant behaviors of NSCLC cells. Besides, circDLG1 interference hindered tumor growth in vivo. CircDLG1 knockdown could impede NSCLC advancement through modulating the miR-630/CENPF axis, manifesting as apromising molecular target for NSCLC treatment.

MiR-1226, mediated by ASCL1, suppresses the progression of non-small cell lung cancer by targeting FGF2

This study aims to elucidate the characteristic and expression level of miR-1226 in non-small cell lung cancer (NSCLC) tissue samples and cell lines, and its potential influence on the malignant progression of NSCLC. The expression level of miR-1226 in tumor tissue and paracancerous tissue of NSCLC patients was detected by qRT-PCR. The influences on clinical features of NSCLC patients were analyzed. The proliferation and metastasis ability in H1299 and SPC-A1 cells were examined by CCK-8 and Transwell assay, respectively. Subsequently, the binding relationship between miR-1226 and FGF2 was verified by dual-luciferase reporter assay. The upstream regulatory factor ASCL1 was identified through database prediction, and the co-regulation on NSCLC cell lines was explored via rescue experiments. miR-1226 was down-regulated in NSCLC tissue samples and cell lines, which suggested that miR-1226 was associated with tumor stage and lymph node metastasis. The transfection of miR-1226 mimic inhibited the proliferation and migration in H1299 and SPC-A1 cells. In addition, the overexpression of miR-1226 suppressed tumor growth in vivo. FGF2 was the downstream target binding miR-1226, which was up-regulated in NSCLC tissue samples. Moreover, ASCL1 was found to be a potential regulator of miR-1226. Finally, the tumor suppressive effect of miR-1226 was reversed by its downstream FGF2 and upstream factor ASCL1. miR-1226, which was regulated by ASCL1, inhibited tumor growth and migration by targeting FGF2, indicating that the ASCL1/miR-1226/FGF2 axis could be a potential therapeutic target for NSCLC.

Upregulation of TRIP13 promotes the malignant progression of lung cancer via the EMT pathway.

Lung cancer is the most common malignant tumor type and it is associated with poor prognosis. The identification of potential biomarkers is of great significance for the early diagnosis and treatment of lung cancer. Non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer. The present study aimed to investigate the mechanism via which thyroid hormone receptor-interacting protein 13 (TRIP13) participates in the malignant progression of NSCLC. Immunohistochemistry, reverse transcription-quantitative PCR and western blotting were used to assess the expression level of TRIP13. According to The Cancer Genome Atlas database, TRIP13 was upregulated in NSCLC tissues compared with adjacent normal tissues. Moreover, TRIP13 knockdown increased apoptosis, induced cell cycle arrest in the S phase and inhibited the proliferation, invasion and migration of H1299 cells in vitro. Furthermore, TRIP13 upregulation was closely associated with tumor metastasis via epithelial-mesenchymal transformation. In conclusion, TRIP13 could promote the malignant progression of lung cancer, and TRIP13 may be a potential biomarker for the early diagnosis and treatment of NSCLC.

TPX2 Promotes Metastasis and Serves as a Marker of Poor Prognosis in Non-Small Cell Lung Cancer

BackgroundMetastasis contributes to the high mortality rate of non-small cell lung cancer (NSCLC), and gaining a better understanding of its metastatic mechanisms would aid in initiating effective clinical treatment.Material/MethodIn this study, bioinformatics analyses of the GEO database and TCGA-LUAD were first used to identify the key node gene regulating NSCLC malignant progression. Further in vitro experiments, including wound healing assay, invasion assay, Western blot assay, and luciferase report assay, were used to clarify the functions and mechanism of TPX2 in NSCLC.ResultsResults of the TCGA analysis showed that TPX2 was significantly positively correlated with tumor metastasis and growth and the clinical stage of NSCLC. In addition, high levels of TPX2 significantly indicated a poor survival rate. In vitro experimental results also revealed that the upregulation of TPX2 significantly promoted NSCLC cell migration and invasion and could affect cell replasticity. Further results indicated that TPX2 significantly activated the epithelial-mesenchymal transition process and promoted the expression and activities of matrix metalloproteinase (MMP)2 and MMP9.ConclusionsThis study demonstrated that TPX2 promotes the metastasis and malignant progression of NSCLC and could thus serve as a marker of poor prognosis in NSCLC.

Increased Expression of TAP Is Predictive of Poor Prognosis in Patients with Non-Small Cell Lung Cancer.

BackgroundThe most common cancer among humans is lung cancer. Non-small cell lung cancer (NSCLC) comprises the majority of these cases. In the development and progression of cancers across the spectrum, tumor abnormal protein (TAP) plays crucial roles. Additionally, in the advancement of the bladder and colorectal cancers, the involvement of glycoproteins like TAP is present. However, it is worth noting that current literature has yet to clarify the clinical significance of the TAP in NSCLC.MethodsIn the present study, to evaluate the relative level of TAP, we utilized a TAP detection agent in 154 cases of NSCLC and normal patients who underwent surgical resection anytime from March 2013 to January 2019 at the People’s Hospital of Chizhou.ResultsOur results demonstrated that in NSCLC patients, the expression level of TAP was significantly higher than in normal patients. Moreover, after surgery, TAP expression was significantly downregulated in NSCLC patients. TAP expression is associated with an array of factors, which include the patient’s sex, history of smoking use, tumor size, pTNM, distant cancer, metastasis of lymph nodes, invasive and aggressive indicator pleural invasion, and differentiation degree of NSCLC. Additionally, TAP has no association with the patient’s age, history of drinking, location of the tumor, hypertension, and diabetes. In NSCLC patients, a poor overall survival rate within 5 years is significantly correlated with the increased TAP expression. For NSCLC patients, an independent prognostic factor is the TAP, which is confirmed using the multivariate survival analysis.ConclusionIn the malignant progression of NSCLC, our results demonstrate how the promoting role of the upregulated TAP expression takes place. Hence, a therapeutic aim for NSCLC and a potential biomarker for NSCLC progress is a TAP.

MiR-182-5p and its target HOXA9 in non-small cell lung cancer: a clinical and in-silico exploration with the combination of RT-qPCR, miRNA-seq and miRNA-chip

BackgroundMiR-182-5p, a cancer-related microRNA (miRNA), modulates tumorigenesis and patient outcomes in various human malignances. This study interroted the clinicopathological significance and molecular mechanisms of miR-182-5p in non-small cell lung cancer (NSCLC).MethodsThe clinical significance of miR-182-5p in NSCLC subtypes was determined based on an analysis of 124 samples (lung adenocarcinomas [LUADs], n = 101; lung squamous cell carcinomas [LUSCs], n = 23) obtained from NSCLC patients and paired noncancer tissues and an analysis of data obtained from public miRNA-seq database, miRNA-chip database, and the scientific literature. The NSCLC samples (n = 124) were analyzed using the real-time quantitative polymerase chain reaction (RT-qPCR). Potential targets of miR-182-5p were identified using lists generated by miRWalk v.2.0, a comprehensive atlas of predicted and validated targets of miRNA-target interactions. Molecular events of miR-182-5p in NSCLC were unveiled based on a functional analysis of candidate targets. The association of miR-182-5p with one of the candidate target genes, homeobox A9 (HOXA9), was validated using in-house RT-qPCR and dual-luciferase reporter assays.ResultsThe results of the in-house RT-qPCR assays analysis of data obtained from public miRNA-seq databases, miRNA-chip databases, and the scientific literature all supported upregulation of the expression level of miR-182-5p level in NSCLC. Moreover, the in-house RT-qPCR data supported the influence of upregulated miR-182-5p on malignant progression of NSCLC. In total, 774 prospective targets of miR-182-5p were identified. These targets were mainly clustered in pathways associated with biological processes, such as axonogenesis, axonal development, and Ras protein signal transduction, as well as pathways involved in axonal guidance, melanogenesis, and longevity regulation, in multiple species. Correlation analysis of the in-house RT-qPCR data and dual-luciferase reporter assays confirmed that HOXA9 was a direct target of miR-182-5p in NSCLC.ConclusionsThe miR-182-5p expression level was upregulated in NSCLC tissues. MiR-182-5p may exert oncogenic influence on NSCLC through regulating target genes such as HOXA9.

ΜicroRNA‑889 plays a suppressive role in cell proliferation and invasion by directly targeting TAB1 in non‑small cell lung cancer.

MicroRNAs (miRNAs) are frequently reported to be aberrantly expressed in non‑small cell lung cancer (NSCLC) and are closely associated with aggressive tumor phenotypes. Hence, identification of cancer‑related miRNAs in NSCLC may be helpful for improving the cure rate of NSCLC treatments. miR‑889 has been demonstrated to be a novel cancer‑associated miRNA that is aberrantly expressed and plays an important role in esophageal squamous cell carcinoma and hepatocellular carcinoma. However, the exact functions and precise molecular mechanisms through which miR‑889 affects NSCLC progression are still unknown. In the present study, we report for the first time that miR‑889 expression is low in NSCLC tissues and cell lines. Clinically, low miR‑889 expression was found to be correlated with the TNM stage and distant metastasis in NSCLC patients. Functionally, miR‑889 overexpression suppressed the proliferation and invasiveness of NSCLC cells invitro and decreased NSCLC xenograft tumor growth in mice. Furthermore, TGF‑β‑activated kinase 1‑binding protein 1 (TAB1) was confirmed as a direct target gene of miR‑889 in NSCLC cells. TAB1 was revealed to be overexpressed in NSCLC tissue samples and was inversely correlated with miR‑889 levels. Moreover, a TAB1 knockdown had effects similar to that of miR‑889 overexpression, whereas restoration of TAB1 expression counteracted the actions of miR‑889 in NSCLC cells. Overall, the present results indicated that miR‑889 inhibits the aggressive behaviors of NSCLC by directly targeting TAB1 mRNA, thus highlighting the importance of the miR‑889/TAB1 pathway in the malignant progression of NSCLC.

The serum level of CC chemokine ligand 18 correlates with the prognosis of non-small cell lung cancer.

CC chemokine ligand 18 (CCL18) is a chemotactic cytokine involved in the pathogenesis and progression of various cancers. Our previous research showed that the expression of CCL18 is obviously higher in non-small cell lung cancer (NSCLC) than in the adjacent normal tissues, suggesting its role in NSCLC. We further examined the serum level of CCL18 in 80 NSCLC patients with enzyme-linked immunosorbent assay and simultaneously analyzed the survival curve of these patients by the Kaplan-Meier method, and then utilized a log-rank test to evaluate the correlation of CCL18 expression with the malignant progression of NSCLC. Our results showed that the median serum concentration of CCL18 was significantly elevated to 436.11 ng/mL in NSCLC patients compared to 41.97 ng/ml in healthy people (P<0.01), which was also positively related to the expression of lung cancer biomarkers carcinoma-embryonic antigen and cytokeratin fragment antigen 21-1. Moreover, correlation analysis showed that an increased level of serum CCL18 was associated with a worse survival time in NSCLC patients. Our findings suggest that the serum CCL18 level of NSCLC patients was negatively correlated with the prognosis, thus suggesting that CCL18 may serve as a potential circulating biomarker for NSCLC diagnosis.

Nrf2 mediates redox adaptation in NOX4-overexpressed non-small cell lung cancer cells

The redox adaptation mechanisms in cancer cells are very complex and remain largely unclear. Our previous studies have confirmed that NADPH oxidase 4 (NOX4) is abundantly expressed in non-small cell lung cancer (NSCLC) and confers apoptosis resistance on NSCLC cells. However, the comprehensive mechanisms for NOX4-mediated oxidative resistance of cancer cells remain still undentified. The present study found that NOX4-derived H2O2 enhanced the nuclear factor erythroid 2-related factor 2 (Nrf2) stability via disruption of redox-dependent proteasomal degradation and stimulated its activity through activation of PI3K signaling. Specifically, the results showed that ectopic NOX4 expression did not induce apoptosis of A549 cells; however, inhibition of Nrf2 resulted in obvious apoptotic death of NOX4-overexpressed A549 cells, accompanied by a significant increase in H2O2 level and decrease in GSH content. Besides, inhibition of Nrf2 could suppress cell growth and efficiently reverse the enhancement effect of NOX4 on cell growth. The in vivo data confirmed that inhibition of Nrf2 could interfere apoptosis resistance in NOX4-overexpressed A549 tumors and led to cell growth inhibition. In conclusion, these results reveal that Nrf2 is critically involved in redox adaptation regulation in NOX4-overexpressed NSCLC cells. Therefore, NOX4 and Nrf2 may be promising combination targets against malignant progression of NSCLC.

Increased Expression of Long Non-Coding RNA BCAR4 Is Predictive of Poor Prognosis in Patients with Non-Small Cell Lung Cancer.

Lung cancer is the most common human cancer, and the majority of lung cancer cases are categorized as non-small cell lung cancer (NSCLC). Long non-coding RNAs (lncRNAs) play key roles in the development and progression of human cancers. LncRNA breast cancer anti-estrogen resistance 4 (BCAR4) has been identified as an oncogenic lncRNA involved in the progression of breast cancer and osteosarcoma. However, the clinical significance of the lncRNA BCAR4 in NSCLC remains largely unclear. In the present study, real-time quantitative reverse transcriptase-polymerase chain reaction was used to examine the relative level of lncRNA BCAR4 in 68 cases of NSCLC tissues and their adjacent non-tumor tissues. Our data showed that the expression level of lncRNA BCAR4 was significantly higher in NSCLC tissues compared to their matched non-tumor tissues. Moreover, BCAR4 expression was significantly upregulated in NSCLC cell lines, when compared to the normal human bronchial epithelial cell line BEAS-2B. In addition, the BCAR4 expression was associated with the lymph node metastasis, distant metastasis and clinical stage, but not with the age, sex, tumor size, histological grade, and histological type. The increased expression of BCAR4 was significantly associated with poorer 5-year overall survival rate of NSCLC patients. Multivariate survival analysis indicated that BCAR4 was an independent prognostic factor for NSCLC patients. Taken together, our study suggests that the upregulation of lncRNA BCAR4 expression plays a promoting role in the malignant progression of NSCLC. Thus, BCAR4 is a potential biomarker for NSCLC progress and a therapeutic target for NSCLC.

MicroRNA-29a plays a suppressive role in non-small cell lung cancer cells via targeting LASP1.

MicroRNA (miR)-29a has been implicated in non-small cell lung cancer (NSCLC), but the mechanism remains largely unclear. LASP1, a cAMP- and cGMP-dependent signaling protein, was recently found to promote proliferation and aggressiveness in NSCLC. However, the regulatory mechanism of LASP1 expression in NSCLC, as well as the relationship between LASP1 and miR-29a, has never been previously studied. In this study, we found that miR-29a was remarkably downregulated and low expression of miR-29a was associated with the malignant progression of NSCLC. Moreover, the expression of LASP1 was markedly increased in NSCLC tissues and cell lines. Bioinformatics analysis and luciferase reporter assay data further identified LASP1 as a target gene of miR-29a, and the expression of LASP1 was negatively mediated by miR-29a at the post-transcriptional level in NSCLC cells. Overexpression of miR-29a reduced the proliferation, migration, and invasion of NSCLC cells, just as the effects of LASP1 knockdown. Moreover, overexpression of LASP1 attenuated the suppressive effect of miR-29a on the malignant phenotypes of NSCLC cells. In addition, upregulation of miR-29a decreased the growth of A549 cells in nude mice and protected the animals from tumor-induced death. Therefore, we demonstrate that miR-29a plays a suppressive role in NSCLC via targeting LASP1, suggesting that the miR-29a/LASP1 axis may become a promising therapeutic target for NSCLC.

Promoter Methylation of the &amp;lt;i&amp;gt;CADM&amp;lt;/i&amp;gt;1 and 4.1&amp;lt;i&amp;gt;B&amp;lt;/i&amp;gt; Genes Occurs Independently of the &amp;lt;i&amp;gt;EGFR&amp;lt;/i&amp;gt; or the &amp;lt;i&amp;gt;KRAS&amp;lt;/i&amp;gt;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 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 CADM1 and 4.1B are independent of the mutation status of the EGFR or KRAS but play roles in the malignant progression of NSCLC. Integration of epigenetic information would be useful for identifying possible modifiers to predict the response or recurrence of lung adenocarcinoma to the EGFR-TKI therapy.