Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis.

Reactive oxygen species (ROS) are mutagenic and may thereby promote cancer. Normally, ROS levels are tightly controlled by an inducible antioxidant program that responds to cellular stressors and is predominantly regulated by the transcription factor Nrf2 (also known as Nfe2l2) and its repressor protein Keap1 (refs 2-5). In contrast to the acute physiological regulation of Nrf2, in neoplasia there is evidence for increased basal activation of Nrf2. Indeed, somatic mutations that disrupt the Nrf2-Keap1 interaction to stabilize Nrf2 and increase the constitutive transcription of Nrf2 target genes were recently identified, indicating that enhanced ROS detoxification and additional Nrf2 functions may in fact be pro-tumorigenic. Here, we investigated ROS metabolism in primary murine cells following the expression of endogenous oncogenic alleles of Kras, Braf and Myc, and found that ROS are actively suppressed by these oncogenes. K-Ras(G12D), B-Raf(V619E) and Myc(ERT2) each increased the transcription of Nrf2 to stably elevate the basal Nrf2 antioxidant program and thereby lower intracellular ROS and confer a more reduced intracellular environment. Oncogene-directed increased expression of Nrf2 is a new mechanism for the activation of the Nrf2 antioxidant program, and is evident in primary cells and tissues of mice expressing K-Ras(G12D) and B-Raf(V619E), and in human pancreatic cancer. Furthermore, genetic targeting of the Nrf2 pathway impairs K-Ras(G12D)-induced proliferation and tumorigenesis in vivo. Thus, the Nrf2 antioxidant and cellular detoxification program represents a previously unappreciated mediator of oncogenesis.

Targeted deletion of MKK4 in cancer cells: a detrimental phenotype manifests as decreased experimental metastasis and suggests a counterweight to the evolution of tumor-suppressor loss.

Tumor-suppressors have commanded attention due to the selection for their inactivating mutations in human tumors. However, relatively little is understood about the inverse, namely, that tumors do not select for a large proportion of seemingly favorable mutations in tumor-suppressor genes. This could be explained by a detrimental phenotype accruing in a cell type-specific manner to most cells experiencing a biallelic loss. For example, MKK4, a tumor suppressor gene distinguished by a remarkably consistent mutational rate across diverse tumor types and an unusually high rate of loss of heterozygosity, has the surprisingly low rate of genetic inactivation of only approximately 5%. To explore this incongruity, we engineered a somatic gene knockout of MKK4 in human cancer cells. Although the null cells resembled the wild-type cells regarding in vitro viability and proliferation in plastic dishes, there was a marked difference in a more relevant in vivo model of experimental metastasis and tumorigenesis. MKK4(-/-) clones injected i.v. produced fewer lung metastases than syngeneic MKK4-competent cells (P = 0.0034). These findings show how cell type-specific detrimental phenotypes can offer a paradoxical and yet key counterweight to the selective advantage attained by cells as they experiment with genetic null states during tumorigenesis, the resultant balance then determining the observed biallelic mutation rate for a given tumor-suppressor gene.

Somatic mutations of GUCY2F, EPHA3, and NTRK3 in human cancers.

Tyrosine kinases are major regulators of signal transduction cascades involved in cellular proliferation and have important roles in tumorigenesis. We have recently analyzed the tyrosine kinase gene family for alterations in human colorectal cancers and identified somatic mutations in seven members of this gene family. In this study we have used high-throughput sequencing approaches to further evaluate this subset of genes for genetic alterations in other human tumors. We identified somatic mutations in GUCY2F, EPHA3, and NTRK3 in breast, lung, and pancreatic cancers. Our results implicate these tyrosine kinase genes in the pathogenesis of other tumor types and suggest that they may be useful targets for diagnostic and therapeutic intervention in selected patients.

A double missense variation of the BUB1 gene and a defective mitotic spindle checkpoint in the pancreatic cancer cell line Hs766T.

To determine sequence variations of the BUB1 and BUB1B genes in pancreatic cancer, the entire coding regions of the BUB1 and BUB1B genes were sequenced in pancreatic cancer cell lines and xenografts. Although only polymorphic alterations were found in the BUB1B gene, the aneuploid pancreatic cell line Hs766T had two novel missense variants (p.[Y259C;H265N]) in the BUB1 gene. These mutations were on the same allele, accompanied by a wild-type BUB1 allele. This change was not found in other samples, the literature, or 110 additional chromosomes from a reference population. Compared to two cell lines having microsatellite instability (MIN), the TP53 wild-type pancreatic cell line Hs766T had a defective mitotic spindle checkpoint, indicative of a cell line with chromosomal instability (CIN). Evidence that this checkpoint pathway can be abrogated by mutations in the BUB1 gene (Cahill et al., 1998) supports the suggestion the missense mutations of the BUB1 gene in the Hs766T cell line may contribute to its observed mitotic checkpoint defect.

Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media.

Current DNA electrophoretic solutions employ high ionic concentrations and require long electrophoretic run times. Here we demonstrate that high and low molecular weight double-stranded DNA, single-stranded DNA (ssDNA), and RNA can be separated rapidly in agarose-based low-molarity conductive media. Separation of small DNA fragments was optimized by substituting 1-mM solutions of alkali metals or a nonbiological amine that distributed voltage with a minute current. These ultra-dilute solutions can separate DNA at least 15-fold faster Low-molarity media at 5-10 mM adequately separated RNA and larger DNA fragments as well. These novel media reduce the Joule heating of the electrophoretic system and allow for easy-to-use, ultra-fast separation of DNA fragments.

FAM190A rearrangements provide a multitude of individualized tumor signatures and neo-antigens in cancer.

We found FAM190A transcripts to have internal rearrangements in 40% (19/48) of unselected human cancers. Most of these tumors (84%) had in-frame structures, 94% of which involved deletion of exon 9. The FAM190A gene is located at 4q22.1 in a region of common fragility, FRA4F. Although normally stable in somatic cells, common fragile sites can be hotspots of rearrangement in cancer. The genomic deletion patterns observed at some sites, including FRA4F at 4q22.1, are proposed to be the result of selection for disrupted tumor-suppressor genes. Our evidence, however, indicated additional patterns for FAM190A. We found genomic deletions accounted for some FAM190A in-frame structures, and cases pre-selected for FAM190A genomic deletions had a yet higher prevalence of FAM190A rearrangements. Our evidence of widespread in-frame heterozygous and homozygous rearrangements affecting this gene in tumors of multiple types leads speculation on structural grounds that the mutant forms may retain, provide new, or possibly convey dominant-negative functions. Although a functionally uncharacterized gene, it is evolutionary conserved across vertebrates. In addition to its potential oncogenic role, the in-frame deletions predict the formation of cancer-specific FAM190A peptide sequences (neo-antigens) with potential diagnostic and therapeutic usefulness.

Core signaling pathways in human pancreatic cancers revealed by global genomic analyses.

There are currently few therapeutic options for patients with pancreatic cancer, and new insights into the pathogenesis of this lethal disease are urgently needed. Toward this end, we performed a comprehensive genetic analysis of 24 pancreatic cancers. We first determined the sequences of 23,219 transcripts, representing 20,661 protein-coding genes, in these samples. Then, we searched for homozygous deletions and amplifications in the tumor DNA by using microarrays containing probes for approximately 10(6) single-nucleotide polymorphisms. We found that pancreatic cancers contain an average of 63 genetic alterations, the majority of which are point mutations. These alterations defined a core set of 12 cellular signaling pathways and processes that were each genetically altered in 67 to 100% of the tumors. Analysis of these tumors' transcriptomes with next-generation sequencing-by-synthesis technologies provided independent evidence for the importance of these pathways and processes. Our data indicate that genetically altered core pathways and regulatory processes only become evident once the coding regions of the genome are analyzed in depth. Dysregulation of these core pathways and processes through mutation can explain the major features of pancreatic tumorigenesis.

Gene-specific selection against experimental fanconi anemia gene inactivation in human cancer.

The Fanconi anemia (FA) gene family comprises at least 12 genes interacting in a common pathway involved in DNA repair. To gain insight into the role of FA gene inactivation occurring in tumors among the general population, we endogenously targeted in cancer cells four FA genes that act at different stages of the FA pathway. After successful mono-allelic deletion of all genes, the sequential homozygous deletion was achieved only for FANCC and FANCG, acting upstream, but not for BRCA2 or FANCD2, acting downstream in the FA pathway. Targeting of the second allele in in BRCA2 and FANCD2 heterozygote clones resulted in redeletion exclusively of the already defective allele in multiple instances (13x concerning BRCA2, 25x concerning FANCD2), strongly suggesting a detrimental phenotype. Unlike complete FANCD2 disruption, the mere reduction of FANCD2 protein levels had no discernible effect. In addition, we confirmed that human cancer cells harboring the Seckel ATR mutation display impaired FANCD2 monoubiquitination and FANCD2 nuclear focus formation, as well as an increased sensitivity to DNA interstrand-crosslinking agents. Nevertheless, these cells were viable, indicating an ATR-independent function of FANCD2, distinct from its major known functions, to be responsible for the detrimental effects of FANCD2 loss. In conclusion, we established the downstream FA genes FANCD2 and BRCA2 to represent particularly vulnerable parts of the FA pathway, providing direct evidence for the paradoxical assumption that their inactivation could be predominantly selected against in cancer cells. This would explain why certain FA gene defects, despite an apparent selection for FA pathway inactivation in cancer, are rarely observed in tumors among the general population.

Identifying allelic loss and homozygous deletions in pancreatic cancer without matched normals using high-density single-nucleotide polymorphism arrays.

Recent advances in oligonucleotide arrays and whole-genome complexity reduction data analysis now permit the evaluation of tens of thousands of single-nucleotide polymorphisms simultaneously for a genome-wide analysis of allelic status. Using these arrays, we created high-resolution allelotype maps of 26 pancreatic cancer cell lines. The areas of heterozygosity implicitly served to reveal regions of allelic loss. The array-derived maps were verified by a panel of 317 microsatellite markers used in a subset of seven samples, showing a 97.1% concordance between heterozygous calls. Three matched tumor/normal pairs were used to estimate the false-negative and potential false-positive rates for identifying loss of heterozygosity: 3.6 regions (average minimal region of loss, 720,228 bp) and 2.3 regions (average heterozygous gap distance, 4,434,994 bp) per genome, respectively. Genomic fractional allelic loss calculations showed that cumulative levels of allelic loss ranged widely from 17.1% to 79.9% of the haploid genome length. Regional increases in "NoCall" frequencies combined with copy number loss estimates were used to identify 41 homozygous deletions (19 first reports), implicating an additional 13 regions disrupted in pancreatic cancer. Unexpectedly, 23 of these occurred in just two lines (BxPc3 and MiaPaCa2), suggesting the existence of at least two subclasses of chromosomal instability (CIN) patterns, distinguished here by allelic loss and copy number changes (original CIN) and those also highly enriched in the genomic "holes" of homozygous deletions (holey CIN). This study provides previously unavailable high-resolution allelotype and deletion breakpoint maps in widely shared pancreatic cancer cell lines and effectively eliminates the need for matched normal tissue to define informative loci.

Targeted disruption of FANCC and FANCG in human cancer provides a preclinical model for specific therapeutic options.

BACKGROUND & AIMS: How specifically to treat pancreatic and other cancers harboring Fanconi anemia gene mutations has raised great interest recently, yet preclinical studies have been hampered by the lack of well-controlled human cancer models. METHODS: We endogenously disrupted FANCC and FANCG in a human adenocarcinoma cell line and determined the impact of these genes on drug sensitivity, irradiation sensitivity, and genome maintenance. RESULTS: FANCC and FANCG disruption abrogated FANCD2 monoubiquitination, confirming an impaired Fanconi anemia pathway function. On treatment with DNA interstrand-cross-linking agents, FANCC and FANCG disruption caused increased clastogenic damage, G2/M arrest, and decreased proliferation. The extent of hypersensitivity varied among agents, with ratios of inhibitory concentration 50% ranging from 2-fold for oxaliplatin to 14-fold for melphalan, a drug infrequently used in solid tumors. No hypersensitivity was observed on gemcitabine, etoposide, 3-aminobenzamide, NU1025, or hydrogen peroxide. FANCC and FANCG disruption also resulted in increased clastogenic damage on irradiation, but only FANCG disruption caused a subsequent decrease in relative survival. Finally, FANCC and FANCG disruption increased spontaneous chromosomal breakage, supporting the role of these genes in genome maintenance and likely explaining why they are mutated in sporadic cancer. CONCLUSIONS: Our human cancer cell model provides optimal controls to elucidate fundamental biologic features of individual Fanconi anemia gene defects and facilitates preclinical studies of therapeutic options. The impact of Fanconi gene defects on drug and irradiation sensitivity renders these genes promising targets for a specific, genotype-based therapy for individual cancer patients, providing a strong rationale for clinical trials.

Comparison of gene expression in squamous cell carcinoma and adenocarcinoma of the uterine cervix.

OBJECTIVES: Microarray expression analysis of cervical tumors has revealed differential expression of genes that may be useful as markers or targets for treatment. We question the application of array findings across the major categories of cervical cancer. We sought to identify differences between normal squamous epithelium (NSQ) and glandular epithelium (NGL) of the uterine cervix and their malignant variants: squamous cell cancer (SCC) and adenocarcinoma (ACA). METHODS: Eight genes were selected: 12-lipoxygenase (12-LOX), keratin 4, trypsinogen 2 (TRY2), Rh glycoprotein C (RhGC), collagen type V alpha 2, integrin alpha 5, integrin alpha 6, and c-myc. Ten cases each of SCC and ACA of the cervix were selected from our tumor bank. NSQ and NGL epithelia were obtained from consecutive patients undergoing surgery for benign disease. RNA extraction, cDNA synthesis, and DNA amplification of all samples were performed according to an established protocol. Electrophoresis of the multiplexed polymerase chain reaction (PCR) products was performed under standard conditions, followed by digital image capture. A ratio of target to control gene (beta-actin) was obtained for each sample. Analysis of variance was applied to the mean ratios for each tissue to establish significant differences. Individual pairwise comparisons were made by Student t tests and verified with the Tukey-Kramer test. RESULTS: Clinically valid comparisons are NSQ to NGL, NSQ to SCC, NGL to ACA, and SCC to ACA. Various expression patterns were observed between the epithelia and their malignant phenotypes. Significant differences in gene expression were observed between benign squamous and glandular epithelium in four of the eight genes and between malignant squamous and glandular epithelium in three of the eight genes. Significant differences in gene expression between benign and malignant tissues were demonstrated in four of the eight genes. CONCLUSIONS: We have defined significant differential expression changes between the two principal cervical tumor types. Differences in genes are demonstrated and must be considered if array technology is applied to the study of the biologic behavior of these tumors as well as their screening and management. The observed differential expression should be a compelling argument to perform type-specific expression analysis for other tumors with histological variants.

Host genetic polymorphism analysis in cervical cancer.

BACKGROUND: The natural history of cervical cancer comprises a latency period that probably involves long-term immunologic tolerance of human papillomavirus infection. Identifying host determinants of viral persistence may help to better understand the mechanisms of tolerance and may lead to the development of tests that can allow more focused follow-up of high-risk individuals. METHODS: Genotypic frequencies of 12 polymorphic loci in four candidate genes from 127 cervical cancer patients were compared with a control group of 108 female blood donors. Genotypes were determined by PCR amplification and direct sequencing of isolated genomic DNA. RESULTS: The tumor necrosis factor-alpha (TNFalpha) -238 polymorphism was significantly underrepresented in cervical cancer patients [heterozygotes (HETs), odds ratio (OR) = 0.33; 95% confidence interval (CI), 0.11-0.96], as was the TNFalpha -376 polymorphism (P = 0.02; 0% for any variant genotype in cases vs 4.7% in controls). The NRAMP1 3' untranslated region STP+86 polymorphism also appeared to be inversely associated with cervical cancer, but this result did not reach statistical significance (HET, OR = 0.57; 95% CI, 0.32-1.02). The p53 codon 72 arginine allele showed a suggestive negative association with cervical cancer (HET, OR = 0.49; 95% CI, 0.14-1.63; homozygotes, OR = 0.35; 95% CI, 0.11-1.17). The remaining alleles tested showed no association with cervical cancer. CONCLUSIONS: We identified host genetic polymorphisms that may be associated with cervical cancer risk, some of which have been linked to potential functional effects on cellular immune responses or antigen processing. We failed to confirm earlier reports of increased cervical cancer susceptibility in women who harbor the p53 P72R allele. Although our findings support the general hypothesis that host immunogenetic determinants other than class II MHC may be important in the development of cervical cancer, further analysis of the HLA gene cluster comprising the implicated TNFalpha single-nucleotide polymorphisms will be required to determine whether their association is linkage independent.

TAP1, TAP2, and HLA-DR2 alleles are predictors of cervical cancer risk.

OBJECTIVE: The likelihood of developing cervical cancer has been shown to be increased in persons with certain HLA alleles. We evaluated immune response genes in the HLA region of chromosome 6 to see if individual or interactive associations with cervical cancer risk could be identified. METHODS: Tissue was obtained from 127 women undergoing surgical treatment for cervical cancer. Blood samples were obtained from 175 control subjects. A combination of polymerase chain reaction (PCR), sequence-specific PCR, and DNA sequencing was used to evaluate polymorphic alleles, including HLA class I B7, TNF alpha, HLA class II DR2, TAP1, and TAP2 genes. Fisher's exact test and logistic regression modeling were used for statistical analysis. RESULTS: A significantly greater proportion of the patients with cervical cancer were found to have the HLA class II DR2 1501 allele (P = 0.023) and the TAP2 A/B heterozygous pattern of alleles (P = 0.0006) than were women without cervical cancer. A proportion of patients with cervical cancer significantly smaller than that of the control women had a polymorphism at the -238 position of the TNF promoter and the TAP1 C/C homozygous pattern of alleles. With logistic modeling, the markers that showed consistent association with the occurrence of cervical cancer were TAP2 A/B, HLA-DR2 1501, and TAP1 C/C. CONCLUSIONS: We demonstrated a significant association between immune response genes and the risk of cervical cancer. Our data create a compelling argument for a gene or a cluster of genes in the HLA region of chromosome 6 that regulates host immune responses to human papillomavirus infection in a manner that results in inherited susceptibility or resistance to the transforming properties of oncogenic papillomaviruses.

BRAF and FBXW7 (CDC4, FBW7, AGO, SEL10) mutations in distinct subsets of pancreatic cancer: potential therapeutic targets.

The recognition of biologically distinct tumor subsets is fundamental to understanding tumorigenesis. This study investigated the mutational status of the serine/threonine kinase BRAF and the cyclin E regulator FBXW7 (CDC4, FBW7, AGO, SEL10) related to two distinct pancreatic carcinoma subsets: the medullary KRAS2-wild-type and the cyclin E overexpressing tumors, respectively. Among KRAS2-wild-type carcinomas, 33% (3 of 9) contained BRAF V599E mutations; one of which was identified in the pancreatic cancer cell line COLO357. Among 74 KRAS2-mutant carcinomas, no BRAF mutations were identified. Among the KRAS2/BRAF wild-type carcinomas, no mutations within pathway members MEK1, MEK2, ERK1, ERK2, RAP1B, or BAD were found. Using pancreatic cancer microarrays and immunohistochemistry, we determined that 6% (4 of 46 and 5 of 100 in two independent panels) of pancreatic adenocarcinomas overexpress cyclin E. We identified two potential mechanisms for this overexpression including the amplification/gain of CCNE1 gene copies in the Panc-1 and Su86.86 cell lines and a novel somatic homozygous mutation (H460R, in one of 11 pancreatic cancer xenografts having allelic loss) in FBXW7, which was accompanied by cyclin E overexpression by immunohistochemistry. Both BRAF and FBXW7 mutations functionally activate kinase effectors important in pancreatic cancer and extend the potential options for therapeutic targeting of kinases in the treatment of phenotypically distinct pancreatic adenocarcinoma subsets.