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Oncogene overexpression activates p53 by a mechanism posited to involve uncharacterized hyperproliferative signals. We determined whether such signals produce metabolic perturbations that generate DNA damage, a known p53 inducer. Biochemical, cytological, cell cycle, and global gene expression analyses revealed that brief c-Myc activation can induce DNA damage prior to S phase in normal human fibroblasts. Damage correlated with induction of reactive oxygen species (ROS) without induction of apoptosis. Deregulated c-Myc partially disabled the p53-mediated DNA damage response, enabling cells with damaged genomes to enter the cycle, resulting in poor clonogenic survival. An antioxidant reduced ROS, decreased DNA damage and p53 activation, and improved survival. We propose that oncogene activation can induce DNA damage and override damage controls, thereby accelerating tumor progression via genetic instability.

High-throughput gene expression profiling has become an important tool for investigating transcriptional activity in a variety of biological samples. To date, the vast majority of these experiments have focused on specific biological processes and perturbations. Here, we have generated and analyzed gene expression from a set of samples spanning a broad range of biological conditions. Specifically, we profiled gene expression from 91 human and mouse samples across a diverse array of tissues, organs, and cell lines. Because these samples predominantly come from the normal physiological state in the human and mouse, this dataset represents a preliminary, but substantial, description of the normal mammalian transcriptome. We have used this dataset to illustrate methods of mining these data, and to reveal insights into molecular and physiological gene function, mechanisms of transcriptional regulation, disease etiology, and comparative genomics. Finally, to allow the scientific community to use this resource, we have built a free and publicly accessible website ( http://expression.gnf.org ) that integrates data visualization and curation of current gene annotations.

The lack of efficacious therapies have hindered the progress in improving the survival of HCC patients worldwide. As a disease that associates with the underlying liver injuries caused by various etiologies and remains asymptomatic during most of its decades long progression, HCC presents challenges to accurately depict its biological landscape and determine suitable therapeutic intervention. An approach to tackle this issue is to define significant biological variants that impact patient survival in each disease stage, then attempt to chart paths of potential therapies. To identify significant biological determinants associated with the overall survival (OS) of early stage, non-metastatic HCC patients, we constructed focused Nanostring panels that are represented by 1,164 genes of validated components and transcriptional outputs of multiple signaling pathways, cellular machineries, and tumor immunity. An unbiased analysis of patient survival and gene transcript levels revealed four distinct groups of genes that are significantly prognostic. The first two (Group 1 & 2) are associated with poorer prognosis when expression is high (hazard ratio > 2), whereas the remaining two (Group 3 & 4) correspond to significantly better OS with elevated transcript levels (hazard ratio Searching for druggable targets associated with poorer prognosis, we identified both EZH2 and CHEK1 as candidates from Group 2. In vitro treatment of three HCC cell lines with EPZ-6438 (EZH2 inhibitor) or GDC-0425 (CHEK1 inhibitor) resulted in significant modulation of H3K27me3 or pCHEK1 levels (s345) respectively. The growth of these cell lines was also dose-dependently suppressed by each inhibitor in colony formation assays . Furthermore, the combination of both inhibitors was able to achieve substantial cell growth inhibition at lower concentrations, suggesting a combinatorial effect of targeting both EZH2 and CHEK1 in HCC cell lines. Xenograft studies are ongoing to assess in vivo efficacy of this combination. In conclusion, utilizing well constructed Nanostring gene panels, we identified significant association of cell cycle machinery and adaptive tumor immunity with HCC patient survival. Our findings not only provided potential rationale for expand testing of cancer immunotherapies to non-metastatic, early stage HCC, but also revealed the potential utility of combining EZH2 and CHEK1 inhibitors to treat HCC. Citation Format: Pei-Zhen Miao, Jeff Cheng, Kwame Okrah, Bonnie Liu, Charlie Sun, Grace Cao, Tony Guo, Shijing Fu, Marie-Claire Wagle, Elizabeth Blackwood, David Shames, Garret Hampton, Lukas Amler, Astrid Kiermaier, Yevgeniya Fridlyand, Mark Lackner, Shih-Min Huang, Jian Zhou, Zhi Dai. Analysis of non-metastatic HCC patient tumors revealed the significance of cell cycle regulation and tumor immunity in association with overall survival and identified clinically relevant druggable targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1750. doi:10.1158/1538-7445.AM2017-1750

Purpose. The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic “hotspot” regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. Methods. We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. Results. Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed “true-positive” gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent “false-positive” calls in clinically druggable oncogenes such as PIK3CA. Conclusion. AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent “false-positive” variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.

Epithelial ovarian cancer is the leading cause of death from gynecologic cancer, in part because of the lack of effective early detection methods. Although alterations of several genes, such as c-erb-B2, c-myc , and p53 , have been identified in a significant fraction of ovarian cancers, none of these mutations are diagnostic of malignancy or predictive of tumor behavior over time. Here, we used oligonucleotide microarrays with probe sets complementary to >6,000 human genes to identify genes whose expression correlated with epithelial ovarian cancer. We extended current microarray technology by simultaneously hybridizing ovarian RNA samples in a highly parallel manner to a single glass wafer containing 49 individual oligonucleotide arrays separated by gaskets within a custom-built chamber (termed “array-of-arrays”). Hierarchical clustering of the expression data revealed distinct groups of samples. Normal tissues were readily distinguished from tumor tissues, and tumors could be further subdivided into major groupings that correlated both to histological and clinical observations, as well as cell type-specific gene expression. A metric was devised to identify genes whose expression could be considered ideal for molecular determination of epithelial ovarian malignancies. The list of genes generated by this method was highly enriched for known markers of several epithelial malignancies, including ovarian cancer. This study demonstrates the rapidity with which large amounts of expression data can be generated. The results highlight important molecular features of human ovarian cancer and identify new genes as candidate molecular markers.

The distal portion of chromosome 1p is frequently deleted in several human cancers, suggesting the presence of one or more putative tumor suppressor genes on this chromosomal arm. In human neuroblastoma, a consistently deleted region at 1p36.1-p36.2 has been defined by comparison of molecular loss of heterozygosity (LOH) analyses. Recently we described the identification of a yeast artificial chromosome, YAC 927G4, that spans a translocation/duplication breakpoint within the minimally defined LOH region at 1p36.1-p36.2 in the neuroblastoma cell line NGP. Here we describe the identification of two overlapping P1 artificial chromosomes comprising 220 kb at the distal end of YAC 927G4, which we have used as hybridization probes under modified conditions to screen a composite, normalized cDNA library (IMAGE cDNA library). Hybridization screening resulted in the rapid and comprehensive identification of partial cDNAs of which a portion comprised two novel candidate genes, termed DNB1/ARPh and DNB5, which encode putative proteins of 1011 and 447 amino acids, respectively. The DNB1/ARPh gene, which was found to be ubiquitously expressed in human adult and fetal tissues, is highly related to the DRPLA gene, in which expansion of a CAG triplet appears to be causal in the dentatorubral and pallidolysian atrophy disease phenotype. The DNB5 sequence, in contrast, which is predominantly expressed in brain tissues and fetal kidney, failed to show any similarity to sequences in the public domain. A preliminary assessment of transcription and sequence of both genes in several neuroblastoma cell lines does not, thus far, support a causal role in neuroblastoma. However, further analyses are required to confirm these results.

First 2 authors contributed equally Hepatocellular carcinoma (HCC) is the second most common cause of death from cancer worldwide with extremely poor prognosis. HCC is known to be closely associated with liver injury-induced cirrhosis caused by various etiologies, including HBV infection. The prolonged timeline and heterogeneous nature of HCC adds complexity to dissecting the biology of this disease in humans. While Sorafenib is approved for the first-line treatment of metastatic HCC, most patients rapidly progress on treatment with Sorafenib. Consequently, alternative therapeutic options for HCC are much needed. The identification of molecular subtypes and reliable biomarkers associated with disease evolution is critical in facilitating development of new therapeutic agents in HCC. To understand the manifestation of early molecular events in HCC disease progression in human, we analyzed genome-wide RNA-seq data derived from 100 paired samples consisting of HCC tumors (most with 60-80% tumor content) and adjacent cirrhotic tissues from early stage patients (TNM system: T1N0M0, T2N0M0, and T3N0M0; similar to BCLC stage A and B). Differential expression analysis revealed a cluster of genes that significantly differentiated HCC from cirrhotic tissues and illustrated a widespread deregulation of cell cycle machinery modulated by probable molecular abnormalities represented by Polo-Like Kinase, Checkpoint kinases, G2/M DNA damage checkpoint regulation, DNA damage-induced 14-3-3σ signaling, ATM signaling, and estrogen-mediated S-phase entry. Prominent down-regulation of FXR/LXR/RXR activation was also observed in HCC tumors. Strikingly, the unsupervised hierarchical clustering of both cirrhotic and HCC tissues revealed 3 groups of genes with mRNA expression closely correlating with disease progression stage-wise from cirrhosis to T1, T2, and T3 stages. Specifically, we made a novel observation illustrating the stage-wise activation of Wnt signaling pathway, but de-activation of MAPK pathway. Upon in-depth analysis, our data also suggests that as HCC progresses, translation machinery and embryonic morphogenesis are stimulated, while angiogenesis, negative regulation of apoptosis, and mesenchymal cell differentiation are possibly impinged. In addition, we found that components of processes crucial for activating immune response appear to be impaired as disease progresses from cirrhosis to stage T3. To confirm the aforementioned finding through focused assessment of immune-microenvironment by gene expression, we utilized Fluidigm platform and corroborated the down-regulation of effector T cell signature. In conclusion, data presented provides a holistic depiction of evolution of HCC and the associated tumor immunity, thus paving a way for future detailed subtyping and therapeutics discovery. Citation Format: Bonnie P. Liu, Kwame Okrah, Jeff Cheng, Maipelo Motlhabi, Charlie Sun, Teiko Sumiyoshi, Shoji Ikeda, Hartmut Koeppen, Zineb Mounir, Craig Cummings, Nadia Haque, Garret Hampton, Lukas Amler, Mark Lackner, Shih-Min A. Huang. Comprehensive RNA-seq transcriptome interrogation of paired hepatocellular carcinoma and cirrhosis tissues revealed significant molecular features of disease evolution and modulation of tumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1837.

Currently there are multiple approaches for measuring gene expression from human tissue samples on the market. These methods range from measuring the expression of a few genes using quantitative RT-PCR to measuring every gene in a sample by whole transcriptome RNA sequencing (RNA-Seq). Although many techniques and products for measuring gene expression are available, the highly degraded RNA isolated from formalin-fixed, paraffin embedded (FFPE) tumor tissues still poses a challenge for many of them. FFPE tumor samples are an abundant source of biomarker information and gene expression analysis in these samples has the potential to identify new signatures, biomarkers, or diagnostics that could predict patient response to treatment. Accurately measuring gene expression in a high throughput manner from this sample type has long been a struggle in the field. To determine the ability of RNA-Seq to accurately measure gene expression from FFPE-derived RNA, we utilized a large collection of matched fresh frozen (FF) and FFPE samples from 12 different tumor indications. By comparing the results to the matching FF sample, we were able to determine the accuracy and sensitivity of each platform when using degraded FFPE-derived RNA. The FFPE samples in the collection had a wide range of RNA quality scores (RIN score: 0-6.8, DV200:0-78) representing what is typically isolated from clinical trial samples. We further compared whole transcriptome RNA-Seq with a hybrid capture method, RNA Access, to determine the accuracy and feasibility of using this technology on degraded RNA. RNA Access selects for protein coding transcripts by hybridization, whereas whole transcriptome RNA-seq removes ribosomal RNA, but analyzes everything else i.e. the global transcriptome. All samples were sequenced to an average of 50 or 25 million paired-end reads for whole transcriptome RNA-Seq and RNA Access, respectively and 100 ng of RNA was used as input in both assays. We conclude that both methods can be used for analyzing FFPE tumor samples. A similar dynamic range was observed for both methods and both show similar correlation between FF and FFPE samples within the method (0.76 vs 0.74). The overall concordance between methods was 0.69 and 0.63 for FF and FFPE, respectively. When looking specifically at the detection of 90 low expressing immune related genes, both assays were able to detect >94% with an inter-platform correlation of 0.66 and 0.56 in FF and FFPE, respectively. Whole transcriptome RNA-seq provides the maximum amount of information from clinical samples including anti-sense and non-coding RNA detection while RNA Access can be applied more cost-efficiently when one is mostly interested in the protein coding transcriptome. Citation Format: Erica B. Schleifman, Anna Kiialainen, Andreas Roller, Sabine Bader, Maipelo Motlhabi, Priti Hegde, Ian McCaffery, Garret Hampton, Michael Cannarile, Craig Cummings, Olivia Spleiss, Eric Peters. Whole transcriptome and exome targeted RNA sequencing for FFPE tumor samples from clinical trials. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3631.

Despite early detection and reduced risk of death by PSA screening, prostate cancer still remains the second leading cause of cancer death in American men. There is currently no cure for advanced prostate cancer. The multistage, stochastic and highly heterogeneous nature of prostate cancer, coupled with genetic and epigenetic alterations that occur during disease progression and response to therapy, represent fundamental challenges in our quest to understand and control this complex and prevalent disease. Recent advances in drug development and breakthroughs in omics technologies have renewed our efforts to identify novel biomarkers for prostate cancer prognosis, prediction and therapeutic response monitoring. In this Perspective article, we overview the current status and highlight future prospects of biomarkers for prostate cancer, a disease that affects millions of men worldwide.

Members of the human epidermal growth factor receptor (HER) family—epidermal growth factor receptor (EGFR, HER1), HER2, HER3, and HER4—are transmembrane tyrosine kinase receptors that are important mediators of cell growth, development, and survival. Activation of the HER tyrosine kinases triggers intracellular signaling pathways, including the MAPK and PI3K-Akt pathways (Olayioye et al., 2000) (Figure 1).

A new and potentially reliable method for the isolation of yeast artificial chromosome (YAC)-insert termini, termed junction trapping, is described. This method is based on simple partial digestions of the YAC-containing yeast strain, ligation to a plasmid vector, and selection of the termini by two rounds of polymerase chain reaction (PCR). To date, the method has generated both terminal sequences from each of nine YACs (100%) that have been examined.

A new strategy for mapping chromosome translocation breakpoints in relation to known genes has been developed. This approach is based on the amplification by the polymerase chain reaction (PCR) of specific target sequences from small numbers of microdissected chromosome fragments. This method has been applied to leukaemia-associated translocations affecting the q23 region of chromosome I I. In two independent leukaemias, the t(6; 11) translocation was distinguished from the t(9; 11) and t(4; 11) translocations by demonstrating that the former breakpoint on chromosome 11 lay proximal to the CD3D gene while the latter breakpoints lay distal to CD3D. All three translocation breakpoints were found to lie proximal to ETSl and THYl. The data suggest that although these leukaemia-associated breakpoints on chromosome 11 are cytogenetically identical they may involve disruption of different genes. This approach offers a rapid alternative to mapping by hybridisation of probes either in situ to chromosomes or to somatic cell hybrids containing the appropriate derivative chromosomes.

A strategy for the rapid isolation from rodent hybrids of human chromosome-specific probes by enzymatic amplification is described. Synthetic oligonucleotide primers based on the consensus Alu sequence were used to amplify inter- Alu sequence from total human genomic DNA and from a somatic cell hybrid, PNTS-1, containing one homolog of chromosome 5 as its only human complement. Direct sequence analysis of the products from human genomic DNA confirmed their inter- Alu structure and provided a novel means for the examination of the 5′ end of the Alu consensus. The amplified sequences from the somatic cell hybrid DNA were cloned into a plasmid vector by blunt-end ligation, yielding clones with inserts in the range 300 to 1000 bp. More than 80% of these clones carried inserts that behaved essentially as single-copy human sequences. Hybridization of a selection of these clones to human DNA, hamster DNA, and the original hybrid DNA confirmed that they were derived from chromosome 5. Direct sequence analysis of the vector/insert boundaries in two clones confirmed that inter- Alu sequences had been cloned. This approach has significant advantages over other methods of isolating chromosome-specific probes from hybrid cells, enabling direct separation and cloning of human DNA probes that can be readily used for mapping studies.

The aim of the study was to identify changes in tumour expression profiling associated with short-term therapy of breast cancer patients with letrozole.Microarray analysis was performed on RNA extracted from paired tumour core biopsies taken before and after 14 days of treatment with letrozole (2.5 mg/daily) in 58 patients. Changes in expression profile were identified by three different approaches on the basis of frequency of changes, magnitude of changes and significance analysis of microarray.No single gene was consistently changed by therapy in all cases. Fifty-two genes, however, were downregulated and 36 upregulated in at least 45 of the 58 cases. In terms of quantitative change, 46 genes showed at least a median 1.5-fold change in expression. Significance analysis of microarray identified 62 genes that were significantly changed by therapy (P0.0001, 56 downregulated and six upregulated). All three approaches showed that greater numbers of genes were downregulated rather than upregulated. Merging data produced a total of 143 genes, which were subject to gene ontology and cluster analysis. The ontology of the 91 downregulated genes showed that they were functionally associated with cell cycle progression, particularly mitosis. In contrast, upregulated genes were associated with organ development, connective tissue extracellular matrix regulation and inflammatory response. Cluster analysis segregated the patients into four groups differing in patterns of gene expression.Genes have been identified which either change markedly or consistently in breast cancer after 14 days treatment with letrozole. These are new important data in understanding letrozole's molecular mechanism of action in breast cancers.

Although the monomeric GTPases RalA and RalB have been shown to regulate a variety of transcription factors, little is known regarding the differences or similarities in transcriptional programs regulated by RalA compared to RalB. Further, the association of these transcriptional pathways to human carcinogenesis and progression remains unclear. Here, we studied the role of RalA and/or RalB in transcriptional regulation by combining short interfering RNA depletion of Ral with gene expression profiling via microarray in the human bladder cancer cell line, UMUC-3. A large number of genes were found to be similarly modulated in cells with RalA and RalB depletion, suggesting that RalA and RalB impinge on overlapping transcriptional signaling pathways. However, smaller sets of genes were modulated by depletion of RalA or RalB, indicating that these closely related proteins also regulate nonoverlapping transcriptional pathways. Computational analysis of upstream sequences of genes modulated by Ral depletion identified Ras-responsive element-binding protein (RREB)-1, as a putative Ral transcriptional target, which we verified experimentally. Importantly, as a group, Ral-regulated probe sets identified here were disproportionally represented among those differentially expressed as a function of human bladder transformation. Taken together, these data strongly suggest that Ral family members mediate both common and specific transcriptional programs that are associated with human cancer and identify RREB-1 as a novel transcriptional effector of Ral.

Purpose: Cancer of the ovary confers the worst prognosis among women with gynecologic malignancies, underscoring the need to develop new biomarkers for detection of early disease, particularly those that can be readily monitored in the blood. Experimental Design: We developed an algorithm to identify secreted proteins encoded among ∼22,500 genes on commercial oligonucleotide arrays and applied it to gene expression profiles of 67 stage I to IV serous papillary carcinomas and 9 crudely enriched normal ovarian tissues, to identify putative diagnostic markers. ELISAs were used to validate increased levels of secreted proteins in patient sera encoded by genes with differentially high expression. Results: We identified 275 genes predicted to encode secreted proteins with increased/decreased expression in ovarian cancers (2-fold, P < 0.001). The serum levels of four of these proteins (matrix metalloproteinase-7, osteopontin, secretory leukoprotease inhibitor, and kallikrein 10) were significantly elevated in a series of 67 independent patients with serous ovarian carcinomas compared with 67 healthy controls (P < 0.001, Wilcoxon rank sum test). Optimized support vector machine classifiers with as few as two of these markers (osteopontin or kallikrein 10/matrix metalloproteinase-7) in combination with CA-125 yielded sensitivity and specificity values ranging from 96% to 98.7% and 99.7% to 100%, respectively, with the ability to discern early-stage disease from normal, healthy controls. Conclusions: Our data suggest that this assay combination warrants further investigation as a multi-analyte diagnostic test for serous ovarian adenocarcinoma.

Overexpression of the ErbB2 receptor tyrosine kinase is common in human cancers and is associated with an increased level of metastasis. To better understand the cellular signaling networks activated by ErbB2, a phosphoproteomic analysis of tyrosine-phosphorylated proteins was carried out in ErbB2-overexpressing breast and ovarian cancer cell lines. A total of 153 phosphorylation sites were assigned on 78 proteins. Treatment of cells with Herceptin, a monoclonal antibody that inhibits ErbB2 activity, significantly reduced the number of detectable protein phosphorylation sites, suggesting that many of these proteins participate in ErbB2-driven cell signaling. Of the 71 proteins that were differentially phosphorylated, only 13 were previously reported to directly associate with ErbB2. The differentially phosphorylated proteins included kinases, adaptor/docking proteins, proteins involved in cell proliferation and migration, and several uncharacterized RNA binding proteins. Selective depletion of some of these proteins, including RNA binding proteins SRRM2, SFRS1, SFRS9, and SFRS10, by siRNAs reduced the rate of migration of ErbB2-overexpressing ovarian cancer cells.

The HE4 (WFDC2) gene encodes a WAP-type four disulphide core domain-containing protein with a presumptive role in natural immunity. Multiple studies have consistently identified upregulation of HE4 gene expression in carcinomas of the ovary; however, the expression in normal and malignant adult tissues has not been examined in detail. Here, we examined the expression of the HE4 gene and protein in a large series of normal and malignant adult tissues by oligonucleotide microarray and tissue microarray, respectively. HE4 gene expression was highest in normal human trachea and salivary gland, and to a lesser extent, lung, prostate, pituitary gland, thyroid, and kidney. In a series of 175 human adult tumors, gene expression was highest in ovarian serous carcinomas. However, adenocarcinomas of the lung, and occasional breast, transitional cell and pancreatic carcinomas had moderate or high levels of HE4 expression. Using tissue microarrays and full tissue sections of normal and 448 neoplastic tissues, HE4 immunoreactivity was found in normal glandular epithelium of the female genital tract and breast, the epididymis and vas deferens, respiratory epithelium, distal renal tubules, colonic mucosa, and salivary glands, consistent with HE4 gene expression. In addition to consistent positivity in ovarian carcinoma, some pulmonary, endometrial, and breast adenocarcinomas, mesotheliomas, and less often, gastrointestinal, renal and transitional cell carcinomas were also positive. Knowledge of the expression patterns of HE4 in our survey is useful for application in histopathologic diagnosis, and should be taken into consideration in future studies that examine the role of HE4 as a serological tumor biomarker or as a target for gene-based therapy.

CCAAT element binding protein beta (C/EBPbeta) is an important regulator of cell growth, differentiation and in promoting tumor invasiveness. C/EBPbeta is located on chromosome 20q, which is amplified in many solid tumors including gastric cancers (GC). We sought to characterize the status of C/EBPbeta expression in GCs, which was recently found to repres TFF1 gene. Microarray analysis revealed overexpression of C/EBPbeta in 25 of 27 (93%) GC when compared to 12 normal gastric tissue samples. RT-PCR analysis confirmed the overexpression of C/EBPbeta transcripts in 54 of 59 (91%) GC. In total, 15 of 18 gastric tumors exhibited at least fivefold higher C/EBPbeta transcript levels compared to their corresponding adjacent normal gastric tissue samples. Moreover, immunohistochemistry analysis demonstrated increased nuclear staining of C/EBPbeta in 10 of 13 GC and at least fourfold overexpression of C/EBPbeta in three primary GC compared to adjacent normal gastric tissue. Furthermore, a striking correlation of decreased TFF1 expression with increased C/EBPbeta was observed in the gastric tumors studied. Microarray analysis demonstrated a loss of TFF1 expression in all 27 GC cases examined, of which 25 exhibited high C/EBPbeta expression compared to normal gastric tissue. RT-PCR analysis revealed loss of TFF1 expression in 56 of 59 gastric tumors in which 54 of these tumors exhibited overexpression of C/EBPbeta. Immunohistochemical analysis revealed overexpression of C/EBPbeta in 10 of 13 gastric tumors that exhibited low expression of TFF1 at the protein level. Thus, overexpression of the transcription factor C/EBPbeta in the majority of GCs is a novel finding.

Adenoid cystic carcinoma (ACC) is a common malignancy of salivary glands, for which the underlying genetic mechanisms of tumorigenesis are poorly understood. Prior studies in ACC have identified deletions in chromosome 12. To further characterize these changes, we performed an extensive LOH analysis in 58 ACC using a panel of 28 microsatellite markers. Results show 66% overall genetic loss. Three markers (D12S1713, D12S2196, D12S398) are contiguous and define a 6.84 Mb region of deletion at 12q13.11-q13.13. Two other markers (D12S2078, D12S1628) are also contiguous and define a 4.5 Mb region of deletion at 12q24.32-q24.33. The three remaining markers, D12S1056 at 12q14.1, D12S1051 at 12q23.1 and D12S1636 at 12q23.3 define smaller regions of deletion. An analysis of microarray gene expression profiling data available for ACC shows several genes with significant transcriptional downregulation that map to these areas of genetic deletion. This combined genetic and genomic analysis provides several candidate genes to test for functional tumor suppressor activity in ACC.

Profiles of gene transcription have begun to delineate the molecular basis of ovarian cancer, including distinctions between carcinomas of differing histology, tumor progression and patient outcome. However, the similarities and differences among the most commonly diagnosed noninvasive borderline (low malignant potential, LMP) lesions and invasive serous carcinomas of varying grade (G1, G2 and G3) have not yet been explored. Here, we used oligonucleotide arrays to profile the expression of 12,500 genes in a series of 57 predominantly stage III serous ovarian adenocarcinomas from 52 patients, eight with borderline tumors and 44 with adenocarcinomas of varying grade. Unsupervised and supervised analyses showed that LMP lesions were distinct from high-grade serous adenocarcinomas, as might be expected; however, well-differentiated (G1) invasive adenocarcinomas showed a strikingly similar profile to LMP tumors as compared to cancers with moderate (G2) or poor (G3) cellular differentiation, which were also highly similar. Comparative genomic hybridization of an independent cohort of five LMP and 63 invasive carcinomas of varying grade demonstrated LMP and G1 were again similar, exhibiting significantly less chromosomal aberration than G2/G3 carcinomas. A majority of LMP and G1 tumors were characterized by high levels of p21/WAF1, with concomitant expression of cell growth suppressors, gadd34 and BTG-2. In contrast, G2/G3 cancers were characterized by the expression of genes associated with the cell cycle and by STAT-1-, STAT-3/JAK-1/2-induced gene expression. The distinction between the LMP-G1 and G2-G3 groups of tumors was highly correlated to patient outcome (chi(2) for equivalence of death rates=7.681189; P=0.0056, log-rank test). Our results are consistent with the recent demonstration of a poor differentiation molecular 'meta-signature' in human cancer, and underscore a number of cell-cycle- and STAT-associated targets that may prove useful as points of therapeutic intervention for those patients with aggressive disease.

Cancer develops through the successive accumulation and selection of genetic and epigenetic alterations, enabling cells to survive, replicate and evade homeostatic control mechanisms such as apoptosis and antiproliferative signals. This transformation process, however, may create vulnerabilities since the accumulation of mutations can expose synthetic lethal gene interactions and oncogene-driven cellular reprogramming ('addiction'), giving rise to new therapeutic avenues. With the completion of the human genome project, it is anticipated that the identification and characterization of genetic networks that regulate cell growth, differentiation, apoptosis and transformation will be fundamental to decoding the complexity of these processes, and ultimately, cancer itself. Genomic methodologies, such as large-scale mRNA profiling using microarrays, have already begun to reveal the molecular basis of cancer heterogeneity and the clinical behavior of tumors. The combination of traditional cell culture techniques with high-throughput screening approaches has given rise to new cellular-genomics methodologies that enable the simultaneous interrogation of thousands of genes in live cells, facilitating true functional profiling of biological processes. Among these, RNA interference (RNAi) has the potential to enable rapid genome-wide loss-of-function (LOF) screens in mammalian systems, which until recently has been the sole domain of lower organisms. Here, we present a broad overview of this maturing technology and explore how, within current technical constraints, large-scale LOF use of RNAi can be exploited to uncover the molecular basis of cancer--from the genetics of synthetic lethality and oncogene-dependent cellular addiction to the acquisition of cancer-associated cellular phenotypes.

Gene expression profiling of anatomically diverse carcinomas and their corresponding normal tissues was used to identify genes with cancer-associated expression. We show here that the ubiquitin conjugase, UbcH10, is significantly overexpressed in many different types of cancers and is associated with the degree of tumor differentiation in carcinomas of the breast, lung, ovary and bladder, as well as in glioblastomas. We also show that UbcH10 overexpression in gastro-esophageal, and probably other carcinomas may be a direct consequence of chromosomal amplification at the UbcH10 locus, 20q13.1, a region known to be amplified in diverse tumors. To evaluate whether inhibition of UbcH10 function may be therapeutically relevant in cancer, we used small interfering RNAs (siRNAs) to silence UbcH10 transcription selectively. Diminution of UbcH10 expression significantly inhibited both tumor and normal cell proliferation without inducing cell death. However, when combined with agonists of the DR5/TRAIL receptor, siRNAs directed against the UbcH10 transcript dramatically enhanced killing of cancer cells, but not of proliferating primary human epithelial cells or fibroblasts. Together, these data demonstrate that UbcH10 plays an important role in tumor development and that its inhibition in combination with agonists of the TRAIL receptor may provide an enhanced therapeutic index.

BACKGROUND The 32-kilodalton dopamine and cyclic adenosine 3′,5′-monophosphate-regulated phosphoprotein (DARPP-32) is the only known protein that can act as a protein phosphatase-1 inhibitor or a protein kinase inhibitor. It is known as a neurogenic protein that plays a major role in dopamine regulation in the central nervous system. Recently, DARPP-32 and a truncated isoform, t-DARPP, have been cloned in gastric carcinoma. The extent of DARPP-32 and t-DARPP expression in human epithelial tissues and carcinomas remained unknown. METHODS Using tissue microarrays and an antibody to DARPP-32, the authors evaluated the immunohistochemical findings in 43 different normal epithelium human tissue samples and in 187 samples of common carcinomas of breast, prostate, colon, and stomach. RESULTS DARPP-32 proteins were expressed at varying levels in several types of normal epithelial tissues outside of the central nervous system. Using quantitative real-time reverse transcriptase-polymerase chain reaction analysis, the authors demonstrated that both DARPP-32 and t-DARPP mRNAs frequently were overexpressed in carcinomas of the breast, prostate, colon, and stomach compared with normal tissue samples. Immunohistochemical analysis of tissue microarrays that contained 187 carcinoma samples confirmed the strong expression of DARPP-32 proteins in these tumor types. CONCLUSIONS The current study provides the first evidence that DARPP-32 expression is not limited to dopamine signaling in normal cells of the central nervous system. The pattern of expression of DARPP-32 proteins in normal epithelial tissues suggests that these proteins play an important role in epithelial signaling that may be tissue specific. Moreover, the observation that DARPP-32 and t-DARPP frequently are overexpressed in common subtypes of human adenocarcinomas suggest that these proteins may be important in tumorigenesis. Cancer 2003;98:1547–51. © 2003 American Cancer Society. DOI 10.1002/cncr.11654

Genetic alterations in tumor cells often lead to the emergence of growth-stimulatory autocrine and paracrine signals, involving overexpression of secreted peptide growth factors, cytokines, and hormones. Increased levels of these soluble proteins may be exploited for cancer diagnosis and management or as points of therapeutic intervention. Here, we combined the use of controlled vocabulary terms and sequence-based algorithms to predict genes encoding secreted proteins from among approximately 12,500 sequences represented on oligonucleotide microarrays. Expression of these genes was queried in 150 carcinomas from 10 anatomic sites of origin and compared with 46 normal tissues derived from the corresponding sites of tumor origin and other body tissues and organs. Of 74 different genes identified as overexpressed in cancer tissues, several encode proteins with demonstrated clinical diagnostic application, such as alpha-fetoprotein in liver carcinoma, and kallikreins 6 and 10 in ovarian cancer, or therapeutic utility, such as gastrin-releasing peptide/bombesin in lung carcinomas. We show that several of the other candidate genes encode proteins with high levels of tumor-associated expression by immunohistochemistry on tissue microarrays and further demonstrate significantly elevated levels of another novel candidate protein, macrophage inhibitory cytokine 1, a distant member of the transforming growth factor-beta superfamily, in the serum of patients with metastatic prostate, breast, and colorectal carcinomas. Our results suggest that the combination of annotation/protein sequence analysis, transcript profiling, immunohistochemistry, and immunoassay is a powerful approach for delineating candidate biomarkers with potential clinical significance and may be broadly applicable to other human diseases.

To discover novel metastasis suppressor genes that are clinically relevant in common human cancers, we used isogenic human bladder cancer cell lines and used DNA microarray technology to identify genes whose expression diminishes as a function of invasive and metastatic competence. We then evaluated the expression profile of such genes in 105 pathologically characterized tumors from seven common organ sites, and we identified one gene, RhoGDI2, whose expression was diminished as a function of primary tumor stage and grade. When RhoGDI2 was transferred back into cells with metastatic ability that lacked its expression, it suppressed experimental lung metastasis but did not affect in vitro growth, colony formation, or in vivo tumorigenicity. In addition, RhoGDI2 reconstitution in these cells blocked invasion in an organotypic assay and led to a reduction of in vitro motility. These results indicate that RhoGDI2 is a metastasis suppressor gene, a marker of aggressive human cancer, and a promising target for therapy.

Salivary gland cancers comprise a heterogeneous group of neoplasms whose biological and clinical characteristics differ considerably from those of mucosal squamous cell carcinomas of the head and neck. One of the most common subtypes, adenoid cystic carcinoma (ACC), is notable for its myoepithelial differentiation, proclivity for hematogenous spread, and slow but progressive clinical course. The molecular alterations that underlie its development and progression are poorly characterized. Here we used oligonucleotide microarray analysis to survey the expression of 8920 different human genes in 15 ACCs, one ACC cell line, and five normal major salivary glands. We observed expression of genes indicative of myoepithelial differentiation, as expected, including those whose protein products are components of basement membranes and extracellular matrix. Other genes that were highly ranked for their expression in ACC were those encoding the transcription factors SOX4 and AP-2 gamma, the latter of which also was overexpressed in ACC relative to 175 other carcinomas from 10 anatomical sites that we had previously profiled. Additional genes, which were highly expressed in ACC compared to the other carcinomas, included casein kinase 1, epsilon and frizzled-7, both members of the Wnt/beta-catenin signaling pathway. Our study documents for the first time the diverse spectrum of genes overexpressed in ACC and highlights gene products and pathways that in the future might be exploited as therapeutic targets for this cancer, which up until now, has shown limited response to chemotherapeutic approaches.

Oncogene overexpression activates p53 by a mechanism posited to involve uncharacterized hyperproliferative signals. We determined whether such signals produce metabolic perturbations that generate DNA damage, a known p53 inducer. Biochemical, cytological, cell cycle, and global gene expression analyses revealed that brief c-Myc activation can induce DNA damage prior to S phase in normal human fibroblasts. Damage correlated with induction of reactive oxygen species (ROS) without induction of apoptosis. Deregulated c-Myc partially disabled the p53-mediated DNA damage response, enabling cells with damaged genomes to enter the cycle, resulting in poor clonogenic survival. An antioxidant reduced ROS, decreased DNA damage and p53 activation, and improved survival. We propose that oncogene activation can induce DNA damage and override damage controls, thereby accelerating tumor progression via genetic instability.

Gene expression profiling using DNA arrays is rapidly becoming an essential tool for research and drug discovery and may soon play a central role in disease diagnosis. Although it is possible to make significant discoveries on the basis of a relatively small number of expression profiles, the full potential of this technology is best realized through more extensive collections of expression measurements. The generation of large numbers of expression profiles can be a time-consuming and labor-intensive process with current one-at-a-time technology. We have developed the ability to obtain expression profiles in a highly parallel yet straightforward format using glass wafers that contain 49 individual high-density oligonucleotide arrays. This arrays of arrays concept is generalizable and can be adapted readily to other types of arrays, including spotted cDNA microarrays. It is also scalable for use with hundreds and even thousands of smaller arrays on a single piece of glass. Using the arrays of arrays approach and parallel preparation of hybridization samples in 96-well plates, we were able to determine the patterns of gene expression in 27 ovarian carcinomas and 4 normal ovarian tissue samples, along with a number of control samples, in a single experiment. This new approach significantly increases the ease, efficiency, and throughput of microarray-based experiments and makes possible new applications of expression profiling that are currently impractical.

We report here the transcriptional profiling of the cell cycle on a genome-wide scale in human fibroblasts. We identified approximately 700 genes that display transcriptional fluctuation with a periodicity consistent with that of the cell cycle. Systematic analysis of these genes revealed functional organization within groups of coregulated transcripts. A diverse set of cytoskeletal reorganization genes exhibit cell-cycle-dependent regulation, indicating that biological pathways are redirected for the execution of cell division. Many genes involved in cell motility and remodeling of the extracellular matrix are expressed predominantly in M phase, indicating a mechanism for balancing proliferative and invasive cellular behavior. Transcripts upregulated during S phase displayed extensive overlap with genes induced by DNA damage; cell-cycle-regulated transcripts may therefore constitute coherent programs used in response to external stimuli. Our data also provide clues to biological function for hundreds of previously uncharacterized human genes.

Objective: To investigate the incidence of microsatellite instability (MI) in cervical carcinoma and its relationship with clinicopathological characteristics. Study design: A retrospective study of 100 cases of cervical carcinoma. Results: MI, defined as tumor-associated alterations in at least one of five dinucleotide microsatellite markers examined, was detected in 25% of the cervical carcinomas which were observed. There was a nonsignificant trend towards MI occurrence in higher grade tumors, more advanced stage and cases with poor clinical outcome. Conclusion: The results suggest that microsatellite instability is present in a subset of cervical carcinoma and may be an independent prognostic factor. Further research with more samples is required.

A previously described genomic library constructed from microdissected DNA has been used to generate a large number of probes around the adenomatous polyposis coli (APC) gene at 5q22. A total of 202 clones were hybridised directly onto a somatic cell hybrid panel containing two APC-related interstitial deletions. Of 75 microclones that gave clear hybridisation signals, 22 independent clones mapped into the region common to both deletions. In addition, 4/22 of the markers are conserved in rodent DNA. These clones should provide a valuable resource for screening cDNA libraries and cloning the DNA around the APC gene in yeast artificial chromosomes.

Nasopharyngeal carcinoma is a malignancy that is prevalent among populations from Southeast Asia. Epidemiological studies indicate that genetic predisposition, Epstein–Barr virus, and environmental conditions may play a role in determining incidence. Molecular studies have implicated a tumor suppressor gene(s) on the short arm of chromosome 3. In this study we provide functional evidence, via monochromosome transfer, for a tumor suppressor gene(s) activity in chromosome 3p21.3.

Introduction: Chronic Myeloid leukemia (CML) is driven by the BCR-ABL oncogene and is initially treated with ABL tyrosine-kinase inhibitors (ABL-TKI), such as Imatinib or Dasatinib. Despite strong initial responses to these drugs, many patients acquire resistance over time through acquisition of BCR-ABL mutations such as T315I, compound BCR-ABL mutations or through BCR-ABL-independent mechanisms. Even though the next generation pan-ABL inhibitor, Ponatinib, targets all know resistant ABL mutations, some patients don't benefit from Ponatinib and in these cases resistance to TKI therapy may be due to BCR-ABL-independent mechanisms. We analyzed the mechanism of ABL-TKI-dual-resistant CML cells independent of new BCR-ABL mutations and tested the sensitivity of these resistant cells to the PI3K inhibitor, GDC-0941 and/or the MEK inhibitor GDC-0973. Methods: We created ABL-TKI-dual-resistant cells and clones through prolonged treatment of K562 and KCL22 cells with Imatinib and then Dasatinib. The cells and clones were tested for sensitivity to Imatinib, Dasatinib, Ponatinib, GDC-0941 and GDC-0973. Whole genome copy number scan (Oncoscan) and targeted deep sequencing using GAII were used to identify BCR-ABL mutations and newly acquired somatic gene alterations. Phosphoproteomic analysis of cell lysates by Reverse Phase Protein Arrays was used to profile cell signaling pathway status associated with resistance and sensitivity to these drugs. Results: The K562 ABL-TKI-dual-resistant cells were insensitive to all three ABL-TKIs including Ponatinib suggesting that resistance was mediated by a BCR-ABL-independent mechanism. Unexpectedly, the resistant cells and clones became more sensitive to GDC-0941 but not GDC-0973 compared to the parental cells. These resistant cells and clones had acquired new somatic mutations in p53, BRCA2, PTEN, RB, SMARCA4 and PBRM1, but not in BCR-ABL. Phosphoprotein profiling showed low phosphorylation of the BCR-ABL substrates c-ABL, SHC and FAK indicative of BCR-ABL-independent mechanisms of resistance. Activity of the PI3K/AKT and MEK/ERK pathways varied across the resistant clones. However, high levels of the AKT substrate, FOXO-1 directly correlated with Dasatinib resistance and with GDC-0941 sensitivity. Sensitivity to GDC-0941 also correlated with modulation of phospho-FOXO-1. Our data suggest that GDC-0941 may be a therapeutic candidate for CML patients who progress on TKI therapy through BCR-ABL-independent mechanisms of resistance. Citation Format: Marie Wagle, Matthew Wongchenko, Shan Lu, Yinghui Guan, Yulei Wang, Mark Lackner, Garret Hampton, Yibing Yan. BCR-Abl independent, Abl-TKI-therapy-resistant CML cells show enhanced sensitivity to GDC0941. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3395. doi:10.1158/1538-7445.AM2013-3395

Detection of loss of heterozygosity (LOH) by comparison of normal and tumor genotypes using PCR-based microsatellite loci provides considerable advantages over traditional Southern blotting-based approaches. However, current methodologies are limited by several factors, including the numbers of loci that can be evaluated for LOH in a single experiment, the discrimination of true alleles versus "stutter bands," and the use of radionucleotides in detecting PCR products. Here we describe methods for high throughput simultaneous assessment of LOH at multiple loci in human tumors; these methods rely on the detection of amplified microsatellite loci by fluorescence-based DNA sequencing technology. Data generated by this approach are processed by several computer software programs that enable the automated linear quantitation and calculation of allelic ratios, allowing rapid ascertainment of LOH. As a test of this approach, genotypes at a series of loci on chromosome 4 were determined for 58 carcinomas of the uterine cervix. The results underscore the efficacy, sensitivity, and remarkable reproducibility of this approach to LOH detection and provide subchromosomal localization of two regions of chromosome 4 commonly altered in cervical tumors.

The gene associated with adenomatous polyposis coli (APC) has been mapped to the long arm of chromosome 5. To saturate the APC region with DNA markers, two independent microdissection libraries with an emphasis on 5q21.2–21.3 and 5q22 have been constructed from GTG-banded human metaphase chromosomes. PCR-amplified insert DNA of the primary amplificate used as a probe in chromosomal in situ suppression (CISS) hybridization of human metaphase spreads revealed region-specific signals at the chromosomal site that was excised for cloning. One hundred forty-two inserts, derived from both libraries, have been characterized in more detail. Deletion mapping analysis was performed with 17 single-copy clones on a hamster-human hybrid cell panel. Seven of these clones were located within two interstitial deletions of 6–8 Mb from APC-affected individuals around chromosome bands 5q21–22. The identification of new microclones mapping into these deletions and their use in isolating YAC clones should contribute to the construction of a contiguous physical map of the APC region.

Using microarray analysis, we have measured changes in gene expression that occurred during the evolution of a primary prostate cancer xenograft, CWR22, from normalcy to relapsed, androgen-independent growth following androgen deprivation therapy. Rather than relying on simple pairwise comparison of the data between successive biological states (for example, normal versus tumor), we clustered the expression data on the basis of behavior in multiple biological states, representative of the entire process of prostate cancer progression. By examining the data in the context of different proliferative conditions, before and after hormonal manipulation in the xenograft model, we could identify groups of genes associated with proliferation, metabolism, hormone responsiveness and malignant transformation, as well as a small number of genes directly associated with androgen-independent growth. Our results imply that the evolution to androgen independence is due, in part, to reactivation of the androgen-response pathway in the absence of androgens, but that this reactivation is probably incomplete.

Carcinomas of the uterine cervix are thought to arise from preinvasive dysplastic lesions, termed cervical intraepithelial neoplasias (CIN), grades I-III. Patients may present clinically with two or more distinct lesions of differing histological severity; however, the genesis of these multifocal lesions is unknown. Despite infection with high-risk human papilloma virus subtypes, which is a major etiological factor in disease pathogenesis, only a small and unpredictable number of dysplastic lesions progress to invasive cancer. Several lines of evidence suggest that additional somatic events, such as tumor suppressor gene inactivation, are required for malignant transformation. In support of this, loss of heterozygosity (LOH) analyses of invasive cervical carcinomas have identified several chromosomal arms likely to harbor tumor suppressor genes, of which regions on 3p, 4p, 4q, and 11q have been validated extensively. To evaluate the potential role of tumor suppressor gene inactivation in dysplastic progression, loci distributed on these four chromosomal regions were assessed for LOH in 42 CIN lesions of varying histological grade obtained from 17 patients. Analysis of at least 16 microsatellite loci in each lesion revealed allelic losses involving one or more of these chromosomal regions in 0% of CIN I lesions; 25% of CIN II lesions; and 88% of CIN III lesions, with 41% of CIN III lesions exhibiting LOH for three or more chromosomal regions. In addition, where LOH was scored for the same locus at a particular chromosomal region in all of the multiple lesions from a single patient, the same allele was lost at each locus, without exception. Statistical analysis of these allele-specific losses strongly suggests that topologically distinct lesions are related and likely arise from a common precursor cell.

A long-range restriction map encompassing the APC (adenomatous polyposis coli) gene has been constructed. The map includes 35 DNA markers and consists of two segments of 10 and 2.5 Mb. Published genetic markers have been connected using additional, nonpolymorphic DNA probes. The map clarifies marker order and allows comparison of physical and genetic data.