Phytosterol and phytostanol-mediated epigenetic changes in cancer and other non-communicable diseases: a systematic review.

Phytosterols/phytostanols are bioactive compounds found in vegetable oils, nuts and seeds and added to a range of commercial food products. Consumption of phytosterols/phytostanols reduces levels of circulating LDL-cholesterol, a causative biomarker of CVD, and is linked to a reduced risk of some cancers. Individuals who consume phytosterols/phytostanols in their diet may do so for many years as part of a non-pharmacological route to lower cholesterol or as part of a healthy diet. However, the impact of long term or high intakes of dietary phytosterols/phytostanols has not been on whole-body epigenetic changes before. The aim of this systematic review was to identify all publications that have evaluated changes to epigenetic mechanisms (post-translation modification of histones, DNA methylation and miRNA expression) in response to phytosterols/phytostanols. A systematic search was performed that returned 226 records, of which eleven were eligible for full-text analysis. Multiple phytosterols were found to inhibit expression of histone deacetylase (HDAC) enzymes and were also predicted to directly bind and impair HDAC activity. Phytosterols were found to inhibit the expression and activity of DNA methyl transferase enzyme 1 and reverse cancer-associated gene silencing. Finally, phytosterols have been shown to regulate over 200 miRNA, although only five of these were reported in multiple publications. Five tissue types (breast, prostate, macrophage, aortic epithelia and lung) were represented across the studies, and although phytosterols/phytostanols alter the molecular mechanisms of epigenetic inheritance in these mammalian cells, studies exploring meiotic or transgenerational inheritance were not found.

Ergothioneine: an underrecognised dietary micronutrient required for healthy ageing?

Ergothioneine is a naturally occurring amino acid and thiol antioxidant found in high amounts in mushrooms and fermented foods. Humans and animals acquire ergothioneine from the diet through the pH-dependent activity of a membrane transporter, the large solute carrier 22A member 4 (SLC22A4), expressed on the apical membrane of the small intestine. The SLC22A4 transporter also functions in the renal reabsorption of ergothioneine in the kidney, with avid absorption and retention of ergothioneine from the diet observed in both animals and humans. Ergothioneine is capable of scavenging a diverse range of reactive oxygen and nitrogen species, has metal chelation properties, and is predicted to directly regulate nuclear factor erythroid 2-related factor 2 (Nrf2) activity. Although not lethal, the genetic knockout of the SLC22A4 gene in multiple organisms increases susceptibility to oxidative stress, damage and inflammation; in agreement with a large body of preclinical data suggesting the physiological function of ergothioneine is as a cellular antioxidant and cytoprotectant agent. In humans, blood levels of ergothioneine decline after the age of 60 years, and lower levels of ergothioneine are associated with more rapid cognitive decline. Conversely, high plasma ergothioneine levels have been associated with significantly reduced cardiovascular mortality and overall mortality risks. In this horizon's manuscript, we review evidence suggesting critical roles for dietary ergothioneine in healthy ageing and the prevention of cardiometabolic disease. We comment on some of the outstanding research questions in the field and consider the question of whether or not ergothioneine should be considered a conditionally essential micronutrient.

NAFLD and vitamin D: Evidence for intersection of microRNA-regulated pathways.

Non-alcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease, worldwide. The molecular pathogenesis of NAFLD is complex, involving numerous signalling molecules, including microRNAs (miRNAs). Dysregulation of miRNA expression is associated with hepatic inflammation, fibrosis and hepatocellular carcinoma. Although miRNAs are also critical to the cellular response to vitamin D, mediating regulation of the vitamin D receptor and vitamin D's anti-cancer effects, the role of vitamin-D-regulated miRNAs in NAFLD pathogenesis has been relatively unexplored. Therefore, this review aims to critically assess the evidence for a potential subset of miRNAs that are both dysregulated in NAFLD and modulated by vitamin D. Comprehensive review of eighty-nine human studies identified twenty-five miRNAs found dysregulated in more than one NAFLD study. In contrast, only seventeen studies, including a protocol for a trial in NAFLD, had examined miRNAs in relation to vitamin D status, response to supplementation, or vitamin D in the context of the liver. This paper summarises these data and reviews the biological roles of six miRNAs (miR-21, miR-30, miR-34, miR-122, miR-146, miR-200) found dysregulated in multiple independent NAFLD studies. While modulation of miRNAs by vitamin D has been understudied, integration of the data suggests seven vitamin-D-modulated miRNAs (miR-27, miR-125, miR-155, miR-192, miR-223, miR-375, miR-378) potentially relevant to NAFLD pathogenesis. Our summary tables provide a significant resource to underpin future hypothesis-driven research, and we conclude that the measurement of serum and hepatic miRNAs in response to vitamin D supplementation in larger trials is warranted.

Phytosterols and phytostanols and the hallmarks of cancer in model organisms: A systematic review and meta-analysis.

Phytosterols and phytostanols are natural products present in vegetable oils, nuts, and seeds, or added to consumer food products whose intake is inversely associated with incidence and prognosis of several cancers. Randomized cancer prevention trials in humans are unfeasible due to time and cost yet the cellular processes and signaling cascades that underpin anti-cancer effects of these phytochemicals have been explored extensively in vitro and in preclinical in vivo models. Here we have performed an original systematic review, meta-analysis, and qualitative interpretation of literature published up to June 2020. MEDLINE, Scopus, and hand-searching identified 408 unique records that were screened leading to 32 original articles that had investigated the effects of phytosterols or phytostanols on cancer biology in preclinical models. Data was extracted from 22 publications for meta-analysis. Phytosterols were most commonly studied and found to reduce primary and metastatic tumor burden in all cancer sites evaluated. Expression of pAKT, and markers of metastasis (alkaline phosphatase, matrix metalloproteases, epithelial to mesenchymal transcription factors, lung and brain colonization), angiogenesis (vascular endothelial growth factor, CD31), and proliferation (Ki67, proliferating cell nuclear antigen) were consistently reduced by phytosterol treatment in breast and colorectal cancer. Very high dose treatment (equivalent to 0.2-1 g/kg body weight not easily achievable through diet or supplementation in humans) was associated with adverse events including poor gut health and intestinal adenoma development. Phytosterols and phytostanols are already clinically recommended for cardiovascular disease risk reduction, and represent promising anti-cancer agents that could be delivered in clinic and to the general population at low cost, with a well understood safety profile, and now with a robust understanding of mechanism-of-action.

Nuclear Receptors and Lipid Sensing.

Fluctuations in concentration of diverse lipid classes occur in response to diet and metabolism. These changes are managed and mediated by a cell network of enzymes, pumps, and carriers under the control of the lipid responsive nuclear receptors. The understanding of how dysregulation of lipid metabolism are causes and indicators of disease beyond the cardiovascular system has developed in the last decade. A particular emphasis on the role of lipids and lipid-sensing nuclear receptors has emerged in the fields of cancer and the immune system's interaction with cancer. The range of known lipid-based ligands has also expanded. Lipids are not just signalling molecules, but also play structural roles in cells and tissues, for example as major constituents of the lipid bilayer - positioning them as integrators and mediators of signaling. This chapter will discuss the major groups of lipid-sensing nuclear receptors focusing on the liver x receptors, farnesoid x receptor, and the peroxisome proliferator-activated receptors. Initially the reader is presented with information on how these receptors behave and function at the molecular biology level, the range of selective modulation of function by endogenous ligands, and examples of how activity is fine-tuned by mechanisms such as miRNA regulation and post-translational modification of the proteins. We then explore the advances in understanding that have positioned these receptors as therapeutic targets in cancer and immuno-oncology. Finally, the chapter explains the gaps in understanding and experimental challenges that should be prioritized in the coming decade.

Inhibition of interferon-signalling halts cancer-associated fibroblast-dependent protection of breast cancer cells from chemotherapy.

BACKGROUND: Triple negative breast cancers (TNBC) have poor prognoses despite aggressive treatment with cytotoxic chemotherapy. Cancer-associated fibroblasts (CAFs) are prominent in tumour stroma. Our hypothesis was that CAFs modulate chemotherapy sensitivity. METHODS: TNBC cells and breast fibroblasts were cultured; survival after chemotherapeutics was assessed using luciferase or clonogenic assays. Signalling was investigated using transcriptomics, reporters, recombinant proteins and blocking antibodies. Clinical relevance was investigated using immunohistochemistry. RESULTS: Breast CAFs dose-dependently protected TNBC cell lines MDA-MB-231 and MDA-MB-157, but not MDA-MB-468s, from chemotherapy. CAF-induced protection was associated with interferon (IFN) activation. CAFs were induced to express IFNß1 by chemotherapy and TNBC co-culture, leading to paracrine activation in cancer cells. Recombinant IFNs were sufficient to protect MDA-MB-231 and MDA-MB-157 but not MDA-MB-468 cells. In TNBC patients, IFNß1 expression in CAFs correlated with cancer cell expression of MX1, a marker of activated IFN signalling. High expression of IFNß1 (CAFs) or MX1 (tumour cells) correlated with reduced survival after chemotherapy, especially in claudin-low tumours (which MDA-MB-231 and MDA-MB-157 cells represent). Antibodies that block IFN receptors reduced CAF-dependent chemoprotection. CONCLUSIONS: CAF-induced activation of IFN signalling in claudin-low TNBCs results in chemoresistance. Inhibition of this pathway represents a novel method to improve breast cancer outcomes.

Transcriptome profiles of stem-like cells from primary breast cancers allow identification of ITGA7 as a predictive marker of chemotherapy response.

BACKGROUND: Breast cancer stem cells (BCSCs) are drivers of therapy-resistance, therefore are responsible for poor survival. Molecular signatures of BCSCs from primary cancers remain undefined. Here, we identify the consistent transcriptome of primary BCSCs shared across breast cancer subtypes, and we examine the clinical relevance of ITGA7, one of the genes differentially expressed in BCSCs. METHODS: Primary BCSCs were assessed using immunohistochemistry and fluorescently labelled using Aldefluor (n = 17). Transcriptomes of fluorescently sorted BCSCs and matched non-stem cancer cells were determined using RNA-seq (n = 6). ITGA7 expression was examined in breast cancers using immunohistochemistry (n = 305), and its functional role was tested using siRNA in breast cancer cells. RESULTS: Proportions of BCSCs varied from 0 to 9.4%. 38 genes were significantly differentially expressed in BCSCs; genes were enriched for functions in vessel morphogenesis, motility, and metabolism. ITGA7 was found to be significantly downregulated in BCSCs, and low expression significantly correlated with reduced survival in patients treated with chemotherapy, and with chemoresistance in breast cancer cells in vitro. CONCLUSIONS: This study is the first to define the molecular profile of BCSCs from a range of primary breast cancers. ITGA7 acts as a predictive marker for chemotherapy response, in accordance with its downregulation in BCSCs.

Identification of candidate mediators of chemoresponse in breast cancer through therapy-driven selection of somatic variants.

PURPOSE: More than a third of primary breast cancer patients are treated with cytotoxic chemotherapy, typically without guidance from predictive markers. Increased use of neoadjuvant chemotherapy provides opportunities for identification of molecules associated with treatment response, by comparing matched tumour samples before and after therapy. Our hypothesis was that somatic variants of increased prevalence after therapy promote resistance, while variants with reduced prevalence cause sensitivity. METHODS: We performed systematic analyses of matched pairs of cancer exomes from primary oestrogen receptor-positive/HER2-negative breast cancers (n = 6) treated with neoadjuvant epirubicin/cyclophosphamide. We identified candidate genes as mediators of chemotherapy response by consistent subclonal changes in somatic variant prevalence through therapy, predicted variant impact on gene function, and enrichment of specific functional pathways. Influence of candidate genes on breast cancer outcome was tested using publicly available breast cancer expression data (n = 1903). RESULTS: We identified 14 genes as the strongest candidate mediators of chemoresponse: TCHH, MUC17, ARAP2, FLG2, ABL1, CENPF, COL6A3, DMBT1, ITGA7, PLXNA1, S100PBP, SYNE1, ZFHX4, and CACNA1C. Genes contained somatic variants showing prevalence changes in up to 4 patients, with up to 3 being predicted as damaging. Genes coding for extra-cellular matrix components or related signalling pathways were significantly over-represented among variants showing prevalence changes. Expression of 5 genes (TCHH, ABL1, CENPF, S100PBP, and ZFHX4) was significantly associated with patient survival. CONCLUSIONS: Genomic analysis of paired pre- and post-therapy samples resulting from neoadjuvant therapy provides a powerful method for identification of mediators of response. Genes we identified should be assessed as predictive markers or targets in chemo-sensitization.

Vitamin D and nonalcoholic fatty liver disease.

PURPOSE OF REVIEW: Vitamin D deficiency may impact disease progression of nonalcoholic fatty liver disease (NAFLD). The aim of this work was to review recent studies examining either vitamin D status or the effects of supplementation in patients with NAFLD, along with investigating the roles of genetic polymorphisms and the gut microbiome. RECENT FINDINGS: Six heterogeneous observational studies of vitamin D status, and four randomized controlled intervention trials of vitamin D supplementation in NAFLD were conflicting. All studies were hampered by the challenges of diagnosing NAFLD, were underpowered, and lacked data on clinically important outcomes. The results of three cross-sectional studies, including a Mendelian randomization study, provide limited evidence for a role for genetic modifiers of vitamin D status in NAFLD. Genetic and experimental evidence suggests that vitamin D and the vitamin D receptor (VDR) may influence the gut microbiome in health and disease. SUMMARY: The evidence relating either lower vitamin D status to the prevalence and severity of NAFLD, or examining vitamin D supplementation in patients with NAFLD is inconclusive. Larger, higher quality trials with relevant endpoints are needed. Further mechanistic studies on the roles of vitamin D and VDR in influencing the gut-liver axis in NAFLD are warranted.

Phytosterols Inhibit Side-Chain Oxysterol Mediated Activation of LXR in Breast Cancer Cells.

Low fruit and vegetable consumption and high saturated fat consumption causes elevated circulating cholesterol and are breast cancer risk factors. During cholesterol metabolism, oxysterols form that bind and activate the liver X receptors (LXRs). Oxysterols halt breast cancer cell proliferation but enhance metastatic colonization, indicating tumour suppressing and promoting roles. Phytosterols and phytostanols in plants, like cholesterol in mammals, are essential components of the plasma membrane and biochemical precursors, and in human cells can alter LXR transcriptional activity. Here, a panel of breast cancer cell lines were treated with four dietary plant sterols and a stanol, alone or in combination with oxysterols. LXR activation and repression were measured by gene expression and LXR-luciferase reporter assays. Oxysterols activated LXR in all cell lines, but surprisingly phytosterols failed to modulate LXR activity. However, phytosterols significantly inhibited the ability of oxysterols to drive LXR transcription. These data support a role for phytosterols in modulating cancer cell behaviour via LXR, and therefore suggest merit in accurate dietary recordings of these molecules in cancer patients during treatment and perhaps supplementation to benefit recovery.

Nuclear receptors and the Warburg effect in cancer.

In 1927 Otto Warburg established that tumours derive energy primarily from the conversion of glucose to lactic acid and only partially through cellular respiration involving oxygen. In the 1950s he proposed that all causes of cancer reflected different mechanisms of disabling cellular respiration in favour of fermentation (now termed aerobic glycolysis). The role of aberrant glucose metabolism in cancer is now firmly established. The shift away from oxidative phosphorylation towards the metabolically expensive aerobic glycolysis is somewhat counter-intuitive given its wasteful nature. Multiple control processes are in place to maintain cellular efficiency and it is likely that these mechanisms are disrupted to facilitate the shift to the reliance on aerobic glycolysis. One such process of cell control is mediated by the nuclear receptor superfamily. This large family of transcription factors plays a significant role in sensing environmental cues and controlling decisions on proliferation, differentiation and cell death for example, to regulate glucose uptake and metabolism and to modulate the actions of oncogenes and tumour suppressors. In this review we highlight mechanisms by which nuclear receptors actions are altered during tumorigenic transformation and can serve to enhance the shift to aerobic glycolysis. At the simplest level, a basic alteration in NR behaviour can serve to enhance glycolytic flux thus providing a basis for enhanced survival within the tumour micro-environment. Ameliorating the enhanced NR activity in this context may help to sensitize cancer cells to Warburg targeted therapies and may provide future drug targets.

Chemotherapy induces Notch1-dependent MRP1 up-regulation, inhibition of which sensitizes breast cancer cells to chemotherapy.

BACKGROUND: Multi-drug Resistance associated Protein-1 (MRP1) can export chemotherapeutics from cancer cells and is implicated in chemoresistance, particularly as is it known to be up-regulated by chemotherapeutics. Our aims in this study were to determine whether activation of Notch signalling is responsible for chemotherapy-induced MRP1 expression Notch in breast cancers, and whether this pathway can be manipulated with an inhibitor of Notch activity. METHODS: MRP1 and Notch1 were investigated in 29 patients treated with neoadjuvant chemotherapy (NAC) for breast cancer, using immunohistochemistry on matched biopsy (pre-NAC) and surgical samples (post-NAC). Breast epithelial cell cultures (T47D, HB2) were treated with doxorubicin in the presence and absence of functional Notch1, and qPCR, siRNA, Western blots, ELISAs and flow-cytometry were used to establish interactions. RESULTS: In clinical samples, Notch1 was activated by neoadjuvant chemotherapy (Wilcoxon signed-rank p < 0.0001) and this correlated with induction of MRP1 expression (rho = 0.6 p = 0.0008). In breast cell lines, doxorubicin induced MRP1 expression and function (non-linear regression p < 0.004). In the breast cancer line T47D, doxorubicin activated Notch1 and, critically, inhibition of Notch1 activation with the γ-secretase inhibitor DAPT abolished the doxorubicin-induced increase in MRP1 expression and function (t-test p < 0.05), resulting in enhanced cellular retention of doxorubicin and increased doxorubicin-induced apoptosis (t-test p = 0.0002). In HB2 cells, an immortal but non-cancer derived breast cell line, Notch1-independent MRP1 induction was noted and DAPT did not enhance doxorubicin-induced apoptosis. CONCLUSIONS: Notch inhibitors may have potential in sensitizing breast cancer cells to chemotherapeutics and therefore in tackling chemoresistance.

Cooperative behavior of the nuclear receptor superfamily and its deregulation in prostate cancer.

The current study aimed to assess the topology of the nuclear receptor (NR) superfamily in normal prostate epithelial cells and its distortion in prostate cancer. Both in vitro and in silico approaches were utilized to profile NRs expressed in non-malignant RWPE-1 cells, which were subsequently investigated by treating cells with 132 binary NR ligand combinations. Nine significant cooperative interactions emerged including both superadditive [22(R)-hydroxycholesterol and eicosatetraenoic acid] and subadditive [1α,25(OH)2D3 and chenodeoxycholic acid] cellular responses, which could be explained in part by cooperative control of cell-cycle progression and candidate gene expression. In addition, publicly available data were employed to assess NR expression in human prostate tissue. Common and significant loss of NR superfamily expression was established in publicly available data from prostate tumors, in part predicting parallel distortion of targeting microRNA. These findings suggest that the NR superfamily in the prostate cooperatively integrates signals from dietary, hormonal and metabolic cues, and is significantly distorted in prostate cancer.

MiR-26b is down-regulated in carcinoma-associated fibroblasts from ER-positive breast cancers leading to enhanced cell migration and invasion.

Carcinoma-associated fibroblasts (CAFs) influence the behaviour of cancer cells but the roles of microRNAs in this interaction are unknown. We report microRNAs that are differentially expressed between breast normal fibroblasts and CAFs of oestrogen receptor-positive cancers, and explore the influences of one of these, miR-26b, on breast cancer biology. We identified differentially expressed microRNAs by expression profiling of clinical samples and a tissue culture model: miR-26b was the most highly deregulated microRNA. Using qPCR, miR-26b was confirmed as down-regulated in fibroblasts from 15 of 18 further breast cancers. Next, we examined whether manipulation of miR-26b expression changed breast fibroblast behaviour. Reduced miR-26b expression caused fibroblast migration and invasion to increase by up to three-fold in scratch-closure and trans-well assays. Furthermore, in co-culture with MCF7 breast cancer epithelial cells, fibroblasts with reduced miR-26b expression enhanced both MCF7 migration in trans-well assays and MCF7 invasion from three-dimensional spheroids by up to five-fold. Mass spectrometry was used to identify expression changes associated with the reduction of miR-26b expression in fibroblasts. Pathway analyses of differentially expressed proteins revealed that glycolysis/TCA cycle and cytoskeletal regulation by Rho GTPases are downstream of miR-26b. In addition, three novel miR-26b targets were identified (TNKS1BP1, CPSF7, COL12A1) and the expression of each in cancer stroma was shown to be significantly associated with breast cancer recurrence. MiR-26b in breast CAFs is a potent regulator of cancer behaviour in oestrogen receptor-positive cancers, and we have identified key genes and molecular pathways that act downstream of miR-26b in CAFs.

Heterochromatin protein 1 gamma and IκB kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages.

IκB kinase α (IKKα) is part of the cytoplasmic IKK complex regulating nuclear factor-κB (NF-κB) release and translocation into the nucleus in response to pro-inflammatory signals. IKKα can also be recruited directly to the promoter of NF-κB-dependent genes by NF-κB where it phosphorylates histone H3 at serine 10, triggering recruitment of the bromodomain-containing protein 4 and the positive transcription elongation factor b. Herein, we report that IKKα travels with the elongating form of ribonucleic acid polymerase II together with heterochromatin protein 1 gamma (HP1γ) at NF-κB-dependent genes in activated macrophages. IKKα binds to and phosphorylates HP1γ, which in turn controls IKKα binding to chromatin and phosphorylation of the histone variant H3.3 at serine 31 within transcribing regions. Downstream of transcription end sites, IKKα accumulates with its inhibitor the CUE-domain containing protein 2, suggesting a link between IKKα inactivation and transcription termination.

Recruitment of NCOR1 to VDR target genes is enhanced in prostate cancer cells and associates with altered DNA methylation patterns.

The current study investigated transcriptional distortion in prostate cancer cells using the vitamin D receptor (VDR) as a tool to examine how epigenetic events driven by corepressor binding and CpG methylation lead to aberrant gene expression. These relationships were investigated in the non-malignant RWPE-1 cells that were 1α,25(OH)(2)D(3) responsive (RWPE-1) and malignant cell lines that were 1α,25(OH)(2)D(3) partially responsive (RWPE-2) and resistant (PC-3). These studies revealed that selective attenuation and repression of VDR transcriptional responses in the cancer cell lines reflected their loss of antiproliferative sensitivity. This was evident in VDR target genes including VDR, CDKN1A (encodes p21( (waf1/cip1) )) and GADD45A; NCOR1 knockdown alleviated this malignant transrepression. ChIP assays in RWPE-1 and PC-3 cells revealed that transrepression of CDKN1A was associated with increased NCOR1 enrichment in response to 1α,25(OH)(2)D(3) treatment. These findings supported the concept that retained and increased NCOR1 binding, associated with loss of H3K9ac and increased H3K9me2, may act as a beacon for the initiation and recruitment of DNA methylation. Overexpressed histone methyltransferases (KMTs) were detectable in a wide panel of prostate cancer cell lines compared with RWPE-1 and suggested that generation of H3K9me2 states would be favored. Cotreatment of cells with the KMT inhibitor, chaetocin, increased 1α,25(OH)(2)D(3)-mediated induction of CDKN1A expression supporting a role for this event to disrupt CDKN1A regulation. Parallel surveys in PC-3 cells of CpG methylation around the VDR binding regions on CDKN1A revealed altered basal and VDR-regulated DNA methylation patterns that overlapped with VDR-induced recruitment of NCOR1 and gene transrepression. Taken together, these findings suggest that sustained corepressor interactions with nuclear-resident transcription factors may inappropriately transform transient-repressive histone states into more stable and repressive DNA methylation events.

Epigenetic control of a VDR-governed feed-forward loop that regulates p21(waf1/cip1) expression and function in non-malignant prostate cells.

In non-malignant RWPE-1 prostate epithelial cells signaling by the nuclear receptor Vitamin D Receptor (VDR, NR1I1) induces cell cycle arrest through targets including CDKN1A (encodes p21((waf1/cip1))). VDR dynamically induced individual histone modification patterns at three VDR binding sites (R1, 2, 3) on the CDKN1A promoter. The magnitude of these modifications was specific to each phase of the cell cycle. For example, H3K9ac enrichment occurred rapidly only at R2, whereas parallel accumulation of H3K27me3 occurred at R1; these events were significantly enriched in G(1) and S phase cells, respectively. The epigenetic events appeared to allow VDR actions to combine with p53 to enhance p21((waf1/cip1)) activation further. In parallel, VDR binding to the MCM7 gene induced H3K9ac enrichment associated with rapid mRNA up-regulation to generate miR-106b and consequently regulate p21((waf1/cip1)) expression. We conclude that VDR binding site- and promoter-specific patterns of histone modifications combine with miRNA co-regulation to form a VDR-regulated feed-forward loop to control p21((waf1/cip1)) expression and cell cycle arrest. Dissection of this feed-forward loop in a non-malignant prostate cell system illuminates mechanisms of sensitivity and therefore possible resistance in prostate and other VDR responsive cancers.

The Second Nutrition and Cancer Networking Meeting Nutrition and Breast Cancer: Translating Evidence into Practice.

The 2nd Nutrition and Cancer Networking Meeting 'Nutrition and Breast Cancer: Translating Evidence into Practice' was held at Newcastle University in May 2022, with support from the Nutrition Society and British Association for Cancer Research. The first meeting in this series was held in Sheffield in 2019. The aim of this joint meeting was to bring together researchers with an interest in nutrition and breast cancer, with the programme spanning topics from risk and prevention to nutrition during treatment and beyond. Several key themes emerged, including the importance of engaging patients in the development of interventions and trials, making trials more accessible to diverse communities; training of clinical staff in nutrition and latest evidence; wider range of compounds should be considered in food composition tables; and alternative trial designs can be considered for prevention research to reduce financial burden and increase power.

Mass spectrometry reveals that oxysterols are secreted from non-alcoholic fatty liver disease induced organoids.

Oxysterols are potential biomarkers for liver metabolism that are altered under disease conditions such as non-alcoholic fatty liver disease (NAFLD). We here apply sterolomics to organoids used for disease modeling of NAFLD. Using liquid chromatography-mass spectrometry with on-line sample clean-up and enrichment, we establish that liver organoids produce and secrete oxysterols. We find elevated levels of 26-hydroxycholesterol, an LXR agonist and the first oxysterol in the acidic bile acid synthesis, in medium from steatotic liver organoids compared to untreated organoids. Other upregulated sterols in medium from steatotic liver organoids are dihydroxycholesterols, such as 7α,26-dihydroxycholesterol, and 7α,25-dihydroxycholesterol. Through 26-hydroxycholesterol exposure to human stem cell-derived hepatic stellate cells, we observe a trend of expressional downregulation of the pro-inflammatory cytokine CCL2, suggesting a protective role of 26-hydroxycholesterol during early-phased NAFLD disease development. Our findings support the possibility of oxysterols serving as NAFLD indicators, demonstrating the usefulness of combining organoids and mass spectrometry for disease modeling and biomarker studies.

Non-Alcoholic Fatty Liver Disease and Vitamin D in the UK Biobank: A Two-Sample Bidirectional Mendelian Randomisation Study.

Evidence for a role for vitamin D in non-alcoholic fatty liver disease (NAFLD) pathogenesis is conflicting. As Mendelian randomisation (MR) avoids many limitations of conventional observational studies, this two-sample bidirectional MR analysis was conducted to determine the following: (i) whether genetically predicted 25-hydroxyvitamin D [25(OH)D] levels are a risk factor for NAFLD, and (ii) whether genetic risk for NAFLD influences 25(OH)D levels. Single-nucleotide polymorphisms (SNPs) associated with serum 25(OH)D levels were obtained from the European ancestry-derived SUNLIGHT consortium. SNPs associated with NAFLD or NASH (p-value < 1 × 10-5) were extracted from previous studies and supplemented by genome-wide association studies (GWASs) performed in the UK Biobank. These GWASs were done both without (primary analysis) and with (sensitivity analysis) the population-level exclusion of other liver diseases (e.g., alcoholic liver diseases, toxic liver diseases, viral hepatitis, etc.). Subsequently, MR analyses were performed to obtain effect estimates using inverse variance weighted (IVW) random effect models. Cochran's Q statistic, MR-Egger regression intercept, MR pleiotropy residual sum and outlier (MR-PRESSO) analyses were used to assess pleiotropy. No causal association of genetically predicted serum 25(OH)D (per standard deviation increase) with risk of NAFLD was identified in either the primary analysis: n = 2757 cases, n = 460,161 controls, odds ratio (95% confidence interval): 0.95 (0.76, -1.18), p = 0.614; or the sensitivity analysis. Reciprocally, no causal association was identified between the genetic risk of NAFLD and serum 25(OH)D levels, OR = 1.00 (0.99, 1.02, p = 0.665). In conclusion, this MR analysis found no evidence of an association between serum 25(OH)D levels and NAFLD in a large European cohort.