Vivek Kumar Mishra

BRD4 localization to lineage-specific enhancers is associated with a distinct transcription factor repertoire

Proper temporal epigenetic regulation of gene expression is essential for cell fate determination and tissue development. The Bromodomain-containing Protein-4 (BRD4) was previously shown to control the transcription of defined subsets of genes in various cell systems. In this study we examined the role of BRD4 in promoting lineage-specific gene expression and show that BRD4 is essential for osteoblast differentiation. Genome-wide analyses demonstrate that BRD4 is recruited to the transcriptional start site of differentiation-induced genes. Unexpectedly, while promoter-proximal BRD4 occupancy correlated with gene expression, genes which displayed moderate expression and promoter-proximal BRD4 occupancy were most highly regulated and sensitive to BRD4 inhibition. Therefore, we examined distal BRD4 occupancy and uncovered a specific co-localization of BRD4 with the transcription factors C/EBPb, TEAD1, FOSL2 and JUND at putative osteoblast-specific enhancers. These findings reveal the intricacies of lineage specification and provide new insight into the context-dependent functions of BRD4.

Histone deacetylase class-I inhibition promotes epithelial gene expression in pancreatic cancer cells in a BRD4- and MYC-dependent manner

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a particularly dismal prognosis. Histone deacetylases (HDAC) are epigenetic modulators whose activity is frequently deregulated in various cancers including PDAC. In particular, class-I HDACs (HDAC 1, 2, 3 and 8) have been shown to play an important role in PDAC. In this study, we investigated the effects of the class I-specific HDAC inhibitor (HDACi) 4SC-202 in multiple PDAC cell lines in promoting tumor cell differentiation. We show that 4SC-202 negatively affects TGFβ signaling and inhibits TGFβ-induced epithelial-to-mesenchymal transition (EMT). Moreover, 4SC-202 markedly induced p21 (CDKN1A) expression and significantly attenuated cell proliferation. Mechanistically, genome-wide studies revealed that 4SC-202-induced genes were enriched for Bromodomain-containing Protein-4 (BRD4) and MYC occupancy. BRD4, a well-characterized acetyllysine reader, has been shown to play a major role in regulating transcription of selected subsets of genes. Importantly, BRD4 and MYC are essential for the expression of a subgroup of genes induced by class-I HDACi. Taken together, our study uncovers a previously unknown role of BRD4 and MYC in eliciting the HDACi-mediated induction of a subset of genes and provides molecular insight into the mechanisms of HDACi action in PDAC.

Krüppel-like Transcription Factor KLF10 Suppresses TGFβ-Induced Epithelial-to-Mesenchymal Transition via a Negative Feedback Mechanism

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.

BRD4 promotes p63 and GRHL3 expression downstream of FOXO in mammary epithelial cells.

Abstract Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression.

Targeted therapy of epigenomic regulatory mechanisms controlling the epithelial to mesenchymal transition during tumor progression

The epithelial-to-mesenchymal transition (EMT) is a reversible change in cell phenotype that plays a crucial role during normal development and cancer metastasis. EMT imparts embryonic epithelial cells with the ability to migrate and to give rise to organs or tissues at distant sites. During cancer progression, the same developmental process is utilized in an analogous manner to enable cancer cells to move to distant organs and form metastases. The reversion of EMT via the mesenchymal-to-epithelial transition (MET) appears to be required for the formation of secondary tumors at distal sites. The plasticity of epigenomic modifications that control the transcriptional program of cells enables cells to switch back and forth from epithelial and mesenchymal phenotypes during these transitions. Here, we review the interplay between complex epigenomic regulatory mechanisms and various transcription factors involved in EMT leading to changes in gene expression and cell phenotype. We also discuss the way that a deeper understanding of the epigenomic regulation of EMT might shed light onto the process of cancer progression and reveal new targets for novel and more specific anticancer epigenomic therapies.

Twist1 induces distinct cell states depending on TGFBR1-activation

Basic helix-loop-helix transcription factor Twist1 is a master regulator of Epithelial-Mesenchymal Transition (EMT), a cellular program implicated in different stages of development as well as metastatic dissemination of carcinomas. Here, we show that Twist1 requires TGF-beta type-I receptor (TGFBR1)-activation to bind an enhancer region of downstream effector ZEB1, thereby inducing ZEB1 transcription and EMT. When TGFBR1-phosphorylation is inhibited, Twist1 generates a distinct cell state characterized by collective invasion, simultaneous proliferation and expression of endothelial markers. By contrast, TGFBR1-activation directs Twist1 to induce stable mesenchymal transdifferentiation through EMT, thereby generating cells that display single-cell invasion, but lose their proliferative capacity. In conclusion, preventing Twist1-induced EMT by inhibiting TGFβ-signaling does not generally block acquisition of invasion, but switches mode from single-cell/non-proliferative to collective/proliferative. Together, these data reveal that transient Twist1-activation induces distinct cell states depending on signaling context and caution against the use of TGFβ-inhibitors as a therapeutic strategy to target invasiveness.

Myc and the Tip60 chromatin remodeling complex control neuroblast maintenance and polarity in Drosophila

Stem cells establish cortical polarity and divide asymmetrically to simultaneously maintain themselves and generate differentiating offspring cells. Several chromatin modifiers have been identified as stemness factors in mammalian pluripotent stem cells, but whether these factors control stem cell polarity and asymmetric division has not been investigated so far. We addressed this question in Drosophila neural stem cells called neuroblasts. We identified the Tip60 chromatin remodeling complex and its interaction partner Myc as regulators of genes required for neuroblast maintenance. Knockdown of Tip60 complex members results in loss of cortical polarity, symmetric neuroblast division, and premature differentiation through nuclear entry of the transcription factor Prospero. We found that aPKC is the key target gene of Myc and the Tip60 complex subunit Domino in regulating neuroblast polarity. Our transcriptome analysis further showed that Domino regulates the expression of mitotic spindle genes previously identified as direct Myc targets. Our findings reveal an evolutionarily conserved functional link between Myc, the Tip60 complex, and the molecular network controlling cell polarity and asymmetric cell division.

Abstract A03: Krüppel-like Transcription Factor-10 (KLF10) suppresses the TGFβ-induced epithelial-to-mesenchymal transition

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)/Kruppel-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. Kruppel-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.