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.

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.

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.

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.

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.

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.

Stratifin Inhibits SCFFBW7 Formation and Blocks Ubiquitination of Oncoproteins during the Course of Lung Adenocarcinogenesis.

PURPOSE: Aberrant overexpression of SFN (stratifin) plays an oncogenic role in lung adenocarcinoma. We have shown previously that SKP1, an adapter component of E3 ubiquitin ligase forming an SCF complex, is a unique SFN-binding protein in lung adenocarcinoma cells. EXPERIMENTAL DESIGN: In silico simulation and in vitro mutagenesis analysis were performed to identify the SFN-binding domain on SKP1. We examined expression, localization, and stability of SKP1 after knockdown of SFN using lung adenocarcinoma cells including A549. In silico library screening and experimental validation were used for drug screening. Daily oral administration of each candidate drugs to A549-injected tumor-bearing mice was performed to evaluate their in vivo antitumor efficacy. RESULTS: Suppression of SFN upregulated the stability of SKP1 and accelerated its cytoplasm-to-nucleus translocation. Consistently, IHC analysis revealed that cytoplasmic expression of SKP1 was significantly associated with SFN positivity, tumor malignancy, and poorer patient outcome. After SFN suppression, ubiquitination of oncoproteins, including p-cyclin E1, p-c-Myc, p-c-Jun, and cleaved Notch 1, which are target proteins of SCFFBW7, was strongly induced. These results indicate that SFN-SKP1 binding results in SCFFBW7 dysfunction and allows several oncoproteins to evade ubiquitination and subsequent degradation. Because inhibition of SFN-SKP1 binding was expected to have antitumor efficacy, we next searched for candidate SFN inhibitors. Aprepitant and ticagrelor were finally selected as potential SFN inhibitors that dose dependently reduced SFN-SKP1 binding and tumor progression in vivo. CONCLUSIONS: As overexpression of SFN is detectable in most adenocarcinoma, we believe that SFN inhibitors would be novel and promising antitumor drugs for lung adenocarcinoma.

High expression of ovarian cancer immunoreactive antigen domain containing 2 (OCIAD2) is associated with poor prognosis in lung adenocarcinoma.

The clinicopathological implications of ovarian cancer immunoreactive antigen domain containing 2 (OCIAD2) in lung adenocarcinoma were investigated. The expression of OCIAD2 in 191 surgically resected lung adenocarcinomas was examined using immunohistochemistry. OCIAD2 expression was quantified using the H-score and dichotomized as high or low. High OCIAD2 protein expression was significantly correlated with vascular invasion (P = 0.0018), lymphatic permeation (P = 0.049), T factor (P = 0.0024), and pathological stage (P = 0.0003). High OCIAD2 expression was significantly associated with poorer overall survival (OS) (n = 191, P = 0.0325). In peripheral-type lung adenocarcinomas (n = 161), high OCIAD2 expression was significantly associated with both poorer OS (P = 0.0214) and poorer disease-free survival (P = 0.0496). Adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) showed weaker OCIAD2 expression than invasive adenocarcinoma. Among small adenocarcinomas measuring 2 cm or less in greatest dimension classified according to the Noguchi's classification (n = 79), invasive adenocarcinomas showed significantly higher OCIAD2 expression than non-invasive adenocarcinomas (P = 0.0007). Interestingly, OCIAD2 was expressed heterogeneously even within a tumor, and its expression was higher in areas of invasion than in areas of in situ spread. Our results suggest that OCIAD2 could be a useful prognostic biomarker of lung adenocarcinoma.

Stratifin regulates stabilization of receptor tyrosine kinases via interaction with ubiquitin-specific protease 8 in lung adenocarcinoma.

Previously we have reported that stratifin (SFN, 14-3-3 sigma) acts as a novel oncogene, accelerating the tumor initiation and progression of lung adenocarcinoma. Here, pull-down assay and LC-MS/MS analysis revealed that ubiquitin-specific protease 8 (USP8) specifically bound to SFN in lung adenocarcinoma cells. Both USP8 and SFN showed higher expression in human lung adenocarcinoma than in normal lung tissue, and USP8 expression was significantly correlated with SFN expression. Expression of SFN, but not of USP8, was associated with histological subtype, pathological stage, and poor prognosis. USP8 stabilizes receptor tyrosine kinases (RTKs) such as EGFR and MET by deubiquitination, contributing to the proliferative activity of many human cancers including non-small cell lung cancer. In vitro, USP8 binds to SFN and they co-localize at the early endosomes in lung adenocarcinoma cells. Moreover, USP8 or SFN knockdown leads to downregulation of tumor cellular proliferation and upregulation of apoptosis, p-EGFR or p-MET, which are related to the degradation pathway, and accumulation of ubiquitinated RTKs, leading to lysosomal degradation. Additionally, mutant USP8, which is unable to bind to SFN, reduces the expression of RTKs and p-STAT3. We also found that interaction with SFN is critical for USP8 to exert its autodeubiquitination function and avoid dephosphorylation by PP1. Our findings demonstrate that SFN enhances RTK stabilization through abnormal USP8 regulation in lung adenocarcinoma, suggesting that SFN could be a more suitable therapeutic target for lung adenocarcinoma than USP8.

Case report of three EGFR TKI naïve lung adenocarcinoma containing double EGFR mutations (L858R/T790M or Exon 19 Deletion/T790M); Comparing genetic information and histology.

EGFR T790M mutation is a crucial gene alteration causing EGFR TKI resistance. However, the implication of T790M mutation is still unknown for the stepwise progression of EGFR TKI naïve lung adenocarcinoma. In this study, we studied site-related EGFR T790M mutation analysis in EGFR TKI naïve lung adenocarcinomas harboring double EGFR mutation (L858R and T790M or Exon 19 deletion (Del.19) and T790M) by droplet digital (dd) PCR method. We examined three resected lung adenocarcinoma cases harboring EGFR double mutation including T790M. These cases didn't receive EGFR TKI treatment. We divided formalin-fixed and paraffin embedded (FFPE) unstained slide tissues into 11-18 areas in each tumor and extracted DNAs from each area separately. The DNAs were analyzed by ddPCR. T790M mutation ratio (T790M/L858R or T790M/Del.19) were calculated. For three cases, we also performed EGFR FISH for analyzing EGFR copy number.In Case 2 and 3, T790M mutation ratio were 100% and 30% homogeneously and showed increased EGFR copy number also homogeneously. However, in case 1, it was different between invasive and non-invasive areas. EGFR copy number was also heterogeneous and showed increasing only in invasive area. We indicated a peculiar case harboring T790M heterogeneity and only invasive area had T790M mutation even though the case was not treated by EGFR TKI. It suggests that T790M is possibly significant not only for EGFR TKI resistance but also the progression in lung adenocarcinoma.

Heterotopic production of ceruloplasmin by lung adenocarcinoma is significantly correlated with prognosis.

OBJECTIVES: Ceruloplasmin (CP) is a well-known copper binding protein synthesized mainly in the liver, but its expression is known to be elevated in the serum of cancer patients and in malignant tumor cells. Lung cancer is the leading cause of cancer-related death worldwide, and adenocarcinoma is the main histological type of lung cancer. However, the role of CP in lung adenocarcinoma is still unclear. Here we examined and compared the expression of CP in various histological subtypes of lung adenocarcinoma and its correlation with clinicopathological parameters. MATERIALS AND METHODS: We examined CP expression in lung adenocarcinoma samples and cell lines using quantitative real-time RT-PCR and Western blot analysis. Immunohistochemistry for CP was carried out using 196 specimens of lung adenocarcinoma. RESULTS: CP expression was significantly higher in invasive adenocarcinoma than in adenocarcinoma in situ (AIS), and was significantly correlated with poorer outcome, pathological stage, pT, and pN. Multivariate analysis showed that CP expression was an independent prognostic factor for lung adenocarcinoma patients. Furthermore, Western blot analysis using protein extracted from lung adenocarcinoma cell lines revealed the secreted form of CP. CONCLUSION: CP is produced heterotopically in lung adenocarcinoma cells and its expression is associated with tumor progression. In view of the presence of the secreted form of CP in tumor cells, CP may be a useful biomarker for lung adenocarcinoma.

Cyclophilin A expression and its prognostic significance in lung adenocarcinoma.

Cyclophilin A (CypA) has been reported to be upregulated in malignant tumors. CypA expression is thought to be associated with acquisition of tumor growth and anti-apoptotic function. Although upregulation of CypA has been reported in lung adenocarcinoma, its clinicopathological significance and roles in malignant progression remain unclear. Here we investigated the implications of CypA expression for outcome in patients with lung adenocarcinoma. Lung adenocarcinoma specimens from 198 cases were selected and reclassified according to the World Health Organization classification (4th edition) and the Noguchi classification. CypA expression was assessed by immunohistochemistry, and the H-score was calculated on the basis of intensity and proportion. The specificity of the antibody used was confirmed by Western blotting and the cut-off point was determined from the ROC curve. Sixty-seven cases (33.8%) had low CypA expression (CypA-L group) and 131 (66.2%) had high CypA expression (CypA-H group). Many cases of adenocarcinoma in situ were CypA-L, and advanced adenocarcinomas tended to be classified as CypA-H. Clinically, patients with CypA-H tumors showed a significantly poorer prognosis than those with CypA-L tumors. This is the first investigation of the implications of the CypA expression level in terms of the clinical characteristics of resected lung adenocarcinomas.

Ubiquitin-specific protease 8 is a novel prognostic marker in early-stage lung adenocarcinoma.

Alterations of epidermal growth factor receptor (EGFR) expression frequently occur in early-stage lung adenocarcinoma. Ubiquitin-specific protease 8 (USP8) has been reported to stabilize EGFR protein at the plasma membrane through the recycling pathway. Here, we examined the correlation between USP8 expression and the expression or mutation status of EGFR, as well as the clinicopathological features of lung adenocarcinoma and patient outcome. Expression of EGFR and USP8 in surgically resected specimens of lung adenocarcinoma (82 cases) was examined by immunohistochemistry. Overexpression of EGFR was mutually correlated with that of USP8, and was also associated with clinicopathological features including pathological subtype, lymphatic permeation, and vascular invasion. Moreover, patients who had USP8-positive tumors had a significantly poorer outcome than those who were USP8-negative, not only overall but also patients who were EGFR-negative. Although EGFR was expressed in invasive adenocarcinoma but not in adenocarcinoma in situ (AIS), USP8 was overexpressed in not only invasive adenocarcinoma but also 38.1% of AIS cases. In vitro, USP8 regulated the expression and half-life of EGFR in immortalized AIS cells, and also cell proliferation. Our findings indicate that overexpression of USP8 in lung adenocarcinoma is an early event during the course of tumor progression, and is related to EGFR expression.

miR-3941: A novel microRNA that controls IGBP1 expression and is associated with malignant progression of lung adenocarcinoma.

Immunoglobulin (CD79a) binding protein 1 (IGBP1) is universally overexpressed in lung adenocarcinoma and exerts an anti-apoptotic effect by binding to PP2Ac. However, the molecular mechanism of IGBP1 overexpression is still unclear. In the present study, we used a microRNA (miRNA) array and TargetScan Human software to detect IGBP1-related miRNAs that regulate IGBP1 expression. The miRNA array analysis revealed more than 100 miRNAs that are dysregulated in early invasive adenocarcinoma. On the other hand, in silico analysis using TargetScan Human revealed 79 miRNAs that are associated with IGBP1 protein expression. Among the miRNAs selected by miRNA array analysis, six (miR-34b, miR-138, miR-374a, miR-374b, miR-1909, miR-3941) were also included among those selected by TargetScan analysis. Real-time reverse transcription PCR (real-time RT-PCR) showed that the six microRNAs were downregulated in invasive adenocarcinoma (IGBP1+) relative to adjacent normal lung tissue (IGBP1-). Among these microRNAs, only miR-34b and miR-3941 depressed luciferase activity by targeting 3'UTR-IGBP1 in the luciferase vector. We transfected miR-34b and miR-3941 into lung adenocarcinoma cell lines (A549, PC-9), and both of them suppressed IGBP1 expression and cell proliferation. Moreover, the transfected miR-34b and miR-3941 induced apoptosis of a lung adenocarcinoma cell line, similarly to the effect of siIGBP1 RNA. As well as miR-34b, we found that miR-3941 targeted IGBP1 specifically and was able to exclusively downregulate IGBP1 expression. These findings indicate that suppression of miR-3941 has an important role in the progression of lung adenocarcinoma at an early stage.

DNMT3a expression pattern and its prognostic value in lung adenocarcinoma.

OBJECTIVES: DNA methyltransferases (DNMTs) are an important part of the methylation pathway that is highly correlated with the pathophysiology of cancers. Several studies have reported overexpression of DNMTs in human lung cancer, but none have compared the expression pattern to pathological features. In this study, we clarified the association of DNMT3a expression pattern with pathological features and prognosis of lung adenocarcinoma. MATERIALS AND METHODS: 135 cases of surgically resected lung adenocarcinoma specimens were used for DNMT3a immunohistochemistry (IHC). IHC score was determined by counting the number of positive nuclei. The ROC curve was drawn to determine the best cut-off point of the score; this was set at 57.5. Western blot also implemented and confirmed the specificity of the antibody. Correlations between expression pattern and clinicopathological features and prognosis were analyzed using chi-squared method and Cox proportional hazards model respectively. RESULT: Seventy-nine of the 135 cases (58.5%) showed strong positive reactivity to anti-DNMT3a. In terms of histological subtypes, among invasive lung adenocarcinomas 41 out of 53 lepidic adenocarcinomas (77%) were strongly positive, while among the other histological subtypes only 23 out of 66 cases (34.8%) showed a positive reaction. Among non-invasive lung adenocarcinomas 15 out of 16 cases (93.8%) were strongly positive. The level of DNMT3a expression was associated with patient outcome, and patients with weak expression of DNMT3a had a poorer outcome than those with strong expression. Multivariate analysis also indicated that DNMT3a is an independent prognostic marker in lung adenocarcinoma. CONCLUSION: Our results indicate that DNMT3a expression in lung adenocarcinoma is associated with the histologically non-invasive type and lepidic subtype, and a favorable prognosis. We also showed that DNMT3a expression is an independent prognostic marker in lung adenocarcinoma. Since lack of DNMT3a is thought to facilitate tumor progression, DNMT3a might be clinically applicable as an indicator of favorable prognosis.

Stratifin accelerates progression of lung adenocarcinoma at an early stage.

BACKGROUNDS: Adenocarcinoma in situ (AIS) of the lung has an extremely favorable prognosis. However, early but invasive adenocarcinoma (eIA) sometimes has a fatal outcome. We had previously compared the expression profiles of AIS with those of eIA showing lymph node metastasis or a fatal outcome, and found that stratifin (SFN, 14-3-3 sigma) was a differentially expressed gene related to cell proliferation. Here, we performed an in vivo study to clarify the role of SFN in initiation and progression of lung adenocarcinoma. FINDINGS: Suppression of SFN expression in A549 (a human lung adenocarcinoma cell line) by siSFN significantly reduced cell proliferation activity and the S-phase subpopulation. In vivo, tumor development or metastasis to the lung was reduced in shSFN-transfected A549 cells. Moreover, we generated SFN-transgenic mice (Tg-SPC-SFN(+/-)) showing lung-specific expression of human SFN under the control of a tissue-specific enhancer, the SPC promoter. We found that Tg-SPC-SFN(+/-) mice developed lung tumors at a significantly higher rate than control mice after administration of chemical carcinogen, NNK. Interestingly, several Tg-SPC-SFN(+/-) mice developed tumors without NNK. These tumor cells showed high hSFN expression. CONCLUSION: These results suggest that SFN facilitates lung tumor development and progression. SFN appears to be a novel oncogene with potential as a therapeutic target.