Hon-Chiu Eastwood Leung

Cell-Specific Gene Expression in Langerhans Cell Histiocytosis Lesions Reveals a Distinct Profile Compared with Epidermal Langerhans Cells

Langerhans cell histiocytosis (LCH) is a rare disease characterized by heterogeneous lesions containing CD207+ Langerhans cells (LCs) and lymphocytes that can arise in almost any tissue and cause significant morbidity and mortality. After decades of research, the cause of LCH remains speculative. A prevailing model suggests that LCH arises from malignant transformation and metastasis of epidermal LCs. In this study, CD207+ cells and CD3+ T cells were isolated from LCH lesions to determine cell-specific gene expression. Compared with control epidermal CD207+ cells, the LCH CD207+ cells yielded 2113 differentially expressed genes (false discovery rate < 0.01). Surprisingly, the expression of many genes previously associated with LCH, including cell-cycle regulators, proinflammatory cytokines, and chemokines, were not significantly different from control LCs in our study. However, several novel genes whose products activate and recruit T cells to sites of inflammation, including SPP1 (osteopontin), were highly overexpressed in LCH CD207+ cells. Furthermore, several genes associated with immature myeloid dendritic cells were overexpressed in LCH CD207+ cells. Compared with the peripheral CD3+ cells from LCH patients, the LCH lesion CD3+ cells yielded only 162 differentially regulated genes (false discovery rate < 0.01), and the expression profile of the LCH lesion CD3+ cells was consistent with an activated regulatory T cell phenotype with increased expression of FOXP3, CTLA4, and SPP1. Results from this study support a model of LCH pathogenesis in which lesions do not arise from epidermal LCs but from accumulation of bone marrow-derived immature myeloid dendritic cells that recruit activated lymphocytes.

Curcumin Modulates DNA Methylation in Colorectal Cancer Cells

Aim Recent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells. Materials and Methods To evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2′-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR. Results As expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean β-values of 0.12), however, non-significant changes in mean β-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines. Conclusions Our data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza-CdR, curcumin-induced methylation changes occurred only in a subset of partially-methylated genes, which provides additional mechanistic insights into the potent chemopreventive effect of this dietary nutraceutical.

Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues.

Although RNA can be retrieved from formalin‐fixed, paraffin‐embedded (FFPE) tissues, the yield is low, and the RNA is fragmented. Recent advances in gene expression profiling underscore the importance of identifying a fixative that preserves histology and mRNA. We demonstrated that, for immersion fixation of brains, 70% ethanol is superior to formalin for mRNA preservation. RNA yield from ethanol‐fixed tissues was 70% of the yield from fresh frozen specimens, but only a negligible quantity was recovered from formalin‐fixed tissues. RNA from ethanol‐fixed brains showed integrity comparable to RNA from fresh frozen tissues, and RT‐PCR using RNA from ethanol‐fixed tissues was consistently successful. RNA from FFPE tissues composed of low‐molecular weight fragments, and their use in RT‐PCR failed repeatedly. The yield and quality of RNA from ethanol‐fixed brains were unaffected after immersion at 4°C for 2 weeks. In a blinded comparison to FFPE tissues, ethanol‐fixed specimens were judged to show comparable histology and superior immunostaining. After laser capture microdissection (LCM), we failed to recover mRNA from FFPE tissues but retrieved mRNA from ethanol‐fixed tissues for RT‐PCR and cDNA microarray analysis. We conclude that 70% ethanol preserves RNA integrity and is suitable for expression profiling of brain tissues by LCM and cDNA microarray.

Genomic profiling in Down syndrome acute lymphoblastic leukemia identifies histone gene deletions associated with altered methylation profiles.

Patients with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) have distinct clinical and biological features. Whereas most DS-ALL cases lack the sentinel cytogenetic lesions that guide risk assignment in childhood ALL, JAK2 mutations and CRLF2 overexpression are highly enriched. To further characterize the unique biology of DS-ALL, we performed genome-wide profiling of 58 DS-ALL and 68 non-DS (NDS) ALL cases by DNA copy number, loss of heterozygosity, gene expression and methylation analyses. We report a novel deletion within the 6p22 histone gene cluster as significantly more frequent in DS-ALL, occurring in 11 DS (22%) and only 2 NDS cases (3.1%) (Fishers exact P=0.002). Homozygous deletions yielded significantly lower histone expression levels, and were associated with higher methylation levels, distinct spatial localization of methylated promoters and enrichment of highly methylated genes for specific pathways and transcription factor-binding motifs. Gene expression profiling demonstrated heterogeneity of DS-ALL cases overall, with supervised analysis defining a 45-transcript signature associated with CRLF2 overexpression. Further characterization of pathways associated with histone deletions may identify opportunities for novel targeted interventions.

Boswellic acid induces epigenetic alterations by modulating DNA methylation in colorectal cancer cells

Accumulating evidence suggests that chemopreventive effects of some dietary polyphenols may in part be mediated by their ability to influence epigenetic mechanisms in cancer cells. Boswellic acids, derived from the plant Boswellia serrata, have long been used for the treatment of various inflammatory diseases due to their potent anti-inflammatory activities. Recent preclinical studies have also suggested that this compound has anti-cancer potential against various malignancies. However, the precise molecular mechanisms underlying their anti-cancer effects remain elusive. Herein, we report that boswellic acids modulate DNA methylation status of several tumor suppressor genes in colorectal cancer (CRC) cells. We treated RKO, SW48 and SW480 CRC cell lines with the active principle present in boswellic acids, acetyl-keto-β-boswellic acid (AKBA). Using genome-wide DNA methylation and gene expression microarray analyses, we discovered that AKBA induced a modest genome-wide demethylation that permitted simultaneous re-activation of the corresponding tumor suppressor genes. The quantitative methylation-specific PCR and RT-PCR validated the gene demethylation and re-expression in several putative tumor suppressor genes including SAMD14 and SMPD3. Furthermore, AKBA inhibited DNMT activity in CRC cells. Taken together, these results lend further support to the growing notion that anti-cancer effect of boswellic acids may in part be due to its ability to demethylate and reactivate methylation-silenced tumor suppressor genes. These results suggest that not only boswellic acid might be a promising epigenetic modulator in the chemoprevention and treatment of CRC, but also provide a rationale for future investigations on the usefulness of such botanicals for epigenetic therapy in other human malignancies.

Histone induced platelet aggregation is inhibited by normal albumin

INTRODUCTION Histones are small, nuclear proteins that serve to package DNA. Recent reports suggest that extracellular histones, including histone H4, may contribute to the pathogenesis of sepsis; they promote platelet aggregation and thrombosis when released into the circulation during inflammation or cell death. The mechanisms by which the body minimizes the deleterious effects of circulating histones are unclear. Because histones can bind to plasma proteins, including albumin, we hypothesized that normal albumin can prevent histones from activating platelets. MATERIALS AND METHODS Platelets and platelet-free plasma were obtained from healthy, adult subjects. The dose-dependent effects of histone H4 on platelet aggregation were studied by optical aggregometry. The effects of native and albumin-depleted plasma (prepared by affinity chromatography) on histone-induced platelet aggregation were also assessed. The effects of normal and surface-neutralized albumin (through modification of carboxyl groups) on histone-induced platelet activation and aggregation were evaluated using flow cytometry and aggregometry. RESULTS Histone H4 induced platelet aggregation in a dose-dependent manner. This histone-induced platelet aggregation was inhibited by both plasma and human serum albumin in a dose-dependent fashion. Furthermore, depletion of albumin from plasma reduced its ability to inhibit aggregation. Finally, surface neutralization of albumin decreased its ability to inhibit histone-induced activation and aggregation. DISCUSSION These data suggest that normal albumin serves a role in preventing histone-induced platelet aggregation in a charge-dependent manner.

Abstract 3936: Identification and validation of the potential biomarker insulin-like growth factor binding protein acid-labile subunit for breast cancer in African American women

Breast cancer is the most frequently diagnosed cancer in women, with an estimated 40, 730 breast cancer deaths in the US in 2015. African American (AA) women have a lower breast cancer incidence rate, but a higher breast cancer death rate, than non-Hispanic White women. Research indicates that breast tumor biology in AA women is different from that in Caucasian women. AA women are more likely to be diagnosed with breast cancer at an earlier age and with more aggressive form of the disease, characterized by higher grade and negative estrogen and progesterone receptor status. Because of the aggressive nature of these tumors and current lack of targeted therapies, identification of novel relevant protein markers is of great importance. The purpose of this study was to validate serum proteins that were previously identified by serum proteomic profiling in 22 serum samples by 2D-DIGE/MS analysis and a subset of samples by shotgun LC/MS technology. Methods and new data: The current study included serum samples from 15 African American breast cancer patients and 12 healthy controls. Patients were grouped into triple negative (TN), HER2 and Luminal A and B subtypes. Proteins of biological significance were validated using western blot analysis. For ceruloplasmin, and insulin-like growth factor binding protein acid-labile subunit (IGFBP-ALS), one-way ANOVA was used to compare mean density among the three groups. For Vitamin D Binding protein (VDB), a two-sample t-test was used to compare the density between the groups. Due to the small sample size, we have also conducted nonparametric tests. IGFBP-ALS was significantly lower in triple negative breast cancer patients (p = 0.016) and in HER2 (p = 0.025) subtypes. There was no significant difference in VDB protein in the luminal A and B subtypes (p = 0.98). Future efforts will focus on validating the identified panel of biomarkers to gain insight into their role(s) in the etiology of aggressive breast tumors. Funded by Susan G Komen for the Cure and SEED grant SPH. Citation Format: Padma P. Tadi Uppala, Carlos Garberoglio, Sharon Lum, Willie Davis, Hon-Chiu Eastwood Leung, Michael Liebman, Keiji Oda, Utkarsh P. Patel. Identification and validation of the potential biomarker insulin-like growth factor binding protein acid-labile subunit for breast cancer in African American women. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3936.

Abstract 4192: Global gene expression profiling confirms the molecular fidelity of primary tumor-based orthotopic xenograft mouse models of medulloblastoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Clinically relevant animal models are needed for understanding biology and testing novel therapies in medulloblastoma (MB), the most common malignant brain tumor that occurs in children. We have recently established a panel of orthotopic xenograft mouse models of MB through direct injection of fresh surgical specimens into the cerebella of Rag2/SCID mice. The xenograft tumors were previously shown to have replicated key histopathological phenotypes and invasive growth characteristics of the original patient tumors. The objective of the current study is to complete the molecular characterization of 11 MB models that have been serially passaged in vivo in mouse brains for at least three generations. Whole genome gene expression profiling was performed in duplicate with Illumina HumanWG6 v3 gene expression profiling beadchip that contains more than 48,000 probes. Our results showed that the xenograft tumors are molecularly accurate. When the nine passage I xenograft tumors were compared with their original patient tumors, high correlation coefficiency were observed (r2 >0.95 in five models, >0.9 in three models, and >0.85 in one model). Serial subtransplantation up to five times did not cause significant changes of gene expression pattern (r2>0.9 in eight models, >0.8 in three models). Using RNAs from 4 normal fetal brains, one child cerebellum, and 5 adult cerebella as control, we further showed that these xenograft models, although molecularly independent displaying distinct gene expression profiles, can be classified into five different subcategories. A selected set of the differentially expressed genes were validated using real-time RT-PCR and Immunohistochemistry. In conclusion, this study provided strong experimental evidence to demonstrate that primary tumor-based orthotopic xenograft mouse models of MBs are molecularly accurate, and our findings of genetic abnormalities should significantly facilitate the rationally designed preclinical drug screenings in the near future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4192.

S1965 Novel Evidence for Soma-Wide MLH1 Hypermethylation and a Global Methylation Defect in Non-Familial Early-Onset Colorectal Cancer

Introduction: Recently soma-wide monoallelic germline epigenetic inactivation of MLH1 has been proposed as a potential mechanism for DNA mismatch repair (MMR) gene inactivation in some individuals with colorectal cancer (CRC). The present study aimed to investigate whether this methylation defect would be limited to the MLH1 promoter, or represents a broader epigenetic defect involving multiple genes. Methods: A 20 year old woman presented with a sigmoid colon cancer, and a negative family history for any cancer. Her tumor was evaluated for DNA mismatch repair (MMR) gene defects, and she was tested for germline mutations in MMR genes. Quantitative pyrosequencing and bisulfite sequencing of cloned PCR products was used to ascertain MLH1 methylation status in all three germ-layers (blood, hair and buccal tissues), as well as in her Epstein-Barr virus (EBV)-transformed lymphoblastoid cells. Genome wide/global methylation status for ~1500 genes/methylation loci was determined in the patients blood and controls (which included both parents, her sibling, and 3 unrelated controls) using Illumina GoldenGate methylation microarrays. Results: The patients tumor showed MSI and loss of expression of MLH1, PMS2 and MSH6 proteins. However, no germline mutations were detected in MLH1, MSH2 or MSH6. A single nucleotide polymorphism (SNP) permitted allele identification; 14%monoallelicMLH1 methylation was found in the tumor, but there was no allelic imbalance at MLH1 in the tumor. MLH1 methylation was observed in the patients blood (10%), EBV transformed lymphocytes (10%), buccal mucosa (22%) and hair follicles (48%). No evidence for MLH1 methylation was present in patients family members or any healthy control (all <1%). Interestingly, MLH1 methylation increased to 24% in the patients blood after 5-FU based chemotherapy treatment. Genome wide methylation analyses showed that aberrant methylation in the blood was not limited to MLH1, but included 43 additional hypermethylated genes (including RUNX3, IGF1, PPARγ and NOTCH4) and another 34 genes which were hypomethylated (including CDK2, STAT5 and IL10). Conclusions: This study reports the youngest patient with soma-wide MLH1 hypermethylation and CRC. The increase in MLH1 methylation following chemotherapy suggests that cells with methylated MLH1 promoters are relatively resistant to the toxic effects of this drug. More importantly, our observation that the epigenetic defects were not restricted to the MLH1 promoter suggests the possibility of a broader epigenetic defect in those individuals with soma-wide methylation of MLH1.