Kari, Vijayalakshmi

Abstract Recent studies have thoroughly described genome-wide expression patterns defining molecular subtypes of pancreatic ductal adenocarcinoma (PDAC), with different prognostic and predictive implications. Although the reversible nature of key regulatory transcription circuits defining the two extreme PDAC subtype lineages “classical” and “basal-like” suggests that subtype states are not permanently encoded but underlie a certain degree of plasticity, pharmacologically actionable drivers of PDAC subtype identity remain elusive. Here, we characterized the mechanistic and functional implications of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) in controlling PDAC plasticity, dedifferentiation, and molecular subtype identity. Utilization of transgenic PDAC models and human PDAC samples linked EZH2 activity to PDAC dedifferentiation and tumor progression. Combined RNA- and chromatin immunoprecipitation sequencing studies identified EZH2 as a pivotal suppressor of differentiation programs in PDAC and revealed EZH2-dependent transcriptional repression of the classical subtype defining transcription factor Gata6 as a mechanistic basis for EZH2-dependent PDAC progression. Importantly, genetic or pharmacologic depletion of EZH2 sufficiently increased GATA6 expression, thus inducing a gene signature shift in favor of a less aggressive and more therapy-susceptible, classical PDAC subtype state. Consistently, abrogation of GATA6 expression in EZH2-deficient PDAC cells counteracted the acquisition of classical gene signatures and rescued their invasive capacities, suggesting that GATA6 derepression is critical to overcome PDAC progression in the context of EZH2 inhibition. Together, our findings link the EZH2-GATA6 axis to PDAC subtype identity and uncover EZH2 inhibition as an appealing strategy to induce subtype-switching in favor of a less aggressive PDAC phenotype. Significance: This study highlights the role of EZH2 in PDAC progression and molecular subtype identity and suggests EZH2 inhibition as a strategy to recalibrate GATA6 expression in favor of a less aggressive disease.

AbstractSchwann cells are the nerve ensheathing cells of the peripheral nervous system. Absence, loss and malfunction of Schwann cells or their myelin sheaths lead to peripheral neuropathies such as Charcot-Marie-Tooth disease in humans. During Schwann cell development and myelination chromatin is dramatically modified. However, impact and functional relevance of these modifications are poorly understood. Here, we analyzed histone H2B monoubiquitination as one such chromatin modification by conditionally deleting the Rnf40 subunit of the responsible E3 ligase in mice. Rnf40-deficient Schwann cells were arrested immediately before myelination or generated abnormally thin, unstable myelin, resulting in a peripheral neuropathy characterized by hypomyelination and progressive axonal degeneration. By combining sequencing techniques with functional studies we show that H2B monoubiquitination does not influence global gene expression patterns, but instead ensures selective high expression of myelin and lipid biosynthesis genes and proper repression of immaturity genes. This requires the specific recruitment of the Rnf40-containing E3 ligase by Egr2, the central transcriptional regulator of peripheral myelination, to its target genes. Our study identifies histone ubiquitination as essential for Schwann cell myelination and unravels new disease-relevant links between chromatin modifications and transcription factors in the underlying regulatory network.

Epigenetic alterations play a central role in cancer development and progression. The acetylation of histone 3 at lysine 27 (H3K27ac) specifically marks active genes. While chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) analyses are commonly performed in cell lines, only limited data are available from primary tumors. We therefore examined whether cancer-specific alterations in H3K27ac occupancy can be identified in primary rectal cancer. Tissue samples from primary rectal cancer and matched mucosa were obtained. ChIP-seq for H3K27ac was performed and differentially occupied regions were identified. The expression of selected genes displaying differential occupancy between tumor and mucosa were examined in gene expression data from an independent patient cohort. Differential expression of four proteins was further examined by immunohistochemistry. ChIP-seq for H3K27ac in primary rectal cancer and matched mucosa was successfully performed and revealed differential binding on 44 regions. This led to the identification of genes with increased H3K27ac, i.e., RIPK2, FOXQ1, KRT23, and EPHX4, which were also highly upregulated in primary rectal cancer in an independent dataset. The increased expression of these four proteins was confirmed by immunohistochemistry. This study demonstrates the feasibility of ChIP-seq-based epigenome mapping of primary rectal cancer and confirms the value of H3K27ac occupancy to predict gene expression differences.

Abstract TGFβ–SMAD signaling exerts a contextual effect that suppresses malignant growth early in epithelial tumorigenesis but promotes metastasis at later stages. Longstanding challenges in resolving this functional dichotomy may uncover new strategies to treat advanced carcinomas. The Krüppel-like transcription factor, KLF10, is a pivotal effector of TGFβ/SMAD signaling that mediates antiproliferative effects of TGFβ. In this study, we show how KLF10 opposes the prometastatic effects of TGFβ by limiting its ability to induce epithelial-to-mesenchymal transition (EMT). KLF10 depletion accentuated induction of EMT as assessed by multiple metrics. KLF10 occupied GC-rich sequences in the promoter region of the EMT-promoting transcription factor SLUG/SNAI2, repressing its transcription by recruiting HDAC1 and licensing the removal of activating histone acetylation marks. In clinical specimens of lung adenocarcinoma, low KLF10 expression associated with decreased patient survival, consistent with a pivotal role for KLF10 in distinguishing the antiproliferative versus prometastatic functions of TGFβ. Our results establish that KLF10 functions to suppress TGFβ-induced EMT, establishing a molecular basis for the dichotomy of TGFβ function during tumor progression. Cancer Res; 77(9); 2387–400. ©2017 AACR.

Abstract The Transforming Growth Factor-β (TGFβ)/SMAD signaling pathway can function as either a tumor suppressor or metastasis promoter during tumor progression. In normal epithelial cells and early stages of epithelial tumorigenesis TGFβ functions as a tumor suppressor to decrease cell proliferation or induce apoptosis. However, during malignant progression tumor cells no longer respond to the anti-proliferative effects of TGFβ, but instead undergo an epithelial-to-mesenchymal transition (EMT) whereby cells acquire a migratory and invasive phenotype which promotes tumor metastasis. Resolution of the dichotomy in TGFβ function and a further understanding of its tumor suppressor and metastasis promoting functions may uncover new strategies for the treatment of epithelial cancers. In previous studies we demonstrated an important role of the TGFβ-Inducible Early Gene-1 (TIEG1)/Krüppel-like Factor-10 (KLF10) as a central regulator of TGFβ/SMAD signaling and the anti-proliferative functions of TGFβ. In this study we examined the role of KLF10 in controlling the TGFβ-induced EMT in human lung adenocarcinoma cells. We show that depletion of KLF10 results in a more pronounced induction of EMT in human A549 lung adenocarcinoma cells as assessed by quantitative real-time PCR, changes in cellular morphology and immunofluorescence microscopy, and transcriptome-wide (RNA-seq) analyses. Moreover, chromatin immunoprecipitation (ChIP) and chromatin immunoprecipitation-sequencing (ChIP-seq) analysis shows that KLF10 directly binds to GC-rich sequences in the promoter region of the EMT-promoting transcription factor SLUG/SNAI2 to repress its transcription. Consistent with these findings, an analysis of KLF10 and SNAI2 expression in lung cancer revealed that while KLF10 levels are decreased in lung cancer vs. normal samples, SNAI2 levels are increased. Additional ChIP studies showed that KLF10 recruits HDAC1 to the SNAI2 promoter and is required for the removal of activating histone acetylation marks. These findings reveal a previously unknown function of KLF10 in suppressing TGFβ-induced EMT and represent a significant advancement in the understanding the molecular dichotomy of TGFβ function during tumor progression. Citation Format: Vivek Kumar Mishra, Vijayalakshmi Kari, Malayannan Subramaniam, Simon J. Baumgart, Sankari Nagarajan, Florian Wegwitz, Thomas C. Spelsberg, John R. Hawse, Steven A. Johnsen. Krüppel-like Transcription Factor-10 (KLF10) suppresses the TGFβ-induced epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A03.