Eric S. Calhoun

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-RasG12D, B-RafV619E and MycERT2 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-RasG12D and B-RafV619E, and in human pancreatic cancer. Furthermore, genetic targeting of the Nrf2 pathway impairs K-RasG12D-induced proliferation and tumorigenesis in vivo. Thus, the Nrf2 antioxidant and cellular detoxification program represents a previously unappreciated mediator of oncogenesis.

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.

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.

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.

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 path¬way. Targeting of the second allele in BRCA2 and FANCD2 heterozygote clones resulted in re-deletion exclusively of the already defective allele in multiple instances (13x concerning BRCA2, 25x concerning FANCD2), strongly suggesting a detrimental phenotype. Unlike com¬plete 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.

Copy-number methods dramatically underestimate loss of heterozygosity in cancer.

In the first seven months of 2006, Genes, Chromosomes and Cancer published eight studies using comparative genomic hybridization (CGH) to analyze genomic gains and losses in cancer (Aarts et al., 2006; Coe et al., 2006; Diep et al., 2006; Horsley et al., 2006; Hosoya et al., 2006; Mendrzyk et al., 2006; Ribeiro et al., 2006; Rossi et al., 2006). Such studies, and those based upon other copy-number methods (Pinkel et al., 1988; Schrock et al., 1996; Lucito et al., 2003), are most informative when copy-number changes closely mimic changes in allelic status, for otherwise they will under-represent the extent of genomic alterations (Cavenee et al., 1983; Huang et al., 2004). Beroukhim and colleagues, using 10K SNP arrays to study prostate cancer, suggested that the extent of under-representation of loss of heterozygosity (LOH) in copy number methods may be very high (Beroukhim et al., 2006). Copy-neutral (i.e., having two copies) and copy-gain (i.e., having more than two copies) forms of allelic loss accounted for a surprising 80% of all LOH identified using paired normal/tumor tissues. Given the interest in deletion assessments by CGH, we felt a need to provide an independent confirmation of these findings. Therefore, we compared copy-number estimates with genotype (allele-discriminating) data obtained from 24 pancreatic cancer cell lines previously evaluated on 100K SNP arrays (Calhoun et al., 2006). Copy-number estimates were determined by the Affymetrix Copy Number Analysis Tool (version 2.2) and variation was smoothed using a moving-average algorithm. All copy-number estimates were rounded to the nearest integer and cataloged with respect to allelic status (i.e., LOH vs. retention of heterozygosity, ROH). Figure 1 summarizes these data, representing more than 2.7 million SNPs. We found that copy-neutral and copy-gain LOH events accounted for 67.5% of all LOH identified in pancreatic cancer (15.0% represented copy-gain LOH), while LOH associated with copy-reduction occurred at a rate of only 32.2% of LOH identified. Before drawing conclusions from these results, an assessment of likely sources of error is in order. For example, *9.5% of ROH SNPs were estimated to have a reduction in copy-number (i.e., having one copy). Such a counter-intuitive result is inherently erroneous and could be due to the failure to correctly classify candidate regions of LOH. Such errors could derive from a number of sources. For example, a previous estimate suggests that 17.5% of LOH went undetected when analyzed in the absence of matched normal tissue (Calhoun et al., 2006). Beroukhim et al., (2006) provided a similar false-negative rate (19.8%) in their study. Such misclassifications would decrease the frequency of copy-reduction LOH. Conversely, an estimated false-positive rate of LOH of 11.1% (ROH incorrectly classified as LOH) would artificially inflate the level of copy-neutral and copy-gain LOH seen in our study. Assuming these represent maximal false-negative and false-positive rates and using our SNP assessment as ‘‘true LOH’’, we did a virtual reassignment of all copy-reduced ROH SNPs as copy-reduced LOH SNPs and reassigned 11.1% of copy-neutral and copy-gain LOH SNPs as ROH SNPs. From this analysis, we predict that methods depending solely upon copy number analysis would theoretically identify a maximum of only 47.2% of all forms of LOH. The results of our study in pancreatic cancer support those of Beroukhim et al., (2006) in prostate cancer and suggest the levels of LOH to be underestimated markedly in recent studies using CGH. In a recent review by Karhu et al., (2006), the authors summarize the spectrum of genetic alterations reported in pancreatic cancer using copy-number methods and reinforce the idea that pancreatic adenocarcinoma is a genetically highly-complex disease. The apparent poor accuracy in illustrating the true level of LOH, however, appears to undermine the effectiveness of these studies in characterizing genomic losses in cancer tissues.

Molecular Genetics of Pancreatic Cancer

In cancer, genome stability is compromised. Ductal adenocarcinoma of the pancreas is no exception. Considerable progress in the identification and characterization of somatic and/or germline genetic alterations has provided the mechanistic foundations of this genetically complex disease. Numerous alterations, including chromosomal copy-number gains, amplifications and homozygous deletions, loss of heterozygosity (LOH) with and without copy number reduction, and balanced and unbalanced structural re-arrangements, are commonly observed ( 1 – 4 ). Gross chromosomal changes are often complemented by smaller, more subtle alterations affecting the open reading frames of proto-oncogenes, tumor suppressors, and genome caretaker genes ( 5 – 8 ). Continued evaluation of these and additional yet-unidentified genetic alterations should translate into rational diagnostic and treatment strategies. This chapter describes the spectrum and frequency of genetic alterations observed in ductal adenocarcinomas of the pancreas, as organized by the underlying presence of two, largely mutually exclusive, types of genome instability: chromosomal instability (CIN) and microsatellite instability (MIN). This distinction is justified, as each tumor type exhibits unique histologic and molecular characteristics ( 9 ).

Abstract 2738: Establishing an in vitro cell culture model for studying the function of FAM190A deletions

A growing number of studies have reported a variety of genetic alterations to FAM190A (CCSER1) in cancer. Interestingly, a large percentage of these are focused on deleting downstream exons producing in-frame arrangements for this protein. An analysis of coding sequences reveals that regions of FAM190A are highly conserved throughout the N-terminus, however, with no recognizable protein domains. Published immunofluorescence and shRNA knockdown studies have since suggested that FAM190A is localized to the midbody during cytokinesis and may function in maintaining genome stability. Unfortunately, attempts to overexpress FAM190A were not successful by these authors. The aim of our current study was to try to generate an in vitro model that would allow us to examine the effect of a C-terminal deletion on cellular division and FAM190A9s ability to associate with the spindle and/or midbody structures. To accomplish this, full length FAM190A and a deletion mutant construct lacking exons 8-11 were separately cloned into mammalian expression vectors that include a N-terminal HaloTag Fusion marker and a modified CMV promoter. Current studies are focused on introducing these constructs into the pancreatic cancer cell line, PL-5, to generate FAM190A stably expressing cell lines. Citation Format: Eric S. Calhoun. Establishing an in vitro cell culture model for studying the function of FAM190A deletions. [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 2738.