Dedifferentiation-the acquisition of an early developmental state-is a hallmark of cancer. However, the underlying mechanisms that lead to cancer-associated dedifferentiation are not fully understood. Transposable elements (TEs) are becoming increasingly recognised as important regulators of development and disease. The recruitment of TE sequences has played an important role in placental evolution, and TE-derived genes play critical roles in placental development. Although important biological differences exist between tumours and the placenta, the placenta shares certain features with tumours, including the capacity to invade surrounding tissue and modulate the maternal immune response. In this regard, TEs have been implicated in cancer development, and are documented to contribute to oncogenesis through multiple different mechanisms. Moreover, cancers reacquire an epigenetic landscape, which is reflective of early development, and which corresponds to increased phenotypic plasticity, including facilitating the activation of early developmental genes. Many cancers can repurpose developmental genes, including TE-associated genes, which may contribute to pathways involved in invasion and metastasis. Determining whether TE activation is a consequence of broader epigenetic reprogramming or actively contributes to dedifferentiation will be important for understanding cancer biology and may facilitate improvements in cancer diagnosis and treatment.

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder caused by mutations in PKD1 or PKD2. The PAX2 and PAX8 genes encode transcription factors required for kidney development, but their role in ADPKD remains unclear. We hypothesized that PAX2 and PAX8 contribute to ADPKD cystogenesis through distinct and overlapping mechanisms. Using immunofluorescence, PAX2 and PAX8 expression was assessed in human ADPKD kidney tissues. Nuclear PAX2 expression, and cytoplasmic PAX8 expression were found to be up-regulated in cyst-lining epithelial cells. Furthermore, using siRNA-mediated knockdown of PAX2 and PAX8, significantly reduced 3D spheroid growth was observed in MDCK, WT9-7, and WT9-12 renal epithelial cell line models. RNA sequencing after individual and combined knockdown of PAX2 and PAX8 in WT9-7 and WT9-12 cells identified differentially expressed genes and enriched pathways, revealing that PAX2 regulates pathways related to embryonic development, endoplasmic reticulum stress, and cilia. In contrast, PAX8 regulates cell cycle and adhesion pathways, while dual knockdown of PAX2 and PAX8 impacted extracellular matrix organization pathways. Several genes, including AGO2 and WWTR1, which were previously found to exhibit expression changes in human ADPKD tissues were co-regulated by both factors, suggesting their upregulation in ADPKD may be due to PAX co-expression. Overall, we found PAX2 and PAX8 regulated both unique and overlapping gene networks known to promote ADPKD cystic epithelial growth. Their differential expression patterns and combined impact on extracellular matrix remodelling highlight their potential as therapeutic targets. These findings advance our understanding of ADPKD pathogenesis, and suggest potential avenues for targeted intervention.

Transposable elements (TEs) play important roles during development and disease, including through transcriptional activation of TE-associated genes during early human development. Moreover, based on the functional and epigenetic similarities between early development and cancer, TE-associated genes contribute not only to early human development, but frequently contribute to cancer progression. In this study, we hypothesised that recruitment of TE-associated genes during cancer onset occurs through epigenetic regulatory processes, especially involving DNA hypomethylation accompanied by transcriptional upregulation of early developmental pathways, such that, when reactivated inappropriately in later life, they may drive malignancy. It is unknown, however, to what extent DNA methylation changes are critically involved in the transcriptional activation of TE-associated genes. Accordingly, to investigate this we used the RepExpress tool to identify developmentally regulated TE-associated genes in placenta and human embryonic stem cells (hESCs), which we then investigated by targeted deep bisulfite sequencing (TDBS) to determine the methylation status of the identified TE-associated genes in placenta, somatic tissues, and melanoma cell lines. Outcomes suggest that DNA methylation may be one of the regulatory factors underscoring transcriptional activation of TE-associated genes, but that methylation is not necessarily the sole factor involved in regulating the transcriptional activation of TE-associated genes during malignant transformation.

Successful immune checkpoint inhibitor (ICI) therapy occurs in only a fraction of melanoma patients, and yet all patients are susceptible to potentially serious ICI-related side-effects. No current biomarkers robustly predict ICI treatment response in melanoma patients. In this study we sought to identify methylome and transcriptome markers which have the potential to predict immunotherapy response in melanoma patients ahead of treatment with anti-PD1 ICI monotherapy. Using Infinium MethylationEPIC microarrays, we analysed DNA methylation profiles of >850,000 CpG sites in pre-treatment melanoma tissues from patients administered anti-PD-1 monotherapy as first-line treatment. In addition, we analysed transcriptomes using RNA-seq. DNA methylation and gene expression data were then statistically compared to patient response to anti-PD1 therapy. We identified 2579 DNA hypomethylation and hypermethylation alterations correlating with melanoma response to anti-PD1 therapy. An integrative analysis of DNA methylomes and transcriptomes identified a subset of 35 loci, 13 of which were significantly differentially methylated in both initial discovery and external validation datasets. Functional enrichment analysis of hypomethylated sites (p-value <0.05) in non-responders was associated with "Formation of the cornified envelope", "Regulation of epithelial cell proliferation", and "Purine-containing compound metabolic process". We have identified novel integrated DNA methylation and gene expression markers, which correlate with anti-PD1 treatment response in melanoma patients. These findings suggest a relationship between tumour-associated genomic DNA methylation, gene expression patterns, and anti-PD1 ICI immunotherapy response in melanoma patients.

This Special Issue focuses on recent advances and innovations that have the potential to improve our understanding of the molecular pathology and therapeutic approaches associated with treating melanoma [...].

Cutaneous melanoma is a highly invasive, heterogeneous and treatment resistant cancer. It's ability to dynamically shift between transcriptional states or phenotypes results in an adaptive cell plasticity that may drive cancer cell invasion or the development of therapy resistance. The expression of peroxidasin (PXDN), an extracellular matrix peroxidase, has been proposed to be associated with the invasive metastatic melanoma phenotype. We have confirmed this association by analysing the transcriptomes of 70 metastatic melanoma cell lines with variable levels of PXDN expression. This analysis highlighted a strong association between high PXDN expression and the undifferentiated invasive melanoma phenotype. To assess the functional role of PXDN in melanoma invasion, we performed a knockout of PXDN in a highly invasive cell line (NZM40). PXDN knockout decreased the invasive potential by ∼50 % and decreased the expression of epithelial-mesenchymal transition and invasive marker genes as determined by RNAseq and substantiated by proteomics analysis. Bioinformatics analysis of differentially expressed genes following PXDN knockout highlighted decreases in genes linked to extracellular matrix formation, organization and degradation as well as signalling pathways such as the WNT pathway. This study provides compelling evidence that PXDN plays a functional role in melanoma invasion by promoting an invasive, mesenchymal-like transcriptional phenotype.

Renal cell carcinoma (RCC) is a significant oncological challenge due to its heterogeneous nature and limited treatment options. The PAX developmental gene family encodes nine highly conserved transcription factors that play crucial roles in embryonic development and organogenesis, which have been implicated in the occurrence and development of RCC. This review explores the molecular landscape of RCC, with a specific focus on the role of the PAX gene family in RCC tumorigenesis and disease progression. Of the various RCC subtypes, clear cell renal cell carcinoma (ccRCC) is the most prevalent, characterized by the loss of the von Hippel–Lindau (VHL) tumor suppressor gene. Here, we review the published literature on the expression patterns and functional implications of PAX genes, particularly PAX2 and PAX8, in the three most common RCC subtypes, including ccRCC, papillary RCC (PRCC), and chromophobe RCC (ChRCC). Further, we review the interactions and potential biological mechanisms involving PAX genes and VHL loss in driving the pathogenesis of RCC, including the key signaling pathways mediated by VHL in ccRCC and associated mechanisms implicating PAX. Lastly, concurrent with our update regarding PAX gene research in RCC, we review and comment on the targeting of PAX towards the development of novel RCC therapies.

Transposable elements (TEs) play a crucial role in placental development and dysfunction. Our study examined TE expression in pre-eclampsia (PE) using RNA-seq datasets. We identified differentially expressed TEs and explored the genomic location of the most significant TEs, investigating their possible regulatory roles. Notably, three TEs overlapped with putative enhancer regions, suggesting a potential regulatory impact on gene expression. These findings highlight the regulatory potential of TEs and their importance in placental development, supporting that TE dysregulation may contribute to PE pathogenesis.

Immune checkpoint inhibitors (ICIs) demonstrate durable responses, long-term survival benefits, and improved outcomes in cancer patients compared to chemotherapy. However, the majority of cancer patients do not respond to ICIs, and a high proportion of those patients who do respond to ICI therapy develop innate or acquired resistance to ICIs, limiting their clinical utility. The most studied predictive tissue biomarkers for ICI response are PD-L1 immunohistochemical expression, DNA mismatch repair deficiency, and tumour mutation burden, although these are weak predictors of ICI response. The identification of better predictive biomarkers remains an important goal to improve the identification of patients who would benefit from ICIs. Here, we review established and emerging biomarkers of ICI response, focusing on epigenomic and genomic alterations in cancer patients, which have the potential to help guide single-agent ICI immunotherapy or ICI immunotherapy in combination with other ICI immunotherapies or agents. We briefly review the current status of ICI response biomarkers, including investigational biomarkers, and we present insights into several emerging and promising epigenomic biomarker candidates, including current knowledge gaps in the context of ICI immunotherapy response in melanoma patients.

Cutaneous melanoma is rapidly on the rise globally, surpassing the growth rate of other cancers, with metastasis being the primary cause of death in melanoma patients. Consequently, understanding the mechanisms behind this metastatic process and exploring innovative treatments is of paramount importance. Recent research has shown promise in unravelling the role of epigenetic factors in melanoma progression to metastasis. While DNA hypermethylation at gene promoters typically suppresses gene expression, we have contributed to establishing the newly understood mechanism of paradoxical activation of genes via DNA methylation, where high methylation coincides with increased gene activity. This mechanism challenges the conventional paradigm that promoter methylation solely silences genes, suggesting that, for specific genes, it might actually activate them. Traditionally, altering DNA methylation in vitro has involved using global demethylating agents, which is insufficient for studying the mechanism and testing the direct consequence of gene methylation changes. To investigate promoter hypermethylation and its association with gene activation, we employed a novel approach utilising a CRISPR-SunTag All-in-one system. Here, we focused on editing the DNA methylation of a specific gene promoter segment (EBF3) in melanoma cells using the All-in-one system. Using bisulfite sequencing and qPCR with RNA-Seq, we successfully demonstrated highly effective methylation and demethylation of the EBF3 promoter, with subsequent gene expression changes, to establish and validate the paradoxical role of DNA methylation. Further, our study provides novel insights into the function of the EBF3 gene, which remains largely unknown. Overall, this study challenges the conventional view of methylation as solely a gene-silencing mechanism and demonstrates a potential function of EBF3 in IFN pathway signalling, potentially uncovering new insights into epigenetic drivers of malignancy and metastasis.

Abstract Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs in melanoma. Deploying a model of adaptive cancer drug tolerance H3K4me3 ChIP-seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo. H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with O-GlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in vivo. Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes. Citation Format: Dinoop Ravindran Menon, Heinz Hammerlindl, Gregory Gimenez, Elmar Zuegner, Christoph Magens, Michael Eccles, Mayumi Fujita, Helmut Schaider. O-GlcNAc transferase drives the proliferative state in adaptive drug tolerance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB293.

"DNA Methylation Profiling of Heterogeneous Sporadic LAM and Matched Lung Tissue." American Journal of Respiratory Cell and Molecular Biology, 70(1), pp. 81–84

Melanoma, a highly heterogeneous tumor, is comprised of a functionally diverse spectrum of cell phenotypes and subpopulations, including stromal cells in the tumor microenvironment (TME). Melanoma has been shown to dynamically shift between different transcriptional states or phenotypes. This is referred to as phenotype switching in melanoma, and it involves switching between quiescent and proliferative cell cycle states, and dramatic shifts in invasiveness, as well as changes in signaling pathways in the melanoma cells, and immune cell composition in the TME. Melanoma cell plasticity is associated with altered gene expression in immune cells and cancer-associated fibroblasts, as well as changes in extracellular matrix, which drive the metastatic cascade and therapeutic resistance. Therefore, resistance to therapy in melanoma is not only dependent on genetic evolution, but it has also been suggested to be driven by gene expression changes and adaptive phenotypic cell plasticity. This review discusses recent findings in melanoma phenotype switching, immunotherapy resistance, and the balancing of the homeostatic TME between the different melanoma cell subpopulations. We also discuss future perspectives of the biology of neural crest-like state(s) in melanoma.

The human placenta is a vital connection between maternal and fetal tissues, allowing for the exchange of molecules and modulation of immune interactions during pregnancy. Interestingly, some of the placenta's unique functions can be attributed to transposable elements (TEs), which are DNA sequences that have mobilised into the genome. Co-option throughout mammalian evolution has led to the generation of TE-derived regulators and TE-derived genes, some of which are expressed in the placenta but silenced in somatic tissues. TE genes encompass both TE-derived genes with a repeat element in the coding region and TE-derived regulatory regions such as alternative promoters and enhancers. Placental-specific TE genes are known to contribute to the placenta's unique functions, and interestingly, they are also expressed in some cancers and share similar functions. There is evidence to support that aberrant activity of TE genes may contribute to placental pathologies, cancer and autoimmunity. In this review, we highlight the crucial roles of TE genes in placental function, and how their dysregulation may lead to pre-eclampsia, a common and dangerous placental condition. We provide a summary of the functional TE genes in the placenta to offer insight into their significance in normal and abnormal human development. Ultimately, this review highlights an opportunity for future research to investigate the potential dysregulation of TE genes in the development of placental pathologies such as pre-eclampsia. Further understanding of TE genes and their role in the placenta could lead to significant improvements in maternal and fetal health.

Circulating tumor cells (CTCs) are precursors of the metastatic cascade, which is responsible for 90% of all cancer-related deaths. CTCs arise from solid tumors and travel through the bloodstream and lymphatic system. Developments in the isolation and analysis of CTCs promise potential biomarker assays for detection and monitoring of cancer through a minimally invasive procedure. Despite this, the precise role of CTCs in metastasis remains poorly characterized, mainly due to the low density of CTCs (1-10 CTCs per 10 mL of blood) present in patient blood and the lack of robust methods for their isolation in a standard laboratory setting. CellSearch is currently the only FDA-approved CTC enrichment protocol, but limitations of this EpCAM-based method include cost, availability, and the use of a single surface marker for capture. To address these limitations, we have optimized an existing method, MetaCell, which exploits the differences in size of CTCs compared to other blood cells for CTC enrichment from blood. MetaCell contains a membrane with 8 μm pores, and blood is filtered through this kit by capillary action and CTCs, which are typically larger than the pores and remain on top of the membrane, while most leukocytes pass through the pores. The membrane along with these CTCs can be detached and transferred to 6-well plates for culturing or directly used for characterization. Here, we provide a detailed protocol for enrichment of CTCs using the filtration device MetaCell and a procedure for characterization of CTCs by immunohistochemical staining.

Aberrant DNA methylation is a universal feature of cancer. Here, we present a protocol for generating high-quality genome-scale DNA methylation sequencing data from a variety of human cancer biospecimens including immortalized cell lines, fresh-frozen surgical resections, and formalin-fixed paraffin-embedded tissues. We describe steps for DNA extraction considerations, reduced representation bisulfite sequencing, data processing and quality control, and downstream data analysis and integration. This protocol is also applicable for other human diseases and methylome profiling in other organisms. For complete details on the use and execution of this protocol, please refer to Rodger et al. (2023).1

Renal cell carcinoma (RCC) is the most common form of kidney cancer, consisting of multiple distinct subtypes. RCC has the highest mortality rate amongst the urogenital cancers, with kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), and kidney chromophobe carcinoma (KICH) being the most common subtypes. The Paired-box (PAX) gene family encodes transcription factors, which orchestrate multiple processes in cell lineage determination during embryonic development and organogenesis. Several PAX genes have been shown to be expressed in RCC following its onset and progression. Here, we performed real-time quantitative polymerase chain reaction (RT-qPCR) analysis on a series of human RCC cell lines, revealing significant co-expression of PAX2, PAX6, and PAX8. Knockdown of PAX2 or PAX8 mRNA expression using RNA interference (RNAi) in the A498 RCC cell line resulted in inhibition of cell proliferation, which aligns with our previous research, although no reduction in cell proliferation was observed using a PAX2 small interfering RNA (siRNA). We downloaded publicly available RNA-sequencing data and clinical histories of RCC patients from The Cancer Genome Atlas (TCGA) database. Based on the expression levels of PAX2, PAX6, and PAX8, RCC patients were categorized into two PAX expression subtypes, PAXClusterA and PAXClusterB, exhibiting significant differences in clinical characteristics. We found that the PAXClusterA expression subgroup was associated with favorable clinical outcomes and better overall survival. These findings provide novel insights into the association between PAX gene expression levels and clinical outcomes in RCC patients, potentially contributing to improved treatment strategies for RCC.

Aims: Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs.Methods: Deploying a model of adaptive cancer drug tolerance, H3K4me3 ChIP-Seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo.Results: H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with OGlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in in vivo models. Upstream of OGT, we identified AMPK as an actionable target. AMPK activation by acetyl salicylic acid down regulated OGT with similar effects on delaying acquired resistance.Conclusion: Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes.

Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide. The majority of CRC deaths are caused by tumor metastasis, even following treatment. There is strong evidence for epigenetic changes, such as DNA methylation, accompanying CRC metastasis and poorer patient survival. Earlier detection and a better understanding of molecular drivers for CRC metastasis are of critical clinical importance. Here, we identify a signature of advanced CRC metastasis by performing whole genome-scale DNA methylation and full transcriptome analyses of paired primary cancers and liver metastases from CRC patients. We observed striking methylation differences between primary and metastatic pairs. A subset of loci showed coordinated methylation-expression changes, suggesting these are potentially epigenetic drivers that control the expression of critical genes in the metastatic cascade. The identification of CRC epigenomic markers of metastasis has the potential to enable better outcome prediction and lead to the discovery of new therapeutic targets.

Supplementary Table 1 from PAX8 Regulates Telomerase Reverse Transcriptase and Telomerase RNA Component in Glioma