Microbiome is an integral part of the gut and is essential for its proper function. Imbalances of the microbiota can be devastating and have been linked with several gastrointestinal conditions. ...Current gastrointestinal models do not fully reflect the in vivo situation. Thus, it is important to establish more advanced in vitro models to study host-microbiome/pathogen interactions. Here, we developed for the first time an apical-out human small intestinal organoid model in hypoxia, where the apical surface is directly accessible and exposed to a hypoxic environment. These organoids mimic the intestinal cell composition, structure and functions and provide easy access to the apical surface. Co-cultures with the anaerobic strains Lactobacillus casei and Bifidobacterium longum showed successful colonization and probiotic benefits on the organoids. These novel hypoxia-tolerant apical-out small intestinal organoids will pave the way for unraveling unknown mechanisms related to host-microbiome interactions and serve as a tool to develop microbiome-related probiotics and therapeutics.
Adaptation to tumor hypoxia is mediated in large part by changes in protein expression. These are driven by multiple pathways, including activation of the hypoxia inducible factor-1 (HIF-1) ...transcription factor and the PKR-like endoplasmic reticulum kinase PERK, a component of the unfolded protein response. Through gene expression profiling we discovered that induction of the HIF-1 target gene CA9 was defective in mouse embryo fibroblasts derived from mice harboring an eIF2α S51A knock-in mutation. This finding was confirmed in two isogenic human cell lines with an engineered defect in eIF2α phosphorylation. We show that impaired CA9 expression was not due to changes in HIF activity or CA9 mRNA stability. Using chromatin immunoprecipitation we show that the eIF2α-dependent translationally regulated gene ATF4 binds directly to the CA9 promoter and is associated with loss of the transcriptional repressive histone 3 lysine 27 tri-methylation mark. Loss or overexpression of ATF4 confirmed its role in CA9 induction during hypoxia. Our data indicate that expression of CA9 is regulated through both the HIF-1 and unfolded protein response hypoxia response pathways in vitro and in vivo.
Abstract Background and purpose EGFRvIII has been described to function as an oncoprotein with constitutive activation promoting neoplastic transformation and tumorigenicity. The present study was ...undertaken to test whether EGFRvIII also contributes to hypoxia tolerance. Material and methods The human glioma cell line U373 was genetically modified to stably express EGFRvIII. Western blotting and immunohistochemistry verified the expression of EGFRvIII. Tumour xenografts were produced by injecting U373 control and EGFRvIII positive cells subcutaneously into the lateral flank of recipient mice. Colony formation assays were performed after ionizing radiation at 4 Gy and after exposure to anoxia for 1–4 days. Results EGFRvIII accelerated tumour growth leading to a 3.5-fold increase in tumour size compared to control tumours at 40 days after cell injection. EGFRvIII promoted clonogenic survival by almost 2-fold and 4-fold after 4 Gy and 4 days of anoxia, respectively. EGFRvIII was also associated with a substantially bigger colony size after anoxic treatment. Conclusions EGFRvIII expression stimulates the growth of tumour xenografts and strongly promotes survival after irradiation and under hypoxic stress.
The epidermal growth factor receptor (EGFR) is amplified or mutated in various human epithelial tumors. Its expression and activation leads to cell proliferation, differentiation, and survival. ...Consistently, EGFR amplification or expression of EGFR variant 3 (EGFRvIII) is associated with resistance to conventional cancer therapy through activation of pro-survival signaling and DNA-repair mechanisms. EGFR targeting has successfully been exploited as strategy to increase treatment efficacy. Nevertheless, these targeting strategies have only been proven effective in a limited percentage of human tumors.
Recent knowledge indicates that EGFR deregulated tumors display differences in autophagy and dependence on autophagy for growth and survival and the use of autophagy to increase resistance to EGFR-targeting drugs. In this review the dependency on autophagy and its role in mediating resistance to EGFR-targeting agents will be discussed. Considering the current knowledge, autophagy inhibition could provide a novel strategy to enhance therapy efficacy in treatment of EGFR deregulated tumors.
Abstract Background and purpose Hypoxia is a hallmark of solid cancers and associated with metastases and treatment failure. During tumor progression epithelial cells often acquire mesenchymal ...features, a phenomenon known as epithelial-to-mesenchymal transition (EMT). Intratumoral hypoxia has been linked to EMT induction. We hypothesized that signals from the tumor microenvironment such as growth factors and tumor oxygenation collaborate to promote EMT and thereby contribute to radioresistance. Materials and methods Gene expression changes under hypoxia were analyzed using microarray and validated by qRT-PCR. Conversion of epithelial phenotype upon hypoxic exposure, TGFβ addition or oncogene activation was investigated by Western blot and immunofluorescence. Cell survival following ionizing radiation was assayed using clonogenic survival. Results Upon hypoxia, TGFβ addition or EGFRvIII expression, MCF7, A549 and NMuMG epithelial cells acquired a spindle shape and lost cell–cell contacts. Expression of epithelial markers such as E-cadherin decreased, whereas mesenchymal markers such as vimentin and N-cadherin increased. Combining hypoxia with TGFβ or EGFRvIII expression, lead to more rapid and pronounced EMT-like phenotype. Interestingly, E-cadherin expression and the mesenchymal appearance were reversible upon reoxygenation. Mesenchymal conversion and E-cadherin loss were associated with radioresistance. Conclusions Our findings describe a mechanism by which the tumor microenvironment may contribute to tumor radioresistance via E-cadherin loss and EMT.
Expression of EGFRvIII is frequently observed in glioblastoma and is associated with increased cellular proliferation, enhanced tolerance to metabolic stresses, accelerated tumor growth, therapy ...resistance and poor prognosis. We observed that expression of EGFRvIII elevates the activation of macroautophagy/autophagy during starvation and hypoxia and explored the underlying mechanism and consequence. Autophagy was inhibited (genetically or pharmacologically) and its consequence for tolerance to metabolic stress and its therapeutic potential in (EGFRvIII
+
) glioblastoma was assessed in cellular systems, (patient derived) tumor xenopgrafts and glioblastoma patients. Autophagy inhibition abrogated the enhanced proliferation and survival advantage of EGFRvIII
+
cells during stress conditions, decreased tumor hypoxia and delayed tumor growth in EGFRvIII
+
tumors. These effects can be attributed to the supporting role of autophagy in meeting the high metabolic demand of EGFRvIII
+
cells. As hypoxic tumor cells greatly contribute to therapy resistance, autophagy inhibition revokes the radioresistant phenotype of EGFRvIII
+
tumors in (patient derived) xenograft tumors. In line with these findings, retrospective analysis of glioblastoma patients indicated that chloroquine treatment improves survival of all glioblastoma patients, but patients with EGFRvIII
+
glioblastoma benefited most. Our findings disclose the unique autophagy dependency of EGFRvIII
+
glioblastoma as a therapeutic opportunity. Chloroquine treatment may therefore be considered as an additional treatment strategy for glioblastoma patients and can reverse the worse prognosis of patients with EGFRvIII
+
glioblastoma.
Abstract Background and purpose Hypoxia is a common feature of solid tumors that is associated with an aggressive phenotype, resistance to therapy and poor prognosis. Major contributors to these ...adverse effects are the transcriptional program activated by the HIF family of transcription factors as well as the translational response mediated by PERK-dependent phosphorylation of eIF2α and inhibition of mTORC1 activity. In this study we determined the relative contribution of both transcriptional and translational responses to changes in hypoxia induced gene expression. Material and methods Total and efficiently translated (polysomal) mRNA was isolated from DU145 prostate carcinoma cells that were exposed for up to 24 h of hypoxia (<0.02% O2 ). Changes in transcription and translation were assessed using affymetrix microarray technology. Results Our data reveal an unexpectedly large contribution of translation control on both induced and repressed gene expression at all hypoxic time points, particularly during acute hypoxia (2–4 h). Gene ontology analysis revealed that gene classes like transcription and signal transduction are stimulated by translational control whereas expression of genes involved in cell growth and protein metabolism are repressed during hypoxic conditions by translational control. Conclusions Our data indicate that translation influences gene expression during hypoxia on a scale comparable to that of transcription.
Abstract Background and purpose The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various human epithelial tumors, and is associated with tumor aggressiveness and ...therapy resistance. Autophagy activation provides a survival advantage for cells in the tumor microenvironment. In the current study, we assessed the potential of autophagy inhibition (using chloroquine (CQ)) in treatment of EGFR expressing tumors. Material and methods Quantitative PCR, immunohistochemistry, clonogenic survival, proliferation assays and in vivo tumor growth were used to assess this potential. Results We show that EGFR overexpressing xenografts are sensitive to CQ treatment and are sensitized to irradiation by autophagy inhibition. In HNSSC xenografts, a correlation between EGFR and expression of the autophagy marker LC3b is observed, suggesting a role for autophagy in EGFR expressing tumors. This observation was substantiated in cell lines, showing high EGFR expressing cells to be more sensitive to CQ addition as reflected by decreased proliferation and survival. Surprisingly high EGFR expressing cells display a lower autophagic flux. Conclusions The EGFR high expressing cells and tumors investigated in this study are highly dependent on autophagy for growth and survival. Inhibition of autophagy may therefore provide a novel treatment opportunity for EGFR overexpressing tumors.
Abstract Background and purpose Tumor hypoxia is associated with therapy resistance and malignancy. Previously we demonstrated that activation of autophagy and the unfolded protein response (UPR) ...promote hypoxia tolerance. Here we explored the importance of ULK1 in hypoxia tolerance, autophagy induction and its prognostic value for recurrence after treatment. Material and methods Hypoxic regulation of ULK1 mRNA and protein was assessed in vitro and in primary human head and neck squamous cell carcinoma (HNSCC) xenografts. Its importance in autophagy induction, mitochondrial homeostasis and tolerance to chronic and acute hypoxia was evaluated in ULK1 knockdown cells. The prognostic value of ULK1 mRNA expression was assessed in 82 HNSCC patients. Results ULK1 enrichment was observed in hypoxic tumor regions. High enrichment was associated with a high hypoxic fraction. In line with these findings, high ULK1 expression in HNSCC patients appeared associated with poor local control. Exposure of cells to hypoxia induced ULK1 mRNA in a UPR and HIF1α dependent manner. ULK1 knockdown decreased autophagy activation, increased mitochondrial mass and ROS exposure and sensitized cells to acute and chronic hypoxia. Conclusions We demonstrate that ULK1 is a hypoxia regulated gene and is associated with hypoxia tolerance and a worse clinical outcome.
Abstract Purpose This phase I/II study sought to determine the safety and maximum tolerated dose (MTD) of the combination of rapamycin, an mTOR inhibitor, with short-course radiotherapy in rectal ...cancer patients. Antitumor activity, changes in metabolic activity and perfusion on imaging, and changes in phosphorylation status of the mTOR pathway were also assessed. Materials and methods Patients with primary resectable rectal cancer were treated with short-course hypofractionated radiotherapy (5 × 5 Gy) combined with oral rapamycin 1 week before and during radiotherapy, followed by surgical resection. Results Thirteen patients were entered in phase I. One patient developed a dose-limiting toxicity, consisting of a grade 4 leak and grade 4 bleeding. Because of an unexpected high rate of grade 3 postoperative toxicity, it was decided to treat patients with delayed surgery in phase II. Primary endpoint for phase II was tumor blood flow ( Ktrans ) assessed by perfusion CT. Thirty-one patients were treated with the MTD of 6 mg rapamycin daily. One patient (3%) developed a pathological complete response (pCR) and 3 patients (10%) had a ypT1N0 tumor at the time of resection. No change in tumor perfusion was observed on perfusion CT, but a significant decrease of metabolic activity was found on PET-scan. Conclusions The combination of short-course radiotherapy and rapamycin turned out to be feasible, provided that the interval between neo-adjuvant treatment and surgical resection is at least 6 weeks. Although from this cohort no clear increase in pCR could be observed, a clear metabolic response after rapamycin run-in was observed, indicating a biological activity of this drug in rectal cancer.
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