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The mammalian target of rapamycin and the integrated stress response are central cellular hubs regulating translation upon stress. The precise proteins and pathway specificity of translation targets of these pathways remained largely unclear. We recently described a new method for quantitative translation proteomics and found that both pathways control translation of the same sets of proteins.
IL-1 family member IL-33 exerts a variety of immune activating and regulating properties and has recently been proposed as a prognostic biomarker for cancer diseases, although its precise role in tumor immunity is unclear. Here we analyzed in vitro conditions influencing the function of IL-33 as an alarmin and a co-factor for the activity of cytotoxic CD8+ T cells in order to explain the widely discussed promiscuous behavior of IL-33 in vivo. Circulating IL-33 detected in the serum of healthy human volunteers was biologically inactive. Additionally, bioactivity of exogenous recombinant IL-33 was significantly reduced in plasma, suggesting local effects of IL-33, and inactivation in blood. Limited availability of nutrients in tissue causes necrosis and thus favors release of IL-33, which—as described before—leads to a locally high expression of the cytokine. The harsh conditions however influence T cell fitness and their responsiveness to stimuli. Nutrient deprivation and pharmacological inhibition of mTOR mediated a distinctive phenotype characterized by expression of IL-33 receptor ST2L on isolated CD8+ T cells, downregulation of CD8, a transitional CD45RAlowROlow phenotype and high expression of secondary lymphoid organ chemokine receptor CCR7. Under nutrient deprivation, IL-33 inhibited an IL-12 induced increase in granzyme B protein expression and increased expression of GATA3 and FOXP3 mRNA. IL-33 enhanced the TCR-dependent activation of CD8+ T cells and co-stimulated the IL-12/TCR-dependent expression of IFNγ. Respectively, GATA3 and FOXP3 mRNA were not regulated during TCR-dependent activation. TCR-dependent stimulation of PBMC, but not LPS, initiated mRNA expression of soluble IL-33 decoy receptor sST2, a control mechanism limiting IL-33 bioactivity to avoid uncontrolled inflammation. Our findings contribute to the understanding of the compartment-specific activity of IL-33. Furthermore, we newly describe conditions, which promote an IL-33-dependent induction of pro- or anti-inflammatory activity in CD8+ T cells during nutrient deprivation.
With an increased understanding of the tumor biology of squamous cell carcinoma of the head and neck (SCCHN), targeted therapies have found their way into the clinical treatment routines against this entity. Nevertheless, to date platinum-based cytostatic agents remain the first line choice and targeting the epidermal growth factor-receptor (EGFR) with combined cetuximab and radiation therapy remains the only targeted therapy approved in the curative setting. Investigation of immune checkpoint inhibitors (ICI), such as antibodies targeting programmed cell death protein 1 (PD-1) and its ligand PD-L1, resulted in a change of paradigms in oncology and in the first approval of new drugs for treating SCCHN. Nivolumab and pembrolizumab, two anti-PD-1 antibodies, were the first agents shown to improve overall survival for patients with metastatic/recurrent tumors in recent years. Currently, several clinical trials investigate the role of ICI in different therapeutic settings. A robust set of biomarkers will be an inevitable tool for future individualized treatment approaches including radiation dose de-escalation and escalation strategies. This review aims to summarize achieved goals, the current status and future perspectives regarding targeted therapies and ICI in the management of SCCHN.
Glioblastomas (GBs) frequently display activation of the epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR). mTOR exists as part of two multiprotein complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2). In GBs, mTORC1 inhibitors such as rapamycin have performed poorly in clinical trials, and in vitro protect GB cells from nutrient and oxygen deprivation. Next generation ATP-competitive mTOR inhibitors with affinity for both mTOR complexes have been developed, but data exploring their effects on GB metabolism are scarce. In this study, we compared the ATP-competitive mTORC1/2 inhibitors torin2, INK-128 and NVP-Bez235 to the allosteric mTORC1 inhibitor rapamycin under conditions that mimic the glioma microenvironment. In addition to inhibiting mTORC2 signaling, INK-128 and NVP-Bez235 more effectively blocked mTORC1 signaling and prompted a stronger cell growth inhibition, partly by inducing cell cycle arrest. However, under hypoxic and nutrient-poor conditions mTORC1/2 inhibitors displayed even stronger cytoprotective effects than rapamycin by reducing oxygen and glucose consumption. Thus, therapies that arrest proliferation and inhibit anabolic metabolism must be expected to improve energy homeostasis of tumor cells. These results mandate caution when treating physiologically or therapeutically induced hypoxic GBs with mTOR inhibitors.
The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-xL, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS.
"Protein Associated with Myc" (PAM) hat aufgrund seiner enormen Größe von 510 kD und der Vielzahl an Proteinbindungsstellen das Potential viele regulatorische und physiologische Prozesse zu regulieren. Mit der Funktion als E3-Ubiquitinligase und davon unabhängigen Proteininteraktionen reguliert es beispielsweise Prozesse der Synaptogenese und der spinalen Schmerzverarbeitung, wie auch die Regulation des Pteridin Stoffwechsels und des cAMP-Signalweges. Im Gegensatz zur spinalen Schmerzverarbeitung war eine Beteiligung von PAM an der peripheren Nozizeption bisher unbekannt und sollte im Rahmen dieser Arbeit untersucht werden. Dazu wurden konditionale PAM-Knockout Mäuse generiert und charakterisiert. Zum einen wurde PAM in Vorläuferzellen von Neuronen und Gliazellen und zum anderen in allen nozizeptiven und thermorezeptiven Neuronen der Dorsalganglia und der Ganglia trigeminale deletiert. Der Knockout in Neuronen und Gliazellen führte zu einer pränatalen Letalität und unterstreicht so die Bedeutung von PAM während der Neuronenentwicklung. Mit Hilfe des spezifischen Knockouts in nozizeptiven Neuronen konnte eine Rolle von PAM bei der Regulation der thermischen Hyperalgesie gezeigt werden. Keinen Einfluss hatte die Deletion von PAM auf basale thermische und mechanische Schmerzschwellen, sowie auf Formalin-induzierte akute Schmerzen. Als potentieller Mechanismus wurde der mTOR- und der p38 MAPK-Signalweg untersucht. Dabei konnte eine Vermittlung der S1P-induzierten mTOR-Aktivierung durch PAM nachgewiesen werden und Rheb als eine Komponente dieser Aktivierung ermittelt werden. Der p38 MAPK Signalweg war in Abwesenheit von PAM konstitutiv aktiviert und einige Proteine des Rezeptortraffickings waren verstärkt exprimiert. Als ursächlich für die beobachtete verlängerte Hyperalgesie in PAM-defizienten Mäusen konnte die Unterbindung der Internalisierung des TRPV1-Rezeptors nachgewiesen werden. Dieser Effekt ist spezifisch für TRPV1, da der verwandte Ionenkanal TRPA1 durch die PAM-Deletion nicht beeinträchtigt wurde. In der vorliegenden Arbeit konnte so zum ersten Mal gezeigt werden, dass PAM in peripheren nozizeptiven Neuronen über die p38 MAPK-vermittelte Internalisierung von TRPV1 die Dauer der thermischen Hyperalgesie reguliert.
Human macrophages infiltrating hypoxic regions alter their metabolism, because oxygen becomes limited. Increased glycolysis is one of the most common cellular adaptations to hypoxia and mostly is regulated via hypoxia-inducible factor (HIF) and RAC-alpha serine/threonine–protein kinase (Akt) signaling, which gets activated under reduced oxygen content. We noticed that micro RNA (miR)-193a-3p enhances Akt phosphorylation at threonine 308 under hypoxia. In detail, miR-193a-3p suppresses the protein abundance of phosphatase PTC7 homolog (PPTC7), which in turn increases Akt phosphorylation. Lowering PPTC7 expression by siRNA or overexpressing miR-193a-3p increases Akt phosphorylation. Vice versa, inhibition of miR-193a-3p attenuates Akt activation and prevents a subsequent increase of glycolysis under hypoxia. Excluding effects of miR-193a-3p and Akt on HIF expression, stabilization, and function, we noticed phosphorylation of 6 phosphofructo-2-kinase/fructose 2,6-bisphosphatase PFKFB3 in response to the PI3K/Akt/mTOR signaling cascade. Inhibition of PFKFB3 blocked an increased glycolytic flux under hypoxia. Apparently, miR-193a-3p balances Akt phosphorylation and dephosphorylation by affecting PPTC7 protein amount. Suppression of PPTC7 increases Akt activation and phosphorylation of PFKFB3, which culminates in higher rates of glycolysis under hypoxia.
This study was designed to investigate whether epigenetic modulation by histone deacetylase (HDAC) inhibition might circumvent resistance towards the mechanistic target of rapamycin (mTOR) inhibitor temsirolimus in a prostate cancer cell model. Parental (par) and temsirolimus-resistant (res) PC3 prostate cancer cells were exposed to the HDAC inhibitor valproic acid (VPA), and tumor cell adhesion, chemotaxis, migration, and invasion were evaluated. Temsirolimus resistance was characterized by reduced binding of PC3res cells to endothelium, immobilized collagen, and fibronectin, but increased adhesion to laminin, as compared to the parental cells. Chemotaxis, migration, and invasion of PC3res cells were enhanced following temsirolimus re-treatment. Integrin α and β receptors were significantly altered in PC3res compared to PC3par cells. VPA significantly counteracted temsirolimus resistance by down-regulating tumor cell–matrix interaction, chemotaxis, and migration. Evaluation of integrin expression in the presence of VPA revealed a significant down-regulation of integrin α5 in PC3res cells. Blocking studies demonstrated a close association between α5 expression on PC3res and chemotaxis. In this in vitro model, temsirolimus resistance drove prostate cancer cells to become highly motile, while HDAC inhibition reversed the metastatic activity. The VPA-induced inhibition of metastatic activity was accompanied by a lowered integrin α5 surface level on the tumor cells.
Regulation of translation is essential during stress. However, the precise sets of proteins regulated by the key translational stress responses—the integrated stress response (ISR) and mTORC1—remain elusive. We developed multiplexed enhanced protein dynamics (mePROD) proteomics, adding signal amplification to dynamic-SILAC and multiplexing, to enable measuring acute changes in protein synthesis. Treating cells with ISR/mTORC1-modulating stressors, we showed extensive translatome modulation with ∼20% of proteins synthesized at highly reduced rates. Comparing translation-deficient sub-proteomes revealed an extensive overlap demonstrating that target specificity is achieved on protein level and not by pathway activation. Titrating cap-dependent translation inhibition confirmed that synthesis of individual proteins is controlled by intrinsic properties responding to global translation attenuation. This study reports a highly sensitive method to measure relative translation at the nascent chain level and provides insight into how the ISR and mTORC1, two key cellular pathways, regulate the translatome to guide cellular survival upon stress.
Patients harboring mutations in the gene DEPDC5 often display variations of neurological diseases including epilepsy, autism spectrum disorders (ASD) and other neuro-architectural alterations. DEPDC5 protein has been identified as an amino acid sensor responsible for negatively regulating the mechanistic target of rapamycin (mTOR), a central regulator in cell growth and cell homeostasis. Often, mutations of the DEPDC5 protein result in mTOR hyperactivity leading to abnormal neuronal phenotypes and the generation of excitatory/inhibitory imbalances in animal models. Complete knockout (KO) of DEPDC5 results in death shortly after birth, while inhibition of mTOR activity recovers postnatal death (Marsan et al. 2016). However, heterozygous DEPDC5-KOs in animals have been variable in their disease phenotypes during adulthood indicating developmental differences between subspecies and early development mechanisms which could be impactful on the outcome of the diseases.
To understand the mechanisms underlying DEPDC5 mutations during early development, a novel primary human neural progenitor cell line extracted from fetal tissue was characterized during proliferation and differentiation. CRISPR-Cas9 induced mutations of the DEPDC5 gene resulted in hyperphosphorylation of mTOR signaling processes and rapid expansion of the neuronal population during differentiation. Analysis of transcriptome data identified deregulation amongst p53 signaling, ribosome biogenesis, nucleotide and lipid synthesis as well as protein degradation pathways due to loss of DEPDC5. Disease gene datasets identified a correlation between Tuberous Sclerosis mutations as being more closely associated with DEPDC5 mutations while also finding overlap with some ASD and epilepsy genes. By using the mTOR inhibitor rapamycin, a substantial amount of the deregulated gene network was recovered while also reversing rapid neuronal differentiation caused by loss of DEPDC5. Though we saw increased dendritic arborization and subsequent decreases in dendrite lengths and soma sizes, rapamycin failed to recover these effects suggesting mTOR independent processes produced by DEPDC5-KO. This study provides new insights on the relationship between mutations in DEPDC5 and the functional, genomic and deregulatory networks it intertwines in humans and highlights that the DEPDC5 associated pathomechanisms are not fully related to mTOR hyperactivation, but include independent processes. This also sheds light on the question why rapamycin treatment only partially restores DEPDC5 related phenotypes and gives insight on treatments for DEPDC5 patients.