Citations

Background Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with a 5-year survival rate of 12%. It has two major molecular subtypes: classical and basal, regulated by the master transcription factors (MTFs) GATA6 and ΔNp63, respectively. Objective This study sought to uncover the transcriptional regulatory mechanisms controlling PDAC subtype identity. Design We integrated primary tumour single-cell RNA-seq, patient-derived xenograft RNA-seq and multispectral imaging to identify MTF-dependent, subtype-specific markers. We created subtype-specific fluorescent reporter systems and conducted drug screenings to find actionable targets. We analysed chromatin accessibility (ATAC-seq), genome-wide occupancy (ChIP-seq) for epigenetic status (H3K27ac), MTFs (GATA6, ΔNp63), RNA polymerase II (Pol II), H3K4me3-anchored chromatin topology (HiChIP) and nascent RNA capture sequencing (PRO-seq). Additionally, we used nuclease-dead Cas9 (dCas9) to manipulate transcriptional regulatory mechanisms. Results Our approach identified glucocorticoid receptor (GR) agonists as agents that suppress the classical transcriptional programme by interacting with GATA6. GATA6 regulates classical-specific transcription through promoter-proximal pause release. Depletion of GATA6 increased Pol II occupancy at GATA6-bound enhancers and transcriptional start sites, stabilising enhancer–promoter interactions. Artificially inducing pausing at GATA6-bound enhancers with dCas9 abrogated target gene expression and induced pausing at both the enhancer and target gene promoter. Conversely, in basal PDAC ΔNp63 promotes Pol II recruitment and stabilises enhancer–promoter interactions. Conclusion This study provides new insights into the transcriptional control and role of GR agonists in controlling PDAC molecular subtype identity.

Abstract Pancreatic ductal adenocarcinoma (PDAC) displays a dismal prognosis due to late diagnosis and high chemoresistance incidence. For advanced disease stages or patients with comorbidities, treatment options are limited to gemcitabine alone or in combination with other drugs. While gemcitabine resistance has been widely attributed to the levels of one of its targets, RRM1, the molecular consequences of gemcitabine resistance in PDAC remain largely elusive. Here we sought to identify genomic, epigenomic, and transcriptomic events associated with gemcitabine resistance in PDAC and their potential clinical relevance. We found that gemcitabine-resistant cells displayed a coamplification of the adjacent RRM1 and STIM1 genes. Interestingly, RRM1, but not STIM1, was required for gemcitabine resistance, while high STIM1 levels caused an increase in cytosolic calcium concentration. Higher STIM1-dependent calcium influx led to an impaired endoplasmic reticulum stress response and a heightened nuclear factor of activated T-cell activity. Importantly, these findings were confirmed in patient and patient-derived xenograft samples. Taken together, our study uncovers previously unknown biologically relevant molecular properties of gemcitabine-resistant tumors, revealing an undescribed function of STIM1 as a rheostat directing the effects of calcium signaling and controlling epigenetic cell fate determination. It further reveals the potential benefit of targeting STIM1-controlled calcium signaling and its downstream effectors in PDAC. Significance: Gemcitabine-resistant and some naïve tumors coamplify RRM1 and STIM1, which elicit gemcitabine resistance and induce a calcium signaling shift, promoting ER stress resistance and activation of NFAT signaling.

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.

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 We aimed to investigate the mechanistic, functional, and therapeutic role of glycogen synthase kinase 3β (GSK-3β) in the regulation and activation of the proinflammatory oncogenic transcription factor nuclear factor of activated T cells (NFATc2) in pancreatic cancer. IHC, qPCR, immunoblotting, immunofluorescence microscopy, and proliferation assays were used to analyze mouse and human tissues and cell lines. Protein–protein interactions and promoter regulation were analyzed by coimmunoprecipitation, DNA pulldown, reporter, and ChIP assays. Preclinical assays were performed using a variety of pancreatic cancer cells lines, xenografts, and a genetically engineered mouse model (GEMM). GSK-3β–dependent SP2 phosphorylation mediates NFATc2 protein stability in the nucleus of pancreatic cancer cells stimulating pancreatic cancer growth. In addition to protein stabilization, GSK-3β also maintains NFATc2 activation through a distinct mechanism involving stabilization of NFATc2–STAT3 complexes independent of SP2 phosphorylation. For NFATc2–STAT3 complex formation, GSK-3β–mediated phosphorylation of STAT3 at Y705 is required to stimulate euchromatin formation of NFAT target promoters, such as cyclin-dependent kinase-6, which promotes tumor growth. Finally, preclinical experiments suggest that targeting the NFATc2–STAT3–GSK-3β module inhibits proliferation and tumor growth and interferes with inflammation-induced pancreatic cancer progression in KrasG12D mice. In conclusion, we describe a novel mechanism by which GSK-3β fine-tunes NFATc2 and STAT3 transcriptional networks to integrate upstream signaling events that govern pancreatic cancer progression and growth. Furthermore, the therapeutic potential of GSK-3β is demonstrated for the first time in a relevant Kras and inflammation-induced GEMM for pancreatic cancer. Mol Cancer Ther; 15(3); 491–502. ©2016 AACR.

Abstract Cancer-associated inflammation is a molecular key feature in pancreatic ductal adenocarcinoma. Oncogenic KRAS in conjunction with persistent inflammation is known to accelerate carcinogenesis, although the underlying mechanisms remain poorly understood. Here, we outline a novel pathway whereby the transcription factors NFATc1 and STAT3 cooperate in pancreatic epithelial cells to promote KrasG12D-driven carcinogenesis. NFATc1 activation is induced by inflammation and itself accelerates inflammation-induced carcinogenesis in KrasG12D mice, whereas genetic or pharmacologic ablation of NFATc1 attenuates this effect. Mechanistically, NFATc1 complexes with STAT3 for enhancer–promoter communications at jointly regulated genes involved in oncogenesis, for example, Cyclin, EGFR and WNT family members. The NFATc1–STAT3 cooperativity is operative in pancreatitis-mediated carcinogenesis as well as in established human pancreatic cancer. Together, these studies unravel new mechanisms of inflammatory-driven pancreatic carcinogenesis and suggest beneficial effects of chemopreventive strategies using drugs that are currently available for targeting these factors in clinical trials. Significance: Our study points to the existence of an oncogenic NFATc1–STAT3 cooperativity that mechanistically links inflammation with pancreatic cancer initiation and progression. Because NFATc1–STAT3 nucleoprotein complexes control the expression of gene networks at the intersection of inflammation and cancer, our study has significant relevance for potentially managing pancreatic cancer and other inflammatory-driven malignancies. Cancer Discov; 4(6); 688–701. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 621