DEP Domain-containing mTOR-interacting Protein Research Articles

Metformin inhibits EV71‑induced pyroptosis by upregulating DEP domain‑containing mTOR‑interacting protein.

Enterovirus 71 (EV71) infection is one of the main causes of severe hand, foot and mouth disease (HFMD), which is usually accompanied by a marked inflammatory response. The excessive inflammatory response has been implicated to serve an important role in EV71-caused HFMD. Pyroptosis is a type of inflammatory programmed cell death. Therefore, a novel treatment strategy against EV71 infection could aim to alleviate the inflammatory response through inhibition of EV71-induced pyroptosis. The present study revealed that metformin had this therapeutic potential. A cell model of EV71 infection was established, cell viability was measured by CCK8 assay, cell damage was measured by LDH release kit, and the dead and dying cells were excluded by propidium iodide staining. The intracellular levels of DEP domain-containing mTOR interacting protein (DEPTOR) and pyroptosis-associated molecules were measured by western blot analysis, the NLRP3 expression was assessed by immunofluorescence labeling, and virus titers in cell culture supernatants were determined by a cell culture infectious dose 50 assay. The results demonstrated that EV71 infection could induce pyroptosis in a time- and dose-dependent manner, and metformin could inhibit EV71-induced pyroptosis. The mechanism of metformin inhibiting EV71-induced pyroptosis was explored next. Subsequent experiments indicated that metformin could increase the levels of DEPTOR, which were decreased by EV71. Finally, overexpression of DEPTOR in cells could reduce EV71-induced pyroptosis. Overall, the present study demonstrated that metformin could exert a novel pharmacodynamic anti-pyroptosis effect in the treatment of EV71 infection by upregulating DEPTOR expression.

DEPTOR loss impairs brown adipocyte development in vitro but has limited impacts in mice

ObjectivesThe mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates growth and metabolism. In mice, activation of mTOR controls cold adaptation by promoting the recruitment and the activation of brown adipose tissue (BAT). DEP-domain containing mTOR-interacting protein (DEPTOR) interacts with mTOR to modulate its activity. Whether DEPTOR levels are modulated by cold in BAT and whether this protein regulates brown adipocyte development and thermogenic activation has never been tested. MethodsDEPTOR levels were measured in mouse tissues upon cold exposure and in brown preadipocytes following the induction of adipogenesis. Lentiviruses expressing short-hairpin RNA were used to repress DEPTOR expression in brown preadipocytes in vitro. Conditional deletion of DEPTOR in brown preadipocytes and in mature brown fat cells was achieved by crossing DEPTOR floxed mice with either Myf5-Cre or Ucp1-CreERT2 mice. These animals were exposed to cold and extensively phenotyped. ResultsDEPTOR is highly expressed in BAT and its levels are induced by chronic cold exposure, a condition that triggers BAT expansion and activation. Supporting a role for DEPTOR in brown fat cell recruitment, we found that DEPTOR is induced during brown adipocyte development and that its depletion impairs adipogenesis in vitro. This adipogenic lesion was associated with defects in both Akt activation and the expression of key adipogenic regulators. Conditional deletion of DEPTOR in brown preadipocytes or mature brown fat cells did not impact BAT recruitment and thermogenesis in mice but slightly reduced the expression of adipogenic and lipogenic genes. ConclusionsDEPTOR is highly expressed in BAT and its levels are dynamically regulated during brown fat cell development and upon cold exposure. Although DEPTOR depletion severely represses brown fat adipogenesis in vitro, its deletion is dispensable for BAT development, recruitment, and thermogenic activation in mice.

Stabilization of DEPTOR sensitizes hypopharyngeal cancer to radiotherapy via targeting degradation

The use of radiotherapy for hypopharyngeal cancer (HC) treatment is increasing, and it is currently the primary treatment option for this cancer. However, radioresistance occurs in a proportion of patients. Here, we found that radiation increased proteasomal gene expression and that proteasome assembly was dependent on the induction of transcription factor NRF1 in HC. Through screening assays, we identified a mechanism by which proteasome-mediated degradation of DEP domain-containing mTOR-interacting protein (DEPTOR) contributes to the elevation of mTORC1 signaling after radiation. Therefore, after treatment with proteasome inhibitors (PIs), stabilization of DEPTOR inhibited mTORC1 signaling elevated by radiation and ultimately sensitized HC to radiotherapy. Mechanically, PIs not only interrupted the deubiquitination and degradation of DEPTOR but also suppressed the ubiquitination of DEPTOR mediated by β-TrCP. Clinically, the high levels of DEPTOR in HC cells were associated with sensitivity to radiotherapy and favorable prognosis. Stabilizing DEPTOR through targeting proteasome-mediated degradation is a potential strategy for sensitizing HC to radiotherapy.

DEPTOR exacerbates bone–fat imbalance in osteoporosis by transcriptionally modulating BMSC differentiation

Bone marrow–derived mesenchymal stem cells (BMSCs) tend to differentiate into adipocytes rather than osteoblasts in osteoporosis and other pathological conditions. Understanding the mechanisms underlying the adipo-osteogenic imbalance greatly contributes to the ability to induce specific MSC differentiation for clinical applications. This study aimed to explore whether DEP-domain containing mTOR-interacting protein (DEPTOR) regulated MSC fate and bone–fat switch, which was indicated to be a key player in bone homeostasis. We found that DEPTOR expression decreased during the osteogenesis of BMSCs but increased during adipogenesis and the shift of cell lineage commitment of BMSCs to adipocytes in mice with osteoporosis. DEPTOR facilitated adipogenic differentiation while preventing the osteogenic differentiation of BMSCs. Deptor ablation in BMSCs alleviated bone loss and reduced marrow fat accumulation in mice with osteoporosis. Mechanistically, DEPTOR binds transcriptional coactivator with a PDZ-binding motif (TAZ) and inhibits its transactivation properties, thereby repressing the transcriptional activity of RUNX2 and elevating gene transcription by peroxisome-proliferator-activated receptor-gamma. TAZ knockdown in BMSCs abolished the beneficial role of Deptor ablation in bone–fat balance in mice. Together, our data indicate that DEPTOR is a molecular rheostat that modulates BMSC differentiation and bone–fat balance, and may represent a potential therapeutic target for age-related bone loss.

The ubiquitin ligase Nedd4-2 mediates the regulation of PepT2 by mTORC1 in bovine mammary epithelial cells.

Peptide transporter 2 (PepT2) transports short peptides from the blood into bovine mammary epithelial cells (BMEC) to stimulate milk protein synthesis. Despite the fact that the effect of PepT2 is acknowledged in BMEC, little is known about its regulation. This study was completed to investigate the role of mammalian target of the rapamycin (mTOR) signaling in regulating the expression and function of PepT2 in BMEC. The regulation of PepT2 by mTOR in BMEC was studied in vitro using peptide transport assay, gene silencing, Western blot. The membrane expression of PepT2 and the uptake of β-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (β-Ala-Lys-AMCA), a model dipeptide, in BMEC were reduced by rapamycin (a mTOR inhibitor) and silencing of either mTOR complex 1 (mTORC1) or mTOR complex 2 (mTORC2), stimulated by DEP domain-containing mTOR-interacting protein (DEPTOR, endogenous inhibitor of mTORC1 and mTORC2) silencing. The trafficking of PepT2 to the membrane and the uptake of β-Ala-Lys-AMCA was promoted by neuronal precursor cell-expressed developmentally down-regulated 4 isoform 2 (Nedd4-2) silencing. The effects of knockdown of mTORC1, but not mTORC2, on cell membrane expression and transport activity of PepT2 was abolished by Nedd4-2 silencing. With immunofluorescence staining, PepT2 was identified to be interacting with Nedd4-2. The Nedd4-2 expression and the interaction between PepT2 and Nedd4-2 was increased through mTORC1 knockdown, indicating an increased ubiquitination of PepT2. The results revealed that mTORC1 can regulate the expression and function of PepT2 through Nedd4-2 in BMEC.

The mixed blessing of AMPK signaling in Cancer treatments

BackgroundNutrient acquisition and metabolism pathways are altered in cancer cells to meet bioenergetic and biosynthetic demands. A major regulator of cellular metabolism and energy homeostasis, in normal and cancer cells, is AMP-activated protein kinase (AMPK). AMPK influences cell growth via its modulation of the mechanistic target of Rapamycin (mTOR) pathway, specifically, by inhibiting mTOR complex mTORC1, which facilitates cell proliferation, and by activating mTORC2 and cell survival. Given its conflicting roles, the effects of AMPK activation in cancer can be counter intuitive. Prior to the establishment of cancer, AMPK acts as a tumor suppressor. However, following the onset of cancer, AMPK has been shown to either suppress or promote cancer, depending on cell type or state.MethodsTo unravel the controversial roles of AMPK in cancer, we developed a computational model to simulate the effects of pharmacological maneuvers that target key metabolic signalling nodes, with a specific focus on AMPK, mTORC, and their modulators. Specifically, we constructed an ordinary differential equation-based mechanistic model of AMPK-mTORC signaling, and parametrized the model based on existing experimental data.ResultsModel simulations were conducted to yield the following predictions: (i) increasing AMPK activity has opposite effects on mTORC depending on the nutrient availability; (ii) indirect inhibition of AMPK activity through inhibition of sirtuin 1 (SIRT1) only has an effect on mTORC activity under conditions of low nutrient availability; (iii) the balance between cell proliferation and survival exhibits an intricate dependence on DEP domain-containing mTOR-interacting protein (DEPTOR) abundance and AMPK activity; (iv) simultaneous direct inhibition of mTORC2 and activation of AMPK is a potential strategy for suppressing both cell survival and proliferation.ConclusionsTaken together, model simulations clarify the competing effects and the roles of key metabolic signalling pathways in tumorigenesis, which may yield insights on innovative therapeutic strategies.

Regulation of human mTOR complexes by DEPTOR.

The vertebrate-specific DEP domain-containing mTOR interacting protein (DEPTOR), an oncoprotein or tumor suppressor, has important roles in metabolism, immunity, and cancer. It is the only protein that binds and regulates both complexes of mammalian target of rapamycin (mTOR), a central regulator of cell growth. Biochemical analysis and cryo-EM reconstructions of DEPTOR bound to human mTOR complex 1 (mTORC1) and mTORC2 reveal that both structured regions of DEPTOR, the PDZ domain and the DEP domain tandem (DEPt), are involved in mTOR interaction. The PDZ domain binds tightly with mildly activating effect, but then acts as an anchor for DEPt association that allosterically suppresses mTOR activation. The binding interfaces of the PDZ domain and DEPt also support further regulation by other signaling pathways. A separate, substrate-like mode of interaction for DEPTOR phosphorylation by mTOR complexes rationalizes inhibition of non-stimulated mTOR activity at higher DEPTOR concentrations. The multifaceted interplay between DEPTOR and mTOR provides a basis for understanding the divergent roles of DEPTOR in physiology and opens new routes for targeting the mTOR-DEPTOR interaction in disease.

Co-treatment With Everolimus, an mTOR-Specific Antagonist, or Downregulation of ELK1 Enhances the Sensitivity of Pancreatic Cancer Cells to Genistein.

Genistein is a natural isoflavone with pharmacological or potentially anti-tumor properties. However, the resistance of cancer cells to genistein remains a major obstacle. This study focused on the mechanism implicated in the resistance of pancreatic cancer (PC) cells to genistein and the mechanism of action. First, key molecules and signaling pathways related to genistein resistance in PC cells were explored using bioinformatics tools. DEP domain containing MTOR interacting protein (DEPTOR), a typical inhibitor of the mammalian target of rapamycin (mTOR) signaling, was predicted to be poorly expressed in the genistein-resistant PC cells. Thereafter, genistein-resistant PC cells (Panc-1 and PaCa) were constructed. Altered expression of DEPTOR was introduced in cells, and everolimus (ELM), an mTOR-specific antagonist, was administrated in cells as well to examine their roles in genistein resistance. The cell apoptosis was examined in vitro and in vivo in mouse xenograft tumors. The upstream regulator of DEPTOR was predicted via bioinformatic tools. The bioinformatic analyses showed that the PI3K/AKT/mTOR signaling pathway was activated in the setting of DEPTOR downregulation in genistein-resistant PC cells. DEPTOR overexpression reduced the 50% inhibiting concentration (IC50) of genistein in PC cells and suppressed mTOR phosphorylation, and it increased caspase-3 activity, LDH release and apoptosis in PC cells. ELM treatment enhanced the sensitivity of PC cells to genistein in vitro and it strengthened the tumor-eliminating role of genistein in mice. ETS transcription factor ELK1 (ELK1), a transcription factor that negatively regulated DEPTOR transcription, was suppressed by genistein. Upregulation of ELK1 suppressed DEPTOR transcription and reduced the genistein sensitivity of cells, and it also blocked the genistein-sensitizing roles of ELM in PC cells. In conclusion, this study demonstrated that ELK1 reduces DEPTOR transcription, leading to mTOR phosphorylation and the drug resistance of PC cells.

Exploring novel and potent molecules for disrupting DEPTOR-mTOR interaction through structure-steered screening, extra-exactitude molecular docking, prime binding free energy estimation and voguish molecular dynamics

Dep domain containing mTOR interacting protein (DEPTOR) has critical implications in the development and progression of human malignancies. Increased expression of DEPTOR promotes the growth of tumor cells by inhibiting the mTORC1, which alleviates the negative feedback inhibition by mTORC1 downstream target S6Ks on PI3K/AKT pathway thereby promotes cell survival and prevents apoptosis. This clearly suggests that targetting DEPTOR-mTOR interactions through small molecules may prove as an effective strategy for circumventing distinct cancers. In this study, we employed a top-down approach for finding three novel molecules which may prove effective in disrupting Deptor–mTOR interaction. Following DEPTOR modelling and validation we performed grid-directed structure-based screening by specifying the residues of DEPTOR known to interact with mTOR. A library of 10,000 protein–protein disrupting molecules was screened against the defined region of DEPTOR. From the screened molecules, 30 molecules with highest binding affinity were chosen for molecular docking. Thirty (30) extra-precision molecular docking experiments and 30 molecular mechanics generalized born surface area (MMGBSA) assays were performed. Following this top 10 molecules in terms of binding affinity were selected and the interaction profile of their corresponding docked files was generated. The top three molecules were finally selected after taking all the three parameters including docking score, binding energy value and interaction profile into consideration. For atomistic insights regarding DEPTOR-topmost hit interactions, molecular dynamics was performed for 100 ns. This molecule after further evaluation may prove as promising candidate for anticancer therapy. Communicated by Ramaswamy H. Sarma

Hyperuricemia Drives Pancreatic β‐cell Death Facilitated by DEPTOR uratylation

Introduction Uric acid (UA) is the final metabolite of the human purine metabolism, also representing the main antioxidant in the blood. However, elevated serum uric acid (SUA) called hyperuricemia and causing gout has been linked to diabetes mellitus (DM). Loss of viable pancreatic β-cells is a hallmark of both development and progression of DM. We aimed to determine how elevated UA levels can induce a loss of cell viability through the AMPK-mTOR pathway, involving the uric acid transporter glucose transporter 9 (GLUT9). Material and methods We employed the human (1.1B4) and mouse (MIN6) pancreatic β-cell lines. Co-immunoprecipitation, siRNA knock-down, western blot analyses, autophagy, MTT and Caspase 3/7 assays were used to determine the effects of chronic hyperuricemia on molecular mechanisms of β-cell viability. Results Hyperuricemia reduced pancreatic β-cell viability by both reduced metabolic activity and increased autophagy/apoptosis, which could be reverted by the GLUT9 inhibitor benzbromarone as well as by GLUT9 knock-down. Phosphorylation of AMP-activated protein kinase (AMPK) was increased upon elevated UA conditions, whereas phosphorylation of mTOR complex-1 (mTORC1) activator Raptor was reduced. However, hyperuricemia increased expression of the mTORC1 negative regulator DEP domain-containing mTOR-interacting protein (DEPTOR). DEPTOR stabilization was mediated by changes in DEPTOR ubiquitination due to the down-regulation of the E3 ligase β-TrCP and upregulation of a newly identified DEPTOR-regulating ubiquitin-specific protease, USP3, in cells exposed to high UA conditions. In addition, we document for the first time that a protein is UA-modified (uratylated) in a cellular context. Interestingly, DEPTOR uratylation seems to contribute to the change of its ubiquitination. We observed this to be different between mouse and human β-cells, where in MIN6 cells only ubiquitin seems to be uratylated, whereas in 1.1B4 cells DEPTOR seems to be directly uratylated. This difference in uratylation of DEPTOR between mouse and human pancreatic β-cells possibly reflects a further evolved, more sophisticated post-transcriptional regulation of proteins based on the difference in UA handling between these species. Our observation also puts into question if mouse models are an accurate representation of the regulatory processes of basic physiological processes such as insulin secretion or β-cell viability in humans. Conclusion Hyperuricemic conditions resembling elevated SUA levels result in a decrease in pancreatic β-cell viability initiated by changes in DEPTOR ubiquitination/uratylation. This results in an increase of autophagy and ultimately apoptosis. Our findings thus suggest a causal basis for the connection between elevated SUA and diabetes mellitus.

DEPTOR Tyrosine Phosphorylation: A Novel Molecular Switch Involved in mTOR Activity

Dysregulated mTOR (mechanistic Target Of Rapamycin) is a potent tumor growth inducer known to promote cancer cell proliferation and survival. Its activity can be regulated by numerous factors composing the PTEN/PI3K/AKT canonical pathway, which are often mutated in cancer. However, in a subset of cancer showing a constitutively activated mTOR, there is no alteration within the canonical activation pathway, suggesting different activation mechanism. We previously discovered several post-translational modifications (PTMs) on DEP domain-containing mTOR-interacting protein (DEPTOR), an endogenous regulator of the two mTOR complexes (mTORC1 and mTORC2). Still, the mechanism by which these PTMs regulate DEPTOR ability to shut down mTOR complexes remains ill-defined. Studies have only shown that DEPTOR is phosphorylated on serine (S286/287/291/293/299) under high energy level condition, leading to its degradation by the ubiquitin/proteasome system. Unlike this irreversible regulatory mechanism, we have recently identified another PTM, a tyrosine phosphorylation that affects DEPTOR inhibitory functions. We found that phosphorylation of DEPTOR tyrosine 289 increase its stability, while promoting DEPTOR dissociation from mTORC1&2, leading to a rapid and sustain increase in mTORC1&2 activity. To identify the upstream signaling pathway causing tyrosine 289 phosphorylation, we performed mass spectrometry analysis, as well as a small drug screening of different tyrosine kinase inhibitors. Using these combined methods, we identify Syk (Spleen tyrosine kinase), whose expression levels correlate with levels of tyrosine 289 phosphorylation. We also found that Syk-induced phosphorylation of DEPTOR was regulated by the EphB2 receptor. Our findings uncovered a new mechanism regulating mTOR activity, which explains the increased mTOR activity in cancer with unaffected PTEN/PI3K/AKT regulatory pathways. Better understanding of this mTOR/DEPTOR regulatory pathway could allow the development of a new therapeutic approach to inhibit mTOR associated cancer progression.

Structural Basis of DEPTOR to Recognize Phosphatidic Acid Using its Tandem DEP Domains

DEP domain containing mTOR-interacting protein (DEPTOR) plays pivotal roles in regulating metabolism, growth, autophagy and apoptosis by functions as an endogenous inhibitor of mTOR signaling pathway. Activated by phosphatidic acid, a second messenger in mTOR signaling, DEPTOR dissociates from mTORC1 complex with unknown mechanism. Here, we present a 1.5 Å resolution crystal structure, which shows that the N-terminal two tandem DEP domains of hDEPTOR fold into a dumbbell-shaped structure, protruding the characteristic β-hairpin arms of DEP domains on each side. An 18 amino acids DDEX motif at the end of DEP2 interacts with DEP1 and stabilizes the structure. Biochemical studies showed that the tandem DEP domains directly interact with phosphatidic acid using two distinct positively charged patches. These results provide insights into mTOR activation upon phosphatidic acid stimulation.

DEPTOR is a direct p53 target that suppresses cell growth and chemosensitivity

DEP-domain containing mTOR-interacting protein (DEPTOR), a natural mTOR inhibitor, has essential roles in several processes, including cell growth, metabolism, apoptosis, and immunity. DEPTOR expression has been shown to be diversely controlled at transcriptional levels in cell- and context-specific manners. However, whether there is a general mechanism for the regulation of DEPTOR expression remains largely unknown. Here, we report that DEPTOR is a downstream target of the tumor suppressor, p53, whose activity is positively correlated with DEPTOR expression both in vitro in cell cultures and in vivo in mouse tissues. Mechanistically, p53 directly binds to the DEPTOR promoter and transactivates its expression. Depletion of the p53-binding site on the DEPTOR promoter by CRISPR-Cas9 technology decreases DEPTOR expression and promotes cell proliferation and survival by activating AKT signaling. Importantly, inhibition of AKT by small molecular inhibitors or genetic knockdown abrogates the induction of cell growth and survival induced by deletion of the p53-binding region on the DEPTOR promoter. Furthermore, p53, upon activation by the genotoxic agent doxorubicin, induces DEPTOR expression, leading to cancer cell resistance to doxorubicin. Together, DEPTOR is a direct p53 downstream target and contributes to p53-mediated inhibition of cell proliferation, survival, and chemosensitivity.

DEPTOR Prevents Osteoarthritis Development Via Interplay With TRC8 to Reduce Endoplasmic Reticulum Stress in Chondrocytes.

Endoplasmic reticulum (ER) stress has been shown to promote chondrocyte apoptosis and osteoarthritis (OA) progression, but the precise mechanisms via which ER stress is modulated in OA remain unclear. Here we report that DEP domain-containing mTOR-interacting protein (DEPTOR) negatively regulated ER stress and OA development independent of mTOR signaling. DEPTOR is ubiquitinated in articular chondrocytes and its expression is markedly reduced along with OA progression. Deletion of DEPTOR in chondrocytes significantly promoted destabilized medial meniscus (DMM) surgery-induced OA development, whereas intra-articular injection of lentivirus-expressing DEPTOR delayed OA progression in mice. Proteomics analysis revealed that DEPTOR interplayed with TRC8, which promoted TRC8 auto-ubiquitination and degraded by the ubiquitin-proteasome system (UPS) in chondrocytes. Loss of DEPTOR led to TRC8 accumulation and excessive ER stress, with subsequent chondrocyte apoptosis and OA progression. Importantly, an inhibitor of ER stress eliminated chondrocyte DEPTOR deletion-exacerbated OA in mice. Together, these findings establish a novel mechanism essential for OA pathogenesis, where decreasing DEPTOR in chondrocytes during OA progression relieves the auto-ubiquitination of TRC8, resulting in TRC8 accumulation, excessive ER stress, and OA progression. Targeting this pathway has promising therapeutic potential for OA treatment. © 2020 American Society for Bone and Mineral Research (ASBMR).

Helicobacter pylori promote inflammation and host defense through the cagA-dependent activation of mTORC1.

Mechanistic target of rapamycin complex 1 (mTORC1) functions as regulating different cellular processes, including cell growth, proliferation, motility, survival, metabolism, autophagy, and protein transcription. Recently, it also found to be associated with many infections and inflammatory diseases, playing complex roles in pathogens growth and inflammation regulation. However, the regulation mechanism of mTORC1 in gastric epithelial cells and its role in Helicobacter pylori (H. pylori) infection and related gastritis remain unclear. Here, we identified that the phosphorylation of mechanistic target of rapamycin (mTOR) and the expression of DEP domain-containing mTOR-interacting protein (DEPTOR) was increased in gastric mucosa of H. pylori-infected patients and mice, as well as in H. pylori-infected gastric epithelial cells, which were largely depended on H. pylori cagA. The expression of DEPTOR was regulated via mTORC1, but, in turn, inhibited mTORC1. Knockdown mTOR significantly decreased expression and secretion of cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6, chemokines CCL7 and CXCL16, and antimicrobial peptide LL37 in vitro, while knockdown DEPTOR had the opposite effect. Similar observations were made using mTOR knockout (KO) mice in vivo, moreover. The gastric inflammation was attenuated, while the bacterial burden was increased in mTOR KO mice during H. pylori infection. These findings supported H. pylori promote gastritis and inhibit bacterial colonization through the cagA-dependent activation of mTORC1.

HIF-mediated Suppression of DEPTOR Confers Resistance to mTOR Kinase Inhibition in Renal Cancer.

SummaryMechanistic target of rapamycin (mTOR) is a fundamental regulator of cell growth, proliferation, and metabolism. mTOR is activated in renal cancer and accelerates tumor progression. Here, we report that the mTOR inhibitor, DEP domain-containing mTOR-interacting protein (DEPTOR), is strikingly suppressed in clear cell renal cell carcinoma (ccRCC) tumors and cell lines. We demonstrate that DEPTOR is repressed by both hypoxia-inducible factors, HIF-1 and HIF-2, which occurs through activation of the HIF-target gene and transcriptional repressor, BHLHe40/DEC1/Stra13. Restoration of DEPTOR- and CRISPR/Cas9-mediated knockout experiments demonstrate that DEPTOR is growth inhibitory in ccRCC. Furthermore, loss of DEPTOR confers resistance to second-generation mTOR kinase inhibitors through deregulated mTORC1 feedback to IRS-2/PI3K/Akt. This work reveals a hitherto unknown mechanism of resistance to mTOR kinase targeted therapy that is mediated by HIF-dependent reprograming of mTOR/DEPTOR networks and suggests that restoration of DEPTOR in ccRCC will confer sensitivity to mTOR kinase therapeutics.

Abstract 2684: Identification of 14 novel genetic loci for testicular germ cell tumor susceptibility

Abstract Testicular germ cell tumors (TGCT) are ideally suited to agnostic genome-wide association methods because of their known high heritability and homogeneous cell of origin. Here we report the most updated results from the international Testicular Cancer Consortium (TECAC) that extends published findings from our recent meta-analysis, which included 3,558 cases and 13,970 controls from five centers. To these existing samples, we added summary statistics from 300 cases and 151,991 controls provided by deCODE combined with imputed genotypes from an additional 5,602 cases and 5,006 controls assembled from 15 TECAC centers, almost all (97%) of which were centrally genotyped using the Illumina Human Core array. A total of 9,458 TGCT cases were included in our current meta-analysis, representing a 66% increase since our last publication. We identified 14 new and confirmed 41 previously reported loci that surpassed genome-wide significance (5 × 10-8). Fifteen previously reported loci did not reach this threshold, although one did so in subgroup analysis. In addition, 57 new loci attained nominal significance (5 × 10-8 ≤ p ≤ 1 × 10-5). As expected, a substantial proportion of top hits continue to map to genetic regions belonging to biological pathways essential to male germ cell development, sex determination, and chromosomal segregation. For example, newly identified genes include anaphase promoting complex subunit 2 (ANAPC2), BCL2 like 11 (BCL211), anti-Mullerian hormone receptor type 2 (AMHR2), and DEP domain containing mTOR interacting protein (DEPTOR). Our findings also further implicate genes located on the X chromosome, specifically the androgen receptor (AR). In analyses stratified by tumor subtype, we identified GATA-binding protein 4 (GATA4) as a susceptibility locus among men diagnosed with seminoma. Despite a similar effect size observed among men diagnosed solely with non-seminoma (i.e. excluding cases with a mixture of both seminoma and non-seminoma tumors), this marker did not surpass genome-wide significance in this subtype likely because of the smaller case sample size. GATA4 previously has been associated with overall risk of TGCT (Litchfield et al., Nat Genet. 2017). Biological mechanisms for all genome-wide significant loci are currently being explored using publicly available data sets, ATAC-seq of four TGCT cell lines, and SPATIaL-Seq (a novel high-resolution chromatin conformation capture (3C) assay) of the NTERA2 pluripotent human embryonal carcinoma cell line. Additional genotyping of top hits in an independent sample set of 1,103 TGCT cases and 1,210 controls is currently ongoing. Citation Format: Louise C. Pyle, John Pluta, Kevin T. Nead, Nandita Mitra, Javier Benitez, D. Timothy Bishop, Victoria Cortessis, Alberto Ferlin, Jourik Gietema, Mark Greene, Tom Grotmol, Ramneek Gupta, Rob Hamilton, Michelle A. Hildebrandt, Trine B. Haugen, Lambertus Kiemeney, Christian Kubisch, Davor Lessel, Paloma Martin, Thorunn Rafnar, Lorenzo Richiardi, Rolf Skotheim, Clare Turnbull, Fredrik Wiklund, Tongzhang Zheng, Ewa Rajpert-De Meyts, Stephen M. Schwartz, Katherine A. McGlynn, Peter A. Kanetsky, Katherine L. Nathanson, Testicular Cancer Consortium (TECAC). Identification of 14 novel genetic loci for testicular germ cell tumor susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2684.

Involvement of Lamin B1 Reduction in Accelerated Cellular Senescence during Chronic Obstructive Pulmonary Disease Pathogenesis.

Downregulation of lamin B1 has been recognized as a crucial step for development of full senescence. Accelerated cellular senescence linked to mechanistic target of rapamycin kinase (MTOR) signaling and accumulation of mitochondrial damage has been implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. We hypothesized that lamin B1 protein levels are reduced in COPD lungs, contributing to the process of cigarette smoke (CS)-induced cellular senescence via dysregulation of MTOR and mitochondrial integrity. To illuminate the role of lamin B1 in COPD pathogenesis, lamin B1 protein levels, MTOR activation, mitochondrial mass, and cellular senescence were evaluated in CS extract (CSE)-treated human bronchial epithelial cells (HBEC), CS-exposed mice, and COPD lungs. We showed that lamin B1 was reduced by exposure to CSE and that autophagy was responsible for lamin B1 degradation in HBEC. Lamin B1 reduction was linked to MTOR activation through DEP domain-containing MTOR-interacting protein (DEPTOR) downregulation, resulting in accelerated cellular senescence. Aberrant MTOR activation was associated with increased mitochondrial mass, which can be attributed to peroxisome proliferator-activated receptor γ coactivator-1β-mediated mitochondrial biogenesis. CS-exposed mouse lungs and COPD lungs also showed reduced lamin B1 and DEPTOR protein levels, along with MTOR activation accompanied by increased mitochondrial mass and cellular senescence. Antidiabetic metformin prevented CSE-induced HBEC senescence and mitochondrial accumulation via increased DEPTOR expression. These findings suggest that lamin B1 reduction is not only a hallmark of lung aging but is also involved in the progression of cellular senescence during COPD pathogenesis through aberrant MTOR signaling.

Repressors of mTORC1 act to blunt the anabolic responseto feeding in the soleus muscle of a cast-immobilized mouse hindlimb.

We recently reported results showing that cast immobilization of a rat hindlimb rapidly leads to development of anabolic resistance as demonstrated by failure of oral leucine administration to activate the mechanistic target of rapamycin complex 1 (mTORC1) and stimulate protein synthesis in the soleus muscle. The goal of this study was to assess the possible contribution of several mTORC1 regulatory proteins to the development of anabolic resistance. To accomplish this, 14‐week‐old male C57BL/6 mice (n = 21) were subjected to unilateral cast immobilization of the hindlimb for either 1 or 3 days, and the immobilized limb was compared to its contralateral control. The mass of the soleus muscle was decreased in the immobilized compared to the non‐immobilized limb within 72‐h in association with diminished protein synthesis. In agreement with our previous report, a 24‐h casting period was sufficient to induce anabolic resistance, as demonstrated by blunted re‐feeding‐induced activation of mTORC1. Moreover, resistance of mTORC1 activation was associated not only with upregulated expression of REDD1, but also with altered expression of other mTORC1 regulatory proteins, that is, Sestrin1 and DEP domain‐containing mTOR interacting protein (DEPTOR). In addition, re‐feeding‐induced phosphorylation of DEPTOR was significantly impaired in the immobilized compared to the non‐immobilized limb. This work builds upon previous discoveries by our laboratory to elucidate the blunted mTORC1 response to stimuli during disuse of skeletal muscle induced by cast immobilization while highlighting new potential therapeutic targets for future countermeasures against muscle atrophy.

DEPTOR regulates osteogenic differentiation via inhibiting MEG3-mediated activation of BMP4 signaling and is involved in osteoporosis

BackgroundThe mammalian target of rapamycin (mTOR) pathway plays a significant role in osteogenic differentiation and bone maintenance. As the only known endogenous inhibitor of mTOR function, DEP domain containing mTOR interacting protein (DEPTOR) is potentially involved in stem cell differentiation, although the pathophysiological significance and its molecular mechanisms remain unclear. The present study aimed to elucidate the effects of DEPTOR on the progress of osteoporosis and investigate the underlying molecular mechanisms of osteogenic regulation.MethodsAn ovariectomy mouse model with decreased bone formation and osteogenic induction with bone marrow mesenchymal stem cells (BMSCs) were used to investigate the relationship between DEPTOR and osteogenic events. A loss-of-function investigation was then performed to explore the role of DEPTOR in the osteogenic differentiation of BMSCs both in vitro and in vivo. Finally, long noncoding RNA (lncRNA) and mRNA sequences were investigated to reveal the underlying mechanisms of DEPTOR in osteogenic regulation. RNA interference, western blotting, and chromatin immunoprecipitation assays were performed for further mechanistic determination.ResultsThe results indicated that DEPTOR contributes to the progress of osteoporosis, and higher expression of Deptor was observed in osteoporotic bones. The expression of DEPTOR was reduced during the osteogenic differentiation of BMSCs, and knockdown of DEPTOR promoted BMSC osteogenesis in vitro and in vivo. lncRNA and mRNA sequences indicated that knockdown of DEPTOR upregulated the expression of maternally expressed 3 (nonprotein coding) (MEG3), which subsequently activated bone morphogenetic protein 4 (BMP4) signaling. Furthermore, DEPTOR could bind to a specific region (− 1000 bp ~ 0) of the MEG3 promoter to regulate its transcription, and inhibition of MEG3 reduced BMP4 activation triggered by DEPTOR knockdown.ConclusionsTaken together, our study revealed a novel function of DEPTOR in osteogenic differentiation by inhibiting MEG3-mediated activation of BMP4 signaling, which suggested that DEPTOR could be a therapeutic target for bone loss diseases and skeletal tissue regeneration.