Search

Age-related DNA methylation is a potential mechanism contributing to breast cancer development. Studies of primarily Caucasian women have identified many CpG sites of age-related methylation in non-diseased breast tissue possibly driving cancer development over time. There is a paucity of studies involving Asian women whose ages at breast cancer onset are usually younger than Caucasians. We identified the 181 most consistent age-related methylation events in non-diseased breast tissue across published studies. Age-related methylation events were measured in adjacent normal and breast tumour tissue in an exclusively Asian population at the previously identified age-related methylation sites. Age-related methylation was found in 118 probes in adjacent normal breast tissue. Methylation of 99% of these sites was increased with age and predominantly located on CpG islands in promoter regions. To ascertain biological relevance to breast cancer, we focused on the 37 sites with overall higher methylation in tumour compared to adjacent normal samples. Some sites positively related to age, including AQP5 and CORO6, inversely correlated with gene expression. Several others have known involvement in suppression of carcinogenesis including GPC5 and SST, suggesting that perturbation of epigenetic regulation at these sites due to ageing may contribute to the progression of carcinogenesis. This study highlights an age-related methylation landscape in non-tumour tissue, consistent not just across studies, but also across different populations. We present candidate age-related methylation sites warranting further investigation as potential epigenetic drivers of breast cancer. They may serve as potential targets of site-specific demethylation intervention strategies for the prevention of age-related breast cancer.

Urinary PGE-M is a stable metabolite of prostaglandin E2 (PGE2). PGE2 is a product of the inflammatory COX signaling pathway and has been associated with cancer incidence and metastasis. Its synthesis can be inhibited by aspirin. We investigated the association of PGE-M with lethal prostate cancer in a case–control study of African American (AA) and European American men. We measured urinary PGE-M using mass-spectrometry. Samples were obtained from 977 cases and 1022 controls at the time of recruitment. We applied multivariable logistic and Cox regression modeling to examine associations of PGE-M with prostate cancer and participant survival. Median survival follow-up was 8.4 years, with 246 deaths among cases. Self-reported aspirin use over the past 5 years was assessed with a questionnaire. Race/ethnicity was self-reported. Urinary PGE-M levels did not differ between men with prostate cancer and population-based controls. We observed no association between PGE-M and aggressive disease nor prostate-cancer-specific survival. However, we observed a statistically significant association between higher (&gt, median) PGE-M and all-cause mortality in AA cases who did not regularly use aspirin (HR = 2.04, 95% CI 1.23–3.37). Among cases who reported using aspirin, there was no association. Our study does not support a meaningful association between urinary PGE-M and prostate cancer. Moreover, PGE-M levels were not associated with aggressive prostate cancer. However, the observed association between elevated PGE-M and all-cause mortality in AA non-aspirin users reinforces the potential benefit of aspirin to reduce mortality among AA men with prostate cancer.

Background: Systemic low-grade inflammation is a candidate risk factor for prostate cancer (PC) in African-American (AA) men potentially contributing to aggressive disease and mortality. Regular use of the anti-inflammatory drug aspirin is associated with decreased risk of advanced stage PC and increased disease-free survival in AA men. The chemopreventive benefits of aspirin have been attributed to inhibition of the cyclooxygenase (COX) pathway. Thromboxane A2 is an eicosanoid produced by the COX 1 enzyme in platelets. Aspirin inhibits cancer metastasis by inhibiting COX1 activity and thromboxane A2 synthesis. Hence, we assessed the role of thromboxane A2 in the development of lethal PC. Because thromboxane A2 is highly unstable, we measured its corresponding primary metabolite, urinary thromboxane b2 (TXB2), to examine the relationship of thromboxane A2 levels with PC. Method: TXB2 was measured in cases (977) and controls (1023) from the NCI-MD PC Case Control study using a mass-spectrometry-based assay. We estimated odds ratios (ORs) and 95% confidence intervals (CI) using unconditional logistic regression, with TXB2 levels modeled by quartiles. Multivariable models were adjusted for PC risk factors. Results: We observed an inverse relationship between aspirin use and levels of TXB2 in both cases and controls. For cases who used aspirin, the odds of having TXB2 urinary levels in the highest quartile (Q4) were significantly reduced when compared to non-users (adjusted OR = 0.29 95%CI 0.19-0.4) and this observation remained significant when stratified by race/ethnicity (AA men: OR 0.21 95%CI 0.11-0.38) (EA men: OR 0.34 95%CI 0.19-0.61), indicating significant inhibition of TXB2 formation by aspirin. The adjusted OR for PC was 1.42 (95%CI 1.09-1.85) for men in Q4 compared to Q1 (P trend 0.002). There was an association between TXB2 and PC in AA men (Q4 adjusted OR 1.95 95%CI 1.31-2.91, P trend Citation Format: Maeve Kiely, Ginger Milne, Tsion Z. Minas, Tiffany H. Dorsey, Wei Tang, Cheryl J. Smith, Michael B. Cook, Clayton Yates, Stefan Ambs. High urinary thromboxane B2 associates with aggressive prostate cancer and inversely correlates with aspirin use [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-157.

Background: Epigenetic age, captured by DNA methylation changes, is thought to be more informative with respect to disease risk and progression, than chronological age. A few studies have associated epigenetic age acceleration (EAA), the difference between DNA methylation age (DNAm age) and chronological age, with BC risk and subtypes. However, most of these studies were conducted in Western populations. In this study, we examined EAA in an Asian population, in which BC incidence rate is lower and age at BC onset is younger compared to most Western populations. Methods: We performed genome-wide DNA methylation profiling of 97 tumor and 89 paired distant normal tissue samples collected from BC patients in Hong Kong (HK) using an Illumina MethylationEPIC array. Two independent datasets were used to compare results: The Cancer Genome Atlas (TCGA, n=525 tumor and n=88 adjacent normal) and healthy women from the Komen tissue bank (n=59). DNAm age was calculated using Horvath's model based on 353 CpGs. The significance of EAA was tested using a simple linear regression model. We also used a multivariate regression model to test for equality of slopes (EAA rates) among different BC subtypes or datasets, considering interaction terms between age and subtype/dataset. Results: The average age at BC diagnosis was 58 years old (range: 33-81) and the distribution of the molecular subtype based on PAM50 was 43.0%, 29.1%, and 27.9% for luminal A, luminal B, and HER2-enriched/basal tumors, respectively. As expected, DNAm age showed a stronger correlation with chronological age in normal tissue (r=0.78, p Conclusion: Consistent with previous studies, we found that EAA in tumor samples varied across tumor subtypes. We also found that HK BC patients' epigenetic age accelerated at a different rate compared to predominantly white TCGA and Komen women, suggesting a potential racial biological difference. Large studies in other Asian populations are warranted to confirm our findings, which may provide biological insight into racial heterogeneity of BC, especially with regard to age at onset. Citation Format: Hela Koka, Bin Zhu, Shelly Lap Ah Tse, Difei Wang, Maeve Kiely, Jennifer Lyn Guida, Priscilla Lee, Feng Wang, Cherry Wu, Koon Ho Tsang, Wing-cheong Chan, Sze Hong Law, Eric Karlins, Bin Zhu, Amy Hutchinson, Belynda Hicks, Xiaohong R. Yang. DNA methylation age of paired tumor-normal breast tissue in Chinese women with breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 146.

peer-reviewed Protein Phosphatase 2A (PP2A) is a major serine/threonine phosphatase in cells. It consists of a catalytic subunit (C), a structural subunit (A), and a regulatory/variable B-type subunit. PP2A has a critical role to play in homeostasis where its predominant function is as a phosphatase that regulates the major cell signaling pathways in cells. Changes in the assembly, activity and substrate specificity of the PP2A holoenzyme have a direct role in disease and are a major contributor to the maintenance of the transformed phenotype in cancer. We have learned a lot about how PP2A functions from specific mutations that disrupt the core assembly of PP2A and from viral proteins that target PP2A and inhibit its effect as a phosphatase. This prompted various studies revealing that restoration of PP2A activity benefits some cancer patients. However, our understanding of the mechanism of action of this is limited because of the complex nature of PP2A holoenzyme assembly and because it acts through a wide variety of signaling pathways. Information on PP2A is also conflicting as there are situations whereby inactivation of PP2A induces apoptosis in many cancer cells. In this review we discuss this relationship and we also address many of the pertinent and topical questions that relate to novel therapeutic strategies aimed at altering PP2A activity. PUBLISHED peer-reviewed

peer-reviewed Conflicting reports implicate the scaffolding protein RACK1 in the progression of breast cancer. RACK1 has been identified as a key regulator downstream of growth factor and adhesion signalling and as a direct binding partner of PP2A. Our objective was to further characterise the interaction between PP2A and RACK1 and to advance our understanding of this complex in breast cancer cells. We examined how the PP2A holoenzyme is assembled on the RACK1 scaffold in MCF-7 cells. We used immobilized peptide arrays representing the entire PP2A-catalytic subunit to identify candidate amino acids on the C subunit of PP2A that might be involved in binding of RACK1. We identified the RACK1 interaction sites on PP2A. Stable cell lines expressing PP2A with FR69/70AA, R214A and Y218F substitutions were generated and it was confirmed that the RACK1/PP2A interaction is essential to stabilise PP2A activity. We used Real-Time Cell Analysis and a series of assays to demonstrate that disruption of the RACK1/PP2A complex also reduces the adhesion, proliferation, migration and invasion of breast cancer cells and plays a role in maintenance of the cancer phenotype. This work has significantly advanced our understanding of the RACK1/PP2A complex and suggests a pro-carcinogenic role for the RACK1/PP2A interaction. This work suggests that approaches to target the RACK1/PP2A complex are a viable option to regulate PP2A activity and identifies a novel potential therapeutic target in the treatment of breast cancer. (C) 2016 Elsevier Inc All rights reserved. ACCEPTED peer-reviewed

In the present paper we have developed, described and validated an in vitro bioassay to monitor skeletal muscle proliferation and differentiation. We have also demonstrated the use of this assay to evaluate factors which may affect muscle protein balance., The importance of growth and maintenance of skeletal muscle is vital for long term health and quality of life. Appropriate nutrition with specific bioactivities relevant to the functionalities of tissues such as skeletal muscle, can assist in maintaining and promoting adaptive responses to biological and environmental stresses which prevent muscle atrophy and promote hypertrophy. The aim of this investigation was to develop a novel in vitro cell-based electric impedance assay to study myoblast to myotube formation on the real time cell analysis (RTCA) platform (xCELLigence™, ACEA) and to validate the system by testing myotube responses to hypertrophic stimuli. C2C12 myoblasts were proliferated until 70% confluent in Dulbecco's Modified Eagles Medium (DMEM) (10% FBS) and subsequently differentiated to myotubes over 8 days in DMEM [2% horse serum (HS)]. Changes in cell behaviour and adhesion properties were monitored by measuring impedance via interdigitated microelectrodes in the base of E-16 cell culture dishes. To establish the suitability of this assay to monitor nutrient regulation of muscle hypertrophy, leucine, a known potent regulator of MPS was then supplemented to the fully formed myotubes in physiologically relevant conditions–0.20 mM, 0.40 mM, 0.6 mM, 0.8 mM and above 1.0 mM, 1.5 mM, 2.0 mM and impedance subsequently monitored. Parallel experiments highlighting alterations in myotube thickness, muscle protein synthesis (MPS) (mammalian target of rapamycin; mTOR) and differentiation (myogenin) were conducted to support RTCA bioassay findings. This in vitro bioassay can be used to monitor skeletal muscle behaviour and identify nutrient compounds with bioactivities promoting skeletal muscle hypertrophy, reducing muscle atrophy and thus inform the development of novel nutrient formulations for the maintenance of skeletal muscle.

Signalling proteins are intrinsic to all biological processes and interact with each other in tightly regulated and orchestrated signalling complexes and pathways. Characterization of protein binding can help to elucidate protein function within signalling pathways. This information is vital for researchers to gain a more comprehensive knowledge of cellular networks which can then be used to develop new therapeutic strategies for disease. However, studying protein–protein interactions (PPIs) can be challenging as the interactions can be extremely transient downstream of specific environmental cues. There are many powerful techniques currently available to identify and confirm PPIs. Choosing the most appropriate range of techniques merits serious consideration. The aim of this review is to provide a starting point for researchers embarking on a PPI study. We provide an overview and point of reference for some of the many methods available to identify interactions from in silico analysis and large scale screening tools through to the methods used to validate potential PPIs. We discuss the advantages and disadvantages of each method and we also provide a workflow chart to highlight the main experimental questions to consider when planning cell lysis to maximize experimental success. * AHA, : L-azidohomoalanine; BIND, : Biomolecular Interaction Network Database; BioGRID, : Biological General Repository of Interacting Datasets; BRET, : bioluminescence resonance energy transfer; CRAPome, : Contaminant Repository for Affinity Purification; DIP, : database of interacting proteins; EMSA, : electrophoretic mobility shift assay; FLIM, : fluorescence lifetime imaging microscopy; MYTH, : membrane yeast two hybrid; PLA, : proximity ligation assay; PPI, : protein–protein interaction; SILAC, : stable-isotope labelling by amino acids in cell culture; TAP, : tandem affinity purification; Y2H, : yeast 2 hybrid

Breast cancer represents 25% of all cancers diagnosed in women globally with almost 1.7 million new cases annually. The average survival rate for patients with early stage breast cancer has increased, however the survival rate for those with metastatic breast cancer has not improved. Metastasis is the number one cause of death in breast cancer patients; it remains a scientific enigma, with no available early detection screen or targeted therapies available to patients. Therefore, understanding how and why cancer cells migrate is of significant biological and clinical interest. Accumulating evidence suggests the metastatic potential of breast cancer is influenced by the composition of the extracellular matrix (ECM). The ECM is composed of fibrous proteins and other non-cellular biomolecules and has a well-documented role in influencing tumour structure and behaviour. We are examining genes that code for several categories of proteins involved in regulating the composition of the ECM. These genes code for proteins that are involved in signal transduction from the cell membrane or for proteins that are secreted into the ECM. We are also examining genes that code for fibrous proteins, glycoproteins, transmembrane proteins, growth factors and proteases. To refine our panel, we used STRING [1], an online database, to examine the relationships between the proteins coded by our genes based on the experimental determination of protein co-expression and protein homology. Using qRT-PCR, we are examining the expression of our refined gene set in three different cell lines, (representing the major sub-categories of breast cancer) which have been cultured on collagen, fibronectin, laminin and stimulated by various growth factors. We extended our study to examine the expression pattern in cells that were maintained in 3-Dimensional culture over a period of 10 days in an attempt to mimic more closely the physiological process. We have determined that there is significant differential gene expression of integrins and growth factor binding proteins as we change the composition of the cells culture environment. This work is helping us to refine a signature set by identifying genes whose expression is dysregulated in response to changes in the stromal environment. We are moving now to determining whether dysregulation of the expression of our gene panel correlates with disease progression by examining matched normal and diseased patient tissue. This could provide the opportunity to generate a novel gene signature that may be used as a prognostic tool to predict invasive cancers. It may also provide information on the design of novel treatment therapies for patients with metastatic cancers. 1. Szklarczyk, D., et al., The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res, 2017. 45(D1): p. D362-d368. Citation Format: Joanne Nolan, Maeve Kiely, Colum P. Dunne, Aoife J. Lowery, Patrick A. Kiely. Examining how the extracellular environment alters gene expression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 189.

Protein translation is one the most energy demanding and highly regulated processes in cells. For this reason, dysregulation of protein synthesis is linked with different types of disease, including cancer. The link between protein synthesis and cancer progression is becoming a central theme among cancer research. Although numerous studies have identified protein synthesis as a process central to cancer progression, further research is required to identify potential novel targets and/or biomarkers. Recently, RACK1 (Receptor for Activated C Kinase 1), a scaffold protein involved in many cell regulatory pathways including cell proliferation and migration, has been found to be part of the ribosomal machinery. Here, RACK1 is able to regulate the synthesis of particular sets of proteins and the translation itself. In fact, RACK1 is located close the mRNA exit channel where is able to recruit kinases, phosphatases and specific mRNA binding proteins. We have identified elongation factor 2 (eEF2) as novel RACK1 interacting protein in our two and three dimensional colon cancer models. Elongation factor 2 is one of the main proteins regulating the elongation phase of protein synthesis, responsible for the translocation of the mRNA along the ribosome. We have confirmed the binding between eEF2 and RACK1 by isolating and purifying a native human form of eEF2 and combining it with a purified human version of RACK1. Interestingly, we have demonstrated that stress conditions and IGF-1 stimulation modulate eEF2 activity in colon cancer cells. Moreover, we have examined the expression of EEF2 and have found that EEF2 gene is dysregulated in our cohort of matched normal and cancer colonic patient tissues. Interestingly, EEF2 expression is upregulated in mucinous cancers (a rare and aggressive type of colorectal cancer) compared to non-mucinous cancers and the expression of EEF2 is as well upregulated in cancers that have recurred within 2 to 3 years after the first surgery. We believe that eEF2 is playing an important role in cancer progression and migration especially by regulating and modulating protein synthesis in certain conditions. A better understanding of the role of this protein will help in the identification of novel targets that can be then translated into therapeutic strategies against colorectal cancer. Citation Format: Beatrice Malacrida, Catríona M. Dowling, Rasmus K. Flygaard, Maeve Kiely, John C. Coffey, Lasse B. Jenner, Patrick A. Kiely. Elongation factor 2: A novel target for cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1914.

Protein Phosphatase 2A (PP2A) is a major serine/threonine phosphatase in cells. It consists of a catalytic (C), structural (A) and regulatory/variable B-type subunits. PP2A has a critical role to play in homeostasis where it functions as a tumour suppressor. Changes in the activity of PP2A have a direct role in maintaining the transformed phenotype in cancer. However, the mechanism of action of PP2A during this process is poorly understood. Our understanding is limited because of the complex nature of PP2A holoenzyme assembly, and because it acts through a wide variety of signalling pathways. We have identified the scaffolding protein RACK1 as a PP2A interacting protein and we showed that RACK1 regulates PP2A activity to mediate crosstalk between growth factor and adhesion signalling by an IGF-I-independent mechanism(1, 2) . Our hypothesis is that RACK1 plays a critical role in determining how PP2A functions in signalling pathways. Using a series of truncation mutations and peptide array technology, we have identified and mapped the binding sites between RACK1 and the catalytic subunit of PP2A. Using a series of specific point mutations of these PP2A sites, we show that the RACK1/PP2A interaction is critical to maintain PP2A activity. Disruption of the RACK1/PP2A interaction has dramatic consequences for cell adhesion, proliferation and migration in MCF-7 cells. Our hypothesis is that RACK1 regulates the cellular distribution of PP2A and scaffolds PP2A to a specific subset of cellular targets. Using 3D in vitro breast cancer models and mass spectroscopy approaches, we have identified several proteins that interact with the RACK1/PP2A complex. One of these proteins, Metadherin has been shown to play a role in drug resistance(3). It has also been linked to the progression of many cancers(4). Using a series of novel approaches we have mapped the Metadherin interaction site on RACK1 and PP2A and confirmed that RACK1 and Metadherin colocalise at the cell membrane. Overexpression of Metadherin promotes IGF-I-mediated cell migration and invasion. Taken together, our data suggest that RACK1 scaffolds PP2A to Metadherin where it regulates Metadherin activity and cell migration in breast cancer. 1. Kiely, PA., et al. “Tyrosine 302 in RACK1 is essential for insulin-like growth factor-I-mediated competitive binding of PP2A and β1 integrin and for tumor cell proliferation and migration.” Journal of Biological Chemistry 283.34 (2008): 22952-22961. 2. Kiely, PA., et al. “Insulin-like growth factor I controls a mutually exclusive association of RACK1 with protein phosphatase 2A and β1 integrin to promote cell migration.” Molecular and cellular biology 26.11 (2006): 4041-4051. 3. Wei, Yong, Guohong Hu, and Yibin Kang. “Metadherin as a link between metastasis and chemoresistance.” Cell Cycle 8.14 (2009): 2131-2137. 4. Brown, DM., and Erkki R. “Metadherin, a cell surface protein in breast tumors that mediates lung metastasis.” Cancer cell 5.4 (2004): 365-374. Citation Format: Maeve L. Kiely, Rosemary O'Connor, Patrick A. Kiely. Identification of metadherin in a complex with RACK1/PP2A in mammary epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5287. doi:10.1158/1538-7445.AM2014-5287