LC3/GABARAP family proteins: autophagy-(un)related functions.

From yeast to mammals, autophagy is an important mechanism for sustaining cellular homeostasis through facilitating the degradation and recycling of aged and cytotoxic components. During autophagy, cargo is captured in double-membraned vesicles, the autophagosomes, and degraded through lysosomal fusion. In yeast, autophagy initiation, cargo recognition, cargo engulfment, and vesicle closure is Atg8 dependent. In higher eukaryotes, Atg8 has evolved into the LC3/GABARAP protein family, consisting of 7 family proteins [LC3A (2 splice variants), LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2]. LC3B, the most studied family protein, is associated with autophagosome development and maturation and is used to monitor autophagic activity. Given the high homology, the other LC3/GABARAP family proteins are often presumed to fulfill similar functions. Nevertheless, substantial evidence shows that the LC3/GABARAP family proteins are unique in function and important in autophagy-independent mechanisms. In this review, we discuss the current knowledge and functions of the LC3/GABARAP family proteins. We focus on processing of the individual family proteins and their role in autophagy initiation, cargo recognition, vesicle closure, and trafficking, a complex and tightly regulated process that requires selective presentation and recruitment of these family proteins. In addition, functions unrelated to autophagy of the LC3/GABARAP protein family members are discussed.-Schaaf, M. B. E., Keulers, T. G, Vooijs, M. A., Rouschop, K. M. A. LC3/GABARAP family proteins: autophagy-(un)related functions.

Enhanced radiation sensitivity, decreased DNA damage repair, and differentiation defects in airway stem cells derived from patients with chronic obstructive pulmonary disease.

Radiation therapy (RT) is a common treatment for lung cancer. Still, it can lead to irreversible loss of pulmonary function and a significant reduction in quality of life for one-third of patients. Preexisting comorbidities, such as chronic obstructive pulmonary disease (COPD), are frequent in patients with lung cancer and further increase the risk of complications. Because lung stem cells are crucial for the regeneration of lung tissue following injury, we hypothesized that airway stem cells from patients with COPD with lung cancer might contribute to increased radiation sensitivity. We used the air-liquid interface model, a three-dimensional (3D) culture system, to compare the radiation response of primary human airway stem cells from healthy and patients with COPD. We found that COPD-derived airway stem cells, compared to healthy airway stem cell cultures, exhibited disproportionate pathological mucociliary differentiation, aberrant cell cycle checkpoints, residual DNA damage, reduced survival of stem cells and self-renewal, and terminally differentiated cells post-irradiation, which could be reversed by blocking the Notch pathway using small-molecule γ-secretase inhibitors. Our findings shed light on the mechanisms underlying the increased radiation sensitivity of COPD and suggest that airway stem cells reflect part of the pathological remodeling seen in lung tissue from patients with lung cancer receiving thoracic RT.

Iron-responsive element of Divalent metal transporter 1 (Dmt1) controls Notch-mediated cell fates.

Notch receptor activation is regulated by the intramembrane protease γ-secretase, which cleaves and liberates the Notch intracellular domain (Nicd) that regulates gene transcription. While γ-secretase cleavage is necessary, we demonstrate it is insufficient for Notch activation and requires vesicular trafficking. Here, we report Divalent metal transporter 1 (Dmt1, Slc11A2) as a novel and essential regulator of Notch signalling. Dmt1-deficient cells are defective in Notch signalling and have perturbed endolysosomal trafficking and function. Dmt1 encodes for two isoforms, with and without an iron response element (ire). We show that isoform-specific silencing of Dmt1-ire and Dmt1+ire has opposite consequences on Notch-dependent cell fates in cell lines and intestinal organoids. Loss of Dmt1-ire suppresses Notch activation and promotes differentiation, whereas loss of Dmt1+ire causes Notch activation and maintains stem-progenitor cell fates. Dmt1 isoform expression correlates with Notch and Wnt signalling in Apc-deficient intestinal organoids and human colorectal cancers. Consistently, Dmt1-ire silencing induces Notch-dependent differentiation in colorectal cancer cells. These data identify Dmt1 isoforms as binary switches controlling Notch cell fate decisions in normal and tumour cells.

Patient-derived glioblastoma organoids reflect tumor heterogeneity and treatment sensitivity.

Background: Treatment resistance and tumor relapse are the primary causes of mortality in glioblastoma (GBM), with intratumoral heterogeneity playing a significant role. Patient-derived cancer organoids have emerged as a promising model capable of recapitulating tumor heterogeneity. Our objective was to develop patient-derived GBM organoids (PGO) to investigate treatment response and resistance. Methods: GBM samples were used to generate PGOs and analyzed using whole-exome sequencing (WES) and single-cell karyotype sequencing. PGOs were subjected to temozolomide (TMZ) to assess viability. Bulk RNA sequencing was performed before and after TMZ. Results: WES analysis on individual PGOs cultured for 3 time points (1-3 months) showed a high inter-organoid correlation and retention of genetic variants (range 92.3%-97.7%). Most variants were retained in the PGO compared to the tumor (range 58%-90%) and exhibited similar copy number variations. Single-cell karyotype sequencing demonstrated preservation of genetic heterogeneity. Single-cell multiplex immunofluorescence showed maintenance of cellular states. TMZ treatment of PGOs showed a differential response, which largely corresponded with MGMT promoter methylation. Differentially expressed genes before and after TMZ revealed an upregulation of the JNK kinase pathway. Notably, the combination treatment of a JNK kinase inhibitor and TMZ demonstrated a synergistic effect. Conclusions: Overall, these findings demonstrate the robustness of PGOs in retaining the genetic and phenotypic heterogeneity in culture and the application of measuring clinically relevant drug responses. These data show that PGOs have the potential to be further developed into avatars for personalized adaptive treatment selection and actionable drug target discovery and as a platform to study GBM biology.

The role of Adams in Notch signaling.

Regulated intramembrane proteolysis (RIP) is a highly conserved signaling paradigm whereby membrane-bound signaling proteins are cleaved in their transmembrane region and then released into the cytoplasm to act as signaling molecules. In most if not all cases intramembrane cleavage is preceded and regulated by a membrane proximal cleavage step called 'ectodomain shedding'. Here we will review the role of ectodomain shedding in RIP of the NOTCH signaling pathway, a highly conserved cell-cell communication pathway that mediates cell fate decisions during development and in adult tissues.

Prognostic and Predictive Value of Integrated Qualitative and Quantitative Magnetic Resonance Imaging Analysis in Glioblastoma.

Glioblastoma (GBM) is the most malignant primary brain tumor for which no curative treatment options exist. Non-invasive qualitative (Visually Accessible Rembrandt Images (VASARI)) and quantitative (radiomics) imaging features to predict prognosis and clinically relevant markers for GBM patients are needed to guide clinicians. A retrospective analysis of GBM patients in two neuro-oncology centers was conducted. The multimodal Cox-regression model to predict overall survival (OS) was developed using clinical features with VASARI and radiomics features in isocitrate dehydrogenase (IDH)-wild type GBM. Predictive models for IDH-mutation, 06-methylguanine-DNA-methyltransferase (MGMT)-methylation and epidermal growth factor receptor (EGFR) amplification using imaging features were developed using machine learning. The performance of the prognostic model improved upon addition of clinical, VASARI and radiomics features, for which the combined model performed best. This could be reproduced after external validation (C-index 0.711 95% CI 0.64-0.78) and used to stratify Kaplan-Meijer curves in two survival groups (p-value < 0.001). The predictive models performed significantly in the external validation for EGFR amplification (area-under-the-curve (AUC) 0.707, 95% CI 0.582-8.25) and MGMT-methylation (AUC 0.667, 95% CI 0.522-0.82) but not for IDH-mutation (AUC 0.695, 95% CI 0.436-0.927). The integrated clinical and imaging prognostic model was shown to be robust and of potential clinical relevance. The prediction of molecular markers showed promising results in the training set but could not be validated after external validation in a clinically relevant manner. Overall, these results show the potential of combining clinical features with imaging features for prognostic and predictive models in GBM, but further optimization and larger prospective studies are warranted.

A lineage-tracing tool to map the fate of hypoxic tumour cells.

Intratumoural hypoxia is a common characteristic of malignant treatment-resistant cancers. However, hypoxia-modification strategies for the clinic remain elusive. To date, little is known on the behaviour of individual hypoxic tumour cells in their microenvironment. To explore this issue in a spatial and temporally controlled manner, we developed a genetically encoded sensor by fusing the O2-labile hypoxia-inducible factor 1α (HIF-1α) protein to eGFP and a tamoxifen-regulated Cre recombinase. Under normoxic conditions, HIF-1α is degraded but, under hypoxia, the HIF-1α-GFP-Cre-ERT2 fusion protein is stabilised and in the presence of tamoxifen activates a tdTomato reporter gene that is constitutively expressed in hypoxic progeny. We visualise the random distribution of hypoxic tumour cells from hypoxic or necrotic regions and vascularised areas using immunofluorescence and intravital microscopy. Once tdTomato expression is induced, it is stable for at least 4 weeks. Using this system, we could show in vivo that the post-hypoxic cells were more proliferative than non-labelled cells. Our results demonstrate that single-cell lineage tracing of hypoxic tumour cells can allow visualisation of their behaviour in living tumours using intravital microscopy. This tool should prove valuable for the study of dissemination and treatment response of post-hypoxic tumour cells in vivo at single-cell resolution.This article has an associated First Person interview with the joint first authors of the paper.

Hypoxic regulation of metastasis via hypoxia-inducible factors.

Metastases formation is a major factor in disease progression and accounts for the majority of cancer deaths. The molecular mechanisms controlling invasion, dissemination to blood or lymphatic systems and spread of tumor cells to distant organs are still poorly understood. Recent observations indicate that the meta-static phenotype may already be present during the angiogenic switch of tumors. Intratumoral hypoxia correlates with poor prognosis and enhanced metastases formation. The Hypoxia Inducible Factors (HIFs) are key molecules in the hypoxic response and play critical roles during tumor cell expansion by regulating energy metabolism and the induction of angiogenesis. Increasing evidence implicates HIF function in metastatic cell characteristics, like epithelial to mesenchymal transition, cell detachment, invasion and tumor cell seeding. Here, we review the link between tumor cell hypoxia and the acquisition of metastatic behavior. We hypothesize that polyclonal tumor selection by hypoxia enhances metastatic capacity by transcriptional control of key regulators of metastasis. This polyclonal hypoxic gene profile potentially develops into a metastatic profile, driving metastasis formation. The hypoxic gene profile in primary tumors may therefore provide a prognostic indicator in clinical decision-making.

Radiation-Induced Lung Injury (RILI).

Radiation pneumonitis (RP) and radiation fibrosis (RF) are two dose-limiting toxicities of radiotherapy (RT), especially for lung, and esophageal cancer. It occurs in 5-20% of patients and limits the maximum dose that can be delivered, reducing tumor control probability (TCP) and may lead to dyspnea, lung fibrosis, and impaired quality of life. Both physical and biological factors determine the normal tissue complication probability (NTCP) by Radiotherapy. A better understanding of the pathophysiological sequence of radiation-induced lung injury (RILI) and the intrinsic, environmental and treatment-related factors may aid in the prevention, and better management of radiation-induced lung damage. In this review, we summarize our current understanding of the pathological and molecular consequences of lung exposure to ionizing radiation, and pharmaceutical interventions that may be beneficial in the prevention or curtailment of RILI, and therefore enable a more durable therapeutic tumor response.

HIF-1α and HIF-2α Differently Regulate the Radiation Sensitivity of NSCLC Cells.

The hypoxia-inducible transcription factors (HIF)-1/2α are the main oxygen sensors which regulate the adaptation to intratumoral hypoxia. The aim of this study was to assess the role of the HIF proteins in regulating the radiation response of a non-small cell lung cancer (NSCLC) in vitro model. To directly assess the unique and overlapping functions of HIF-1α and HIF-2α, we use CRISPR gene-editing to generate isogenic H1299 non-small cell lung carcinoma cells lacking HIF-1α, HIF-2α or both. We found that in HIF1 knockout cells, HIF-2α was strongly induced by hypoxia compared to wild type but the reverse was not seen in HIF2 knockout cells. Cells lacking HIF-1α were more radiation resistant than HIF2 knockout and wildtype cells upon hypoxia, which was associated with a reduced recruitment of γH2AX foci directly after irradiation and not due to differences in proliferation. Conversely, double-HIF1/2 knockout cells were most radiation sensitive and had increased γH2AX recruitment and cell cycle delay. Compensatory HIF-2α activity in HIF1 knockout cells is the main cause of this radioprotective effect. Under hypoxia, HIF1 knockout cells uniquely had a strong increase in lactate production and decrease in extracellular pH. Using genetically identical HIF-α isoform-deficient cells we identified a strong radiosensitizing of HIF1, but not of HIF2, which was associated with a reduced extracellular pH and reduced glycolysis.

Moving Breast Cancer Therapy up a Notch.

Breast cancer is the second most common malignancy, worldwide. Treatment decisions are based on tumor stage, histological subtype, and receptor expression and include combinations of surgery, radiotherapy, and systemic treatment. These, together with earlier diagnosis, have resulted in increased survival. However, initial treatment efficacy cannot be guaranteed upfront, and these treatments may come with (long-term) serious adverse effects, negatively affecting a patient's quality of life. Gene expression-based tests can accurately estimate the risk of recurrence in early stage breast cancers. Disease recurrence correlates with treatment resistance, creating a major need to resensitize tumors to treatment. Notch signaling is frequently deregulated in cancer and is involved in treatment resistance. Preclinical research has already identified many combinatory therapeutic options where Notch involvement enhances the effectiveness of radiotherapy, chemotherapy or targeted therapies for breast cancer. However, the benefit of targeting Notch has remained clinically inconclusive. In this review, we summarize the current knowledge on targeting the Notch pathway to enhance current treatments for breast cancer and to combat treatment resistance. Furthermore, we propose mechanisms to further exploit Notch-based therapeutics in the treatment of breast cancer.

EGFRvIII expression triggers a metabolic dependency and therapeutic vulnerability sensitive to autophagy inhibition.

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.

GABARAPL1 is required for increased EGFR membrane expression during hypoxia.

BACKGROUND AND PURPOSE: The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various human epithelial tumors and hypoxia is a common feature of solid tumors. Both EGFR and hypoxia are associated with therapy resistance and poor treatment outcome. To survive hypoxia, cells adapt by activation of hypoxia responsive pathways and expression of hypoxia-induced plasma membrane proteins. We observed that GABAA receptor associated protein like1 (GABARAPL1) and plasma membrane expression of EGFR were increased during hypoxia. Here we explored the role of the GABARAPL1 in EGFR membrane expression during hypoxia. MATERIAL AND METHODS: Quantitative qPCR, immunoblot analysis, flow cytometry and cytochemistry were used to assess this interplay. RESULTS: GABARAPL1 mRNA and protein levels are increased during hypoxia in vitro and correlate with tumor hypoxia in a panel of primary HNSCC xenografts. High GABARAPL1 mRNA is associated with poor outcome of HNSCC patients. During hypoxia, EGFR membrane expression is increased and GABARAPL1 and EGFR colocalize at the plasma membrane. GABARAPL1 knockdown inhibits EGFR membrane expression during hypoxia. CONCLUSION: GABARAPL1 is required for increased membrane expression of EGFR during hypoxia, suggesting a role for GABARAPL1 in the trafficking of these membrane proteins.

EGFR overexpressing cells and tumors are dependent on autophagy for growth and survival.

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.

The role of hypoxia inducible factor-1alpha in gynecological cancer.

Understanding the mechanisms of carcinogenesis and progression of gynecological tumors is important as these insights might lead to improved diagnostic tools for the pathologist, improved prediction of prognosis, response to therapy, and eventually better biology-based disease management, thereby improving prognosis and quality of life for the individual patient. Hypoxia is an important event in carcinogenesis because it renders a more aggressive phenotype with increased invasiveness and proliferation, formation of metastases and poorer survival. Although selecting patients with hypoxic tumors may therefore be clinically important, there is no consensus as to the method best suited for routine assessment of hypoxia. One of the potential tumor hypoxia markers is hypoxia inducible factor 1 (HIF-1). HIF-1 is the key cellular survival protein under hypoxia, and is associated with tumor progression and metastasis in various solid tumors. In this review, we show that in gynecological cancers, HIF-1A is emerging as an important factor in carcinogenesis, and that overexpression of HIF-1A and its target genes CA9 and SLC2A1 seems associated with shorter progression free- and overall survival. Since hypoxia and HIF-1A expression are associated with treatment failure, targeting HIF-1A could be an attractive therapeutic strategy with the potential for disrupting multiple pathways crucial for tumor growth. Currently, HIF-1A inhibitors are being studied in clinical trials in recurrent ovarian- and cervical cancer, and trials in other gynecological cancers are expected.