CDKN2A-p16 Deletion and Activated KRASG12D Drive Barrett's-Like Gland Hyperplasia-Metaplasia and Synergize in the Development of Dysplasia Precancer Lesions.

BACKGROUND & AIMS: Barrett's esophagus is the precursor of esophageal dysplasia and esophageal adenocarcinoma. CDKN2A-p16 deletions were reported in 34%-74% of patients with Barrett's esophagus who progressed to dysplasia and esophageal adenocarcinoma, suggesting that p16 loss may drive neoplastic progression. KRAS activation frequently occurs in esophageal adenocarcinoma and precancer lesions. LGR5+ stem cells in the squamocolumnar-junction (SCJ) of mouse stomach contribute as Barrett's esophagus progenitors. We aimed to determine the functional effects of p16 loss and KRAS activation in Barrett's-like metaplasia and dysplasia development. METHODS: We established mouse models with conditional knockout of CDKN2A-p16 (p16KO) and/or activated KRASG12D expression targeting SCJ LGR5+ cells in interleukin 1b transgenic mice and characterized histologic alterations (mucous-gland hyperplasia/metaplasia, inflammation, and dysplasia) in mouse SCJ. Gene expression was determined by microarray, RNA sequencing, and immunohistochemistry of SCJ tissues and cultured 3-dimensional organoids. RESULTS: p16KO mice exhibited increased mucous-gland hyperplasia/metaplasia versus control mice (P = .0051). Combined p16KO+KRASG12D resulted in more frequent dysplasia and higher dysplasia scores (P = .0036), with 82% of p16KO+KRASG12D mice developing high-grade dysplasia. SCJ transcriptome analysis showed several activated pathways in p16KO versus control mice (apoptosis, tumor necrosis factor-α/nuclear factor-kB, proteasome degradation, p53 signaling, MAPK, KRAS, and G1-to-S transition). CONCLUSIONS: p16 deletion in LGR5+ cell precursors triggers increased SCJ mucous-gland hyperplasia/metaplasia. KRASG12D synergizes with p16 deletion resulting in higher grades of SCJ glandular dysplasia, mimicking Barrett's high-grade dysplasia. These genetically modified mouse models establish a functional role of p16 and activated KRAS in the progression of Barrett's-like lesions to dysplasia in mice, representing an in vivo model of esophageal adenocarcinoma precancer. Derived 3-dimensional organoid models further provide in vitro modeling opportunities of esophageal precancer stages.

Downregulation of Friend Leukemia Integration 1 (FLI1) follows the stepwise progression to gastric adenocarcinoma.

Gastric adenocarcinoma (GC) is a leading cause of cancer-related deaths worldwide. The transcription factor gene Friend Leukemia Integration 1 (FLI1) is methylated and downregulated in human GC tissues. Using human GC samples, we determined which cells downregulate FLI1, when FLI1 downregulation occurs, if FLI1 downregulation correlates with clinical-pathologic characteristics, and whether FLI1 plays a role in invasion and/or proliferation of cultured cells. We analyzed stomach tissues from 98 patients [8 normal mucosa, 8 intestinal metaplasia (IM), 7 dysplasia, 91 GC] by immunohistochemistry for FLI1. Epithelial cells from normal, IM, and low-grade dysplasia (LGD) showed strong nuclear FLI1 staining. GC epithelial cells showed significantly less nuclear FLI1 staining as compared to normal epithelium, IM and LGD (P=1.2×10-5, P=1.4×10-6 and P=0.006, respectively). FLI1 expression did not correlate with tumor stage or differentiation, but was associated with patient survival, depending on tumor differentiation. We tested the functional role of FLI1 by assaying proliferation and invasion in cultured GC cells. Lentiviral-transduced FLI1 overexpression in GC AGS cells inhibited invasion by 73.5% (P = 0.001) and proliferation by 31.5% (P = 0.002), as compared to controls. Our results support a combined role for FLI1 as a suppressor of invasiveness and proliferation in gastric adenocarcinoma, specifically in the transition from pre-cancer lesions and dysplasia to invasive adenocarcinoma, and suggest that FLI1 may be a prognostic biomarker of survival in gastric cancers.

High-resolution genomic alterations in Barrett's metaplasia of patients who progress to esophageal dysplasia and adenocarcinoma.

The main risk factor for esophageal dysplasia and adenocarcinoma (DAC) is Barrett's esophagus (BE), characterized by intestinal metaplasia. The critical genomic mechanisms that lead to progression of nondysplastic BE to DAC remain poorly understood and require analyses of longitudinal patient cohorts and high-resolution assays. We tested BE tissues from 74 patients, including 42 nonprogressors from two separate groups of 21 patients each and 32 progressors (16 in a longitudinal cohort before DAC/preprogression-BE and 16 with temporally concurrent but spatially separate DAC/concurrent-BE). We interrogated genome-wide somatic copy number alterations (SCNAs) at the exon level with high-resolution SNP arrays in DNA from formalin-fixed samples histologically confirmed as nondysplastic BE. The most frequent abnormalities were SCNAs involving FHIT exon 5, CDKN2A/B or both in 88% longitudinal BE progressors to DAC vs. 24% in both nonprogressor groups (p = 0.0004). Deletions in other genomic regions were found in 56% of preprogression-BE but only in one nonprogressor-BE (p = 0.0004). SCNAs involving FHIT exon 5 and CDKN2A/B were also frequently detected in BE temporally concurrent with DAC. TP53 losses were detected in concurrent-BE but not earlier in preprogression-BE tissues of patients who developed DAC. CDKN2A/p16 immunohistochemistry showed significant loss of expression in BE of progressors vs. nonprogressors, supporting the genomic data. Our data suggest a role for CDKN2A/B and FHIT in early progression of BE to dysplasia and adenocarcinoma that warrants future mechanistic research. Alterations in CDKN2A/B and FHIT by high-resolution assays may serve as biomarkers of increased risk of progression to DAC when detected in BE tissues.

A precision oncology approach to the pharmacological targeting of mechanistic dependencies in neuroendocrine tumors.

We introduce and validate a new precision oncology framework for the systematic prioritization of drugs targeting mechanistic tumor dependencies in individual patients. Compounds are prioritized on the basis of their ability to invert the concerted activity of master regulator proteins that mechanistically regulate tumor cell state, as assessed from systematic drug perturbation assays. We validated the approach on a cohort of 212 gastroenteropancreatic neuroendocrine tumors (GEP-NETs), a rare malignancy originating in the pancreas and gastrointestinal tract. The analysis identified several master regulator proteins, including key regulators of neuroendocrine lineage progenitor state and immunoevasion, whose role as critical tumor dependencies was experimentally confirmed. Transcriptome analysis of GEP-NET-derived cells, perturbed with a library of 107 compounds, identified the HDAC class I inhibitor entinostat as a potent inhibitor of master regulator activity for 42% of metastatic GEP-NET patients, abrogating tumor growth in vivo. This approach may thus complement current efforts in precision oncology.

The number of titrated microRNA species dictates ceRNA regulation.

microRNAs (miRNAs) play key roles in cancer, but their propensity to couple their targets as competing endogenous RNAs (ceRNAs) has only recently emerged. Multiple models have studied ceRNA regulation, but these models did not account for the effects of co-regulation by miRNAs with many targets. We modeled ceRNA and simulated its effects using established parameters for miRNA/mRNA interaction kinetics while accounting for co-regulation by multiple miRNAs with many targets. Our simulations suggested that co-regulation by many miRNA species is more likely to produce physiologically relevant context-independent couplings. To test this, we studied the overlap of inferred ceRNA networks from four tumor contexts-our proposed pan-cancer ceRNA interactome (PCI). PCI was composed of interactions between genes that were co-regulated by nearly three-times as many miRNAs as other inferred ceRNA interactions. Evidence from expression-profiling datasets suggested that PCI interactions are predictive of gene expression in 12 independent tumor- and non-tumor contexts. Biochemical assays confirmed ceRNA couplings for two PCI subnetworks, including oncogenes CCND1, HIF1A and HMGA2, and tumor suppressors PTEN, RB1 and TP53. Our results suggest that PCI is enriched for context-independent interactions that are coupled by many miRNA species and are more likely to be context independent.

High-definition CpG methylation of novel genes in gastric carcinogenesis identified by next-generation sequencing.

Gastric cancers are the most frequent gastric malignancy and usually arise in the sequence of Helicobacter pylori-associated chronic gastritis. CpG methylation is a central mechanism of epigenetic gene regulation affecting cancer-related genes, and occurs early in gastric carcinogenesis. DNA samples from non-metaplastic gastric mucosa with variable levels of gastritis (non-metaplastic mucosa), intestinal metaplasia, or gastric cancer were screened with methylation arrays for CpG methylation of cancer-related genes and 30 gene targets were further characterized by high-definition bisulfite next-generation sequencing. In addition, data from The Cancer Genome Atlas were analyzed for correlation of methylation with gene expression. Overall, 13 genes had significantly increased CpG methylation in gastric cancer vs non-metaplastic mucosa (BRINP1, CDH11, CHFR, EPHA5, EPHA7, FGF2, FLI1, GALR1, HS3ST2, PDGFRA, SEZ6L, SGCE, and SNRPN). Further, most of these genes had corresponding reduced expression levels in gastric cancer compared with intestinal metaplasia, including novel hypermethylated genes in gastric cancer (FLI1, GALR1, SGCE, and SNRPN), suggesting that they may regulate neoplastic transformation from non-malignant intestinal metaplasia to cancer. Our data suggest a tumor-suppressor role for FLI1 in gastric cancer, consistent with recently reported data in breast cancer. For the genes with strongest methylation/expression correlation, namely FLI1, the expression was lowest in microsatellite-unstable tumors compared with other gastric cancer molecular subtypes. Importantly, reduced expression of hypermethylated BRINP1 and SGCE was significantly associated with favorable survival in gastric cancer. In summary, we report novel methylation gene targets that may have functional roles in discrete stages of gastric carcinogenesis and may serve as biomarkers for diagnosis and prognosis of gastric cancer.

The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser³59 Mediate the Redox-Dependent Increase in Protein Kinase C-δ Activity.

The diverse roles of protein kinase C-δ (PKCδ) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKCδ signaling responses. PKCδ exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Tyr(313)-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an interaction between PKCδ's Tyr(313)-phosphorylated hinge region and its phosphotyrosine-binding C2 domain that controls PKCδ's enzymology indirectly by decreasing phosphorylation in the kinase domain ATP-positioning loop at Ser(359). We show that wild-type (WT) PKCδ displays a strong preference for substrates with serine as the phosphoacceptor residue at the active site when it harbors phosphomimetic or bulky substitutions at Ser(359.) In contrast, PKCδ-S359A displays lipid-independent activity toward substrates with either a serine or threonine as the phosphoacceptor residue. Additional studies in cardiomyocytes show that oxidative stress decreases Ser(359) phosphorylation on native PKCδ and that PKCδ-S359A overexpression increases basal levels of phosphorylation on substrates with both phosphoacceptor site serine and threonine residues. Collectively, these studies identify a C2 domain-pTyr(313) docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKCδ catalytic activity.

Role of the urokinase plasminogen activator receptor in mediating impaired efferocytosis of anti-SSA/Ro-bound apoptotic cardiocytes: Implications in the pathogenesis of congenital heart block.

RATIONALE: Binding of maternal anti-Ro/La antibodies to cognate antigen expressed on apoptotic cardiocytes decreases clearance by healthy cardiocytes, which may contribute to the development of autoimmune associated congenital heart block and fatal cardiomyopathy. OBJECTIVE: Given recent evidence implicating the urokinase plasminogen activator receptor (uPAR) as a "don't eat me" signal during efferocytosis, experiments addressed whether surface bound anti-Ro antibodies inhibit apoptotic cell removal via an effect on the expression/function of the urokinase-type plasminogen activator protease uPA/uPAR system. METHODS AND RESULTS: As assessed by flow cytometry and confocal microscopy, uPAR colocalizes and interacts with Ro60 on the surface of apoptotic human fetal cardiocytes. Blocking of uPAR enhances phagocytosis of apoptotic cardiocytes by healthy cardiocytes and reverses the anti-Ro60-dependent impaired clearance of apoptotic cardiocytes. Binding of anti-Ro60 antibodies to apoptotic cardiocytes results in increased uPAR expression, as well as enhanced uPA activity. The binding of anti-Ro60 did not alter other surface molecules involved in cell recognition (calreticulin, CD31, or CD47). CONCLUSIONS: These data suggest that increased uPAR expression and uPA activity induced by anti-Ro60 binding to the apoptotic fetal cardiocyte provide a molecular basis by which these antibodies inhibit efferocytosis and ultimately lead to scar of the fetal conduction system and working myocardium.

Evaluation of fetuses in a study of intravenous immunoglobulin as preventive therapy for congenital heart block: Results of a multicenter, prospective, open-label clinical trial.

OBJECTIVE: The recurrence rate of anti-SSA/Ro-associated congenital heart block (CHB) is 17%. Sustained reversal of third-degree block has never been achieved. Based on potential reduction of maternal autoantibody titers as well as fetal inflammatory responses, intravenous immunoglobulin (IVIG) was evaluated as preventive therapy for CHB. METHODS: A multicenter, prospective, open-label study based on Simon's 2-stage optimal design was initiated. Enrollment criteria included the presence of anti-SSA/Ro antibodies in the mother, birth of a previous child with CHB/neonatal lupus rash, current treatment with < or = 20 mg/day of prednisone, and <12 weeks pregnant. IVIG (400 mg/kg) was given every 3 weeks from week 12 to week 24 of gestation. The primary outcome was the development of second-degree or third-degree CHB. RESULTS: Twenty mothers completed the IVIG protocol before the predetermined stopping rule of 3 cases of advanced CHB in the study was reached. CHB was detected at 19, 20, and 25 weeks; none of the cases occurred following the finding of an abnormal PR interval on fetal Doppler monitoring. One of these mothers had 2 previous children with CHB. One child without CHB developed a transient rash consistent with neonatal lupus. Sixteen children had no manifestations of neonatal lupus at birth. No significant changes in maternal titers of antibody to SSA/Ro, SSB/La, or Ro 52 kd were detected over the course of therapy or at delivery. There were no safety issues. CONCLUSION: This study establishes the safety of IVIG and the feasibility of recruiting pregnant women who have previously had a child with CHB. However, IVIG at low doses consistent with replacement does not prevent the recurrence of CHB or reduce maternal antibody titers.

Arsenite induced poly(ADP-ribosyl)ation of tumor suppressor P53 in human skin keratinocytes as a possible mechanism for carcinogenesis associated with arsenic exposure.

Arsenite is an environmental pollutant. Exposure to inorganic arsenic in drinking water is associated with elevated cancer risk, especially in skin. Arsenite alone does not cause skin cancer in animals, but arsenite can enhance the carcinogenicity of solar UV. Arsenite is not a significant mutagen at non-toxic concentrations, but it enhances the mutagenicity of other carcinogens. The tumor suppressor protein P53 and nuclear enzyme PARP-1 are both key players in DNA damage response. This laboratory demonstrated earlier that in cells treated with arsenite, the P53-dependent increase in p21(WAF1/CIP1) expression, normally a block to cell cycle progression after DNA damage, is deficient. Here we show that although long-term exposure of human keratinocytes (HaCaT) to a nontoxic concentration (0.1 microM) of arsenite decreases the level of global protein poly(ADP-ribosyl)ation, it increases poly(ADP-ribosyl)ation of P53 protein and PARP-1 protein abundance. We also demonstrate that exposure to 0.1 microM arsenite depresses the constitutive expression of p21 mRNA and P21 protein in HaCaT cells. Poly(ADP-ribosyl)ation of P53 is reported to block its activation, DNA binding and its functioning as a transcription factor. Our results suggest that arsenite's interference with activation of P53 via poly(ADP-ribosyl)ation may play a role in the comutagenic and cocarcinogenic effects of arsenite.

Gene expression levels in normal human lymphoblasts with variable sensitivities to arsenite: identification of GGT1 and NFKBIE expression levels as possible biomarkers of susceptibility.

Drinking arsenic-contaminated water is associated with increased risk of neoplasias of the skin, lung, bladder and possibly other sites, as well as other diseases. Earlier, we showed that human lymphoblast lines from different normal unexposed donors showed variable sensitivities to the toxic effects of arsenite. In the present study, we used microarray analysis to compare the basal gene expression profiles between two arsenite-resistant (GM02707, GM00893) and two arsenite-sensitive lymphoblast lines (GM00546, GM00607). A number of genes were differentially expressed in arsenite-sensitive and arsenite-resistant cells. Among these, gamma-glutamyltranspeptidase 1 (GGT1) and NF kappa B inhibitor-epsilon (NFKBIE) showed higher expression levels in arsenite-resistant cells. RT-PCR analysis with gene-specific primers confirmed these results. Reduction of GGT1 expression level in arsenite-resistant lymphoblasts with GGT1-specific siRNA resulted in increased cell sensitivity to arsenite. In conclusion, we have demonstrated for the first time that expression levels of GGT1 and possibly NFKBIE might be useful as biomarkers of genetic susceptibility to arsenite. Expression microarrays can thus be exploited for identifying additional biomarkers of susceptibility to arsenite and to other toxicants.

Dead or dying: the importance of time in cytotoxicity assays using arsenite as an example.

Arsenite is a toxicant and environmental pollutant associated with multisite neoplasias and other health effects. The wide range of doses used and the claims that some high doses are "not toxic" in some assays have confounded studies on its mechanism of action. The purpose of this study is to determine whether the treatment time and particularly the duration between treatment and assay are important factors in assessing arsenite toxicity. We compared three commonly used assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR), and clonal survival, using human osteogenic sarcoma (HOS) cell line U-2OS. Results from the assays were well correlated only when the factor of time was taken into account. In both the MTT and NR assays, exposure to arsenite for 24 h induced much less toxicity than exposure for 48 or 72 h, which gave similar results. In contrast, results in clonal survival assays showed only a small difference between 24-h exposure and longer exposure times. Arsenite demonstrated delayed cytotoxicity, killing the cells even after its removal from the medium in NR assay. Apoptosis was assessed by TUNEL staining and caspase-3 activation. After treatment for 24 h with 0.1 and 1 microM arsenite, no apoptosis was seen. However, after an additional 24 h in arsenite-free medium, a small amount of apoptosis could be detected, and much more apoptosis was seen after 48 h. In contrast, 10 microM arsenite triggered rapid necrosis and failed to activate caspase 3 or cause TUNEL staining. We also confirmed previous reports that exposure to low concentrations of arsenite caused transient stimulation of cell growth. Our finding of delayed toxicity by arsenite suggests that to avoid underestimation of toxicity, the duration between treatment and assay should be taken into account in choosing appropriate doses for arsenite as well as for other toxicants that may show similar delayed toxicity. The NR and MTT assays should be performed only after an interval of at least 48 h after a 24-h exposure to arsenite.

Variability in sensitivity to arsenite does not correlate with arsenic accumulation rate in normal human lymphoblasts.

Arsenic is a common environmental contaminant of our air, water and food, but not every individual who drinks arsenic-contaminated water shows clinical signs of toxicity. Large inter-individual variations are also found in arsenite-induced aneuploidy, chromosome aberrations and sister chromatid exchanges in peripheral blood lymphocytes from different human donors. Lymphoblasts are virally immortalized lymphocytes that retain most of the properties of lymphocytes. Individual lymphoblast cell lines retained their arsenite sensitivity after cryopreservation and subsequent revival. We measured the accumulation of 73[As]-arsenite into lymphoblast lines derived from 11 normal individuals. Arsenite accumulation rate varied 6.3 fold between the slowest and the fastest subjects. Assays in 14 lymphoblast lines showed variability to the toxic effects of arsenite, as measured by growth inhibition. Lymphoblast lines also vary with regard to their growth rates, but there is no relationship between growth rate and arsenite sensitivity. Surprisingly, we also found no correlation between arsenite accumulation rate and cellular sensitivity to growth inhibition, suggesting that the arsenite accumulation rate may not be the main determinant of cellular sensitivity to arsenic. We were also unable to detect evidence for a human homolog for the yeast arsenite efflux gene ACR3, using RT-PCR.

fau and its ubiquitin-like domain (FUBI) transforms human osteogenic sarcoma (HOS) cells to anchorage-independence.

Arsenite is the most likely carcinogenic form of arsenic in the environment. Previously, expression cloning for cDNAs whose overexpression confers arsenite-resistance in Chinese hamster V79 cells identified two genes: fau and a novel gene, asr2. The fau gene encodes a ubiquitin-like protein (here called FUBI) fused to the ribosomal S30 protein. Since the expression of the fox sequence (antisense to fau) increased the tumorigenicity of a mouse sarcoma virus, it was proposed that fau might be a tumor suppressor gene. We intended to test its ability to block arsenite-induced transformation of human osteogenic sarcoma (HOS) cells to anchorage-independence. Instead, we found that overexpressing fau itself was able to transform HOS cells. When the two domains were expressed separately, only FUBI was transforming and only the S30 domain conferred arsenite resistance. An incidental finding was the transforming activity of the selectable marker, hyg. FUBI belongs to the ubiquitin-like protein group that is capable of forming conjugates to other proteins, although none have so far been identified. Alternatively, FUBI may act as a substitute or inhibitor of ubiquitin, to which it is most closely related, or to close ubiquitin-like relatives UCRP, FAT10, and/or Nedd8.