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Autophagy in PanNET cell lines and human PanNET tissue

Increasing the efficacy of approved systemic treatments in metastasized pancreatic neuroendocrine tumors (PanNET) is an unmet medical need. The antiangiogenic tyrosine kinase inhibitor sunitinib is approved for PanNET treatment. In addition, sunitinib is a lysosomotropic drug and such drugs can induce lysosomal membrane permeabilization as well as autophagy. We investigated sunitinib-induced autophagy as a possible mechanism of PanNET therapy resistance. Sunitinib accumulated in lysosomes and induced autophagy in PanNET cell lines. Adding the autophagy inhibitor chloroquine reduced cell viability in cell lines and in primary cells isolated from PanNET patients. The same treatment combination reduced tumor burden in the Rip1Tag2 transgenic PanNET mouse model. The combination of sunitinib and chloroquine reduced recovery and induced apoptosis in vitro, whereas single treatments did not. Knockdown of key autophagy proteins in combination with sunitinib showed similar effect as chloroquine. Sunitinib also induced lysosomal membrane permeabilization, which further increased in the presence of chloroquine or knockdown of lysosome-associated membrane protein (LAMP2). Both combinations led to cell death. Our data indicate that chloroquine increases sunitinib efficacy in PanNET treatment via autophagy inhibition and lysosomal membrane permeabilization. We suggest that adding chloroquine to sunitinib treatment will increase efficacy of PanNET treatment and that such patients should be included in respective ongoing clinical trials. Mol Cancer Ther; 16(11); 2502–15. ©2017 AACR.

Protocol for intracellular metabolite extraction from different cell lines for targeted metabolomics measurement using a 6-well or 24-well plate format.

Updated version of DMP at midterm

Initial version of DMP

Solute carrier (SLC) transporters control fluxes of nutrients and metabolites across membranes and thereby represent a critical interface between the microenvironment and cellular and subcellular metabolism. Because of substantial functional overlap, the interplay and relative contributions of members of this family in response to environmental stresses remain poorly elucidated. In order to infer functional relationships between SLCs and metabolites, we developed a strategy to identify human SLCs able to sustain cell viability and proliferation under growth-limiting concentrations of essential nutrients. One-by-one depletion of 13 amino acids required for cell proliferation enabled gain-of-function genetic screens using a SLC-focused CRISPR/Cas9-based transcriptional activation approach to uncover transporters relieving cells from the growth-limiting metabolic bottleneck. We identified the cationic amino acid transporter SLC7A3 as a gene that, when upregulated, overcame low availability of arginine and lysine by increasing their uptake. SLC7A5 (LAT1), on the other hand, was able to sustain cellular fitness upon deprivation of several neutral amino acids. A genome-wide screen identified SLC7A3 as the single main gene product able to rescue cell survival in the limiting arginine conditions tested, demonstrating the potentially decisive role of transporters in overcoming nutrient limitations. Moreover, we identified metabolic compensation mediated by the glutamate/aspartate transporters SLC1A2 and SLC1A3 under glutamine-limiting conditions. Overall, this gain-of-function approach using human cells led to the definition of functional transporter-nutrient relationships and revealed that upregulation of transport activity may be sufficient to overcome environmental metabolic restrictions.

The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.

This article belongs to the Special Issue Autophagy in Cancer., Autophagy is a highly conserved degradation mechanism that is essential for maintaining cellular homeostasis. In human disease, autophagy pathways are frequently deregulated and there is immense interest in targeting autophagy for therapeutic approaches. Accordingly, there is a need to determine autophagic activity in human tissues, an endeavor that is hampered by the fact that autophagy is characterized by the flux of substrates whereas histology informs only about amounts and localization of substrates and regulators at a single timepoint. Despite this challenging task, considerable progress in establishing markers of autophagy has been made in recent years. The importance of establishing clear-cut autophagy markers that can be used for tissue analysis cannot be underestimated. In this review, we attempt to summarize known techniques to quantify autophagy in human tissue and their drawbacks. Furthermore, we provide some recommendations that should be taken into consideration to improve the reliability and the interpretation of autophagy biomarkers in human tissue samples., This work was supported by grants from the Bernese Cancer League, “Stiftung für klinisch-experimentelle Tumorforschung”, and the Werner and Hedy Berger-Janser Foundation for Cancer Research (to M.H.); by Institute of Health Carlos III (ISCIII) and FEDER funds from the EU (PI14/01085 and PI17/00093) and supported by Miguel Servet contract by ISCIII and FSE funds (CPII16/00023) (to M.M.); from the Spanish Ministry of Science, Innovation and Universities (RTI2018-096748-B-100 to N.A.); from the University Professor Training Fellowship, Ministry of Science, Innovation and University, Government of Spain (FPU17/00026) (to P.C.O); from the ISCIII (PI16/00090 and PI19/01266) and the Andalusian Government (Consejería de Igualdad, Salud y Políticas Sociales, PI-0198-2016) for their financial support, and from the Biomedical Research Network Center for Liver and Digestive Diseases (CIBERehd) founded by the ISCIII and co-financed by European Development Regional Fund (EDRF) “A way to achieve Europe” for their financial support (to J.M.), from Breakthrough Cancer Research, Ireland funding (to S.L.M); from the PI18/00442 grant integrated into the State Plan for R & D + I2013-2016 and funded by the ISCIII and the ERDF, a way to make Europe (to G.V.); from the Luxembourg National Research Fund (C18/BM/12670304/COMBATIC to B.J.); from the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, by the European Regional Development Fund (FEDER), through the Competitiveness Factors Operational Programme (COMPETE) (NORTE-01-0145-FEDER-000013) and from the projects POCI-01-0145-FEDER-028159 and POCI-01-0145-FEDER-030782 by FEDER, through the COMPETE (to P.L.); from National funds, through the Foundation for Science and Technology (FCT) (to P.L.); from ARRS—the Slovenian research agency, programme P1-0140: Proteolysis and its regulation (led by B. Turk) (to E.Ž.); from the Swiss Cancer Research (KFS-3360-02-2014) (to A.P, and M.P.T.) (KFS-3409-02-2014), and the Swiss National Science Foundation (31003A_173219) (to M.P.T.).

Neuroendocrine tumours (NET) of the gastrointestinal tract and the lung are a rare and heterogeneous group of tumours. The molecular characterization and the clinical classification of these tumours have been evolving slowly and show differences according to organs of origin. Novel technologies such as next-generation sequencing revealed new molecular aspects of NET over the last years. Notably, whole-exome/genome sequencing (WES/WGS) approaches underlined the very low mutation rate of well-differentiated NET of all organs compared to other malignancies, while the engagement of epigenetic changes in driving NET evolution is emerging. Indeed, mutations in genes encoding for proteins directly involved in chromatin remodelling, such asDAXXandATRXare a frequent event in NET. Epigenetic changes are reversible and targetable; therefore, an attractive target for treatment. The discovery of the mechanisms underlying the epigenetic changes and the implication on gene and miRNA expression in the different subgroups of NET may represent a crucial change in the diagnosis of this disease, reveal new therapy targets and identify predictive markers. Molecular profiles derived from omics data including DNA mutation, methylation, gene and miRNA expression have already shown promising results in distinguishing clinically and molecularly different subtypes of NET. In this review, we recapitulate the major genetic and epigenetic characteristics of pancreatic, lung and small intestinal NET and the affected pathways. We also discuss potential epigenetic mechanisms leading to NET development.

DAXX and or ATRX loss occur in 40% of pancreatic neuroendocrine tumors (PanNETs). PanNETs negative for DAXX or ATRX show an increased risk of relapse. The tumor-associated pathways activated upon DAXX or ATRX loss and how this event may induce chromosomal instability (CIN) and alternative lengthening telomeres (ALT) are still unknown. Both DAXX and ATRX are involved in DNA methylation regulation. DNA methylation of heterochromatin and of non-coding sequences is extremely important for the maintenance of genomic stability. We analyzed the association of DAXX and/or ATRX loss and CIN with global DNA methylation in human PanNET samples and the effect of DAXX knock-down on methylation and cell proliferation. We assessedLINE1as well as global DNA methylation in 167 PanNETs, and we found that DAXX and or ATRX-negative tumors and tumors with CIN were hypomethylated. DAXX knock-down in PanNET cell lines blocked cells in G1/G0 phase and seemed to increase CIN in QGP-1 cells. However, no direct changes in DNA methylation were observed after DAXX knock-downin vitro. In conclusion, our data indicate that epigenetic changes are crucial steps in the progression of PanNETs loss and suggest that DNA methylation is the mechanism via which CIN is induced, allowing clonal expansion and selection.

Sunitinib, an anti-angiogenic tyrosine kinase inhibitor, is approved for treatment of pancreatic neuroendocrine tumors (pNETs). However, its efficacy is greatly limited due to resistance. Sunitinib is a lysosomotropic drug, thus accumulates in lysosomes, leading to their destabilization and to lysosomal membrane permeabilization (LMP), which in turn can lead to cell death. Autophagy might be activated for clearance of damaged lysosomes, thus promote survival and act as a mechanism of resistance. We found that the autophagy inhibitor chloroquine increases sunitinib efficacy in pNET cell lines and in a transgenic mouse model of pNETs. Interestingly, chloroquine is a lysosomotropic drug as well and the response towards sunitinib and chloroquine in pNET cell lines correlated with lysosome-associated membrane protein 2 (LAMP2) levels, which influence lysosome stability. We hypothesized that sunitinib and the combination with chloroquine induce LMP in pNETs and that LMP leads to activation of the transcription factor EB (TFEB), master regulator of genes involved in lysosomal biogenesis and autophagy, leading to therapy resistance. We found that LMP increased upon combined treatment of sunitinib and chloroquine compared to single treatment in pNET cell lines. Treatment of pNET cell lines with sunitinib or chloroquine led to activation of TFEB as assessed by nuclear/cytoplasmic fractionation and western blotting. Additionally, sunitinib significantly increased the expression of TFEB target genes, which were further upregulated upon combination with chloroquine. Interestingly, activation of TFEB and upregulation of TFEB target genes was more pronounced in the more resistant pNET cell line with respect to reduced viability and increased apoptosis. Our data indicate that sunitinib and chloroquine activate TFEB in pNET cell lines, leading to autophagy as a survival mechanism. Upon massive LMP or if autophagy is dysfunctional, cell death is induced. Based on our data, we suggest the combination of sunitinib and chloroquine as a treatment option for pNET patients and that TFEB could be an interesting therapeutic target in combination with lysosomotropic drugs. Citation Format: Tabea Wiedmer, Rasmus M. Frank, Mario P. Tschan, Aurel Perren, Ilaria Marinoni. Lysosomal membrane permeabilization as potential mediator of resistance in pancreatic neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3159. doi:10.1158/1538-7445.AM2017-3159

LC/GC-MS coupled to novel deconvolution routines for determining the approximately 3,000 molecules typically observable in serum