BRAF Expression and Copy Number Alterations Predict Unfavorable Tumor Features and Adverse Outcomes in Patients With Breast Cancer.

Background: The role of BRAF in breast cancer pathogenesis is still unclear. To address this knowledge gap, this study is aimed at evaluating the impact of BRAF gene expression and copy number alterations (CNAs) on clinicopathologic characteristics and survival in patients with breast cancer. Methods: The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset was obtained from the cBioPortal public domain. Tumoral BRAF mRNA expression and CNAs along with demographic and tumor data for patients with breast cancer were retrieved. The association of BRAF expression and CNAs with breast cancer clinicopathologic characteristics was analyzed. The impact of BRAF mRNA expression on the overall survival of patients was assessed using Kaplan-Meier survival analysis. Results: BRAF gene mRNA log intensity expression was positively correlated with tumor size and the Nottingham Prognostic Index (NPI) (p < 0.001). Alternatively, BRAF gene expression was negatively correlated with the age at diagnosis (p = 0.003). The average BRAF mRNA expression was significantly higher in premenopausal patients, patients with high tumor grade, hormone receptor-negative status, and non-luminal tumors compared to postmenopausal patients, patients with low-grade, hormone receptor-positive, and luminal disease. BRAF gain and high-level amplification copy numbers were significantly associated with higher NPI scores and larger tumor sizes compared to neutral copy number status. Survival analysis revealed no discernible differences in overall survival for patients with low and high BRAF mRNA expression. Conclusion: High BRAF mRNA expression as well as the gain and high-level amplification copy numbers were associated with advanced tumor characteristics and unfavorable prognostic factors in breast cancer. BRAF could be an appealing target for the treatment of premenopausal patients with hormone receptor-negative breast cancer.

Pharmacogenomics of lipid-lowering agents: the impact on efficacy and safety.

Hyperlipidemia is a significant risk factor for cardiovascular disease morbidity and mortality. The lipid-lowering drugs are considered the cornerstone of primary and secondary prevention of atherosclerotic cardiovascular disease. Unfortunately, the lack of efficacy and associated adverse effects, ranging from mild-to-moderate to potentially life-threatening, lead to therapy discontinuation. Numerous reports support the role of gene polymorphisms in drugs' pharmacokinetic parameters and their associated adverse reactions. Therefore, this study aims to understand the pharmacogenomics of lipid-lowering drugs and the impact of genetic variants of key genes on the drugs' efficacy and toxicity. Indeed, genetically guided lipid-lowering therapy enhances overall safety, improves drug adherence and achieves long-term therapy.

Lipid-lowering drugs treat elevated lipid levels in the blood and reduce the risk of cardiovascular diseases and death. There are differences in the genetic makeup of people, which explains why different people may respond differently to lipid-lowering drugs. These genetic differences affect how the body absorbs, transports, metabolizes or eliminates lipid-lowering drugs. Some other genes may also control how much the body can absorb cholesterol and how much it can synthesize or metabolize it. As a result, people's risk of cardiovascular diseases varies due to differences in their genetic composition and how their bodies handle lipid-lowering drugs. Therefore, understanding the pharmacogenomics of lipid-lowering agents improves efficacy, enhances overall safety and achieves long-term therapy.

Estrogen-Related Receptors Gene Expression and Copy Number Alteration Association With the Clinicopathologic Characteristics of Breast Cancer.

Purpose: It has been suggested that dysregulation of transcription factors expression or activity plays significant roles in breast cancer (BC) severity and poor prognosis. Therefore, our study aims to thoroughly evaluate the estrogen-related receptor isoforms (ESRRs) expression and copy number alteration (CNA) status and their association with clinicopathologic characteristics in BC. Methods: A METABRIC dataset consist of 2509 BC patients' samples was obtained from the cBioPortal public domain. The gene expression, putative CNA, and relevant tumor information of ESRRs were retrieved. ESRRs messenger RNA (mRNA) expression in BC cell lines was obtained from the Cancer Cell Line Encyclopedia (CCLE). Association and correlation analysis of ESRRs expression with BC clinicopathologic characteristics and molecular subtype were performed. Kaplan-Meier survival analysis was conducted to evaluate the prognostic value of ESRRs expression on patient survival. Results: ESRRα expression correlated negatively with patients' age and overall survival, whereas positively correlated with tumor size, the number of positive lymph nodes, and Nottingham prognostic index (NPI). Conversely, ESRRγ expression was positively correlated with patients' age and negatively correlated with NPI. ESRRα and ESRRγ expression were significantly associated with tumor grade, expression of hormone receptors, human epidermal growth factor receptor 2 (HER2), and molecular subtype, whereas ESRRß was only associated with tumor stage. A significant and distinct association of each of ESRRs CNA with various clinicopathologic and prognostic factors was also observed. Kaplan-Meier survival analysis demonstrated no significant difference for survival curves among BC patients with high or low expression of ESRRα, ß, or γ. On stratification, high ESRRα expression significantly reduced survival among premenopausal patients, patients with grade I/II, and early-stage disease. In BC cell lines, only ESRRα expression was significantly higher in HER2-positive cells. No significant association was observed between ESRRß expression and any of the clinicopathologic characteristics examined. Conclusions: In this clinical dataset, ESRRα and ESRRγ mRNA expression and CNA show a significant correlation and association with distinct clinicopathologic and prognostic parameters known to influence treatment outcomes; however, ESRRß failed to show a robust role in BC pathogenesis. ESRRα and ESRRγ can be employed as therapeutic targets in BC-targeted therapy. However, the role of ESRRß in BC pathogenesis remains unclear.

Differential Histone Posttranslational Modifications Induced by DNA Hypomethylating Agents.

INTRODUCTION: The prototype DNA hypomethylating agents 5-azacytidine (5AC) and decitabine (DAC) are currently FDA-approved for treatment of blood and bone marrow disorders like myelodysplastic syndrome. 5AC and DAC are considered similar drugs and were shown to induce histone modifications that modulate gene expression. The aim of this study is to compare the effect of both drugs on histone acetylation and methylation at multiple histone amino acids residues. METHODS: Mass spectrometry was used to compare the effect of both drugs on 95 different histone posttranslational modifications (PTMs) in leukemia cells. ChIP-Seq analysis was used to compare the impact of both drugs on the genome-wide acetylation of the H3K9 mark using primary leukemia cells from six de-identified AML patients. RESULTS: Both DAC and 5AC induced histone PTMs in different histone isoforms like H1.4, H2A, H3, H3.1, and H4. Changes in both histone methylation and acetylation were observed with both drugs; however, there were distinct differences in the histone modifications induced by the two drugs. Since both drugs were shown to increase the activity of the HDAC SIRT6 previously, we tested the effect of 5AC on the acetylation of H3K9, the physiological substrate SIRT6, using ChIP-Seq analysis and compared it to the previously published DAC-induced changes. Significant H3K9 acetylation changes (P< .05) were detected at 925 genes after 5AC treatment vs only 182 genes after DAC treatment. Nevertheless, the gene set modified by 5AC was different from that modified by DAC with only ten similar genes modulated by both drugs. CONCLUSION: Despite similarity in chemical structure and DNA hypomethylating activity, 5AC and DAC induced widely different histone PTMs and considering them interchangeable should be carefully evaluated. The mechanism of these histone PTM changes is not clear and may involve modulation of the activity or the expression of the enzymes inducing histone PTMs.

Roles of the tumor suppressor inhibitor of growth family member 4 (ING4) in cancer.

Inhibitor of growth family member 4 (ING4) is best known as a tumor suppressor that is frequently downregulated, deleted, or mutated in many cancers. ING4 regulates a broad array of tumor-related processes including proliferation, apoptosis, migration, autophagy, invasion, angiogenesis, DNA repair and chromatin remodeling. ING4 alters local chromatin structure by functioning as an epigenetic reader of H3K4 trimethylation histone marks (H3K4Me3) and regulating gene transcription through directing histone acetyltransferase (HAT) and histone deacetylase (HDAC) protein complexes. ING4 may serve as a useful prognostic biomarker for many cancer types and help guide treatment decisions. This review provides an overview of ING4's central functions in gene expression and summarizes current literature on the role of ING4 in cancer and its possible use in therapy.

ING4 Expression Landscape and Association With Clinicopathologic Characteristics in Breast Cancer.

BACKGROUND: Breast cancer (BC) development and progression is complex and still not fully understood. The expression or dysregulation of a variety of transcription factors has been suggested as contributing to disease severity and a poor prognosis. Therefore, the present study was designed to systematically outline ING4 expression and characteristics in clinical samples and cell lines of BC. MATERIALS AND METHODS: A METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) dataset was obtained from a cBioPortal public domain. ING4 gene expression, putative copy number alterations, and pertinent tumor information were retrieved. ING4 gene expression was identified for 1904 BC patients. ING4 mRNA expression data in BC cell lines were obtained from the Cancer Cell Line Encyclopedia. Analyses were conducted for associations between ING4 expression and age at diagnosis, tumor clinicopathologic characteristics, and molecular subtypes. The prognostic value of ING4 in BC patients was evaluated using Kaplan-Meier survival analysis. RESULTS: The ING4 mRNA expression log intensity mean was 6.95, and 1005 (52.8%) of patients were determined to have high ING4 mRNA expression (mRNA log intensity > 6.95). Although ING4 gene expression correlated significantly and negatively with the Nottingham Prognostic Index and the number of positive lymph nodes (P < .05) and positively with overall survival time (P < .001). However, these correlations were weak in magnitude (r ∼ 0.1). The expression of ING4 was significantly associated with tumor grade, hormone receptor expression, human epidermal growth factor receptor 2, and molecular subtype. ING4 copy number alteration was also significantly associated with several clinicopathologic and prognostic factors. Kaplan-Meier survival analysis demonstrated greater overall survival for BC patients with high ING4 expression (P = .0046). Stratification of the data by menopausal status and tumor characteristics revealed a significant effect of ING4 expression on survival among premenopausal patients and women with a nonluminal subtype. Furthermore, the expression of ING4 in BC cell lines was significantly greater in luminal A and basal-like cells compared with human epidermal growth factor receptor 2-positive cells, which was also observed in the clinical samples. CONCLUSIONS: The present study showed that ING4 gene expression is modulated in BC. High ING4 gene expression was associated with favorable prognostic parameters and positive clinical outcomes in our series of BC patients. ING4 could be used as a potential therapeutic target in BC-targeted therapy.

Author Correction: Activation of SIRT6 by DNA hypomethylating agents and clinical consequences on combination therapy in leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pharmacogenomics Instruction Depth, Extent, and Perception in US Medical Curricula.

INTRODUCTION: This descriptive study aimed to evaluate the depth, extent, and perception of pharmacogenomics instruction in schools and colleges of medicine in the United States. Changes in medical pharmacogenomics instruction over the past decade were also assessed by comparing our results with those of a previous study. METHODS: An electronic survey was emailed to all accredited allopathic and osteopathic medical schools across the US using Qualtrics online survey software. Multiple email reminders were sent to increase the response rate. RESULTS: Of 151 targeted eligible medical schools across the United States, 22 responded to the survey. One invalid response was excluded, resulting in a response rate of 13.9%. Of responding schools, 85.7% cover pharmacogenomics in their curriculum, mainly in the second year, however, none teach pharmacogenomics as a stand-alone course. The depth and the extent of pharmacogenomics coverage varied among responding programs. Although 66.7% of respondents believe that neither physicians nor other health care professionals possess appropriate knowledge in pharmacogenomics, only 23.8% plan to increase pharmacogenomics instruction in their curricula in the near future. CONCLUSIONS: Most medical schools surveyed include some pharmacogenomics instruction in their curricula, although the depth and the extent of the instruction varies. Most respondents believe that physicians and other health care professionals today do not possess an appropriate level of knowledge in pharmacogenomics; however, few institutions report short-term plans to increase pharmacogenomics instruction. Pharmacogenomics plays a significant role in personalized medicine; greater efforts by medical school decision-makers are needed to improve the level of pharmacogenomics instruction in medical curricula.

Activation of SIRT6 by DNA hypomethylating agents and clinical consequences on combination therapy in leukemia.

The FDA-approved DNA hypomethylating agents (DHAs) like 5-azacytidine (5AC) and decitabine (DAC) demonstrate efficacy in the treatment of hematologic malignancies. Despite previous reports that showed histone acetylation changes upon using these agents, the exact mechanism underpinning these changes is unknown. In this study, we investigated the relative potency of the nucleoside analogs and non-nucleoside analogs DHAs on DNA methylation reversal using DNA pyrosequencing. Additionally, we screened their effect on the enzymatic activity of the histone deacetylase sirtuin family (SIRT1, SIRT2, SIRT3, SIRT5 and SIRT6) using both recombinant enzymes and nuclear lysates from leukemia cells. The nucleoside analogs (DAC, 5AC and zebularine) were the most potent DHAs and increased the enzymatic activity of SIRT6 without showing any significant increase in other sirtuin isoforms. ChIP-Seq analysis of bone marrow cells derived from six acute myeloid leukemia (AML) patients and treated with the nucleoside analog DAC induced genome-wide acetylation changes in H3K9, the physiological substrate for SIRT6. Data pooling from the six patients showed significant acetylation changes in 187 gene loci at different chromosomal regions including promoters, coding exons, introns and distal intergenic regions. Signaling pathway analysis showed that H3K9 acetylation changes are linked to AML-relevant signaling pathways like EGF/EGFR and Wnt/Hedgehog/Notch. To our knowledge, this is the first report to identify the nucleoside analogs DHAs as activators of SIRT6. Our findings provide a rationale against the combination of the nucleoside analogs DHAs with SIRT6 inhibitors or chemotherapeutic agents in AML due to the role of SIRT6 in maintaining genome integrity and DNA repair.

Identification of the inhibitor of growth protein 4 (ING4) as a potential target in prostate cancer therapy.

INhibitor of Growth protein 4 (ING4) is a potential chromatin modifier that has been implicated in several cancer-related processes. However, the role of ING4 in prostate cancer (PC) is largely unknown. This study aimed to assess ING4's role in global transcriptional regulation in PC cells to identify potential cellular processes associated with ING4 loss. RNA-Seq using next-generation sequencing (NGS) was used to identify altered genes in LNCaP PC cells following ING4 depletion. Ingenuity pathways analysis (IPA®) was applied to the data to highlight candidates, ING4-regulated pathways, networks and cellular processes. Selected genes were validated using RT-qPCR. RNA-Seq of LNCaP cells revealed a total of 159 differentially expressed genes (fold change ≥ 1.5 or ≤ - 1.5, FDR ≤ 0.05) following ING4 knockdown. RT-qPCR used to validate the expression level of selected genes was in agreement with RNA-Seq results. Key genes, unique pathways, and biological networks were identified using IPA® analysis. This is the first report of global gene regulation in PC cells by ING4. The resultant differential expression profile revealed the potential role of ING4 in PC pathogenesis possibly through modulation of key genes, pathways and biological networks that are central drivers of the disease. Collectively, these findings shed light on a novel transcriptional regulator of PC that ultimately may influence the disease progression and as a potential target in the disease therapy.

A comparative study of the depth, breadth, and perception of pharmacogenomics instruction in a subgroup of US pharmacy curricula.

INTRODUCTION: This study was designed to assess the depth, breadth, and perception of pharmacogenomics education in pharmacy curricula in the United States (US). METHODS: A modified, online questionnaire from previous studies was sent to all accredited US schools and colleges of pharmacy. The survey covered three distinct areas related to the schools' educational environments, the depth and the extent of pharmacogenomics core competencies and topics taught, and the institutions' perceptions of the importance of pharmacogenomics in the curriculum and future plans for expanded pharmacogenomics education. Multiple approaches were used to increase the response rate, and results were analyzed using descriptive statistics. RESULTS: Of the 133 eligible programs, 32 participated in the survey. Six invalid surveys were excluded from our study, resulting in a 19.6% response rate. Results revealed that all responding schools included pharmacogenomics in the curriculum. Interestingly, 76.9% of the respondents believed pharmacists do not have the appropriate knowledge of pharmacogenomics. However, only 30.7% indicated that their programs planned to expand pharmacogenomics in their curriculum. CONCLUSIONS: The responding schools all included some pharmacogenomics in their curriculum. However, the depth and the extent of pharmacogenomics topics covered varied. Respondents perceived that pharmacists today do not possess the appropriate level of pharmacogenomics knowledge. Despite this, there is limited emphasis on expanding pharmacogenomics instruction in the responding schools' curriculums.

A qualitative assessment of West Virginia pharmacist activities and attitude in diabetes management.

AIMS AND OBJECTIVES: The role of pharmacists in chronic disease state management has been shown to significantly improve patient health outcomes and reduce overall health care costs. The current study is designed to assess the roles and attitudes of West Virginia (WV) pharmacists toward diabetes, evaluate services provided, address pharmacist clinical understanding and training, and demonstrate the challenges that limit pharmacists ability to deliver an efficient disease state management. METHODS: We invited 435 preceptors affiliated with the University of Charleston School of Pharmacy to participate in the study using Qualtrics online survey software. The survey was divided into sections related to pharmacists, practice environment, pharmacist's roles in diabetes management, and challenges faced that limit their ability to deliver effective care to diabetic patients. Data were analyzed using 1-way analysis of variance, and a P value ≤.05 was considered statistically significant. RESULTS: Of all eligible invited preceptors, 104 accessed the online survey based on the Qualtrics tracking tool, while 58 participated in the survey with a 56% response rate. Generally, WV pharmacists have positive attitudes regarding the provision of primary activities related to drug use and its associated problems. However, we report that WV pharmacists are less involved in providing education or recommendations regarding diabetes-associated risk factors such as nephropathy, retinopathy, foot care, and gastroparesis. In addition, the majority of pharmacists indicated that they face many challenges related to patient and the practice site environment that limit their ability to provide optimum diabetes patient care services. CONCLUSION: Despite the mounting evidence that pharmacists can improve diabetic patient outcomes while significantly reducing overall costs, WV pharmacists are less involved in providing education or counseling in a variety of areas related to disease state management. In addition, identifying pharmacist challenges provides significant information for future planning toward improving diabetic patient care.

Molecular and genetic crosstalks between mTOR and ERRα are key determinants of rapamycin-induced nonalcoholic fatty liver.

mTOR and ERRα are key regulators of common metabolic processes, including lipid homeostasis. However, it is currently unknown whether these factors cooperate in the control of metabolism. ChIP-sequencing analyses of mouse liver reveal that mTOR occupies regulatory regions of genes on a genome-wide scale including enrichment at genes shared with ERRα that are involved in the TCA cycle and lipid biosynthesis. Genetic ablation of ERRα and rapamycin treatment, alone or in combination, alter the expression of these genes and induce the accumulation of TCA metabolites. As a consequence, both genetic and pharmacological inhibition of ERRα activity exacerbates hepatic hyperlipidemia observed in rapamycin-treated mice. We further show that mTOR regulates ERRα activity through ubiquitin-mediated degradation via transcriptional control of the ubiquitin-proteasome pathway. Our work expands the role of mTOR action in metabolism and highlights the existence of a potent mTOR/ERRα regulatory axis with significant clinical impact.

Hormone depletion-insensitivity of prostate cancer cells is supported by the AR without binding to classical response elements.

A need for androgen response elements (AREs) for androgen receptor (AR)-dependent growth of hormone depletion-insensitive prostate cancer is generally presumed. In such cells, androgen-independent activation by AR of certain genes has been attributed to selective increases in basal associations of AR with putative enhancers. We examined the importance of AR binding to DNA in prostate cancer cells in which proliferation in the absence of hormone was profoundly (∼ 90%) dependent on endogenous AR and where the receptor was not up-regulated or mutated but was predominantly nuclear. Here, ARE-mediated promoter activation and the binding of AR to a known ARE in the chromatin remained entirely androgen dependent, and the cells showed an androgen-responsive gene expression profile with an unaltered sensitivity to androgen dose. In the same cells, a different set of genes primarily enriched for cell division functions was activated by AR independently of hormone and significantly overlapped the signature gene overexpression profile of hormone ablation-insensitive clinical tumors. After knockdown of endogenous AR, hormone depletion-insensitive cell proliferation and AR apoprotein-dependent gene expression were rescued by an AR mutant that was unable to bind to ARE but that could transactivate through a well-established AR tethering protein. Hormone depletion-insensitive AR binding sites in the chromatin were functional, binding, and responding to both the wild-type and the mutant AR and lacked enrichment for canonical or noncanonical ARE half-sites. Therefore, a potentially diverse set of ARE-independent mechanisms of AR interactions with target genes must underlie truly hormone depletion-insensitive gene regulation and proliferation in prostate cancer.

R5020 and RU486 act as progesterone receptor agonists to enhance Sp1/Sp4-dependent gene transcription by an indirect mechanism.

It has been suggested that ligand-dependent gene activation by the progesterone receptor (PR) can result from recruitment of PR by the promoter bound Sp1. A detailed investigation of the Sp1-dependent agonistic activity of RU486 and R5020 on the folate receptor (FR) type alpha, p27, thymidine kinase 1 and p21 genes reveals a different mechanism. The FR-alpha P4 promoter and the endogenous FR-alpha gene were up-regulated by the PR agonist R5020 through either PR-A or PR-B. The classical antagonist RU486 also activated the promoter but only through PR-B. The most proximal (essential) G/C-rich (Sp1 binding) element and the initiator region constituted the minimal promoter responsive to PR regulation; substitution with a stronger cluster of G/C-rich elements enhanced the magnitude of the PR response. In contrast, substitution of the G/C-rich element with a TATA box resulted in the loss of regulation by PR. Overexpression of Sp1 and Sp4 but not Sp3 enhanced activation of the FR-alpha promoter by PR, knocking down Sp1 decreased the activation in a manner that was reversed by ectopic Sp1 or Sp4. The ligand-dependent action of PR on the promoter was delayed compared with its activation of a classical glucocorticoid response element-driven promoter and activation of both the promoter and the endogenous FR-alpha gene by PR required new protein synthesis. Activation by PR paralleled RNA polymerase II recruitment but was not accompanied by either association of PR or a change in the association of Sp1 with the endogenous FR-alpha P4 promoter. Similar observations were made for PR regulation of the genes encoding p27, thymidine kinase 1, and p21. The results contradict the current view of Sp1-dependent gene regulation by PR and point to the existence of one or more PR target genes whose promoter and cell context(s) must thus be key determinants of the agonistic activity of RU486 on a large group of important Sp1-dependent downstream target genes.

Enhancement of folate receptor alpha expression in tumor cells through the glucocorticoid receptor: a promising means to improved tumor detection and targeting.

The utility of the folate receptor (FR) type alpha, in a broad range of targeted therapies and as a diagnostic serum marker in cancer, is confounded by its variable tumor expression levels. FR-alpha, its mRNA and its promoter activity were coordinately up-regulated by the glucocorticoid receptor (GR) agonist, dexamethasone. Optimal promoter activation which occurred at <50 nmol/L dexamethasone was inhibited by the GR antagonist, RU486, and was enhanced by coactivators, supporting GR mediation of the dexamethasone effect. The dexamethasone response of the FR-alpha promoter progressed even after dexamethasone was withdrawn, but this delayed effect required prior de novo protein synthesis indicating an indirect regulation. The dexamethasone effect was mediated by the G/C-rich (Sp1 binding) element in the core P4 promoter and was optimal in the proper initiator context without associated changes in the complement of major Sp family proteins. Histone deacetylase (HDAC) inhibitors potentiated dexamethasone induction of FR-alpha independent of changes in GR levels. Dexamethasone/HDAC inhibitor treatment did not cause de novo FR-alpha expression in a variety of receptor-negative cells. In a murine HeLa cell tumor xenograft model, dexamethasone treatment increased both tumor-associated and serum FR-alpha. The results support the concept of increasing FR-alpha expression selectively in the receptor-positive tumors by brief treatment with a nontoxic dose of a GR agonist, alone or in combination with a well-tolerated HDAC inhibitor, to increase the efficacy of various FR-alpha-dependent therapeutic and diagnostic applications. They also offer a new paradigm for cancer diagnosis and combination therapy that includes altering a marker or a target protein expression using general transcription modulators.