Whole-exome sequencing of human pancreatic cancers and characterization of genomic instability caused by MLH1 haploinsufficiency and complete deficiency.

Whole-exome sequencing (Exome-seq) has been successfully applied in several recent studies. We here sequenced the exomes of 15 pancreatic tumor cell lines and their matched normal samples. We captured 162,073 exons of 16,954 genes and sequenced the targeted regions to a mean coverage of 56-fold. This study identified a total of 1517 somatic mutations and validated 934 mutations by transcriptome sequencing. We detected recurrent mutations in 56 genes. Among them, 41 have not been described. The mutation rates varied widely among cell lines. The diversity of the mutation rates was significantly correlated with the distinct MLH1 copy-number status. Exome-seq revealed intensive genomic instability in a cell line with MLH1 homozygous deletion, indicated by a dramatically elevated rate of somatic substitutions, small insertions/deletions (indels), as well as indels in microsatellites. Notably, we found that MLH1 expression was decreased by nearly half in cell lines with an allelic loss of MLH1. While these cell lines were negative in conventional microsatellite instability assay, they showed a 10.5-fold increase in the rate of somatic indels, e.g., truncating indels in TP53 and TGFBR2, indicating MLH1 haploinsufficiency in the correction of DNA indel errors. We further analyzed the exomes of 15 renal cell carcinomas and confirmed MLH1 haploinsufficiency. We observed a much higher rate of indel mutations in the affected cases and identified recurrent truncating indels in several cancer genes such as VHL, PBRM1, and JARID1C. Together, our data suggest that MLH1 hemizygous deletion, through increasing the rate of indel mutations, could drive the development and progression of sporadic cancers.

Prospective randomized phase II study determines the clinical usefulness of genetic biomarkers for sensitivity to primary chemotherapy with paclitaxel in breast cancer.

In patients with breast cancer, taxane as well as anthracycline play central roles in systemic chemotherapy. By evaluating the pathological response, we can gauge sensitivity to primary chemotherapy. However, biomarkers that would predict a response to taxane have not yet been established. We conducted a prospective randomized trial to evaluate whether selecting patients using sensitivity testing based on the gene expression of the tumor might enhance the probability of the pathological response. Five genes were identified as biomarkers derived from a microarray of DNA gene profiles from microdisected breast tumors. In the experimental arm (B1), 12 cycles of weekly paclitaxel, 80 mg/m(2) , were preoperatively given when the sensitivity test was positive and therefore judged to be sensitive to paclitaxel. When the test was negative, meaning insensitive to paclitaxel, four cycles of FEC100 were given (arm B2). In the control arm (A), paclitaxel was administered weekly without the use of the sensitivity test. A total of 92 patients were enrolled and 86 patients were analyzed. The pathological response rate (pRR) of each arm was 36.4% in B1 (expected sensitive to paclitaxel), 21.1% in A (control) and 12.5% in B2, respectively. Weekly paclitaxel-treated patients selected by the sensitivity test did not enhance the pRR. The study failed to validate sensitivity testing using five gene expressions for primary chemotherapy with paclitaxel in patients with breast cancer. However, this study suggests that a randomized phase II study is a robust tool for obtaining a rapid conclusion on the usefulness of biomarkers and could be the foundation for further large clinical trials.

Function of EWS-POU5F1 in sarcomagenesis and tumor cell maintenance.

POU5F1 is a transcription factor essential for the self-renewal activity and pluripotency of embryonic stem cells and germ cells. We have previously reported that POU5F1 is fused to EWSR1 in a case of undifferentiated sarcoma with chromosomal translocation t(6;22)(p21;q12). In addition, the EWS-POU5F1 chimeras have been recently identified in human neoplasms of the skin and salivary glands. To clarify the roles of the EWS-POU5F1 chimera in tumorigenesis and tumor cell maintenance, we used small-interfering RNA-mediated gene silencing. Knockdown of EWS-POU5F1 in the t(6;22) sarcoma-derived GBS6 cell line resulted in a significant decrease of cell proliferation because of G1 cell cycle arrest associated with p27(Kip1) up-regulation. Moreover, senescence-like morphological changes accompanied by actin polymerization were observed. In contrast, EWS-POU5F1 down-regulation markedly increased the cell migration and invasion as well as activation of metalloproteinase 2 and metalloproteinase 14. The results indicate that the proliferative activity of cancer cells and cell motility are discrete processes in multistep carcinogenesis. These findings reveal the functional role of the sarcoma-related chimeric protein as well as POU5F1 in the development and progression of human neoplasms.

Reduced levels of ATF-2 predispose mice to mammary tumors.

Transcription factor ATF-2 is a nuclear target of stress-activated protein kinases, such as p38, which are activated by various extracellular stresses, including UV light. Here, we show that ATF-2 plays a critical role in hypoxia- and high-cell-density-induced apoptosis and the development of mammary tumors. Compared to wild-type cells, Atf-2(-/-) mouse embryonic fibroblasts (MEFs) were more resistant to hypoxia- and anisomycin-induced apoptosis but remained equally susceptible to other stresses, including UV. Atf-2(-/-) and Atf-2(+/-) MEFs could not express a group of genes, such as Gadd45alpha, whose overexpression can induce apoptosis, in response to hypoxia. Atf-2(-/-) MEFs also had a higher saturation density than wild-type cells and expressed lower levels of Maspin, the breast cancer tumor suppressor, which is also known to enhance cellular sensitivity to apoptotic stimuli. Atf-2(-/-) MEFs underwent a lower degree of apoptosis at high cell density than wild-type cells. Atf-2(+/-) mice were highly prone to mammary tumors that expressed reduced levels of Gadd45alpha and Maspin. The ATF-2 mRNA levels in human breast cancers were lower than those in normal breast tissue. Thus, ATF-2 acts as a tumor susceptibility gene of mammary tumors, at least partly, by activating a group of target genes, including Maspin and Gadd45alpha.

Novel anti-filamin-A antibody detects a secreted variant of filamin-A in plasma from patients with breast carcinoma and high-grade astrocytoma.

Identification of tumor-derived proteins in the circulation may allow for early detection of cancer and evaluation of therapeutic responses. To identify circulating tumor-derived proteins, mice were immunized with concentrated culture medium conditioned by human breast cancer cells. Antibodies generated by hybridomas were screened against conditioned media from both normal epithelial cells and tumor cells. Antibody selectively reacting with tumor cell-conditioned media was further characterized. This led to the development of a monoclonal antibody (Alper-p280) that reacts with a newly identified 280-kDa secreted variant of human filamin-A. Circulating filamin-A was detected in patient plasma samples using Alper-p280 in an ELISA assay. Human plasma samples from 134 patients with brain, breast, or ovarian cancer, 15 patients with active arthritis, and 76 healthy controls were analyzed. Filamin-A protein levels in human cell lines and tissues were analyzed by western blotting, immunohistochemistry, and electron and confocal microscopy. Circulating filamin-A was detected in the plasma of 109 of 143 patients with breast cancer and primary brain tumors. Plasma levels of filamin-A showed 89.5% sensitivity (95% confidence interval [CI] = 0.67% to 0.99%) and 97.8% specificity (95% CI = 0.88% to 0.99%) for glioblastoma at a cut-off of 21.0 ng/mL. Plasma levels of filamin-A (>36.0 ng/mL) had 96.7% sensitivity (95% CI = 0.80% to 0.99%) and 67.8% specificity (95% CI = 0.54% to 0.79%) for metastatic breast cancer. Filamin-A levels were increased in malignant breast or brain tissues, but not in normal control tissues. Filamin-A localized to lysosomes in MDA.MB.231 breast cancer cells, but not in normal human mammary epithelial cells, suggesting that filamin-A may undergo cancer-specific processing. Plasma filamin-A appears to be a specific and sensitive marker for patients with high-grade astrocytoma or metastatic breast cancer. Additional novel cancer biomarkers have been identified and are being developed alongside Alper-p280 for use in diagnosis of breast carcinoma and high-grade astrocytoma, and for use in the evaluation of therapeutic responses.

Identification of novel deletion polymorphisms in breast cancer.

Breast cancer is the most frequent cancer in females worldwide and it has long been known that multiple genetic rearrangements correlate with complex biology and clinical behavior. In addition, copy number variations (CNVs) of DNA sequences account for a significant proportion of normal phenotypic variation and may have an important role in human pathological variation. In this study, we carried out a high-density oligonucleotide array comparative genomic hybridization (CGH) analyses in a series of breast cancer cell lines to identify novel homozygous deletion loci. The results were confirmed by quantitative PCR (Q-PCR) and 4 genes, the REV1L, ZNF14, NPAS1 and APOBEC3B genes, were selected. Analyses of 30 microdissected human breast tumors and paired normal mammary tissue samples indicated that these homozygous deletions are small-scale deletion polymorphisms. The variation in copy number at the loci of the 4 genes in blood-derived DNA demonstrated the frequency of deletions including homozygous deletions and single copy variants to be higher in breast cancer patients than healthy females. Notably, the homozygous deletion of APOBEC3B involved part of exon 5 and seemed to be cancer-specific in some patients, indicating that this is a functionally important structural variant. These copy number changes may play an important role in breast cancer and array-CGH analyses can thus be expected to provide new insight into the genetic background of breast cancer.

Gene expression profiling of breast cancer.

Numerous genes are controlled by complex regulatory networks and involved in the development and progression of breast cancer, and these genes are the key factors determining each characteristic of the tumor. Gene expression profiling, a large scale analysis of gene expression, has created new possibilities for the molecular characterization of cancer. Systematic analysis of expression patterns of thousands of genes in tumor cells using DNA microarrays and correlation of these patterns to specific features of phenotypic variation may provide the basis for an improved taxonomy of cancer. These profiles have the potential to explain the genetic heterogeneity of breast cancer and allow treatment strategies to be planned in accordance with their probability of success in individual patients.

[Gene expression profiling for prediction of response to chemotherapy].

Numerous genes whose expression is controlled by complex regulatory networks are involved in the development and progression of each cancer,and those genes will be the key factors for determining each characteristic of the tumor. The recent development of DNA microarray and related technologies provides an opportunity to perform more detailed characterization( profiling) of individual tumor cells. Indeed, the gene expression profile of a tumor provides a unique molecular portrait or signature that can be correlated with clinical behavior and drug responsiveness. The development of personalized( or customized) medicine and molecularly targeted drugs is enthusiastically awaited based on the results of research examining genomic diversity such as SNPs( single nucleotide polymorphisms), gene expression profiling with DNA microarrays, and analysis of protein expression and interactions.

Amplification of the BCAS2 gene at chromosome 1p13.3-21 in human primary breast cancer.

BCAS2 is a novel gene isolated from breast cancer cell line by differential display technique. Previously we reported that BCAS2 gene is localized on chromosome 1p13.3-21 and is up-regulated by gene amplification in breast cancer cell lines MCF-7 and BT-20. In this study, we investigated the amplification of the BCAS2 gene in a series of 104 gynecological primary tumors by means of Southern blot analysis. The BCAS2 gene was amplified in two of 60 primary breast cancer tissues, whereas no amplification was detected in any of endometrial (0/26) and cervical (0/18) tumor tissues. Gene amplification was also not detected in a series of pancreatic (0/9) and gastric (0/6) cancer cell lines. An enhanced green fluorescent protein assay revealed that BCAS2 protein seems to be translocated into the nucleus. Although frequent deletions of the proximal region of chromosome 1p13.3-21 have been found in primary breast cancer, our results support first evidence of amplification within this region and indicate that BCAS2 gene codes for a nuclear protein.

Expression and regulation of tumor suppressor gene maspin in human bladder cancer.

Maspin is a member of serine protease inhibitor family with tumor suppressing activity for breast and prostate cancers, acting at the level of tumor invasion and metastasis. However, there have been no published data regarding the role of maspin in human bladder cancer. We evaluated maspin expression in 65 series of bladder cancer samples (22 transurethral resection (TUR) and 43 radical cystectomy) and studied the regulatory mechanism of maspin gene activation in bladder cancer cells. Maspin expression was immunohistochemically detected in four (18.2%) patients with TUR and 22 (51.2%) patients with radical cystectomy whereas no expression was observed in normal transitional cells located at tumor-free area in bladder. The maspin expression was significantly correlated with the development of muscle invasive bladder cancer (P=0.00008). Using a luciferase reporter system, maspin promoter activity was induced in the maspin-positive bladder cancer cell lines as well as maspin-negative RT4 cells. Furthermore, treatment with the DNA methyltransferase inhibitor, 5-aza-2' deoxycytidine, and histone deacetylase inhibitor, trichostatin A, led to re-expression of maspin in RT4 cells. Our results indicate that maspin may contribute to bladder cancer development and that DNA methylation and histone deacetylation may be important for regulating maspin gene activation in bladder cancer cells.

Clinicopathological significance and molecular regulation of maspin expression in ductal adenocarcinoma of the pancreas.

We evaluated the biological relevance of maspin expression in pancreatic ductal adenocarcinoma and studied regulatory mechanisms of maspin gene activation in pancreatic carcinoma cell lines. Maspin expression was immunohistochemically detected in a series of 57 pancreatic ductal adenocarcinomas, 51 (90%) of which were classified as high-expressers. In lymph node metastases, maspin expression was somewhat decreasingly found in 39/49 (80%). Maspin high-expressers showed predominantly a low histological grade (p=0.013). Moreover, maspin expression was found in two mixed ductal-endocrine carcinomas, but not in 10 endocrine tumors and the surrounding normal pancreatic tissues. Using a luciferase reporter system, maspin promoter activity was induced in the maspin-positive pancreatic cancer cell lines as well as maspin-negative PANC-1 cells. Additionally, treatment with the DNA methyltransferase inhibitor, 5-aza-2' deoxycytidine, and histone deacetylase inhibitor, trichostatin A, led to re-expression of maspin mRNA in PANC-1 cells. Our results indicate that maspin expression is up-regulated in most if not all pancreatic ductal adenocarcinomas and may be related to the development and differentiation, and that DNA methylation and histone deacetylation may suppress maspin gene activation in pancreatic cancer cells.

Prognostic significance of the maspin tumor suppressor gene in pulmonary adenocarcinoma.

PURPOSE: Maspin is a member of the serpin family and has tumor suppressor activity. We evaluated maspin expression in pulmonary adenocarcinoma in relation to a number of clinicopathological features. METHODS: Maspin expression was examined immunohistochemically in a series of 78 pulmonary adenocarcinomas by the EnVision ChemMate method. RESULTS: . Thirty-seven of 78 cases (47%) showed distinct maspin expression (maspin-positive group) and 41 (53%) did not (maspin-negative group). Maspin expression was not associated significantly with most clinicopathological variables including sex, age, tumor size, primary tumor, lymph node metastasis, visceral pleural invasion, pulmonary metastasis, and disease stage. However, the maspin-positive group had a better 5-year survival rate (62%) than did the maspin-negative group (42%). The difference in the 5-year survival rate was greatest in stage II patients (maspin-positive group, 69%; maspin-negative group, 17%; P = 0.048). CONCLUSION: Our data indicate that maspin has prognostic significance for pulmonary adenocarcinoma. A better understanding of the role of maspin in tumor suppression may be helpful for development of novel chemotherapies for patients with this deadly tumor.

Expression and regulation of tumor suppressor gene maspin in breast cancer.

Maspin (mammary serpin) is an inhibitor of serine proteases with tumor suppressor activity in breast cancer. Maspin was originally identified by subtractive hybridization in normal breast epithelial cells, but its expression decreased during tumor progression. The loss of maspin gene expression with increasing malignancy is by transcriptional regulation. Maspin is known to be involved in invasion and metastasis, interact with the p53 tumor suppressor pathway, and act as an inhibitor of angiogenesis. The immunohistochemical analysis and reverse-transcription polymerase chain reaction of maspin in normal human breast tissue and breast carcinoma indicated a stepwise reduction of maspin expression during the progression from ductal carcinoma in situ to invasive carcinoma to lymph node metastasis. The lack of maspin expression in breast cancer seems to be associated with a short disease-free survival and supports maspin's function as an indicator for tumor aggressiveness and metastatic potential. New studies on the gene regulation of maspin provide evidence for promising potential of possible re-expression of maspin in tumor cells. The function of maspin as a tumor suppressor gene involved in tumor invasion, metastasis, and angiogenesis may not be limited to breast cancer.

Development of detection method for novel fusion gene using GeneChip exon array.

BACKGROUND: Fusion genes have been recognized to play key roles in oncogenesis. Though, many techniques have been developed for genome-wide analysis of fusion genes, a more efficient method is desired. RESULTS: We introduced a new method of detecting the novel fusion gene by using GeneChip Exon Array that enables exon expression analysis on a whole-genome scale and TAIL-PCR. To screen genes with abnormal exon expression profiles, we developed computational program, and confirmed that the program was able to search the fusion partner gene using Exon Array data of T-cell acute lymphocytic leukemia (T-ALL) cell lines. It was reported that the T-ALL cell lines, ALL-SIL, BE13 and LOUCY, harbored the fusion gene NUP214-ABL1, NUP214-ABL1 and SET-NUP214, respectively. The program extracted the candidate genes with abnormal exon expression profiles: 1 gene in ALL-SIL, 1 gene in BE13, and 2 genes in LOUCY. The known fusion partner gene NUP214 was included in the genes in ALL-SIL and LOUCY. Thus, we applied the proposed program to the detection of fusion partner genes in other tumors. To discover novel fusion genes, we examined 24 breast cancer cell lines and 20 pancreatic cancer cell lines by using the program. As a result, 20 and 23 candidate genes were obtained for the breast and pancreatic cancer cell lines respectively, and seven genes were selected as the final candidate gene based on information of the EST data base, comparison with normal cell samples and visual inspection of Exon expression profile. Finding of fusion partners for the final candidate genes was tried by TAIL-PCR, and three novel fusion genes were identified. CONCLUSIONS: The usefulness of our detection method was confirmed. Using this method for more samples, it is thought that fusion genes can be identified.

Prognostic value of matrix Gla protein in breast cancer.

To assess the prognostic value of matrix Gla protein (MGP) expression in cases of breast cancer, 9 samples from patients diagnosed with breast cancer who were followed up for more than 10 years were microdissected and then analyzed using Affymetrix U133 Plus 2.0 Arrays. Genes that exhibited significant differences in expression between patients with a good prognosis and those with a poor prognosis were identified. The MGP gene was among the genes up-regulated in cases where the prognosis was poor, indicating that the mRNA levels of MGP are a potential prognostic indicator of breast cancer. However, immunohistostaining of breast tissue microarrays (n=207) did not reveal a correlation between the protein expression of MGP and overall survival, neither was there a correlation between the protein expression of MGP and ER status or bone metastasis. In breast cancer cases, the mRNA level of MGP may be a marker indicating poor prognosis; however, protein expression determined by immunohistostaining is not.

SKI and MEL1 cooperate to inhibit transforming growth factor-beta signal in gastric cancer cells.

Chromosomal amplification occurs frequently in solid tumors and is associated with poor prognosis. Several reports demonstrated the cooperative effects of oncogenic factors in the same amplicon during cancer development. However, the functional correlation between the factors remains unclear. Transforming growth factor (TGF)-beta signaling plays important roles in cytostasis and normal epithelium differentiation, and alterations in TGF-beta signaling have been identified in many malignancies. Here, we demonstrated that transcriptional co-repressors of TGF-beta signaling, SKI and MDS1/EVI1-like gene 1 (MEL1), were aberrantly expressed in MKN28 gastric cancer cells by chromosomal co-amplification of 1p36.32. SKI and MEL1 knockdown synergistically restored TGF-beta responsiveness in MKN28 cells and reduced tumor growth in vivo. MEL1 interacted with SKI and inhibited TGF-beta signaling by stabilizing the inactive Smad3-SKI complex on the promoter of TGF-beta target genes. These findings reveal a novel mechanism where distinct transcriptional co-repressors are co-amplified and functionally interact, and provide molecular targets for gastric cancer treatment.

Molecular prediction of the therapeutic response to neoadjuvant chemotherapy in breast cancer.

Breast cancer is considered to be relatively sensitive to chemotherapy, and multiple combinations of cytotoxic agents are used as standard therapy. Chemotherapy is applied empirically despite the observation that not all regimens are equally effective across the population of patients. Up to date clinical tests for predicting cancer chemotherapy response are not available, and individual markers have shown little predictive value. A number of microarray studies have demonstrated the use of genomic data, particularly gene expression signatures, as clinical prognostic factors in breast cancer. The identification of patient subpopulations most likely to respond to therapy is a central goal of recent personalized medicine. We have designed experiments to identify gene sets that will predict treatment-specific response in breast cancer. Taken together with our recent trial about the construction of a high-throughput functional screening system for chemo-sensitivity related genes, studies for drug sensitivity will provide rational strategies for establishment of the prediction system with high accuracy, and identification of ideal targets for drug intervention.

Functional pathway characterized by gene expression analysis of supraclavicular lymph node metastasis-positive breast cancer.

The outcome of breast cancer patients with supraclavicular lymph node metastasis is generally poor, but some patients do survive for a long time. Consequently, the ability to predict the outcome is important in terms of choosing the appropriate therapy for breast cancer patients with supraclavicular lymph node metastasis. In this study, we attempted to identify functional pathways that determine the outcome of breast cancer patients with supraclavicular lymph node metastasis by profiling cDNA microarrays. Thirty-one breast cancer patients with supraclavicular lymph node metastasis without distant metastasis comprised the study cohort; these were divided into three groups based on prognosis - poor, intermediate, and good. Two functional pathways, the Wnt signaling pathway and the mitochondrial apoptosis pathway, were constructed using six genes (DVL1, VDAC2, BIRC5, Stathmin1, PARP1, and RAD21) that were differently expressed between the good and poor outcome groups. Our results indicate that these two functional pathways may play an important role in determining the outcome of breast cancer patients with supraclavicular lymph node metastasis. We also determined that immunohistostaining for the Stathmin1 gene product is a potential tool for predicting the outcome of breast cancer patients with supraclavicular lymph node metastasis.

Common peak approach using mass spectrometry data sets for predicting the effects of anticancer drugs on breast cancer.

We propose a method for biomarker discovery from mass spectrometry data, improving the common peak approach developed by Fushiki et al. (BMC Bioinformatics, 7:358, 2006). The common peak method is a simple way to select the sensible peaks that are shared with many subjects among all detected peaks by combining a standard spectrum alignment and kernel density estimates. The key idea of our proposed method is to apply the common peak approach to each class label separately. Hence, the proposed method gains more informative peaks for predicting class labels, while minor peaks associated with specific subjects are deleted correctly. We used a SELDI-TOF MS data set from laser microdissected cancer tissues for predicting the treatment effects of neoadjuvant therapy using an anticancer drug on breast cancer patients. The AdaBoost algorithm is adopted for pattern recognition, based on the set of candidate peaks selected by the proposed method. The analysis gives good performance in the sense of test errors for classifying the class labels for a given feature vector of selected peak values.