Kevin C. Johnson

Genome-wide DNA methylation profiles in progression to in situ and invasive carcinoma of the breast with impact on gene transcription and prognosis

BackgroundDuctal carcinoma in situ (DCIS) of the breast is a precursor of invasive breast carcinoma. DNA methylation alterations are thought to be an early event in progression of cancer, and may prove valuable as a tool in clinical decision making and for understanding neoplastic development.ResultsWe generate genome-wide DNA methylation profiles of 285 breast tissue samples representing progression of cancer, and validate methylation changes between normal and DCIS in an independent dataset of 15 normal and 40 DCIS samples. We also validate a prognostic signature on 583 breast cancer samples from The Cancer Genome Atlas. Our analysis reveals that DNA methylation profiles of DCIS are radically altered compared to normal breast tissue, involving more than 5,000 genes. Changes between DCIS and invasive breast carcinoma involve around 1,000 genes. In tumors, DNA methylation is associated with gene expression of almost 3,000 genes, including both negative and positive correlations. A prognostic signature based on methylation level of 18 CpGs is associated with survival of breast cancer patients with invasive tumors, as well as with survival of patients with DCIS and mixed lesions of DCIS and invasive breast carcinoma.ConclusionsThis work demonstrates that changes in the epigenome occur early in the neoplastic progression, provides evidence for the possible utilization of DNA methylation-based markers of progression in the clinic, and highlights the importance of epigenetic changes in carcinogenesis.

Age-related DNA methylation in normal breast tissue and its relationship with invasive breast tumor methylation

Age is a key risk factor for breast cancer and epigenetic alterations may contribute to age-related increases in breast cancer risk, though the relation of age-related methylation in normal breast tissues with altered methylation in breast tumors is unclear. We investigated the relation of age with DNA methylation in normal breast tissues genome-wide using two data sets from the Gene Expression Omnibus (GEO) database (GSE32393 and GSE31979). We validated our observations in an independent set of normal breast tissues, examined age-related methylation in normal breast for enrichment of genomic features, and compared age-related methylation in normal tissue with methylation alterations in breast tumors. Between the two array-based methylation data sets, there were 204 CpG loci with significant (P < 0.05) and consistent age-related methylation, 97% of which were increases in methylation. Our validation sets confirmed the direction of age-related DNA methylation changes in all measured regions. Among the 204 age-related CpG loci, we observed a significant enrichment for CpG islands (P = 8.7E-6) and polycomb group protein target genes (P = 0.03). In addition, 24 of the 204 CpGs with age-related methylation in normal breast were significantly differentially methylated between normal and breast tumor tissues. We identified consistent age-related methylation changes in normal breast tissue that are further altered in breast tumors and may represent early events contributing to breast carcinogenesis. This work identifies age-related methylation in normal breast tissue and begins to deconstruct the contribution of aging to epigenetic alterations present in breast tumors.

Evidence for tankyrases as antineoplastic targets in lung cancer

BackgroundNew pharmacologic targets are urgently needed to treat or prevent lung cancer, the most common cause of cancer death for men and women. This study identified one such target. This is the canonical Wnt signaling pathway, which is deregulated in cancers, including those lacking adenomatous polyposis coli or β-catenin mutations. Two poly-ADP-ribose polymerase (PARP) enzymes regulate canonical Wnt activity: tankyrase (TNKS) 1 and TNKS2. These enzymes poly-ADP-ribosylate (PARsylate) and destabilize axin, a key component of the β-catenin phosphorylation complex.MethodsThis study used comprehensive gene profiles to uncover deregulation of the Wnt pathway in murine transgenic and human lung cancers, relative to normal lung. Antineoplastic consequences of genetic and pharmacologic targeting of TNKS in murine and human lung cancer cell lines were explored, and validated in vivo in mice by implantation of murine transgenic lung cancer cells engineered with reduced TNKS expression relative to controls.ResultsMicroarray analyses comparing Wnt pathway members in malignant versus normal tissues of a murine transgenic cyclin E lung cancer model revealed deregulation of Wnt pathway components, including TNKS1 and TNKS2. Real-time PCR assays independently confirmed these results in paired normal-malignant murine and human lung tissues. Individual treatments of a panel of human and murine lung cancer cell lines with the TNKS inhibitors XAV939 and IWR-1 dose-dependently repressed cell growth and increased cellular axin 1 and tankyrase levels. These inhibitors also repressed expression of a Wnt-responsive luciferase construct, implicating the Wnt pathway in conferring these antineoplastic effects. Individual or combined knockdown of TNKS1 and TNKS2 with siRNAs or shRNAs reduced lung cancer cell growth, stabilized axin, and repressed tumor formation in murine xenograft and syngeneic lung cancer models.ConclusionsFindings reported here uncovered deregulation of specific components of the Wnt pathway in both human and murine lung cancer models. Repressing TNKS activity through either genetic or pharmacological approaches antagonized canonical Wnt signaling, reduced murine and human lung cancer cell line growth, and decreased tumor formation in mouse models. Taken together, these findings implicate the use of TNKS inhibitors to target the Wnt pathway to combat lung cancer.

5-Hydroxymethylcytosine localizes to enhancer elements and is associated with survival in glioblastoma patients

Glioblastomas exhibit widespread molecular alterations including a highly distorted epigenome. Here, we resolve genome-wide 5-methylcytosine and 5-hydroxymethylcytosine in glioblastoma through parallel processing of DNA with bisulfite and oxidative bisulfite treatments. We apply a statistical algorithm to estimate 5-methylcytosine, 5-hydroxymethylcytosine and unmethylated proportions from methylation array data. We show that 5-hydroxymethylcytosine is depleted in glioblastoma compared with prefrontal cortex tissue. In addition, the genomic localization of 5-hydroxymethylcytosine in glioblastoma is associated with features of dynamic cell-identity regulation such as tissue-specific transcription and super-enhancers. Annotation of 5-hydroxymethylcytosine genomic distribution reveal significant associations with RNA regulatory processes, immune function, stem cell maintenance and binding sites of transcription factors that drive cellular proliferation. In addition, model-based clustering results indicate that patients with low-5-hydroxymethylcytosine patterns have significantly poorer overall survival. Our results demonstrate that 5-hydroxymethylcytosine patterns are strongly related with transcription, localizes to disease-critical genes and are associated with patient prognosis.

DNA methylation in ductal carcinoma in situ related with future development of invasive breast cancer

BackgroundDuctal carcinoma in situ (DCIS) is a heterogeneous, pre-invasive lesion associated with an increased risk for future invasive ductal carcinoma. However, accurate risk stratification for development of invasive disease and appropriate treatment decisions remain clinical challenges. DNA methylation alterations are early events in the progression of cancer and represent emerging molecular markers that may predict invasive recurrence more accurately than traditional measures of DCIS prognosis.ResultsWe measured DNA methylation using the Illumina HumanMethylation450K array of estrogen-receptor positive DCIS (n = 40) and adjacent-normal (n = 15) tissues from subjects in the New Hampshire Mammography Network longitudinal breast imaging registry. We identified locus-specific methylation differences between DCIS and matched adjacent-normal tissue (95,609 CpGs, Q < 0.05). Among 40 DCIS cases, 13 later developed invasive disease and we identified 641 CpG sites that exhibited differential DNA methylation (P < 0.01 and median |∆β| > 0.1) in these cases compared with age-matched subjects without invasive disease. The set of differentially methylated CpG loci associated with disease progression was enriched in homeobox-containing genes (P = 1.3E-09) and genes involved with limb morphogenesis (P = 1.0E-05). In an independent cohort, a subset of genes with progression-related differential methylation between DCIS and invasive breast cancer were confirmed. Further, the functional relevance of these genes’ regulation by methylation was demonstrated in early stage breast cancers from The Cancer Genome Atlas database.ConclusionsThis work contributes to the understanding of epigenetic alterations that occur in DCIS and illustrates the potential of DNA methylation as markers of DCIS progression.

Effect of estrogen receptor α binding on functional DNA methylation in breast cancer

Epigenetic modifications introduce an additional layer of regulation that drastically expands the instructional capability of the human genome. The regulatory consequences of DNA methylation is context dependent; it can induce, enhance, and suppress gene expression, or have no effect on gene regulation. Therefore, it is essential to account for the genomic location of its occurrence and the protein factors it associates with to improve our understanding of its function and effects. Here, we use ENCODE ChIP-seq and DNase I hypersensitivity data, along with large-scale breast cancer genomic data from The Cancer Genome Atlas (TCGA) to computationally dissect the intricacies of DNA methylation in regulation of cancer transcriptomes. In particular, we identified a relationship between estrogen receptor α (ERα) activity and DNA methylation patterning in breast cancer. We found compelling evidence that methylation status of DNA sequences at ERα binding sites is tightly coupled with ERα activity. Furthermore, we predicted several transcription factors including FOXA1, GATA1, and SUZ12 to be associated with breast cancer by examining the methylation status of their binding sites in breast cancer. Lastly, we determine that methylated CpGs highly correlated with gene expression are enriched in regions 1kb or more downstream of TSSs, suggesting more significant regulatory roles for CpGs distal to gene TSSs. Our study provides novel insights into the role of ERα in breast cancers.

Hydroxymethylation is uniquely distributed within term placenta, and is associated with gene expression

The conversion of cytosine to 5‐methylcystosine (5mC) is an important regulator of gene expression. 5mC may be enzymatically converted to 5‐hydroxymethylcytosine (5hmC), with a potentially distinct regulatory function. We sought to investigate these cytosine modifications and their effect on gene expression by parallel processing of genomic DNA using bisulfite and oxidative bisulfite conversion in conjunction with RNA sequencing. Although values of 5hmC across the placental genome were generally low, we identified ~21,000 loci with consistently elevated levels of 5‐hydroxymethycytosine. Absence of 5hmC was observed in CpG islands and, to a greater extent, in non‐CpG island‐associated regions. 5hmC was enriched within poised enhancers, and depleted within active enhancers, as defined by H3K27ac and H3K4me1 measurements. 5hmC and 5mC were significantly elevated in transcriptionally silent genes when compared with actively transcribed genes. 5hmC was positively associated with transcription in actively transcribed genes only. Our data suggest that dynamic cytosine regulation, associated with transcription, provides the most complete epigenomic landscape of the human placenta, and will be useful for future studies of the placental epigenome.—Green, B. B., Houseman, E. A., Johnson, K. C., Guerin, D. J., Armstrong, D. A., Christensen, B. C., Marsit, C. J. Hydroxymethylation is uniquely distributed within term placenta, and is associated with gene expression. FASEB J. 30, 2874‐2884 (2016). www.fasebj.org

OxyBS: estimation of 5-methylcytosine and 5-hydroxymethylcytosine from tandem-treated oxidative bisulfite and bisulfite DNA.

UNLABELLED The use of sodium bisulfite (BS) treatment followed by hybridization to an Illumina Infinium BeadChip (HumanMethylation450 and MethylationEPIC) is a common method for interrogating 5-methylcytosine (5mC) at single nucleotide resolution. However, standard treatment of DNA with BS does not allow disambiguation of 5mC from an additional cytosine modification, 5-hydroxymethylcytosine (5hmC). Recently, it has been demonstrated that paired BS and oxidative bisulfite (oxBS) treatment on the same sample followed by hybridization to an Infinium microarray permits the differentiation of 5hmC from 5mC. Nevertheless, estimation of 5hmC and 5mC from tandem-treated arrays has been shown to produce irregular estimates of cytosine modifications. RESULTS We present a novel method using maximum likelihood estimation to accurately estimate the parameters of unmethylated cytosine (5C), 5mC and 5hmC from Infinium microarray data given the signal intensities from the oxBS and BS replicates. AVAILABILITY AND IMPLEMENTATION OxyBS is an R package available on CRAN. CONTACT Andres.Houseman@oregonstate.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Response to inhibition of smoothened in diverse epithelial cancer cells that lack smoothened or patched 1 mutations

Hedgehog (HH) pathway Smoothened (Smo) inhibitors are active against Gorlin syndrome-associated basal cell carcinoma (BCC) and medulloblastoma where Patched (Ptch) mutations occur. We interrogated 705 epithelial cancer cell lines for growth response to the Smo inhibitor cyclopamine and for expressed HH pathway-regulated species in a linked genetic database. Ptch and Smo mutations that respectively conferred Smo inhibitor response or resistance were undetected. Previous studies revealed HH pathway activation in lung cancers. Therefore, findings were validated using lung cancer cell lines, transgenic and transplantable murine lung cancer models, and human normal-malignant lung tissue arrays in addition to testing other Smo inhibitors. Cyclopamine sensitivity most significantly correlated with high cyclin E (P=0.000009) and low insulin-like growth factor binding protein 6 (IGFBP6) (P=0.000004) levels. Gli family members were associated with response. Cyclopamine resistance occurred with high GILZ (P=0.002) expression. Newer Smo inhibitors exhibited a pattern of sensitivity similar to cyclopamine. Gain of cyclin E or loss of IGFBP6 in lung cancer cells significantly increased Smo inhibitor response. Cyclin E-driven transgenic lung cancers expressed a gene profile implicating HH pathway activation. Cyclopamine treatment significantly reduced proliferation of murine and human lung cancers. Smo inhibition reduced lung cancer formation in a syngeneic mouse model. In human normal-malignant lung tissue arrays cyclin E, IGFBP6, Gli1 and GILZ were each differentially expressed. Together, these findings indicate that Smo inhibitors should be considered in cancers beyond those with activating HH pathway mutations. This includes tumors that express genes indicating basal HH pathway activation.

Genome-wide DNA methylation profiles in progression to

BACKGROUND Ductal carcinoma in situ (DCIS) of the breast is a precursor of invasive breast carcinoma. DNA methylation alterations are thought to be an early event in progression of cancer, and may prove valuable as a tool in clinical decision making and for understanding neoplastic development. RESULTS We generate genome-wide DNA methylation profiles of 285 breast tissue samples representing progression of cancer, and validate methylation changes between normal and DCIS in an independent dataset of 15 normal and 40 DCIS samples. We also validate a prognostic signature on 583 breast cancer samples from The Cancer Genome Atlas. Our analysis reveals that DNA methylation profiles of DCIS are radically altered compared to normal breast tissue, involving more than 5,000 genes. Changes between DCIS and invasive breast carcinoma involve around 1,000 genes. In tumors, DNA methylation is associated with gene expression of almost 3,000 genes, including both negative and positive correlations. A prognostic signature based on methylation level of 18 CpGs is associated with survival of breast cancer patients with invasive tumors, as well as with survival of patients with DCIS and mixed lesions of DCIS and invasive breast carcinoma. CONCLUSIONS This work demonstrates that changes in the epigenome occur early in the neoplastic progression, provides evidence for the possible utilization of DNA methylation-based markers of progression in the clinic, and highlights the importance of epigenetic changes in carcinogenesis.