Reproducibility2020: Progress and priorities.

The preclinical research process is a cycle of idea generation, experimentation, and reporting of results. The biomedical research community relies on the reproducibility of published discoveries to create new lines of research and to translate research findings into therapeutic applications. Since 2012, when scientists from Amgen reported that they were able to reproduce only 6 of 53 "landmark" preclinical studies, the biomedical research community began discussing the scale of the reproducibility problem and developing initiatives to address critical challenges. Global Biological Standards Institute (GBSI) released the "Case for Standards" in 2013, one of the first comprehensive reports to address the rising concern of irreproducible biomedical research. Further attention was drawn to issues that limit scientific self-correction, including reporting and publication bias, underpowered studies, lack of open access to methods and data, and lack of clearly defined standards and guidelines in areas such as reagent validation. To evaluate the progress made towards reproducibility since 2013, GBSI identified and examined initiatives designed to advance quality and reproducibility. Through this process, we identified key roles for funders, journals, researchers and other stakeholders and recommended actions for future progress. This paper describes our findings and conclusions.

Matched preclinical designs for improved translatability.

A more stratified preclinical design strategy will increase the statistical power and reproducibility of animal studies and their translatability.

[Letter to the Editor] The need for improved education and training in research antibody usage and validation practices.

Address correspondence to Leonard P. Freedman, Global Biological Standards Institute, 1020 19th Street, NW, Suite 550, Washington, DC, 20036. E-mail: lfreedman@gbsi.org.

The Economics of Reproducibility in Preclinical Research.

Low reproducibility rates within life science research undermine cumulative knowledge production and contribute to both delays and costs of therapeutic drug development. An analysis of past studies indicates that the cumulative (total) prevalence of irreproducible preclinical research exceeds 50%, resulting in approximately US$28,000,000,000 (US$28B)/year spent on preclinical research that is not reproducible-in the United States alone. We outline a framework for solutions and a plan for long-term improvements in reproducibility rates that will help to accelerate the discovery of life-saving therapies and cures.

The increasing urgency for standards in basic biologic research.

Research advances build upon the validity and reproducibility of previously published data and findings. Yet irreproducibility in basic biologic and preclinical research is pervasive in both academic and commercial settings. Lack of reproducibility has led to invalidated research breakthroughs, retracted articles, and aborted clinical trials. Concerns and requirements for transparent, reproducible, and translatable research are accelerated by the rapid growth of "post-publication peer review," open access publishing, and data sharing that facilitate the identification of irreproducible data/studies; they are magnified by the explosion of high-throughput technologies, genomics, and other data-intensive disciplines. Collectively, these changes and challenges are decreasing the effectiveness of traditional research quality mechanisms and are contributing to unacceptable-and unsustainable-levels of irreproducibility. The global oncology and basic biologic research communities can no longer tolerate or afford widespread irreproducible research. This article discusses (i) how irreproducibility in preclinical research can ultimately be traced to an absence of a unifying life science standards framework, and (ii) makes an urgent case for the expanded development and use of consensus-based standards to both enhance reproducibility and drive innovations in cancer research.

Liver X receptor-retinoid X receptor (LXR-RXR) heterodimer cistrome reveals coordination of LXR and AP1 signaling in keratinocytes.

Liver X receptors (LXRs) play a critical role in regulating lipid synthesis and transport in numerous tissues. In the skin, activation of LXR induces keratinocyte differentiation and improves epidermal permeability barrier homeostasis. To elucidate the mechanism of LXR action in skin, we mapped its cistrome by identifying LXRß-RXRα binding sites using ChIP-on-chip in normal human epidermal keratinocytes (NHEKs). The cistrome was integrated with transcription data to obtain a global view of LXR action in keratinocyte biology. Here, we identify 2035 LXRß-RXRα binding sites containing 4794 LXR response elements in NHEKs and show the presence of consensus heterodimer active regions in genes involved in keratinocyte lipid transport/synthesis and terminal differentiation. Bioinformatics analysis of the cistrome revealed an enrichment of AP1 cis-regulatory motifs in the vicinity of the LXRß-RXRα binding sites. Importantly, we have demonstrated a direct interaction between LXR and Jun/Fos, indicating that the cooperation between LXR and AP1 may orchestrate keratinocyte differentiation. Finally, we corroborated these results by genome-wide mapping of the c-Fos and c-Jun cistromes in NHEKs, demonstrating that 77% of all the LXRß-RXRα binding regions show the presence of AP1 motifs at adjacent locations. Our findings provide new insight into the mechanism of LXR action in keratinocyte differentiation, lipid production and barrier formation, further strengthening the validation of LXR as a potential therapeutic target for skin disorders including skin aging, psoriasis, and atopic dermatitis.

Identification of 5α, 6α-epoxycholesterol as a novel modulator of liver X receptor activity.

The liver X receptors (LXRα and LXRß) are members of the nuclear receptor superfamily that function as key transcriptional regulators of a number of biological processes, including cholesterol homeostasis, lipid metabolism, and keratinocyte differentiation. Natural ligands that activate LXRs include oxysterol derivatives such as 25-hydroxycholesterol, 27-hydroxycholesterol, 22(R)-hydroxycholesterol, 20(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. Related oxysterols, such as 5α,6α-epoxycholesterol (5,6-EC) are present in a number of foods and have been shown to induce atherosclerosis in animal models. Intriguingly, these oxysterols have also been detected in atherosclerotic plaques. Using a variety of biochemical and cellular assays, we demonstrate that 5,6-EC is the first dietary modulator and an endogenous LXR ligand with cell and gene context-dependent antagonist, agonist, and inverse agonist activities. In a multiplexed LXR-cofactor peptide interaction assay, 5,6-EC induced the recruitment of a number of cofactor peptides onto both LXRα and LXRß and showed an EC(50) of approximately 2 µM in peptide recruitment. Furthermore, 5,6-EC bound to LXRα in a radiolabeled ligand displacement assay (EC(50) = 76 nM), thus demonstrating it to be one of the most potent natural LXRα ligands known to date. Analysis of endogenous gene expression in various cell-based systems indicated the potential of 5,6-EC to antagonize LXR-mediated gene expression. Furthermore, it also induced the expression of some LXR-responsive genes in keratinocytes. These results clearly demonstrate that 5,6-EC is an LXR modulator that may play a role in the development of lipid disorders, such as atherosclerosis, by antagonizing the agonistic action of endogenous LXR ligands.

Discovery of the selective androgen receptor modulator MK-0773 using a rational development strategy based on differential transcriptional requirements for androgenic anabolism versus reproductive physiology.

Selective androgen receptor modulators (SARMs) are androgen receptor (AR) ligands that induce anabolism while having reduced effects in reproductive tissues. In various experimental contexts SARMs fully activate, partially activate, or even antagonize the AR, but how these complex activities translate into tissue selectivity is not known. Here, we probed receptor function using >1000 synthetic AR ligands. These compounds produced a spectrum of activities in each assay ranging from 0 to 100% of maximal response. By testing different classes of compounds in ovariectomized rats, we established that ligands that transactivated a model promoter 40-80% of an agonist, recruited the coactivator GRIP-1 <15%, and stabilized the N-/C-terminal interdomain interaction <7% induced bone formation with reduced effects in the uterus and in sebaceous glands. Using these criteria, multiple SARMs were synthesized including MK-0773, a 4-aza-steroid that exhibited tissue selectivity in humans. Thus, AR activated to moderate levels due to reduced cofactor recruitment, and N-/C-terminal interactions produce a fully anabolic response, whereas more complete receptor activation is required for reproductive effects. This bimodal activation provides a molecular basis for the development of SARMs.

Estrogen receptor beta is a novel therapeutic target for photoaging.

One of the many harmful factors faced by the skin is solar UV radiation, which damages skin by inducing chronic low-grade inflammation through increased expression of proinflammatory cytokines, metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). Estrogen receptors (ERs) alpha and beta are ligand-dependent transcription factors that are expressed in skin, and an ERbeta agonist has previously shown efficacy in vivo in models of pain and inflammation. Because ERbeta does not carry the breast and uterine proliferation liabilities of ERalpha, we decided to explore the possibility of using ERbeta as a target for photoaging. We show that ERbeta-selective compounds suppressed the expression of cytokines and MMPs in activated keratinocytes and fibroblast-based in vitro models of photoaging. Furthermore, in activated dermal fibroblasts, ERbeta-selective compounds also inhibited COX-2. These activities of ERbeta ligands in skin cells correlated with the expression levels of ERbeta and showed reversal by treatment with a potent synthetic ER antagonist. Furthermore, the pharmacology of ERbeta-selective compound was observed in wild-type but not in skin cells obtained from ERbeta knockout mice. Finally, we demonstrate that a synthetic ERbeta agonist inhibited UV-induced photodamage and skin wrinkle formation in a murine model of photoaging. Therefore, the potential of an ERbeta ligand to regulate multiple pathways underlying the cause of photoaging suggests ERbeta to be a novel therapeutic target for the prevention and treatment of photoaging.

Androgen-mediated improvement of body composition and muscle function involves a novel early transcriptional program including IGF1, mechano growth factor, and induction of {beta}-catenin.

Androgens promote anabolism in the musculoskeletal system while generally repressing adiposity, leading to lean body composition. Circulating androgens decline with age, contributing to frailty, osteoporosis, and obesity; however, the mechanisms by which androgens modulate body composition are largely unknown. Here, we demonstrate that aged castrated rats develop increased fat mass, reduced muscle mass and strength, and lower bone mass. Treatment with testosterone or 5alpha-dihydrotestosterone (DHT) reverses the effects on muscle and adipose tissues while only aromatizable testosterone increased bone mass. During the first week, DHT transiently increased soleus muscle nuclear density and induced expression of IGF1 and its splice variant mechano growth factor (MGF) without early regulation of the myogenic factors MyoD, myogenin, monocyte nuclear factor, or myostatin. A genome-wide microarray screen was also performed to identify potential pro-myogenic genes that respond to androgen receptor activation in vivo within 24 h. Of 24 000 genes examined, 70 candidate genes were identified whose functions suggest initiation of remodeling and regeneration, including the type II muscle genes for myosin heavy chain type II and parvalbumin and the chemokine monocyte chemoattractant protein-1. Interestingly, Axin and Axin2, negative regulators of beta-catenin, were repressed, indicating modulation of the beta-catenin pathway. DHT increased total levels of beta-catenin protein, which accumulated in nuclei in vivo. Likewise, treatment of C2C12 myoblasts with both IGF1Ea and MGF C-terminal peptide increased nuclear beta-catenin in vitro. Thus, we propose that androgenic anabolism involves early downregulation of Axin and induction of IGF1, leading to nuclear accumulation of beta-catenin, a pro-myogenic, anti-adipogenic stem cell regulatory factor.

Differential biochemical and cellular actions of Premarin estrogens: distinct pharmacology of bazedoxifene-conjugated estrogens combination.

The use of estrogen-based therapies and the selective estrogen receptor (ER) modulator (SERM), raloxifene, which are approved for postmenopausal osteoporosis, is associated with side effects such as uterine/breast hyperproliferation, thromboembolism, and hot flashes. A combination of a new SERM, bazedoxifene (BZA), and Premarin (conjugated estrogens; CE) is under investigation to mitigate the estrogen/SERM side effects with promising results in Phase III clinical trials. To explore the mechanism of BZA/CE action, we investigated the recruitment of cofactor peptides to ERalpha by components of CE and a mixture containing the 10 major components of CE with or without three different SERMs. Here, we demonstrate differential recruitment of cofactor peptides to ERalpha by the individual CE components using a multiplex nuclear receptor-cofactor peptide interaction assay. We show that estrone and equilin are partial agonists in comparison with 17beta-estradiol in recruiting cofactor peptides to ERalpha. Further, CE was more potent than 17beta-estradiol in mediating ERalpha interaction with cofactor peptides. Interestingly, BZA was less potent than other SERMs in antagonizing the CE-mediated cofactor peptide recruitment to ERalpha. Finally, in accordance with these biochemical findings, 17beta-estradiol and CE, as well as SERM/CE combinations, showed differential gene regulation patterns in MCF-7 cells. In addition, BZA showed antagonism of a unique set of CE-regulated genes and did not down-regulate the expression of a number of CE-regulated genes, the expression of which was effectively antagonized by the other two SERMs. These results indicate that SERMs in combination with CE exhibit differential pharmacology, and therefore, combinations of other SERMs and estrogen preparations may not yield the same beneficial effects that are observed in clinic by pairing BZA with CE.

Liver X receptor is a therapeutic target for photoaging and chronological skin aging.

Liver X receptors (LXRalpha and -beta) are liposensors that exert their metabolic effects by orchestrating the expression of macrophage genes involved in lipid metabolism and inflammation. LXRs are also expressed in other tissues, including skin, where their natural oxysterol ligands induce keratinocyte differentiation and improve epidermal barrier function. To extend the potential use of LXR ligands to dermatological indications, we explored the possibility of using LXR as a target for skin aging. We demonstrate that LXR signaling is down-regulated in cell-based models of photoaging, i.e. UV-activated keratinocytes and TNFalpha-activated dermal fibroblasts. We show that a synthetic LXR ligand inhibits the expression of cytokines and metalloproteinases in these in vitro models, thus indicating its potential in decreasing cutaneous inflammation associated with the etiology of photoaging. Furthermore, a synthetic LXR ligand induces the expression of differentiation markers, ceramide biosynthesis enzymes, and lipid synthesis and transport genes in keratinocytes. Remarkably, LXRbeta-null mouse skin showed some of the molecular defects that are observed in chronologically aged human skin. Finally, we demonstrate that a synthetic LXR agonist inhibits UV-induced photodamage and skin wrinkle formation in a murine model of photoaging. Therefore, the ability of an LXR ligand to modulate multiple pathways underlying the etiology of skin aging suggests that LXR is a novel target for developing potential therapeutics for photoaging and chronological skin aging indications.

MNAR plays an important role in ERa activation of Src/MAPK and PI3K/Akt signaling pathways.

Estrogens play a critical role in the regulation of cellular proliferation, differentiation, and apoptosis. Evidence indicates that this regulation is mediated by a complex interface of direct control of gene expression (so-called "genomic action") and by regulation of cell-signaling/phosphorylation cascades (referred to as the "non-genomic", or "extranuclear" action). However, the mechanisms of the non-genomic action of estrogens are not well defined. We have recently described the identification of a novel scaffold protein termed MNAR (modulator of non-genomic action of estrogen receptor), that couples conventional steroid receptors with extranuclear signal transduction pathways, thus potentially providing additional and tissue- or cell-specific level of steroid hormone regulation of cell functions. We have demonstrated that the MNAR is required for ER alpha (ERa) interaction with p60(src) (Src), which leads to activation of Src/MAPK pathway. Our new data also suggest that activation of cSrc in response to E2 leads to MNAR phosphorylation, interaction with p85, and activation of the PI3 and Akt kinases. These data therefore suggest that MNAR acts as an important scaffold that integrates ERa action in regulation of important signaling pathways. ERa non-genomic action has been suggested to play a key role in estrogen-induced cardio-, neuro-, and osteo-protection. Therefore, evaluation of the molecular crosstalk between MNAR and ERa may lead to development of functionally selective ER modulators that can separate between beneficial, prodifferentiative effects in bone, the cardiovascular system and the CNS and the "detrimental", proliferative effects in reproductive tissues and organs.