Supplementary Tables S1 & S2 from Reprimo Methylation Is a Potential Biomarker of Barrett's-Associated Esophageal Neoplastic Progression

Supplementary Figure S1 from Reprimo Methylation Is a Potential Biomarker of Barrett's-Associated Esophageal Neoplastic Progression

Supplementary Table S1 from Hypermethylation of Tachykinin-1 Is a Potential Biomarker in Human Esophageal Cancer

Supplementary Table 1 from Pituitary Tumor-Transforming 1 Increases Cell Motility and Promotes Lymph Node Metastasis in Esophageal Squamous Cell Carcinoma

Supplementary Figure Legends 1-2 from A Multicenter, Double-Blinded Validation Study of Methylation Biomarkers for Progression Prediction in Barrett's Esophagus

Subsets of non-acute promyelocytic leukemia (APL) acute myelogenous leukemia (AML) exhibit aberrant retinoid signaling and demonstrate sensitivity to retinoids in vitro. We present the results of a phase 1 dose-escalation study that evaluated the safety, pharmacodynamics, and efficacy of IRX195183, a novel retinoic acid receptor α agonist, in patients with relapsed or refractory myelodysplastic syndrome (MDS) or AML. In this single center, single arm study, eleven patients with relapsed or refractory MDS/AML were enrolled and treated. Oral IRX195183 was administered at two dose levels: 50 mg daily or 75 mg daily for a total of two 28-day cycles. Patients with stable disease or better were allowed to continue on the drug for four additional 28-day cycles. Common adverse events included hypertriglyceridemia, fatigue, dyspnea, and edema. Three patients at the first dose level developed asymptomatic Grade 3 hypertriglyceridemia. The maximally tolerated dose was not reached. Four of the eleven patients had (36%) stable disease or better. One had a morphological complete remission with incomplete hematologic recovery while on the study drug. Two patients had evidence of in vivo leukemic blast maturation, as reflected by increased CD38 expression. In a pharmacodynamics study, plasma samples from four patients treated at the lowest dose level demonstrated the capacity to differentiate leukemic cells from the NB4 cell line in vitro. These results suggest that IRX195183 is safe, achieves biologically meaningful plasma concentrations and may be efficacious in a subset of patients with MDS/AML. Clinical Trial Registration: clinicaltrials.gov, identifier NCT02749708.

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.

Abstract Studies of cells harboring Fanconi anemia (FA) pathway defects have aided clinical understanding of inherited cancer risks and therapeutic strategies. Here, we observed a novel and large (27X) hypersensitivity of BRCA2- and PALB2-null genotypes to the epidemiologically important ethanol metabolite, acetaldehyde. This prominent acetaldehyde sensitivity may hold evolutionary and clinical significance. Interrogation of a novel panel of cells engineered to be null for various FA genes also revealed two classes of chemical hypersensitivities: the shared and divergent phenotypes. Prominent chemical hypersensitivities to various interstrand crosslinking (ICL) agents in vitro (melphalan, mitomycin C, and cisplatin), to γ-radiation in vitro, and to mitomycin C in vivo were essentially similar among the tested genotypes. A large divergence of responsiveness existed, however, between the cell lines when using the PARP inhibitor KU0058948, the topoisomerase II inhibitor etoposide, and acetaldehyde. These results indicate that, toward some agents, not all FA defects are necessarily equivalent; this divergence among phenotypes may dissect functions differing among FA genes and may presage differing clinical and epidemiological implications. We additionally present the first engineered PALB2-null human cancer cells. The results suggest new applications in cancer epidemiology, prevention, and targeted therapy. Citation Format: Soma Ghosh, Surojit Sur, Sashidhar R. Yerram, Carlo Rago, Anil K. Bhunia, M. Zulfiquer Hossain, Bogdan C. Paun, Yunzhao R. Ren, Christine A. Iacobuzio-Donahue, Nilofer A. Azad, Scott E. Kern. Acetaldehyde and drug hypersensitivities of Fanconi anemia defects: Implications for cancer initiation, prevention, and therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3580. doi:10.1158/1538-7445.AM2013-3580

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 gamma-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.

Chronic obstructive pulmonary disease appears to occur slowly and progressively over many years, with both genetic factors and environmental modifiers contributing to its pathogenesis. Although the c-Jun/activator protein 1 transcriptional factor regulates cell proliferation, apoptosis, and inflammatory responses, its role in lung pathogenesis is largely unknown. In this study, we report decreased expression levels of c-Jun mRNA and protein in the lung tissues of patients with advanced chronic obstructive pulmonary disease, and the genetic deletion of c-Jun specifically in alveolar epithelial cells causes progressive emphysema with lung inflammation and alveolar air space enlargement, which are cardinal features of emphysema. Although mice lacking c-Jun specifically in lung alveolar epithelial cells appear normal at the age of 6 weeks, when exposed to long-term cigarette smoke, c-Jun-mutant mice display more lung inflammation with perivascular and peribronchiolar infiltrates compared with controls. These results demonstrate that the c-Jun/activator protein 1 pathway is critical for maintaining lung alveolar cell homeostasis and that loss of its expression can contribute to lung inflammation and progressive emphysema.

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.

Polo-like kinase 1 (PLK1) is overexpressed in various human cancers. However, the biological functions and the post-transcriptional regulations of PLK1 in esophageal cancer (EC) are still unknown. The purposes of our study are to determine whether PLK1 can be a molecular target of EC therapy and to identify a microRNA (miRNA) targeting PLK1. We performed loss-of-function and gain-of-function experiments regarding cell proliferation, cell cycle, apoptosis, in vivo tumor formation and luciferase reporter assays, using siRNAs against PLK1 and miRNA. PLK1 protein was expressed in all 11 EC cell lines, but not in normal esophageal epithelial cells (HEEpiC). Knockdown of PLK1 in EC cells induced G2/M arrest (p < 0.001) in cell cycle assay and reduced cell proliferation (p = 0.019) and tumor formation ability in vivo (p < 0.0001). MiR-593*, identified as a miRNA targeting PLK1 by a database search, was less expressed especially in six EC cell lines than HEEpiC cells. Moreover, miR-593* expression level was inversely correlated with PLK1 mRNA level in 48 clinical tissue specimens of EC (p = 0.006). Introduction of synthetic miR-593* suppressed PLK1 expression by 69-73%, reduced cell proliferation (p = 0.008) and increased cell proportion of G2/M phase (p = 0.01) in HSA/c (an EC cells), whereas a miR-593* inhibitor upregulated PLK1 expression by 11-55%. Additionally, luciferase assay demonstrated that miR-593* interacted two binding sites in the PLK1 3'-UTR and reduced 56.8-71.5% of luciferase activity by degrading luciferase mRNA in HSA/c cells. In conclusion, PLK1 is post-transcriptionally regulated by miR-593* and could be a promising molecular target for EC treatment.

BACKGROUND: Colorectal cancer (CRC) is 1 of the leading causes of death in the Western world. CRC develops from premalignant lesions, chiefly colorectal adenomas. Currently, the most accurate and recommended screening method for finding colorectal adenomas is colonoscopy performed on all individuals aged >50 years. However, the costs and risks associated with this procedure are relatively high. The objectives of the current study were to correlate epigenetic alterations that occur in normal rectal mucosa, smoking status, and age with the presence or absence of concomitant colorectal adenomas and to assess the potential clinical value of methylation in normal rectal biopsies as a screening assay for the presence of polyps and, hence, the need for a full colonoscopy. METHODS: One hundred thirteen normal rectal mucosal biopsies from 113 patients were studied. DNA was extracted, modified with sodium bisulfite, and subjected to real-time quantitative, methylation-specific polymerase chain reaction analysis for the following genes: adenomatous polyposis coli (APC); cadherin 1, type 1, E-cadherin (epithelial) (CDH1); estrogen receptor 1 (ESR1); cytokine high in normal 1 (HIN1); hyperplastic polyposis protein 1 (HPP1); O-6 methylguanine-DNA methyltransferase (MGMT); neural epidermal growth factor-like 1 (NELL1); splicing factor 3B, 14-kDa subunit (p14); cyclin-dependent kinase (CDK) inhibitor 2B (inhibits CDK4) (p15); retinoic acid receptor beta (RAR beta); somatostatin (SST); tachykinin, precursor 1 (TAC1); and tissue inhibitor of metalloproteinase (TIMP) metallopeptidase inhibitor 3 (TIMP3). Data were then analyzed using several proprietary software programs. RESULTS: By using several sets of genes, clinical characteristics, and multivariate analyses, the authors developed a prediction model for the presence of concomitant colorectal adenomas at the time of rectal biopsy. They also observed strong correlations between smoking status and rectal methylation pattern and between smoking status and the presence or risk of concomitant adenomas. CONCLUSIONS: A prediction model was developed for the presence of colorectal adenomas based on gene methylation patterns in the normal rectum. The results indicated that these genes may be involved in early stages of adenoma formation. The observed epigenetic alterations in these markers may be caused in part by the effects of smoking and/or age. Normal rectal methylation may be useful as a biomarker for narrowing the population in need of screening colonoscopy. Cancer 2010;116:4495-501. (C) 2010 American Cancer Society.

Background. Cholangiocarcinoma (CCA) is an aggressive epithelial cancer of the biliary tree with survival measured in months. The only potentially curative treatment for CCA is surgical resection. Unfortunately, the vast majority of patients are diagnosed at late stages, when surgery is not an option. MicroRNAs (miRs) are short single stranded RNA sequences, recently shown to have major regulatory functions in cancer. TIMP3 is one of 4 members of the metalloproteinases family and its role was associated with cancer invasion and metastasis. We aimed at elucidating the regulatory mechanisms of TIMP3 in CCA. Methods. Fifteen histologically confirmed CCAs and 9 normal liver specimens were collected at surgery at Johns Hopkins Hospital after informed consent was obtained from all patients. The NLs were obtained at the time of surgery for CCA from normal liver. Quantitative RT-PCR (qRTPCR) for messenger RNA (mRNA) and miRs and quantitative Methylation Specific PCR (qMSP) were performed as previously described. Results. We measured the qRT-PCR level of TIMP3 mRNA and found it to be underexpressed in CCA vs. NL specimens, suggesting a tumor-suppressive role for TIMP3 in the biliary tree. In contrast, our previous work revealed that miR-21 is overexpressed in CCA vs. NL, suggesting a possible negative regulation of TIMP3 by miR-21. In silico searches suggested that TIMP3 is indeed a putative target of miR-21. The CCA cell lines CAK1, TFK1 and HuCCT1 were transfected with a miR-21 inhibitor and TIMP3 protein levels were measured. TIMP3 was significantly elevated in all 3 cell lines transfected with the inhibitor, strongly suggesting that miR-21 downregulates TIMP3 protein levels. Interestingly, miR-21 does not directly bind to TIMP3 mRNA, as evinced by luciferase assays, suggesting the presence of an intermediary molecule or a different mechanism of miR-21 regulation of TIMP3. To investigate a potential co-regulation of TIMP3 in CCA by hypermethylation, we measured the promoter methylation in all 24 specimens. TIMP3 promoter was found to be hypermethylated in only 1/15 CCA (6.6%), 0/3 CCA cell lines and 0/9 NL specimens. Conclusions: TIMP3 mRNA is elevated in NL and decreased in CCA specimens implicating TIMP3 as a tumor suppressor gene in the biliary tree. Our data also suggests that TIMP3 promoter hypermethylation does not appear to play a major role in its downregulation in human CCA specimens. In contrast, miR-21 is a major regulator of TIMP3 in CCA. Furthermore, miR-21 may exert at least part of its oncogenic, pro-invasive effects in CCA by inhibiting TIMP3.

Background & Aims: Barrett's esophagus (BE) is a highly premalignant disease that predisposes to the development of esophageal adenocarcinoma (EAC); however, the involvement of microRNAs (miRs) in BE-EAC carcinogenic progression is not known. Methods: Esophageal cultured cells (HEEpiC, QhTRT, ChTRT, GihTRT, and OE-33) and esophageal tissues (22 normal epithelia, 24 BE, and 22 EAC) were studied. MiR microarrays and quantitative reverse-transcription polymerase chain reaction (RT-PCR) were employed to explore and verify differentially expressed miRs. Quantitative genomic PCR was performed to study copy number variation at the miR-106b-25 polycistron and MCM7 gene locus on chromosome 7q22.1. In vitro cell proliferation, cell cycle, and apoptosis assays and in vivo tumorigenesis experiments were performed to elucidate biologic effects of the miR-106b-25 polycistron. Western blotting and luciferase assays were performed to confirm direct messenger RNA (mRNA) targeting by the miR-106b-25 polycistron. Results: The miR-106b-25 polycistron exerted potential proliferative, antiapoptotic, cell cycle-promoting effects in vitro and tumorigenic activity in vivo. MiRs-93 and -106b targeted and inhibited p21, whereas miR-25 targeted and inhibited Bim. This polycistron was upregulated progressively at successive stages of neoplasia, in association with genomic amplification and overexpression of MCM7. In addition, miRs-93 and -106b decreased p21 mRNA, whereas miR-25 did not alter Bim mRNA, suggesting the following discrete miR effector mechanisms: (1) for p21, mRNA degradation; (2) for Bim, translational inhibition. Conclusions: The miR-106b-25 polycistron is activated by genomic amplification and is potentially involved in esophageal neoplastic progression and proliferation via suppression of 2 target genes: p21 and Bim.

Abstract Esophageal adenocarcinoma risk in Barrett's esophagus (BE) is increased 30- to 125-fold versus the general population. Among all BE patients, however, neoplastic progression occurs only once per 200 patient-years. Molecular biomarkers are therefore needed to risk-stratify patients for more efficient surveillance endoscopy and to improve the early detection of progression. We therefore performed a retrospective, multicenter, double-blinded validation study of eight BE progression prediction methylation biomarkers. Progression or nonprogression were determined at 2 years (tier 1) and 4 years (tier 2). Methylation was assayed in 145 nonprogressors and 50 progressors using real-time quantitative methylation-specific PCR. Progressors were significantly older than nonprogressors (70.6 versus 62.5 years; P < 0.001). We evaluated a linear combination of the eight markers, using coefficients from a multivariate logistic regression analysis. Areas under the ROC curve (AUC) were high in the 2-year, 4-year, and combined data models (0.843, 0.829, and 0.840; P < 0.001, <0.001, and <0.001, respectively). In addition, even after rigorous overfitting correction, the incremental AUCs contributed by panels based on the 8 markers plus age versus age alone were substantial (Δ-AUC = 0.152, 0.114, and 0.118, respectively) in all 3 models. A methylation biomarker–based panel to predict neoplastic progression in BE has potential clinical value in improving both the efficiency of surveillance endoscopy and the early detection of neoplasia. [Cancer Res 2009;69(10):4112–5]

Background: Coding region microsatellite instability (MSI) results in loss of gene products and promotion of microsatellite-unstable (MSI-H) carcinogenesis. Recent studies have indicated that MSI within 3'-untranslated regions (3'UTRs) may post-transcriptionally dysregulate gene products. Within this context, we conducted a broad mutational survey of 42 short 3'UTR microsatellites (MSs) in 45 MSI-H colorectal tumors and their corresponding normal colonic mucosae.Methodology/Principal Findings: In order to estimate the overall susceptibility of MSs to MSI in MSI-H tumors, the observed MSI frequency of each MS was correlated with its length, interspecies sequence conservation level, and distance from some genetic elements (i.e., stop codon, polyA signal, and microRNA binding sites). All MSs were stable in normal colonic mucosae. The MSI frequency at each MS in MSI-H tumors was independent of sequence conservation level and distance from other genetic elements. In contrast, MS length correlated significantly with MSI frequency in MSI-H tumors (r = 0.86, p = 7.2x10(-13)). 3'UTR MSs demonstrated MSI frequencies in MSI-H tumors higher than the 99% upper limit predicted by MS length for RB1-inducible coiled-coil 1(RB1CC1, mutation frequency 68.4%), NUAK family SNF1-like kinase 1(NUAK1, 31.0%), and Rtf1, Paf1/RNA polymerase II complex component, homolog (RTF1, 25.0%). An in silico prediction of RNA structure alterations was conducted for these MSI events to gauge their likelihood of affecting post-transcriptional regulation. RB1CC1 mutant was predicted to lose a microRNA-accessible loop structure at a putative binding site for the tumor-suppressive microRNA, miR-138. In contrast, the predicted 3'UTR structural change was minimal for NUAK1- and RTF1 mutants. Notably, real-time quantitative RT-PCR analysis revealed significant RB1CC1 mRNA overexpression vs. normal colonic mucosae in MSI-H cancers manifesting RB1CC1 3'UTR MSI (9.0-fold; p = 3.6x10(-4)).Conclusions: This mutational survey of well-characterized short 3'UTR MSs confirms that MSI incidence in MSI-H colorectal tumors correlates with MS length, but not with sequence conservation level or distance from other genetic elements. This study also identifies RB1CC1 as a novel target of frequent mutation and aberrant upregulation in MSI-H colorectal tumors. The predicted loss of a microRNA-accessible structure in mutant RB1CC1 RNA fits the hypothesis that 3'UTR MSI involves in aberrant RB1CC1 posttranscriptional upregulation. Further direct assessments are indicated to investigate this possibility.