Warwick J. Locke

Carcinogenesis is accompanied by widespread DNA methylation changes within the cell. These changes are characterized by a globally hypomethylated genome with focal hypermethylation of numerous 5’-cytosine-phosphate-guanine-3’ (CpG) islands, often spanning gene promoters and first exons. Many of these epigenetic changes occur early in tumorigenesis and are highly pervasive across a tumor type. This allows DNA methylation cancer biomarkers to be suitable for early detection and also to have utility across a range of areas relevant to cancer detection and treatment. Such tests are also simple in construction, as only one or a few loci need to be targeted for good test coverage. These properties make cancer-associated DNA methylation changes very attractive for development of cancer biomarker tests with substantive clinical utility. Across the patient journey from initial detection, to treatment and then monitoring, there are several points where DNA methylation assays can inform clinical practice. Assays on surgically removed tumor tissue are useful to determine indicators of treatment resistance, prognostication of outcome, or to molecularly characterize, classify, and determine the tissue of origin of a tumor. Cancer-associated DNA methylation changes can also be detected with accuracy in the cell-free DNA present in blood, stool, urine, and other biosamples. Such tests hold great promise for the development of simple, economical, and highly specific cancer detection tests suitable for population-wide screening, with several successfully translated examples already. The ability of circulating tumor DNA liquid biopsy assays to monitor cancer in situ also allows for the ability to monitor response to therapy, to detect minimal residual disease and as an early biomarker for cancer recurrence. This review will summarize existing DNA methylation cancer biomarkers used in clinical practice across the application domains above, discuss what makes a suitable DNA methylation cancer biomarker, and identify barriers to translation. We discuss technical factors such as the analytical performance and product-market fit, factors that contribute to successful downstream investment, including geography, and how this impacts intellectual property, regulatory hurdles, and the future of the marketplace and healthcare system.

Clinical relevance of the transcriptional signature regulated by CDC42 in colorectal cancer Clinical relevance of the transcriptional signature regulated by CDC42 in colorectal cancer

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with no targeted treatment available. Our previous study identified 38 TNBC-specific genes with altered expression comparing tumour to normal samples. This study aimed to establish whether DNA methylation contributed to these expression changes in the same cohort as well as disease progression from primary breast tumour to lymph node metastasis associated with changes in the epigenome. We obtained DNA from 23 primary TNBC samples, 12 matched lymph node metastases, and 11 matched normal adjacent tissues and assayed for differential methylation profiles using Illumina HumanMethylation450 BeadChips. The results were validated in an independent cohort of 70 primary TNBC samples. The expression of 16/38 TNBC-specific genes was associated with alteration in DNA methylation. Novel methylation changes between primary tumours and lymph node metastases, as well as those associated with survival were identified. Altered methylation of 18 genes associated with lymph node metastasis were identified and validated. This study reveals the important role DNA methylation plays in altered gene expression of TNBC-specific genes and lymph node metastases. The novel insights into progression of TNBC to secondary disease may provide potential prognostic indicators for this hard-to-treat breast cancer subtype.

Oncogene is one of the world’s leading cancer journals. It is published weekly and covers all aspects of the structure and function of Oncogenes.

Expression of oestrogen receptor (ESR1) determines whether a breast cancer patient receives endocrine therapy, but does not guarantee patient response. The molecular factors that define endocrine response in ESR1-positive breast cancer patients remain poorly understood. Here we characterize the DNA methylome of endocrine sensitivity and demonstrate the potential impact of differential DNA methylation on endocrine response in breast cancer. We show that DNA hypermethylation occurs predominantly at oestrogen-responsive enhancers and is associated with reduced ESR1 binding and decreased gene expression of key regulators of ESR1 activity, thus providing a novel mechanism by which endocrine response is abated in ESR1-positive breast cancers. Conversely, we delineate that ESR1-responsive enhancer hypomethylation is critical in transition from normal mammary epithelial cells to endocrine-responsive ESR1-positive cancer. Cumulatively, these novel insights highlight the potential of ESR1-responsive enhancer methylation to both predict ESR1-positive disease and stratify ESR1-positive breast cancer patients as responders to endocrine therapy.

Dysregulation of the epigenome is a common event in malignancy; however, deciphering the earliest cancer-associated epigenetic events remains a challenge. Cancer epigenome studies to date have primarily utilised cancer cell lines or clinical samples, where it is difficult to identify the initial epigenetic lesions from those that occur over time. Here, we analysed the epigenome of human mammary epithelial cells (HMEC) and a matched variant cell population (vHMEC) that have spontaneously escaped senescence and undergone partial carcinogenic transformation. Using this model of basal-like breast carcinogenesis, we provide striking new insights into the very first epigenetic changes that occur during the initial stages of malignancy. The first phase of malignancy is defined by coordinated changes in the epigenome. At the chromatin level, this is embodied in long-range epigenetic deregulation, which involves the concomitant but atypical acquisition or loss of active and repressive histone modifications across large regional blocks. Changes in DNA methylation also occurs in a highly coordinated manner. We identified differentially methylated regions (DMRs) in the very earliest passages of vHMECs. Notably, we find that differential methylation targets loci regulated by key transcription factors including p53, AHR and E2F family members suggesting that epigenetic deregulation of transcription factor binding is a key event in breast carcinogenesis. Interestingly, DMRs identified in vHMEC are extensively methylated in breast cancer, with hypermethylation frequently encroaching into neighbouring regions. A subset of vHMEC DMRs exhibited a strong basal-like cancer specific hypermethylation. Here, we generated epigenome-wide maps of the earliest phase of breast malignancy and show long-range epigenetic deregulation and coordinated DNA hypermethylation targets loci regulated by key transcription factors. These findings support a model where induction of breast cancer occurs through epigenetic disruption of transcription factor binding leading to deregulation of cancer-associated transcriptional networks. With their stability and very early occurrence, vHMECs hypermethylated loci could serve as excellent biomarkers for the initial detection of basal breast cancer.

Epigenetic alterations in the cancer methylome are common in breast cancer and provide novel options for tumour stratification. Here, we perform whole-genome methylation capture sequencing on small amounts of DNA isolated from formalin-fixed, paraffin-embedded tissue from triple-negative breast cancer (TNBC) and matched normal samples. We identify differentially methylated regions (DMRs) enriched with promoters associated with transcription factor binding sites and DNA hypersensitive sites. Importantly, we stratify TNBCs into three distinct methylation clusters associated with better or worse prognosis and identify 17 DMRs that show a strong association with overall survival, including DMRs located in the Wilms tumour 1 (WT1) gene, bi-directional-promoter and antisense WT1-AS. Our data reveal that coordinated hypermethylation can occur in oestrogen receptor-negative disease, and that characterizing the epigenetic framework provides a potential signature to stratify TNBCs. Together, our findings demonstrate the feasibility of profiling the cancer methylome with limited archival tissue to identify regulatory regions associated with cancer.

Epigenetic gene regulation has influence over a diverse range of cellular functions, including the maintenance of pluripotency, differentiation, and cellular identity, and is deregulated in many diseases, including cancer. Whereas the involvement of epigenetic dysregulation in cancer is well documented, much of the mechanistic detail involved in triggering these changes remains unclear. In the current age of genomics, the development of new sequencing technologies has seen an influx of genomic and epigenomic data and drastic improvements in both resolution and coverage. Studies in cancer cell lines and clinical samples using next-generation sequencing are rapidly delivering spectacular insights into the nature of the cancer genome and epigenome. Despite these improvements in technology, the timing and relationship between genetic and epigenetic changes that occur during the process of carcinogenesis are still unclear. In particular, what changes to the epigenome are playing a driving role during carcinogenesis and what influence the temporal nature of these changes has on cancer progression are not known. Understanding the early epigenetic changes driving breast cancer has the exciting potential to provide a novel set of therapeutic targets or early-disease biomarkers or both. Therefore, it is important to find novel systems that permit the study of initial epigenetic events that potentially occur during the first stages of breast cancer. Non-malignant human mammary epithelial cells (HMECs) provide an exciting in vitro model of very early breast carcinogenesis. When grown in culture, HMECs are able to temporarily escape senescence and acquire a pre-malignant breast cancer-like phenotype (variant HMECs, or vHMECs). Cultured HMECs are composed mainly of cells from the basal breast epithelial layer. Therefore, vHMECs are considered to represent the basal-like subtype of breast cancer. The transition from HMECs to vHMECs in culture recapitulates the epigenomic phenomena that occur during the progression from normal breast to pre-malignancy. Therefore, the HMEC model system provides the unique opportunity to study the very earliest epigenomic aberrations occurring during breast carcinogenesis and can give insight into the sequence of epigenomic events that lead to breast malignancy. This review provides an overview of epigenomic research in breast cancer and discusses in detail the utility of the HMEC model system to discover early epigenomic changes involved in breast carcinogenesis.

GATA-2, a member of the GATA family of transcription factors, is involved in androgen receptor (AR) signaling, however, little is known regarding its role in prostate cancer. Here, we report that GATA-2 is expressed in a substantial proportion of prostate cancers and that high expression of GATA-2 is associated with biochemical recurrence and distant metastatic progression in a validation set of 203 cancers. In vitro data show that GATA-2 is directly recruited to the promoter region of the AR upon androgen stimulation of LNCaP prostate cancer cells with 5α-dihydroxytestosterone (DHT) for 24 h. Ectopic GATA-2 expression causes the induction of AR transcript levels under androgen-depleted conditions (PAZGP1, is induced upon androgen stimulation and this effect is repressed by GATA-2. In contrast, GATA-2 significantly increases transcript levels of KLK2, which increases further in a time-dependent manner on DHT treatment and in the presence of GATA-2. These results indicate that upregulation of GATA-2 may contribute to the progression to aggressive prostate cancer through modulation of expression of AR and key androgen-regulated genes, one of which, AZGP1, is associated with the progression to metastatic disease.