Epigenetic promoter alterations in GI tumour immune-editing and resistance to immune checkpoint inhibition.

OBJECTIVES: Epigenomic alterations in cancer interact with the immune microenvironment to dictate tumour evolution and therapeutic response. We aimed to study the regulation of the tumour immune microenvironment through epigenetic alternate promoter use in gastric cancer and to expand our findings to other gastrointestinal tumours. DESIGN: Alternate promoter burden (APB) was quantified using a novel bioinformatic algorithm (proActiv) to infer promoter activity from short-read RNA sequencing and samples categorised into APBhigh, APBint and APBlow. Single-cell RNA sequencing was performed to analyse the intratumour immune microenvironment. A humanised mouse cancer in vivo model was used to explore dynamic temporal interactions between tumour kinetics, alternate promoter usage and the human immune system. Multiple cohorts of gastrointestinal tumours treated with immunotherapy were assessed for correlation between APB and treatment outcomes. RESULTS: APBhigh gastric cancer tumours expressed decreased levels of T-cell cytolytic activity and exhibited signatures of immune depletion. Single-cell RNAsequencing analysis confirmed distinct immunological populations and lower T-cell proportions in APBhigh tumours. Functional in vivo studies using 'humanised mice' harbouring an active human immune system revealed distinct temporal relationships between APB and tumour growth, with APBhigh tumours having almost no human T-cell infiltration. Analysis of immunotherapy-treated patients with GI cancer confirmed resistance of APBhigh tumours to immune checkpoint inhibition. APBhigh gastric cancer exhibited significantly poorer progression-free survival compared with APBlow (median 55 days vs 121 days, HR 0.40, 95% CI 0.18 to 0.93, p=0.032). CONCLUSION: These findings demonstrate an association between alternate promoter use and the tumour microenvironment, leading to immune evasion and immunotherapy resistance.

Inhibition of LINE-1 retrotransposon-encoded reverse transcriptase modulates the expression of cell differentiation genes in breast cancer cells.

Long Interspersed Elements (L1 elements) are biologically active retrotransposons that are capable of autonomous replication using their own reverse transcriptase (RT) enzyme. Expression of the normally repressed RT has been implicated in cancer cell growth. However, at present, little is known about the expression of L1-encoded RT activity or the molecular changes that are associated with RT activity in the development of breast cancer. Here, we report that RT activity is widespread in breast cancer cells. The expression of RT protein decreased markedly in breast cancer cells after treatment with the antiretroviral drug, efavirenz. While the majority of cells showed a significant reduction in proliferation, inhibition of RT was also accompanied by cell-specific differences in morphology. MCF7 cells displayed elongated microtubule extensions that adhered tightly to their substrate, while a large fraction of the T47D cells that we studied formed long filopodia projections. These morphological changes were reversible upon cessation of RT inhibition, confirming their dependence on RT activity. We also carried out gene expression profiling with microarrays and determined the genes that were differentially expressed during the process of cellular differentiation. Genes involved in proliferation, cell migration, and invasive activity were repressed in RT-inhibited cells. Concomitantly, genes involved in cell projection, formation of vacuolar membranes, and cell-to-cell junctions were significantly upregulated in RT-inhibited cells. qRT-PCR examination of the mRNA expression of these genes in additional cell lines yielded close correlation between their differential expression and the degree of cellular differentiation. Our study demonstrates that the inhibition of L1-encoded RT can reduce the rate of proliferation and promote differentiation of breast cancer cells. Together, these results provide a direct functional link between the expression of L1 retrotransposons and the development of breast cancer.

Candidate gene association studies: a comprehensive guide to useful in silico tools.

The candidate gene approach has been a pioneer in the field of genetic epidemiology, identifying risk alleles and their association with clinical traits. With the advent of rapidly changing technology, there has been an explosion of in silico tools available to researchers, giving them fast, efficient resources and reliable strategies important to find casual gene variants for candidate or genome wide association studies (GWAS). In this review, following a description of candidate gene prioritisation, we summarise the approaches to single nucleotide polymorphism (SNP) prioritisation and discuss the tools available to assess functional relevance of the risk variant with consideration to its genomic location. The strategy and the tools discussed are applicable to any study investigating genetic risk factors associated with a particular disease. Some of the tools are also applicable for the functional validation of variants relevant to the era of GWAS and next generation sequencing (NGS).

Genetic polymorphisms in the human tissue kallikrein (KLK) locus and their implication in various malignant and non-malignant diseases.

The Kallikrein ( KLK ) gene locus encodes a family of serine proteases and is the largest contiguous cluster of protease-encoding genes attributed an evolutionary age of 330 million years. The KLK locus has been implicated asa high susceptibility risk loci in numerous cancer studies through the last decade. The KLK3 gene already has established clinical relevance as a biomarker in prostate cancer prognosis through its encoded protein, prostate-specific antigen. Data mined through genome-wide association studies (GWAS) and next-generation sequencing point to many important candidate single nucleotide polymorphisms(SNPs) in KLK3 and other KLK genes. SNPs in the KLK locus have been found to be associated with several diseases including cancer, hypertension, cardiovascular disease and atopic dermatitis. Moreover, introducing a model incorporating SNPs to improve the efficiency of prostate-specific antigen in detecting malignant states of prostate cancer has been recently suggested. Establishing the functional relevance of these newly-discovered SNPs, and their interactions with each other, through in silico investigations followed by experimental validation,can accelerate the discovery of diagnostic and prognostic biomarkers. In this review, we discuss the various genetic association studies on the KLK loci identified either through candidate gene association studies or at the GWAS and post-GWAS front to aid researchers in streamlining their search for the most significant, relevant and therapeutically promising candidate KLK gene and/or SNP for future investigations.

Recent progress in therapeutic strategies for microglia-mediated neuroinflammation in neuropathologies.

INTRODUCTION: Chronic activation of microglia is the hallmark of numerous neuropathologies such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. The activated microglia perpetuate inflammation by releasing an array of pro-inflammatory and neurotoxic factors, which eventually exacerbate neurotoxicity and neurodegeneration upon chronic activation of these cells. However, under acute conditions, activated microglia elicit pro-inflammatory as well as anti-inflammatory responses that are associated with neuroprotection. Given the role of microglia in neuroinflammation, recent studies have attempted to unravel the mechanisms that aid to establish microglial cell-based therapy. Areas covered: While total suppression of microglial activation may compromise its beneficial role in tissue repair in the aftermath of an insult, the benefits of modulating microglial activation and promoting microglia polarization to a neuroprotective phenotype have been highlighted recently. Expert opinion: So far, the therapeutic strategy focussed on neutralizing microglia-mediated neuroinflammation using drugs that block the release of pro-inflammatory mediators has limitations, such as unwarranted side effects. Recent advances reveal several alternative molecular targets and potential epi-drugs that are capable of modulating microglial function and promoting neuroprotection. This review discusses the recent progress made in understanding the mechanisms of microglia-mediated neuroinflammation in various neuropathologies, and the emerging anti-inflammatory therapeutic strategies in this field.

HDAC Inhibitor Sodium Butyrate-Mediated Epigenetic Regulation Enhances Neuroprotective Function of Microglia During Ischemic Stroke.

Cerebral ischemia leads to neuroinflammation and activation of microglia which further contribute to stroke pathology. Understanding regulation of microglial activation will aid in the development of therapeutic strategies that mitigate microglia-mediated neurotoxicity in neuropathologies, including ischemia. In this study, we investigated the epigenetic regulation of microglial activation by studying histone modification histone 3-lysine 9-acetylation (H3K9ac) and its regulation by histone deacetylase (HDAC) inhibitors. In vitro analysis of activated microglia showed that HDAC inhibitor, sodium butyrate (SB), alters H3K9ac enrichment and transcription at the promoters of pro-inflammatory (Tnf-α, Nos2, Stat1, Il6) and anti-inflammatory (Il10) genes while inducing the expression of genes downstream of the IL10/STAT3 anti-inflammatory pathway. In an experimental mouse (C57BL/6NTac) model of middle cerebral artery occlusion (MCAO), we observed that SB mediates neuroprotection by epigenetically regulating the microglial inflammatory response, via downregulating the expression of pro-inflammatory mediators, TNF-α and NOS2, and upregulating the expression of anti-inflammatory mediator IL10, in activated microglia. Interestingly, H3K9ac levels were found to be upregulated in activated microglia distributed in the cortex, striatum, and hippocampus of MCAO mice. A similar upregulation of H3K9ac was detected in lipopolysaccharide (LPS)-activated microglia in the Wistar rat brain, indicating that H3K9ac upregulation is consistently associated with microglial activation in vivo. Altogether, these results show evidence of HDAC inhibition being a promising molecular switch to epigenetically modify microglial behavior from pro-inflammatory to anti-inflammatory which could mitigate microglia-mediated neuroinflammation.

MicroRNAs: Key Players in Microglia and Astrocyte Mediated Inflammation in CNS Pathologies.

The significance of microglia and astrocytes in neural development, in maintaining synaptic connections and homeostasis in the healthy brain is well established. Microglia are dynamic immune cells of the brain that elicit an immune response during brain damage and also participate in tissue repair and regeneration, while astrocytes contribute to the local inflammatory response by producing proinflammatory cytokines and resolving neuronal damage through production of anti-inflammatory cytokines and neurotrophic factors. Recent efforts have focused on elucidating the epigenetic mechanisms which regulate glial cell behavior in normal and pathologic states. An important class of epigenetic regulators is microRNAs (miRNAs) which are small non-coding RNA molecules that regulate gene expression posttranscriptionally. Certain dysregulated miRNAs contribute to chronic microglial inflammation in the brain, thereby leading to progression of neurological diseases like Alzheimer's disease, traumatic injury, amyotrophic lateral sclerosis and stroke. Further, several miRNAs are differentially expressed in astrocytes after ischemia and spinal cord injury. Despite knowledge about miRNAs in neuroinflammation, little is known about effective delivery routes and pharmacokinetic data for miRNA based therapeutics. This review summarizes the current research on the role of miRNAs in promoting and inhibiting inflammatory response of microglia and astrocytes in a disease-specific manner. In addition, miRNA delivery as a therapeutic strategy to treat neuroinflammation is discussed.