Combined effects of asbestos and cigarette smoke on the development of lung adenocarcinoma: different carcinogens may cause different genomic changes.

The carcinogens in cigarette smoke are distinct from asbestos. However, an understanding of their differential effects on lung adenocarcinoma development remains elusive. We investigated loss of heterozygosity (LOH) and the p53 mutation in 132 lung adenocarcinomas, for which asbestos body burden (AB; in numbers per gram of dry lung) was measured using adjacent normal lung. All cases were classified into 9 groups based on a matrix of cumulative smoking (CS in pack­years; CS=0, 00 groups, LOH frequency increased as AB and/or CS was elevated and was significantly higher in the ≥1,000 AB, ≥25 CS group (p=0.032). p53 mutation frequency was the lowest in the AB=0, CS=0 group, increased as AB and/or CS rose, and was significantly higher in the ≥1,000 AB, ≥25 CS group (p=0.039). p53 mutations characteristic of smoking were frequently observed in the CS>0 groups contrary to non-specific mutations in the CS=0, AB>0 groups. Combined effects of asbestos and smoking were suggested by LOH and p53 analyses. Sole exposure to asbestos did not increase LOH frequency but increased non­specific p53 mutations. These findings indicate that the major carcinogenic mechanism of asbestos may be tumor promotion, acting in an additive or synergistic manner, contributing to the genotoxic effect of smoking. Since this study was based on a general cancer center's experience, the limited sample size did not permit the consideration that the result was conclusive. Further investigation with a large sample size is needed to establish the mechanism of asbestos-induced lung carcinogenesis.

Cell division cycle-associated protein 1 overexpression is essential for the malignant potential of colorectal cancers.

To identify new cancer biomarkers and therapeutic targets for colorectal cancers (CRCs), we performed immunohistochemical analysis using tissue microarrays covering archival tumor tissue samples from 434 CRC patients and antibodies to cell division cycle-associated protein 1 (CDCA1) that was originally identified as an oncoantigen by our gene expression profile database, and compared its expression with several clinicopathological factors. Strong CDCA1 positivity was associated with poorer prognosis for patients with CRC (P=0.019) and multivariate analysis confirmed its independent prognostic value. In addition, transfection of siRNAs against CDCA1 suppressed its expression and induced apoptosis of CRC cells. These results suggest that CDCA1 could be a prognostic biomarker and a potential therapeutic target for CRCs.

RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

UNLABELLED: Genome-wide gene expression profiling revealed that the Ras and EF-hand domain containing (RASEF) transcript was significantly transactivated in the majority of lung cancers. Using lung cancer cells, transient expression of RASEF promoted cell growth, whereas RASEF knockdown not only reduced its expression but resulted in growth suppression of the cancer cells. Immunohistochemical staining using tumor tissue microarrays consisting of 341 archived non-small cell lung cancers (NSCLC) revealed the association of strong RASEF positivity with poor prognosis (P = 0.0034 by multivariate analysis). Mechanistically, RASEF interacted with extracellular signal-regulated kinase (ERK) 1/2 and enhanced ERK1/2 signaling. Importantly, inhibiting the interaction between RASEF and ERK1/2 using a cell-permeable peptide that corresponded to the ERK1/2-interacting site of RASEF, suppressed growth of lung cancer cells. This study demonstrates that elevated RASEF promoted cell growth via enhanced ERK signaling and is associated with poor prognosis of NSCLC. IMPLICATIONS: RASEF may play an important role in lung carcinogenesis and could serve as a vaiable prognostic biomarker and target for the development of new molecular therapies.

Enhanced HSP70 lysine methylation promotes proliferation of cancer cells through activation of Aurora kinase B.

Although heat-shock protein 70 (HSP70), an evolutionarily highly conserved molecular chaperone, is known to be post-translationally modified in various ways such as phosphorylation, ubiquitination and glycosylation, physiological significance of lysine methylation has never been elucidated. Here we identify dimethylation of HSP70 at Lys-561 by SETD1A. Enhanced HSP70 methylation was detected in various types of human cancer by immunohistochemical analysis, although the methylation was barely detectable in corresponding non-neoplastic tissues. Interestingly, methylated HSP70 predominantly localizes to the nucleus of cancer cells, whereas most of the HSP70 protein locates to the cytoplasm. Nuclear HSP70 directly interacts with Aurora kinase B (AURKB) in a methylation-dependent manner and promotes AURKB activity in vitro and in vivo. We also find that methylated HSP70 has a growth-promoting effect in cancer cells. Our findings demonstrate a crucial role of HSP70 methylation in human carcinogenesis.

Regulation of histone modification and chromatin structure by the p53-PADI4 pathway.

Histone proteins are modified in response to various external signals; however, their mechanisms are still not fully understood. Citrullination is a post-transcriptional modification that converts arginine in proteins into citrulline. Here we show in vivo and in vitro citrullination of the arginine 3 residue of histone H4 (cit-H4R3) in response to DNA damage through the p53-PADI4 pathway. We also show DNA damage-induced citrullination of Lamin C. Cit-H4R3 and citrullinated Lamin C localize around fragmented nuclei in apoptotic cells. Ectopic expression of PADI4 leads to chromatin decondensation and promotes DNA cleavage, whereas Padi4(-/-) mice exhibit resistance to radiation-induced apoptosis in the thymus. Furthermore, the level of cit-H4R3 is negatively correlated with p53 protein expression and with tumour size in non-small cell lung cancer tissues. Our findings reveal that cit-H4R3 may be an 'apoptotic histone code' to detect damaged cells and induce nuclear fragmentation, which has a crucial role in carcinogenesis.

Characterization of an Opa interacting protein 5 involved in lung and esophageal carcinogenesis.

To identify potential molecular targets for diagnosis, treatment and/or prevention of lung and esophageal carcinomas, we screened for genes that were overexpressed in tumors through gene expression analyses of 120 lung cancers and 19 esophageal squamous-cell carcinomas using a cDNA microarray consisting of 27,648 cDNA or expressed sequence tags. In this process, we identified a gene, Opa interacting protein 5 (OIP5), to be highly transactivated in the majority of lung and esophageal cancers. Immunohistochemical staining using 336 archived non-small cell lung cancers and 305 esophageal squamous-cell carcinomas specimens demonstrated that OIP5 expression was significantly associated with poor prognosis of lung and esophageal cancer patients (P = 0.0053 and 0.0168, respectively), and multivariate analysis confirmed its independent prognostic value for non-small cell lung cancers (P = 0.0112). Suppression of OIP5 expression with siRNA effectively suppressed the growth of cancer cells, whereas the exogenous expression of OIP5 enhanced the growth of cancer cells. In addition, OIP5 protein is likely to be stabilized through its interaction with Raf1. OIP5 is a promising target for developing new prognostic biomarkers and anti-cancer drugs.

Chondrolectin is a novel diagnostic biomarker and a therapeutic target for lung cancer.

PURPOSE: This study aims to identify molecules that might be useful as diagnostic/prognostic biomarkers and as targets for the development of new molecular therapies for lung cancer. EXPERIMENTAL DESIGN: We screened for genes that were highly transactivated in a large proportion of 120 lung cancers by means of a cDNA microarray representing 27,648 genes and found chondrolectin (CHODL) as a candidate. Tumor tissue microarray was applied to examine the expression of CHODL protein and its clinicopathologic significance in archival non-small cell lung cancer (NSCLC) tissues from 295 patients. A role of CHODL in cancer cell growth and/or survival was examined by siRNA experiments. Cellular invasive effect of CHODL on mammalian cells was examined by Matrigel assays. RESULTS: Immunohistochemical staining revealed that strong positivity of CHODL protein was associated with shorter survival of patients with NSCLC (P = 0.0006), and multivariate analysis confirmed it to be an independent prognostic factor. Treatment of lung cancer cells with siRNAs against CHODL suppressed growth of the cancer cells. Furthermore, induction of exogenous expression of CHODL conferred growth and invasive activity of mammalian cells. CONCLUSIONS: CHODL is likely to be a prognostic biomarker in the clinic and targeting CHODL might be a strategy for the development of anticancer drugs.

Histone lysine methyltransferase Wolf-Hirschhorn syndrome candidate 1 is involved in human carcinogenesis through regulation of the Wnt pathway.

A number of histone methyltransferases have been identified and biochemically characterized, but the pathologic roles of their dysfunction in human diseases like cancer are not well understood. Here, we demonstrate that Wolf-Hirschhorn syndrome candidate 1 (WHSC1) plays important roles in human carcinogenesis. Transcriptional levels of this gene are significantly elevated in various types of cancer including bladder and lung cancers. Immunohistochemical analysis using a number of clinical tissues confirmed significant up-regulation of WHSC1 expression in bladder and lung cancer cells at the protein level. Treatment of cancer cell lines with small interfering RNA targeting WHSC1 significantly knocked down its expression and resulted in the suppression of proliferation. Cell cycle analysis by flow cytometry indicated that knockdown of WHSC1 decreased the cell population of cancer cells at the S phase while increasing that at the G(2)/M phase. WHSC1 interacts with some proteins related to the WNT pathway including ß-catenin and transcriptionally regulates CCND1, the target gene of the ß-catenin/Tcf-4 complex, through histone H3 at lysine 36 trimethylation. This is a novel mechanism for WNT pathway dysregulation in human carcinogenesis, mediated by the epigenetic regulation of histone H3. Because expression levels of WHSC1 are significantly low in most normal tissue types, it should be feasible to develop specific and selective inhibitors targeting the enzyme as antitumor agents that have a minimal risk of adverse reaction.

Identification of Epstein-Barr virus-induced gene 3 as a novel serum and tissue biomarker and a therapeutic target for lung cancer.

PURPOSE: This study aims to identify novel biomarkers and therapeutic targets for lung cancer. EXPERIMENTAL DESIGN: We carried out gene expression profile analysis of 120 lung cancers to screen for genes encoding transmembrane/secretory molecules that are commonly transactivated in lung cancers. Epstein-Barr virus-induced gene 3 (EBI3), which encodes a secretory glycoprotein, was selected as a good candidate. Immunohistochemical staining using tissue microarray consisting of 414 non-small cell lung cancers was applied to examine the expression level and prognostic value of EBI3. Serum EBI3 levels in 400 individuals for training assays (274 lung cancers and 126 healthy volunteers) and those in 173 individuals for validation analysis (132 lung cancers and 41 healthy volunteers) were measured by ELISA. The role of EBI3 in cancer cell growth was examined by siRNA and cell growth assays, using cells stably expressing exogenous EBI3. RESULTS: Immunohistochemical staining of EBI3 using tissue microarrays revealed that a high level of EBI3 expression was associated with a poor prognosis of lung cancer (P = 0.0014) and multivariate analysis confirmed it to be an independent prognostic factor (P = 0.0439). Serum levels of EBI3 in the training set were found to be significantly higher in lung cancer patients than in healthy volunteers; this result was also observed in the validation set. Furthermore, reduction in EBI3 expression by siRNA suppressed cancer cell proliferation whereas induction of exogenous EBI3 conferred growth-promoting activity. CONCLUSIONS: EBI3 is a potential serum and tissue biomarker as well as therapeutic target for lung cancer.

Characterization of a cleavage stimulation factor, 3' pre-RNA, subunit 2, 64 kDa (CSTF2) as a therapeutic target for lung cancer.

PURPOSE: This study aims to discover novel biomarkers and therapeutic targets for lung cancers. EXPERIMENTAL DESIGN: We screened for genes showing elevated expression in the majority of lung cancers by genome-wide gene expression profile analysis of 120 lung cancers obtained by cDNA microarray representing 27,648 genes or expressed sequence tags. In this process, we detected a gene encoding cleavage stimulation factor, 3' pre-RNA, subunit 2, 64 kDa (CSTF2) as a candidate. Immunohistochemical staining using tissue microarray consisting of 327 lung cancers was applied to examine the expression of CSTF2 protein and its prognostic value. A role of CSTF2 in cancer cell growth was examined by siRNA experiments. RESULTS: Northern blot and immunohistochemical analyses detected the expression of CSTF2 only in testis among 16 normal tissues. Immunohistochemical analysis using tissue microarray showed an association of strong CSTF2 expression with poor prognosis of patients with non-small cell lung cancer (P = 0.0079), and multivariate analysis showed that CSTF2 positivity is an independent prognostic factor. In addition, suppression of CSTF2 expression by siRNAs suppressed lung cancer cell growth, whereas exogenous expression of CSTF2 promoted growth and invasion of mammalian cells. CONCLUSIONS: CSTF2 is likely to play an important role in lung carcinogenesis and be a prognostic biomarker in the clinic.

Minichromosome Maintenance Protein 7 is a potential therapeutic target in human cancer and a novel prognostic marker of non-small cell lung cancer.

BACKGROUND: The research emphasis in anti-cancer drug discovery has always been to search for a drug with the greatest antitumor potential but fewest side effects. This can only be achieved if the drug used is against a specific target located in the tumor cells. In this study, we evaluated Minichromosome Maintenance Protein 7 (MCM7) as a novel therapeutic target in cancer. RESULTS: Immunohistochemical analysis showed that MCM7 was positively stained in 196 of 331 non-small cell lung cancer (NSCLC), 21 of 29 bladder tumor and 25 of 70 liver tumor cases whereas no significant staining was observed in various normal tissues. We also found an elevated expression of MCM7 to be associated with poor prognosis for patients with NSCLC (P = 0.0055). qRT-PCR revealed a higher expression of MCM7 in clinical bladder cancer tissues than in corresponding non-neoplastic tissues (P < 0.0001), and we confirmed that a wide range of cancers also overexpressed MCM7 by cDNA microarray analysis. Suppression of MCM7 using specific siRNAs inhibited incorporation of BrdU in lung and bladder cancer cells overexpressing MCM7, and suppressed the growth of those cells more efficiently than that of normal cell strains expressing lower levels of MCM7. CONCLUSIONS: Since MCM7 expression was generally low in a number of normal tissues we examined, MCM7 has the characteristics of an ideal candidate for molecular targeted cancer therapy in various tumors and also as a good prognostic biomarker for NSCLC patients.

Rare MDM4 gene amplification in colorectal cancer: The principle of a mutually exclusive relationship between MDM alteration and TP53 inactivation is not applicable.

MDM4, a homolog of MDM2, is considered a key negative regulator of p53. Gene amplification of MDM4 has been identified in a variety of tumors. MDM2 or MDM4 gene amplification is only associated with the wild-type TP53 gene in retinoblastomas, thus the amplification of the two genes is mutually exclusive. Previously, we demonstrated that MDM2 amplification and TP53 alteration were not mutually exclusive in colorectal cancer, and we identified a subset of colorectal cancer patients without alterations in either the TP53 or the MDM2 gene. In this study, we investigated the gene amplification status of MDM4 in the same set of colorectal cancer cases. Unexpectedly, MDM4 amplification was rare, detected in only 1.4% (3 out of 211) of colorectal cancer cases. All the three gene-amplified tumors also harbored TP53-inactivating mutations. This contradicts the simple mutually exclusive relationship observed in retinoblastomas. Surprisingly, two of the three MDM4-amplified tumors also demonstrated MDM2 amplification. Paradoxically, the MDM4 protein levels were decreased in the tumor tissue of the gene-amplified cases compared with levels in the matched normal mucosa. We speculate that MDM4 might play a role in colorectal carcinogenesis that is not limited to negative regulation of p53 in combination with MDM2. The functional significance of MDM4 is still unclear and further studies are needed.

ETS family-associated gene fusions in Japanese prostate cancer: analysis of 194 radical prostatectomy samples.

The incidence and clinical significance of the TMPRSS2:ERG gene fusion in prostate cancer has been investigated with contradictory results. It is now common knowledge that significant variability in gene alterations exists according to ethnic background in various kinds of cancer. In this study, we evaluated gene fusions involving the ETS gene family in Japanese prostate cancer. Total RNA from 194 formalin-fixed and paraffin-embedded prostate cancer samples obtained by radical prostatectomy was subjected to reverse-transcriptase polymerase chain reaction to detect the common TMPRSS2:ERG T1-E4 and T1-E5 fusion transcripts and five other non-TMPRSS2:ERG fusion transcripts. We identified 54 TMPRSS2:ERG-positive cases (54/194, 28%) and two HNRPA2B1:ETV1-positive cases (2/194, 1%). The SLC45A3-ELK4 transcript, a fusion transcript without structural gene rearrangement, was detectable in five cases (5/194, 3%). The frequencies of both TMPRSS2:ERG- and non-TMPRSS2:ERG-positive cases were lower than those reported for European, North American or Brazilian patients. Internodular heterogeneity of TMPRSS2:ERG was observed in 5 out of 11 multifocal cases (45%); a frequency similar to that found in European and North American cases. We found a positive correlation between the TMPRSS2:ERG fusion and a Gleason score of ≤7 and patient age, but found no relationship with pT stage or plasma prostate-specific antigen concentration. To exclude the possibility that Japanese prostate cancer displays novel TMPRSS2:ERG transcript variants or has unique 5' fusion partners for the ETS genes, we performed 5' RACE using fresh-frozen prostate cancer samples. We identified only the normal 5' cDNA ends for ERG, ETV1 and ETV5 in fusion-negative cases. Because we identified a relatively low frequency of TMPRSS2:ERG and other fusions, further evaluation is required before this promising molecular marker should be introduced into the management of Japanese prostate cancer patients.

Proteomic screening of completely resected tumors in relation to survival in patients with stage I non-small cell lung cancer.

To investigate the early changes in protein function that induce micro-metastasis in early-stage non-small cell lung cancer, we conducted proteomic analysis of tissue that had been completely resected. We selected sixteen patients whose tumors were pathological stage I adenocarcinoma with hobnail cell morphology. We compared their proteomic profiles between patients whose cancers recurred and did not recur after 5 years of follow-up. Proteins extracted from frozen tumor tissue were saturated by CyDye labeling and subjected to two-dimensional difference gel electrophoresis (2D-DIGE). We found approximately 2500 protein spots by DeCyder-DIA software analysis. A random forest classifier to the spot volume data sets revealed 30 extracted spots that were potentially effective marker proteins for distinguishing the recurrence and non-recurrence groups. Among them, Mann-Whitney U-test analysis showed that 15 spots were candidate marker proteins. Finally, 11 unique proteins corresponding to the 15 candidate spots were found using an improved in-gel digestion method and MALDI-TOF MS and nanoLC-ESI MS analysis. Among them, aldehyde dehydrogenase and tropomyosin were considered to have undergone protein modifications such as phosphorylation, acetylation, and glycosylation. We have thus identified some biomarkers that can predict tumor recurrence in patients with early-stage non-small cell lung cancer including hobnail-type adenocarcinoma. A further study is required to confirm the utility of these biomarkers.

Phosphorylation and activation of cell division cycle associated 5 by mitogen-activated protein kinase play a crucial role in human lung carcinogenesis.

We analyzed the gene expression profiles of clinical lung carcinomas using a cDNA microarray containing 27,648 genes or expressed sequence tags, and identified CDCA5 (cell division cycle associated 5) to be upregulated in the majority of lung cancers. Tumor tissue microarray analysis of 262 non-small cell lung cancer patients revealed that CDCA5 positivity was an independent prognostic factor for lung cancer patients. Suppression of CDCA5 expression with siRNAs inhibited the growth of lung cancer cells; concordantly, induction of exogenous expression of CDCA5 conferred growth-promoting activity in mammalian cells. We also found that extracellular signal-regulated kinase (ERK) kinase phosphorylated CDCA5 at Ser79 and Ser209 in vivo. Exogenous expression of phospho-mimicking CDCA5 protein whose Ser209 residue was replaced with glutamine acid further enhanced the growth of cancer cells. In addition, functional inhibition of the interaction between CDCA5 and ERK kinase by a cell-permeable peptide corresponding to a 20-amino-acid sequence part of CDCA5, which included the Ser209 phosphorylation site by ERK, significantly reduced phosphorylation of CDCA5 and resulted in growth suppression of lung cancer cells. Our data suggest that transactivation of CDCA5 and its phosphorylation at Ser209 by ERK play an important role in lung cancer proliferation, and that the selective suppression of the ERK-CDCA5 pathway could be a promising strategy for cancer therapy.

Wnt inhibitor Dickkopf-1 as a target for passive cancer immunotherapy.

Dickkopf-1 (DKK1) is an inhibitor of Wnt/beta-catenin signaling that is overexpressed in most lung and esophageal cancers. Here, we show its utility as a serum biomarker for a wide range of human cancers, and we offer evidence favoring the potential application of anti-DKK1 antibodies for cancer treatment. Using an original ELISA system, high levels of DKK1 protein were found in serologic samples from 906 patients with cancers of the pancreas, stomach, liver, bile duct, breast, and cervix, which also showed elevated expression levels of DKK1. Additionally, anti-DKK1 antibody inhibited the invasive activity and the growth of cancer cells in vitro and suppressed the growth of engrafted tumors in vivo. Tumor tissues treated with anti-DKK1 displayed significant fibrotic changes and a decrease in viable cancer cells without apparent toxicity in mice. Our findings suggest DKK1 as a serum biomarker for screening against a variety of cancers, and anti-DKK1 antibodies as potential theranostic tools for diagnosis and treatment of cancer.

Predictive advantage of a cell type classification for pulmonary adenocarcinoma coupled with data for p53, K-ras and EGFR alterations.

We analyzed relationships between histological subtypes of pulmonary adenocarcinomas and three gene alterations (p53, K-ras, and epidermal growth factor receptor gene), or thyroid transcription factor-1 (TTF-1) expression, and also studied prognoses by the subtypes, with or without combined multiple gene mutation status. Our purpose was to clearly determine pathogenesis, along with the best predictive value for biology and therapy-related traits. A total of 223 consecutively resected pulmonary adenocarcinomas were sub-classified using either the World Health Organization (WHO) or our five-cell type (FCT) classification system (hobnail, columnar/cuboidal, mixed, polygonal/oval, and goblet cell types). DNAs extracted from frozen samples of the adenocarcinomas were examined for gene alterations, and TTF-1 expressions were determined using immunohistochemistry. Next, relationships among the various data and clinicopathological factors were analyzed. The most striking result was: while almost 70% of adenocarcinomas were sub-classified as a mixed subtype by WHO, the FCT classified many of them as other cell subtypes. The FCT closely reflected differences in etiological factors, cellular lineages, and frequencies of gene mutations; and whether the data from combined gene mutations were used or not, differences among the cell types in postoperative survivals appeared. In contrast, subtypes of WHO did not show any association with the gene alteration or prognosis, and the FCT more suitably indicated sensitivity to gefitinib therapy than did WHO. The FCT combined with multiple gene mutation status appears to be useful in indicating pathogenesis and predicting the biological nature of pulmonary adenocarcinomas, and it could facilitate development of new therapies for each subtype.

MDM2 gene amplification in colorectal cancer is associated with disease progression at the primary site, but inversely correlated with distant metastasis.

MDM2 is a crucial negative regulator of the TP53 tumor suppressor and almost 10% of human tumors exhibit MDM2 amplification. Although TP53 pathway perturbation has been extensively examined in colorectal cancer (CRC), only one previous report has evaluated MDM2 amplification in relation to clinicopathological factors. In that report, MDM2 amplification was shown to be associated with disease progression from Dukes' Stages A to D. In this study, we investigated MDM2 amplification by quantitative PCR and fluorescence in situ hybridization (FISH) together with the SNP309 genotypes, and analyzed the correlations with TP53 and KRAS mutations and clinicopathological features in 211 Japanese CRC patients. MDM2 amplification was detected in 8% of the specimens and its incidence was significantly higher in Dukes' stage C than in the combined earlier Stages A and B (P = 0.025). Unexpectedly, the incidence was significantly decreased in Stage D metastatic disease (P = 0.043). The copy number gain ranged from four to eight copies and was generally concordant with gain of centromere 12 using FISH analysis. Together with the results of centromere 1 FISH and TP53 copy number assessment, the MDM2 increment most likely resulted from chromosome 12 gain. The mechanism of the copy number gain and incidence in Dukes' Stage D differed considerably from the previous report. Ethnic or geographic factors could be responsible for these differences. Several promising therapeutic strategies targeting the TP53-MDM2 system are being developed. Further understanding of the significance of MDM2 and MDM2 amplification in CRC is required to facilitate personalized treatment for CRC patients.

Lung adenocarcinoma cells floating in lymphatic vessels resist anoikis by expressing phosphorylated Src.

The ability to resist anoikis is critical for carcinoma cells to metastasize. Although several lung adenocarcinoma cell lines were shown to repress anoikis through the activation of Src, it remains unknown whether Src actually plays a crucial role in anoikis resistance in lung adenocarcinoma tissues. We examined 20 human lung adenocarcinoma tissues with lymphatic permeation and nine cell lines to investigate whether intralymphatic floating carcinoma cells in the tissues, used as an in vivo model of anoikis resistance, actually suppressed anoikis and whether cell lines in suspension culture, an in vitro model of anoikis resistance, survived through Src activation. We observed that the intralymphatic carcinoma cells aggregated tightly to form nests expressing E-cadherin and phosphorylated Src (p-Src). The apoptotic indices of these cells were comparable to those of extracellular matrix adhesive cells in all tissues, indicating that the intralymphatic cells actually evaded anoikis. Next, we found that the nine cell lines in suspension aggregated loosely (five cell lines) or tightly (four cell lines), and all cells resisted anoikis. Upon detachment, four cell lines (LC-KJ, HCC827, H1650, and H1975) formed compact spheroids that expressed E-cadherin and p-Src. The spheroids were similar to intralymphatic tumour nests and were thus considered to be a suitable model of the nests. The spheroids of the four cell lines underwent apoptosis after treatment with the Src/Abl/Kit inhibitor PP1 or Src/Abl inhibitor bosutinib. On the other hand, the Abl/Kit inhibitor imatinib did not affect cell growth or apoptosis in the four types of spheroids. These results indicate that Src, but not Abl or Kit, plays an essential role in the development of anoikis resistance in lung adenocarcinomas.

Activation of an oncogenic TBC1D7 (TBC1 domain family, member 7) protein in pulmonary carcinogenesis.

To develop novel biomarkers and therapeutic agents for lung cancers, we screened molecules that were highly expressed in lung cancers by means of cDNA microarray analysis and found an elevated expression of TBC1 domain family, member 7 (TBC1D7) in the majority of lung cancers. Northern-blot analysis using mRNAs from 16 normal tissues detected its expression only in testis. Immunohistochemical staining using tumor tissue microarrays consisting of 261 archived non-small cell lung cancer (NSCLC) specimens suggested an association of TBC1D7 expression with poor prognosis for NSCLC patients (P = 0.0063). Treatment of lung cancer cells using siRNA against TBC1D7, suppressed its expression and resulted in inhibition of the cell growth. Furthermore, the induction of exogenous expression of TBC1D7 conferred growth-promoting activity at in vitro and in vivo conditions. We also identified TBC1D7 to interact with TSC1 protein in lung cancer cells. TSC1 introduction into cells increased the level of TBC1D7 protein, whereas knockdown of TSC1 expression decreased the level of TBC1D7 protein, suggesting that TBC1D7 is stabilized probably through interaction with TSC1. In addition, inhibition of the binding between TBC1D7 and TSC1 by a TBC1D7-derived 20-amino acid cell-permeable peptide (11R-TBC1D7(152-171)), which corresponded to the binding domain to TSC1, effectively suppressed growth of lung cancer cells. Selective suppression of TBC1D7 and/or inhibition of the TBC1D7-TSC1 complex formation could be promising therapeutic strategies for lung cancer therapy.