Pancreatic cancer pathology viewed in the light of evolution.

One way to understand ductal adenocarcinoma of the pancreas (pancreatic cancer) is to view it as unimaginably large numbers of evolving living organisms interacting with their environment. This "evolutionary view" creates both expected and surprising perspectives in all stages of neoplastic progression. Advances in the field will require greater attention to this critical evolutionary prospective.

The genetics of ductal adenocarcinoma of the pancreas in the year 2020: dramatic progress, but far to go.

The publication of the "Pan-Cancer Atlas" by the Pan-Cancer Analysis of Whole Genomes Consortium, a partnership formed by The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC), provides a wonderful opportunity to reflect on where we stand in our understanding of the genetics of pancreatic cancer, as well as on the opportunities to translate this understanding to patient care. From germline variants that predispose to the development of pancreatic cancer, to somatic mutations that are therapeutically targetable, genetics is now providing hope, where there once was no hope, for those diagnosed with pancreatic cancer.

Genome annotation by shotgun inactivation of a native gene in hemizygous cells: application to BRCA2 with implication of hypomorphic variants.

The greatest interpretive challenge of modern medicine may be to functionally annotate the vast variation of human genomes. Demonstrating a proposed approach, we created a library of BRCA2 exon 27 shotgun-mutant plasmids including solitary and multiplex mutations to generate human knockin clones using homologous recombination. This 55-mutation, 13-clone syngeneic variance library (SyVaL) comprised severely affected clones having early-stop nonsense mutations, functionally hypomorphic clones having multiple missense mutations emphasizing the potential to identify and assess hypomorphic mutations in novel proteomic and epidemiologic studies, and neutral clones having multiple missense mutations. Efficient coverage of nonessential amino acids was provided by mutation multiplexing. Severe mutations were distinguished from hypomorphic or neutral changes by chemosensitivity assays (hypersensitivity to mitomycin C and acetaldehyde), by analysis of RAD51 focus formation, and by mitotic multipolarity. A multiplex unbiased approach of generating all-human SyVaLs in medically important genes, with random mutations in native genes, would provide databases of variants that could be functionally annotated without concerns arising from exogenous cDNA constructs or interspecies interactions, as a basis for subsequent proteomic domain mapping or clinical calibration if desired. Such gene-irrelevant approaches could be scaled up for multiple genes of clinical interest, providing distributable cellular libraries linked to public-shared functional databases.

Salivary α-amylase, serum albumin, and myoglobin protect against DNA-damaging activities of ingested dietary agents in vitro.

Potent DNA-damaging activities were seen in vitro from dietary chemicals found in coffee, tea, and liquid smoke. A survey of tea varieties confirmed genotoxic activity to be widespread. Constituent pyrogallol-like polyphenols (PLPs) such as epigallocatechin-3-gallate (EGCG), pyrogallol, and gallic acid were proposed as a major source of DNA-damaging activities, inducing DNA double-strand breaks in the p53R assay, a well characterized assay sensitive to DNA strand breaks, and comet assay. Paradoxically, their consumption does not lead to the kind of widespread cellular toxicity and acute disease that might be expected from genotoxic exposure. Existing physiological mechanisms could limit DNA damage from dietary injurants. Serum albumin and salivary α-amylase are known to bind EGCG. Salivary α-amylase, serum albumin, and myoglobin, but not salivary proline-rich proteins, reduced damage from tea, coffee, and PLPs, but did not inhibit damage from the chemotherapeutics etoposide and camptothecin. This represents a novel function for saliva in addition to its known functions including protection against tannins. Cell populations administered repeated pyrogallol exposures had abatement of measured DNA damage by two weeks, indicating an innate cellular adaptation. We suggest that layers of physiological protections may exist toward natural dietary products to which animals have had high-level exposure over evolution.

Hypersensitivities for acetaldehyde and other agents among cancer cells null for clinically relevant Fanconi anemia genes.

Large-magnitude numerical distinctions (>10-fold) among drug responses of genetically contrasting cancers were crucial for guiding the development of some targeted therapies. Similar strategies brought epidemiological clues and prevention goals for genetic diseases. Such numerical guides, however, were incomplete or low magnitude for Fanconi anemia pathway (FANC) gene mutations relevant to cancer in FANC-mutation carriers (heterozygotes). We generated a four-gene FANC-null cancer panel, including the engineering of new PALB2/FANCN-null cancer cells by homologous recombination. A characteristic matching of FANCC-null, FANCG-null, BRCA2/FANCD1-null, and PALB2/FANCN-null phenotypes was confirmed by uniform tumor regression on single-dose cross-linker therapy in mice and by shared chemical hypersensitivities to various inter-strand cross-linking agents and γ-radiation in vitro. Some compounds, however, had contrasting magnitudes of sensitivity; a strikingly high (19- to 22-fold) hypersensitivity was seen among PALB2-null and BRCA2-null cells for the ethanol metabolite, acetaldehyde, associated with widespread chromosomal breakage at a concentration not producing breaks in parental cells. Because FANC-defective cancer cells can share or differ in their chemical sensitivities, patterns of selective hypersensitivity hold implications for the evolutionary understanding of this pathway. Clinical decisions for cancer-relevant prevention and management of FANC-mutation carriers could be modified by expanded studies of high-magnitude sensitivities.

"Selective cell death mediated by small conditional RNAs" is not selective.

Small conditional RNAs were used to kill cells selectively in a prior report. The method utilized the cellular innate immune response to dsRNA, causing PKR activation and cell death. We designed small conditional RNAs specific to a highly restricted transcript, that of the mesothelin gene expressed in various cancer lines and specific to a tpc/hpr fusion transcript expressed in a published "control" line. Hairpins of small conditional RNAs were functionally active in cell-free conditions. Hairpins were transfected into 6 types of cells. We observed non-specific killing of cells after transfection of the hairpins targeting the reported fusion transcript or of the mesothelin transcript. Thus when attempting to use this system for a special purpose, to target cancer mutations, the results were not satisfactory. Specifically when repeating the work described in the publication, we could not replicate the results using the methods described. Recently the publication was retracted but without comment on the validity of the reported method. Here we provide scientific basis to consider the method impaired or invalid.

FAM190A deficiency creates a cell division defect.

Like the p16, SMAD4, and RB1 genes, FAM190A (alias CCSER1) lies at a consensus site of homogeneous genomic deletions in human cancer. FAM190A transcripts in 40% of cancers also contain in-frame deletions of evolutionarily conserved exons. Its gene function was unknown. We found an internal deletion of the FAM190A gene in a pancreatic cancer having prominent focal multinuclearity. The experimental knockdown of FAM190A expression by shRNA caused focal cytokinesis defects, multipolar mitosis, and multinuclearity as observed in time-lapse microscopy. FAM190A was localized to the γ-tubulin ring complex of early mitosis and to the midbody in late cytokinesis by immunofluorescence assay and was present in the nuclear fraction of unsynchronized cells by immunoblot. FAM190A interacted with EXOC1 and Ndel1, which function in cytoskeletal organization and the cell division cycle. Levels of FAM190A protein peaked 12 hours after release from thymidine block, corresponding to M-phase. Slower-migrating phosphorylated forms accumulated toward M-phase and disappeared after release from a mitotic block and before cytokinesis. Studies of FAM190A alterations may provide mechanistic insights into mitotic dysregulation and multinuclearity in cancer. We propose that FAM190A is a regulator or structural component required for normal mitosis and that both the rare truncating mutations and common in-frame deletion alteration of FAM190A may contribute to the chromosomal instability of cancer.

DNA-intercalators causing rapid re-expression of methylated and silenced genes in cancer cells.

Epigenetic inactivation of tumor-suppressor and other regulatory genes plays a critical role in carcinogenesis. Transcriptional silencing is often maintained by DNA methyl transferase (DNMT)-mediated hypermethylation of CpG islands in promoter DNA. Nucleoside analogs including azacytidine and decitabine have been used to inhibit DNMT and re-activate genes, and are clinically used. Their shortcomings include a short half-life and a slow onset of action due to required nucleotide incorporation during DNA replication, which may limit clinical utility. It might be useful to begin to identify lead compounds having novel properties, specifically distinct and fast-acting gene desilencing. We previously identified chemicals augmenting gene expression in multiple reporter systems. We now report that a subset of these compounds that includes quinacrine re-expresses epigenetically silenced genes implicated in carcinogenesis. p16, TFPI2, the cadherins E-cadherin and CDH13, and the secreted frizzle-related proteins (SFRPs) SFRP1 and SFRP5 were desilenced in cancer cell lines. These lead compounds were fast-acting: re-expression occurred by 12-24 hours. Reactivation of silenced genes was accompanied by depletion of DNMT1 at the promoters of activated genes and demethylation of DNA. A model compound, 5175328, induced changes more rapidly than decitabine. These gene desilencing agents belonged to a class of acridine compounds, intercalated into DNA, and inhibited DNMT1 activity in vitro. Although to define the mechanism would be outside the scope of this initial report, this class may re-activate silenced genes in part by intercalating into DNA and subsequently inhibiting full DNMT1 activity. Rapid mechanisms for chemical desilencing of methylated genes therefore exist.

Biological clues to potent DNA-damaging activities in food and flavoring.

Population differences in age-related diseases and cancer could stem from differences in diet. To characterize DNA strand-breaking activities in selected foods/beverages, flavorings, and some of their constituent chemicals, we used p53R cells, a cellular assay sensitive to such breaks. Substances testing positive included reference chemicals: quinacrine (peak response, 51×) and etoposide (33×); flavonoids: EGCG (19×), curcumin (12×), apigenin (9×), and quercetin (7×); beverages: chamomile (11×), green (21×), and black tea (26×) and coffee (3-29×); and liquid smoke (4-28×). Damage occurred at dietary concentrations: etoposide near 5µg/ml produced responses similar to a 1:1000 dilution of liquid smoke, a 1:20 dilution of coffee, and a 1:5 dilution of tea. Pyrogallol-related chemicals and tannins are present in dietary sources and individually produced strong activity: pyrogallol (30×), 3-methoxycatechol (25×), gallic acid (21×), and 1,2,4-benzenetriol (21×). From structure-activity relationships, high activities depended on specific orientations of hydroxyls on the benzene ring. Responses accompanied cellular signals characteristic of DNA breaks such as H2AX phosphorylation. Breaks were also directly detected by comet assay. Cellular toxicological effects of foods and flavorings could guide epidemiologic and experimental studies of potential disease risks from DNA strand-breaking chemicals in diets.

Why your new cancer biomarker may never work: recurrent patterns and remarkable diversity in biomarker failures.

Less than 1% of published cancer biomarkers actually enter clinical practice. Although best practices for biomarker development are published, optimistic investigators may not appreciate the statistical near-certainty and diverse modes by which the other 99% (likely including your favorite new marker) do indeed fail. Here, patterns of failure were abstracted for classification from publications and an online database detailing marker failures. Failure patterns formed a hierarchical logical structure, or outline, of an emerging, deeply complex, and arguably fascinating science of biomarker failure. A new cancer biomarker under development is likely to have already encountered one or more of the following fatal features encountered by prior markers: lack of clinical significance, hidden structure in the source data, a technically inadequate assay, inappropriate statistical methods, unmanageable domination of the data by normal variation, implausibility, deficiencies in the studied population or in the investigator system, and its disproof or abandonment for cause by others. A greater recognition of the science of biomarker failure and its near-complete ubiquity is constructive and celebrates a seemingly perpetual richness of biologic, technical, and philosophical complexity, the full appreciation of which could improve the management of scarce research resources.

Unbiased discovery of interactions at a control locus driving expression of the cancer-specific therapeutic and diagnostic target, mesothelin.

Although significant effort is expended on identifying transcripts/proteins that are up-regulated in cancer, there are few reports on systematic elucidation of transcriptional mechanisms underlying such druggable cancer-specific targets. The mesothelin (MSLN) gene offers a promising subject, being expressed in a restricted pattern normally, yet highly overexpressed in almost one-third of human malignancies and a target of cancer immunotherapeutic trials. CanScript, a cis promoter element, appears to control MSLN cancer-specific expression; its related genomic sequences may up-regulate other cancer markers. CanScript is a 20-nt bipartite element consisting of an SP1-like motif and a consensus MCAT sequence. The latter recruits TEAD (TEA domain) family members, which are universally expressed. Exploration of the active CanScript element, especially the proteins binding to the SP1-like motif, thus could reveal cancer-specific features having diagnostic or therapeutic interest. The efficient identification of sequence-specific DNA-binding proteins at a given locus, however, has lagged in biomarker explorations. We used two orthogonal proteomics approaches--unbiased SILAC (stable isotope labeling by amino acids in cell culture)/DNA affinity-capture/mass spectrometry survey (SD-MS) and a large transcription factor protein microarray (TFM)--and functional validation to explore systematically the CanScript interactome. SD-MS produced nine candidates, and TFM, 18. The screens agreed in confirming binding by TEAD proteins and by newly identified NAB1 and NFATc. Among other identified candidates, we found functional roles for ZNF24, NAB1 and RFX1 in MSLN expression by cancer cells. Combined interactome screens yield an efficient, reproducible, sensitive, and unbiased approach to identify sequence-specific DNA-binding proteins and other participants in disease-specific DNA elements.

A thymidylate synthase ternary complex-specific antibody, FTS, permits functional monitoring of fluoropyrimidines dosing.

5-Fluorouracil (5FU) and similar fluoropyrimidines induce covalent modification of thymidylate synthase (TS) and inhibit its activity. They are often used to treat solid cancers, but drug resistance and toxicity are drawbacks. Therefore, there is an unmet need for a functional assay to quantify fluorouracil activity in tissues, so as to individually tailor dosing. It is cumbersome to separately quantify unmodified and 5FU-modified TS using currently available commercial anti-TS antibodies because they recognize both forms. We report here the first monoclonal antibody (FTS) specific to 5FU-modified TS. By immunoblot assay, the FTS antibody specifically recognizes modified TS in a dose-dependent manner in 5FU-treated cells, in cancer xenograft tissues of 5FU-treated mice, and in the murine tissues. In the same assay, the antibody is nonreactive with unmodified TS in untreated or treated cells and tissues. Speculatively, a high-throughput assay could be enabled by pairing anti-TS antibodies of two specificities, one recognizing only modified TS and another recognizing both forms, to structurally quantify the TS-inhibiting effect of fluorouracil at a cellular or tissue level without requiring prior protein separation. Such a development might aid preclinical analytic studies or make practical the individual tailoring of dosing.

Analysis of polymorphisms and haplotype structure of the human thymidylate synthase genetic region: a tool for pharmacogenetic studies.

5-Fluorouracil (5FU), a widely used chemotherapeutic drug, inhibits the DNA replicative enzyme, thymidylate synthase (Tyms). Prior studies implicated a VNTR (variable numbers of tandem repeats) polymorphism in the 5'-untranslated region (5'-UTR) of the TYMS gene as a determinant of Tyms expression in tumors and normal tissues and proposed that these VNTR genotypes could help decide fluoropyrimidine dosing. Clinical associations between 5FU-related toxicity and the TYMS VNTR were reported, however, results were inconsistent, suggesting that additional genetic variation in the TYMS gene might influence Tyms expression. We thus conducted a detailed genetic analysis of this region, defining new polymorphisms in this gene including mononucleotide (poly A:T) repeats and novel single nucleotide polymorphisms (SNPs) flanking the VNTR in the TYMS genetic region. Our haplotype analysis of this region used data from both established and novel genetic variants and found nine SNP haplotypes accounting for more than 90% of the studied population. We observed non-exclusive relationships between the VNTR and adjacent SNP haplotypes, such that each type of VNTR commonly occurred on several haplotype backgrounds. Our results confirmed the expectation that the VNTR alleles exhibit homoplasy and lack the common ancestry required for a reliable marker of a linked adjacent locus that might govern toxicity. We propose that it may be necessary in a clinical trial to assay multiple types of genetic polymorphisms in the TYMS region to meaningfully model linkage of genetic markers to 5FU-related toxicity. The presence of multiple long (up to 26 nt), polymorphic monothymidine repeats in the promoter region of the sole human thymidylate synthetic enzyme is intriguing.

Reexamining a proposal: thymidylate synthase 5'-untranslated region as a regulator of translation efficiency.

The DNA replicative gene, thymidylate synthase (TYMS), is inhibited upon treatment with the anticancer drug 5-fluorouracil (5FU). TYMS has 28-bp tandem repeat sequences or VNTR (variable numbers of tandem repeats) in the 5'-untranslated region (5'-UTR). The number of these repeats is variable in any given population, but the most prevalent are double (2R) and triple (3R) repeat sequences. A single G/C nucleotide polymorphism in the triple repeat sequence gives rise to a 3Rc or a 3Rg triple repeat structure. A widely cited literature used plasmid constructs of the 5'-UTR and proposed that genotyping the TYMS UTRs would predict the efficiency of Tyms protein translation, justifying altered therapeutic dosage of 5FU. Prior studies had unusual features in experimental design, such as using the firefly Kozak sequence in place of the native human TYMS Kozak sequence to determine the ribosomal translational efficiency of TYMS mRNA. Our results using transient transfection, antibiotic-selected pools of transfected cells, and stably transfected clones, while using plasmids having native human Kozak sequence, refute the earlier results.

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.

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.

Structural analysis of the cancer-specific promoter in mesothelin and in other genes overexpressed in cancers.

Mesothelin (MSLN) may be the most "dramatic" of the tumor markers, being strongly overexpressed in nearly one-third of human malignancies. The biochemical cause is unclear. We previously ascribed this cancer-specific overexpression to an element, Canscript, residing around 50 bp 5' of the transcription start site in cancer (Hucl, T., Brody, J. R., Gallmeier, E., Iacobuzio-Donahue, C. A., Farrance, I. K., and Kern, S. E. (2007) Cancer Res. 67, 9055-9065). Herein, we found a Canscript promoter activity elevated over 100-fold in cancer cells. In addition to a highly conserved TEAD1 (TEA domain family member 1)-binding MCAT motif, nucleotide substitution revealed the consensus core sequence (WCYCCACCC) of an SP1-like motif in Canscript. The unknown transcription factor binding to the SP1-like motif may hold the key for the cancer specificity of Canscript. SP1, GLI1, and RUNX1, -2, and -3 appeared unlikely to be the direct transcription factors acting at the SP1-like motif, but KLF6 had some features of such a candidate. YAP1, a TEAD1-binding protein, appeared necessary, but not sufficient, for Canscript activity; knockdown of YAP1 by small interfering RNAs greatly reduced MSLN levels in MSLN-overexpressing cells, but overexpressing YAP1 in MSLN-negative cells did not induce MSLN expression. Cansript-like sequences were found in other genes up-regulated in pancreatic cancer; reporters driven by the sequences from FXYD3, MUC1, and TIMP1 had activities more than 2 times that of the control. This suggested that the cause of MSLN overexpression might also contribute mechanistically to the overexpression of other tumor markers.

The complexity of pancreatic ductal cancers and multidimensional strategies for therapeutic targeting.

The directions of differentiation and the molecular features of ductal pancreatic cancer have by now been explored in reasonable detail. Already, diagnoses and therapeutic strategies benefit from observations distinguishing the major variant types of pancreatic cancer and the differing stages of disease at presentation. Additionally, individual patients differ within each variant type. In certain high-risk groups, this permits focused screening efforts. The tumorigenic influences that characterize individual patients are increasingly considered appropriate in defining clinical treatment plans. As a result, multiple variables affect success when individualizing screening or therapy. These competing variables often limit the potential for success: some variables dominate and should receive greater consideration than others. Simplistic expectations, often falsely optimistic, for individualized care may fail to 'pan out' in the real world. The development of individualized care will be efficient only when the full complexity of the disease is embraced.