microRNAs have emerged as powerful regulators of many biological processes, and their expression in many cancer tissues has been shown to correlate with clinical parameters such as cancer type and prognosis. Present in a variety of biological fluids, microRNAs have been described as a ‘gold mine’ of potential noninvasive biomarkers. Release of microRNA content of blood cells upon hemolysis dramatically alters the microRNA profile in blood, potentially affecting levels of a significant number of proposed biomarker microRNAs and, consequently, accuracy of serum or plasma-based tests. Several methods to detect low levels of hemolysis have been proposed; however, a direct comparison assessing their sensitivities is currently lacking. In this study, we evaluated the sensitivities of four methods to detect hemolysis in serum (listed in the order of sensitivity): measurement of hemoglobin using a Coulter® AcT diff™ Analyzer, visual inspection, the absorbance of hemoglobin measured by spectrophotometry at 414 nm and the ratio of red blood cell-enriched miR-451a to the reference microRNA miR-23a-3p. The miR ratio detected hemolysis down to approximately 0.001%, whereas the Coulter® AcT diff™ Analyzer was unable to detect hemolysis lower than 1%. The spectrophotometric method could detect down to 0.004% hemolysis, and correlated with the miR ratio. Analysis of hemolysis in a cohort of 86 serum samples from cancer patients and healthy controls showed that 31 of 86 (36%) were predicted by the miR ratio to be hemolyzed, whereas only 8 of these samples (9%) showed visible pink discoloration. Using receiver operator characteristic (ROC) analyses, we identified absorbance cutoffs of 0.072 and 0.3 that could identify samples with low and high levels of hemolysis, respectively. Overall, this study will assist researchers in the selection of appropriate methodologies to test for hemolysis in serum samples prior to quantifying expression of microRNAs.

The HER2 (ERBB2) and MYC genes are commonly amplified in breast cancer, yet little is known about their molecular and clinical interaction. Using a novel chimeric mammary transgenic approach and in vitro models, we demonstrate markedly increased self-renewal and tumour-propagating capability of cells transformed with Her2 and c-Myc. Coexpression of both oncoproteins in cultured cells led to the activation of a c-Myc transcriptional signature and acquisition of a self-renewing phenotype independent of an epithelial-mesenchymal transition programme or regulation of conventional cancer stem cell markers. Instead, Her2 and c-Myc cooperated to induce the expression of lipoprotein lipase, which was required for proliferation and self-renewal in vitro. HER2 and MYC were frequently coamplified in breast cancer, associated with aggressive clinical behaviour and poor outcome. Lastly, we show that in HER2(+) breast cancer patients receiving adjuvant chemotherapy (but not targeted anti-Her2 therapy), MYC amplification is associated with a poor outcome. These findings demonstrate the importance of molecular and cellular context in oncogenic transformation and acquisition of a malignant stem-like phenotype and have diagnostic and therapeutic consequences for the clinical management of HER2(+) breast cancer.

Epithelial ovarian cancer has the highest mortality of the gynecological malignancies. High grade serous epithelial ovarian cancer (SEOC) is the most common subtype, with the majority of women presenting with advanced disease where 5-year survival is around 25%. Platinum-based chemotherapy in combination with paclitaxel remains the most effective treatment despite platinum therapies being introduced almost 40 years ago. Advances in molecular medicine are underpinning new strategies for the treatment of cancer. Major advances have been made by international initiatives to sequence cancer genomes. For SEOC, with the exception of TP53 that is mutated in virtually 100% of these tumors, there is no other gene mutated at high frequency. There is extensive copy number variation, as well as changes in methylation patterns that will influence gene expression. To date, the role of histones and their post-translational modifications in ovarian cancer is a relatively understudied field. Post-translational histone modifications play major roles in gene expression as they direct the configuration of chromatin and so access by transcription factors. Histone modifications include methylation, acetylation, and monoubiquitination, with involvement of enzymes including histone methyltransferases, histone acetyltransferases/deacetylases, and ubiquitin ligases/deubiquitinases, respectively. Complexes such as the Polycomb repressive complex also play roles in the control of histone modifications and more recently roles for long non-coding RNA and microRNAs are emerging. Epigenomic-based therapies targeting histone modifications are being developed and offer new approaches for the treatment of ovarian cancer. Here, we discuss histone modifications and their aberrant regulation in malignancy and specifically in ovarian cancer. We review current and upcoming histone-based therapies that have the potential to inform and improve treatment strategies for women with ovarian cancer.

Colorectal cancer (CRC) results from a transformation of colonic epithelial cells into adenocarcinoma cells due to genetic and epigenetic instabilities, alongside remodelling of the surrounding stromal tumour microenvironment. Epithelial-specific epigenetic variations escorting this process include chromatin remodelling, histone modifications and aberrant DNA methylation, which influence gene expression, alternative splicing and function of non-coding RNA. In this review, we first highlight epigenetic modulators, modifiers and mediators in CRC, then we elaborate on causes and consequences of epigenetic alterations in CRC pathogenesis alongside an appraisal of the complex feedback mechanisms realized through alternative splicing and non-coding RNA regulation. An emphasis in our review is put on how this intricate network of epigenetic and post-transcriptional gene regulation evolves during the initiation, progression and metastasis formation in CRC.

Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1 + cells. Id1 + cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or pre-leukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represents a novel mechanism of resistance to BH3-mimetics and a potential barrier to longer-term efficacy of drugs targeting BCL-2 in AML.

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Vast transcriptomics and epigenomics changes are characteristic of human cancers, including leukaemia. At remission, we assume that these changes normalise so that omics-profiles resemble those of healthy individuals. However, an in-depth transcriptomic and epigenomic analysis of cancer remission has not been undertaken. A striking exemplar of targeted remission induction occurs in chronic myeloid leukaemia (CML) following tyrosine kinase inhibitor (TKI) therapy. Using RNA sequencing and whole-genome bisulfite sequencing, we profiled samples from chronic-phase CML patients at diagnosis and remission and compared these to healthy donors. Remarkably, our analyses revealed that abnormal splicing distinguishes remission samples from normal controls. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Most remarkable are the high intron retention (IR) levels that even exceed those observed in the diagnosis samples. Increased IR affects cell cycle regulators at diagnosis and splicing regulators at remission. We show that aberrant splicing in CML is associated with reduced expression of specific splicing factors, histone modifications and reduced DNA methylation. Our results provide novel insights into the changing transcriptomic and epigenomic landscapes of CML patients during remission. The conceptually unanticipated observation of widespread aberrant alternative splicing after remission induction warrants further exploration. These results have broad implications for studying CML relapse and treating minimal residual disease.