Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation.

Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status.

GABARAPL1 is essential in extracellular vesicle cargo loading and metastasis development.

BACKGROUND AND PURPOSE: Hypoxia is a common feature of tumours, associated with poor prognosis due to increased resistance to radio- and chemotherapy and enhanced metastasis development. Previously we demonstrated that GABARAPL1 is required for the secretion of extracellular vesicles (EV) with pro-angiogenic properties during hypoxia. Here, we explored the role of GABARAPL1+ EV in the metastatic cascade. MATERIALS AND METHODS: GABARAPL1 deficient or control MDA-MB-231 cells were injected in murine mammary fat pads. Lungs were dissected and analysed for human cytokeratin 18. EV from control and GABARAPL1 deficient cells exposed to normoxia (21% O2) or hypoxia (O2 < 0.02%) were isolated and analysed by immunoblot, nanoparticle tracking analysis, high resolution flow cytometry, mass spectrometry and next-generation sequencing. Cellular migration and invasion were analysed using scratch assays and transwell-invasion assays, respectively. RESULTS: The number of pulmonary metastases derived from GABARAPL1 deficient tumours decreased by 84%. GABARAPL1 deficient cells migrate slower but display a comparable invasive capacity. Both normoxic and hypoxic EV contain proteins and miRNAs associated with metastasis development and, in line, increase cancer cell invasiveness. Although GABARAPL1 deficiency alters EV content, it does not alter the EV-induced increase in cancer cell invasiveness. CONCLUSION: GABARAPL1 is essential for metastasis development. This is unrelated to changes in migration and invasion and suggests that GABARAPL1 or GABARAPL1+ EV are essential in other processes related to the metastatic cascade.

ATG12 deficiency results in intracellular glutamine depletion, abrogation of tumor hypoxia and a favorable prognosis in cancer.

Hypoxia is a common feature of solid tumors and is associated with increased tumor progression, resistance to therapy and increased metastasis. Hence, tumor hypoxia is a prognostic factor independent of treatment modality. To survive hypoxia, cells activate macroautophagy/autophagy. Paradoxically, in several cancer types, mutations or loss of essential autophagy genes have been reported that are associated with earlier onset of tumor growth. However, to our knowledge, the phenotypic and therapeutic consequences of autophagy deficiency have remained unexplored. In this study, we determined autophagy-defects in head and neck squamous cell carcinoma (HNSCC) and observed that expression of ATG12 (autophagy related 12) was lost in 25%-40% of HNSCC. In line, ATG12 loss is associated with absence of hypoxia, as determined by pimonidazole immunohistochemistry. Hence, ATG12 loss is associated with improved prognosis after therapy in two independent HNSCC cohorts and 7 additional cancer types. In vivo, ATG12 targeting resulted in decreased hypoxia tolerance, increased necrosis and sensitivity of the tumor to therapy, but in vitro ATG12-deficient cells displayed enhanced survival in nutrient-rich culture medium. Besides oxygen, delivery of glucose was hampered in hypoxic regions in vivo, which increases the reliance of cells on other carbon sources (e.g., L-glutamine). We observed decreased intracellular L-glutamine levels in ATG12-deficient cells during hypoxia and increased cell killing after L-glutamine depletion, indicating a central role for ATG12 in maintaining L-glutamine homeostasis. Our results demonstrate that ATG12low tumors represent a phenotypically different subtype that, due to the lowered hypoxia tolerance, display a favorable outcome after therapy.Abbreviations: ARCON:accelerated radiotherapy with carbogen and nicotinamide; ATG: autophagy related; BrdUrd: bromodeoxyuridine; CA9/CAIX: carbonic anhydrase 9; HIF1A/HIF1α: hypoxia inducible factor 1 subunit alpha; HNSCC: head and neck squamous cell carcinoma; HPV: human papilloma virus; HR: hazard ratio; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; mRNA: messenger ribonucleic acid; PCR: polymerase chain reaction; SLC2A1/GLUT1: solute carrier family 2 member 1; TCGA: the Cancer Genome Atlas; TME: tumor microenvironment; UTR: untranslated region; VEGF: vascular endothelial growth factor.

Tumors Responsive to Autophagy-Inhibition: Identification and Biomarkers.

Recent advances in cancer treatment modalities reveal the limitations of the prevalent "one-size-fits-all" therapies and emphasize the necessity to develop personalized approaches. In this perspective, identification of predictive biomarkers and intrinsic vulnerabilities are an important advancement for further therapeutic strategies. Autophagy is an important lysosomal degradation and recycling pathway that provides energy and macromolecular precursors to maintain cellular homeostasis. Although all cells require autophagy, several genetic and/or cellular changes elevate the dependence of cancer cells on autophagy for their survival and indicates that autophagy inhibition in these tumors could provide a favorable addition to current therapies. In this context, we review the current literature on tumor (sub)types with elevated dependence on autophagy for their survival and highlight an exploitable vulnerability. We provide an inventory of microenvironmental factors, genetic alterations and therapies that may be exploited with autophagy-targeted approaches to improve efficacy of conventional anti-tumor therapies.

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.

The anti-malarial drug chloroquine sensitizes oncogenic NOTCH1 driven human T-ALL to γ-secretase inhibition.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from T-cell progenitors. Although current treatments, including chemotherapy and glucocorticoids, have significantly improved survival, T-ALL remains a fatal disease and new treatment options are needed. Since more than 60% of T-ALL cases bear oncogenic NOTCH1 mutations, small molecule inhibitors of NOTCH1 signalling; γ-secretase inhibitors (GSI), are being actively investigated for the treatment of T-ALL. Unfortunately, GSI have shown limited clinical efficacy and dose-limiting toxicities. We hypothesized that by combining known drugs, blocking NOTCH activity through another mechanism, may synergize with GSI enabling equal efficacy at a lower concentration. Here, we show that the clinically used anti-malarial drug chloroquine (CQ), an inhibitor of lysosomal function and autophagy, decreases T-ALL cell viability and proliferation. This effect of CQ was not observed in GSI-resistant T-ALL cell lines. Mechanistically, CQ impairs the redox balance, induces ds DNA breaks and activates the DNA damage response. CQ also interferes with intracellular trafficking and processing of oncogenic NOTCH1. Interestingly, we show for the first time that the addition of CQ to γ-secretase inhibition has a synergistic therapeutic effect on T-ALL and reduces the concentration of GSI required to obtain a reduction in cell viability and a block of proliferation. Overall, our results suggest that CQ may be a promising repurposed drug in the treatment of T-ALL, as a single treatment or in combination with GSI, increasing the therapeutic ratio.

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.

Synergistic Effects of NOTCH/γ-Secretase Inhibition and Standard of Care Treatment Modalities in Non-small Cell Lung Cancer Cells.

Background: Lung cancer is the leading cause of cancer death worldwide. More effective treatments are needed to increase durable responses and prolong patient survival. Standard of care treatment for patients with non-operable stage III-IV NSCLC is concurrent chemotherapy and radiation. An activated NOTCH signaling pathway is associated with poor outcome and treatment resistance in non-small cell lung cancer (NSCLC). NOTCH/γ-secretase inhibitors have been effective in controlling tumor growth in preclinical models but the therapeutic benefit of these inhibitors as monotherapy in patients has been limited so far. Because NOTCH signaling has been implicated in treatment resistance, we hypothesized that by combining NOTCH inhibitors with chemotherapy and radiotherapy this could result in an increased therapeutic effect. A direct comparison of the effects of NOTCH inhibition when combined with current treatment combinations for NSCLC is lacking. Methods: Using monolayer growth assays, we screened 101 FDA-approved drugs from the Cancer Therapy Evaluation Program alone, or combined with radiation, in the H1299 and H460 NSCLC cell lines to identify potent treatment interactions. Subsequently, using multicellular three-dimensional tumor spheroid assays, we tested a selection of drugs used in clinical practice for NSCLC patients, and combined these with a small molecule inhibitor, currently being tested in clinical trials, of the NOTCH pathway (BMS-906024) alone, or in combination with radiation, and measured specific spheroid growth delay (SSGD). Statistical significance was determined by one-way ANOVA with post-hoc Bonferroni correction, and synergism was assessed using two-way ANOVA. Results: Monolayer assays in H1299 and H460 suggest that 21 vs. 5% were synergistic, and 17 vs. 11% were additive chemoradiation interactions, respectively. In H1299 tumor spheroids, significant SSGD was obtained for cisplatin, etoposide, and crizotinib, which increased significantly after the addition of the NOTCH inhibitor BMS-906024 (but not for paclitaxel and pemetrexed), and especially in triple combination with radiation. Synergistic interactions were observed when BMS-906024 was combined with chemoradiation (cisplatin, paclitaxel, docetaxel, and crizotinib). Similar results were observed for H460 spheroids using paclitaxel or crizotinib in dual combination treatment with NOTCH inhibition and triple with radiation. Conclusions: Our findings point to novel synergistic combinations of NOTCH inhibition and chemoradiation that should be tested in NSCLC in vivo models for their ability to achieve an improved therapeutic ratio.

NOTCH blockade combined with radiation therapy and temozolomide prolongs survival of orthotopic glioblastoma.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. The current standard of care includes surgery followed by radiotherapy (RT) and chemotherapy with temozolomide (TMZ). Treatment often fails due to the radiation resistance and intrinsic or acquired TMZ resistance of a small percentage of cells with stem cell-like behavior (CSC). The NOTCH signaling pathway is expressed and active in human glioblastoma and NOTCH inhibitors attenuate tumor growth in vivo in xenograft models. Here we show using an image guided micro-CT and precision radiotherapy platform that a combination of the clinically approved NOTCH/γ-secretase inhibitor (GSI) RO4929097 with standard of care (TMZ + RT) reduces tumor growth and prolongs survival compared to dual combinations. We show that GSI in combination with RT and TMZ attenuates proliferation, decreases 3D spheroid growth and results into a marked reduction in clonogenic survival in primary and established glioma cell lines. We found that the glioma stem cell marker CD133, SOX2 and Nestin were reduced following combination treatments and NOTCH inhibitors albeit in a different manner. These findings indicate that NOTCH inhibition combined with standard of care treatment has an anti-glioma stem cell effect which provides an improved survival benefit for GBM and encourages further translational and clinical studies.

An image guided small animal radiation therapy platform (SmART) to monitor glioblastoma progression and therapy response.

BACKGROUND AND PURPOSE: Glioblastoma multiforme is the most common malignant brain tumor. Standard treatment including surgery, radiotherapy and chemotherapy with temozolomide is not curative. There is a great need for in vitro and in vivo models closely mimicking clinical practice to ensure better translation of novel preclinical findings. METHODS AND MATERIALS: A 3D spheroid model was established using the U87MG cell line. The efficacy of temozolomide, RT and combinations was assessed using growth delay assays. Orthotopic glioblastoma tumors were established, different radiation doses delivered based on micro-CT based treatment planning (SmART-plan) and dose volume histograms (DVH) were determined. Tumor growth was monitored using bioluminescent imaging. RESULTS: 3D spheroid cultures showed a dose-dependent growth delay upon single and combination treatments. Precise uniform radiation was achieved in all in vivo treatment groups at all doses tested, and DVHs showed accurate dose coverage in the planning target volume which resulted in tumor growth delay. CONCLUSION: We demonstrate that 3D spheroid technology can be reliably used for treatment efficacy evaluation and that mimicking a clinical setting is also possible in small animals. Both these in vitro and in vivo techniques can be combined for clinically relevant testing of novel drugs combined with radiation.