SAMD1 suppresses epithelial-mesenchymal transition pathways in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) poses a significant threat due to its tendency to evade early detection, frequent metastasis, and the subsequent challenges in devising effective treatments. Processes that govern epithelial-mesenchymal transition (EMT) in PDAC hold promise for advancing novel therapeutic strategies. SAMD1 (SAM domain-containing protein 1) is a CpG island-binding protein that plays a pivotal role in the repression of its target genes. Here, we revealed that SAMD1 acts as a repressor of genes associated with EMT. Upon deletion of SAMD1 in PDAC cells, we observed significantly increased migration rates. SAMD1 exerts its effects by binding to specific genomic targets, including CDH2, encoding N-cadherin, which emerged as a driver of enhanced migration upon SAMD1 knockout. Furthermore, we discovered the FBXO11-containing E3 ubiquitin ligase complex as an interactor and negative regulator of SAMD1, which inhibits SAMD1 chromatin-binding genome-wide. High FBXO11 expression in PDAC is associated with poor prognosis and increased expression of EMT-related genes, underlining an antagonistic relationship between SAMD1 and FBXO11. In summary, our findings provide insights into the regulation of EMT-related genes in PDAC, shedding light on the intricate role of SAMD1 and its interplay with FBXO11 in this cancer type.

Synthetic lethality between ATR and POLA1 reveals a potential new target for individualized cancer therapy.

The ATR-CHK1 pathway plays a fundamental role in the DNA damage response and is therefore an attractive target in cancer therapy. The antitumorous effect of ATR inhibitors is at least partly caused by synthetic lethality between ATR and various DNA repair genes. In previous studies, we have identified members of the B-family DNA polymerases as potential lethal partner for ATR, i.e. POLD1 and PRIM1. In this study, we validated and characterized the synthetic lethality between ATR and POLA1. First, we applied a model of ATR-deficient DLD-1 human colorectal cancer cells to confirm synthetic lethality by using chemical POLA1 inhibition. Analyzing cell cycle and apoptotic markers via FACS and Western blotting, we were able to show that apoptosis and S phase arrest contributed to the increased sensitivity of ATR-deficient cancer cells towards POLA1 inhibitors. Importantly, siRNA-mediated POLA1 depletion in ATR-deficient cells caused similar effects in regard to impaired cell viability and cumulation of apoptotic markers, thus excluding toxic effects of chemical POLA1 inhibition. Conversely, we demonstrated that siRNA-mediated POLA1 depletion sensitized several cancer cell lines towards chemical inhibition of ATR and its main effector kinase CHK1. In conclusion, the synthetic lethality between ATR/CHK1 and POLA1 might represent a novel and promising approach for individualized cancer therapy: First, alterations of POLA1 could serve as a screening parameter for increased sensitivity towards ATR and CHK1 inhibitors. Second, alterations in the ATR-CHK1 pathway might predict in increased sensitivity towards POLA1 inhibitors.

BAG6 restricts pancreatic cancer progression by suppressing the release of IL33-presenting extracellular vesicles and the activation of mast cells.

Recent studies reveal a critical role of tumor cell-released extracellular vesicles (EVs) in pancreatic cancer (PC) progression. However, driver genes that direct EV function, the EV-recipient cells, and their cellular response to EV uptake remain to be identified. Therefore, we studied the role of Bcl-2-associated-anthanogene 6 (BAG6), a regulator of EV biogenesis for cancer progression. We used a Cre recombinase/LoxP-based reporter system in combination with single-cell RNA sequencing to monitor in vivo EV uptake and tumor microenvironment (TME) changes in mouse models for pancreatic ductal adenocarcinoma (PDAC) in a Bag6 pro- or deficient background. In vivo data were validated using mouse and human organoids and patient samples. Our data demonstrated that Bag6-deficient subcutaneous and orthotopic PDAC tumors accelerated tumor growth dependent on EV release. Mechanistically, this was attributed to mast cell (MC) activation via EV-associated IL33. Activated MCs promoted tumor cell proliferation and altered the composition of the TME affecting fibroblast polarization and immune cell infiltration. Tumor cell proliferation and fibroblast polarization were mediated via the MC secretome containing high levels of PDGF and CD73. Patients with high BAG6 gene expression and high protein plasma level have a longer overall survival indicating clinical relevance. The current study revealed a so far unknown tumor-suppressing activity of BAG6 in PDAC. Bag6-deficiency allowed the release of EV-associated IL33 which modulate the TME via MC activation promoting aggressive tumor growth. MC depletion using imatinib diminished tumor growth providing a scientific rationale to consider imatinib for patients stratified with low BAG6 expression and high MC infiltration. EVs derived from BAG6-deficient pancreatic cancer cells induce MC activation via IL33/Il1rl1. The secretome of activated MCs induces tumor proliferation and changes in the TME, particularly shifting fibroblasts into an inflammatory cancer-associated fibroblast (iCAF) phenotype. Blocking EVs or depleting MCs restricts tumor growth.

T-bet+ B cells are activated by and control endogenous retroviruses through TLR-dependent mechanisms.

Endogenous retroviruses (ERVs) are an integral part of the mammalian genome. The role of immune control of ERVs in general is poorly defined as is their function as anti-cancer immune targets or drivers of autoimmune disease. Here, we generate mouse-strains where Moloney-Murine Leukemia Virus tagged with GFP (ERV-GFP) infected the mouse germline. This enables us to analyze the role of genetic, epigenetic and cell intrinsic restriction factors in ERV activation and control. We identify an autoreactive B cell response against the neo-self/ERV antigen GFP as a key mechanism of ERV control. Hallmarks of this response are spontaneous ERV-GFP+ germinal center formation, elevated serum IFN-γ levels and a dependency on Age-associated B cells (ABCs) a subclass of T-bet+ memory B cells. Impairment of IgM B cell receptor-signal in nucleic-acid sensing TLR-deficient mice contributes to defective ERV control. Although ERVs are a part of the genome they break immune tolerance, induce immune surveillance against ERV-derived self-antigens and shape the host immune response.

Feasibility of the chick chorioallantoic membrane model for preclinical studies on tumor radiofrequency ablation.

BACKGROUND: We evaluated the feasibility of a chick chorioallantoic membrane (CAM) tumor model for preclinical research on tumor radiofrequency ablation (RFA). METHODS: Fertilized chicken eggs were incubated and divided into five cohorts: RFA for 30 s (n = 5), RFA for 60 s (n = 5), RFA for 120 s (n = 4), sham (n = 8), and controls (n = 6). Xenografting using pancreatic neuroendocrine tumor cells of the BON-1 cell line was performed on embryonic day (ED) 8. The RFA was performed on ED 12. Survival, stereomicroscopic observations, and histological observations using hematoxylin-eosin (H&E) and Ki67 staining were evaluated. RESULTS: The survival rates in the 30-s, 60-s, and 120-s, sham and control cohort were 60%, 60%, 0%, 100%, and 50%, respectively. Signs of bleeding and heat damage were common findings in the evaluation of stereomicroscopic observations. Histological examination could be performed in all but one embryo. Heat damage, bleeding, thrombosis, and leukocyte infiltration and hyperemia were regular findings in H&E-stained cuts. A complete absence of Ki67 staining was recorded in 33.3% and 50% of embryos in the 30-s and 60-s cohorts that survived until ED 14, respectively. CONCLUSIONS: The CAM model is a feasible and suiting research model for tumor RFA with many advantages over other animal models. It offers the opportunity to conduct in vivo research under standardized conditions. Further studies are needed to optimize this model for tumor ablations in order to explore promising but unrefined strategies like the combination of RFA and immunotherapy. RELEVANCE STATEMENT: The chick chorioallantoic membrane model allows in vivo research on tumor radiofrequency ablation under standardized conditions that may enable enhanced understanding on combined therapies while ensuring animal welfare in concordance with the "Three Rs." KEY POINTS: • The chorioallantoic membrane model is feasible and suiting for tumor radiofrequency ablation. • Radiofrequency ablation regularly achieved reduction but not eradication of Ki67 staining. • Histological evaluation showed findings comparable to changes in humans after RFA. • The chorioallantoic membrane model can enable studies on combined therapies after optimization.

Toll-like receptor 5 tunes hepatic and pancreatic stellate cells activation.

OBJECTIVE: Stellate cells are responsible for liver and pancreas fibrosis and strictly correlate with tumourigenesis. Although their activation is reversible, an exacerbated signalling triggers chronic fibrosis. Toll-like receptors (TLRs) modulate stellate cells transition. TLR5 transduces the signal deriving by the binding to bacterial flagellin from invading mobile bacteria. DESIGN: Human hepatic and pancreatic stellate cells were activated by the administration of transforming growth factor-beta (TGF-ß). TLR5 was transiently knocked down by short-interference RNA transfection. Reverse Transcription-quantitativePCR and western blot were performed to analyse the transcript and protein level of TLR5 and the transition players. Fluorescence microscopy was performed to identify these targets in spheroids and in the sections of murine fibrotic liver. RESULTS: TGF-ß-activated human hepatic and pancreatic stellate cells showed an increase of TLR5 expression. TLR5 knockdown blocked the activation of those stellate cells. Furthermore, TLR5 busted during murine liver fibrosis and co-localised with the inducible Collagen I. Flagellin suppressed TLR5, COL1A1 and ACTA2 expression after the administration of TGF-ß. Instead, the antagonist of TLR5 did not block the effect of TGF-ß. Wortmannin, a specific AKT inhibitor, induced TLR5 but not COL1A1 and ACTA2 transcript and protein level. CONCLUSION: TGF-ß-mediated activation of hepatic and pancreatic stellate cells requires the over-expression of TLR5. Instead, its autonomous signalling inhibits the activation of the stellate cells, thus prompting a signalling through different regulatory pathways.

A systems biology approach to define mechanisms, phenotypes, and drivers in PanNETs with a personalized perspective.

Pancreatic neuroendocrine tumors (PanNETs) are a rare tumor entity with largely unpredictable progression and increasing incidence in developed countries. Molecular pathways involved in PanNETs development are still not elucidated, and specific biomarkers are missing. Moreover, the heterogeneity of PanNETs makes their treatment challenging and most approved targeted therapeutic options for PanNETs lack objective responses. Here, we applied a systems biology approach integrating dynamic modeling strategies, foreign classifier tailored approaches, and patient expression profiles to predict PanNETs progression as well as resistance mechanisms to clinically approved treatments such as the mammalian target of rapamycin complex 1 (mTORC1) inhibitors. We set up a model able to represent frequently reported PanNETs drivers in patient cohorts, such as Menin-1 (MEN1), Death domain associated protein (DAXX), Tuberous Sclerosis (TSC), as well as wild-type tumors. Model-based simulations suggested drivers of cancer progression as both first and second hits after MEN1 loss. In addition, we could predict the benefit of mTORC1 inhibitors on differentially mutated cohorts and hypothesize resistance mechanisms. Our approach sheds light on a more personalized prediction and treatment of PanNET mutant phenotypes.

Corrigendum: Clinical impact of molecular subtyping of pancreatic cancer.

[This corrects the article DOI: 10.3389/fcell.2021.743908.].

IL18 Receptor Signaling Regulates Tumor-Reactive CD8+ T-cell Exhaustion via Activation of the IL2/STAT5/mTOR Pathway in a Pancreatic Cancer Model.

Intratumoral cytotoxic CD8+ T cells (CTL) enter a dysfunctional state characterized by expression of coinhibitory receptors, loss of effector function, and changes in the transcriptional landscape. Even though several regulators of T-cell exhaustion have been identified, the molecular mechanisms inducing T-cell exhaustion remain unclear. Here, we show that IL18 receptor (IL18R) signaling induces CD8+ T-cell exhaustion in a murine pancreatic cancer model. Adoptive transfer of Il18r-/- OT-1 CD8+ CTLs resulted in enhanced rejection of subcutaneous tumors expressing ovalbumin (OVA) as a model antigen (PancOVA), compared with wild-type OT-1 CTLs. Transferred intratumoral IL18R-deficient CTLs expressed higher levels of effector cytokines TNF and IFNγ and had reduced expression of coinhibitory receptors (PD-1, TIM-3, 2B4, LAG-3) and the transcription factors Eomes and TOX. Lower expression of coinhibitory receptors and TOX on IL18R-deficient versus IL18R-sufficient CD8+ T cells were confirmed in an orthotopic KPC model. IL18R-induced T-cell exhaustion was regulated by IL2/STAT5 and AKT/mTOR pathways, as demonstrated in an in vitro exhaustion assay. Concordantly, mice deficient in NLRP3, the molecular complex activating IL18, had decreased expression of coinhibitory receptors on intratumoral T cells and similar changes in signaling pathways at the transcriptome level. Thus, molecular pathways promoting T-cell exhaustion indicate an involvement of an NLRP3-expressing tumor microenvironment, which mediates IL18 release. The Cancer Genome Atlas analysis of patients with pancreatic carcinoma showed an association between NLRP3-mediated IL18 signaling and shorter survival. These findings indicate NLRP3-mediated IL18R signaling as a regulator of intratumoral T-cell exhaustion and a possible target for immunotherapy. See related Spotlight by Stromnes, p. 400.

IL-17A-producing CD8+ T cells promote PDAC via induction of inflammatory cancer-associated fibroblasts.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic stroma composed of cancer-associated fibroblasts (CAF) and interspersed immune cells. A non-canonical CD8+ T-cell subpopulation producing IL-17A (Tc17) promotes autoimmunity and has been identified in tumours. Here, we evaluated the Tc17 role in PDAC. DESIGN: Infiltration of Tc17 cells in PDAC tissue was correlated with patient overall survival and tumour stage. Wild-type (WT) or Il17ra-/- quiescent pancreatic stellate cells (qPSC) were exposed to conditional media obtained from Tc17 cells (Tc17-CM); moreover, co-culture of Tc17-CM-induced inflammatory (i)CAF (Tc17-iCAF) with tumour cells was performed. IL-17A/F-, IL-17RA-, RAG1-deficient and Foxn1nu/nu mice were used to study the Tc17 role in subcutaneous and orthotopic PDAC mouse models. RESULTS: Increased abundance of Tc17 cells highly correlated with reduced survival and advanced tumour stage in PDAC. Tc17-CM induced iCAF differentiation as assessed by the expression of iCAF-associated genes via synergism of IL-17A and TNF. Accordingly, IL-17RA controlled the responsiveness of qPSC to Tc17-CM. Pancreatic tumour cells co-cultured with Tc17-iCAF displayed enhanced proliferation and increased expression of genes implicated in proliferation, metabolism and protection from apoptosis. Tc17-iCAF accelerated growth of mouse and human tumours in Rag1-/- and Foxn1nu/nu mice, respectively. Finally, Il17ra-expressed by fibroblasts was required for Tc17-driven tumour growth in vivo. CONCLUSIONS: We identified Tc17 as a novel protumourigenic CD8+ T-cell subtype in PDAC, which accelerated tumour growth via IL-17RA-dependent stroma modification. We described a crosstalk between three cell types, Tc17, fibroblasts and tumour cells, promoting PDAC progression, which resulted in poor prognosis for patients.

IL18 Receptor Signaling Inhibits Intratumoral CD8+ T-Cell Migration in a Murine Pancreatic Cancer Model.

In pancreatic ductal adenocarcinoma (PDAC), the infiltration of CD8+ cytotoxic T cells (CTLs) is an important factor in determining prognosis. The migration pattern and interaction behavior of intratumoral CTLs are pivotal to tumor rejection. NLRP3-dependent proinflammatory cytokines IL-1ß and IL-18 play a prominent role for CTL induction and differentiation. Here, we investigate the effects of T-cellular IL-1R and IL-18R signaling for intratumoral T-cell motility. Murine adenocarcinoma cell line Panc02 was stably transfected with ovalbumin (OVA) and fluorophore H2B-Cerulean to generate PancOVA H2B-Cerulean tumor cells. Dorsal skinfold chambers (DSFC) were installed on wild-type mice, and PancOVA H2B-Cerulean tumor cells were implanted into the chambers. PancOVA spheroids were formed using the Corning® Matrigel®-based 3D cell culture technique. CTLs were generated from OT-1 mice, Il1r-/- OT-1 mice, or Il18r-/- OT-1 mice and were marked with fluorophores. This was followed by the adoptive transfer of CTLs into tumor-bearing mice or the application into tumor spheroids. After visualization with multiphoton microscopy (MPM), Imaris software was used to perform T-cell tracking. Imaris analysis indicates a significantly higher accumulation of Il18r-/- CTLs in PancOVA tumors and a significant reduction in tumor volume compared to wild-type CTLs. Il18r-/- CTLs covered a longer distance (track displacement length) in comparison to wild-type (WT) CTLs, and had a higher average speed (mean track speed). The analysis of instantaneous velocity suggests a higher percentage of arrested tracks (arrests: <4 µm/min) for Il18r-/- CTLs. Our data indicate the contribution of IL-18R signaling to T-cell effector strength, warranting further investigation on phenomena such as intratumoral T-cell exhaustion.

Dissecting the role of toll-like receptor 7 in pancreatic cancer.

BACKGROUND: Toll-like receptors (TLRs) are gaining attention for their potential to influence tumor biology both on the level of the tumor cells as well as on the level of the surrounding inflammatory stroma. Previous studies resulted in partly conflicting data on the expression of TLR7 in healthy and neoplastic pancreatic tissues as well as its role in pancreatic tumor biology. METHODS: We used qRT-PCR and immunohistochemistry to asses TLR7 expression in primary patient material and cell lines. Cell viability was analyzed by MTT assay upon incubation with TLR7 agonist/antagonist. Mouse models were used to investigate the role of TLR7 in vivo. RESULTS: TLR7 is overexpressed in more than 50% of primary human pancreatic ductal adenocarcinoma (PDAC). High TLR7 expression was associated with shorter patient survival, and TLR7 inhibition in cell lines reduced viability in a dose-dependent manner. In contrast, global TLR7 deficiency did not alter survival or overall histopathological tumor features in genetic mouse models of PDAC. CONCLUSIONS: TLR7 may have opposing functions in tumor versus stroma cells. Further work is required to more precisely dissect the roles of TLR7 and its ligands in different populations of epithelial and stromal cells and to understand their relative contributions to tumor progression.

IRF4 deficiency vulnerates B-cell progeny for leukemogenesis via somatically acquired Jak3 mutations conferring IL-7 hypersensitivity.

The processes leading from disturbed B-cell development to adult B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) remain poorly understood. Here, we describe Irf4-/- mice as prone to developing BCP-ALL with age. Irf4-/- preB-I cells exhibited impaired differentiation but enhanced proliferation in response to IL-7, along with reduced retention in the IL-7 providing bone marrow niche due to decreased CXCL12 responsiveness. Thus selected, preB-I cells acquired Jak3 mutations, probably following irregular AID activity, resulting in malignant transformation. We demonstrate heightened IL-7 sensitivity due to Jak3 mutants, devise a model to explain it, and describe structural and functional similarities to Jak2 mutations often occurring in human Ph-like ALL. Finally, targeting JAK signaling with Ruxolitinib in vivo prolonged survival of mice bearing established Irf4-/- leukemia. Intriguingly, organ infiltration including leukemic meningeosis was selectively reduced without affecting blood blast counts. In this work, we present spontaneous leukemogenesis following IRF4 deficiency with potential implications for high-risk BCP-ALL in adult humans.

Exploring the MEN1 dependent modulation of caspase 8 and caspase 3 in human pancreatic and murine embryo fibroblast cells.

MEN1 mutation causes pancreatic neuroendocrine neoplasia and benign malignancies of the parathyroid, the adrenal cortex and pituitary gland. The transcriptional activity of its product menin promotes the expression of genes deputed to several cellular mechanism including cell death. Here, we focused on its implication in the activation of the initiator and executioner caspases after staurosporine mediated cell death in 2D and 3D human and murine cell models. The administration of staurosporine, a well-known inducer of apoptotic cell death, caused a significant reduction of BON1, QGP1 and HPSC2.2 cell viability. The transient knockdown of MEN1, performed by using a specific siRNA, caused a significant down-regulation of CDKN1A and TP53 transcripts. The treatment with 1 µM of staurosporine caused also a significant down-regulation of MEN1 and was able to restore the basal expression of TP53 only in QGP1 cells. Transient or permanent MEN1 inactivation caused a decrease of caspase 8 activity in BON1, HPSC2.2 cells and MEN1-/- MEFs treated with staurosporine. Caspase 3/7 activity was suppressed after administration of staurosporine in MEN1 knocked down HPSC2.2 and MEN1-/- MEFs as well. The cleaved caspase 8 and caspase 3 decreased in human cells after MEN1 knockdown and in MEN1-/- MEFs. The treatment with staurosporine caused a reduction of the size of MEN1+/+ MEFs spheroids. Instead, MEN1-/- MEFs spheroids did not show any significant reduction of their size. In conclusion, MEN1 controls the activity of the initiator caspase 8 and the executioner caspase 3 in human and murine cells. Restoring of a functional MEN1 and interfering with the apoptotic mechanism could represent a future strategy for the treatment of MEN1-related malignancies.

Clinical Impact of Molecular Subtyping of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma is a highly lethal malignancy, which has now become the seventh most common cause of cancer death in the world, with the highest mortality rates in Europe and North America. In the past 30 years, there has been some progress in 5-year survival (rates increasing from 2.5 to 10%), but this is still extremely poor compared to all other common cancer types. Targeted therapies for advanced pancreatic cancer based on actionable mutations have been disappointing, with only 3-5% showing even a short clinical benefit. There is, however, a molecular diversity beyond mutations in genes responsible for producing classical canonical signaling pathways. Pancreatic cancer is almost unique in promoting an excess production of other components of the stroma, resulting in a complex tumor microenvironment that contributes to tumor development, progression, and response to treatment. Various transcriptional subtypes have also been described. Most notably, there is a strong alignment between the Classical/Pancreatic progenitor and Quasi-mesenchymal/Basal-like/Squamous subtype signatures of Moffit, Collinson, Bailey, Puleo, and Chan-Seng-Yue, which have potential clinical impact. Sequencing of epithelial cell populations enriched by laser capture microscopy combined with single-cell RNA sequencing has revealed the potential genomic evolution of pancreatic cancer as being a consequence of a gene expression continuum from mixed Basal-like and Classical cell populations within the same tumor, linked to allelic imbalances in mutant KRAS, with metastatic tumors being more copy number-unstable compared to primary tumors. The Basal-like subtype appears more chemoresistant with reduced survival compared to the Classical subtype. Chemotherapy and/or chemoradiation will also enrich the Basal-like subtype. Squamous/Basal-like programs facilitate immune infiltration compared with the Classical-like programs. The immune infiltrates associated with Basal and Classical type cells are distinct, potentially opening the door to differential strategies. Single-cell and spatial transcriptomics will now allow single cell profiling of tumor and resident immune cell populations that may further advance subtyping. Multiple clinical trials have been launched based on transcriptomic response signatures and molecular subtyping including COMPASS, Precision Promise, ESPAC6/7, PREDICT-PACA, and PASS1. We review several approaches to explore the clinical relevance of molecular profiling to provide optimal bench-to-beside translation with clinical impact.

MicroRNA-124 Alleviates Retinal Vasoregression via Regulating Microglial Polarization.

Microglial activation is implicated in retinal vasoregression of the neurodegenerative ciliopathy-associated disease rat model (i.e., the polycystic kidney disease (PKD) model). microRNA can regulate microglial activation and vascular function, but the effect of microRNA-124 (miR-124) on retinal vasoregression remains unclear. Transgenic PKD and wild-type Sprague Dawley (SD) rats received miR-124 at 8 and 10 weeks of age intravitreally. Retinal glia activation was assessed by immunofluorescent staining and in situ hybridization. Vasoregression and neuroretinal function were evaluated by quantitative retinal morphometry and electroretinography (ERG), respectively. Microglial polarization was determined by immunocytochemistry and qRT-PCR. Microglial motility was examined via transwell migration assays, wound healing assays, and single-cell tracking. Our data showed that miR-124 inhibited glial activation and improved vasoregession, as evidenced by the reduced pericyte loss and decreased acellular capillary formation. In addition, miR-124 improved neuroretinal function. miR-124 shifted microglial polarization in the PKD retina from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype by suppressing TNF-α, IL-1ß, CCL2, CCL3, MHC-II, and IFN-γ and upregulating Arg1 and IL-10. miR-124 also decreased microglial motility in the migration assays. The transcriptional factor of C/EBP-α-PU.1 signaling, suppressed by miR-124 both in vivo (PKD retina) and in vitro (microglial cells), could serve as a key regulator in microglial activation and polarization. Our data illustrate that miR-124 regulates microglial activation and polarization. miR-124 inhibits pericyte loss and thereby alleviates vasoregression and ameliorates neurovascular function.

Unraveling the Molecular Tumor-Promoting Regulation of Cofilin-1 in Pancreatic Cancer.

Cofilin-1 (CFL1) overexpression in pancreatic cancer correlates with high invasiveness and shorter survival. Besides a well-documented role in actin remodeling, additional cellular functions of CFL1 remain poorly understood. Here, we unraveled molecular tumor-promoting functions of CFL1 in pancreatic cancer. For this purpose, we first show that a knockdown of CFL1 results in reduced growth and proliferation rates in vitro and in vivo, while apoptosis is not induced. By mechanistic modeling we were able to predict the underlying regulation. Model simulations indicate that an imbalance in actin remodeling induces overexpression and activation of CFL1 by acting on transcription factor 7-like 2 (TCF7L2) and aurora kinase A (AURKA). Moreover, we could predict that CFL1 impacts proliferation and apoptosis via the signal transducer and activator of transcription 3 (STAT3). These initial model-based regulations could be substantiated by studying protein levels in pancreatic cancer cell lines and human datasets. Finally, we identified the surface protein CD44 as a promising therapeutic target for pancreatic cancer patients with high CFL1 expression.

Modulation of Pancreatic Neuroendocrine Neoplastic Cell Fate by Autophagy-Mediated Death.

INTRODUCTION: Autophagic cell death in cancer cells can be mediated by inhibition of deacetylases. Although extensive studies have focused on the autophagic process in cancer, little is known about the role of autophagy in degrading cytosolic and nuclear components of pancreatic neuroendocrine neoplastic (pNEN) cells leading to cell death, thus improving the therapy of patients affected by pNEN. METHODS: 2D and 3D human pNEN and pancreatic stellate cells were treated with panobinostat and bafilomycin. Autophagy markers were detected by RT-qPCR, immunofluorescence, and Western blot. Autophagosomes were detected by electron microscopy and their maturation by real-time fluorescence of LC3B stable transfected cells. ChIP was performed at the cAMP responsive element. Immunofluorescence was performed in murine pancreatic tissue. RESULTS: We observed that pan-deacetylase inhibitor panobinostat treatment causes autophagic cell death in pNEN cells. We also found that although AMPK-α phosphorylation is counterbalanced by phosphorylated AKT, it is not capable to inhibiting autophagic cell death. However, the binding activity of the cAMP responsive element is prompted by panobinostat. Although autophagy inhibition prevented autophagosome synthesis, maturation, and cell death, panobinostat treatment induced the accumulation of mature autophagosomes in the cytosol and the nucleus, leading to disruption of the organelles, cellular digestion, and decay. Observation of autophagosome membrane proteins Beclin1 and LC3B aggregation in murine pancreatic islets indicates that autophagy restoration may also lead to autophagosome aggregation in murine insulinoma cells. A basal low expression of autophagy markers was detectable in patients affected by pNEN, and, interestingly, the expression of these markers was significantly lower in metastatic pNEN. DISCUSSION/CONCLUSION: Our study highlights that the autophagy functional restoration and prolongation of this catabolic process, mediated by inhibition of deacetylase, is responsible for the reduction of pNEN cells. Prompting of autophagy cell death could be a promising strategy for the therapy of pNEN.

The POLD1R689W variant increases the sensitivity of colorectal cancer cells to ATR and CHK1 inhibitors.

Inhibition of the kinase ATR, a central regulator of the DNA damage response, eliminates subsets of cancer cells in certain tumors. As previously shown, this is at least partly attributable to synthetic lethal interactions between ATR and POLD1, the catalytic subunit of the polymerase δ. Various POLD1 variants have been found in colorectal cancer, but their significance as therapeutic targets for ATR pathway inhibition remains unknown. Using CRISPR/Cas9 in the colorectal cancer cell line DLD-1, which harbors four POLD1 variants, we established heterozygous POLD1-knockout clones with exclusive expression of distinct variants to determine the functional relevance of these variants individually by assessing their impact on ATR pathway activation, DNA replication, and cellular sensitivity to inhibition of ATR or its effector kinase CHK1. Of the four variants analyzed, only POLD1R689W affected POLD1 function, as demonstrated by compensatory ATR pathway activation and impaired DNA replication. Upon treatment with ATR or CHK1 inhibitors, POLD1R689W strongly decreased cell survival in vitro, which was attributable at least partly to S phase impairment and apoptosis. Similarly, treatment with the ATR inhibitor AZD6738 inhibited growth of murine xenograft tumors, harboring the POLD1R689W variant, in vivo. Our POLD1-knockout model thus complements algorithm-based models to predict the pathogenicity of tumor-specific variants of unknown significance and illustrates a novel and potentially clinically relevant therapeutic approach using ATR/CHK1 inhibitors in POLD1-deficient tumors.

The Immune Microenvironment in Pancreatic Cancer.

The biology of solid tumors is strongly determined by the interactions of cancer cells with their surrounding microenvironment. In this regard, pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) represents a paradigmatic example for the multitude of possible tumor-stroma interactions. PDAC has proven particularly refractory to novel immunotherapies, which is a fact that is mediated by a unique assemblage of various immune cells creating a strongly immunosuppressive environment in which this cancer type thrives. In this review, we outline currently available knowledge on the cross-talk between tumor cells and the cellular immune microenvironment, highlighting the physiological and pathological cellular interactions, as well as the resulting therapeutic approaches derived thereof. Hopefully a better understanding of the complex tumor-stroma interactions will one day lead to a significant advancement in patient care.