Inhibition Of mTOR Signaling Research Articles

Targeting lncRNA DDIT4-AS1 Sensitizes Triple Negative Breast Cancer to Chemotherapy via Suppressing of Autophagy.

In this study, it is found that the lncRNA, DNA damage inducible transcript 4 antisense RNA1 (DDIT4-AS1), is highly expressed in triple-negative breast cancer (TNBC) cell lines and tissues due to H3K27 acetylation in the promoter region, and promotes the proliferation, migration, and invasion of TNBC cells via activating autophagy. Mechanistically, it is shown that DDIT4-AS1 induces autophagy by stabilizing DDIT4 mRNA via recruiting the RNA binding protein AUF1 and promoting the interaction between DDIT4 mRNA and AUF1, thereby inhibiting mTOR signaling pathway. Furthermore, silencing of DDIT4-AS1 enhances the sensitivity of TNBC cells to chemotherapeutic agents such as paclitaxel both in vitro and in vivo. Using a self-activatable siRNA/drug core-shell nanoparticle system, which effectively deliver both DDIT4-AS1 siRNA and paclitaxel to the tumor-bearing mice, a significantly enhanced antitumor activity is achieved. Importantly, the codelivery nanoparticles exert a stronger antitumor effect on breast cancer patient-derived organoids. These findings indicate that lncRNA DDIT4-AS1-mediated activation of autophagy promotes progression and chemoresistance of TNBC, and targeting of DDIT4-AS1 may be exploited as a new therapeutic approach to enhancing the efficacy of chemotherapy against TNBC.

A traditional prescription comprising Astragali Radix and Schisandra chinensis Fructus induces apoptosis and protective autophagy in hepatocellular carcinoma cells

Ethnopharmacological relevanceHepatocellular carcinoma (HCC) poses a growing challenge to global health efforts. The 5-year survival rate of HCC patients is still dismal. A traditional prescription Qi-Wei-Wan (QWW) comprising Astragali Radix and Schisandra chinensis Fructus has traditionally been used for HCC treatment according to traditional Chinese medicine theory, but the pharmacological basis is not clear. Aim of the studyThis study aims to investigate the anti-HCC effects of an ethanolic extract of QWW (hereafter, QWWE) and the mechanism of action. Materials and methodsAn UPLC-Q-TOF-MS/MS method was developed to control the quality of QWWE. Two human HCC cell lines (HCCLM3 and HepG2) and a HCCLM3 xenograft mouse model were employed to investigate the anti-HCC effects of QWWE. The anti-proliferative effect of QWWE in vitro was determined by MTT, colony formation and EdU staining assays. Apoptosis and protein levels were examined by flow cytometry and Western blotting, respectively. Nuclear presence of signal transducer and activator of transcription 3 (STAT3) was examined by immunostaining. Transient transfection of pEGFP-LC3 and STAT3C plasmids was performed to assess autophagy and determine the involvement of STAT3 signaling in QWWE’s anti-HCC effects, respectively. ResultsWe found that QWWE inhibited the proliferation of and triggered apoptosis in HCC cells. Mechanistically, QWWE inhibited the activation of SRC and STAT3 at Tyr416 and Tyr705, respectively; inhibited the nuclear translocation of STAT3; lowered Bcl-2 protein levels, while increased Bax protein levels in HCC cells. Over-activating STAT3 attenuated the cytotoxic and apoptotic effects of QWWE in HCC cells. Moreover, QWWE induced autophagy in HCC cells by inhibiting mTOR signaling. Blocking autophagy with autophagy inhibitors (3-methyladenine and chloroquine) enhanced the cytotoxicity, apoptotic effect and the inhibitory effect on STAT3 activation of QWWE. Intragastric administration of QWWE at 10 mg/kg and 20 mg/kg potently repressed tumor growth and inhibited STAT3 and mTOR signaling in tumor tissues, but did not significantly affect mouse body weight. ConclusionQWWE exhibited potent anti-HCC effects. Inhibiting the STAT3 signaling pathway is involved in QWWE-mediated apoptosis, while blocking mTOR signaling contributes to QWWE-mediated autophagy induction. Blockade of autophagy enhanced the anti-HCC effects of QWWE, indicating that the combination of an autophagy inhibitor and QWWE might be a promising therapeutic strategy for HCC management. Our findings provide pharmacological justifications for the traditional use of QWW in treating HCC.

Functional characterization of novel NPRL3 mutations identified in three families with focal epilepsy.

Focal epilepsy accounts for 60% of all forms of epilepsy, but the pathogenic mechanism is not well understood. In this study, three novel mutations in NPRL3 (nitrogen permease regulator-like 3), c.937_945del, c.1514dupC and 6,706-bp genomic DNA (gDNA) deletion, were identified in three families with focal epilepsy by linkage analysis, whole exome sequencing (WES) and Sanger sequencing. NPRL3 protein is a component of the GATOR1 complex, a major inhibitor of mTOR signaling. These mutations led to truncation of the NPRL3 protein and hampered the binding between NPRL3 and DEPDC5, which is another component of the GATOR1 complex. Consequently, the mutant proteins enhanced mTOR signaling in cultured cells, possibly due to impaired inhibition of mTORC1 by GATOR1. Knockdown of nprl3 in Drosophila resulted in epilepsy-like behavior and abnormal synaptic development. Taken together, these findings expand the genotypic spectrum of NPRL3-associated focal epilepsy and provide further insight into how NPRL3 mutations lead to epilepsy.

Deciphering the mechanism of PSORI-CM02 in suppressing keratinocyte proliferation through the mTOR/HK2/glycolysis axis.

Hyperplasia of epidermal keratinocytes that depend on glycolysis is a new hallmark of psoriasis pathogenesis. Our previous studies demonstrated that PSORI-CM02 could halt the pathological progression of psoriasis by targeting inflammatory response and angiogenesis, but its effect(s) and mechanism(s) on proliferating keratinocytes remained unclear. In this study, we aim to identify components of PSORI-CM02 that are absorbed into the blood and to determine the effect(s) of PSORI-CM02 on keratinocyte proliferation and its molecular mechanism(s). We used the immortalized human epidermal keratinocyte cell line, HaCaT, as an in vitro model of proliferating keratinocytes and the imiquimod-induced psoriasis mouse (IMQ) as an in vivo model. Metabolite profiles of vehicle pharmaceutic serum (VPS), PSORI-CM02 pharmaceutic serum (PPS), and water extraction (PWE) were compared, and 23 components of PSORI-CM02 were identified that were absorbed into the blood of mice. Both PPS and PWE inhibited the proliferation of HaCaT cells and consequently reduced the expression of the proliferation marker ki67. Additionally, PPS and PWE reduced phosphorylation levels of mTOR pathway kinases. Seahorse experiments demonstrated that PPS significantly inhibited glycolysis, glycolytic capacity, and mitochondrial respiration, thus reducing ATP production in HaCaT cells. Upon treatments of PPS or PWE, hexokinase 2 (HK2) expression was significantly decreased, as observed from the set of glycolytic genes we screened. Finally, in the IMQ model, we observed that treatment with PSORI-CM02 or BPTES, an inhibitor of mTOR signaling, reduced hyperproliferation of epidermal keratinocytes, inhibited the expression of p-S6 and reduced the number of proliferating cell nuclear antigen (PCNA)-positive cells in lesioned skin. Taken together, we demonstrate that PSORI-CM02 has an anti-proliferative effect on psoriatic keratinocytes, at least in part, by inhibiting the mTOR/HK2/glycolysis axis.

Targeting mTOR for Anti-Aging and Anti-Cancer Therapy

The balance between anabolism and catabolism is disrupted with aging, with the rate of anabolism being faster than that of catabolism. Therefore, mTOR, whose major function is to enhance anabolism and inhibit catabolism, has become a potential target of inhibition for anti-aging therapy. Interestingly, it was found that the downregulation of the mTOR signaling pathway had a lifespan-extending effect resembling calorie restriction. In addition, the mTOR signaling pathway promotes cell proliferation and has been regarded as a potential anti-cancer target. Rapamycin and rapalogs, such as everolimus, have proven to be effective in preventing certain tumor growth. Here, we reviewed the basic knowledge of mTOR signaling, including both mTORC1 and mTORC2. Then, for anti-aging, we cited a lot of evidence to discuss the role of targeting mTOR and its anti-aging mechanism. For cancer therapy, we also discussed the role of mTOR signaling in different types of cancers, including idiopathic pulmonary fibrosis, tumor immunity, etc. In short, we discussed the research progress and both the advantages and disadvantages of targeting mTOR in anti-aging and anti-cancer therapy. Hopefully, this review may promote more ideas to be generated for developing inhibitors of mTOR signaling to fight cancer and extend lifespan.

Rapamycin Alleviates Protein Aggregates, Reduces Neuroinflammation, and Rescues Demyelination in Globoid Cell Leukodystrophy

We have shown in vivo and in vitro previously that psychosine causes dysfunction of autophagy and the ubiquitin-proteasome system underlying the pathogenesis of globoid cell leukodystrophy (GLD), a devastating lysosomal storage disease complicated by global demyelination. Here, we investigated the therapeutic efficacy of the mTOR inhibitor rapamycin in twitcher mice, a murine model of infantile GLD, in biochemical, histochemical, and clinical aspects. Administration of rapamycin to twitcher mice inhibited mTOR signaling in the brains, and significantly reduced the accumulation of insoluble ubiquitinated protein and the formation of ubiquitin aggregates. The astrocytes and microglia reactivity were attenuated in that reactive astrocytes, ameboid microglia, and globoid cells were reduced in the brains of rapamycin-treated twitcher mice. Furthermore, rapamycin improved the cortical myelination, neurite density, and rescued the network complexity in the cortex of twitcher mice. The therapeutic action of rapamycin on the pathology of the twitcher mice's brains prolonged the longevity of treated twitcher mice. Overall, these findings validate the therapeutic efficacy of rapamycin and highlight enhancing degradation of aggregates as a therapeutic strategy to modulate neuroinflammation, demyelination, and disease progression of GLD and other leukodystrophies associated with intracellular aggregates.

Re-organization of nucleolar architecture in myogenic differentiation.

Myogenesis, the process of muscle differentiation, requires an extensive remodeling of the cellular transcriptome and proteome. Whereas the transcriptional program underpinning myogenesis is well characterized, the required adaptation in protein synthesis is incompletely understood. Enhanced protein synthesis necessitates ribosome biogenesis at the nucleolus. Nucleolar size and activity are inextricably linked with altered gene expression. Here, we report changes in nucleolar morphology and function during myogenic differentiation. Immunofluorescence analysis revealed alterations in nucleolar morphology that were dependent on the cellular state - proliferative or quiescent myogenic progenitors (myoblasts or reserve cells) contained multiple small nucleoli with a characteristic spherical shape, whereas multinucleated myotubes typically contained one large, often irregularly shaped nucleolus. These morphological alterations are consistent with changes to nucleolar phase separation properties. Re-organization of the nucleolar structure was correlated with enhanced rRNA production and protein translation. Inhibition of mTOR signaling with rapamycin perturbed nucleolar re-organization. Conversely, hyperactivated mTOR enhanced alterations in nucleolar morphology. These findings support the idea that there is an mTOR dependent re-organization of nucleolar structure during myogenesis, enhancing our understanding of myogenesis and possibly facilitating new approaches to therapeutic interventions in muscle pathologies.

Pharmacological vitamin C inhibits mTOR signaling and tumor growth by degrading Rictor and inducing HMOX1 expression.

Pharmacological vitamin C (VC) is a potential natural compound for cancer treatment. However, the mechanism underlying its antitumor effects remains unclear. In this study, we found that pharmacological VC significantly inhibits the mTOR (including mTORC1 and mTORC2) pathway activation and promotes GSK3-FBXW7-mediated Rictor ubiquitination and degradation by increasing the cellular ROS. Moreover, we identified that HMOX1 is a checkpoint for pharmacological-VC-mediated mTOR inactivation, and the deletion of FBXW7 or HMOX1 suppresses the regulation of pharmacological VC on mTOR activation, cell size, cell viability, and autophagy. More importantly, it was observed that the inhibition of mTOR by pharmacological VC supplementation in vivo produces positive therapeutic responses in tumor growth, while HMOX1 deficiency rescues the inhibitory effect of pharmacological VC on tumor growth. These results demonstrate that VC influences cellular activities and tumor growth by inhibiting the mTOR pathway through Rictor and HMOX1, which may have therapeutic potential for cancer treatment.

Kavalactone Kawain Impedes Urothelial Tumorigenesis in UPII-Mutant Ha-Ras Mice via Inhibition of mTOR Signaling and Alteration of Cancer Metabolism.

UPII-mutant Ha-ras transgenic mice develop urothelial hyperplasia and low-grade papillary carcinoma, which mimics human non-muscle invasive bladder cancer (NMIBC). We investigated the effects and mechanisms of kawain, a main kavalactone in the kava plant, on oncogenic Ha-ras-driven urothelial carcinoma in these mice. The mice were fed at six weeks of age with vehicle control or kawain (6 g/kg) formulated food for approximately five months. Seventy-eight percent of the mice or more fed with kawain food survived more than six months of age, whereas only 32% control food-fed male mice survived, (p = 0.0082). The mean wet bladder weights (a surrogate for tumor burden) of UPII-mutant Ha-ras transgenic mice with kawain diet was decreased by approximately 56% compared to those fed with the control diet (p = 0.035). The kawain diet also significantly reduced the occurrence of hydronephrosis and hematuria in UPII-mutant Ha-ras transgenic mice. Histological examination and immunohistochemistry analysis revealed that vehicle control-treated mice displayed more urothelial carcinoma and Ki67-positive cells in the bladder compared to kawain treated mice. Global metabolic profiling of bladder tumor samples from mice fed with kawain food showed significantly more enrichment of serotonin and less abundance of xylulose, prostaglandin A2, D2 and E2 compared to those from control diet-fed mice, suggesting decreased shunting of glucose to the pentose phosphate pathway (PPP) and reduced inflammation. In addition, kawain selectively inhibited the growth of human bladder cancer cell lines with a significant suppression of 4E-BP1 expression and rpS6 phosphorylation. These observations indicate a potential impact of kawain consumption on bladder cancer prevention by rewiring the metabolic programs of the tumor cells.

The Role of Green Tea Catechin Epigallocatechin Gallate (EGCG) and Mammalian Target of Rapamycin (mTOR) Inhibitor PP242 (Torkinib) in the Treatment of Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating condition that has physical and psychological consequences for patients. SCI is accompanied by scar formation and systemic inflammatory response leading to an intense degree of functional loss. The catechin, epigallocatechin gallate (EGCG), an active compound found in green tea, holds neuroprotective features and is known for its anti-inflammatory potential. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two functionally distinct complexes termed mTOR complex 1 and 2 (mTORC1; mTORC2). Inhibition of mTORC1 by rapamycin causes neuroprotection, leading to partial recovery from SCI. In this study the effects of EGCG, PP242 (an inhibitor of both complexes of mTOR), and a combination of EGCG and PP242 in SCI have been examined. It has been found that both EGCG and PP242 significantly improved sensory/motor functions following SCI. However, EGCG appeared to be more effective (BBB motor test, from 2 to 8 weeks after SCI, p = 0.019, p = 0.007, p = 0.006, p = 0.006, p = 0.05, p = 0.006, and p = 0.003, respectively). The only exception was the Von Frey test, where EGCG was ineffective, while mTOR inhibition by PP242, as well as PP242 in combination with EGCG, significantly reduced withdrawal latency starting from week three (combinatorial therapy (EGCG + PP242) vs. control at 3, 5, and 7 weeks, p = 0.011, p = 0.007, and p = 0.05, respectively). It has been found that EGCG was as effective as PP242 in suppressing mTOR signaling pathways, as evidenced by a reduction in phosphorylated S6 expression (PP242 (t-test, p < 0.0001) or EGCG (t-test, p = 0.0002)). These results demonstrate that EGCG and PP242 effectively suppress mTOR pathways, resulting in recovery from SCI in rats, and that EGCG acts via suppressing mTOR pathways.

Streptococcal arginine deiminase regulates endothelial inflammation, mTOR pathway and autophagy

Endothelial cells (EC) are active participants in the inflammation process. During the infection, the change in endothelium properties provides the leukocyte infiltrate formation and restrains pathogen dissemination due to coagulation control. Pathogenic microbes are able to change the endothelium properties and functions in order to invade the bloodstream and disseminate in the host organism. Arginine deiminase (ADI), a bacterial arginine-hydrolyzing enzyme, which causes the amino acid deficiency, important for endothelium biology. Previous research implicates altered metabolism of arginine in the development of endothelial dysfunction and inflammation. It was shown that arginine deficiency, as well as overabundance affects the balance of mechanical target of rapamycin (mTOR)/S6 kinase (S6K) pathway, arginase and endothelial nitric oxide synthase (eNOS) resulted in reactive oxygen species (ROS) production and EC activation. ADI creating a deficiency of arginine can interfere cellular arginine-dependent processes. Thus, this study was aimed at investigation of the influence of streptococcal ADI on the metabolism and inflammations of human umbilical vein endothelial cells (HUVEC). The action of ADI was studied by comparing the effect Streptococcus pyogenes M49-16 paternal strain expressing ADI and its isogenic mutant M49-16delArcA with the inactivated gene ArcA. Based on comparison of the parental and mutant strain effects, it can be concluded, that ADI suppressed mTOR signaling pathway and enhanced autophagy. The processes failed to return to the basic level with arginine supplement. Our study also demonstrates that ADI suppressed endothelial proliferation, disrupted actin cytoskeleton structure, increased phospho-NF-κB p65, CD62P, CD106, CD54, CD142 inflammatory molecules expression, IL-6 production and lymphocytes-endothelial adhesion. In spite of the ADI-mediated decrease in arginine concentration in the cell-conditioned medium, the enzyme enhanced the production of nitric oxide in endothelial cells. Arginine supplementation rescued proliferation, actin cytoskeleton structure, brought NO production to baseline and prevented EC activation. Additional evidence for the important role of arginine bioavailability in the EC biology was obtained. The results allow us to consider bacterial ADI as a pathogenicity factor that can potentially affect the functions of endothelium.

MTOR signaling inhibition decreases lysosome migration and impairs the success of Trypanosoma cruzi infection and replication in cardiomyocytes

Chagas disease is caused by the parasite Trypanosoma cruzi (T. cruzi) and, among all the chronic manifestations of the disease, Chronic Chagas Cardiomyopathy (CCC) is the most severe outcome. Despite high burden and public health importance in Latin America, there is a gap in understanding the molecular mechanisms that results in CCC development. Previous studies showed that T. cruzi uses the host machinery for infection and replication, including the repurposing of the responses to intracellular infection such as mitochondrial activity, vacuolar membrane, and lysosomal activation in benefit of parasite infection and replication. One common signaling upstream to many responses to parasite infection is mTOR pathway, previous associated to several downstream cellular mechanisms including autophagy, mitophagy and lysosomal activation. Here, using human iPSC derived cardiomyocytes (hiPSCCM), we show the mTOR pathway is activated in hiPSCCM after T. cruzi infection, and the inhibition of mTOR with rapamycin reduced number of T. cruzi 48 h post infection (hpi). Rapamycin treatment also reduced lysosome migration from nuclei region to cell periphery resulting in less T. cruzi inside the parasitophorous vacuole (PV) in the first hour of infection. In addition, the number of parasites leaving the PV to the cytoplasm to replicate in later times of infection was also lower after rapamycin treatment. Altogether, our data suggest that host's mTOR activation concomitant with parasite infection modulates lysosome migration and that T. cruzi uses this mechanism to achieve infection and replication. Modulating this mechanism with rapamycin impaired the success of T. cruzi life cycle independent of mitophagy.

Responses to Many Anti-Aging Interventions Are Sexually Dimorphic.

There is increasing appreciation that sex differences are not limited to reproductive organs or traits related to reproduction and that sex is an important biological variable in most characteristics of a living organism. The biological process of aging and aging-related traits are no exception and exhibit numerous, often major, sex differences. This article explores one aspect of these differences, namely sex differences in the responses to anti-aging interventions. Aging can be slowed down and/or postponed by a variety of environmental ("lifestyle"), genetic or pharmacological interventions. Although many, particularly older studies utilized only one sex of experimental animals, there is considerable evidence that responses to these interventions can be very different in females and males. Calorie restriction (CR), that is reducing food intake without malnutrition can extend longevity in both sexes, but specific metabolic alterations and health benefits induced by CR are not the same in women and men. In laboratory mice, several of the genetic alterations that reduce insulin-like growth factor I (IGF-1) signaling extend longevity more effectively in females or in females only. Beneficial effects of rapamycin, an inhibitor of mTOR signaling, on mouse longevity are greater in females. In contrast, several anti-aging compounds, including a weak estrogen, 17 alpha estradiol, extend longevity of male, but not female, mice. Apparently, fundamental mechanisms of aging are not identical in females and males and it is essential to use both sexes in studies aimed at identifying novel anti-aging interventions. Recommendations for lifestyle modifications, drugs, and dietary supplements to maintain good health and functionality into advanced age and to live longer will likely need to be tailored to the sex of the user.

Autophagy activation in breast cancer cells in vitro after the treatment with PI3K/AKT/mTOR inhibitors

Introduction. Current chemotherapy of breast cancer has a wide range of disadvantages, in particular, the development of therapy-related infections and hormonal imbalance. Combination of main cytostatic with glucocorticoids allows to broaden its therapeutic interval and to decrease the total toxicity of the treatment. However, long-term treatment with glucocorticoids leads to the development of severe side effects via activation of multiple molecular mechanisms. Thus, glucocorticoids activate prosurvival mTOR-dependent autophagy. Therefore, the evaluation of PI3K (phosphoinositide 3-kinases) / Akt (protein kinase B) / mTOR (mammalian target of rapamycin) inhibitors as adjuvants for breast cancer therapy is important for optimization of treatment protocol.Aim. Analysis of the effects of PI3K/Akt/mTOR inhibitors, rapamycin, wortmannin and LY-294002 in combination with glucocorticoids in breast cancer cell lines of different subtypes.Materials and methods. We demonstrated the inhibition of PI3K/Akt/mTOR signaling and the autophagy induction after the treatment of breast cancer cells with rapamycin, wortmannin and LY-294002 by Western blotting analysis of Beclin-1, phospho-Beclin-1 (Ser93 and Ser30).Conclusion. PI3K/Akt/mTOR inhibitors in combination with Dexamethasone cooperatively inhibited mTOR signaling and activated autophagy in breast cancer cells in vitro.

G3BP1 coordinates lysophagy activity to protect against compression-induced cell ferroptosis during intervertebral disc degeneration.

Lysophagy is a form of selective autophagy to remove unwanted lysosomes. However, its role in the pathogenesis of intervertebral disc degeneration (IDD) remains unclear. We intended to investigate the relationship between lysophagy and ferroptosis, as well as the potential involved molecules during IDD. Human nucleus pulposus (NP) cells were obtained from clinical patients. The protein levels, protein colocalization and cellular reactive oxygen species levels were assessed by western blotting, immunofluorescence analysis, immunoprecipitation and flow cytometry, respectively. The in vivo experiments were conducted based on the needle puncture-induced IDD model in rats. Compression pressure induces the lysophagy inactivation and lysosomal damage, resulting in iron overload and ferroptosis in human NP cells. Notably, Ras GTPase-activating protein-binding proteins 1 (G3BP1) resides at lysosomes to coordinate lysophagy activity mainly via the function of G3BP1/TSC2 complex. Dysfunction of G3BP1/TSC2 complex accelerates the lysosomal damage and ferroptosis in NP cells. Besides, inhibition of mTOR signalling ameliorates lysosomal damage and protects against cell ferroptosis. The in vivo experiments also demonstrate that the G3BP1/mTOR signalling is involved in the progression of IDD. These findings illustrate the relationship between lysophagy and compression-induced cell ferroptosis. It also indicates the positive role of G3BP1 and may provide potential targets for IDD treatment.

Endothelial mTOR Preserves Hematopoietic Stem Cell Fitness By Suppressing Thrombospondin1

Hematopoietic stem cells (HSC) reside in close proximity to blood vessels within the bone marrow (BM) termed 'perivascular niches' which provide instructive cues for maintaining HSC self-renewal potential. The perivascular niche is comprised of endothelial cells (ECs) lining the BM blood vessels (BMECs), and an array of perivascular stromal cells that collectively serve to regulate balanced HSC self-renewal and differentiation. While young HSCs are endowed with robust self-renewal potential, aging is associated with a significant decline in HSC fitness including a loss of their self-renewal ability and a biased differentiation towards the myeloid lineages at the expense of lymphopoiesis. These aging related HSC defects result in anemias, impaired response to immunizations, and an increased risk for developing clonal disorders including acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS). However, mechanisms that drive HSC defects during aging remain poorly understood, and represents a critical barrier towards developing strategies for preservation of hematopoietic fitness during aging. While HSC aging was generally assumed as a predominantly cell-intrinsic process, recent studies have identified that aging of the BM niche plays an instructive role in promoting age-related HSC defects. Supporting this, we recently discovered that aging is associated with a significant decline in mTOR activity within BMECs that impairs their capacity to serve as a HSC-supportive niche. Our findings showed that deletion of mTOR specifically within ECs of young (4 month old) mice (Endothelial Cell specific mTOR Knock Out; mTORECKO) is sufficient to recapitulate HSC aging phenotypes including loss of HSC self-renewal, and a myeloid-biased differentiation. Here, we sought to identify the mechanisms by which endothelial mTOR preserves HSC fitness. To identify candidate factors that promote HSC aging in mTORECKO mice, we performed a plasma proteomic analysis that revealed a putative role for Thrombospondin1 (Thbs1) wherein mTORECKO mice displayed elevated levels of Thbs1. Analysis of cultured BMECs revealed that inhibition of mTOR signaling within BMECs was sufficient to increase Thbs1 expression. Thbs1 is a well-defined antiangiogenic factor that has recently been identified to play a critical role in promoting a multitude of age-related cardiovascular and inflammatory disorders. However, whether Thbs1 regulates HSC activity is not known. To this end, we performed ex vivo HSC expansion transplantation assays which revealed that Thbs1 causes a significant decline in long term HSC engraftment. RNA Seq analysis of HSC expansions revealed that Thbs1 activates chronic inflammatory responses within HSCs that have been suggested to promote aging related HSC defects. To determine whether Thbs1 promotes HSC aging in mTORECKO mice, we crossed them with Thbs1 global knockout mice (Thbs1KO) to generate mTORECKOThbs1KO mice. Analysis of mTORECKOThbs1KO mice revealed that deletion of Thbs1 was sufficient to restore hematopoietic defects in mTORECKO mice including restoration of peripheral blood myeloid bias, BM HSC frequency and hematopoietic progenitor activity. Furthermore, serial HSC transplantation analysis confirmed that deletion of Thbs1 was sufficient to restore HSC self-renewal and lymphoid output in mTORECKO mice, identifying a putative role for Thbs1 as a pro-geronic factor for HSCs. To determine whether Thbs1 promotes HSC dysfunction during physiological aging, we aged Thbs1KO mice for 18 months and analyzed their hematopoietic parameters. Our analysis revealed that deletion of Thbs1 was able to prevent the age-associated increase in peripheral blood myeloid bias and BM HSC frequency. Notably, HSCs derived from aged (18 month old) Thbs1KO mice demonstrated a suppression of inflammatory responses by RNA Seq analysis, and a restoration of self-renewal ability and balanced lineage output in serial HSC transplantation assays, that are indistinguishable from HSCs derived from young (3 month old) mice. Our findings identify Thbs1 as a novel pro-geronic factor for HSC aging that can be targeted to preserve hematopoietic healthspan.

Blockade of Ceramide-Dependent Extracellular Vesicle Secretion Improves Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell (HSC) transplantation serves as a curative therapy for numerous benign and malignant hematopoietic diseases, and as a platform for HSC gene therapy. A main constraint is the limited quantity of HSCs collected from patients or donors and the inherent risk of graft failure, or oligocolonal reconstitution, especially after ex vivo manipulation. Current efforts to mitigate these limitations have predominantly focused on the discovery of extrinsic factors to maintain or expand hematopoietic stem and progenitor cells (HSPCs), whereas the role of the HSPC secretome in modulating niche function and improving occupancy have not been extensively studied. Recently, classes of nanometer-sized extracellular vesicles (EVs) have been shown to regulate tissue function by providing potent proangiogenic signals and remodeling cellular microenvironments. EV secretion additionally regulates homeostasis and stemness properties of HSPCs; however, in-depth studies into HSPC EV biogenesis and the impact on homeostatic and regenerative potency have been hindered by numerous technical challenges. Here, we utilize an optimized ex vivo expansion protocol for human and murine HSPCs to examine the impact of different EV biogenesis pathways on vesicle secretion. Our studies rapidly focused on ceramide-dependent vesicle secretion; blockade of ceramide biosynthesis with a widely used neutral sphingomyelinase inhibitor, GW4869 significantly reduced HSPC EV secretion. Treatment of HSPCs with GW4869 further inhibited mTOR signaling, altered autophagic flux, and decreased cell cycle entry. Increased ratios of lymphoid (MPP4):myeloid (MPP2/3) primed progenitors accumulated in culture. Upon competitive transplantation into myeloablated recipients, GW4869-exposed murine HSPCs demonstrated superior long-term engraftment potential (>8 steady-state fold change, p < 0.001), independent of bone marrow or splenic homing. Ceramide inhibition did not significantly alter long-term lineage output from transplanted HSPCs. Confirmatory genetic knockout (KO) of neutral sphingomyelinase (nSMase2) in murine and human HSPCs using Cas9 ribonucleoprotein complex delivery similarly demonstrated enhanced colony forming potential. To understand the impact of ceramide inhibition on vesicle cargo, comparative mass spectrometry was performed on HSPC nSMase2 KO EVs (EVnSmase2-KO). Nearly 900 proteins were depleted (fold change <2) in EVnSmase2-KO; these underrepresented proteins were noted to be involved in myeloid differentiation and activation, immune response, and exocytosis, suggesting vesicle modulation of these processes. These results suggest that ceramide inhibition not only leads to a reduction in EV release, but also impacts selective trafficking of EV cargo, ultimately altering HSPC function. While other EV biogenesis pathways have been demonstrated to promote HSC-self renewal, these findings implicate ceramide-dependent EV secretion as a novel negative regulator of HSPC repopulation. Transient disruption of ceramide-dependent EV secretion results in a more quiescent and less metabolically active HSPC pool, and additionally prevents bias toward a myeloid-primed progenitor pool seen with ex vivo expansion. These effects culminate in dramatically improved repopulation potential of HSPCs. Furthermore, unlike other small molecular inhibitors proposed to enhance engraftment of HSPCs, including rapamycin, no significant hinderance of ex vivo expansion was seen with GW4869 treatment. We therefore propose broadened exploration of ceramide inhibition for ex vivo maintenance of HSPCs prior to transplantation.

Interactive contribution of hyperinsulinemia, hyperglycemia, and mammalian target of rapamycin signaling to valvular interstitial cell differentiation and matrix remodeling.

Diabetes and its major key determinants insulin resistance and hyperglycemia are known risk factors for calcific aortic valve disease (CAVD). The processes leading to molecular and structural alterations of the aortic valve are yet not fully understood. In previous studies, we could show that valvular interstitial cells (VIC) display canonical elements of classical insulin signaling and develop insulin resistance upon hyperinsulinemia and hyperglycemia accompanied by impaired glucose metabolism. Analyses of cultured VIC and aortic valve tissue revealed extracellular matrix remodeling and degenerative processes. Since PI3K signaling through mammalian target of rapamycin (mTOR) is involved in fibrotic processes of the heart, we aim at further functional investigation of this particular Akt-downstream signaling pathway in the context of diabetes-induced CAVD. Primary cultures of VIC were treated with hyperinsulinemia and hyperglycemia. Phosphorylation of mTOR(Ser2448) was determined by Western blot analysis after acute insulin stimulus. Inhibition of mTOR phosphorylation was performed by rapamycin. Phosphorylation of mTOR complex 1 (MTORC1) downstream substrates 4E-BP1(Thr37/46) and P70S6K(Thr389), and MTORC2 downstream substrate Akt(Ser473) as well as the PDK1-dependent phosphorylation of Akt(Thr308) was investigated. Markers for extracellular matrix remodeling, cell differentiation and degenerative changes were analyzed by Western blot analysis, semi-quantitative real-time PCR and colorimetric assays. Hyperinsulinemia and hyperglycemia lead to alterations of VIC activation, differentiation and matrix remodeling as well as to an abrogation of mTOR phosphorylation. Inhibition of mTOR signaling by rapamycin leads to a general downregulation of matrix molecules, but to an upregulation of α-smooth muscle actin expression and alkaline phosphatase activity. Comparison of expression patterns upon diabetic conditions and rapamycin treatment reveal a possible regulation of particular matrix components and key degeneration markers by MTORC1 downstream signaling. The present findings broaden the understanding of mitogenic signaling pathways in VIC triggered by hyperinsulinemia and hyperglycemia, supporting the quest for developing strategies of prevention and tailored treatment of CAVD in diabetic patients.

Discovery of EMD37, a 1,2,4-oxadiazole derivative, as a novel endoplasmic reticulum stress inducer with potent anticancer activity

Targeting endoplasmic reticulum (ER) stress presents a promising strategy in cancer therapy. We previously reported a series of 1,2,4-oxadiazole derivatives that induced the degradation of EGFR and c-Met which are implicated in tumorigenesis. Based on our previous SAR studies, herein, we report the discovery of EMD37, a novel 1,2,4-oxadiazole derivative, which demonstrated potent anticancer activity against NCI-60 cancer cell lines panel compared to its parent/lead compounds. Anti-proliferative assays revealed preferential cytotoxicity of EMD37 on cancer cells compared to normal cells. Delving deeper, we exploited unbiased genome-wide transcriptome profiling of EMD37-treated cancer cells. Gene Ontology and gene set enrichment analyses revealed that EMD37 promoted ER stress and unfolded protein response (UPR) machinery which was confirmed using RT-qPCR. Mining drug signature databases also confirmed the enrichment of the signature of canonical UPR inducers. Knocking down ER stress transcription factors compromised at least partly the anticancer activity of EMD37. Immunoblot analysis showed that EMD37 induced the accumulation of polyubiquitinated proteins and inhibited mTOR signaling. EMD37 induced G2/M cell cycle arrest and apoptosis of human cancer cells. Inhibiting apoptosis evidently abrogated the anticancer efficacy of EMD37. Altogether, this study introduces EMD37 as a novel ER inducer which warrants further investigation as a potentially relevant anti-cancer therapy.

Inhibition of mTOR signaling protects human glioma cells from hypoxia-induced cell death in an autophagy-independent manner

Although malignant gliomas frequently show aberrant activation of the mammalian target of rapamycin (mTOR), mTOR inhibitors have performed poorly in clinical trials. Besides regulating cell growth and translation, mTOR controls the initiation of autophagy. By recycling cellular components, autophagy can mobilize energy resources, and has thus been attributed cancer-promoting effects. Here, we asked whether the activation of autophagy represents an escape mechanism to pharmacological mTOR inhibition in glioma cells, and explored co-treatment with mTOR and autophagy inhibitors as a therapeutic strategy. Mimicking conditions of the glioma microenvironment, glioma cells were exposed to nutrient starvation and hypoxia. We analyzed autophagic activity, cell growth, viability and oxygen consumption following (co-)treatment with the mTOR inhibitors torin2 or rapamycin, and autophagy inhibitors bafilomycin A1 or MRT68921. Changes in global proteome were quantified by mass spectrometry. In the context of hypoxia and starvation, autophagy was strongly induced in glioma cells and further increased by mTOR inhibition. While torin2 enhanced glioma cell survival, co-treatment with torin2 and bafilomycin A1 failed to promote cell death. Importantly, treatment with bafilomycin A1 alone also protected glioma cells from cell death. Mechanistically, both compounds significantly reduced cell growth and oxygen consumption. Quantitative proteomics analysis showed that bafilomycin A1 induced broad changes in the cellular proteome. More specifically, proteins downregulated by bafilomycin A1 were associated with the mitochondrial respiratory chain and ATP synthesis. Taken together, our results show that activation of autophagy does not account for the cytoprotective effects of mTOR inhibition in our in vitro model of the glioma microenvironment. Our proteomic findings suggest that the pharmacological inhibition of autophagy induces extensive changes in the cellular proteome that can support glioma cell survival under nutrient-deplete and hypoxic conditions. These findings provide a novel perspective on the complex role of autophagy in gliomas.