The Patient's Point of View: COVID-19 and Neuroendocrine Tumor Disease.

The assessment of cancer patient care during the COVID-19 pandemic has been mainly reported from a physician's perspective. Patients with rare tumor entities such as neuroendocrine tumors (NET), which require a complex and specialized care infrastructure, were highly affected by the COVID-19 crisis. Using a structured questionnaire consisting of a general section on the disease and a special COVID-19 section to record medical care, vaccination behavior as well as social and psycho-emotional parameters were collected from NET patients. The survey was distributed via direct medical contact and via the patient organization NETZWERK NeT. A total of 684 patients participated in the survey and 79.2% (n = 542) of the participants answered the questionnaire completely (54 questions). Patient characteristics were comparable to those in large NET registries. The majority of participants were patients with pancreatic and small bowel NET on somatostatin analogue (SSA) therapy. Medical care under COVID-19 was adequate and appointment cancellations and postponements were not common. Nevertheless, the majority of patients were worried about adequate treatment for their tumor disease during the crisis. Most of the participants considered themselves to be at risk of severe COVID-19 infection and were therefore very concerned. This was accompanied by an extremely high vaccination readiness rate of 90%. Increased distress in the social and psycho-emotional domains in the course of the crisis reflected a need for optimization in the medical care of NET patients, although the rate of COVID-19 positive participants was low (3.7%). Therefore, patient-reported measurements are required to identify and address all areas of medical care. Overall, our survey provides an essential contribution to the care of NET patients during the COVID-19 pandemic from the patient's perspective.

The Selective 5-HT1A Agonist SR57746A Protects Intestinal Epithelial Cells and Enteric Glia Cells and Promotes Mucosal Recovery in Experimental Colitis.

BACKGROUND: Neurotrophic growth factors can stabilize the intestinal barrier by preventing the apoptosis of enteric glial cells (EGCs) and enterocytes. We reasoned that a selective 5-HT1A receptor agonist may have neuroprotective properties in the gut and that topical application of SR57746A might be an effective treatment strategy in inflammatory bowel disease (IBD). METHODS: The therapeutic potential of 5-HT1A receptor agonist SR57746A in IBD was evaluated in vitro (nontransformed NCM460 colonic epithelial cells, SW480 colorectal carcinoma cells) and in vivo (murine dextran sulfate sodium [DSS] colitis and CD4-T-cell transfer colitis). In vitro, we analyzed the effect of SR57746A on apoptosis in intestinal epithelial cells (IECs) and EGCs, and upon proliferation, migration, and intracellular signaling in IECs. In vivo, the effect of topical application of SR57746 on disease activity and on histological and endoscopic findings was compared with intraperitoneal infliximab and placebo, respectively. RESULTS: The SR57746A activates PI3-K/AKT- and ERK-signaling in IECs. Depending on ERK- and AKT activation, SR57746A potently prevents apoptosis of IECs without inducing proliferation or migration in these cells. Moreover, SR57746A prevented apoptosis in EGCs in vitro. Topical SR57746A treatment significantly reduced mucosal injury in 2 experimental murine colitis models and was as effective as intraperitoneal infliximab treatment. CONCLUSIONS: Treatment with SR57746A prevents inflammatory cell damage and apoptosis in IECs and EGCs, similar to the neurotrophic effects of EGCs on IECs. Topical treatment with SR57746A could be a candidate for clinical evaluation in the treatment of IBD.

Targeting HDACs in Pancreatic Neuroendocrine Tumor Models.

Compared to pancreatic adenocarcinoma (PDAC), pancreatic neuroendocrine tumors (PanNET) represent a rare and heterogeneous tumor entity. In addition to surgical resection, several therapeutic approaches, including biotherapy, targeted therapy or chemotherapy are applicable. However, primary or secondary resistance to current therapies is still challenging. Recent genome-wide sequencing efforts in PanNET identified a large number of mutations in pathways involved in epigenetic modulation, including acetylation. Therefore, targeting epigenetic modulators in neuroendocrine cells could represent a new therapeutic avenue. Detailed information on functional effects and affected signaling pathways upon epigenetic targeting in PanNETs, however, is missing. The primary human PanNET cells NT-3 and NT-18 as well as the murine insulinoma cell lines beta-TC-6 (mouse) and RIN-T3 (rat) were treated with the non-selective histone-deacetylase (HDAC) inhibitor panobinostat (PB) and analyzed for functional effects and affected signaling pathways by performing Western blot, FACS and qPCR analyses. Additionally, NanoString analysis of more than 500 potentially affected targets was performed. In vivo immunohistochemistry (IHC) analyses on tumor samples from xenografts and the transgenic neuroendocrine Rip1Tag2-mouse model were investigated. PB dose dependently induced cell cycle arrest and apoptosis in neuroendocrine cells in human and murine species. HDAC inhibition stimulated redifferentiation of human primary PanNET cells by increasing mRNA-expression of somatostatin receptors (SSTRs) and insulin production. In addition to hyperacetylation of known targets, PB mediated pleitropic effects via targeting genes involved in the cell cycle and modulation of the JAK2/STAT3 axis. The HDAC subtypes are expressed ubiquitously in the existing cell models and in human samples of metastatic PanNET. Our results uncover epigenetic HDAC modulation using PB as a promising new therapeutic avenue in PanNET, linking cell-cycle modulation and pathways such as JAK2/STAT3 to epigenetic targeting. Based on our data demonstrating a significant impact of HDAC inhibition in clinical relevant in vitro models, further validation in vivo is warranted.

CUX1-Transcriptional Master Regulator of Tumor Progression in Pancreatic Neuroendocrine Tumors.

Recently, we identified the homeodomain transcription factor Cut homeobox 1 (CUX1) as mediator of tumour de-differentiation and metastatic behaviour in human insulinoma patients. In insulinomas, CUX1 enhanced tumour progression by stimulating proliferation and angiogenesis in vitro and in vivo. In patients with non-functional pancreatic neuroendocrine tumours (PanNET), however, the impact of CUX1 remains to be elucidated. Here, we analysed CUX1 expression in two large independent cohorts (n = 43 and n = 141 tissues) of non-functional treatment-naïve and pre-treated PanNET patients, as well as in the RIP1Tag2 mouse model of pancreatic neuroendocrine tumours. To further assess the functional role of CUX1, expression profiling of DNA damage-, proliferation- and apoptosis-associated genes was performed in CUX1-overexpressing Bon-1 cells. Validation of differentially regulated genes was performed in Bon-1 and QGP1 cells with knock-down and overexpression strategies. CUX1 expression assessed by a predefined immunoreactivity score (IRS) was significantly associated with shorter progression-free survival (PFS) of pre-treated PanNET patients (23 vs. 8 months; p = 0.005). In treatment-naïve patients, CUX1 was negatively correlated with grading and recurrence-free survival (mRFS of 39 versus 8 months; p = 0.022). In both groups, high CUX1 levels indicated a metastatic phenotype. Functionally, CUX1 upregulated expression of caspases and death associated protein kinase 1 (DAPK1), known as mediators of tumour progression and resistance to cytotoxic drugs. This was also confirmed in both cell lines and human tissues. In the RIP1Tag2 mouse model, CUX1 expression was associated with advanced tumour stage and resistance to apoptosis. In summary, we identified the transcription factor CUX1 as mediator of tumour progression in non-functional PanNET in vitro and in vivo, indicating that the CUX1-dependent signalling network is a promising target for future therapeutic intervention.

Regulation of MRTF-A by JMY via a nucleation-independent mechanism.

BACKGROUND: MRTF-A (myocardin-related transcription factor A) is a coactivator for SRF-mediated gene expression. The activity of MRTF-A is critically dependent on the dissociation of G-actin from N-terminal RPEL motifs. MRTF-SRF induction often correlates with enhanced polymerization of F-actin. Here we investigate MRTF regulation by the multifunctional JMY protein, which contains three WASP/verprolin homology 2 (WH2/V) domains and facilitates Arp2/3-dependent and -independent actin nucleation. METHODS: Co-immunoprecipitation experiments, immunofluorescence and luciferase reporter assays were combined with selective inhibitors to investigate the effect of JMY and its domains on MRTF-A in NIH 3 T3 mouse fibroblasts. RESULTS: JMY induced MRTF-A transcriptional activity and enhanced its nuclear translocation. Unexpectedly, MRTF-A was hyperactivated when the Arp2/3-recruiting CA region of JMY was deleted or mutated, suggesting an autoinhibitory mechanism for full-length JMY. Moreover, isolated WH2/V domains which are unable to nucleate actin were sufficient for nuclear accumulation and SRF activation. Recombinant WH2/V regions of JMY biochemically competed with MRTF-A for actin binding. Activation of MRTF-A by JMY was unaffected by Arp3 knockdown, by an Arp2/3 inhibitor, and by latrunculin which disassembles cellular F-actin. Restriction of JMY to the nucleus abrogated its MRTF-A activation. Finally, JMY RNAi reduced basal and stimulated transcriptional activation via MRTF-A. CONCLUSIONS: Our results suggest that JMY activates MRTF-SRF independently of F-actin via WH2/V-mediated competition with the RPEL region for G-actin binding in the cytoplasm. Furthermore, the C-terminal region facilitates an autoinhibitory effect on full-length JMY, possibly by intramolecular folding.

Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding.

The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin-MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin-RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation.

Analysis of nuclear actin by overexpression of wild-type and actin mutant proteins.

Compared to the cytoplasmic F-actin abundance in cells, nuclear F-actin levels are generally quite low. However, nuclear actin is present in certain cell types including oocytes and under certain cellular conditions including stress or serum stimulation. Currently, the architecture and polymerization status of nuclear actin networks has not been analyzed in great detail. In this study, we investigated the architecture and functions of such nuclear actin networks. We generated nuclear actin polymers by overexpression of actin proteins fused to a nuclear localization signal (NLS). Raising nuclear abundance of a NLS wild-type actin, we observed phalloidin- and LifeAct-positive actin bundles forming a nuclear cytoskeletal network consisting of curved F-actin. In contrast, a polymer-stabilizing actin mutant (NLS-G15S-actin) deficient in interacting with the actin-binding protein cofilin generated a nuclear actin network reminiscent of straight stress fiber-like microfilaments in the cytoplasm. We provide a first electron microscopic description of such nuclear actin polymers suggesting bundling of actin filaments. Employing different cell types from various species including neurons, we show that the morphology of and potential to generate nuclear actin are conserved. Finally, we demonstrate that nuclear actin affects cell function including morphology, serum response factor-mediated gene expression, and herpes simplex virus infection. Our data suggest that actin is able to form filamentous structures inside the nucleus, which share architectural and functional similarities with the cytoplasmic F-actin.