Centrosome amplification promotes cell invasion via cell-cell contact disruption and Rap-1 activation.

Centrosome amplification (CA) is a prominent feature of human cancers linked to tumorigenesis in vivo. Here, we report mechanistic contributions of CA induction alone to tumour architecture and extracellular matrix (ECM) remodelling. CA induction in non-tumorigenic breast cells MCF10A causes cell migration and invasion, with underlying disruption of epithelial cell-cell junction integrity and dysregulation of expression and subcellular localisation of cell junction proteins. CA also elevates expression of integrin ß-3, its binding partner fibronectin-1 and matrix metalloproteinase enzymes, promoting cell-ECM attachment, ECM degradation, and a migratory and invasive cell phenotype. Using a chicken embryo xenograft model for in vivo validation, we show that CA-induced (+CA) MCF10A cells invade into the chick mesodermal layer, with inflammatory cell infiltration and marked focal reactions between chorioallantoic membrane and cell graft. We also demonstrate a key role of small GTPase Rap-1 signalling through inhibition using GGTI-298, which blocked various CA-induced effects. These insights reveal that in normal cells, CA induction alone (without additional oncogenic alterations) is sufficient to confer early pro-tumorigenic changes within days, acting through Rap-1-dependent signalling to alter cell-cell contacts and ECM disruption.

Profiling leadership: Attitudes, knowledge and training in the biological sciences.

The development and practice of good leadership skills (distinct from management skills) enhances both an individual's career development, and their organization. However, universities are known to present unique issues around the development, and practice, of good leadership. Good leadership skills should be considered essential for university staff who train (and mentor) staff or students. Currently, there is no clear evidence that staff in the biological (life) sciences undergo formal (routine) leadership skills training (or appraisal). Furthermore, what leadership training this group needs, or wants, is unknown. A questionnaire was designed to explore leadership dimensions (roles, training, perceptions, and attitudes), and incorporated the Leadership Attitudes and Belief scale (LABS) instrument. Including LABS allows evaluation of leadership attitudes as either Systemic (individual responsibility) or Hierarchical (chain-of-command). Self-selecting biological science academics and staff were recruited using an online survey. Analysis focused on academic staff (lecturer/Assistant professor, and above), and explored the relationship of leadership dimensions with key categories (career stage, gender, age, role, and professional experience). Staff were found to be knowledgeable about what leadership is, but strongly desire formal training in leadership skills and practice. Importantly, staff did not have access to specific leadership training (but did have access to management training), but felt strongly that gaining leadership skills would improve their professional skill set. Analysis found that academics in the biological sciences were oriented towards Systemic leadership, a more collective and supportive approach. It was clear that while good leadership skills are highly valued by academic staff, in practice these skills are underprovided in the biological sciences workplace. This work provides a profile, and benchmark, of leadership (current skills, and desired needs) in the biological sciences. These results provide evidence for the need to embed specific leadership skills training into professional development (and teaching) programmes in the biological sciences.

Peripheral Blood Cell Ratios as Prognostic Indicators in a Neoadjuvant Chemotherapy-Treated Breast Cancer Cohort.

Breast cancer represents a heterogeneous condition in which the interaction between host immune response and primary oncogenic events can impact disease progression. Ratios of systemic blood-based immunocytes have emerged as clinically-relevant prognostic biomarkers in cancer patients. The NLR (neutrophil-to-lymphocyte ratio) has been shown to be prognostic in a variety of cancers, including breast cancer. However, evaluation of the prognostic value for overall survival (OS) and disease-free survival (DFS) of other key immunocyte ratios-neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), neutrophil-to-white cell count ratio (NWR), lymphocyte-to-white cell count ratio (LWR), monocyte-to-white cell count ratio (MWR), platelet-to-lymphocyte (PLR)-by breast cancer subtypes in a neoadjuvant chemotherapy (NAC) cohort remains to be fully explored. An NAC-treated breast cancer cohort, comprised of Luminal A, Luminal B, HER2-positive, and triple negative/basal breast cancers, treated at a tertiary referral center (minimum 3-year follow-up), was used to calculate immunocyte ratios and immunocyte cut-off values, calculated with >80% specificity (using decision tree modeling). The association with subtype-specific OS, DFS, and tumor grade was analyzed using cut offs calculated using both receiver operating characteristic curves and decision tree modelling. Decision tree calculated ratios showed that LMR (5.29) and MWR (0.06) were significantly associated with Luminal A OS (p = 0.004 and p = 0.022) and DFS (p = 0.004 and p = 0.022), and Luminal B OS (p = 0.027 and p = 0.008) and DFS (p = 0.005 and p = 0.007). NLR (1.79) and LWR (0.30) were significantly associated with HER2-positive OS (p = 0.013 and p = 0.043). NLR (1.79) and NWR (0.62) were significantly associated with DFS (p = 0.035 and p = 0.021). No significant association we observed between any immunocyte ratio in the triple negative cohort. Our results demonstrate the subtype-specific prognostic value of immunocyte ratios in NAC-treated breast cancer patients. Further validation of immunocyte ratios will provide clinicians with a new prognostic aid for disease management and monitoring.

Recent Advances in the Role of Discoidin Domain Receptor Tyrosine Kinase 1 and Discoidin Domain Receptor Tyrosine Kinase 2 in Breast and Ovarian Cancer.

Discoidin domain receptor tyrosine kinases (DDRs) are a class of receptor tyrosine kinases (RTKs), and their dysregulation is associated with multiple diseases (including cancer, chronic inflammatory conditions, and fibrosis). The DDR family members (DDR1a-e and DDR2) are widely expressed, with predominant expression of DDR1 in epithelial cells and DDR2 in mesenchymal cells. Structurally, DDRs consist of three regions (an extracellular ligand binding domain, a transmembrane domain, and an intracellular region containing a kinase domain), with their kinase activity induced by receptor-specific ligand binding. Collagen binding to DDRs stimulates DDR phosphorylation activating kinase activity, signaling to MAPK, integrin, TGF-ß, insulin receptor, and Notch signaling pathways. Abnormal DDR expression is detected in a range of solid tumors (including breast, ovarian, cervical liver, gastric, colorectal, lung, and brain). During tumorigenesis, abnormal activation of DDRs leads to invasion and metastasis, via dysregulation of cell adhesion, migration, proliferation, secretion of cytokines, and extracellular matrix remodeling. Differential expression or mutation of DDRs correlates with pathological classification, clinical characteristics, treatment response, and prognosis. Here, we discuss the discovery, structural characteristics, organizational distribution, and DDR-dependent signaling. Importantly, we highlight the key role of DDRs in the development and progression of breast and ovarian cancer.

A Novel Mechanism of Ataxia Telangiectasia Mutated Mediated Regulation of Chromatin Remodeling in Hypoxic Conditions.

The effects of genotoxic stress can be mediated by activation of the Ataxia Telangiectasia Mutated (ATM) kinase, under both DNA damage-dependent (including ionizing radiation), and independent (including hypoxic stress) conditions. ATM activation is complex, and primarily mediated by the lysine acetyltransferase Tip60. Epigenetic changes can regulate this Tip60-dependent activation of ATM, requiring the interaction of Tip60 with tri-methylated histone 3 lysine 9 (H3K9me3). Under hypoxic stress, the role of Tip60 in DNA damage-independent ATM activation is unknown. However, epigenetic changes dependent on the methyltransferase Suv39H1, which generates H3K9me3, have been implicated. Our results demonstrate severe hypoxic stress (0.1% oxygen) caused ATM auto-phosphorylation and activation (pS1981), H3K9me3, and elevated both Suv39H1 and Tip60 protein levels in FTC133 and HCT116 cell lines. Exploring the mechanism of ATM activation under these hypoxic conditions, siRNA-mediated Suv39H1 depletion prevented H3K9me3 induction, and Tip60 inhibition (by TH1834) blocked ATM auto-phosphorylation. While MDM2 (Mouse double minute 2) can target Suv39H1 for degradation, it can be blocked by sirtuin-1 (Sirt1). Under severe hypoxia MDM2 protein levels were unchanged, and Sirt1 levels depleted. SiRNA-mediated depletion of MDM2 revealed MDM2 dependent regulation of Suv39H1 protein stability under these conditions. We describe a novel molecular circuit regulating the heterochromatic state (H3K9me3 positive) under severe hypoxic conditions, showing that severe hypoxia-induced ATM activation maintains H3K9me3 levels by downregulating MDM2 and preventing MDM2-mediated degradation of Suv39H1. This novel mechanism is a potential anti-cancer therapeutic opportunity, which if exploited could target the hypoxic tumor cells known to drive both tumor progression and treatment resistance.

Prospective Assessment of Systemic MicroRNAs as Markers of Response to Neoadjuvant Chemotherapy in Breast Cancer.

Neoadjuvant chemotherapy (NACT) is used in locally advanced breast cancer to reduce tumour burden prior to surgical resection. However, only a subset of NACT treated patients will respond to treatment or achieve a pathologic complete response (pCR). This multicenter, prospective study (CTRIAL-IE (ICORG) 10-11 study) evaluated circulating microRNA as novel non-invasive prognostic biomarkers of NACT response in breast cancer. Selected circulating microRNAs (Let-7a, miR-21, miR-145, miR-155, miR-195) were quantified from patients undergoing standard of care NACT treatment (n = 114) from whole blood at collected at diagnosis, and the association with NACT response and clinicopathological features evaluated. NACT responders had significantly lower levels of miR-21 (p = 0.036) and miR-195 (p = 0.017), compared to non-responders. Evaluating all breast cancer cases miR-21 was found to be an independent predictor of response (OR 0.538, 95% CI 0.308-0.943, p < 0.05). Luminal cancer NACT responders were found to have significantly decreased levels of miR-145 (p = 0.033) and miR-21 (p = 0.048), compared to non-responders. This study demonstrates the prognostic ability of miR-21, miR-195 and miR-145 as circulating biomarkers stratifying breast cancer patients by NACT response, identifying patients that will derive the maximum benefit from chemotherapy.

Recent Advances in Understanding FOXN3 in Breast Cancer, and Other Malignancies.

FOXN3 (forkhead box N3; CHES1: check point suppressor 1) belongs to the forkhead box (FOX) protein family. FOXN3 displays transcriptional inhibitory activity, and is involved in cell cycle regulation and tumorigenesis. FOXN3 is a tumor suppresser and alterations in FOXN3 are found in of a variety of cancers including melanoma, osteosarcoma, and hepatocellular carcinoma. While the roles of FOXN3 role in some cancers have been explored, its role in breast cancer remains unclear. Here we describe current state of knowledge of FOXN3 functions, and focus on its roles (known and potential) in breast cancer.

Circulating microRNAs miR-331 and miR-195 differentiate local luminal a from metastatic breast cancer.

BACKGROUND: Breast cancer is the leading cause of cancer related death in women, with metastasis the principle cause of mortality. New non-invasive prognostic markers are needed for the early detection of metastasis, facilitating treatment decision optimisation. MicroRNA (miRNA) are small, non-coding RNAs regulating gene expression and involved in many cellular processes, including metastasis. As biomarkers, circulating miRNAs (in blood) hold great promise for informing diagnosis or monitoring treatment responses. METHODS: Plasma extracted RNA from age matched local Luminal A (n = 4) or metastatic disease (n = 4) were profiled using Next Generation Sequencing. Selected differentially expressed miRNA were validated on a whole blood extracted miRNA cohort [distant metastatic disease (n = 22), local disease (n = 31), healthy controls (n = 21)]. Area Under the Curve (AUC) in Receiver Operating Characteristic (ROC) analyses was performed. RESULTS: Of 4 miRNA targets tested (miR-181a, miR-329, miR-331, miR-195), mir-331 was significantly over-expressed in patients with metastatic disease, compared to patients with local disease (p < 0.001) or healthy controls (p < 0.001). miR-195 was significantly under-expressed in patients with metastatic disease, compared to patients with local disease (p < 0.001) or healthy controls (p = 0.043). In combination, miR-331 and miR-195 produced an AUC of 0.902, distinguishing metastatic from local breast cancer. CONCLUSIONS: We identified and validated two circulating miRNAs differentiating local Luminal A breast cancers from metastatic breast cancers. Further investigation will reveal the molecular role of these miRNAs in metastasis, and determine if they are subtype specific. This work demonstrates the ability of circulating miRNA to identify metastatic disease, and potentially inform diagnosis or treatment effectiveness.

Clinically Applicable Inhibitors Impacting Genome Stability.

Advances in technology have facilitated the molecular profiling (genomic and transcriptomic) of tumours, and has led to improved stratification of patients and the individualisation of treatment regimes. To fully realize the potential of truly personalised treatment options, we need targeted therapies that precisely disrupt the compensatory pathways identified by profiling which allow tumours to survive or gain resistance to treatments. Here, we discuss recent advances in novel therapies that impact the genome (chromosomes and chromatin), pathways targeted and the stage of the pathways targeted. The current state of research will be discussed, with a focus on compounds that have advanced into trials (clinical and pre-clinical). We will discuss inhibitors of specific DNA damage responses and other genome stability pathways, including those in development, which are likely to synergistically combine with current therapeutic options. Tumour profiling data, combined with the knowledge of new treatments that affect the regulation of essential tumour signalling pathways, is revealing fundamental insights into cancer progression and resistance mechanisms. This is the forefront of the next evolution of advanced oncology medicine that will ultimately lead to improved survival and may, one day, result in many cancers becoming chronic conditions, rather than fatal diseases.

Correction to: Breast cancer subtype discordance: impact on post-recurrence survival and potential treatment options.

It has been highlighted that the original manuscript [1] contains a typesetting error regarding the authorship.

Breast cancer subtype discordance: impact on post-recurrence survival and potential treatment options.

BACKGROUND: Recent studies have shown that breast cancer subtype can change from the primary tumour to the recurrence. Discordance between primary and recurrent breast cancer has implications for further treatment and ultimately prognosis. The aim of the study was to determine the rate of change between primary and recurrence of breast cancer and to assess the impact of these changes on survival and potential treatment options. METHODS: Patient demographics were collected on those who underwent surgery for breast cancer between 2001 and 2014 and had a recurrence with biopsy results and pathology scoring of both the primary and recurrence. RESULTS: One hundred thirty two consecutive patients were included. There were 31 (23.5%) changes in subtype. Discordance occurred most frequently in luminal A breast cancer (n = 20), followed by triple negative (n = 4), luminal B (n = 3) and HER2 (n = 3). Patients who changed from luminal A to triple negative (n = 18) had a significantly worse post-recurrence survival (p < 0.05) with overall survival approaching significance (p = 0.064) compared to concordant luminal A cases (n = 46). Overall receptor discordance rates were: estrogen receptor 20.4% (n = 27), progesterone receptor 37.7% (n = 50) and HER2 3% (n = 4). Loss of estrogen receptor and progesterone receptor was more common than gain (21 vs. 6 (p = 0.04) and 44 vs. 6 (p = 0.01) respectively). Nine patients (6.8%) gained receptor status potentially impacting treatment options. CONCLUSION: Discordance in subtype and receptor status occurs between primary and recurrent breast cancer, ultimately affecting survival and potentially impacting treatment options.

In Vitro Histone Acetylation Assay.

Acetylation is a core cellular process involved in maintaining genomic integrity, gene regulation, and metabolism. Histone acetyltransferases (HATs) are an enzyme family that regulates these processes by catalyzing the transfer of an acetyl moiety onto target proteins. Perturbations of cellular acetylation profiles have been associated with a variety of disease states, including cancer. Changes in acetylation profiles can be achieved by mechanisms associated with acetyltransferases, such as gene down-regulation or alterations in the activity of key acetyltransferase enzymes. An important set of tools for quantifying enzyme activity are in vitro histone acetylation assays, using either endogenous or tagged overexpressed proteins. Detailed in this unit is an in vitro acetylation assay used to quantify HAT activity. © 2017 by John Wiley & Sons, Inc.

Advances in Precision Medicine: Tailoring Individualized Therapies.

The traditional bench-to-bedside pipeline involves using model systems and patient samples to provide insights into pathways deregulated in cancer. This discovery reveals new biomarkers and therapeutic targets, ultimately stratifying patients and informing cohort-based treatment options. Precision medicine (molecular profiling of individual tumors combined with established clinical-pathological parameters) reveals, in real-time, individual patient's diagnostic and prognostic risk profile, informing tailored and tumor-specific treatment plans. Here we discuss advances in precision medicine presented at the Irish Association for Cancer Research Annual Meeting, highlighting examples where personalized medicine approaches have led to precision discovery in individual tumors, informing customized treatment programs.

Differential impact of hormone receptor status on survival and recurrence for HER2 receptor-positive breast cancers treated with Trastuzumab.

INTRODUCTION: Hormone receptor status has major implications for treatment and survival of breast cancer. Yet the impact of hormone receptor status on outcome after Trastuzumab has received little attention. The objective here was to explore any differential effects of Trastuzumab treatment (Trast +ve) on Luminal B HER2 or HER2+(ER-) breast cancer subtypes. METHODS: A cohort of 469 HER2 receptor-positive breast cancers was categorised by molecular subtype and Trastuzumab treatment. Effects of Trastuzumab treatment on survival, locoregional recurrence and distant metastasis were investigated by subtype, using univariate and multivariate analysis. RESULTS: Trast +ve Luminal B HER2 patients had significant improvements in 5-year DFS (p < 0.001) and OS (p < 0.001), while Trast +ve HER2+(ER-) patients had significant improvements in 5-year DFS (p = 0.012) alone. Only Trast +ve Luminal B HER2 cancers displayed a significant reduction in LRR rates (p < 0.001). A significant reduction in distant metastasis rates was seen in Trast +ve Luminal B HER2 (p < 0.001) and HER2+(ER-) (p = 0.009) cancers. Interestingly, bone metastasis rates in Trast +ve Luminal B HER2 cancers demonstrated the greatest reduction (36.2-6.7%). Multivariate analysis of Trast +ve patients found no difference in distant metastasis rates (p = 0.96) between subtypes. Significantly, lower LRR rates were seen in Trast +ve Luminal B HER2 cancers, compared to Trast +ve HER2+(ER-) (p = 0.018). CONCLUSION: An enhanced response to Trastuzumab was seen in Luminal B HER2 cancers. We highlight how Trastuzumab treatment changed the natural history of the HER2 receptor-positive breast cancer, demonstrating improved efficacy in changing the outcome of hormone receptor-positive patients.

Circulating Nucleosomes and Nucleosome Modifications as Biomarkers in Cancer.

Traditionally the stratification of many cancers involves combining tumour and clinicopathological features (e.g., patient age; tumour size, grade, receptor status and location) to inform treatment options and predict recurrence risk and survival. However, current biomarkers often require invasive excision of the tumour for profiling, do not allow monitoring of the response to treatment and stratify patients into broad heterogeneous groups leading to inconsistent treatment responses. Here we explore and describe the benefits of using circulating biomarkers (nucleosomes and/or modifications to nucleosomes) as a non-invasive method for detecting cancer and monitoring response to treatment. Nucleosomes (DNA wound around eight core histone proteins) are responsible for compacting our genome and their composition and post-translational modifications are responsible for regulating gene expression. Here, we focus on breast and colorectal cancer as examples where utilizing circulating nucleosomes as biomarkers hold real potential as liquid biopsies. Utilizing circulating nucleosomes as biomarkers is an exciting new area of research that promises to allow both the early detection of cancer and monitoring of treatment response. Nucleosome-based biomarkers combine with current biomarkers, increasing both specificity and sensitivity of current tests and have the potential to provide individualised precision-medicine based treatments for patients.

Practical Bioinformatics Analysis of MiRNA Data Using Online Tools.

The purpose of this chapter is to provide a starting point for the analysis of miRNA array data, using freely available online suites of tools. This chapter does not describe how to perform analysis of primary array data, rather how to use the top differentially regulated miRNA (returned from comparing one miRNA group to another) as the starting point for further practical analysis.Here we describe the methods and tools required to identify targets worthy of additional investigation, using the identified miRNA as a starting point. Importantly, this additional information (pathways targeted, gene expression, mRNA targets, miRNA families) can be used to positively inform any project.

Screening of exosomal microRNAs from colorectal cancer cells.

BACKGROUND: Cells release extracellular membrane vesicles including microvesicles known as exosomes. Exosomes contain microRNAs (miRNAs) however the full range within colorectal cancer cell secreted exosomes is unknown. OBJECTIVE: To identify the full range of exosome encapsulated miRNAs secreted from 2 colorectal cancer cell lines and to investigate engineering of exosomes over-expressing miRNAs. METHODS: Exosomes were isolated from HCT-116 and HT-29 cell lines. RNA was extracted from exosomes and microRNA array performed. Cells were engineered to express miR-379 (HCT-116-379) or a non-targeting control (HCT-116-NTC) and functional effects were determined. Exosomes secreted by engineered cells were transferred to recipient cells and the impact examined. RESULTS: Microvesicles 40-100 nm in size secreted by cell lines were visualised and confirmed to express exosomal protein CD63. HT-29 exosomes contained 409 miRNAs, HCT-116 exosomes contained 393, and 338 were common to exosomes from both cell lines. Selected targets were validated. HCT-116-379 cells showed decreased proliferation (12-15% decrease, p < 0.001) and decreased migration (32-86% decrease, p < 0.001) compared to controls. HCT-116-379 exosomes were enriched for miR-379. Confocal microscopy visualised transfer of HCT-116-379 exosomes to recipient cells. CONCLUSIONS: Colorectal cancer cells secrete a large number of miRNAs within exosomes. miR-379 decreases cell proliferation and migration, and miR-379 enriched exosomes can be engineered.

Big Data-Led Cancer Research, Application, and Insights.

Insights distilled from integrating multiple big-data or "omic" datasets have revealed functional hierarchies of molecular networks driving tumorigenesis and modifiers of treatment response. Identifying these novel key regulatory and dysregulated elements is now informing personalized medicine. Crucially, although there are many advantages to this approach, there are several key considerations to address. Here, we examine how this big data-led approach is impacting many diverse areas of cancer research, through review of the key presentations given at the Irish Association for Cancer Research Meeting and importantly how the results may be applied to positively affect patient outcomes. Cancer Res; 76(21); 6167-70. ©2016 AACR.