Gene amplification-associated overexpression of the RNA editing enzyme ADAR1 enhances human lung tumorigenesis.

The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis.

Translating the Diabetes Prevention Program in Native Hawaiian and Pacific Islander communities: the PILI 'Ohana Project.

Native Hawaiians/Pacific Islanders experience a high prevalence of overweight/obesity. The Diabetes Prevention Program Lifestyle Intervention (DPP-LI) was translated into a 3-month community-based intervention to benefit these populations. The weight loss and other clinical and behavioral outcomes of the translated DPP-LI and the socio-demographic, behavioral, and biological factors associated with the weight loss were examined. A total of 239 Native Hawaiian/Pacific Islander adults completed the translated DPP-LI through four community-based organizations (CBOs). Changes from pre- to post-intervention assessments in weight, blood pressure, physical functioning, exercise frequency, and fat in diet were measured. Significant improvements on all variables were found, with differences observed across the four CBOs. CBOs with predominately Native Hawaiian and ethnically homogenous intervention groups had greater weight loss. General linear modeling indicated that larger baseline weight and CBO predicted weight loss. The translated DPP-LI can be effective for Native Hawaiians/Pacific Islanders, especially when socio-cultural, socio-economic, and CBO-related contextual factors are taken into account.

Possible involvement of LKB1-AMPK signaling in non-homologous end joining.

LKB1/STK11 is a tumor suppressor gene responsible for Peutz-Jeghers syndrome, an inherited cancer disorder associated with genome instability. The LKB1 protein functions in the regulation of cell proliferation, polarization and differentiation. Here, we suggest a role of LKB1 in non-homologous end joining (NHEJ), a major DNA double-strand break (DSB) repair pathway. LKB1 localized to DNA ends upon the generation of micro-irradiation and I-SceI endonuclease-induced DSBs. LKB1 inactivation either by RNA interference or by kinase-dead mutation compromised NHEJ-mediated DNA repair by suppressing the accumulation of BRM, a catalytic subunit of the SWI/SNF complex, at DSB sites, which promotes the recruitment of an essential NHEJ factor, KU70. AMPK2, a major substrate of LKB1 and a histone H2B kinase, was recruited to DSBs in an LKB1-dependent manner. AMPK2 depletion and a mutation of H2B that disrupted the AMPK2 phoshorylation site impaired KU70 and BRM recruitment to DSB sites. LKB1 depletion induced the formation of chromosome breaks and radials. These results suggest that LKB1-AMPK signaling controls NHEJ and contributes to genome stability.

Histone acetylation by CBP and p300 at double-strand break sites facilitates SWI/SNF chromatin remodeling and the recruitment of non-homologous end joining factors.

Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs) generated by ionizing radiation (IR) and anti-cancer drugs. Therefore, inhibiting the activity of proteins involved in this pathway is a promising way of sensitizing cancer cells to both radiotherapy and chemotherapy. In this study, we developed an assay for evaluating NHEJ activity against DSBs in chromosomal DNA in human cells to identify the chromatin modification/remodeling proteins involved in NHEJ. We showed that ablating the activity of the homologous histone acetyltransferases, CBP and p300, using inhibitors or small interfering RNAs-suppressed NHEJ. Ablation of CBP or p300 impaired IR-induced DSB repair and sensitized lung cancer cells to IR and the anti-cancer drug, etoposide, which induces DSBs that are repaired by NHEJ. The CBP/p300 proteins were recruited to sites of DSBs and their ablation suppressed acetylation of lysine 18 within histone H3, and lysines 5, 8, 12, and 16 within histone H4, at the DSB sites. This then suppressed the recruitment of KU70 and KU80, both key proteins for NHEJ, to the DSB sites. Ablation of CBP/p300 also impaired the recruitment of BRM, a catalytic subunit of the SWI/SNF complex involved in chromatin remodeling at DSB sites. These results indicate that CBP and p300 function as histone H3 and H4 acetyltransferases at DSB sites in NHEJ and facilitate chromatin relaxation. Therefore, inhibition CBP and p300 activity may sensitize cancer cells to radiotherapy and chemotherapy.

Unbalanced translocation, a major chromosome alteration causing loss of heterozygosity in human lung cancer.

Loss of heterozygosity (LOH) is a major genetic event causing inactivation of tumor suppressor genes in human carcinogenesis. To elucidate chromosomal mechanisms causing LOH, 201 LOHs in 10 cases of human lung cancer, which were detected by a genome-wide single nucleotide polymorphism array analysis, were investigated for responsible chromosome alterations by integrating information on breakpoints for DNA copy number changes obtained by array-comparative genome hybridization and on numerical and structural chromosomal alterations obtained by spectral karyotyping. The majority (80%) of LOHs were partial chromosome LOHs caused by structural chromosomal alterations, while the remaining (20%) were whole chromosome LOHs caused by whole chromosome deletions. Unbalanced translocation was defined as the most frequent alteration, and it accounted for 30% of all LOHs. Three other structural alterations-interstitial deletion (19%), mitotic recombination (9%) and gene conversion (6%)-also contributed to the occurrence of LOH, while terminal deletion contributed to only a small subset (1%). Since unbalanced translocation is a common chromosomal alteration in lung cancer cells, the results in the present study strongly indicate that a considerable fraction of LOHs detected in lung cancer cells are caused by unbalanced translocation.

Prevalence and specificity of LKB1 genetic alterations in lung cancers.

Germline LKB1 mutations cause Peutz-Jeghers syndrome, a hereditary disorder that predisposes to gastrointestinal hamartomatous polyposis and several types of malignant tumors. Somatic LKB1 alterations are rare in sporadic cancers, however, a few reports showed the presence of somatic alterations in a considerable fraction of lung cancers. To determine the prevalence and the specificity of LKB1 alterations in lung cancers, we examined a large number of lung cancer cell lines and lung adenocarcinoma (AdC) specimens for the alterations. LKB1 genetic alterations were frequently detected in the cell lines (21/70, 30%), especially in non-small cell lung cancers (NSCLCs) (20/51, 39%), and were significantly more frequent in cell lines with KRAS mutations. Point mutations were detected only in AdCs and large cell carcinomas, whereas homozygous deletions were detected in all histological types of lung cancer. Among lung AdC specimens, LKB1 mutations were found in seven (8%) of 91 male smokers but in none of 64 females and/or nonsmokers, and were significantly more frequent in poorly differentiated tumors. The difference in the frequency of LKB1 alterations between cell lines and tumor specimens was likely to be owing to masking of deletions by the contamination of noncancerous cells in the tumor specimens. These results indicate that somatic LKB1 genetic alterations preferentially occur in a subset of poorly differentiated lung AdCs that appear to correlate with smoking males.

Identification of tumor markers and differentiation markers for molecular diagnosis of lung adenocarcinoma.

To identify tumor markers and differentiation markers for lung adenocarcinoma (AdC), we analysed expression profiles of 14,500 genes against three cases of type II alveolar epithelial cells, bronchiolar epithelial cells, and bronchial epithelial cells, respectively, and 10 cases of AdC cells isolated by laser capture microdissection. Hierarchical clustering analysis indicated that AdC cells and noncancerous lung epithelial cells are significantly different in their expression profiles, and that different sets of differentiation markers are expressed among alveolar, bronchiolar and bronchial epithelial cells. Nine genes were identified as being highly expressed in AdC cells, but not expressed in noncancerous lung epithelial cells. Sixteen genes were identified as differentiation markers for lung epithelial cells. Real-time RT-PCR analysis of 45 lung AdC cases further revealed that expression of four tumor markers in AdC cells was significantly higher than that in noncancerous lung cells and that expression of ten differentiation markers was retained in a considerable fraction of lung AdC cases. Five tumor markers and seven differentiation markers were not expressed in peripheral blood cells. Similarities and differences in expression profiles between normal epithelial cells from different lung respiratory compartments and AdC cells demonstrated in this study will be informative for the molecular diagnosis of lung AdC.

Retinoic acid suppresses endothelin-1 gene expression at the transcription level in endothelial cells.

Retinoids have been shown to inhibit cell growth, which can result in an anti-atherosclerotic action in the vasculature. Endothelin-1 (ET-1), a potent vasoconstrictor peptide produced in endothelial cells, plays an important role in inducing proliferation of vascular smooth muscle cells. In this study, we investigated the effect of retinoids on the mRNA expression and transcriptional activity of the ET-1 gene in endothelial cells. All-trans retinoic acid (ATRA) suppressed ET-1 mRNA expression in cultured endothelial cells. Synthetic retinoids, Ch55 and Am580 (retinoic acid receptor (RAR) agonists) markedly enhanced this effect, and an RAR antagonist, LE540, blocked this inhibitory effect on ET-1 gene expression. ATRA did not change ET-1 mRNA half-life. Transfection experiments using 5 kb of the ET-1 promoter-reporter gene construct which contains 5 kb of the preproET-1 promoter revealed that ATRA and Ch55 suppressed ET-1 promoter activity, resulting in down-regulation of ET-1 gene transcription. Taken together, retinoids may be another modulator of endothelial cell function through regulation of vasoactive substances at the transcription level.

Inhibition of lung cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor gene SEMA3B.

Semaphorins SEMA3B and its homologue SEMA3F are 3p21.3 candidate tumor suppressor genes (TSGs), the expression of which is frequently lost in lung cancers. To test the TSG candidacy of SEMA3B and SEMA3F, we transfected them into lung cancer NCI-H1299 cells, which do not express either gene. Colony formation of H1299 cells was reduced 90% after transfection with wild-type SEMA3B compared with the control vector. By contrast, only 30-40% reduction in colony formation was seen after the transfection of SEMA3F or SEMA3B variants carrying lung cancer-associated single amino acid missense mutations. H1299 cells transfected with wild-type but not mutant SEMA3B underwent apoptosis. We found that lung cancers (n = 34) always express the neuropilin-1 receptor for secreted semaphorins, whereas 82% expressed the neuropilin-2 receptor. Because SEMA3B and SEMA3F are secreted proteins, we tested conditioned medium from COS-7 cells transfected with SEMA3B and SEMA3F and found that medium from wild-type SEMA3B transfectants reduced the growth of several lung cancer lines 30-90%, whereas SEMA3B mutants or SEMA3F had little effect in the same assay. Sequencing of sodium bisulfite-treated DNA showed dense methylation of CpG sites in the SEMA3B 5' region of lung cancers not expressing SEMA3B but no methylation in SEMA3B-expressing tumors. These results are consistent with SEMA3B functioning as a TSG, the expression of which is inactivated frequently in lung cancers by allele loss and promoter region methylation.

Overexpression of candidate tumor suppressor gene FUS1 isolated from the 3p21.3 homozygous deletion region leads to G1 arrest and growth inhibition of lung cancer cells.

Recently we identified FUS1 as a candidate tumor suppressor gene (TSG) in the 120 kb 3p21.3 critical region contained in nested lung and breast cancer homozygous deletions. Mutation of FUS1 is infrequent in lung cancers which we have confirmed in 40 other primary lung cancers. In addition, we found no evidence for FUS1 promoter region methylation. Because haploinsufficiency or low expression of Fus1 may play a role in lung tumorigenesis, we tested the effect of exogenously induced overexpression of Fus1 protein and found 60-80% inhibition of colony formation for non-small cell lung cancer lines NCI-H1299 (showing allele loss for FUS1) and NCI-H322 (containing only a mutated FUS1 allele) in vitro. By contrast, a similar level of expression of a tumor-acquired mutant form of FUS1 protein did not significantly suppress colony formation. Also, induced expression of Fus1 under the control of an Ecdysone regulated promoter decreased colony formation 75%, increased the doubling time twofold, and arrested H1299 cells in G1. In conclusion, our data are consistent with the hypothesis that FUS1 may function as a 3p21.3 TSG, warranting further studies of its function in the pathogenesis of human cancers.

The OGG1 gene encodes a repair enzyme for oxidatively damaged DNA and is involved in human carcinogenesis.

8-Hydroxyguanine (oh8G) is a major base lesion produced by reactive oxygen species. oh8G in DNA causes G:C to T:A transversions and, thus, could be responsible for mutations that lead to carcinogenesis. A human DNA glycosylase/AP lyase encoded by the OGG1 gene has an activity to remove directly oh8G from DNA, and suppresses the mutagenic effect of oh8G. OGG1 protein has a helix-hairpin-helix-GPD motif as a domain for both DNA binding and catalysis, a nuclear localization signal, and a mitochondria targeting signal. Among multiple OGG1 isoforms, OGG1-type la is expressed predominantly in human cells and repairs chromosomal DNA in the nucleus. Inactivation of the OGG1 gene in yeast and mice leads to elevated spontaneous mutation frequency in the cells. The human OGG1 gene maps to chromosome 3p26.2, and allelic deletions of this region occur frequently in a variety of human cancers. Moreover, the OGG1 gene is somatically mutated in some cancer cells and is highly polymorphic among human populations. Repair activities of some mutated and polymorphic OGG1 proteins are lower than those of wild-type OGG1-type la-Ser326 protein and, thus, could be involved in human carcinogenesis.

OGG1 protein suppresses G:C-->T:A mutation in a shuttle vector containing 8-hydroxyguanine in human cells.

8-Hydroxyguanine (8-OHG) is an oxidatively damaged mutagenic base which causes G:C-->T:A transversions in DNA. OGG1 was cloned as a human gene encoding a DNA glycosylase that specifically excises 8-OHG from DNA in vitro. However, it was not clear whether OGG1 protein suppresses G:C-->T:A transversions caused by 8-OHG in human cells in vivo. In the present study we have examined the ability of OGG1 protein to suppress G:C-->T:A transversions caused by 8-OHG in human cells by bacterial suppressor tRNA (supF) forward mutation assay using a shuttle vector DNA, pMY189. Introduction of a single 8-OHG residue at position 159 of the supF gene in plasmid pMY189 resulted in a 130-fold increase in mutation frequency compared with untreated plasmid pMY189 after replication in the NCI-H1299 human lung cancer cell line. G:C-->T:A transversions at position 159 were detected in >90% of the supF mutants from the 8-OHG-containing plasmid. The mutation frequency of the 8-OHG-containing plasmid was significantly reduced by overexpression of OGG1 protein in NCI-H1299 cells and, in particular, the occurrence of G:C-->T:A transversion at position 159 in the supF gene was suppressed. Furthermore, frequencies and spectra of mutations of the untreated pMY189 plasmid did not differ significantly with overexpression of OGG1 protein. These results indicate that OGG1 protein has the ability to suppress G:C-->T:A transversions caused by 8-OHG in human cells in vivo.

DNA damage-inducible gene p33ING2 negatively regulates cell proliferation through acetylation of p53.

The p33ING1 protein is a regulator of cell cycle, senescence, and apoptosis. Three alternatively spliced transcripts of p33ING1 encode p47ING1a, p33ING1b, and p24ING1c. We cloned an additional ING family member, p33ING2/ING1L. Unlike p33ING1b, p33ING2 is induced by the DNA-damaging agents etoposide and neocarzinostatin. p33ING1b and p33ING2 negatively regulate cell growth and survival in a p53-dependent manner through induction of G(1)-phase cell-cycle arrest and apoptosis. p33ING2 strongly enhances the transcriptional-transactivation activity of p53. Furthermore, p33ING2 expression increases the acetylation of p53 at Lys-382. Taken together, p33ING2 is a DNA damage-inducible gene that negatively regulates cell proliferation through activation of p53 by enhancing its acetylation.

Germline p53 mutation in a patient with multiple primary cancers.

We report a case of multiple primary cancers having a germline missense mutation of the p53 gene. The patient was a Japanese female and had a history of five different types of cancers. PCR/direct sequencing analysis revealed the presence of a nucleotide substitution, AGC (Ser) to AGG (Arg), at codon 106 of the p53 gene in DNA from non-cancerous breast tissue. This is the first case of germline p53 mutation at codon 106, and could contribute to establishing correlations between the types and locations of germline p53 mutations and their phenotypical consequences.

Alterations in expression of E2F-1 and E2F-responsive genes by RB, p53 and p21(Sdi1/WAF1/Cip1) expression.

RB, p53 and p21(Sdi1/WAF1/Cip1) interact in the induction of G1 arrest. We established osteosarcoma cell lines in which a tetracycline-regulatable promoter controls the induction of RB, p53 and p21. By using these cell lines, we investigated whether RB, p53 or p21 regulates, in the same manner or differently, expression and function of E2F-1 and its responsive genes. E2F-1 gene products and transcripts of the E2F-responsive genes decreased in response to RB. Similar changes occurred to p53 and p21 when RB is present. However, in the absence of RB, some of the E2F-responsive genes decreased in response to p53 but not to p21. Thus, RB is a critical component for regulating the E2F-responsive genes, while p53 alone affects only a subset of these genes.

hOGG1 exon7 polymorphism and gastric cancer in case-control studies of Japanese Brazilians and non-Japanese Brazilians.

Polymorphism of hOGG1 may be capable of serving as a genetic marker for individual susceptibility to various cancers because of its role in the repair of oxyradical DNA damage. We examined the distribution of the hOGG1 Ser326Cys polymorphism and its presumed correlation with gastric cancer risk in two case-control studies of different ethnic groups in São Paulo, Brazil. Potentially eligible Japanese (JB) and non-Japanese Brazilian (NJB) case subjects were defined as patients with newly diagnosed malignant neoplasms of the stomach in 13 hospitals in São Paulo. Ninety-six JBs and 236 NJBs were adopted as subjects. Two controls were matched for each JB case, and one control for each NJB case. The subjects were interviewed using a questionnaire and their blood samples were collected. A significant difference in the distribution of this polymorphism between the two ethnic groups was observed (chi(2)=58.3, P<0.01). The mutant type (Ser/Cys or Cys/Cys) was predominant (approximately 65%) in the JBs, but was only present in approximately 40% of the NJBs. Logistic regression analysis showed no significant increased risk for either the Ser/Cys or Cys/Cys type in either group. The odds ratios of the Cys allele for gastric cancer were 1.01 (95% confidence interval (CI): 0.52-1.93) in the JBs and 0.85 (95% CI: 0.57-1.26) in the NJBs. In the NJBs, a significant increased risk of smoking was shown only in the Ser/Ser type, and no increased risk was shown in the genotypes with the Cys allele. However, no statistically significant interactions were observed with smoking or other possible confounding factors. No statistically significant difference in the distribution of the polymorphism was observed between the intestinal type and diffuse type of gastric cancer in either the JBs or the NJBs. The ethnic difference in hOGG1 Ser326Cys polymorphism was much greater than the case-control difference, and this polymorphism is unlikely to be associated with gastric cancer.