Novel insights into the GCN2 pathway and its targeting. Therapeutic value in cancer and lessons from lung fibrosis development.

Defining the mechanisms that allow cells to adapt to environmental stress is critical for understanding the progression of chronic diseases and identifying relevant drug targets. Among these, activation of the pathway controlled by the eIF2-alpha kinase GCN2 is critical for translational and metabolic reprogramming of the cell in response to various metabolic, proteotoxic, and ribosomal stressors. However, its role has frequently been investigated through the lens of a stress pathway signaling via the eIF2α-activating transcription factor 4 (ATF4) downstream axis, while recent advances in the field have revealed that the GCN2 pathway is more complex than previously thought. Indeed, this kinase can be activated through a variety of mechanisms, phosphorylate substrates other than eIF2α, and regulate cell proliferation in a steady state. This review presents recent findings regarding the fundamental mechanisms underlying GCN2 signaling and function, as well as the development of drugs that modulate its activity. Furthermore, by comparing the literature on GCN2's antagonistic roles in two challenging pathologies, cancer and pulmonary diseases, the benefits, and drawbacks of GCN2 targeting, particularly inhibition, are discussed.

Soft extracellular matrix drives endoplasmic reticulum stress-dependent S quiescence underlying molecular traits of pulmonary basal cells.

Cell culture on soft matrix, either in 2D and 3D, preserves the characteristics of progenitors. However, the mechanism by which the mechanical microenvironment determines progenitor phenotype, and its relevance to human biology, remains poorly described. Here we designed multi-well hydrogel plates with a high degree of physico-chemical uniformity to reliably address the molecular mechanism underlying cell state modification driven by physiological stiffness. Cell cycle, differentiation and metabolic activity could be studied in parallel assays, showing that the soft environment promotes an atypical S-phase quiescence and prevents cell drift, while preserving the differentiation capacities of human bronchoepithelial cells. These softness-sensitive responses are associated with calcium leakage from the endoplasmic reticulum (ER) and defects in proteostasis and enhanced basal ER stress. The analysis of available single cell data of the human lung also showed that this non-conventional state coming from the soft extracellular environment is indeed consistent with molecular feature of pulmonary basal cells. Overall, this study demonstrates that mechanical mimicry in 2D culture supports allows to maintain progenitor cells in a state of high physiological relevance for characterizing the molecular events that govern progenitor biology in human tissues. STATEMENT OF SIGNIFICANCE: This study focuses on the molecular mechanism behind the progenitor state induced by a soft environment. Using innovative hydrogel supports mimicking normal human lung stiffness, the data presented demonstrate that lung mechanics prevent drift while preserving the differentiation capabilities of lung epithelial cells. Furthermore, we show that the cells are positioned in a quiescent state in the atypical S phase. Mechanistically, we demonstrate that this quiescence: i) is driven by calcium leakage from the endoplasmic reticulum (ER) and basal activation of the PERK branch of ER stress signalling, and ii) protects cells from lethal ER stress caused by metabolic stress. Finally, we validate using human single-cell data that these molecular features identified on the soft matrix are found in basal lung cells. Our results reveal original and relevant molecular mechanisms orchestrating cell fate in a soft environment and resistance to exogenous stresses, thus providing new fundamental and clinical insights into basal cell biology.

SUrface SEnsing of Translation (SUnSET), a Method Based on Western Blot Assessing Protein Synthesis Rates in vitro.

As the most energy- and metabolite-consuming process, protein synthesis is under the control of several intrinsic and extrinsic factors that determine its fine-tuning to the cellular microenvironment. Consequently, variations in protein synthesis rates occur under various physiological and pathological conditions, enabling an adaptive response by the cell. For example, global protein synthesis increases upon mitogenic factors to support biomass generation and cell proliferation, while exposure to low concentrations of oxygen or nutrients require translational repression and reprogramming to avoid energy depletion and cell death. To assess fluctuations in protein synthesis rates, radioactive isotopes or radiolabeled amino acids are often used. Although highly sensitive, these techniques involve the use of potentially toxic radioactive compounds and require specific materials and processes for the use and disposal of these molecules. The development of alternative, non-radioactive methods that can be easily and safely implemented in laboratories has therefore been encouraged to avoid handling radioactivity. In this context, the SUrface SEnsing of Translation (SUnSET) method, based on the classical western blot technique, was developed by Schmidt et al. in 2009. The SUnSET is nowadays recognized as a simple alternative to radioactive methods assessing protein synthesis rates. Key features • As a structural analogue of aminoacyl-transfer RNA, puromycin incorporates into the elongating peptide chain. • Detection of puromycin-labeled peptides by western blotting reflects translation rates without the need for radioactive isotopes. • The protocol described here for in vitro applications is derived from the SUnSET method originally published by Schmidt et al. (2009).

Tailoring a specific medical leadership development program for faculty members: the Lyon-Ottawa experience.

The development of leadership skills has been the topic of several position statements over recent decades, and the need of medical leaders for a specific training was emphasized during the COVID-19 crisis, to enable them to adequately collaborate with governments, populations, civic society, organizations, and universities. However, differences persist as to the way such skills are taught, at which step of training, and to whom. From these observations and building on previous experience at the University of Ottawa, a team of medical professors from Lyon (France), Ottawa, and Montreal (Canada) universities decided to develop a specific medical leadership training program dedicated to faculty members taking on leadership responsibilities. This pilot training program was based on a holistic vision of a transformation model for leadership development, the underlying principle of which is that leaders are trained by leaders. All contributors were eminent French and Canadian stakeholders. The model was adapted to French faculty members, following an inner and outer analysis of their specific needs, both contextual and related to their time constraints. This pilot program, which included 10 faculty members from Lyon, was selected to favor interactivity and confidence in older to favor long-term collaborations between them and contribute to institutional changes from the inner; it combined several educational methods mixing interactive plenary sessions and simulation exercises during onescholar year. All the participants completed the program and expressed global satisfaction with it, validating its acceptability by the target. Future work will aim to develop the program, integrate evaluation criteria, and transform it into a graduating training.

The stress sensor GCN2 differentially controls ribosome biogenesis in colon cancer according to the nutritional context.

Nutrient availability is a key determinant of tumor cell behavior. While nutrient-rich conditions favor proliferation and tumor growth, scarcity, and particularly glutamine starvation, promotes cell dedifferentiation and chemoresistance. Here, linking ribosome biogenesis plasticity with tumor cell fate, we uncover that the amino acid sensor general control non-derepressible 2 (GCN2; also known as eIF-2-alpha kinase 4) represses the expression of the precursor of ribosomal RNA (rRNA), 47S, under metabolic stress. We show that blockade of GCN2 triggers cell death by an irremediable nucleolar stress and subsequent TP53-mediated apoptosis in patient-derived models of colon adenocarcinoma (COAD). In nutrient-rich conditions, a cell-autonomous GCN2 activity supports cell proliferation by stimulating 47S rRNA transcription, independently of the canonical integrated stress response (ISR) axis. Impairment of GCN2 activity prevents nuclear translocation of methionyl-tRNA synthetase (MetRS), resulting in nucleolar stress, mTORC1 inhibition and, ultimately, autophagy induction. Inhibition of the GCN2-MetRS axis drastically improves the cytotoxicity of RNA polymerase I (RNA pol I) inhibitors, including the first-line chemotherapy oxaliplatin, on patient-derived COAD tumoroids. Our data thus reveal that GCN2 differentially controls ribosome biogenesis according to the nutritional context. Furthermore, pharmacological co-inhibition of the two GCN2 branches and RNA pol I activity may represent a valuable strategy for elimination of proliferative and metabolically stressed COAD cells.

Improved NGS-based detection of microsatellite instability using tumor-only data.

Microsatellite instability (MSI) is a molecular signature of mismatch repair deficiency (dMMR), a predictive marker of immune checkpoint inhibitor therapy response. Despite its recognized pan-cancer value, most methods only support detection of this signature in colorectal cancer. In addition to the tissue-specific differences that impact the sensitivity of MSI detection in other tissues, the performance of most methods is also affected by patient ethnicity, tumor content, and other sample-specific properties. These limitations are particularly important when only tumor samples are available and restrict the performance and adoption of MSI testing. Here we introduce MSIdetect, a novel solution for NGS-based MSI detection. MSIdetect models the impact of indel burden and tumor content on read coverage at a set of homopolymer regions that we found are minimally impacted by sample-specific factors. We validated MSIdetect in 139 Formalin-Fixed Paraffin-Embedded (FFPE) clinical samples from colorectal and endometrial cancer as well as other more challenging tumor types, such as glioma or sebaceous adenoma or carcinoma. Based on analysis of these samples, MSIdetect displays 100% specificity and 96.3% sensitivity. Limit of detection analysis supports that MSIdetect is sensitive even in samples with relatively low tumor content and limited microsatellite instability. Finally, the results obtained using MSIdetect in tumor-only data correlate well (R=0.988) with what is obtained using tumor-normal matched pairs, demonstrating that the solution addresses the challenges posed by MSI detection from tumor-only data. The accuracy of MSI detection by MSIdetect in different cancer types coupled with the flexibility afforded by NGS-based testing will support the adoption of MSI testing in the clinical setting and increase the number of patients identified that are likely to benefit from immune checkpoint inhibitor therapy.

The Kynurenine Pathway and Cancer: Why Keep It Simple When You Can Make It Complicated.

The kynurenine pathway has been highlighted as a gatekeeper of immune-privileged sites through its ability to generate from tryptophan a set of immunosuppressive metabolic intermediates. It additionally constitutes an important source of cellular NAD+ for the organism. Hijacking of its immunosuppressive functions, as recurrently observed in multiple cancers, facilitates immune evasion and promotes tumor development. Based on these observations, researchers have focused on characterizing indoleamine 2,3-dioxygenase (IDO1), the main enzyme catalyzing the first and limiting step of the pathway, and on developing therapies targeting it. Unfortunately, clinical trials studying IDO1 inhibitors have thus far not met expectations, highlighting the need to unravel this complex signaling pathway further. Recent advances demonstrate that these metabolites additionally promote tumor growth, metastatic dissemination and chemoresistance by a combination of paracrine and autocrine effects. Production of NAD+ also contributes to cancer progression by providing cancer cells with enhanced plasticity, invasive properties and chemoresistance. A comprehensive survey of this complexity is challenging but necessary to achieve medical success.

The hexosamine pathway and coat complex II promote malignant adaptation to nutrient scarcity.

The glucose-requiring hexosamine biosynthetic pathway (HBP), which produces UDP-N-acetylglucosamine for glycosylation reactions, promotes lung adenocarcinoma (LUAD) progression. However, lung tumor cells often reside in low-nutrient microenvironments, and whether the HBP is involved in the adaptation of LUAD to nutrient stress is unknown. Here, we show that the HBP and the coat complex II (COPII) play a key role in cell survival during glucose shortage. HBP up-regulation withstood low glucose-induced production of proteins bearing truncated N-glycans, in the endoplasmic reticulum. This function for the HBP, alongside COPII up-regulation, rescued cell surface expression of a subset of glycoproteins. Those included the epidermal growth factor receptor (EGFR), allowing an EGFR-dependent cell survival under low glucose in anchorage-independent growth. Accordingly, high expression of the HBP rate-limiting enzyme GFAT1 was associated with wild-type EGFR activation in LUAD patient samples. Notably, HBP and COPII up-regulation distinguished LUAD from the lung squamous-cell carcinoma subtype, thus uncovering adaptive mechanisms of LUAD to their harsh microenvironment.

Pemetrexed Hinders Translation Inhibition upon Low Glucose in Non-Small Cell Lung Cancer Cells.

Genetic alterations in non-small cell lung cancers (NSCLC) stimulate the generation of energy and biomass to promote tumor development. However, the efficacy of the translation process is finely regulated by stress sensors, themselves often controlled by nutrient availability and chemotoxic agents. Yet, the crosstalk between therapeutic treatment and glucose availability on cell mass generation remains understudied. Herein, we investigated the impact of pemetrexed (PEM) treatment, a first-line agent for NSCLC, on protein synthesis, depending on high or low glucose availability. PEM treatment drastically repressed cell mass and translation when glucose was abundant. Surprisingly, inhibition of protein synthesis caused by low glucose levels was partially dampened upon co-treatment with PEM. Moreover, PEM counteracted the elevation of the endoplasmic reticulum stress (ERS) signal produced upon low glucose availability, providing a molecular explanation for the differential impact of the drug on translation according to glucose levels. Collectively, these data indicate that the ERS constitutes a molecular crosstalk between microenvironmental stressors, contributing to translation reprogramming and proteostasis plasticity.

ATF4-Dependent NRF2 Transcriptional Regulation Promotes Antioxidant Protection during Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress generates reactive oxygen species (ROS) that induce apoptosis if left unabated. To limit oxidative insults, the ER stress PKR-like endoplasmic reticulum Kinase (PERK) has been reported to phosphorylate and activate nuclear factor erythroid 2-related factor 2 (NRF2). Here, we uncover an alternative mechanism for PERK-mediated NRF2 regulation in human cells that does not require direct phosphorylation. We show that the activation of the PERK pathway rapidly stimulates the expression of NRF2 through activating transcription factor 4 (ATF4). In addition, NRF2 activation is late and largely driven by reactive oxygen species (ROS) generated during late protein synthesis recovery, contributing to protecting against cell death. Thus, PERK-mediated NRF2 activation encompasses a PERK-ATF4-dependent control of NRF2 expression that contributes to the NRF2 protective response engaged during ER stress-induced ROS production.

Rapid detection of EGFR mutations in decalcified lung cancer bone metastasis.

Detection of molecular alterations in lung cancer bone metastasis (LCBM) is particularly difficult when decalcification procedure is needed. The Idylla™ real-time (RT)-PCR is compared to the routine method used in our laboratory, which combines next generation and Sanger sequencing, for the detection of EGFR mutations in LCBM. LCBM subjected to EDTA or formic acid decalcification were analysed for EGFR mutational status using two methods: first, the Ion Torrent Ampliseq next generation sequencing (NGS) assay +/- Sanger sequencing was used prospectively; then, the fully-automated, RT-PCR based molecular testing system Idylla™ EGFR Mutation Test was applied retrospectively. Out of the 34 LCBM assayed, 14 (41.2%) were unsuitable for NGS analysis and five remained unsuitable after additional Sanger EGFR sequencing (5/34, 14.7%). Using Idylla™, valid results were observed for 33/34 samples (97.1%). The concordance between the NGS +/- Sanger sequencing method and the RT-PCR method was 89.7% (26/29), one false positive EGFR S768I mutation and two false negative results were observed using Idylla™; one of these false negative cases was diagnosed by Sanger sequencing with a rare exon 19 EGFR mutation not covered by the Idylla™ EGFR Mutation Test design. Detection of EGFR mutations in decalcified LCBM is challenging using NGS, more than half of samples showing invalid results. Alternative methods should thus be preferred to spare clinical samples and decrease delay. The Idylla™ EGFR Mutation Test shows a good performance on decalcified bone samples and could be used as a first step. In case of negative results, a sequencing approach is mandatory to check the presence of rare EGFR mutations sensitive to EGFR tyrosine kinase inhibitors.

A Multiplex Quantitative Reverse Transcription Polymerase Chain Reaction Assay for the Detection of KIAA1549-BRAF Fusion Transcripts in Formalin-Fixed Paraffin-Embedded Pilocytic Astrocytomas.

BACKGROUND AND OBJECTIVE: Genomic duplications and fusion involving BRAF and KIAA1549 that create fusion proteins with constitutive B-RAF kinase activity are a hallmark of pilocytic astrocytomas (PAs). The detection of KIAA1549-BRAF fusion transcripts is of paramount importance to classify these tumors and to identify patients who could benefit from BRAF inhibitors. In a clinical setting, the available material for molecular analysis from these pediatric tumors is often limited to formalin-fixed paraffin-embedded (FFPE) tissue. The aim of the present study was to develop a new method to detect the three most frequent KIAA1549-BRAF fusion transcripts, 15-9, 16-11, and 16-9, where numbers refer to the exons fused together, using a FFPE-compatible multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR). METHODS: We compared performance of the assay to a reference singleplex method on a collection of 46 FFPE PAs. RESULTS: The results showed that both methods are comparable. The multiplex method had an overall 97% sensitivity and 100% specificity compared to the singleplex method, and agreement between the two techniques was almost perfect (Cohen's kappa: 0.97). There was no evidence of a significant difference between the qRT-PCR efficiencies of the multiplex technique and of the singleplex assay for all fusion transcripts and for GAPDH, the latter used as a reference gene. The multiplex method consumed four times less complementary DNA (cDNA), cost less, and required half the hands-on technical time. CONCLUSION: The results show that it could be beneficial to implement the multiplex method in a clinical setting, where samples presenting low quantity of degraded RNA are not unusual.

Derivation of Breast Cancer Cell Lines Under Physiological (5%) Oxygen Concentrations.

Background: Most human breast cancer cell lines currently in use were developed and are cultured under ambient (21%) oxygen conditions. While this is convenient in practical terms, higher ambient oxygen could increase oxygen radical production, potentially modulating signaling pathways. We have derived and grown a series of four human breast cancer cell lines under 5% oxygen, and have compared their properties to those of established breast cancer lines growing under ambient oxygen. Methods: Cell lines were characterized in terms of appearance, cellular DNA content, mutation spectrum, hormone receptor status, pathway utilization and drug sensitivity. Results: Three of the four lines (NZBR1, NZBR2, and NZBR4) were triple negative (ER-, PR-, HER2-), with NZBR1 also over-expressing EGFR. NZBR3 was HER2+ and ER+ and also over-expressed EGFR. Cell lines grown in 5% oxygen showed increased expression of the hypoxia-inducible factor 1 (HIF-1) target gene carbonic anhydrase 9 (CA9) and decreased levels of ROS. As determined by protein phosphorylation, NZBR1 showed low AKT pathway utilization while NZBR2 and NZBR4 showed low p70S6K and rpS6 pathway utilization. The lines were characterized for sensitivity to 7-hydroxytamoxifen, doxorubicin, paclitaxel, the PI3K inhibitor BEZ235 and the HER inhibitors lapatinib, afatinib, dacomitinib, and ARRY-380. In some cases they were compared to established breast cancer lines. Of particular note was the high sensitivity of NZBR3 to HER inhibitors. The spectrum of mutations in the NZBR lines was generally similar to that found in commonly used breast cancer cell lines but TP53 mutations were absent and mutations in EVI2B, LRP1B, and PMS2, which have not been reported in other breast cancer lines, were detected. The results suggest that the properties of cell lines developed under low oxygen conditions (5% O2) are similar to those of commonly used breast cancer cell lines. Although reduced ROS production and increased HIF-1 activity under 5% oxygen can potentially influence experimental outcomes, no difference in sensitivity to estrogen or doxorubicin was observed between cell lines cultured in 5 vs. 21% oxygen.

Cross-platform comparison for the detection of RAS mutations in cfDNA (ddPCR Biorad detection assay, BEAMing assay, and NGS strategy).

CfDNA samples from colon (mCRC) and non-small cell lung cancers (NSCLC) (CIRCAN cohort) were compared using three platforms: droplet digital PCR (ddPCR, Biorad); BEAMing/OncoBEAM™-RAS-CRC (Sysmex Inostics); next-generation sequencing (NGS, Illumina), utilizing the 56G oncology panel (Swift Biosciences). Tissue biopsy and time matched cfDNA samples were collected at diagnosis in the mCRC cohort and during 1st progression in the NSCLC cohort. Excellent matches between cfDNA/FFPE mutation profiles were observed. Detection thresholds were between 0.5-1% for cfDNA samples examined using ddPCR and NGS, and 0.03% with BEAMing. This high level of sensitivity enabled the detection of KRAS mutations in 5/19 CRC patients with negative FFPE profiles. In the mCRC cohort, comparison of mutation results obtained by testing FFPE to those obtained by testing cfDNA by ddPCR resulted in 47% sensitivity, 77% specificity, 70% positive predictive value (PPV) and 55% negative predictive value (NPV). For BEAMing, we observed 93% sensitivity, 69% specificity, 78% PPV and 90% NPV. Finally, sensitivity of NGS was 73%, specificity was 77%, PPV 79% and NPV 71%. Our study highlights the complementarity of different diagnostic approaches and variability of results between OncoBEAM™-RAS-CRC and NGS assays. While the NGS assay provided a larger breadth of coverage of the major targetable alterations of 56 genes in one run, its performance for specific alterations was frequently confirmed by ddPCR results.

Combinatorial Treatment with mTOR Inhibitors and Streptozotocin Leads to Synergistic In Vitro and In Vivo Antitumor Effects in Insulinoma Cells.

Streptozotocin-based chemotherapy is the first-line chemotherapy recommended for advanced pancreatic neuroendocrine tumors (pNETs), whereas targeted therapies, including mTOR inhibitors, are available in second-line treatment. Unfortunately, objective response rates to both treatments are limited. Because mTOR pathway activation, commonly observed in pNETs, has been reported as one of the major mechanisms accounting for chemoresistance, we investigated the potential benefit of mTOR inhibition combined with streptozotocin treatment in a subset of pNETs, namely insulinomas. To evaluate the potential of mTOR inhibition in combination with streptozotocin, we selected four different inhibitors acting at various levels of the pathway (everolimus: inhibition of mTORC1; MK-2206: inhibition of AKT; BKM120: inhibition of PI3K, mTORC1, and mTORC2; and BEZ235: inhibition of mTORC1 and mTORC2). Effects on cell viability and apoptosis were assessed in insulinoma cell lines INS-1E (rat) and MIN6 (mouse) in vitro and were confirmed in vivo by using a mouse model of hepatic tumor dissemination after intrasplenic xenograft. In vitro, all four combinations display synergistic effects. These combinations lead to heterogeneous mTOR pathway inhibition, in agreement with their respective target, and increased apoptosis. In vivo, tumor growth in the liver was significantly inhibited by combining streptozotocin with everolimus (P = 0.0014), BKM120 (P = 0.0092), or BEZ235 (P = 0.008) as compared to each agent alone. These results suggest that targeting the mTOR pathway in combination with streptozotocin could be of potential benefit for insulinomas and pNET patients and thus support further clinical investigations. Mol Cancer Ther; 17(1); 60-72. ©2017 AACR.

Endocrine Therapy of Estrogen Receptor-Positive Breast Cancer Cells: Early Differential Effects on Stem Cell Markers.

INTRODUCTION: Endocrine therapy of breast cancer, which either deprives cancer tissue of estrogen or prevents estrogen pathway signaling, is the most common treatment after surgery and radiotherapy. We have previously shown for the estrogen-responsive MCF-7 cell line that exposure to tamoxifen, or deprivation of estrogen, leads initially to inhibition of cell proliferation, followed after several months by the emergence of resistant sub-lines that are phenotypically different from the parental line. We examined the early responses of MCF-7 cells following either exposure to 4-hydroxytamoxifen or deprivation of estrogen for periods of 2 days-4 weeks. METHODS: Endocrine-sensitive or -resistant breast cancer cell lines were used to examine the expression of the stem cell gene SOX2, and the Wnt effector genes AXIN2 and DKK1 using quantitative PCR analysis. Breast cancer cell lines were used to assess the anti-proliferative effects (as determined by IC50 values) of Wnt pathway inhibitors LGK974 and IWP-2. RESULTS: Hormone therapy led to time-dependent increases of up to 10-fold in SOX2 expression, up to threefold in expression of the Wnt target genes AXIN2 and DKK1, and variable changes in NANOG and OCT4 expression. The cells also showed increased mammosphere formation and increased CD24 surface protein expression. Some but not all hormone-resistant MCF-7 sub-lines, emerging after long-term hormonal stress, showed up to 50-fold increases in SOX2 expression and smaller increases in AXIN2 and DKK1 expression. However, the increase in Wnt target gene expression was not accompanied by an increase in sensitivity to Wnt pathway inhibitors LGK974 and IWP-2. A general trend of lower IC50 values was observed in 3-dimensional spheroid culture conditions (which allowed enrichment of cells with cancer stem cell phenotype) relative to monolayer cultures. The endocrine-resistant cell lines showed no significant increase in sensitivity to Wnt inhibitors. CONCLUSION: Hormone treatment of cultured MCF-7 cells leads within 2 days to increased expression of components of the SOX2 and Wnt pathways and to increased potential for mammosphere formation. We suggest that these responses are indicative of early adaptation to endocrine stress with features of stem cell character and that this facilitates the survival of emerging hormone-resistant cell populations.

mTOR inhibitors activate PERK signaling and favor viability of gastrointestinal neuroendocrine cell lines.

mTOR and Unfolded Protein Response (UPR) are two signaling pathways frequently activated in cancer cells. The mTOR pathway has been shown to be up-regulated in most gastroenteropancreatic neuroendocrine tumors. In contrast, little is known about the UPR status in neoplastic neuroendocrine cells. However, these hormone-producing cells are likely to present distinctive adaptations of this pathway, as other secretory cells. We therefore analyzed the status of the three axes of UPR and their relation to mTOR pathway in two gastrointestinal neuroendocrine tumors (GI-NET) cell lines STC-1 and GluTag. At baseline, pharmacological inducers activate the three arms of UPR: PERK, ATF6 and IRE1. Although hypoxia stimulates the PERK, ATF6 and IRE-1 pathways in both cell lines, glucose depletion activates UPR only in STC-1 cell line. Strikingly, P-p70S6K1 increases concomitantly to P-PERK and BiP in response to thapsigargin treatment, glucose depletion or hypoxia. We found that different mTOR inhibitors activate the PERK signaling pathway. To confirm that mTOR inhibition modulates PERK activation, we inhibited PERK and showed that it decreased cell viability when associated to mTOR inhibition, indicating that mTOR drives a PERK-dependent survival pathway. In conclusion, in GI-NET cell lines, UPR signaling is functional and PERK arm is induced by mTOR inhibition. These observations open up new perspectives for therapeutic strategies: the crosstalk between mTOR and UPR might contribute to the resistance to mTOR inhibitors and could be targeted by mTOR and PERK inhibitors in combination therapy.

Nutrient shortage triggers the hexosamine biosynthetic pathway via the GCN2-ATF4 signalling pathway.

The hexosamine biosynthetic pathway (HBP) is a nutrient-sensing metabolic pathway that produces the activated amino sugar UDP-N-acetylglucosamine, a critical substrate for protein glycosylation. Despite its biological significance, little is known about the regulation of HBP flux during nutrient limitation. Here, we report that amino acid or glucose shortage increase GFAT1 production, the first and rate-limiting enzyme of the HBP. GFAT1 is a transcriptional target of the activating transcription factor 4 (ATF4) induced by the GCN2-eIF2α signalling pathway. The increased production of GFAT1 stimulates HBP flux and results in an increase in O-linked ß-N-acetylglucosamine protein modifications. Taken together, these findings demonstrate that ATF4 provides a link between nutritional stress and the HBP for the regulation of the O-GlcNAcylation-dependent cellular signalling.

Antitumor effect of everolimus in preclinical models of high-grade gastroenteropancreatic neuroendocrine carcinomas.

BACKGROUND/AIMS: While the range of therapeutic options for well-differentiated gastroenteropancreatic neuroendocrine tumors has recently increased with the emergence of targeted therapies, such as mTOR inhibitors, there is no recent progress in the treatment of poorly differentiated neuroendocrine carcinomas (PDNECs). Since PDNECs have been shown to strongly express mTOR pathway components, the aim of the present study was to assess the antitumor effect of the mTOR inhibitor everolimus in preclinical models of PDNECs. METHODS: The expression of mTOR pathway components and their response to everolimus were assessed in two neuroendocrine cell lines: STC-1 and GluTag. A xenograft model of intrahepatic dissemination in the nude mouse, based on the intrasplenic injection of either STC-1 and GluTag tumor cells, was used. Animals were started on everolimus treatment 3 days after injection. The effects of treatment on tumor growth, proliferative capacities, apoptosis and in situ expression of mTOR pathway components were assessed. RESULTS: The expression of mTOR pathway components was comparable in STC-1 and GluTag cells and in human PDNECs and could be inhibited in vitro by everolimus. In vivo, the tumor volume of STC-1 and GluTag xenografts was significantly reduced in treated animals (6.05 ± 1.84% as compared to 21.76 ± 3.88% in controls). Everolimus treatment also induced a significant decrease in Ki67 index and in the phosphorylation levels of the two major effectors of mTOR, p70S6K and 4E-BP1. CONCLUSION: Our experimental data suggest that mTOR inhibition could be considered a therapeutic option for high-grade gastroenteropancreatic neuroendocrine tumors.

Molecular characteristics of papillary thyroid carcinomas without BRAF mutation or RET/PTC rearrangement: relationship with clinico-pathological features.

About 60-70% of papillary thyroid carcinomas (PTC) present a BRAF(T1799A) gene mutation or a rearrangement of RET gene (RET/PTC). In this study, we examined whether PTC without BRAF(T1799A) mutation and without RET/PTC rearrangement named PTC-ga(-) were distinguishable from PTC-ga(+) (with one or the other gene alteration) on the basis of gene expression characteristics. We analyzed the mutational state of 116 PTC and we compared gene expression profiles of PTC-ga(+) and PTC-ga(-) from data of a 200 gene macroarray and quantitative PCR. Seventy five PTC were PTC-ga(+) and 41 were PTC-ga(-). Unsupervised analyses of macroarray data by hierarchical clustering led to a complete segregation of PTC-ga(+) and PTC-ga(-). In a series of 42 genes previously recognized as PTC 'marker' genes, 22 were found to be expressed at a comparable level in PTC-ga(-) and normal tissue. Thyroid-specific genes, TPO, TG, DIO1, and DIO2 were under-expressed in PTC-ga(+) but expressed at a normal level in PTC-ga(-). A few genes including DUOX1 and DUOX2 were selectively dys-regulated in PTC-ga(-). Tumor grade of PTC-ga(-) was lower than that of PTC-ga(+). There was a strong association between the mutational state and histiotype of PTC; 81% of PTC follicular variants were corresponded to PTC-ga(-), whereas 84% of PTC of classical form were PTC-ga(+). In conclusion, we show that PTC without BRAF(T1799A) mutation or RET/PTC rearrangement, mainly corresponding to follicular variants, maintain a thyroid differentiation expression level close to that of normal tissue and should be of better prognosis than PTC with one or the other gene alteration.