Intra-tumor heterogeneity is one of the biggest challenges in cancer treatment today. Here we investigate tissue-wide gene expression heterogeneity throughout a multifocal prostate cancer using the ...spatial transcriptomics (ST) technology. Utilizing a novel approach for deconvolution, we analyze the transcriptomes of nearly 6750 tissue regions and extract distinct expression profiles for the different tissue components, such as stroma, normal and PIN glands, immune cells and cancer. We distinguish healthy and diseased areas and thereby provide insight into gene expression changes during the progression of prostate cancer. Compared to pathologist annotations, we delineate the extent of cancer foci more accurately, interestingly without link to histological changes. We identify gene expression gradients in stroma adjacent to tumor regions that allow for re-stratification of the tumor microenvironment. The establishment of these profiles is the first step towards an unbiased view of prostate cancer and can serve as a dictionary for future studies.
Iterative liver injury results in progressive fibrosis disrupting hepatic architecture, regeneration potential, and liver function. Hepatic stellate cells (HSCs) are a major source of pathological ...matrix during fibrosis and are thought to be a functionally homogeneous population. Here, we use single-cell RNA sequencing to deconvolve the hepatic mesenchyme in healthy and fibrotic mouse liver, revealing spatial zonation of HSCs across the hepatic lobule. Furthermore, we show that HSCs partition into topographically diametric lobule regions, designated portal vein-associated HSCs (PaHSCs) and central vein-associated HSCs (CaHSCs). Importantly we uncover functional zonation, identifying CaHSCs as the dominant pathogenic collagen-producing cells in a mouse model of centrilobular fibrosis. Finally, we identify LPAR1 as a therapeutic target on collagen-producing CaHSCs, demonstrating that blockade of LPAR1 inhibits liver fibrosis in a rodent NASH model. Taken together, our work illustrates the power of single-cell transcriptomics to resolve the key collagen-producing cells driving liver fibrosis with high precision.
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•scRNA-seq reveals spatial zonation of hepatic stellate cells (HSCs)•HSCs partition into topographically diametric lobule regions•Functional zonation of HSCs during centrilobular injury-induced fibrosis is uncovered•LPAR1 is a therapeutic target on pathological central vein-associated HSC
Dobie et al. use scRNA-seq to reveal spatial and functional zonation of hepatic stellate cells (HSCs) across the hepatic lobule, identifying central vein-associated HSCs as the dominant pathogenic collagen-producing cells during centrilobular injury-induced fibrosis. This illustrates the power of scRNA-seq to resolve the key collagen-producing cells driving liver fibrosis.
Signaling factors including retinoic acid (RA) and thyroid hormone (T3) promote neuronal, oligodendrocyte, and astrocyte differentiation of cortical neural stem cells (NSCs). However, the functional ...specificity of transcriptional repressor checkpoints controlling these differentiation programs remains unclear. Here, we show by genome-wide analysis that histone deacetylase (HDAC)2 and HDAC3 show overlapping and distinct promoter occupancy at neuronal and oligodendrocyte-related genes in NSCs. The absence of HDAC3, but not HDAC2, initiated a neuronal differentiation pathway in NSCs. The ablation of the corepressor NCOR or HDAC2, in conjunction with T3 treatment, resulted in increased expression of oligodendrocyte genes, revealing a direct HDAC2-mediated repression of Sox8 and Sox10 expression. Interestingly, Sox10 was required also for maintaining the more differentiated state by repression of stem cell programming factors such as Sox2 and Sox9. Distinct and nonredundant actions of NCORs and HDACs are thus critical for control of lineage progression and differentiation programs in neural progenitors.
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•ChIP-seq reveals distinct and overlapping occupancy of HDAC2 and HDAC3 in NSCs•Absence of NCOR promotes oligodendrocyte differentiation of NSCs•HDAC2 controls Sox10 expression in OL differentiation via a SOX2-occupied enhancer•Sox10 is required for maintaining the differentiated state in late OL precursors
ChIP-seq in neural stem cells revealed that HDAC2 and HDAC3 show overlapping and distinct promoter occupancy at neuronal and oligodendrocyte-related genes. HDAC3 knockdown initiated neuronal differentiation. Hermanson, Castelo-Branco, and colleagues show that ablation of Ncor or, in the presence of T3, Hdac2 resulted in increased expression of oligodendrocyte genes, unveiling an HDAC2-mediated repression of Sox10 expression. Sox10 was required for maintaining a more differentiated state by repression of stem cell factors including Sox2 and Sox9.
Rett syndrome (RTT) is a neurodevelopmental disorder with neurological symptoms, such as motor disorders and mental retardation. In most cases, RTT is caused by mutations in the DNA binding protein ...MeCP2. In mice, MeCP2 gene deletion has been reported to result in genome-wide increased histone acetylation. Transcriptional regulation of neurotrophic factor BDNF and transcription factor DLX5, essential for proper neurogenesis, is further altered in MeCP2-deleted animals. We therefore investigated the chromatin environment of MeCP2 target genes BDNF and DLX5 in lymphocytes from RTT patients and human controls, and analyzed the density of histones H3, H2B and H1, as well as the levels of methylation and acetylation on selected lysines of histone H3. Notably, we found a general increase in the density of histone H3 in RTT patients' lymphocytes compared with controls, and decreased levels of trimethylation of lysine 4 on histone H3 (H3K4me3), a modification associated with transcriptional activation. The levels of acetylation of lysine 9 (H3K9ac) and 27 (H3K27ac) did not show any statistically significant changes when normalized to the decreased histone H3 levels; nevertheless, an average decrease in acetylation was noted. Our results reveal an unexpected alteration of the chromatin state of established MeCP2 target genes in lymphocytes of human subjects with RTT.
Glioblastoma multiforme (GBM) is the most common and lethal adult brain tumor. Resistance to standard radiation and chemotherapy is thought to involve survival of GBM cancer stem cells (CSCs). To ...date, no single marker for identifying GBM CSCs has been able to capture the diversity of CSC populations, justifying the needs for additional CSC markers for better characterization. Employing targeted mass spectrometry, here we present five cell‐surface markers HMOX1, SLC16A1, CADM1, SCAMP3, and CLCC1 which were found to be elevated in CSCs relative to healthy neural stem cells (NSCs). Transcriptomic analyses of REMBRANDT and TCGA compendiums also indicated elevated expression of these markers in GBM relative to controls and non‐GBM diseases. Two markers SLC16A1 and HMOX1 were found to be expressed among pseudopalisading cells that reside in the hypoxic region of GBM, substantiating the histopathological hallmarks of GBM. In a prospective study (N = 8) we confirmed the surface expression of HMOX1 on freshly isolated primary GBM cells (P0). Employing functional assays that are known to evaluate stemness, we demonstrate that elevated HMOX1 expression is associated with stemness in GBM and can be modulated through TGFβ. siRNA‐mediated silencing of HMOX1 impaired GBM invasion—a phenomenon related to poor prognosis. In addition, surgical resection of GBM tumors caused declines (18% ± 5.1SEM) in the level of plasma HMOX1 as measured by ELISA, in 8/10 GBM patients. These findings indicate that HMOX1 is a robust predictor of GBM CSC stemness and pathogenesis. Further understanding of the role of HMOX1 in GBM may uncover novel therapeutic approaches. Stem Cells 2016;34:2276–2289
Targeted proteomic analyses revealed elevated expressions of transmembrane proteinsHMOX1 and SLC16A1on the surface of glioblastoma cancer stem cells (CSCs) relative to healthy neural stem cells (NSCs). In the hypoxic region of the tumor, these proteins were found to be expressed among pseudopalisading glioma cells that also express known stem cell factors. From biological assays that are known to evaluate stemness, HMOX1 expression was found to be associated with stemness that could be regulated through TGFβ and PTEN signaling networks. Additionally, siRNA mediated inhibition of HMOX1 expression impaired cancer cell invasion. Together, this study demonstrates the association of HMOX1 expression with cancer stemness in glioblastoma and suggests a possible regulatory mechanism of CSC invasion and survival in the hypoxic region of the tumor.
Glioblastoma multiforme (GBM) is the most common and lethal adult brain tumor. Resistance to standard radiation and chemotherapy is thought to involve survival of GBM cancer stem cells (CSCs). To ...date, no single marker for identifying GBM CSCs has been able to capture the diversity of CSC populations, justifying the needs for additional CSC markers for better characterization. Employing targeted mass spectrometry, here we present five cell-surface markers HMOX1, SLC16A1, CADM1, SCAMP3 and CLCC1 which were found to be elevated in CSCs relative to healthy neural stem cells (NSCs). Transcriptomic analyses of REMBRANDT and TCGA compendiums also indicated elevated expression of these markers in GBM relative to controls and non-GBM diseases. Two markers SLC16A1 and HMOX1 were found to be expressed among pseudopalisading cells that reside in the hypoxic region of GBM, substantiating the histopathological hallmarks of GBM. In a prospective study (N=8) we confirmed the surface expression of HMOX1 on freshly isolated primary GBM cells (P0). Employing functional assays that are known to evaluate stemness, we demonstrate that elevated HMOX1 expression is associated with stemness in GBM and can be modulated through TGFβ.
si
RNA-mediated silencing of HMOX1 impaired GBM invasion- a phenomenon related to poor prognosis. In addition, surgical resection of GBM tumors caused declines (18%±5.1SEM) in the level of plasma HMOX1 as measured by ELISA, in 8/10 GBM patients. These findings indicate that HMOX1 is a robust predictor of GBM CSC stemness and pathogenesis. Further understanding of the role of HMOX1 in GBM may uncover novel therapeutic approaches.
Glioblastoma multiforme (GBM) is the most common and lethal adult brain tumor. Resistance to standard radiation and chemotherapy is thought to involve survival of GBM cancer stem cells (CSCs). To ...date, no single marker for identifying GBM CSCs has been able to capture the diversity of CSC populations, justifying the needs for additional CSC markers for better characterization. Employing targeted mass spectrometry, here we present five cell-surface markers HMOX1, SLC16A1, CADM1, SCAMP3, and CLCC1 which were found to be elevated in CSCs relative to healthy neural stem cells (NSCs). Transcriptomic analyses of REMBRANDT and TCGA compendiums also indicated elevated expression of these markers in GBM relative to controls and non-GBM diseases. Two markers SLC16A1 and HMOX1 were found to be expressed among pseudopalisading cells that reside in the hypoxic region of GBM, substantiating the histopathological hallmarks of GBM. In a prospective study (N=8) we confirmed the surface expression of HMOX1 on freshly isolated primary GBM cells (P0). Employing functional assays that are known to evaluate stemness, we demonstrate that elevated HMOX1 expression is associated with stemness in GBM and can be modulated through TGFbeta. siRNA-mediated silencing of HMOX1 impaired GBM invasion--a phenomenon related to poor prognosis. In addition, surgical resection of GBM tumors caused declines (18%±5.1SEM) in the level of plasma HMOX1 as measured by ELISA, in 8/10 GBM patients. These findings indicate that HMOX1 is a robust predictor of GBM CSC stemness and pathogenesis. Further understanding of the role of HMOX1 in GBM may uncover novel therapeutic approaches. Stem Cells 2016;34:2276-2289
Red wine contains antioxidants and is at moderate amounts believed to exert certain positive health effects. Resveratrol is one of the most studied antioxidants in red wine and has been suggested to ...activate the longevity- and metabolism-associated histone deacetylase SIRT1. Here we show that relatively low concentrations of resveratrol (0.5–3 μM) specifically inhibited neuronal differentiation of neural stem cells in a SIRT1-dependent manner whereas higher concentrations of resveratrol (≥
10 μM) induced a SIRT1-independent cell death. Surprisingly, using a cell based assay, we found that small amounts of red wine (1–5% v/v) – but not white wine – induced a massive and rapid cell death of various cell types, including neural stem cells and several cancer cell lines. This red wine-induced cell death was ethanol-, SIRT1- and resveratrol-independent but associated with increased oxidative stress and inhibition of thioredoxin reductase (TrxR) activity. The TrxR inhibition correlated with the red color (absorbance at 520 nm) of the wines demonstrating that pigment components of red wine can exert profound cellular effects. Our results unveil important roles for SIRT1 and TrxR in resveratrol and red wine-mediated effects on progenitor and cancer cells, and demonstrate that cellular responses to red wine may be more complex than generally appreciated.
Development of an organism requires correct spatial and temporal regulation of gene expression. Epigenetic regulation of gene expression has been shown to be an important part of many cellular ...events. Nucleosomes consist of two subunits of each core histone proteins H2A,H2B, H3, H4, and 146 base pairs of DNA, wrapped around the histones. In this way, DNA can be tightly packaged in the cell. The N-terminal tails, that protrude out from the histone surfaces can be subjected to different covalent modifications, including, acetylation, methylation, phosphorylation, and ubiquitination. These modifications take part in gene regulation by changing the structure of chromatin and by recruiting gene regulatory proteins. Addition of acetyl-groups to the N-terminal of histones is catalyzed by histone acetyltransferases (HATs) where as removal of the acetyl-groups is carried out by histone deacetylases(HDACs). Increased acetylation has been correlated with increased gene expression, and decreased acetylation has been correlated with transcriptional repression. HDACs are divided into four families: class I, class II, and class IV histone deacetylases and the class IIINAD-dependent enzymes of the sirtu in family. HDACs are expressed early in development and specific HDAC gene deletions show that they are important for many cellular events, such as proliferation, growth, and differentiation. Putative roles for HDACs in neural development come mostly from work done using inhibitors of class I and class II HDACs, and from the association of HDACs to protein complexes that are known to repress neuronal differentiation. HDACs have also been shown to play an important part in cancer development and HDAC inhibitors have been shown to block cell proliferation, promote differentiation, and induce or facilitate apoptosis. The aim of this thesis was to investigate the role of histone deacetylases in neural stem- and cancer cells. In paper I, we show that HDAC3 is an essential repressor of neuronal differentiation in embryonic neural stem cells, required for repression of BDNF expression and histone H3K9 acetylation. Paper II and III address the use of HDAC inhibitors in cancer treatment and their effect on apoptotic and epigenetic mechanisms. We show that trichost at in A in combination with etoposide, VP16, induce apoptosis via caspase-dependent path ways and the mitochondrial AIF dependent pathway in multi-resistant cancer cells. In addition, we show that cell death promoting effects of valproic acid and trichost at in A depend on the regulation of histoneH4K16 acetylation by the histone modifying enzymes hMOF and SIRT1. In paper IV, we show that resveratrol inhibits neuronal differentiation of embryonic neural stem cells in a SIRT1-dependent fashion and that the effects of red wine on embryonic NSCs and cancer cells are toxic and are linked to inhibition of thioredoxin reductase in are sveratrol /SIRT1-independent manner. In conclusion, this thesis shows that chromatin modifying proteins play essential roles in neural stem cell differentiation and cancer cell characteristics, and contributes to the understanding of epigenetic mechanisms in the regulation of neuralistem and cancer cell fate.
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