Characterizing the tumor suppressor activity of FLCN in Birt-Hogg-Dubé syndrome through transcriptiomic and proteomic analysis.

Birt-Hogg-Dubé (BHD) syndrome patients are uniquely susceptible to all renal tumour subtypes. The underlying mechanism of carcinogenesis is unclear. To study cancer development in BHD, we used human proximal kidney (HK2) cells and found that long-term folliculin (FLCN) knockdown was required to increase their tumorigenic potential, forming larger spheroids in non-adherent conditions. Transcriptomic and proteomic analysis uncovered links between FLCN, cell cycle control and the DNA damage response (DDR) machinery. HK2 cells lacking FLCN had an altered transcriptome profile with cell cycle control gene enrichment. G1/S cell cycle checkpoint signaling was compromised with heightened protein levels of cyclin D1 (CCND1) and hyperphosphorylation of retinoblastoma 1 (RB1). A FLCN interactome screen uncovered FLCN binding to DNA-dependent protein kinase (DNA-PK). This novel interaction was reversed in an irradiation-responsive manner. Knockdown of FLCN in HK2 cells caused a marked elevation of γH2AX and RB1 phosphorylation. Both CCND1 and RB1 phosphorylation remained raised during DNA damage, showing an association with defective cell cycle control with FLCN knockdown. Furthermore, Flcn-knockdown C. elegans were defective in cell cycle arrest by DNA damage. This work implicates that long-term FLCN loss and associated cell cycle defects in BHD patients could contribute to their increased risk of cancer.

Tuberous Sclerosis Complex cell-derived EVs have an altered protein cargo capable of regulating their microenvironment and have potential as disease biomarkers.

Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) is a feature of many solid tumours and is a key pathogenic driver in the inherited condition Tuberous Sclerosis Complex (TSC). Modulation of the tumour microenvironment by extracellular vesicles (EVs) is known to facilitate the development of various cancers. The role of EVs in modulating the tumour microenvironment and their impact on the development of TSC tumours, however, remains unclear. This study, therefore, focuses on the poorly defined contribution of EVs to tumour growth in TSC. We characterised EVs secreted from TSC2-deficient and TSC2-expressing cells and identified a distinct protein cargo in TSC2-deficient EVs, containing an enrichment of proteins thought to be involved in tumour-supporting signalling pathways. We show EVs from TSC2-deficient cells promote cell viability, proliferation and growth factor secretion from recipient fibroblasts within the tumour microenvironment. Rapalogs (mTORC1 inhibitors) are the current therapy for TSC tumours. Here, we demonstrate a previously unknown intercellular therapeutic effect of rapamycin in altering EV cargo and reducing capacity to promote cell proliferation in the tumour microenvironment. Furthermore, EV cargo proteins have the potential for clinical applications as TSC biomarkers, and we reveal three EV-associated proteins that are elevated in plasma from TSC patients compared to healthy donor plasma.

Phosphorylation of the novel mTOR substrate Unkempt regulates cellular morphogenesis.

Mechanistic target of rapamycin (mTOR) is a protein kinase that integrates multiple inputs to regulate anabolic cellular processes. For example, mTOR complex 1 (mTORC1) has key functions in growth control, autophagy, and metabolism. However, much less is known about the signaling components that act downstream of mTORC1 to regulate cellular morphogenesis. Here, we show that the RNA-binding protein Unkempt, a key regulator of cellular morphogenesis, is a novel substrate of mTORC1. We show that Unkempt phosphorylation is regulated by nutrient levels and growth factors via mTORC1. To analyze Unkempt phosphorylation, we immunoprecipitated Unkempt from cells in the presence or the absence of the mTORC1 inhibitor rapamycin and used mass spectrometry to identify mTORC1-dependent phosphorylated residues. This analysis showed that mTORC1-dependent phosphorylation is concentrated in a serine-rich intrinsically disordered region in the C-terminal half of Unkempt. We also found that Unkempt physically interacts with and is directly phosphorylated by mTORC1 through binding to the regulatory-associated protein of mTOR, Raptor. Furthermore, analysis in the developing brain of mice lacking TSC1 expression showed that phosphorylation of Unkempt is mTORC1 dependent in vivo. Finally, mutation analysis of key serine/threonine residues in the serine-rich region indicates that phosphorylation inhibits the ability of Unkempt to induce a bipolar morphology. Phosphorylation within this serine-rich region thus profoundly affects the ability of Unkempt to regulate cellular morphogenesis. Taken together, our findings reveal a novel molecular link between mTORC1 signaling and cellular morphogenesis.

Drug Inhibition of Redox Factor-1 Restores Hypoxia-Driven Changes in Tuberous Sclerosis Complex 2 Deficient Cells.

Therapies with the mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for tuberous sclerosis complex (TSC) patients. Here, we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses a redox signaling activity that stimulates the transcriptional activity of STAT3, NF-kB, and HIF-1α, which are involved in inflammation, proliferation, angiogenesis, and hypoxia, respectively. Here, we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor APX3330 was effective at blocking the hyperactivity of STAT3, NF-kB, and HIF-1α. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors such as STAT3, NF-kB, and HIF-1α as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits compared to using mTORC1 inhibitors alone.

The Role of Mitochondria-Linked Fatty-Acid Uptake-Driven Adipogenesis in Graves Orbitopathy.

CONTEXT: Depot-specific expansion of orbital adipose tissue (OAT) in Graves orbitopathy (GO; an autoimmune condition producing proptosis, visual impairment and reduced quality of life) is associated with fatty acid (FA)-uptake-driven adipogenesis in preadipocytes/fibroblasts (PFs). OBJECTIVE: This work sought a role for mitochondria in OAT adipogenesis in GO. METHODS: Confluent PFs from healthy OAT (OAT-H), OAT from GO (OAT-GO) and white adipose tissue in culture medium compared with culture medium containing a mixed hormonal cocktail as adipogenic medium (ADM), or culture-medium containing FA-supplementation, oleate:palmitate:linoleate (45:30:25%) with/without different concentration of mitochondrial biosubstrate adenosine 5'-diphosphate/guanosine 5'-diphosphate (ADP/GDP), AICAR (adenosine analogue), or inhibitor oligomycin-A for 17 days. Main outcome measures included oil-red-O staining and foci count of differentiated adipocytes for in vitro adipogenesis, flow cytometry, relative quantitative polymerase chain reaction, MTS-assay/106 cells, total cellular-ATP detection kit, and Seahorse-XFe96-Analyzer for mitochondria and oxidative-phosphorylation (OXPHOS)/glycolysis-ATP production analysis. RESULTS: During early adipogenesis before adipocyte formation (days 0, 4, and7), we observed OAT-specific cellular ATP production via mitochondrial OXPHOS in PFs both from OAT-H and OAT-GO, and substantially disrupted OXPHOS-ATP/glycolysis-ATP production in PFs from OAT-GO, for example, a 40% reduction in OXPHOS-ATP and trend-increased glycolysis-ATP production on days 4 and 7 compared with day 0, which contrasted with the stable levels in OAT-H. FA supplementation in culture-medium triggered adipogenesis in PFs both from OAT-H and OAT-GO, which was substantially enhanced by 1-mM GDP reaching 7% to 18% of ADM adipogenesis. The FA-uptake-driven adipogenesis was diminished by oligomycin-A but unaffected by treatment with ADP or AICAR. Furthermore, we observed a significant positive correlation between FA-uptake-driven adipogenesis by GDP and the ratios of OXPHOS-ATP/glycolysis-ATP through adipogenesis of PFs from OAT-GO. CONCLUSION: Our study confirmed that FA uptake can drive OAT adipogenesis and revealed a fundamental role for mitochondria-OXPHOS in GO development, which provides potential for therapeutic interventions.

The zinc finger/RING domain protein Unkempt regulates cognitive flexibility.

Correct orchestration of nervous system development is a profound challenge that involves coordination of complex molecular and cellular processes. Mechanistic target of rapamycin (mTOR) signaling is a key regulator of nervous system development and synaptic function. The mTOR kinase is a hub for sensing inputs including growth factor signaling, nutrients and energy levels. Activation of mTOR signaling causes diseases with severe neurological manifestations, such as tuberous sclerosis complex and focal cortical dysplasia. However, the molecular mechanisms by which mTOR signaling regulates nervous system development and function are poorly understood. Unkempt is a conserved zinc finger/RING domain protein that regulates neurogenesis downstream of mTOR signaling in Drosophila. Unkempt also directly interacts with the mTOR complex I component Raptor. Here we describe the generation and characterisation of mice with a conditional knockout of Unkempt (UnkcKO) in the nervous system. Loss of Unkempt reduces Raptor protein levels in the embryonic nervous system but does not affect downstream mTORC1 targets. We also show that nervous system development occurs normally in UnkcKO mice. However, we find that Unkempt is expressed in the adult cerebellum and hippocampus and behavioural analyses show that UnkcKO mice have improved memory formation and cognitive flexibility to re-learn. Further understanding of the role of Unkempt in the nervous system will provide novel mechanistic insight into the role of mTOR signaling in learning and memory.

Exploring transcriptional regulators Ref-1 and STAT3 as therapeutic targets in malignant peripheral nerve sheath tumours.

BACKGROUND: MPNST is a rare soft-tissue sarcoma that can arise from patients with NF1. Existing chemotherapeutic and targeted agents have been unsuccessful in MPNST treatment, and recent findings implicate STAT3 and HIF1-α in driving MPNST. The DNA-binding and transcriptional activity of both STAT3 and HIF1-α is regulated by Redox factor-1 (Ref-1) redox function. A first-generation Ref-1 inhibitor, APX3330, is being tested in cancer clinical trials and could be applied to MPNST. METHODS: We characterised Ref-1 and p-STAT3 expression in various MPNST models. Tumour growth, as well as biomarkers of apoptosis and signalling pathways, were measured by qPCR and western blot following treatment with inhibitors of Ref-1 or STAT3. RESULTS: MPNSTs from Nf1-Arfflox/floxPostnCre mice exhibit significantly increased positivity of p-STAT3 and Ref-1 expression when malignant transformation occurs. Inhibition of Ref-1 or STAT3 impairs MPNST growth in vitro and in vivo and induces apoptosis. Genes highly expressed in MPNST patients are downregulated following inhibition of Ref-1 or STAT3. Several biomarkers downstream of Ref-1 or STAT3 were also downregulated following Ref-1 or STAT3 inhibition. CONCLUSIONS: Our findings implicate a unique therapeutic approach to target important MPNST signalling nodes in sarcomas using new first-in-class small molecules for potential translation to the clinic.

The RNA-helicase DDX21 upregulates CEP55 expression and promotes neuroblastoma.

Approximately 25% of human neuroblastoma is caused by amplification of the MYCN oncogene, which leads to overexpression of N-Myc oncoprotein. The survival rate for this patient subtype is <50%. Here, we show that N-Myc protein bound to the DEAD-box RNA helicase DDX21 gene promoter and upregulated DDX21 mRNA and protein expression. Genome-wide differential gene expression studies identified centrosomal protein CEP55 as one of the genes most dramatically downregulated after DDX21 knockdown in MYCN-amplified neuroblastoma cells. Knocking down DDX21 or CEP55 reduced neuroblastoma cell cytoskeleton stability and cell proliferation and all but abolished clonogenic capacity. Importantly, DDX21 knockdown initially induced tumor regression in neuroblastoma-bearing mice and suppressed tumor progression. In human neuroblastoma tissues, a high level of DDX21 expression correlated with a high level of N-Myc expression and with CEP55 expression, and independently predicted poor patient prognosis. Taken together, our data show that DDX21 induces CEP55 expression, MYCN-amplified neuroblastoma cell proliferation, and tumorigenesis, and that DDX21 and CEP55 are valid therapeutic targets for the treatment of MYCN-amplified neuroblastoma.

Targeted Therapy of TERT-Rearranged Neuroblastoma with BET Bromodomain Inhibitor and Proteasome Inhibitor Combination Therapy.

PURPOSE: TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. EXPERIMENTAL DESIGN: Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. RESULTS: The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. CONCLUSIONS: OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.

Reciprocal signaling between mTORC1 and MNK2 controls cell growth and oncogenesis.

eIF4E plays key roles in protein synthesis and tumorigenesis. It is phosphorylated by the kinases MNK1 and MNK2. Binding of MNKs to eIF4G enhances their ability to phosphorylate eIF4E. Here, we show that mTORC1, a key regulator of mRNA translation and oncogenesis, directly phosphorylates MNK2 on Ser74. This suppresses MNK2 activity and impairs binding of MNK2 to eIF4G. These effects provide a novel mechanism by which mTORC1 signaling impairs the function of MNK2 and thereby decreases eIF4E phosphorylation. MNK2[S74A] knock-in cells show enhanced phosphorylation of eIF4E and S6K1 (i.e., increased mTORC1 signaling), enlarged cell size, and increased invasive and transformative capacities. MNK2[Ser74] phosphorylation was inversely correlated with disease progression in human prostate tumors. MNK inhibition exerted anti-proliferative effects in prostate cancer cells in vitro. These findings define a novel feedback loop whereby mTORC1 represses MNK2 activity and oncogenic signaling through eIF4E phosphorylation, allowing reciprocal regulation of these two oncogenic pathways.

Distinctive Features of Orbital Adipose Tissue (OAT) in Graves' Orbitopathy.

Depot specific expansion of orbital-adipose-tissue (OAT) in Graves' Orbitopathy (GO) is associated with lipid metabolism signaling defects. We hypothesize that the unique adipocyte biology of OAT facilitates its expansion in GO. A comprehensive comparison of OAT and white-adipose-tissue (WAT) was performed by light/electron-microscopy, lipidomic and transcriptional analysis using ex vivo WAT, healthy OAT (OAT-H) and OAT from GO (OAT-GO). OAT-H/OAT-GO have a single lipid-vacuole and low mitochondrial number. Lower lipolytic activity and smaller adipocytes of OAT-H/OAT-GO, accompanied by similar essential linoleic fatty acid (FA) and (low) FA synthesis to WAT, revealed a hyperplastic OAT expansion through external FA-uptake via abundant SLC27A6 (FA-transporter) expression. Mitochondrial dysfunction of OAT in GO was apparent, as evidenced by the increased mRNA expression of uncoupling protein 1 (UCP1) and mitofusin-2 (MFN2) in OAT-GO compared to OAT-H. Transcriptional profiles of OAT-H revealed high expression of Iroquois homeobox-family (IRX-3&5), and low expression in HOX-family/TBX5 (essential for WAT/BAT (brown-adipose-tissue)/BRITE (BRown-in-whITE) development). We demonstrated unique features of OAT not presented in either WAT or BAT/BRITE. This study reveals that the pathologically enhanced FA-uptake driven hyperplastic expansion of OAT in GO is associated with a depot specific mechanism (the SLC27A6 FA-transporter) and mitochondrial dysfunction. We uncovered that OAT functions as a distinctive fat depot, providing novel insights into adipocyte biology and the pathological development of OAT expansion in GO.

Combination therapy with the CDK7 inhibitor and the tyrosine kinase inhibitor exerts synergistic anticancer effects against MYCN-amplified neuroblastoma.

Patients with neuroblastoma due to MYCN oncogene amplification and consequent N-Myc oncoprotein overexpression have very poor prognosis. The cyclin-dependent kinase 7 (CDK7)/super-enhancer inhibitor THZ1 suppresses MYCN gene transcription, reduces neuroblastoma cell proliferation, but does not cause significant cell death. The protein kinase phosphatase 1 nuclear targeting subunit (PNUTS) has recently been shown to interact with c-Myc protein and suppresses c-Myc protein degradation. Here we screened the U.S. Food and Drug Administration-Approved Oncology Drugs Set V from the National Cancer Institute, and identified tyrosine kinase inhibitors (TKIs), including ponatinib and lapatinib, as the Approved Oncology Drugs exerting the best synergistic anticancer effects with THZ1 in MYCN-amplified neuroblastoma cells. Combination therapy with THZ1 and ponatinib or lapatinib synergistically induced neuroblastoma cell apoptosis, while having little effects in normal nonmalignant cells. Differential gene expression analysis identified PNUTS as one of the genes most synergistically reduced by the combination therapy. Reverse transcription polymerase chain reaction and immunoblot analyses confirmed that THZ1 and the TKIs synergistically downregulated PNUTS mRNA and protein expression and reduced N-Myc protein but not N-Myc mRNA expression. In addition, PNUTS knockdown resulted in decreased N-Myc protein but not mRNA expression and decreased MYCN-amplified neuroblastoma cell proliferation and survival. As CDK7 inhibitors are currently under clinical evaluation in patients, our data suggest the addition of the TKI ponatinib or lapatinib in CDK7 inhibitor clinical trials in patients.

The long noncoding RNA lncNB1 promotes tumorigenesis by interacting with ribosomal protein RPL35.

The majority of patients with neuroblastoma due to MYCN oncogene amplification and consequent N-Myc oncoprotein over-expression die of the disease. Here our analyses of RNA sequencing data identify the long noncoding RNA lncNB1 as one of the transcripts most over-expressed in MYCN-amplified, compared with MYCN-non-amplified, human neuroblastoma cells and also the most over-expressed in neuroblastoma compared with all other cancers. lncNB1 binds to the ribosomal protein RPL35 to enhance E2F1 protein synthesis, leading to DEPDC1B gene transcription. The GTPase-activating protein DEPDC1B induces ERK protein phosphorylation and N-Myc protein stabilization. Importantly, lncNB1 knockdown abolishes neuroblastoma cell clonogenic capacity in vitro and leads to neuroblastoma tumor regression in mice, while high levels of lncNB1 and RPL35 in human neuroblastoma tissues predict poor patient prognosis. This study therefore identifies lncNB1 and its binding protein RPL35 as key factors for promoting E2F1 protein synthesis, N-Myc protein stability and N-Myc-driven oncogenesis, and as therapeutic targets.

JMJD6 is a tumorigenic factor and therapeutic target in neuroblastoma.

Chromosome 17q21-ter is commonly gained in neuroblastoma, but it is unclear which gene in the region is important for tumorigenesis. The JMJD6 gene at 17q21-ter activates gene transcription. Here we show that JMJD6 forms protein complexes with N-Myc and BRD4, and is important for E2F2, N-Myc and c-Myc transcription. Knocking down JMJD6 reduces neuroblastoma cell proliferation and survival in vitro and tumor progression in mice, and high levels of JMJD6 expression in human neuroblastoma tissues independently predict poor patient prognosis. In addition, JMJD6 gene is associated with transcriptional super-enhancers. Combination therapy with the CDK7/super-enhancer inhibitor THZ1 and the histone deacetylase inhibitor panobinostat synergistically reduces JMJD6, E2F2, N-Myc, c-Myc expression, induces apoptosis in vitro and leads to neuroblastoma tumor regression in mice, which are significantly reversed by forced JMJD6 over-expression. Our findings therefore identify JMJD6 as a neuroblastoma tumorigenesis factor, and the combination therapy as a treatment strategy.

Finding a cure for tuberous sclerosis complex: From genetics through to targeted drug therapies.

Tuberous sclerosis complex (TSC) is a rare, autosomal dominant genetic condition caused by a mutation in either the TSC1 or TSC2 gene. Phenotypically, this leads to aberrant cell growth and the formation of benign tumors called hamartomas in multiple organs. Understanding the mechanisms of pathology that are caused through the presence of disease causing mutations is a real hurdle for many rare genetic disorders; a limiting factor that restricts knowledge of the disease and any hope of a future cure. Through the discovery of the TSC1 and TSC2 genes and the signaling pathways responsible for the pathology of TSC, a new drug target called mechanistic target of rapamycin complex 1 (mTORC1) was discovered. Rapamycin, an mTORC1 inhibitor, is now the only pharmacological therapy approved for the treatment of TSC. This chapter summarizes the success story of TSC and explores the future possibilities of finding a cure.

Correction: Loss of tuberous sclerosis complex 2 sensitizes tumors to nelfinavir-bortezomib therapy to intensify endoplasmic reticulum stress-induced cell death.

This article was originally published under standard licence, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly.

The histone chaperone complex FACT promotes proliferative switch of G0 cancer cells.

Cancer cell repopulation through cell cycle re-entry by quiescent (G0 ) cell is thought to be an important mechanism behind treatment failure and cancer recurrence. Facilitates Chromatin Transcription (FACT) is involved in DNA repair, replication and transcription by eviction of histones or loosening their contact with DNA. While FACT expression is known to be high in a range of cancers, the biological significance of the aberrant increase is not clear. We found that in prostate and lung cancer cells FACT mRNA and protein levels were low at G0 compared to the proliferating state but replenished upon cell cycle re-entry. Silencing of FACT with Dox-inducible shRNA hindered cell cycle re-entry by G0 cancer cells, which could be rescued by ectopic expression of FACT. An increase in SKP2, c-MYC and PIRH2 and a decrease in p27 protein levels seen upon cell cycle re-entry were prevented or diminished when FACT was silenced. Further, using mVenus-p27K- infected cancer cells to measure p27 degradation capacity, we confirm that inhibition of FACT at release from quiescence suppressed the p27 degradation capacity resulting in an increased mVenus-p27K- signal. In conclusion, FACT plays an important role in promoting the transition from G0 to the proliferative state and can be a potential therapeutic target to prevent prostate and lung cancer from progression and recurrence.