Robust estimation of cancer and immune cell-type proportions from bulk tumor ATAC-Seq data

African green monkeys (AGMs), Chlorocebus pygerythrus, are a natural host for a lentivirus related to HIV, SIV. SIV-infected AGMs rarely progress to AIDS despite robust viral replication. Though multiple mechanisms are involved, a primary component is the animals' ability to downregulate CD4 expression on mature CD4+ Th cells, rendering these cells resistant to infection by SIV. These CD8αα+ T cells retain functional characteristics of CD4+ Th cells while simultaneously acquiring abilities of cytotoxic CD8αβ+ T cells. To determine mechanisms underlying functional differences between T cell subsets in AGMs, chromatin accessibility in purified populations was determined by assay for transposase-accessible chromatin sequencing. Differences in chromatin accessibility alone were sufficient to cluster cells by subtype, and accessibility at the CD4 locus reflected changes in CD4 expression. DNA methylation at the CD4 locus also correlated with inaccessible chromatin. By associating accessible regions with nearby genes, gene expression was found to correlate with accessibility changes. T cell and immune system activation pathways were identified when comparing regions that changed accessibility from CD4+ T cells to CD8αα+ T cells. Different transcription factor binding sites are revealed as chromatin accessibility changes, and these differences may elicit downstream changes in differentiation. This comprehensive description of the epigenetic landscape of AGM T cells identified genes and pathways that could have translational value in therapeutic approaches recapitulating the protective effects CD4 downregulation.

Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is the leading cause of death in patients with SSc with unclear pathogenesis and limited treatment options. Evidence strongly supports an important role for profibrotic secreted phosphoprotein 1 (SPP1)-expressing macrophages in SSc-ILD. This study was undertaken to define the transcriptome and chromatin structural changes of SPP1 SSc-ILD macrophages in order to better understand their role in promoting fibrosis and to identify transcription factors associated with open chromatin driving their altered phenotype. We performed single-cell RNA sequencing (scRNA-Seq) on 11 explanted SSc-ILD and healthy control lung samples, as well as single-cell assay for transposase-accessible chromatin sequencing on 5 lung samples to define altered chromatin accessibility of SPP1 macrophages. We predicted transcription factors regulating SPP1 macrophages using single-cell regulatory network inference and clustering (SCENIC) and determined transcription factor binding sites associated with global alterations in SPP1 chromatin accessibility using Signac/Seurat. We identified distinct macrophage subpopulations using scRNA-Seq analysis in healthy and SSc-ILD lungs and assessed gene expression changes during the change of healthy control macrophages into SPP1 macrophages. Analysis of open chromatin validated SCENIC predictions, indicating that microphthalmia-associated transcription factor, transcription factor EB, activating transcription factor 6, sterol regulatory element binding transcription factor 1, basic helix-loop-helix family member E40, Kruppel-like factor 6, ETS variant transcription factor 5, and/or members of the activator protein 1 family of transcription factors regulate SPP1 macrophage differentiation. Our findings shed light on the underlying changes in chromatin structure and transcription factor regulation of profibrotic SPP1 macrophages in SSc-ILD. Similar alterations in SPP1 macrophages may underpin fibrosis in other organs involved in SSc and point to novel targets for the treatment of SSc-ILD, specifically targeting profibrotic macrophages.

Type 1 diabetes in children is heralded by a preclinical phase defined by circulating autoantibodies to pancreatic islet antigens. How islet autoimmunity is initiated and then progresses to clinical diabetes remains poorly understood. Only one study has reported gene expression in specific immune cells of children at risk associated with progression to islet autoimmunity. We analyzed gene expression with RNA sequencing in CD4+ and CD8+ T cells, natural killer (NK) cells, and B cells, and chromatin accessibility by assay for transposase-accessible chromatin sequencing (ATAC-seq) in CD4+ T cells, in five genetically at risk children with islet autoantibodies who progressed to diabetes over a median of 3 years ("progressors") compared with five children matched for sex, age, and HLA-DR who had not progressed ("nonprogressors"). In progressors, differentially expressed genes (DEGs) were largely confined to CD4+ T cells and enriched for cytotoxicity-related genes/pathways. Several top-ranked DEGs were validated in a semi-independent cohort of 13 progressors and 11 nonprogressors. Flow cytometry confirmed that progression was associated with expansion of CD4+ cells with a cytotoxic phenotype. By ATAC-seq, progression was associated with reconfiguration of regulatory chromatin regions in CD4+ cells, some linked to differentially expressed cytotoxicity-related genes. Our findings suggest that cytotoxic CD4+ T cells play a role in promoting progression to type 1 diabetes.

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal developmental disorder of lung morphogenesis caused by insufficiency of FOXF1 (forkhead box F1) transcription factor function. The cellular and transcriptional mechanisms by which FOXF1 deficiency disrupts human lung formation are unknown. Objectives: To identify cell types, gene networks, and cell-cell interactions underlying the pathogenesis of ACDMPV. Methods: We used single-nucleus RNA and assay for transposase-accessible chromatin sequencing, immunofluorescence confocal microscopy, and RNA in situ hybridization to identify cell types and molecular networks influenced by FOXF1 in ACDMPV lungs. Measurements and Main Results: Pathogenic single-nucleotide variants and copy-number variant deletions involving the FOXF1 gene locus in all subjects with ACDMPV (n = 6) were accompanied by marked changes in lung structure, including deficient alveolar development and a paucity of pulmonary microvasculature. Single-nucleus RNA and assay for transposase-accessible chromatin sequencing identified alterations in cell number and gene expression in endothelial cells (ECs), pericytes, fibroblasts, and epithelial cells in ACDMPV lungs. Distinct cell-autonomous roles for FOXF1 in capillary ECs and pericytes were identified. Pathogenic variants involving the FOXF1 gene locus disrupt gene expression in EC progenitors, inhibiting the differentiation or survival of capillary 2 ECs and cell-cell interactions necessary for both pulmonary vasculogenesis and alveolar type 1 cell differentiation. Loss of the pulmonary microvasculature was associated with increased VEGFA (vascular endothelial growth factor A) signaling and marked expansion of systemic bronchial ECs expressing COL15A1 (collagen type XV α 1 chain). Conclusions: Distinct FOXF1 gene regulatory networks were identified in subsets of pulmonary endothelial and fibroblast progenitors, providing both cellular and molecular targets for the development of therapies for ACDMPV and other diffuse lung diseases of infancy.

Skeletal muscle accounts for the largest proportion of human body mass, on average, and is a key tissue in complex diseases and mobility. It is composed of several different cell and muscle fiber types. Here, we optimize single-nucleus ATAC-seq (snATAC-seq) to map skeletal muscle cell–specific chromatin accessibility landscapes in frozen human and rat samples, and single-nucleus RNA-seq (snRNA-seq) to map cell-specific transcriptomes in human. We additionally perform multi-omics profiling (gene expression and chromatin accessibility) on human and rat muscle samples. We capture type I and type II muscle fiber signatures, which are generally missed by existing single-cell RNA-seq methods. We perform cross-modality and cross-species integrative analyses on 33,862 nuclei and identify seven cell types ranging in abundance from 59.6% to 1.0% of all nuclei. We introduce a regression-based approach to infer cell types by comparing transcription start site–distal ATAC-seq peaks to reference enhancer maps and show consistency with RNA-based marker gene cell type assignments. We find heterogeneity in enrichment of genetic variants linked to complex phenotypes from the UK Biobank and diabetes genome-wide association studies in cell-specific ATAC-seq peaks, with the most striking enrichment patterns in muscle mesenchymal stem cells (∼3.5% of nuclei). Finally, we overlay these chromatin accessibility maps on GWAS data to nominate causal cell types, SNPs, transcription factor motifs, and target genes for type 2 diabetes signals. These chromatin accessibility profiles for human and rat skeletal muscle cell types are a useful resource for nominating causal GWAS SNPs and cell types.

417 Background: Gastric cancer is the 4th leading cause of cancer death worldwide with high incidence in East Asia. Despite recent developments in treatment options, gastric cancer survival rate remains low, partially attribute to tumor heterogeneity. Here we performed a comprehensive single cell analysis of malignant cells from gastric tumor samples. In addition to characterizing heterogenous transcriptional patterns, we developed a framework to identify HER2+ malignant cells, which may complement standard diagnostics for treatment decisions. Methods: Published and in-house generated 10x Genomics single cell RNAseq data in tumor and normal samples from gastric cancer patients were aggregated, including 64 tumor samples and 27 pre-cancerous samples and 12 adjacent normal samples. The R package Seurat (version 4.0) was applied for data QC and downstream analysis. High quality cells were clustered using highly variable genes and cell types were annotated by curated marker genes. To identify malignant epithelial cells, a gastric cancer signature was derived using matched tumor and normal samples from TCGA gastric cancer patients. Gastric tumor samples were also leveraged to develop a HER2 signature and TCGA breast tumor samples were used to validate the signature. HER2+ malignant cells were identified by re-clustering malignant epithelial cells using the HER2 signature. Results: 345,361 high-quality single cells were profiled, representing major cell types within tumor microenvironment. Among 59,861 epithelial cells, 22,770 malignant cells were identified with high expression of a gastric cancer signature. Copy number alterations (aneuploid) were highly enriched in inferred malignant cells compared with non-malignant cells (48.6% vs 1.4%, p<2.2×10-16, Fisher’s exact test). Further clustering analysis identified 10 subclusters with divergent expression of cancer oncogenic pathways, highlighting the vast heterogeneity of tumor origin. A Her2 amplification signature was derived using TCGA gastric cancer cohort (AUC=0.99) and validated in breast cancer cohort (AUC=0.93). Applying this signature to malignant cells, HER2+ cells were identified in a majority of tumors, with various proportions. In an independent data set, the percentage of HER2+ malignant cells is highly concordant with clinical HER2+ status, indicating the potential use of single cell data to supplement HER2+ clinical testing for treatment selection. Conclusions: This meta-analysis of single cell RNAseq data provides a comprehensive snapshot of tumor heterogeneity in gastric cancers, with aberrance in multiple oncogenic pathways observed in patients. The HER2+ cell classifier developed here holds the potential to complement standard clinical testing and enable personalized medicine, warranting further validation.

The tumor microenvironment is a complex milieu relevant to the progression of cancer. A detailed analysis needs to be performed for each cancer type to identify relevant cell types and secreted factors promoting metastasis. Fibrolamellar carcinoma (FLC) is an aggressive liver cancer that predominantly afflicts adolescents and young adults who lack predisposing conditions, and patients with FLC are characterized by a heterozygous deletion on chromosome 19 that creates an oncogenic gene fusion, DNAJB1-PRKACA. The extracellular environment of FLC tumors is poorly characterized and may contribute to cancer progression, metastasis, and/or drug resistance. To bridge this knowledge gap, we performed experiments to assess pathways relevant to proteoglycans, a major component of the extracellular matrix. We first analyzed gene expression data from FLC and non-malignant liver tissue (n=27) to identify changes in pathways pertinent to glycosaminoglycan (GAG) biosynthesis. We then developed and implemented a novel chemical analytics method, utilizing custom molecular standards, to quantify the abundance of different types of GAGs in patient tumor samples by liquid chromatography with tandem mass spectrometry. We then measured the mRNA and protein levels of different GAG-associated proteins, followed by immunohistochemical confirmation. Finally, we performed the first single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on FLC tumors to define the heterogeneous cellular landscape and to identify cell types likely responsible for producing the most dominant GAG-associated protein in FLC. We found that chondroitin sulfate (CS) but not heparan sulfate (HS) or hyaluronic acid (HA) biosynthesis genes are dramatically increased in FLC, especially the rate-limiting enzyme CS GalNAc transferase 1 (CSGALNACT1). We also determined that CS chains are significantly elevated in FLC. Further analysis showed that versican (VCAN) is the primary CS-associated protein in FLC, that CSGALNACT1 and VCAN are correlated with DNAJB1-PRKACA levels, and that VCAN is more highly expressed in FLC compared to almost all other cancer types. Finally, the single cell ATAC-seq analyses demonstrated for the first time the cellular heterogeneity of FLC and revealed that the VCAN locus is active in tumor epithelial cells but is most active in proliferating stellate cells. This study merges chemical, molecular, and single-cell analyses to uncover the marked elevation of chondroitin sulfate proteoglycans in FLC tumors. Our results suggest that VCAN may play a critical role in FLC tumor growth and/or metastasis. We also identified the presence of activated stellate cells in FLC tumors and suggest a role in propagating fibrosis. Future studies are required to identify whether and how attenuation of CS and/or VCAN mitigates proliferative and/or metastatic potential in FLC. Citation Format: Adam Francisco, Jine Li, Alaa Farghili, Matt Kanke, Bo Shui, Wencheng Zhang, Paul Soloway, Zhangjie Wang, Lola Reid, Praveen Sethupathy. Chemical, molecular, and single cell analysis reveal chondroitin sulfate proteoglycan aberrancy in fibrolamellar carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3830.

BackgroundDespite affecting millions of people worldwide, psychiatric disorders are inadequately treated. Understanding how adversity, one of the major risk factors for these disorders, raises risk for psychopathology is essential for identifying new treatments. The orbitofrontal cortex (OFC), a sub-region of prefrontal cortex (PFC), is involved in higher cognitive processes and is highly vulnerable to adverse experiences. However, the impacts of adversity on various cell-types across the cortical layers of the OFC are largely unknown. Here, we interrogated human postmortem OFC (BA11) samples to unravel adversity-induced cell-type-specific transcriptomic changes in psychiatric disorders. Explorations of single-cell epigenomic, as well as, spatial transcriptomic alterations within the same samples are currently underway.Aims & ObjectivesThe primary objective of this study is to disentangle adversity-induced cell-type- and cortical layer-specific molecular changes in the OFC of psychiatric patients. The overarching goal is to determine if adversity is associated with a transdiagnostic subtype of psychiatric disorders that may benefit from targeted therapy.MethodWe examined 86 individuals (n=32 controls vs n=54 cases with mixed major psychiatric disorders: schizophrenia, major depression and bipolar disorder) with or without history of profound psychological adversity exposure (n=23 with severe adversity and n=31 without). To capture the cell-type-specific effects, we generated single-nucleus Assay for Transposase-Accessible Chromatin sequencing (snATAC-seq) and single-nucleus RNA sequencing (snRNA-seq) datasets using 10x Chromium (10x Genomics, USA). To dissect spatial heterogeneity, we produced spatial transcriptomic dataset using Visium (10x Genomics, USA). We performed compositional and differential gene expression analyses, followed by gene ontology and disease enrichment analyses, on the snRNA-seq data.ResultsAfter filtering out low-quality nuclei from the snRNA-seq dataset, about ~800,000 nuclei from 15 different cell types were retained. The proportions of inhibitory neurons and astrocytes were significantly lower in individuals exposed to adversity vs those without adversity, while the proportion of oligodendrocytes was higher (Log2FoldChange of -0.51, -1.02 and 0.33, respectively). We identified a large number of differentially expressed genes (DEGs) in oligodendrocytes and reelin-expressing inhibitory neurons (160 and 265, respectively) only when the contrast was performed among cases based on their history of adversity. Overall, the DEGs showed significant enrichment of psychopathology-associated metabolic processes and phenotypes, including synaptic signaling and inflammatory responses.Discussion & ConclusionsThese results highlight the striking cell-type-specific impacts of adverse experiences on gene expressions and cellular abundances in the OFC of individuals with psychiatric disorders. The glial cells such as astrocytes and oligodendrocytes, which are known to be involved in stress response, as well as inhibitory neuronal subtypes, known to be affected in stress-related disorders, showed marked impacts of adversity in the context of psychopathology. In general, synaptic signaling, transmembrane transports, energy metabolism and immune response pathways were affected, all of which are implicated in psychopathology.Experiments and analyses are underway to further disentangle the regulatory changes affecting transcription by delineating epigenomic modifications and gene co-expression networks within these cell types and to unmask the changes in transcript and cellular abundances across the cortical layers. The study will thus help decompose adversity-induced transcriptomic and gene regulatory changes into cell-type-specific alterations across cortical layers.

Single-cell chromatin analysis provides new opportunities to identify homologous cells that regulate gene expression in a similar manner in different species. The aim of this study is to analyze chromatin accessibility of embryonic brain cells in chicken and turkey. Chromatin accessibility was profiled in single nuclei isolated from day-14 embryonic brain of chicken and turkey using the 10X Genomics Chromium ATAC (Assay for Transposase Accessible Chromatin) sequencing (data availability: GEO accession # GSE269838). Cluster analysis of the single-nuclei ATAC-seq data showed differential cluster patterns of embryonic brain cells between the two species based on the variation of chromatin accessibility. Data analysis further identified a total of 60,376 peaks associated with 22,382 genes in the chicken brain. These open chromatins were identified more frequently in the distal regulatory regions (n=43,224) compared to promoter regions (n=17,152). In the turkey brain, the analysis identified a total of 60,375 peaks associated with 14,516 genes. The majority of these open chromatins, similar to chicken brain, were also identified more frequently in the distal regulatory regions (n=56,834) compared to promoter regions (n=7,871). However, there was a 14-fold greater number of intergenic peaks in the turkey brain compared to chicken brain. Motif enrichment analysis of the peak sequences showed that the binding motif of the transcription factor ARID3A (AT-Rich Interaction Domain 3A) was commonly enriched as a distal cis-regulatory element of specific genes such as AGT, ANO4, ANO5, GPR139, GRIK1, GRM3, HAAO, LRRTM3, MGAT4C, RHCG, SLC25A21, TAL2, and TRPC5 in the brain cells of both chicken and turkey. Bulk RNA-seq analysis further showed that these predicted ARID3 target genes were expressed in the embryonic brain of chicken and turkey. The chorioallantoic membrane plays vital functions for embryonic development in chicken and turkey unlike the placental functions that modulate embryo development in mice. The expression of the predicted ARID3 target genes was quantified in the placenta and fetal brain of mouse. This comparative analysis showed that the majority of the ARID3 target genes were expressed in the mouse fetal brain but suppressed in the placenta. However, three genes AGT, HAAO and SLC25A21 were expressed in the placenta but suppressed in the fetal brain. The findings of this study suggest that ARID3A plays a common regulatory role in the brain development in avian and mammalian embryos. While role of ARID3A in placenta development is documented, further research is needed to understand the role of ARID3A in the development of chorioallantoic membrane of avian embryos.

A Novel Single Cell Model of Tamoxifen Response in Primary Human Breast Tumors Austin Whitman, Hyunsoo Kim, Kamila Wisniewska, Rasha Kakati, Susana Garcia Recio, Hector Franco, Charles Perou, Philip Spanheimer Background: Resistance to endocrine therapy is a primary cause of treatment failure and death in patients with estrogen receptor (ER)-positive breast cancer. Intratumor heterogeneity is associated with resistance to therapy across tumors, and specifically in ER+/HER2- breast cancer, heterogeneity in ER and PR expression is associated with a worse response to endocrine therapy. We hypothesize that subpopulations within and across ER+/HER2- human breast tumors have distinct responses to tamoxifen and that discerning heterogeneity in response will improve understanding of inherent and emerging resistance to endocrine therapy. Methods: We developed an operating room-to-laboratory pipeline immediately after surgical resection for studies using alive tissue. Tissue samples were obtained and single cell suspensions created using physical and enzymatic dissociation. Cells were treated with tamoxifen (10 M) or control media for 12 hours in suspension and single cell RNA libraries generated using the 10X Genomics droplet-based kit and sequenced using the Illumina NextSeq2000. Results: We obtained normal breast tissue from 2 women undergoing reduction mammoplasty and tumor tissue from 10 women with ER+/HER2- invasive breast carcinoma. In tamoxifen treated and control matched pairs, a total of 22,195 cells from normal breast and 94,558 cells from tumor samples were sequenced. Computational analysis using consensus clustering was performed and cell types assigned using canonical correlation. Both tumor and normal samples identified clustering by cell type and not by patient revealing significant variability in cell type abundance between samples. In the normal breast samples, we performed differentially expressed genes (DEG) analysis comparing tamoxifen treatment to control for each cell type (Immune cells, fibroblasts, basal epithelial cells, luminal progenitor cells, and mature luminal cells) and enrichment analysis of up- and down-regulated genes performed. Strong depletion of estrogen induced genes was observed in tamoxifen-treated normal luminal progenitor and mature luminal cells, but not in basal epithelial cells or fibroblasts, demonstrating distinct, subpopulation-specific response to tamoxifen. In the 10 tumor matched pairs, 4 had a high epithelial proportion and tumor cells identified using inferred copy number variation. Tumor cells in 3 of these 4 samples showed significant down regulation of estrogen response genes with tamoxifen treatment. Using scBCSubtype to assign PAM50 subtype to individual tumor cells, the 3 responsive tumors were comprised primarily of LumA cells while the unresponsive tumor was predominantly LumB. Finally, we developed a novel score to quantify responsiveness at the single cell level based on downregulation of estrogen response genes with tamoxifen treatment relative to matched cluster-specific untreated expression. This analysis demonstrated heterogeneity in response to tamoxifen in tumor cells and identified distinct subpopulations of responsive and unresponsive tumor cells to tamoxifen treatment. Conclusion: We developed a novel ex vivo model to determine heterogeneity in therapeutic response to tamoxifen in normal human breast tissue and primary human breast tumors. We demonstrate differences in tamoxifen response by cell type and identify distinctly responsive and resistant subpopulations within human tumors. This provides a foundation to define features of responsive and resistant populations on the individual cell and specimen basis, and should allow us to develop precise, single cell-based predictors of response to endocrine therapy, and to identify genes and pathways driving resistance to therapy. Citation Format: Hyunsoo Kim, Austin Whitman, Kamila Wisniewska, Susana Garcia-Recio, Rasha Kakati, Hector L. Franco, Charles M. Perou, Philip Spanheimer. PD4-08 A Novel Single Cell Model of Tamoxifen Response in Primary Human Breast Tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-08.

Single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) provides an opportunity to look deeply into the gene regulation mechanism at the single-cell resolution. With the rapid accumulation of scATAC-seq data, there is an urgent need for automatic cell type annotations using scATAC-seq data. Most existing methods rely on creating artificial gene activity matrix, due to the extreme sparsity nature of the scATAC-seq data. However, these methods fail to exploit the intrinsic information inherent in the scATAC-seq peaks. We present scDIFF, a diffusive transformer-based method that integrates bulk-level genomic and epigenomic information with scATAC-seq data to annotate cell types without creating artificial gene activity matrix. Our scDIFF performed constantly better than state-of-the-art methods on all 46 benchmarking pairs of reference and query datasets across different sequencing platforms. The complete implementation of scDIFF is well-documented and freely available on GitHub (https://github.com/haoyu-wangg/scDIFF).

<div><p>Fibrolamellar carcinoma (FLC) is an aggressive liver cancer with no effective therapeutic options. The extracellular environment of FLC tumors is poorly characterized and may contribute to cancer growth and/or metastasis. To bridge this knowledge gap, we assessed pathways relevant to proteoglycans, a major component of the extracellular matrix. We first analyzed gene expression data from FLC and nonmalignant liver tissue (<i>n</i> = 27) to identify changes in glycosaminoglycan (GAG) biosynthesis pathways and found that genes associated with production of chondroitin sulfate, but not other GAGs, are significantly increased by 8-fold. We then implemented a novel LC/MS-MS based method to quantify the abundance of different types of GAGs in patient tumors (<i>n</i> = 16) and found that chondroitin sulfate is significantly more abundant in FLC tumors by 6-fold. Upon further analysis of GAG-associated proteins, we found that versican (<i>VCAN</i>) expression is significantly upregulated at the mRNA and protein levels, the latter of which was validated by IHC. Finally, we performed single-cell assay for transposase-accessible chromatin sequencing on FLC tumors (<i>n</i> = 3), which revealed for the first time the different cell types in FLC tumors and also showed that <i>VCAN</i> is likely produced not only from FLC tumor epithelial cells but also activated stellate cells. Our results reveal a pathologic aberrancy in chondroitin (but not heparan) sulfate proteoglycans in FLC and highlight a potential role for activated stellate cells.</p>Significance:<p>This study leverages a multi-disciplinary approach, including state-of-the-art chemical analyses and cutting-edge single-cell genomic technologies, to identify for the first time a marked chondroitin sulfate aberrancy in FLC that could open novel therapeutic avenues in the future.</p></div>

BackgroundPancreatic ductal adenocarcinoma (PDAC) is resistant to many available therapies including immunotherapy because of its highly complex tumor microenvironment (TME). PDAC TME consists of a significant proportion of stromal cells,...

Estrogen and progesterone are critically involved in the development of breast cancer and their effects are mediated by the estrogen receptor (ER) and progesterone receptor (PR) respectively. The activities of ER and PR are regulated at multiple steps of steroid hormone receptor (SHR) signaling pathway. Post-translational modifications (PTMs) play pivotal roles in regulating SHR signaling, enable quick responses of these receptors to stimuli without the need for new protein synthesis. Initial mass spectrometry analysis of PTMs on ER identified multiple methylated arginines, suggesting that arginine methylation is an important player regulating ER activity. The objectives of the first part of the thesis were to identify protein arginine methyltransferases (PRMTs) that methylate ER and PR and to determine the functional relevance of the methylation. This study showed that PRMT6 binds to ER, PR and androgen receptor (AR), and methylates them independent of ligand. The site of the interaction and mode of regulation by PRMT6 seems to vary among the receptors but the role of PRMT6 in regulating ER activity has been further defined. PRMT6 binds to ER at the C-terminus domains (LBO-region F) and proximity ligation assays indicated that PRMT6 interacts with ER exclusively in the nucleus. PRMT6 overexpression enhances ER ligand-independent and ligand-induced activity via different mechanisms. PRMT6 significantly enhances the ligand-independent activity of ER without the need for its methyltransferase activity. We present evidences suggesting that the ligand-independent activation of ER by PRMT6 is mediated through its binding to ER which may lead to dissociation of chaperone heat shock protein 70 (Hsp70) and Hsp90. On the other hand, enhancement of ligang-induced ER activity requires enzymatically active PRMT6 and possible arginine residues targeted by PRMT6 have been identified. In light of the recent report that PRMT6 dysfunction is associated with worse prognosis in ER-positive breast cancers, our results suggest that PRMT6 may promote the mitogenic effect of ER both in the presence and absence of estrogen. The increase in ligand-independent ER signaling with high PRMT6 expression level may result in hormone-independent tumor growth which is resistant to the inhibitory effect of tamoxifen. In contrast to the mitogenic role of ER in breast cancer, PR can exert inhibitory effect on breast cancer growth. Progesterone-induced growth inhibition in PR-transfected MDAMB-231 cells was associated with a remarkable upregulation of tripartite motif-containing 22 (TRIM22), a p53 and interferon (lFN) target gene. The objective of the second part of the thesis work was to understand the regulation of TRIM22 gene expression and its clinical significance. TRIM22 protein level is consistently downregulated in breast cancer cell lines and breast tumor tissues as compared to non-malignant mammary epithelial cells and normal breast tissues, making it a potential marker for breast malignancy. TRIM22 expression also correlates with p53 expression in normal breast tissue and it can be induced upon p53 activation in non-malignant cell. However, in breast cancer cells, TRIM22 expression is not inducible in response to p53 activation by genotoxic drugs, suggesting that regulation of TRIM22 by p53 is specifically disrupted during tumorigenesis. In addition, TRIM22 is also a regulator of PR activity. It upregulates both the expression level and ligand induced activity of full length PR but inhibits ligandindependent activity of LBO truncation PRo Together with the reported growth inhibitory role of TRIM22 for leukemic cells, this study presents evidence that TRIM22 may inhibit the growth of breast cancer cells via p53 and PR signaling pathway. Further correlation of TRIM22 expression in clinical breast cancer samples with clinicopathological parameters will shed light on the prognostic value of TRIM22 in breast cancer.

Developmental trajectory of prehematopoietic stem cell formation from endothelium