The Arp2/3-activator Wiskott-Aldrich syndrome protein and Scar homologue (WASH) is suggested to regulate actin-dependent membrane scission during endosomal sorting, but its cellular roles have not ...been fully elucidated. To investigate WASH function, we generated tamoxifen-inducible WASH-knockout mouse embryonic fibroblasts (WASHout MEFs). Of interest, although EEA1(+) endosomes were enlarged, collapsed, and devoid of filamentous-actin and Arp2/3 in WASHout MEFs, we did not observe elongated membrane tubules emanating from these disorganized endomembranes. However, collapsed WASHout endosomes harbored segregated subdomains, containing either retromer cargo recognition complex-associated proteins or EEA1. In addition, we observed global collapse of LAMP1(+) lysosomes, with some lysosomal membrane domains associated with endosomes. Both epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR) exhibited changes in steady-state cellular localization. EGFR was directed to the lysosomal compartment and exhibited reduced basal levels in WASHout MEFs. However, although TfnR was accumulated with collapsed endosomes, it recycled normally. Moreover, EGF stimulation led to efficient EGFR degradation within enlarged lysosomal structures. These results are consistent with the idea that discrete receptors differentially traffic via WASH-dependent and WASH-independent mechanisms and demonstrate that WASH-mediated F-actin is requisite for the integrity of both endosomal and lysosomal networks in mammalian cells.
The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, is a strong and selective inhibitor of Gq proteins, making it an indispensable pharmacological tool to study ...Gq‐related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as shown by in vivo experiments in mice, its affinity to insect Gq proteins, and insect toxicity studies. The uncultured endosymbiont of A. crenata was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. We here provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies, and the novel analogue AC‐1, which demonstrates the flexibility of the FR starter condensation domains. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time.
The nonribosomal peptide FR900359, a potent Gq protein inhibitor made by an obligate plant symbiont, was heterologously produced in E. coli. The biosynthesis of FR was investigated in vitro and by computations, and an ecological role as a defense chemical against herbivores was defined for FR by investigating the effects of the compound on insects and mice.
Abstract
Maintaining cancer patients’ exercise capacity and therefore patients’ ability to live a self-determined life is of huge importance, but little is known about major determinants. We sought ...to identify determinants of exercise capacity in patients with a broad spectrum of cancer types, who were already receiving cancer treatment or about to commence such therapy. Exercise capacity was assessed in 253 consecutive patients mostly suffering from advanced cancer using the 6-min walk test (6-MWT). All patients underwent echocardiography, physical examination, resting electrocardiogram, hand grip strength (HGS) measurement, and laboratory assessments. Patients were divided into two groups according to the median distance in the 6-MWT (459 m). Patients with lower exercise capacity were older, had significantly lower HGS and haemoglobin and higher values of high sensitive (hs) Troponin T and NT-proBNP (all
p
< 0.05). Whilst the co-morbidity burden was significantly higher in this group, no differences were detected for sex, body mass index, tumor type, or cachexia (all
p
> 0.2). Using multivariable logistic regression, we found that the presence of anaemia (odds ratio (OR) 6.172, 95% confidence interval (CI) 1.401–27.201,
p
= 0.016) as well as an increase in hs Troponin T (OR 3.077, 95% CI 1.202–5.301,
p
= 0.019) remained independent predictors of impaired exercise capacity. Increasing HGS was associated with a reduced risk of a lower exercise capacity (OR 0.896, 95% CI 0.813–0.987,
p
= 0.026). Screening patients for elevated hs troponin levels as well as reduced HGS may help to identify patients at risk of lower exercise capacity during cancer treatment.
The debate is ongoing regarding the potential role of preoperative chemoradiotherapy (CRT) for patients with pancreatic ductal adenocarcinoma (PDAC), and whether it should be reserved for borderline ...resectable or unresectable tumors. However, treatment response is heterogeneous, implicating the need to unveil and overcome the underlying mechanisms of resistance. Activation of the transcription factor STAT3 was recently linked to CRT resistance in other gastrointestinal cancers such as rectal and esophageal cancers, but its role in PDAC needs to be clarified. Protein expression and phosphorylation of STAT3 was determined in PDAC cell lines and connected to transcriptional activity measured by dual-luciferase reporter gene assays. Inhibition of STAT3 signaling was achieved by RNAi or the small-molecule inhibitor napabucasin. We observed a positive correlation between STAT3 signaling activity and CRT resistance. Importantly, genetical and pharmacological perturbation of the IL-6/STAT3 pathway resulted in CRT sensitization specifically in those cell lines, in which STAT3 activity was augmented by IL-6. In conclusion, our data underscore the general importance of IL-6/STAT3 signaling for CRT resistance and suggest that pathway inhibition may represents a putative treatment strategy in order to increase the fraction of patients with PDAC who are candidates for surgical approaches.
In patients with RAS wild-type metastatic colorectal cancer, depth of response (DpR) has gained importance as a novel end-point in clinical trials. We investigated the overall DpR, as well as the ...prognostic and predictive impact of DpR to induction therapy (six cycles of 5-fluorouracil, leucovorin FU/FA, oxaliplatin FOLFOX and panitumumab Pmab) on consecutive maintenance therapy (FU/FA plus Pmab or FU/FA alone) in patients treated within the PanaMa trial.
Central radiological assessment was performed according to RECIST 1.1. DpR was defined as percentage change in tumour diameter within defined time intervals (induction therapy, maintenance therapy, total course of therapy). For prognostic and predictive analyses, median DpR (</≥) served as threshold.
Out of 248 patients receiving maintenance therapy, 211 were evaluable for DpR analyses (FU/FA + Pmab, n = 106; FU/FA alone, n = 105). The overall DpR in all patients was 56.5%. DpR of induction therapy (42.5%) accounted for the largest proportion (75.2%) of the overall DpR. While greater DpR to induction therapy was significantly associated with prolonged PFS (HR 0.70, 95% CI 0.52–0.93, p = 0.013) and OS (HR 0.38, 95% CI 0.28–0.51, p < 0.001), there was no significant correlation of DpR and maintenance treatment arm.
In the PanaMa trial, the overall DpR was similar to DpR achieved by other epidermal growth factor receptor-based regimens. DpR to induction therapy accounted for three quarters of the total tumour shrinkage potentially suggesting that FOLFOX plus Pmab can be de-escalated following induction without substantially compromising efficacy. DpR to induction therapy was prognostic but not predictive for efficacy of consecutive maintenance therapy.
NCT01991873.
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•In the PanaMa trial, the median overall DpR was 58.1% versus 54.9%, respectively.•Approximately 75% of the overall DpR was achieved during induction therapy.•Additional DpR achieved by maintenance therapy was numerically greater with Pmab.•DpR to induction was prognostic for PFS and OS of maintenance therapy.
Despite molecular selection, patients (pts) with RAS wildtype mCRC represent a heterogeneous population including diversity in metastatic spread. We investigated metastatic patterns for their ...prognostic and predictive impact on maintenance therapy with 5‐fluorouracil/folinic acid ± panitumumab. The study population was stratified according to (1) number of involved metastatic sites (single vs multiple organ metastasis), liver‐limited disease vs (2) liver metastasis plus one additional site, and (3) vs liver metastasis plus ≥two additional sites. Kaplan‐Meier method and Cox regressions were used to correlate efficacy endpoints. Single organ metastasis was observed in 133 pts (53.6%) with 102 pts (41.1%) presenting with liver‐limited disease, while multiple organ metastases were reported in 114 pts (46.0). Multiple compared to single organ metastases were associated with less favorable PFS (HR 1.48, 95% CI 1.13‐1.93; P = .004) and OS (HR 1.37, 95% CI 0.98‐1.93; P = .068) of maintenance therapy. While metastatic spread involving one additional extrahepatic site was not associated with clearly impaired survival compared to liver‐limited disease, pts with liver metastasis plus ≥two additional sites demonstrated less favorable PFS (HR 1.92, 95% CI 1.30‐2.83; P < .001), and OS (HR 2.38, 95% CI 1.51‐3.76; P < .001) of maintenance therapy. Pmab‐containing maintenance therapy appeared active in both pts with multiple (HR 0.58; 95% CI, 0.39‐0.86; P = .006) as well as to a lesser numerical extent in pts with single organ metastasis (HR 0.83; 95% CI, 0.57‐1.21; P = .332; Interaction P = .183). These data may support clinical decisions when EGFR‐based maintenance therapy is considered.
What's new?
A variety of patterns in metastasis can occur in RAS wildtype metastatic colorectal cancer (mCRC). Heterogeneity in metastatic spread, however, challenges prognostic evaluation for patients with these tumors. Here, the prognostic and therapeutic significance of different metastatic patterns in RAS wildtype mCRC was investigated in patients on maintenance therapy with 5‐fluorouracil/folinic acid, with or without panitumumab. In patients who presented with liver‐limited disease, spread to one additional organ had limited impact on survival. Survival was less favorable for metastasis to multiple organs. Maintenance therapy involving panitumumab had greater effect in RAS wildtype mCRC patients with multiple organ metastasis.
Tumor grading is a robust prognostic predictor in patients with neuroendocrine neoplasms (NEN) and guides therapy, especially in tumors with high proliferation. NEN can be separated into ...well-differentiated and poorly differentiated types. The more aggressive NEN have been further separated into neuroendocrine tumors (NET G3) with a better prognosis and neuroendocrine carcinomas (NEC) with a worse prognosis. Despite this distinction’s tremendous clinical and therapeutic relevance, optimal diagnostic biomarkers are still lacking. In this study, we analyzed the protein expression and prognostic impact of Enhancer of Zeste Homolog 2 (EZH2) by immunohistochemistry in 219 tissue samples of gastroenteropancreatic (GEP-NEN) and pulmonary NEN (P-NEN). EZH2 was almost exclusively expressed in NEN with a proliferation rate above 20% (G3), while all low-grade tumors were nearly negative. Among high-grade NEN, 65% showed high and 35% low expression of EZH2. In this group, the high expression of EZH2 was significantly associated with poor overall survival and NEC histology. Interestingly, EZH2 seems to act independently of Polycomb Repressive Complex 2 (PRC2) in NEN. In conclusion, we propose EZH2 as a robust biomarker for distinguishing between NET G3 and NEC among gastroenteropancreatic and pulmonary NEN.
Background & Aims The ability of exocrine pancreatic cells to change the cellular phenotype is required for tissue regeneration upon injury, but also contributes to their malignant transformation and ...tumor progression. We investigated context-dependent signaling and transcription mechanisms that determine pancreatic cell fate decisions toward regeneration and malignancy. In particular, we studied the function and regulation of the inflammatory transcription factor nuclear factor of activated T cells 1 (NFATC1) in pancreatic cell plasticity and tissue adaptation. Methods We analyzed cell plasticity during pancreatic regeneration and transformation in mice with pancreas-specific expression of a constitutively active form of NFATC1, or depletion of enhancer of zeste 2 homologue 2 (EZH2), in the context of wild-type or constitutively activate Kras, respectively. Acute and chronic pancreatitis were induced by intraperitoneal injection of caerulein. EZH2-dependent regulation of NFATC1expression was studied in mouse in human pancreatic tissue and cells by immunohistochemistry, immunoblotting, and quantitative reverse transcription polymerase chain reaction. We used genetic and pharmacologic inhibition of EZH2 and NFATC1 to study the consequences of pathway disruption on pancreatic morphology and function. Epigenetic modifications on the NFATC1 gene were investigated by chromatin immunoprecipitation assays. Results NFATC1 was rapidly and transiently induced in early adaptation to acinar cell injury in human samples and in mice, where it promoted acinar cell transdifferentiation and blocked proliferation of metaplastic pancreatic cells. However, in late stages of regeneration, Nfatc1 was epigenetically silenced by EZH2-dependent histone methylation, to enable acinar cell redifferentiation and prevent organ atrophy and exocrine insufficiency. In contrast, oncogenic activation of KRAS signaling in pancreatic ductal adenocarcinoma cells reversed the EZH2-dependent effects on NFATC1 gene and was required for EZH2-mediated transcriptional activation of NFATC1. Conclusions In studies of human and mouse pancreatic cells and tissue, we identified context-specific epigenetic regulation of NFATc1 activity an important mechanism of pancreatic cell plasticity. Inhibitors of EZH2 might therefore interfere with oncogenic activity of NFATC1 and be used in treatment of pancreatic ductal adenocarcinoma.
The marine alga-derived fungus Coniothyrium cereale is a prolific producer of phenalenones. These polyketides were shown to possess antimicrobial effects and inhibitory activity towards the protease ...human leucocyte elastase (HLE). The current study focused on the biosynthesis of eight different structural types of phenalenones, comprising the natural products rousselianone A' (1), coniosclerodin (3), cereolactam (12), cereoaldomine (15), and trypethelone (16). Solid agar cultures of C. cereale were used to follow up the incorporation of 1-(13)C labeled acetate into these metabolites. Taking the respective mechanisms of polyketide metabolism into account, the labeling pattern was interpreted, thus providing a hypothesis for the biosynthetic formation of the phenalenones. The polyketide skeleton of the phenanthrene-based compound cereolactam is proposed to be formed through degradation of a heptaketide by loss of two carbon atoms.
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