MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch.

The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.

Stellate cell expression of SPARC-related modular calcium-binding protein 2 is associated with human non-alcoholic fatty liver disease severity.

Background & Aims: Histological assessment of liver biopsies is the gold standard for diagnosis of non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease (NAFLD), despite its well-established limitations. Therefore, non-invasive biomarkers that can offer an integrated view of the liver are needed to improve diagnosis and reduce sampling bias. Hepatic stellate cells (HSCs) are central in the development of hepatic fibrosis, a hallmark of NASH. Secreted HSC-specific proteins may, therefore, reflect disease state in the NASH liver and serve as non-invasive diagnostic biomarkers. Methods: We performed RNA-sequencing on liver biopsies from a histologically characterised cohort of obese patients (n = 30, BMI >35 kg/m2) to identify and evaluate HSC-specific genes encoding secreted proteins. Bioinformatics was used to identify potential biomarkers and their expression at single-cell resolution. We validated our findings using single-molecule fluorescence in situ hybridisation (smFISH) and ELISA to detect mRNA in liver tissue and protein levels in plasma, respectively. Results: Hepatic expression of SPARC-related modular calcium-binding protein 2 (SMOC2) was increased in NASH compared to no-NAFLD (p.adj <0.001). Single-cell RNA-sequencing data indicated that SMOC2 was primarily expressed by HSCs, which was validated using smFISH. Finally, plasma SMOC2 was elevated in NASH compared to no-NAFLD (p <0.001), with a predictive accuracy of AUROC 0.88. Conclusions: Increased SMOC2 in plasma appears to reflect HSC activation, a key cellular event associated with NASH progression, and may serve as a non-invasive biomarker of NASH. Impact and implications: Non-alcoholic fatty liver disease (NAFLD) and its progressive form, non-alcoholic steatohepatitis (NASH), are the most common forms of chronic liver diseases. Currently, liver biopsies are the gold standard for diagnosing NAFLD. Blood-based biomarkers to complement liver biopsies for diagnosis of NAFLD are required. We found that activated hepatic stellate cells, a cell type central to NAFLD pathogenesis, upregulate expression of the secreted protein SPARC-related modular calcium-binding protein 2 (SMOC2). SMOC2 was elevated in blood samples from patients with NASH and may hold promise as a blood-based biomarker for the diagnosis of NAFLD.

Collagen Density Modulates the Immunosuppressive Functions of Macrophages.

Tumor-associated macrophages (TAMs) support tumor growth by suppressing the activity of tumor-infiltrating T cells. Consistently, TAMs are considered a major limitation for the efficacy of cancer immunotherapy. However, the molecular reason behind the acquisition of an immunosuppressive TAM phenotype is not fully clarified. During tumor growth, the extracellular matrix (ECM) is degraded and substituted with a tumor-specific collagen-rich ECM. The collagen density of this tumor ECM has been associated with poor patient prognosis but the reason for this is not well understood. In this study, we investigated whether the collagen density could modulate the immunosuppressive activity of TAMs. The murine macrophage cell line RAW 264.7 was three-dimensionally cultured in collagen matrices of low and high collagen densities mimicking healthy and tumor tissue, respectively. Collagen density did not affect proliferation or viability of the macrophages. However, whole-transcriptome analysis revealed a striking response to the surrounding collagen density, including the regulation of immune regulatory genes and genes encoding chemokines. These transcriptional changes were shown to be similar in murine bone marrow-derived macrophages and TAMs isolated from murine tumors. Strikingly, coculture assays with primary T cells showed that macrophages cultured in high-density collagen were less efficient at attracting cytotoxic T cells and capable of inhibiting T cell proliferation more than macrophages cultured in low-density collagen. Our study demonstrates that a high collagen density can instruct macrophages to acquire an immunosuppressive phenotype. This mechanism could reduce the efficacy of immunotherapy and explain the link between high collagen density and poor prognosis.

Collagen density regulates the activity of tumor-infiltrating T cells.

BACKGROUND: Tumor progression is accompanied by dramatic remodeling of the surrounding extracellular matrix leading to the formation of a tumor-specific ECM, which is often more collagen-rich and of increased stiffness. The altered ECM of the tumor supports cancer growth and metastasis, but it is unknown if this effect involves modulation of T cell activity. To investigate if a high-density tumor-specific ECM could influence the ability of T cells to kill cancer cells, we here studied how T cells respond to 3D culture in different collagen densities. METHODS: T cells cultured in 3D conditions surrounded by a high or low collagen density were imaged using confocal fluorescent microscopy. The effects of the different collagen densities on T cell proliferation, survival, and differentiation were examined using flow cytometry. Cancer cell proliferation in similar 3D conditions was also measured. Triple-negative breast cancer specimens were analyzed for the number of infiltrating CD8+ T cells and for the collagen density. Whole-transcriptome analyses were applied to investigate in detail the effects of collagen density on T cells. Computational analyses were used to identify transcription factors involved in the collagen density-induced gene regulation. Observed changes were confirmed by qRT-PCR analysis. RESULTS: T cell proliferation was significantly reduced in a high-density matrix compared to a low-density matrix and prolonged culture in a high-density matrix led to a higher ratio of CD4+ to CD8+ T cells. The proliferation of cancer cells was unaffected by the surrounding collagen-density. Consistently, we observed a reduction in the number of infiltrating CD8+ T-cells in mammary tumors with high collagen-density indicating that collagen-density has a role in regulating T cell abundance in human breast cancer. Whole-transcriptome analysis of 3D-cultured T cells revealed that a high-density matrix induces downregulation of cytotoxic activity markers and upregulation of regulatory T cell markers. These transcriptional changes were predicted to involve autocrine TGF-ß signaling and they were accompanied by an impaired ability of tumor-infiltrating T cells to kill autologous cancer cells. CONCLUSIONS: Our study identifies a new immune modulatory mechanism, which could be essential for suppression of T cell activity in the tumor microenvironment.

Pharmacological Inhibition of Protein Kinase G1 Enhances Bone Formation by Human Skeletal Stem Cells Through Activation of RhoA-Akt Signaling.

Development of novel approaches to enhance bone regeneration is needed for efficient treatment of bone defects. Protein kinases play a key role in regulation of intracellular signal transduction pathways, and pharmacological targeting of protein kinases has led to development of novel treatments for several malignant and nonmalignant conditions. We screened a library of kinase inhibitors to identify small molecules that enhance bone formation by human skeletal (stromal or mesenchymal) stem cells (hMSC). We identified H-8 (known to inhibit protein kinases A, C, and G) as a potent enhancer of ex vivo osteoblast (OB) differentiation of hMSC, in a stage- and cell type-specific manner, without affecting adipogenesis or osteoclastogenesis. Furthermore, we showed that systemic administration of H-8 enhances in vivo bone formation by hMSC, using a preclinical ectopic bone formation model in mice. Using functional screening of known H-8 targets, we demonstrated that inhibition of protein kinase G1 (PRKG1) and consequent activation of RhoA-Akt signaling is the main mechanism through which H-8 enhances osteogenesis. Our studies revealed PRKG1 as a novel negative regulator of OB differentiation and suggest that pharmacological inhibition of PRKG1 in hMSC implanted at the site of bone defect can enhance bone regeneration. Stem Cells 2015;33:2219-2231.