Increased oxidative stress in AOA3 cells disturbs ATM-dependent DNA damage responses.

Ataxia telangiectasia (AT) is caused by a mutation in the ataxia-telangiectasia-mutated (ATM) gene; the condition is associated with hyper-radiosensitivity, abnormal cell-cycle checkpoints, and genomic instability. AT patients also show cerebellar ataxia, possibly due to reactive oxygen species (ROS) sensitivity in neural cells. The ATM protein is a key regulator of the DNA damage response. Recently, several AT-like disorders have been reported. The genes responsible for them are predicted to encode proteins that interact with ATM in the DNA-damage response. Ataxia with oculomotor apraxia types 1-3 (AOA1, 2, and 3) result in a neurodegenerative and cellular phenotype similar to AT; however, the basis of this phenotypic similarity is unclear. Here, we show that the cells of AOA3 patients display aberrant ATM-dependent phosphorylation and apoptosis following γ-irradiation. The ATM-dependent response to H2O2 treatment was abrogated in AOA3 cells. Furthermore, AOA3 cells had reduced ATM activity. Our results suggest that the attenuated ATM-related response is caused by an increase in endogenous ROS in AOA3 cells. Pretreatment of cells with pyocyanin, which induces endogenous ROS production, abolished the ATM-dependent response. Moreover, AOA3 cells had decreased homologous recombination (HR) activity, and pyocyanin pretreatment reduced HR activity in HeLa cells. These results indicate that excess endogenous ROS represses the ATM-dependent cellular response and HR repair in AOA3 cells. Since the ATM-dependent cell-cycle checkpoint is an important block to carcinogenesis, such inactivation of ATM may lead to tumorigenesis as well as neurodegeneration.

WRN participates in translesion synthesis pathway through interaction with NBS1.

Werner syndrome (WS), caused by mutation of the WRN gene, is an autosomal recessive disorder associated with premature aging and predisposition to cancer. WRN belongs to the RecQ DNA helicase family, members of which play a role in maintaining genomic stability. Here, we demonstrate that WRN rapidly forms discrete nuclear foci in an NBS1-dependent manner following DNA damage. NBS1 physically interacts with WRN through its FHA domain, which interaction is important for the phosphorylation of WRN. WRN subsequently forms DNA damage-dependent foci during the S phase, but not in the G1 phase. WS cells exhibit an increase in spontaneous focus formation of poleta and Rad18, which are important for translesion synthesis (TLS). WRN also interacts with PCNA in the absence of DNA damage, but DNA damage induces the dissociation of PCNA from WRN, leading to the ubiquitination of PCNA, which is essential for TLS. This dissociation correlates with ATM/NBS1-dependent degradation of WRN. Moreover, WS cells show constitutive ubiquitination of PCNA and interaction between PCNA and Rad18 E3 ligase in the absence of DNA damage. Taken together, these results indicate that WRN participates in the TLS pathway to prevent genomic instability in an ATM/NBS1-dependent manner.

Correlating phosphatidylinositol 3-kinase inhibitor efficacy with signaling pathway status: in silico and biological evaluations.

The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers, and several agents targeting this pathway including PI3K/Akt/mammalian target of rapamycin inhibitors have recently entered clinical trials. One question is whether the efficacy of a PI3K pathway inhibitor can be predicted based on the activation status of pathway members. In this study, we examined the mutation, expression, and phosphorylation status of PI3K and Ras pathway members in a panel of 39 pharmacologically well-characterized human cancer cell lines (JFCR39). Additionally, we evaluated the in vitro efficacy of 25 PI3K pathway inhibitors in addition to conventional anticancer drugs, combining these data to construct an integrated database of pathway activation status and drug efficacies (JFCR39-DB). In silico analysis of JFCR39-DB enabled us to evaluate correlations between the status of pathway members and the efficacy of PI3K inhibitors. For example, phospho-Akt and KRAS/BRAF mutations prominently correlated with the efficacy and the inefficacy of PI3K inhibitors, respectively, whereas PIK3CA mutation and PTEN loss did not. These correlations were confirmed in human tumor xenografts in vivo, consistent with their ability to serve as predictive biomarkers. Our findings show that JFCR39-DB is a useful tool to identify predictive biomarkers and to study the molecular pharmacology of the PI3K pathway in cancer.

Correlation between morphology and EGFR mutations in lung adenocarcinomas Significance of the micropapillary pattern and the hobnail cell type.

The presence of epidermal growth factor receptor (EGFR) tyrosine kinase (TK) mutations significantly correlates with tumor sensitivity to TK inhibitors, particularly in lung adenocarcinomas, the predominant histological subtype in Japan and the United States. To clarify links between EGFR mutations and pathological findings in Japanese lung cancer, detailed pathological features of adenocarcinomas were examined using the WHO criteria as well as our cell type classification (hobnail, columnar and polygonal). Medical records were reviewed for a total of 107 surgically resected tumors. Clinicopathological factors were examined and correlations with EGFR status were evaluated. EGFR mutations were found in 63 patients (59%) distributed through all four exons examined (through exons 18-21). EGFR mutations were significantly associated with female gender (P=0.003), non-smoker status (P=0.008) and hobnail cell morphology (P<0.00001). In addition, detailed pathological examination showed significant associations with bronchioloalveolar carcinoma (BAC) component and a micropapillary pattern (MPP) (P=0.012 and 0.043, respectively). We conclude that characteristic histological features, i.e. the hobnail cell morphology and the presence of BAC component and MPP are good predictors of EGFR mutations in lung adenocarcinoma.

EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers.

INTRODUCTION: Very recently, we have found a novel fusion product between the echinoderm microtubule-associated protein-like4 (EML4) and the anaplastic lymphoma kinase (ALK) in non-small cell lung cancers (NSCLCs). Tumors featuring EML4-ALK fusion constitute one subtype of NSCLC that might be highly sensitive to ALK inhibitors. Herein, we present results of a first large scale study of EML4-ALK fusion in lung cancers. METHODS: Using reverse transcription-polymerase chain reaction for EML4-ALK fusion mRNA, we investigated 149 lung adenocarcinomas, 48 squamous cell carcinomas, 3 large-cell neuroendocrine carcinomas, and 21 small-cell carcinomas. For EML4-ALK-positive cancers, we further investigated the presence of ALK fusion proteins by immunohistochemistry. RESULTS: Five of 149 adenocarcinomas (3.4%) showed EML4-ALK fusion mRNA, this being totally lacking in carcinomas of other types (0/72). In all the fusion-positive cases, ALK fusion protein could be detected in the cytoplasm immunohistochemically. The five fusion cases featured two EML4-ALK variant 1 fusions and three variant 2 fusions. Histologically, both variant 1 cases were mixed type adenocarcinomas, showing papillary with bronchioloalveolar components. Interestingly, all three variant 2 cases were acinar adenocarcinomas, the link being statistically significant (p = 0.00018). None of the five fusion-positive cases demonstrated any mutations of EGFR or KRAS, pointing to a mutually exclusive relationship (p = 0.014). There was no association with smoking habits. CONCLUSIONS: In the present first investigation of EML4-ALK fusion in a large study of lung cancers (5/221), we found an interesting histotype-genotype relationship. Furthermore, we could detect the fusion protein by immunohistochemistry, pointing to possible clinical applications.

HOXB2 as a novel prognostic indicator for stage I lung adenocarcinomas.

BACKGROUND: Outcomes of patients with lung adenocarcinomas can be predicted to some extent from the pathologic stage (p-stage). Although all attempts are made to fully remove cancer lesions, still a number of p-stage I patients without metastatic disease at the time of surgery develop recurrences and die of cancer. It is thus very important to identify p-stage I patients who are at risk of recurrence. METHODS: Previously, using microdissected samples, we identified metastasis-related genes. Using real-time reverse-transcriptase polymerase chain reaction analysis, we investigated the transcriptional levels of the top metastasis-related genes using 96 independent test lung adenocarcinoma samples and investigated their correlations with the prognosis. RESULTS AND CONCLUSIONS: We document evidence that p-stage I patients with HOXB2 up-regulation have a worse prognosis than those with HOXB2 down-regulation (p = 0.0065), whereas the HOXB2 status has no prognostic significance for p-stage II-IV patients. Comparing tumors and corresponding normal lung tissue, we confirmed HOXB2 up-regulated lesions to have much higher HOXB2 expression than the corresponding normal tissue. Confirmation with a larger number of samples is needed, with further research to clarify the molecular functions of HOXB2.

Up-regulation of PTEN at the transcriptional level is an adverse prognostic factor in female lung adenocarcinomas.

In lung adenocarcinomas, genetic alterations of PTEN are relatively rare and little has been reported concerning the relationship between PTEN transcriptional level and clinicopathologic features or genetic changes. This study was conducted to gain insight into clinicopathologic correlations. The transcriptional levels of PTEN were examined using real time RT-PCR and analyzed for correlations with clinicopathologic features and the mutation status of EGFR and KRAS. After confirming significant correlation for PTEN levels between macrodissected and microdissected materials (p<0.01), macrodissected samples from 115 lung adenocarcinomas were examined. There were no significant difference between the PTEN levels, divided into three ranges, and the mutation status of EGFR or KRAS. Noteworthy clinicopathologic correlations between PTEN transcriptional up/down-regulation and young age (p=0.0081, 61.7+/-8.7years versus 66.1+/-8.1years), smoking (p=0.032) and less differentiated adenocarcinomas (p=0.013) were identified. Whereas male patients demonstrated no prognostic association with PTEN levels, female cases with up-regulated PTEN expression had significantly worse survival compared with those with normal PTEN levels (p=0.0027). This study revealed distinct clinicopathologic correlations with PTEN transcriptional up/down-regulation.

A metastatic signature in entire lung adenocarcinomas irrespective of morphological heterogeneity.

Recent microarray expression studies support the hypothesis that metastatic potential is acquired early in tumorigenesis and that most tumor cells have the potential to metastasize. To assess this possibility, we investigated invasive lung adenocarcinomas, which characteristically display morphological heterogeneity with a less malignant appearance at the periphery as a model. In lymph node-positive lesions, gene expression profiles were compared among moderately differentiated components with an aggressive appearance, peripheral well-differentiated components with a less malignant appearance, and patient-matched lymph node metastases. We also compared these with node-negative lung adenocarcinomas, which are morphologically indistinguishable from node-positive tumors. Striking similarities were observed between pairs of primary and metastatic tumors, even within primary well-differentiated components. We generated a 75-gene signature separating primary lung adenocarcinomas according to lymph node status. Hierarchical clustering using this gene set identified a distinct independent group composed of node-positive cases, clearly separate from node-negative tumors and normal lung tissue. The results suggest that the metastatic signature is maintained throughout progression, implying that the entirety of a primary tumor, including the morphologically less malignant components, might have metastatic potential. This finding has profound clinical implications. In the future, the metastatic potential of tumors may be predicted by biopsy, helping to avoid unnecessary lymph node dissection in low-risk patients.

Transcription factor single-minded 2 (SIM2) is ubiquitinated by the RING-IBR-RING-type E3 ubiquitin ligases.

Human single-minded 2 (SIM2) is a member of the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors and is associated with the etiology of Down syndrome phenotype. Here, we examined a possibility of the post-translational modification of SIM2 protein by transfecting various expression constructs followed by the analysis with immunoprecipitation and Western blotting. In fact, transient expression of SIM2 cDNA in HEK293 cells revealed poly-ubiquitination of SIM2 protein. In the stable transfectants, a proteasome inhibitor MG132 protected the poly-ubiquitinated SIM2 protein from degradation. Furthermore, in the cells co-transfected with SIM2 and each of four different E3 ubiquitin ligases, SIM2 was immunoprecipitated with the RING-IBR-RING-type E3 ubiquitin ligases, Parkin and HHARI, but it was not immunoprecipitated with other E3 ligases, such as one RING-type Siah-1 and the PHD type AIRE. A series of deletion constructs revealed that Parkin actually binds to SIM2 with the IBR (294-377)-RING2 (378-465) domains and that the sites for poly-ubiquitination of SIM2 reside within the PAS1-PAS2 region (aa 141-289). We postulated that transcription factor SIM2 and E3 ubiquitin ligase Parkin may interact each other to play an important physiological role in the brain development which is controlled by ubiquitination.

Identification of a novel zinc finger protein gene (ZNF298) in the GAP2 of human chromosome 21q.

We have isolated a novel zinc finger protein gene, designated ZNF298, as a candidate gene for a particular phenotype of Down syndrome or bipolar affective disorder (BPAD) which maps to human chromosome 21q22.3. ZNF298 gene consists of 25 exons spanning approximately 80kb in a direction from the telomere to centromere. There are four kinds of transcripts that harbor three types of 3' UTR. These four transcripts (ZNF298a, ZNF298b, ZNF298c, and ZNF298d) contain putative open reading frames encoding 1178, 1198, 555, and 515 amino acids, respectively. ZNF298 gene was ubiquitously expressed in various tissues at very low level. The protein motif analysis revealed that ZNF298 proteins contain a SET [Su(var)3-9, Enhancer-of-zeste, Trithorax] domain, multiple C2H2-type zinc finger (ZnF_C2H2) domains, several nuclear localization signals (NLSs), and PEST sequences. Nuclear localization of ZNF298 protein was confirmed by transfection of expression vector of GFP-tagged protein into two human cell lines. Interestingly, this gene crosses over a clone gap (GAP2) remaining in the band 21q22.3. We obtained the DNA fragments corresponding to GAP2 using ZNF298 cDNA sequence as anchor primers for PCR and determined its genomic DNA sequence.

Initial characterization of an uromodulin-like 1 gene on human chromosome 21q22.3.

We have isolated a novel gene, designated UMODL1, similar to uromodulin (UMOD)/Tamm-Horsfall glycoprotein, on human chromosome 21q22.3. Uromodulin like-1 (UMODL1) consists of 22 exons and spans approximately 80 kb in a direction from centromere to telomere. Two major transcripts produced by alternative splicing have been identified. These transcripts contain open reading frames of 4125 and 3741 bp encoding proteins of 1374 and 1246 amino acids, respectively. Expression of UMODL1 mRNA was detected only in 14 human tissues, e.g., kidney, testis, and fetal thymus at low level. Interestingly, two gene products (UMODL1L and UMODL1S) contain multiple domains including whey acidic protein, sea urchin sperm protein, enterokinase, and agrin, zona pellucida domain, and so on. Both proteins seemed to localize in cytoplasm, but UMODL1 is likely to be ubiquitinated and rapidly degraded in HEK293 cells. This gene may be a potent candidate for Down syndrome or bipolar affective disorder.

The transmembrane serine protease (TMPRSS3) mutated in deafness DFNB8/10 activates the epithelial sodium channel (ENaC) in vitro.

TMPRSS3 encodes a transmembrane serine protease that contains both LDLRA and SRCR domains and is mutated in non-syndromic autosomal recessive deafness (DFNB8/10). To study its function, we cloned the mouse ortholog which maps to Mmu17, which is structurally similar to the human gene and encodes a polypeptide with 88% identity to the human protein. RT-PCR and RNA in situ hybridization on rat and mouse cochlea revealed that Tmprss3 is expressed in the spiral ganglion, the cells supporting the organ of Corti and the stria vascularis. RT-PCR on mouse tissues showed expression in the thymus, stomach, testis and E19 embryos. Transient expression of wild-type or tagged TMPRSS3 protein showed a primary localization in the endoplasmic reticulum. The epithelial amiloride-sensitive sodium channel (ENaC), which is expressed in many sodium-reabsorbing tissues including the inner ear and is regulated by membrane-bound channel activating serine proteases (CAPs), is a potential substrate of TMPRSS3. In the Xenopus oocyte expression system, proteolytic processing of TMPRSS3 was associated with increased ENaC mediated currents. In contrast, 6 TMPRSS3 mutants (D103G, R109W, C194F, W251C, P404L, C407R) causing deafness and a mutant in the catalytic triad of TMPRSS3 (S401A), failed to undergo proteolytic cleavage and activate ENaC. These data indicate that important signaling pathways in the inner ear are controlled by proteolytic cleavage and suggest: (i) the existence of an auto-catalytic processing by which TMPRSS3 would become active, and (ii) that ENaC could be a substrate of TMPRSS3 in the inner ear.