Tomas Hucl

High Cancer-Specific Expression of Mesothelin (MSLN) Is Attributable to an Upstream Enhancer Containing a Transcription Enhancer Factor–Dependent MCAT Motif

Identification of genes with cancer-specific overexpression offers the potential to efficiently discover cancer-specific activities in an unbiased manner. We apply this paradigm to study mesothelin (MSLN) overexpression, a nearly ubiquitous, diagnostically and therapeutically useful characteristic of pancreatic cancer. We identified an 18-bp upstream enhancer, termed CanScript, strongly activating transcription from an otherwise weak tissue-nonspecific promoter and operating selectively in cells having aberrantly elevated cancer-specific MSLN transcription. Introducing mutations into CanScript showed two functionally distinct sites: an Sp1-like site and an MCAT element. Gel retardation and chromatin immunoprecipitation assays showed the MCAT element to be bound by transcription enhancer factor (TEF)-1 (TEAD1) in vitro and in vivo. The presence of TEF-1 was required for MSLN protein overexpression as determined by TEF-1 knockdown experiments. The cancer specificity seemed to be provided by a putative limiting cofactor of TEF-1 that could be outcompeted by exogenous TEF-1 only in a MSLN-overexpressing cell line. A CanScript concatemer offered enhanced activity. These results identify a TEF family member as a major regulator of MSLN overexpression, a fundamental characteristic of pancreatic and other cancers, perhaps due to an upstream and highly frequent aberrant cellular activity. The CanScript sequence represents a modular element for cancer-specific targeting, potentially suitable for nearly a third of human malignancies.

Evaluating the drug-target relationship between thymidylate synthase expression and tumor response to 5-fluorouracil. Is it time to move forward?

Thymidylate synthase is a target of 5-fluoruracil, a pyrimidine analog used to treat gastrointestinal and other cancers. The 5-fluorouracil metabolite, fluoro-deoxyuridine monophosphate, forms a ternary complex with thymidylate synthase and 5,10-methylene tetrahydrofolate. The purpose of this study was to evaluate the time-honored connection between thymidylate synthase and 5-fluorouracil. From our literature search spanning reports from 1995 to 2007 published in journals having an impact factor greater than 2, we stratified the tumors within each article, according to low versus high thymidylate synthase expression. These groups were subdivided into responders, stable disease, or disease progression. The relationship between thymidylate synthase expression and 5-fluorouracil response was analyzed for the overall group, as well as for subsets. Overall, the literature supported an approximately 2-fold inverse relationship between thymidylate synthase expression and response to 5-fluoruracil. We found no change in the trend for a relationship between thymidylate synthase and 5-fluorouracil when the literature was stratified by date of publication, impact factor of the journal in which the report was published, or substrate (mRNA versus protein) for measuring thymidylate synthase expression. Of note, there is no significant change in the trend when comparing 5-fluorouracil treatment alone or in combination with leucovorin. We found a decline of this trend when certain chemotherapeutics were used in combination with 5-fluorouracil. In sum, the connection between thymidylate synthase expression and patient response to 5-fluorouracil does not satisfy expectations for an effective drug-target relationship; and thus, studies of the thymidylate synthase tandem repeat status might only be clinically valuable in regards to patient toxicity. Thus, we question the reliability of thymidylate synthase expression as a clinical predictor of 5-fluorouracil response. Future research could perhaps be directed towards alternate targets and metabolites of 5-fluorouracil, in an effort to find a clinically relevant biomarker panel for response and to optimize fluoropyrimidine-based therapy.

A syngeneic variance library for functional annotation of human variation: application to BRCA2.

The enormous scope of natural human genetic variation is now becoming defined. To accurately annotate these variants, and to identify those with clinical importance, is often difficult to assess through functional assays. We explored systematic annotation by using homologous recombination to modify a native gene in hemizygous (wt/Deltaexon) human cancer cells, generating a novel syngeneic variance library (SyVaL). We created a SyVaL of BRCA2 variants: nondeleterious, proposed deleterious, deleterious, and of uncertain significance. We found that the null states BRCA2(Deltaex11/Deltaex11) and BRCA2(Deltaex11/Y3308X) were deleterious as assessed by a loss of RAD51 focus formation on genotoxic damage and by acquisition of toxic hypersensitivity to mitomycin C and etoposide, whereas BRCA2(Deltaex11/Y3308Y), BRCA2(Deltaex11/P3292L), and BRCA2(Deltaex11/P3280H) had wild-type function. A proposed phosphorylation site at codon 3291 affecting function was confirmed by substitution of an acidic residue (glutamate, BRCA2(Deltaex11/S3291E)) for the native serine, but in contrast to a prior report, phosphorylation was dispensable (alanine, BRCA2(Deltaex11/S3291A)) for BRCA2-governed cellular phenotypes. These results show that SyVaLs offer a means to comprehensively annotate gene function, facilitating numerical and unambiguous readouts. SyVaLs may be especially useful for genes in which functional assays using exogenous expression are toxic or otherwise unreliable. They also offer a stable, distributable cellular resource for further research.

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.

Pancreas divisum does not modify the natural course of chronic pancreatitis

BackgroundPancreas divisum is the most common congenital variant of the pancreas; however, its clinical significance remains controversial. The purpose of our study was to determine the role of pancreas divisum in the development of chronic pancreatitis.MethodsWe compared the clinical presentation, morphological findings, and course of disease of 30 patients with chronic pancreatitis associated with pancreas divisum (there was coexisting chronic alcohol abuse in 18 cases) to those of 57 patients with chronic pancreatitis and no evidence of pancreas divisum (15 with nonalcoholic pancreatitis and 42 with alcoholic pancreatitis).ResultsSex distribution, age at onset of disease, clinical presentation, course of disease, and frequency of complications were not affected by the presence of pancreas divisum. Although the etiology of pancreatitis in patients with pancreas divisum may be attributed to impaired drainage of the majority of the gland through the minor papilla, we observed a relatively low frequency of isolated dorsal duct involvement in our patients irrespective of alcohol use (25% and 28% in patients with and without a history of alcohol abuse, respectively). However, involvement of the ventral duct was commonly observed (75% and 72%, respectively).ConclusionsThe presence of pancreas divisum in our study did not modify the natural course of chronic nonalcoholic or alcoholic pancreatitis. Pancreas divisum is not likely to play a dominant role in the etiopathogenesis of chronic pancreatitis.

Non-erosive and erosive gastroesophageal reflux diseases: No difference with regard to reflux pattern and motility abnormalities

Objective. To investigate whether gastroesophageal reflux disease (GERD) with mild to moderate erosive esophagitis (ERD) is a more severe disease regarding the amount of acid reflux, motor abnormalities, and the presence/absence of hiatus hernia than non-erosive reflux disease (NERD). Material and methods. The study comprised a retrospective analysis of prospectively collected data on 313 consecutive subjects referred for endoscopy, pH-metry, and esophageal manometry. The patients were divided into four groups: Group I (n=92), subjects without GERD with normal pH-metry, without esophagitis and without reflux symptoms; Group II (n=111), patients with NERD (no esophagitis, abnormal pH-metry); Group III (n=77), patients with mild to moderate ERD (LA A, B; abnormal pH-metry); Group IV (n=33), patients with severe or complicated esophagitis (LA C, D; Barretts esophagus). All data are expressed as medians with 5th–95th percentiles. Results. No difference was found in the amount of acid reflux between patients with mild to moderate ERD and those with NERD. The DeMeester score was 34.5 (17–105) in NERD patients and 31.6 (15–102) in ERD patients. No significant differences were found between NERD and ERD patients regarding lower esophageal sphincter (LES) basal pressure (11.6 mmHg; 3–25 in NERD versus 10.7 mmHg; 3–22 in ERD). Similar proportions of patients with NERD and ERD had low LES basal pressure (20.7% in NERD versus 24.7% in ERD; NS) and hiatus hernia (44% and 56%; NS). A relatively high proportion of patients without GERD (Group I) had ineffective esophageal motility (39%) and hiatus hernia (30%). Conclusions. No difference was found between NERD and mild to moderate ERD in terms of acid exposure time and esophageal motor abnormalities. To a certain extent, ERD is a “more complicated” form of the disease. Host factors related to a particular patient (e.g. mucosal defense, genetics, acid clearance) might be responsible for the development of esophagitis.

Limits to Thymidylate Synthase and TP53 Genes as Predictive Determinants for Fluoropyrimidine Sensitivity and Further Evidence for RNA-Based Toxicity as a Major Influence

The major determinants of 5-flurouracil (5-FU) response would seem, based on accumulated literature, to be thymidylate synthase (TYMS, TS) expression levels, TS gene modifications, and TP53 status. We tested 5-FU sensitivity in yeast and human cancer cell models in which TS or TP53 alleles and expression were varied. Polymorphic TS tandem repeat status, TS expression levels reported, TS intragenic mutations, and TP53 status in outbred and experimental cancer cell lines did not predict 5-FU sensitivity or resistance. Novel observations included a dose-resistant persistence of unbound TS protein in many cancers and, upon 5-FU treatment of the colon cancer cell line, HCT116, evidence of allelic switching favoring transcripts of the mutant TS allele. The reported alleles having an intragenic mutation could not be causally associated with major degrees of 5-FU sensitivity. In yeast, TS protein was altered upon treatment with FdUMP, but 5-FU toxicity seemed to be largely RNA-based, being rescued by uridine rather than by thymidine. Cancer cell lines were also rescued from 5-FU toxicity with uridine rather than thymidine. Additionally, a TS (CDC21) knockout yeast strain, obviating any potential role for TS protein as a target, was hypersensitive to 5-FU. When denatured proteins from cancer cells treated with radiolabeled 5-FU were labeled, species with alternative molecular weights other than TS were visualized, providing further evidence for alternative 5-FU protein targets. These data emphasize that TS and TP53 status do not consistently explain the variance in responses of fluoropyrimidine-treated cancer cells, in part due to RNA-based toxicity.

Prevalence and risk factors of steatosis after liver transplantation and patient outcomes

Steatosis occurs frequently after liver transplantation (LT). We aimed to determine the prevalence of steatosis in adult LT recipients, to determine the effects of significant (>33%; grades 2‐3) steatosis on patient survival, and to identify risk factors for the development of significant steatosis and its effect on fibrosis progression. We retrospectively examined 2360 posttransplant biopsies of 548 LT recipients. Survival was compared between patients with significant steatosis and those with grades 0‐1 steatosis. Patients with significant steatosis were compared to controls without steatosis (grade 0) for clinical and laboratory factors and fibrosis progression. Steatosis was found in 309 (56.4%) patients, including 93 (17.0%) patients with significant steatosis. Steatohepatitis (nonalcoholic fatty liver disease activity score ≥ 5) was diagnosed in 57 (10.4%) patients. The prevalence of steatosis increased from 30.3% at 1 year to 47.6% at 10 years after LT (P < 0.001). Survival times did not differ between groups (P = 0.29). On multivariate analysis of pretransplant factors and initial immunosuppression (IS), alcohol‐induced cirrhosis (P < 0.001) and high body mass index (BMI; P = 0.002) were associated with the development of significant steatosis, whereas increased levels of alkaline phosphatase (P = 0.01) and mycophenolate mofetil given initially (P = 0.009) appeared to protect against significant steatosis. On multivariate analysis of posttransplant factors, high BMI (P < 0.001), serum triglycerides (P < 0.001), alcohol consumption (P = 0.005), and type 2 diabetes mellitus (P = 0.048) were associated with significant steatosis, whereas high creatinine (P = 0.02) appeared to protect against significant steatosis. Significant steatosis was not associated with a higher fibrosis stage (P = 0.62). Posttransplant steatosis affects 56.4% of LT recipients, and the prevalence increases with time after LT. Recipient factors and types of IS affect the risk for significant steatosis, which is not associated with a higher fibrosis stage or worse patient survival. Liver Transplantation 22 644‐655 2016 AASLD.

Distinguishing rational from irrational applications of pharmacogenetic synergies from the bench to clinical trials.

Single therapeutic agents very often fail in unselected patients. It is therefore commonplace to combine an agent specifically with a selected patient subgroup or with another agent. To support such efforts, it is useful to clarify the distinctions between the terms and the mathematical models used in analyzing combinations. To incorporate molecular disease classifications, the familiar concept of the therapeutic window is modified to define a pharmacogenetic window, which is an unambiguous numerical measure of the magnitude of interaction produced by a combination, and to define a test of pharmacogenetic synergy. In contrast, certain common comparative methods, such as vertical windows (comparing effects at a given dose) and animal models of mutational targets may be dominated by undesirable features. Although this discussion is oriented towards cancer therapy, an extension of these concepts to other comparative biologic assays is feasible and advisable.

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.