Histologic transformation of non-small-cell lung cancer in response to tyrosine kinase inhibitors: Current knowledge of genetic changes and molecular mechanisms.

Lung cancer is the leading cause of cancer death and includes two major types: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), accounting for 85% and 15% of cases, respectively. Non-small-cell lung cancer harboring actionable driver mutations is generally treated with tyrosine kinase inhibitors (TKIs) molecularly targeting individual oncogenes. Although TKIs have greatly contributed to better clinical outcomes, acquired resistance to them inevitably occurs. Histologic or lineage transformation is a rare but well-documented off-target mechanism associated with acquired resistance, and has been identified in settings following treatment with multiple different TKIs and other drugs. It includes neuroendocrine transformation, squamous cell transformation, and epithelial-to-mesenchymal transition. Here, we review the clinicopathologic features of transformed tumors and current understanding of the key genetic alterations and biologic mechanism of lineage transformation in NSCLC, particularly TKI-triggered transformation.

Novel therapeutic strategies targeting bypass pathways and mitochondrial dysfunction to combat resistance to RET inhibitors in NSCLC.

RET fusion is an oncogenic driver in 1-2 % of patients with non-small cell lung cancer (NSCLC). Although RET-positive tumors have been treated with multikinase inhibitors such as vandetanib or RET-selective inhibitors, ultimately resistance to them develops. Here we established vandetanib resistance (VR) clones from LC-2/ad cells harboring CCDC6-RET fusion and explored the molecular mechanism of the resistance. Each VR clone had a distinct phenotype, implying they had acquired resistance via different mechanisms. Consistently, whole exome-seq and RNA-seq revealed that the VR clones had unique mutational signatures and expression profiles, and shared only a few common remarkable events. AXL and IGF-1R were activated as bypass pathway in different VR clones, and sensitive to a combination of RET and AXL inhibitors or IGF-1R inhibitors, respectively. SMARCA4 loss was also found in a particular VR clone and 55 % of post-TKI lung tumor tissues, being correlated with higher sensitivity to SMARCA4/SMARCA2 dual inhibition and shorter PFS after subsequent treatments. Finally, we detected an increased number of damaged mitochondria in one VR clone, which conferred sensitivity to mitochondrial electron transfer chain inhibitors. Increased mitochondria were also observed in post-TKI biopsy specimens in 13/20 cases of NSCLC, suggesting a potential strategy targeting mitochondria to treat resistant tumors. Our data propose new promising therapeutic options to combat resistance to RET inhibitors in NSCLC.

Expression patterns of HNF4α, TTF-1, and SMARCA4 in lung adenocarcinomas: impacts on clinicopathological and genetic features.

INTRODUCTION: HNF4α expression and SMARCA4 loss were thought to be features of non-terminal respiratory unit (TRU)-type lung adenocarcinomas, but their relationships remained unclear. MATERIALS AND METHODS: HNF4α-positive cases among 241 lung adenocarcinomas were stratified based on TTF-1 and SMARCA4 expressions, histological subtypes, and driver mutations. Immunohistochemical analysis was performed using xenograft tumors of lung adenocarcinoma cell lines with high HNF4A expression. RESULT: HNF4α-positive adenocarcinomas(n = 33) were divided into two groups: the variant group(15 mucinous, 2 enteric, and 1 colloid), where SMARCA4 was retained in all cases, and the conventional non-mucinous group(6 papillary, 5 solid, and 4 acinar), where SMARCA4 was lost in 3/15 cases(20%). All variant cases were negative for TTF-1 and showed wild-type EGFR and frequent KRAS mutations(10/18, 56%). The non-mucinous group was further divided into two groups: TRU-type(n = 7), which was positive for TTF-1 and showed predominantly papillary histology(6/7, 86%) and EGFR mutations(3/7, 43%), and non-TRU-type(n = 8), which was negative for TTF-1, showed frequent loss of SMARCA4(2/8, 25%) and predominantly solid histology(4/8, 50%), and never harbored EGFR mutations. Survival analysis of 230 cases based on histological grading and HNF4α expression revealed that HNF4α-positive poorly differentiated (grade 3) adenocarcinoma showed the worst prognosis. Among 39 cell lines, A549 showed the highest level of HNF4A, immunohistochemically HNF4α expression positive and SMARCA4 lost, and exhibited non-mucinous, high-grade morphology in xenograft tumors. CONCLUSION: HNF4α-positive non-mucinous adenocarcinomas included TRU-type and non-TRU-type cases; the latter tended to exhibit the high-grade phenotype with frequent loss of SMARCA4, and A549 was a representative cell line.

Mutational landscape of primary breast angiosarcoma with repeated resection and recurrence over a 15-year period: A case report.

Angiosarcoma is a rare malignant tumor derived from vascular endothelial cells and has a poor prognosis. We have experienced a case of multiple breast angiosarcoma for which multiple resections had been performed during the course of its progression over a period of more than 15 years, allowing comprehensive genetic mutation analysis. Somatic mutations in several cancer-related genes were detected, but no previously reported driver gene mutations of angiosarcoma were evident. Several germline mutations associated with malignancy, such as single nucleotide polymorphisms in Fibroblast Growth Factor Receptor 4 (FGFR4) (p.Gly388Arg, rs351855), Kinase Insert Domain Receptor (KDR) (Gln472His, rs1870377) and tumor protein p53 (TP53) (p.Pro72Arg, rs1042522) were detected. Common signatures and genetic mutations were scarce in the tumor samples subjected to genetic mutational analysis. These findings suggested that this case was very probably a multiprimary angiosarcoma.

Relevance of A Disintegrin and Metalloproteinase Domain-Containing (ADAM)9 Protein Expression to Bladder Cancer Malignancy.

We evaluated the effect of A Disintegrin and Metalloproteinase Domain-Containing (ADAM)9 protein on exacerbation in bladder cancer KK47 and T24. First, we knocked down ADAM9 and investigated cell proliferation, migration, cell cycle, and the epithelial-mesenchymal transition (EMT)-related proteins expression in vitro. We then investigated the expression level of ADAM9 in clinical urine cytology samples and the Cancer Genome Atlas (TCGA) data. Cell proliferation was significantly reduced in both cell lines after ADAM9 knockdown. In the cell-cycle assay, the percentage of G0/G1 cells was significantly increased in ADAM9 knockdown T24. Migration of T24 was more strongly suppressed than KK47. The expression level of EMT-related proteins suggested that EMT was suppressed in ADAM9 knockdown T24. TCGA analysis revealed that ADAM9 mRNA expression was significantly higher in stage IV and high-grade cancer than in other stages and low-grade cancer. Moreover, in the gene expression omnibus (GEO) study, bladder cancer with surrounding carcinoma and invasive carcinoma showed significantly high ADAM9 mRNA expression. We found that ADAM9 knockdown suppressed cell proliferation and migration in bladder cancer and that high-grade bladder cancer is correlated with higher expression of ADAM9.

Analysis of lorlatinib analogs reveals a roadmap for targeting diverse compound resistance mutations in ALK-positive lung cancer.

Lorlatinib is currently the most advanced, potent and selective anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor for the treatment of ALK-positive non-small cell lung cancer in the clinic; however, diverse compound ALK mutations driving therapy resistance emerge. Here, we determine the spectrum of lorlatinib-resistant compound ALK mutations in patients, following treatment with lorlatinib, the majority of which involve ALK G1202R or I1171N/S/T. We further identify structurally diverse lorlatinib analogs that harbor differential selective profiles against G1202R versus I1171N/S/T compound ALK mutations. Structural analysis revealed increased potency against compound mutations through improved inhibition of either G1202R or I1171N/S/T mutant kinases. Overall, we propose a classification of heterogenous ALK compound mutations enabling the development of distinct therapeutic strategies for precision targeting following sequential tyrosine kinase inhibitors.

Ovarian carcinoma immunoreactive antigen domain 2 controls mitochondrial apoptosis in lung adenocarcinoma.

Ovarian carcinoma immunoreactive antigen domain 2 (OCIAD2) has been reported to show significantly higher expression in invasive lung adenocarcinoma than in lung adenocarcinoma in situ, and its abnormal expression is associated with poorer prognosis of the patients. However, the cellular function of OCIAD2 in this tumor remains poorly understood. In the present study, we first validated that OCIAD2 showed higher expression in human lung adenocarcinoma tissues or cell lines than in normal lung tissue or immortalized normal bronchial epithelial cells. OCIAD2 was localized predominantly at the mitochondrial membrane in lung adenocarcinoma cells. Interestingly, suppression of OCIAD2 led to loss of mitochondrial structure and a reduction in the number of mitochondria. Moreover, OCIAD2 suppression led to downregulation of cellular growth, proliferation, migration, and invasion, and upregulation of mitochondria-related apoptosis. We also showed that OCIAD2 suppression induced a decrease in mitochondrial membrane potential and release of cytochrome c. Transcriptional profiling using RNA sequencing revealed a total of 137 genes whose expression was commonly altered after OCIAD2 knockdown in three lung adenocarcinoma cell lines (A549, HCC827, and PC9). Pathway enrichment analysis of those genes demonstrated significant enrichment in apoptotic signaling or endoplasmic reticulum (ER) stress pathways. Our data suggest that OCIAD2 inhibits the mitochondria-initiated apoptosis and thus promotes the survival of lung cancer cells. Therefore, OCIAD2 may be an effective target for treatment of lung adenocarcinoma.

Significance of stratifin in early progression of lung adenocarcinoma and its potential therapeutic relevance.

Lung cancer is the most common cause of global cancer-related mortality, and the main histological type is adenocarcinoma, accounting for 50% of non-small cell lung cancer. In 2015, the World Health Organization (WHO) histological classification defined the concepts of "adenocarcinoma in situ" (AIS) and "minimally invasive adenocarcinoma" (MIA), which are considered to be adenocarcinomas at a very early stage. Although AIS and MIA have a very favorable outcome, once they progress to early but invasive adenocarcinoma (eIA), they can sometimes have a fatal outcome. We previously compared the expression profiles of eIA and AIS, and identified stratifin (SFN; 14-3-3 sigma) as a protein showing significantly higher expression in eIA than in AIS. Expression of SFN is controlled epigenetically by DNA demethylation, and its overexpression is significantly correlated with poorer outcome. In vitro and in vivo analyses have shown that SFN facilitates early progression of adenocarcinoma by enhancing cell proliferation. This review summarizes genetic and epigenetic abnormalities that can occur in early-stage lung adenocarcinoma and introduces recent findings regarding the biological significance of SFN overexpression during the course of lung adenocarcinoma progression. Therapeutic strategies for targeting SFN are also discussed.

Nanaomycin K inhibited epithelial mesenchymal transition and tumor growth in bladder cancer cells in vitro and in vivo.

Nanaomycin K, derived from Streptomyces rosa subsp. notoensis OS-3966T, has been discovered to have inhibitory bioactivity on epithelial-mesenchymal transition (EMT), an important mechanism of cancer cell invasion and migration. In this study, we examined the anti-EMT and anti-tumor effect of nanaomycin K in bladder cancer, where EMT has important roles in progression. We treated two bladder cancer lines, non-muscle-invasive KK47 and muscle-invasive T24, with nanaomycin K to determine the effects on cell proliferation, apoptosis and expression of EMT markers in vitro. Wound-healing assays were performed to assess cell invasion and migration. We conducted an in vivo xenograft study in which mice were inoculated with bladder cancer cells and treated with intratumoral administration of nanaomycin K to investigate its anti-tumor and EMT inhibition effects. As the results, nanaomycin K (50 µg/mL) significantly inhibited cell proliferation in KK47 (p < 0.01) and T24 (p < 0.01) in the presence of TGF-ß, which is an EMT-inducer. Nanaomycin K (50 µg/mL) also significantly inhibited cell migration in KK47 (p < 0.01) and T24 (p < 0.01), and induced apoptosis in both cell lines in the presence of TGF-ß (p < 0.01). Nanaomycin K increased the expression of E-cadherin and inhibited the expression of N-cadherin and vimentin in both cell lines. Nanaomycin K also decreased expression of Snail, Slug, phospho-p38 and phospho-SAPK/JNK especially in T24. Intratumoral administration of nanaomycin K significantly inhibited tumor growth in both KK47 and T24 cells at high dose (1.0 mg/body) (p = 0.009 and p = 0.003, respectively) with no obvious adverse events. In addition, nanaomycin K reversed EMT and significantly inhibited the expression of Ki-67 especially in T24. In conclusion, we demonstrated that nanaomycin K had significant anti-EMT and anti-tumor effects in bladder cancer cells, suggesting that nanaomycin K may be a therapeutic candidate for bladder cancer treatment.

Differential effects of chromosome and plasmid blaCTX-M-15 genes on antibiotic susceptibilities in extended-spectrum beta-lactamase-producing Escherichia coli isolates from patients with urinary tract infection.

OBJECTIVES: To compare antibiotic susceptibilities between chromosomal and plasmid blaCTX-M-15 locations in urinary tract infection-causing extended-spectrum ß-lactamases-producing Escherichia coli blaCTX-M-15 isolated in Indonesia. METHODS: A total of 84 strains identified as extended-spectrum ß-lactamases-producing E. coli were isolated from patients with urinary tract infection in Indonesia in 2015. Antimicrobial susceptibility tests were performed on these strains using 18 antibiotics, and extended-spectrum ß-lactamase bla genes were detected by polymerase chain reaction. Gene localization of blaCTX-M-15 -positive strains was confirmed by Southern blot hybridization, and epidemiological typing was conducted using multilocus sequence typing. RESULTS: Of 54 strains harboring the blaCTX-M-15 gene, 27 showed localization on chromosome, 20 on plasmid, and seven on chromosome and plasmid. Most multilocus sequence typing sequence types of the 27 strains with chromosomal blaCTX-M-15 were ST405 (25.9%) and ST131 (22.2%) strains, whereas the 20 strains with plasmid-blaCTX-M-15 were mostly ST410 (55.0%). CONCLUSIONS: Extended-spectrum ß-lactamases-producing E. coli blaCTX-M-15 with plasmid genes show significantly higher resistant rates against piperacillin-tazobactam but lower resistant rates against chloramphenicol compared to chromosomal strains in Indonesian patients with urinary tract infection. Mechanistic investigations will be necessary to advance our knowledge of antimicrobial resistance in urinary tract infection.

Cross-Resistance and the Mechanisms of Cephalosporin-Resistant Bacteria in Urinary Tract Infections Isolated in Indonesia.

Urinary tract infection (UTI) by antibiotic-resistant strains has become increasingly problematic, with trends that differ from country to country. This study examined cross-resistance and the mechanisms of cephalosporin resistance in UTI-causative bacteria isolated in Indonesia. Antibiotic susceptibility tests based on Clinical Laboratory Standards Institute (CLSI) standards were done for UTI-causative strains (n = 50) isolated from patients in Indonesia in 2015-2016 and showed resistance against the third-generation cephalosporin. Mechanistic studies were carried out to confirm the presence of extended-spectrum ß-lactamase (ESBL) genes, carbapenemase-related genes, the fosA3 gene related to fosfomycin resistance, and mutations of quinolone-resistance-related genes. Isolated UTI-causative bacteria included Escherichia coli (64.0%), Pseudomonas aeruginosa (16.0%), Klebsiella pneumoniae (10.0%), and others (10.0%). These strains showed 96.0% susceptibility to amikacin, 76.0% to fosfomycin, 90.0% to imipenem, 28.0% to levofloxacin, 92.0% to meropenem, and 74.0% to tazobactam/piperacillin. ESBL was produced by 68.0% of these strains. Mechanistic studies found no strains with carbapenemase genes but 6.0% of strains had the fosA3 gene. Seventy-two % of the strains had mutations in the gyrA gene and 74.0% in the parC gene. Most E. coli strains (87.5%) had Ser-83 → Leu and Asp-87 → Asn in gyrA and 93.8% of E. coli had Ser-80 → Ile in parC. There were significant correlations among mutations in gyrA and parC, and fosA3 gene detection (P < 0.05), respectively. To our knowledge, this is the first mechanistic study of antibiotic-cross-resistant UTI-causative bacteria in Indonesia. Further studies with a longer period of observation are necessary, especially for changes in carbapenem resistance without carbapenemase-related genes.

Gene expression profiles of the original tumors influence the generation of PDX models of lung squamous cell carcinoma.

Patient-derived xenograft (PDX) murine models are employed for preclinical research on cancers, including non-small cell lung cancers (NSCLCs). Even though lung squamous cell carcinomas (LUSCs) show the highest engraftment rate among NSCLCs, half of them nevertheless show PDX failure in immunodeficient mice. Here, using immunohistochemistry and RNA sequencing, we evaluated the distinct immunohistochemical and gene expression profiles of resected LUSCs that showed successful engraftment. Among various LUSCs, including the basal, classical, secretory, and primitive subtypes, those in the non-engrafting (NEG) group showed gene expression profiles similar to the pure secretory subtype with positivity for CK7, whereas those in the engrafting (EG) group were similar to the mixed secretory subtype with positivity for p63. Pathway analysis of 295 genes that demonstrated significant differences in expression between NEG and EG tumors revealed that the former had enriched expression of genes related to the immune system, whereas the latter had enriched expression of genes related to the cell cycle and DNA replication. Interestingly, NEG tumors showed higher infiltration of B cells (CD19+) and follicular dendritic cells (CD23+) in lymph follicles than EG tumors. Taken together, these findings suggest that the PDX cancer model of LUSC represents only a certain population of LUSCs and that CD19- and CD23-positive tumor-infiltrating immune cells in the original tumors may negatively influence PDX engraftment in immunodeficient mice.

Genetic analysis of ESBL-producing Klebsiella pneumoniae isolated from UTI patients in Indonesia.

INTRODUCTION: Extended spectrum beta-lactamase (ESBL)-producing Klebsiellapneumoniae is a serious concern for nosocomial infection and the emergence rate in Indonesia is higher than that in developed countries. The purpose of this study was to investigate the genetic characteristics of ESBL-producing K. pneumoniae isolated from UTI patients in Indonesia. MATERIALS AND METHODS: We collected K. pneumoniae resistant to ceftazidime or cefotaxime isolated from UTI patients in Dr. Soetomo's Academic Hospital in Surabaya, Indonesia in 2015. Ninety-four strains were identified as ESBL-producing bacteria by confirmation tests. The isolates were investigated by antimicrobial susceptibility testing with 20 drugs and ESBL gene detection, plasmid replicon typing and virulence genes as hypermucoviscous (HMV) strains were tested by the string test. RESULTS: High rates of resistance to ciprofloxacin (86.2%), tetracycline (80.9%) and nalidixic acid (78.7%) were observed. CTX-M-15 was the most common ESBL gene (89.4%), 33 of which also carried SHV-type ESBL. IncF was the most prevalent plasmid replicon typing (47.6%). Sixteen (17.0%) strains were judged as HMV, all of which had rmpA and more than half of which had fimH, uge, and wab. IncL/M was the most common replicon plasmid in the HMV strains, and the difference in the positive rate was statistically significant (p = 0.0024). CONCLUSION: This study showed the high prevalence of multiple-drug resistant and predominately CTX-M-15-positive ESBL-producing K. pneumoniae in Indonesia. There was a correlation between IncL/M and the HMV phenotype in this study. As such hypervirulent strains continue to emerge, studying their dissemination with resistance determinants is an urgent priority.

Anti-tumor Effect of Hedgehog Signaling Inhibitor, Vismodegib, on Castration-resistant Prostate Cancer.

BACKGROUND/AIM: Epithelial-mesenchymal transition (EMT) via Sonic Hedgehog (Shh) signaling may be one of the mechanisms of progression of castration-resistant prostate cancer (CRPC). In this study, we investigated the possible therapeutic effect of vismodegib, a new Shh inhibitor, in a mouse CRPC model. MATERIALS AND METHODS: We determined cell proliferation, apoptosis and the expression of EMT-related genes for three prostate cancer cell lines; androgen-dependent LNCaP and independent C4-2B and PC-3 in the presence of vismodegib in vitro. Fifty mg/kg of vismodegib were orally administered into mice bearing C4-2B and PC-3 tumors, respectively every other week for 3 weeks. RESULTS: Vismodegib significantly inhibited cell proliferation and induced cell apoptosis in all cell lines in vitro (p<0.05). Vismodegib significantly inhibited EMT in CRPC cells and tumor growth in C4-2B-bearing mice compared to controls in vivo (p<0.05). Higher expression of caspase-3 and lower expression of vimentin in PC-3 and C4-2B tumors were induced by vismodegib in immunohistochemical analysis. CONCLUSION: Vismodegib inhibited cell proliferation via apoptosis and also suppressed EMT, showing anti-tumor effects in mice. Further mechanistic studies are needed to investigate the feasibility of vismodegib for CRPC treatment.

Increased tumor-associated macrophages in the prostate cancer microenvironment predicted patients' survival and responses to androgen deprivation therapies in Indonesian patients cohort.

BACKGROUND: Tumor-associated macrophages (TAMs) and microvessel density (MVD) play an essential role for tumor progression in prostate cancer (PCa). In this study, we evaluated the association between TAMs, the infiltration with tumor angiogenesis and the response to androgen deprivation therapies (ADTs) in PCa to evaluate TAM infiltration as a predictive factor for PCa survival. MATERIALS AND METHODS: Fifty-four specimens were collected and stained with CD 68 antibody to investigated TAM infiltration in tumor. Von Willebrand factor was stained to evaluate MVD around the cancer foci. We assessed the association between patient's age, preoperative serum prostate-specific antigen, pathologic Gleason sum (GS), TAM infiltration, MVD, and the response to ADT for 5 years after PCa diagnosis. RESULTS: The median TAM was observed in 28 (6-76)/high power field (x400). Increasing TAM correlated with increasing tumor angiogenesis (P < 0.001, r = 0.61), and the response to ADT was significantly better in patients with fewer TAMs (<28) than in patients with higher TAMs (>28) (P = 0.003). TAM infiltration was significantly higher in those with higher serum prostate-specific antigen, higher GS, and metastasis. Multivariate analysis showed that GS, ADT type, and MVD number were significant prognostic factors for response to ADT in PCa (P < 0.0001). An increased infiltration of TAM [hazards ratio (HR) = 4.47; 95% confidence interval (CI): 1.97-10.15], MVD (HR = 2.66; 95% CI: 1.27-5.61), metastatic status (HR = 2.29; 95% CI: 0.14-0.60), and prostate volume (HR = 2.19; 95% CI: 1.27-3.12) significantly correlated with shorter survival in PCa patients by univariate analysis (P < 0.05). Multivariate analyses revealed that TAM and metastatic status significantly correlated with poor overall survival. CONCLUSIONS: TAM infiltration is associated with response to ADT and increased tumor angiogenesis in PCa. GS, ADT type, and MVD in PCa specimens are useful predictive factors for poor response to ADT. Increasing TAM and positive metastatic status were prognostic factors for a poorer survival in PCa patients.

Can ultrasound irradiation be a therapeutic option for prostate cancer?

BACKGROUND: Focal therapies for prostate cancer (PC) can reduce adverse events and do not lead to androgen-independent progression. Ultrasound could be used for cancer treatments if the repetition frequency is fitted to the purpose. We investigated the possible therapeutic effect of ultrasound irradiation on PC cells. MATERIALS AND METHODS: We irradiated two PC cell lines, androgen-dependent LNCaP and -independent PC-3 with ultrasound (3.0 W/cm2 , 3 MHz, irradiation time rate: 20%) for 2 minutes for 1 day or 3 consecutive days at a repetition frequency of 1, 10, or 100 Hz in vitro. Cell proliferation and apoptosis were determined after irradiation. RESULTS: Cell proliferation of PC-3 was significantly inhibited after 1 day (P < .0001) and 3 days (P < .0001) of 10 Hz ultrasound irradiation, and that of LNCaP after 1 day (P < .0001) and 3 days (P < .0001) of irradiation. LNCaP was more sensitive to ultrasound at both lower and higher cell density but PC-3 was only sensitive at a lower cell density (P < .01). Irradiation with 10 Hz ultrasound-induced significantly more PC-3 apoptotic cells than control (1 day, P = .0137; 3 days, P = .0386) rather than irradiation with 1 Hz. Apoptosis via caspase-3 was induced at 10 Hz in 1-day (P < .05) irradiation in both cell lines. CONCLUSIONS: Ultrasound irradiation with even 1 day of 10 Hz significantly inhibited cell proliferation in both LNCaP and PC-3, especially by the remarkable induction of apoptosis in vitro. Our study indicated that ultrasound irradiation can be a therapeutic option for PC and further studies in vivo will be undertaken.

Carcinogen-induced tumors in SFN-transgenic mice harbor a characteristic mutation spectrum of human lung adenocarcinoma.

The landscape of genetic alterations in disease models such as transgenic mice or mice with carcinogen-induced tumors has provided a huge amount of information that has shed light on the process of tumorigenesis in human non-small-cell lung cancer (NSCLC). We have previously identified stratifin (SFN) as a potent oncogene, and generated SFN-transgenic (Tg-SPC-SFN+/- ) mice, which express human SFN (hSFN) only in the lung. Here, we have found that carcinogen nicotine-derived nitrosaminoketone (NNK)-induced tumors developing in Tg-SPC-SFN+/- mice show a similar histology to human lung adenocarcinoma and exhibit high hSFN expression. In order to compare the genetic characteristics of Tg-SPC-SFN+/- tumors and human lung adenocarcinoma, the former were subjected to whole-exome sequencing. Interestingly, Tg-SPC-SFN+/- tumors showed the distinct distribution of exonic mutations and high number of mutated genes and transversion. Moreover, Tg-SPC-SFN+/- tumors showed 73 genes that were commonly detected in more than 2 tumors, mutations of which were also found in human lung adenocarcinoma. The expression levels of some of these genes were significantly associated with the clinical outcome of lung adenocarcinoma patients. Additionally, mutated genes in Tg-SPC-SFN+/- tumors were closely associated with key canonical pathways such as PI3K/AKT signaling and apoptosis signaling. These results suggest that SFN overexpression is a universal abnormality in human lung adenocarcinogenesis and Tg-SPC-SFN+/- tumors recapitulate key features of major human lung adenocarcinoma. Therefore, Tg-SPC-SFN+/- mice provide a useful model for clarifying the molecular mechanism underlying lung adenocarcinogenesis.

DNA hypomethylation-related overexpression of SFN, GORASP2 and ZYG11A is a novel prognostic biomarker for early stage lung adenocarcinoma.

Although alteration of DNA methylation in advanced cancer has been extensively investigated, few data for early-stage lung adenocarcinoma are available. Here, we compared DNA methylation profiles between adenocarcinoma in situ (AIS) and early invasive adenocarcinoma using the Infinium methylation array to investigate methylation abnormalities causing early progression of adenocarcinomas. We focused on differentially methylated sites which were located in promoter CpG islands or shore regions, and identified 579 hypermethylated sites and 23 hypomethylated sites in early invasive adenocarcinoma relative to AIS and normal lung. These hypermethylated genes were significantly associated with neuronal pathways such as the GABA receptor and serotonin signaling pathways. Among the hypomethylated genes, we found that GORASP2, ZYG11A, and SFN had significantly lower methylation rates at the shore regions and significantly higher protein expression in invasive adenocarcinoma. Moreover, overexpression of those proteins was strongly associated with patient's poor outcome. Despite DNA demethylation at the promoter region might be rare relative to DNA hypermethylation, we identified 2 new genes, GORASP2 and ZYG11A, which show hypomethylation and overexpression in invasive adenocarcinoma, suggesting that they have important functions in tumor cells. These genes may be clinically applicable as prognostic indicators and could be potential novel target molecules for drug development.