Microenvironmental Determinants of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) belongs to the most lethal solid tumors in humans. A histological hallmark feature of PDAC is the pronounced tumor microenvironment (TME) that dynamically evolves during tumor progression. The TME consists of different non-neoplastic cells such as cancer-associated fibroblasts, immune cells, endothelial cells, and neurons. Furthermore, abundant extracellular matrix components such as collagen and hyaluronic acid as well as matricellular proteins create a highly dynamic and hypovascular TME with multiple biochemical and physical interactions among the various cellular and acellular components that promote tumor progression and therapeutic resistance. In recent years, intensive research efforts have resulted in a significantly improved understanding of the biology and pathophysiology of the TME in PDAC, and novel stroma-targeted approaches are emerging that may help to improve the devastating prognosis of PDAC patients. However, none of anti-stromal therapies has been approved in patients so far, and there is still a large discrepancy between multiple successful preclinical results and subsequent failure in clinical trials. Furthermore, recent findings suggest that parts of the TME may also possess tumor-restraining properties rendering tailored therapies even more challenging.

An NFATc1/SMAD3/cJUN Complex Restricted to SMAD4-Deficient Pancreatic Cancer Guides Rational Therapies.

BACKGROUND & AIMS: The highly heterogeneous cellular and molecular makeup of pancreatic ductal adenocarcinoma (PDAC) not only fosters exceptionally aggressive tumor biology, but contradicts the current concept of one-size-fits-all therapeutic strategies to combat PDAC. Therefore, we aimed to exploit the tumor biological implication and therapeutic vulnerabilities of a clinically relevant molecular PDAC subgroup characterized by SMAD4 deficiency and high expression of the nuclear factor of activated T cells (SMAD4-/-/NFATc1High). METHODS: Transcriptomic and clinical data were analyzed to determine the prognostic relevance of SMAD4-/-/NFATc1High cancers. In vitro and in vivo oncogenic transcription factor complex formation was studied by immunoprecipitation, proximity ligation assays, and validated cross model and species. The impact of SMAD4 status on therapeutically targeting canonical KRAS signaling was mechanistically deciphered and corroborated by genome-wide gene expression analysis and genetic perturbation experiments, respectively. Validation of a novel tailored therapeutic option was conducted in patient-derived organoids and cells and transgenic as well as orthotopic PDAC models. RESULTS: Our findings determined the tumor biology of an aggressive and chemotherapy-resistant SMAD4-/-/NFATc1High subgroup. Mechanistically, we identify SMAD4 deficiency as a molecular prerequisite for the formation of an oncogenic NFATc1/SMAD3/cJUN transcription factor complex, which drives the expression of RRM1/2. RRM1/2 replenishes nucleoside pools that directly compete with metabolized gemcitabine for DNA strand incorporation. Disassembly of the NFATc1/SMAD3/cJUN complex by mitogen-activated protein kinase signaling inhibition normalizes RRM1/2 expression and synergizes with gemcitabine treatment in vivo to reduce the proliferative index. CONCLUSIONS: Our results suggest that PDAC characterized by SMAD4 deficiency and oncogenic NFATc1/SMAD3/cJUN complex formation exposes sensitivity to a mitogen-activated protein kinase signaling inhibition and gemcitabine combination therapy.

NFATc1 signaling drives chronic ER stress responses to promote NAFLD progression.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) can persist in the stage of simple hepatic steatosis or progress to steatohepatitis (NASH) with an increased risk for cirrhosis and cancer. We examined the mechanisms controlling the progression to severe NASH in order to develop future treatment strategies for this disease. DESIGN: NFATc1 activation and regulation was examined in livers from patients with NAFLD, cultured and primary hepatocytes and in transgenic mice with differential hepatocyte-specific expression of the transcription factor (Alb-cre, NFATc1c.a . and NFATc1Δ/Δ ). Animals were fed with high-fat western diet (WD) alone or in combination with tauroursodeoxycholic acid (TUDCA), a candidate drug for NAFLD treatment. NFATc1-dependent ER stress-responses, NLRP3 inflammasome activation and disease progression were assessed both in vitro and in vivo. RESULTS: NFATc1 expression was weak in healthy livers but strongly induced in advanced NAFLD stages, where it correlates with liver enzyme values as well as hepatic inflammation and fibrosis. Moreover, high-fat WD increased NFATc1 expression, nuclear localisation and activation to promote NAFLD progression, whereas hepatocyte-specific depletion of the transcription factor can prevent mice from disease acceleration. Mechanistically, NFATc1 drives liver cell damage and inflammation through ER stress sensing and activation of the PERK-CHOP unfolded protein response (UPR). Finally, NFATc1-induced disease progression towards NASH can be blocked by TUDCA administration. CONCLUSION: NFATc1 stimulates NAFLD progression through chronic ER stress sensing and subsequent activation of terminal UPR signalling in hepatocytes. Interfering with ER stress-responses, for example, by TUDCA, protects fatty livers from progression towards manifest NASH.

Antithetical NFATc1-Sox2 and p53-miR200 signaling networks govern pancreatic cancer cell plasticity.

In adaptation to oncogenic signals, pancreatic ductal adenocarcinoma (PDAC) cells undergo epithelial-mesenchymal transition (EMT), a process combining tumor cell dedifferentiation with acquisition of stemness features. However, the mechanisms linking oncogene-induced signaling pathways with EMT and stemness remain largely elusive. Here, we uncover the inflammation-induced transcription factor NFATc1 as a central regulator of pancreatic cancer cell plasticity. In particular, we show that NFATc1 drives EMT reprogramming and maintains pancreatic cancer cells in a stem cell-like state through Sox2-dependent transcription of EMT and stemness factors. Intriguingly, NFATc1-Sox2 complex-mediated PDAC dedifferentiation and progression is opposed by antithetical p53-miR200c signaling, and inactivation of the tumor suppressor pathway is essential for tumor dedifferentiation and dissemination both in genetically engineered mouse models (GEMM) and human PDAC. Based on these findings, we propose the existence of a hierarchical signaling network regulating PDAC cell plasticity and suggest that the molecular decision between epithelial cell preservation and conversion into a dedifferentiated cancer stem cell-like phenotype depends on opposing levels of p53 and NFATc1 signaling activities.

Stapled EGFR peptide reduces inflammatory breast cancer and inhibits additional HER-driven models of cancer.

BACKGROUND: The human epidermal growth factor receptor (HER) family of transmembrane tyrosine kinases is overexpressed and correlates with poor prognosis and decreased survival in many cancers. The receptor family has been therapeutically targeted, yet tyrosine kinase inhibitors (TKIs) do not inhibit kinase-independent functions and antibody-based targeting does not affect internalized receptors. We have previously demonstrated that a peptide mimicking the internal juxtamembrane domain of HER1 (EGFR; EJ1) promotes the formation of non-functional HER dimers that inhibit kinase-dependent and kinase-independent functions of HER1 (ERBB1/EGFR), HER2 (ERBB2) and HER3 (ERBB3). Despite inducing rapid HER-dependent cell death in vitro, EJ1 peptides are rapidly cleared in vivo, limiting their efficacy. METHOD: To stabilize EJ1 activity, hydrocarbon staples (SAH) were added to the active peptide (SAH-EJ1), resulting in a 7.2-fold increase in efficacy and decreased in vivo clearance. Viability assays were performed across HER1 and HER2 expressing cell lines, therapeutic-resistant breast cancer cells, clinically relevant HER1-mutated lung cancer cells, and patient-derived glioblastoma cells, in all cases demonstrating improved efficacy over standard of care pan-HER therapeutics. Tumor burden studies were also performed in lung, glioblastoma, and inflammatory breast cancer mouse models, evaluating tumor growth and overall survival. RESULTS: When injected into mouse models of basal-like and inflammatory breast cancers, EGFRvIII-driven glioblastoma, and lung adenocarcinoma with Erlotinib resistance, tumor growth is inhibited and overall survival is extended. Studies evaluating the toxicity of SAH-EJ1 also demonstrate a broad therapeutic window. CONCLUSIONS: Taken together, these data indicate that SAH-EJ1 may be an effective therapeutic for HER-driven cancers with the potential to eliminate triple negative inflammatory breast cancer.

What affects utilization of malaria control services? A qualitative approach to understanding community perception in highly malarious Gadchiroli district, Maharashtra, India.

National Vector Borne Disease Control Programme (NVBDCP) data have shown that nearly half of all malaria deaths in India occur in tribal-dominated areas. The present study took a qualitative approach to understanding community perceptions and practices related to malarial infection and anti-malarial programmes. Twelve focus group discussions and 26 in-depth interviews of Accredited Social Health Activists (ASHAs) were conducted in nine villages in the district of Gadchiroli, Maharashtra state in India in June 2016. A total of 161 village residents (94 males and 67 females) participated in the focus group discussions and 26 health workers participated in the in-depth interviews. Data were analysed using the content analysis approach. The findings revealed widespread misconceptions about malaria among village residents, and low use of preventive measures and anti-malarial services. Ignorance and treatment by unqualified traditional healers delay effective treatment seeking. Furthermore, failure to maintain drug compliance adds to the gravity of the problem. The study identified the social and behavioural factors affecting treatment uptake and use of treatment facilities in the study area. These should help the development of the behavioural change communication arm of any control strategy for malaria through improving community participation, so improving preventive practices and optimizing utilization of anti-malarial services.

Senescence in pancreatic carcinogenesis: from signalling to chromatin remodelling and epigenetics.

Mutational activation of K-Ras is a key genetic event involved in the initiation of pancreatic carcinogenesis. However, K-Ras generally fails to transform precursor lesions into invasive cancers due to activation of powerful fail-safe programmes that counteract transformation and growth. The importance of cellular senescence, a permanent cell growth arrest, is increasingly being recognised as a critical fail-safe programme in pancreatic carcinogenesis. Emerging evidence suggests that oncogene-induced senescence requires transcriptional induction of the CDKN2A gene locus as well as comprehensive chromatin modifications involved in epigenetic silencing of pro-proliferative genes. Moreover, recent work in pancreatic cancer mouse models proposes that inactivation of the CDKN2A tumour suppressor locus is the molecular switch required for senescence evasion and unleashed K-Ras driven malignant transformation in the pancreas.

Innate and adaptive immune-directed tumour microenvironment in pancreatic ductal adenocarcinoma.

One of the most deadly and aggressive cancers in the world, pancreatic ductal adenocarcinoma (PDAC), typically manifests at an advanced stage. PDAC is becoming more common, and by the year 2030, it is expected to overtake lung cancer as the second greatest cause of cancer-related death. The poor prognosis can be attributed to a number of factors, including difficulties in early identification, a poor probability of curative radical resection, limited response to chemotherapy and radiotherapy, and its immunotherapy resistance. Furthermore, an extensive desmoplastic stroma that surrounds PDAC forms a mechanical barrier that prevents vascularization and promotes poor immune cell penetration. Phenotypic heterogeneity, drug resistance, and immunosuppressive tumor microenvironment are the main causes of PDAC aggressiveness. There is a complex and dynamic interaction between tumor cells in PDAC with stromal cells within the tumour immune microenvironment. The immune suppressive microenvironment that promotes PDAC aggressiveness is contributed by a range of cellular and humoral factors, which itself are modulated by the cancer. In this review, we describe the role of innate and adaptive immune cells, complex tumor microenvironment in PDAC, humoral factors, innate immune-mediated therapeutic advances, and recent clinical trials in PDAC.

Restricted heterochromatin formation links NFATc2 repressor activity with growth promotion in pancreatic cancer.

BACKGROUND & AIMS: Transcriptional silencing of the p15(INK4b) tumor suppressor pathway overcomes cellular protection against unrestrained proliferation in cancer. Here we show a novel pathway involving the oncogenic transcription factor nuclear factor of activated T cells (NFAT) c2 targeting a p15(INK4b)-mediated failsafe mechanism to promote pancreatic cancer tumor growth. METHODS: Immunohistochemistry, real-time polymerase chain reaction, immunoblotting, and immunofluorescence microscopy were used for expression studies. Cancer growth was assessed in vitro by [(3)H]thymidine incorporation, colony formation assays, and in vivo using xenograft tumor models. Protein-protein interactions, promoter regulation, and local histone modifications were analyzed by immunoprecipitation, DNA pull-down, reporter, and chromatin immunoprecipitation assays. RESULTS: Our study uncovered induction of NFATc2 in late-stage pancreatic intraepithelial neoplasia lesions with increased expression in tumor cell nuclei of advanced cancers. In the nucleus, NFATc2 targets the p15(INK4b) promoter for inducible heterochromatin formation and silencing. NFATc2 binding to its cognate promoter site induces stepwise recruitment of the histone methyltransferase Suv39H1, causes local H3K9 trimethylation, and allows docking of heterochromatin protein HP1γ to the repressor complex. Conversely, inactivation of NFATc2 disrupts this repressor complex assembly and local heterochromatin formation, resulting in restoration of p15(INK4b) expression and inhibition of pancreatic cancer growth in vitro and in vivo. CONCLUSIONS: Here we describe a novel mechanism for NFATc2-mediated gene regulation and identify a functional link among its repressor activity, the silencing of the suppressor pathway p15(INK4b), and its pancreatic cancer growth regulatory functions. Thus, we provide evidence that inactivation of oncogenic NFATc2 might be an attractive strategy in treatment of pancreatic cancer.

NFATc1 Is a Central Mediator of EGFR-Induced ARID1A Chromatin Dissociation During Acinar Cell Reprogramming.

BACKGROUND & AIMS: Loss of AT-rich interactive domain-containing protein 1A (ARID1A) fosters acinar-to-ductal metaplasia (ADM) and pancreatic carcinogenesis by down-regulating transcription programs controlling acinar cell identity. However, how ARID1A reacts to metaplasia-triggering environmental cues remains elusive. Here, we aimed to elucidate the role of ARID1A in controlling ductal pancreatic gene signatures and deciphering hierarchical signaling cues determining ARID1A-dependent chromatin regulation during acinar cell reprogramming. METHODS: Acinar cell explants with differential ARID1A status were subjected to genome-wide expression analyses. The impact of epidermal growth factor receptor (EGFR) signaling, NFATc1 activity, and ARID1A status on acinar reprogramming processes were characterized by ex vivo ADM assays and transgenic mouse models. EGFR-dependent ARID1A chromatin binding was studied by chromatin immunoprecipitation sequencing analysis and cellular fractionation. RESULTS: EGFR signaling interferes with ARID1A-dependent transcription by inducing genome-wide ARID1A displacement, thereby phenocopying ARID1A loss-of-function mutations and inducing a shift toward ADM permissive ductal transcription programs. Moreover, we show that EGFR signaling is required to push ARID1A-deficient acinar cells toward a metaplastic phenotype. Mechanistically, we identified the transcription factor nuclear factor of activated T cells 1 (NFATc1) as the central regulatory hub mediating both EGFR signaling-induced genomic ARID1A displacement and the induction of ADM-promoting gene signatures in the absence of ARID1A. Consequently, pharmacologic inhibition of NFATc1 or its depletion in transgenic mice not only preserves genome-wide ARID1A occupancy, but also attenuates acinar metaplasia led by ARID1A loss. CONCLUSIONS: Our data describe an intimate relationship between environmental signaling and chromatin remodeling in orchestrating cell fate decisions in the pancreas, and illustrate how ARID1A loss influences transcriptional regulation in acinar cell reprogramming.

Disruption of a nuclear NFATc2 protein stabilization loop confers breast and pancreatic cancer growth suppression by zoledronic acid.

The aminobisphosphonate zoledronic acid has elicited significant attention due to its remarkable anti-tumoral activity, although its detailed mechanism of action remains unclear. Here, we demonstrate the existence of a nuclear GSK-3ß-NFATc2 stabilization pathway that promotes breast and pancreatic cancer growth in vitro and in vivo and serves as a bona fide target of zoledronic acid. Specifically, the serine/threonine kinase GSK-3ß stabilizes nuclear NFATc2 through phosphorylation of the serine-rich SP2 domain, thus protecting the transcription factor from E3-ubiquitin ligase HDM2-mediated proteolysis. Zoledronic acid disrupts this NFATc2 stabilization pathway through two mechanisms, namely GSK-3ß inhibition and induction of HDM2 activity. Upon nuclear accumulation, HDM2 targets unphosphorylated NFATc2 for ubiquitination at acceptor lysine residues Lys-684/Lys-897 and hence labels the factor for subsequent proteasomal degradation. Conversely, mutagenesis-induced constitutive serine phosphorylation (Ser-215, Ser-219, and Ser-223) of the SP2 domain prevents NFATc2 from HDM2-mediated ubiquitination and degradation and consequently rescues cancer cells from growth suppression by zoledronic acid. In conclusion, this study demonstrates a critical role of the GSK-3ß-HDM2 signaling loop in the regulation of NFATc2 protein stability and growth promotion and suggests that double targeting of this pathway is responsible, at least to a significant part, for the potent and reliable anti-tumoral effects of zoledronic acid.

Mechanisms of PDAC subtype heterogeneity and therapy response.

Pancreatic ductal adenocarcinoma (PDAC) is clinically challenging due to late diagnosis and resistance to therapy. Two major PDAC subtypes have been defined based on malignant epithelial cell gene expression profiles; the basal-like/squamous subtype is associated with a worse prognosis and therapeutic resistance as opposed to the classical subtype. Subtype specification is not binary, consistent with plasticity of malignant cell phenotype. PDAC heterogeneity and plasticity reflect partly malignant cell-intrinsic transcriptional and epigenetic regulation. However, the stromal and immune compartments of the tumor microenvironment (TME) also determine disease progression and therapy response. It is evident that integration of intrinsic and extrinsic factors can dictate subtype heterogeneity, and thus, delineating the pathways involved can help to reprogram PDAC towards a classical/druggable subtype.

TP53-Status-Dependent Oncogenic EZH2 Activity in Pancreatic Cancer.

Pancreatic Ductal Adenocarcinoma (PDAC) represents a lethal malignancy with a consistently poor outcome. Besides mutations in PDAC driver genes, the aggressive tumor biology of the disease and its remarkable therapy resistance are predominantly installed by potentially reversible epigenetic dysregulation. However, epigenetic regulators act in a context-dependent manner with opposing implication on tumor progression, thus critically determining the therapeutic efficacy of epigenetic targeting. Herein, we aimed at exploring the molecular prerequisites and underlying mechanisms of oncogenic Enhancer of Zeste Homolog 2 (EZH2) activity in PDAC progression. Preclinical studies in EZH2 proficient and deficient transgenic and orthotopic in vivo PDAC models and transcriptome analysis identified the TP53 status as a pivotal context-defining molecular cue determining oncogenic EZH2 activity in PDAC. Importantly, the induction of pro-apoptotic gene signatures and processes as well as a favorable PDAC prognosis upon EZH2 depletion were restricted to p53 wildtype (wt) PDAC subtypes. Mechanistically, we illustrate that EZH2 blockade de-represses CDKN2A transcription for the subsequent posttranslational stabilization of p53wt expression and function. Together, our findings suggest an intact CDKN2A-p53wt axis as a prerequisite for the anti-tumorigenic consequences of EZH2 depletion and emphasize the significance of molecular stratification for the successful implementation of epigenetic targeting in PDAC.

Axon guidance receptor ROBO3 modulates subtype identity and prognosis via AXL-associated inflammatory network in pancreatic cancer.

Metastatic pancreatic cancer (PDAC) has a poor clinical outcome with a 5-year survival rate below 3%. Recent transcriptome profiling of PDAC biopsies has identified 2 clinically distinct subtypes - the "basal-like" (BL) subtype with poor prognosis and therapy resistance compared with the less aggressive and drug-susceptible "classical" (CLA) subtype. However, the mechanistic events and environmental factors that promote the BL subtype identity are not very clear. Using preclinical models, patient-derived xenografts, and FACS-sorted PDAC patient biopsies, we report here that the axon guidance receptor, roundabout guidance receptor 3 (ROBO3), promotes the BL metastatic program via a potentially unique AXL/IL-6/phosphorylated STAT3 (p-STAT3) regulatory axis. RNA-Seq identified a ROBO3-mediated BL-specific gene program, while tyrosine kinase profiling revealed AXL as the key mediator of the p-STAT3 activation. CRISPR/dCas9-based ROBO3 silencing disrupted the AXL/p-STAT3 signaling axis, thereby halting metastasis and enhancing therapy sensitivity. Transcriptome analysis of resected patient tumors revealed that AXLhi neoplastic cells associated with the inflammatory stromal program. Combining AXL inhibitor and chemotherapy substantially restored a CLA phenotypic state and reduced disease aggressiveness. Thus, we conclude that a ROBO3-driven hierarchical network determines the inflammatory and prometastatic programs in a specific PDAC subtype.

Organoids at the PUB: The Porcine Urinary Bladder Serves as a Pancreatic Niche for Advanced Cancer Modeling.

Despite intensive research and progress in personalized medicine, pancreatic ductal adenocarcinoma remains one of the deadliest cancer entities. Pancreatic duct-like organoids (PDLOs) derived from human pluripotent stem cells (PSCs) or pancreatic cancer patient-derived organoids (PDOs) provide unique tools to study early and late stage dysplasia and to foster personalized medicine. However, such advanced systems are neither rapidly nor easily accessible and require an in vivo niche to study tumor formation and interaction with the stroma. Here, the establishment of the porcine urinary bladder (PUB) is revealed as an advanced organ culture model for shaping an ex vivo pancreatic niche. This model allows pancreatic progenitor cells to enter the ductal and endocrine lineages, while PDLOs further mature into duct-like tissue. Accordingly, the PUB offers an ex vivo platform for earliest pancreatic dysplasia and cancer if PDLOs feature KRASG12D mutations. Finally, it is demonstrated that PDOs-on-PUB i) resemble primary pancreatic cancer, ii) preserve cancer subtypes, iii) enable the study of niche epithelial crosstalk by spiking in pancreatic stellate and immune cells into the grafts, and finally iv) allow drug testing. In summary, the PUB advances the existing pancreatic cancer models by adding feasibility, complexity, and customization at low cost and high flexibility.

Sequential activation of NFAT and c-Myc transcription factors mediates the TGF-beta switch from a suppressor to a promoter of cancer cell proliferation.

Transforming growth factor beta (TGF-beta) has a dual role in carcinogenesis, acting as a growth inhibitor in early tumor stages and a promoter of cell proliferation in advanced diseases. Although this cellular phenomenon is well established, the underlying molecular mechanisms remain elusive. Here, we report that sequential induction of NFAT and c-Myc transcription factors is sufficient and required for the TGF-beta switch from a cell cycle inhibitor to a growth promoter pathway in cancer cells. Mechanistically, TGF-beta induces in a calcineurin-dependent manner the expression and activation of NFAT factors, which then translocate into the nucleus to promote c-Myc expression. In response to TGF-beta, activated NFAT factors bind to and displace Smad3 repressor complexes from the previously identified TGF-beta inhibitory element (TIE) to transactivate the c-Myc promoter. c-Myc in turn stimulates cell cycle progression and growth through up-regulation of D-type cyclins. Most importantly, NFAT knockdown not only prevents c-Myc activation and cell proliferation, but also partially restores TGF-beta-induced cell cycle arrest and growth suppression. Taken together, this study provides the first evidence for a Smad-independent master regulatory pathway in TGF-beta-promoted cell growth that is defined by sequential transcriptional activation of NFAT and c-Myc factors.

The Gain-of-Function p53 R248W Mutant Promotes Migration by STAT3 Deregulation in Human Pancreatic Cancer Cells.

Missense p53 mutations (mutp53) occur in approx. 70% of pancreatic ductal adenocarcinomas (PDAC). Typically, mutp53 proteins are aberrantly stabilized by Hsp90/Hsp70/Hsp40 chaperone complexes. Notably, stabilization is a precondition for specific mutp53 alleles to acquire powerful neomorphic oncogenic gain-of-functions (GOFs) that promote tumor progression in solid cancers mainly by increasing invasion and metastasis. In colorectal cancer (CRC), we recently established that the common hotspot mutants mutp53R248Q and mutp53R248W exert GOF activities by constitutively binding to and hyperactivating STAT3. This results in increased proliferation and invasion in an autochthonous CRC mouse model and correlates with poor survival in patients. Comparing a panel of p53 missense mutations in a series of homozygous human PDAC cell lines, we show here that, similar to CRC, the mutp53R248W protein again undergoes a strong Hsp90-mediated stabilization and selectively promotes migration. Highly stabilized mutp53 is degradable by the Hsp90 inhibitors Onalespib and Ganetespib, and correlates with growth suppression, possibly suggesting therapeutic vulnerabilities to target GOF mutp53 proteins in PDAC. In response to mutp53 depletion, only mutp53R248W harboring PDAC cells show STAT3 de-phosphorylation and reduced migration, again suggesting an allele-specific GOF in this cancer entity, similar to CRC. Moreover, mutp53R248W also exhibits the strongest constitutive complex formation with phosphorylated STAT3. The selective mutp53R248W GOF signals through enhancing the STAT3 axis, which was confirmed since targeting STAT3 by knockdown or pharmacological inhibition phenocopied mutp53 depletion and reduced cell viability and migration preferentially in mutp53R248W-containing PDAC cells. Our results confirm that mutp53 GOF activities are allele specific and can span across tumor entities.

Chromatin-Independent Interplay of NFATc1 and EZH2 in Pancreatic Cancer.

BACKGROUND: The Nuclear Factor of Activated T-cells 1 (NFATc1) transcription factor and the methyltransferase Enhancer of Zeste Homolog 2 (EZH2) significantly contribute to the aggressive phenotype of pancreatic ductal adenocarcinoma (PDAC). Herein, we aimed at dissecting the mechanistic background of their interplay in PDAC progression. METHODS: NFATc1 and EZH2 mRNA and protein expression and complex formation were determined in transgenic PDAC models and human PDAC specimens. NFATc1 binding on the Ezh2 gene and the consequences of perturbed NFATc1 expression on Ezh2 transcription were explored by Chromatin Immunoprecipitation (ChIP) and upon transgenic or siRNA-mediated interference with NFATc1 expression, respectively. Integrative analyses of RNA- and ChIP-seq data was performed to explore NFATc1-/EZH2-dependent gene signatures. RESULTS: NFATc1 targets the Ezh2 gene for transcriptional activation and biochemically interacts with the methyltransferase in murine and human PDAC. Surprisingly, our genome-wide binding and expression analyses do not link the protein complex to joint gene regulation. In contrast, our findings provide evidence for chromatin-independent functions of the NFATc1:EZH2 complex and reveal posttranslational EZH2 phosphorylation at serine 21 as a prerequisite for robust complex formation. CONCLUSION: Our findings disclose a previously unknown NFATc1-EZH2 axis operational in the pancreas and provide mechanistic insights into the conditions fostering NFATc1:EZH2 complex formation in PDAC.

Comprehensive Wet-Bench and Bioinformatics Workflow for Complex Microbiota Using Oxford Nanopore Technologies.

The advent of high-throughput sequencing techniques has recently provided an astonishing insight into the composition and function of the human microbiome. Next-generation sequencing (NGS) has become the gold standard for advanced microbiome analysis; however, 3rd generation real-time sequencing, such as Oxford Nanopore Technologies (ONT), enables rapid sequencing from several kilobases to >2 Mb with high resolution. Despite the wide availability and the enormous potential for clinical and translational applications, ONT is poorly standardized in terms of sampling and storage conditions, DNA extraction, library creation, and bioinformatic classification. Here, we present a comprehensive analysis pipeline with sampling, storage, DNA extraction, library preparation, and bioinformatic evaluation for complex microbiomes sequenced with ONT. Our findings from buccal and rectal swabs and DNA extraction experiments indicate that methods that were approved for NGS microbiome analysis cannot be simply adapted to ONT. We recommend using swabs and DNA extractions protocols with extended washing steps. Both 16S rRNA and metagenomic sequencing achieved reliable and reproducible results. Our benchmarking experiments reveal thresholds for analysis parameters that achieved excellent precision, recall, and area under the precision recall values and is superior to existing classifiers (Kraken2, Kaiju, and MetaMaps). Hence, our workflow provides an experimental and bioinformatic pipeline to perform a highly accurate analysis of complex microbial structures from buccal and rectal swabs. IMPORTANCE Advanced microbiome analysis relies on sequencing of short DNA fragments from microorganisms like bacteria, fungi, and viruses. More recently, long fragment DNA sequencing of 3rd generation sequencing has gained increasing importance and can be rapidly conducted within a few hours due to its potential real-time sequencing. However, the analysis and correct identification of the microbiome relies on a multitude of factors, such as the method of sampling, DNA extraction, sequencing, and bioinformatic analysis. Scientists have used different protocols in the past that do not allow us to compare results across different studies and research fields. Here, we provide a comprehensive workflow from DNA extraction, sequencing, and bioinformatic workflow that allows rapid and accurate analysis of human buccal and rectal swabs with reproducible protocols. This workflow can be readily applied by many scientists from various research fields that aim to use long-fragment microbiome sequencing.

TNF-α-producing macrophages determine subtype identity and prognosis via AP1 enhancer reprogramming in pancreatic cancer.

Large-scale genomic profiling of pancreatic cancer (PDAC) has revealed two distinct subtypes: 'classical' and 'basal-like'. Their variable coexistence within the stromal immune microenvironment is linked to differential prognosis; however, the extent to which these neoplastic subtypes shape the stromal immune landscape and impact clinical outcome remains unclear. By combining preclinical models, patient-derived xenografts, as well as FACS-sorted PDAC patient biopsies, we show that the basal-like neoplastic state is sustained via BRD4-mediated cJUN/AP1 expression, which induces CCL2 to recruit tumor necrosis factor (TNF)-α-secreting macrophages. TNF-α+ macrophages force classical neoplastic cells into an aggressive phenotypic state via lineage reprogramming. Integration of ATAC-, ChIP- and RNA-seq data revealed distinct JUNB/AP1 (classical) and cJUN/AP1 (basal-like)-driven regulation of PDAC subtype identity. Pharmacological inhibition of BRD4 led to suppression of the BRD4-cJUN-CCL2-TNF-α axis, restoration of classical subtype identity and a favorable prognosis. Hence, patient-tailored therapy for a cJUNhigh/TNF-αhigh subtype is paramount in overcoming highly inflamed and aggressive PDAC states.