Rapamycin-insensitive Companion Of mTOR Research Articles

MTORc2 in the distal convoluted tubule (DCT) stimulates renal K+ excretion by inhibiting basolateral Kir4.1/Kir5.1 and Na-Cl−cotransporter (NCC) during increasing dietary K+ intake

Background: The mTORc2 is a multiprotein complex and the core components of mTORc2 contain mTOR (a serine/threonine protein kinase) and RICTOR (rapamycin insensitive companion of mTOR). Previous studies have strongly suggested the role of mTORc2 in the regulation of renal K+ excretion by stimulating epithelial Na+ channels (ENaC) and ROMK (Kir1.1) in the aldosterone-sensitive distal nephron (ASDN). The mice with deletion of RICTOR in the distal nephron or in the renal tubules are hyperkalemic during increasing dietary K+ intake. A large body of evidence has demonstrated that HK-induced inhibition of NCC is essential for stimulating renal K+ excretion and this is achieved by inhibiting the basolateral Kir4.1/Kir5.1 heterotetramer in the DCT. However, the role of mTORc2 in the regulation of basolateral Kir4.1/Kir5.1 heterotetramer in the DCT and NCC is not explored. Thus, the aim of the present study is to explore whether mTORc2 in the DCT regulates the basolateral Kir4.1/Kir5.1 of the DCT, NCC activity and renal K+ excretion. Method: We have generated DCT-specific RICTOR KO mice (DCT-RICTOR-KO) with C57BL6/J background by crossing the mice expressing Slc12a3-Cre-ERT2 with Rictorflox/flox mice. We used patch-clamp-technique to examine basolateral Kir4.1/Kir5.1 in early-DCT, immunoblotting and immunofluorescence to examine NCC expression and in vivo measurement of urinary K+-excretion to determine baseline renal K+-excretion (EK). We also used metabolic cage to examine the body weight (BW), food/water intakes, urinary Na+ (UNa) /K+ (UK) excretion rate, and plasma Na+ and K+ concentrations in male(m)/female(f) DCT-RICTOR KO mice and Slc12a3-Cre-ERT2 -Rictorflox/flox mice (control). Results: Overnight-HK intake stimulates RICTOR expression in the kidney and increases Rictor mRNA in the DCT. The deletion of RICTOR in the DCT was confirmed by immunofluorescence staining showing that RICTOR is expressed in parvalbumin-positive tubule (DCT) of control mice but is almost absent in DCT-RICTOR-KO mice. Deletion of RICTOR in the DCT increased the Kir4.1/Kir5.1-mediated K+-currents, hyperpolarized DCT membrane and increased the expression of pWNK4 and pNCC. While the two genotypes did not have significant difference regarding BW, food/water intakes, UNa and plasma Na+ concentrations, m/f DCT-RICTOR KO mice had a significantly lower UK and higher plasma K+ concentrations than their corresponding control. In vivo measurement of baseline renal K+ excretion (EK) also shows that renal-EK was lower and plasma K+ was higher in DCT-RICTOR-KO mice than corresponding control mice. Also, overnight-HK did not inhibit Kir4.1/Kir5.1 activity in the DCT and failed to inhibit the expression of pNCC in DCT-RICTOR-KO mice. Overnight-HK stimulated renal-EK in control mice, but this effect was attenuated in DCT-RICTOR-KO mice. Thus, overnight-HK induced hyperkalemia in DCT-RICTOR-KO mice but not in control mice. Conclusion: The mTORc2 of the DCT plays a significant role in mediating the effect of HK intake on Kir4.1/Kir5.1 activity and NCC expression. Since mTORC2 also stimulates ENaC and ROMK in the ASDN, an increase in Na+-delivery is expected to stimulate ENaC-ROMK-dependent K+-excretion and flow-stimulated renal K+-excretion. The work is supported by NIH Grants DK136491 (LDH) and DK133220 (WHW). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Anti-cancer effects of alpha lipoic acid, cisplatin and paclitaxel combination in the OVCAR-3 ovarian adenocarcinoma cell line.

Ovarian cancer is the leading cause of gynecological cancer deaths. One of the major challenges in treating ovarian cancer with chemotherapy is managing the resistance developed by cancer cells to drugs, while also minimizing the side effects caused by these agents In the present study, we aimed to examine the effects of a combination of alpha lipoic acid (ALA), with cisplatin and paclitaxel in ovarian cancer(OVCAR-3). The cytotoxic effects of ALA, cisplatin and paclitaxel on OVCAR-3 cells were determined. Four groups were formed: Control, ALA, Cisplatin + Paclitaxel, ALA + Cisplatin + Paclitaxel. The effects of single and combined therapy on cell migration, invasion and colony formation were analyzed. Changes in the expression of genes related to apoptosis, cell adhesion and cell cycle were analyzed with Real-time polymerase chain reaction(RT-PCR). The oxidative stress index and The Annexin V test were performed. The reduction in rapamycin-insensitive companion of mTOR(RICTOR) expression in the ALA + Cisplatin + Paclitaxel group was found statistically significant(p < 0.05). The decrease in MMP-9 and - 11 expressions the ALA + Cisplatin + Paclitaxel group was statistically significant(p < 0.05). The lowest values for mitogen-activated protein kinase(MAPK) proteins were found in the ALA + Cisplatin + Paclitaxel group. No colony formation was observed in the Cisplatin + Paclitaxel and ALA + Cisplatin + Paclitaxel groups. The lowest wound healing at 24h was seen in the ALA + Cisplatin + Paclitaxel group. This study is the first one to investigate the combined treatment of ALA, Cisplatin, Paclitaxel on OVCAR-3. While ALA alone was not effective, combined therapy with ALA, has been found to reduce cell invasion, especially wound healing in the first 24h, along with tumor cell adhesion.

MTOR hyperactivity and RICTOR amplification as targets for personalized treatments in malignancies.

The increasing knowledge of molecular alterations in malignancies, including mutations and regulatory failures in the mTOR (mechanistic target of rapamycin) signaling pathway, highlights the importance of mTOR hyperactivity as a validated target in common and rare malignancies. This review summarises recent findings on the characterization and prognostic role of mTOR kinase complexes (mTORC1 and mTORC2) activity regarding differences in their function, structure, regulatory mechanisms, and inhibitor sensitivity. We have recently identified new tumor types with RICTOR (rapamycin-insensitive companion of mTOR) amplification and associated mTORC2 hyperactivity as useful potential targets for developing targeted therapies in lung cancer and other newly described malignancies. The activity of mTOR complexes is recommended to be assessed and considered in cancers before mTOR inhibitor therapy, as current first-generation mTOR inhibitors (rapamycin and analogs) can be ineffective in the presence of mTORC2 hyperactivity. We have introduced and proposed a marker panel to determine tissue characteristics of mTOR activity in biopsy specimens, patient materials, and cell lines. Ongoing phase trials of new inhibitors and combination therapies are promising in advanced-stage patients selected by genetic alterations, molecular markers, and/or protein expression changes in the mTOR signaling pathway. Hopefully, the summarized results, our findings, and the suggested characterization of mTOR activity will support therapeutic decisions.

SUMO modifies GβL and mediates mTOR signaling

The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications; however, underlying mechanisms remain unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GβL (G protein β-subunit-like protein, also known as mLST8), is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GβL is SUMOylated at lysine sites K86, K215, K245, K261, and K305. We found that SUMO depletion reduces mTOR-Raptor (regulatory protein associated with mTOR) and mTOR-Rictor (rapamycin-insensitive companion of mTOR) complex formation and diminishes nutrient-induced mTOR signaling. Reconstitution with WT GβL but not SUMOylation-defective KR mutant GβL promotes mTOR signaling in GβL-depleted cells. Taken together, we report for the very first time that SUMO modifies GβL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.

Effects and mechanism of Rictor interference in podocyte injury induced by high glucose.

Rapamycin-insensitive companion of mTOR (Rictor) is a critical effector of mTOR protein complex 2 (mTORC2). The aim of the present study was to investigate the effect of Rictor in the mTORC2 signaling pathway in high glucose (HG)-induced diabetic podocyte injury by silencing the expression of Rictor. In the present study, mouse podocytes were treated with glucose (150 mM) and mannitol (200 mM), the Rictor gene was silenced using small interfering RNA (siRNA). Apoptosis was detected by flow cytometry, whereas podocyte cytoskeletal protein expression was detected by western blotting (WB) and immunofluorescence staining. The results demonstrated that, compared with that in the control group, the podocyte apoptotic rate was significantly increased in the mannitol group (negative group) and the groups that were treated with glucose (model groups). The podocyte apoptotic rate in the model + Rictor siRNA group was significantly decreased compared with that in the negative, model and the model glucose + siRNA negative control (NC) groups. WB indicated that the protein expression levels of podocalyxin and synaptopodin were reduced in the model and model + siRNA NC groups compared with those in the normal control and negative groups. Additionally, the protein expression levels of α-smooth muscle actin (α-SMA) and P-AKT/AKT were increased in the model and model + siRNA NC groups compared with the those in control and negative groups. Compared with those the model and model + siRNA NC groups, the protein expression levels of podocalyxin and synaptopodin were increased, whilst those of the α-SMA and P-AKT/AKT proteins were decreased, in the model + Rictor siRNA group. Results from immunofluorescence analysis were basically consistent with those of WB. Therefore, results of the present study suggest that silencing of the Rictor gene may reduce the damage to podocytes induced by HG, such that the Rictor/mTORC2 signaling pathway may be involved in the remodeling of podocyte actin cytoskeletal in diabetes.

A Comprehensive Pan-Cancer Analysis of the Potential Biological Functions and Prognosis Values of RICTOR.

The importance of the network defined by phosphatidylinositol-3-kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) downstream of Receptor Tyrosine Kinase (RTK) has been recognized for many years. However, the central role of RICTOR (rapamycin-insensitive companion of mTOR) in this pathway has only recently come to light. The function of RICTOR in pan-cancer still needs to be systematically elucidated. In this study, we examined RICTOR's molecular characteristics and clinical prognostic value by pan-cancer analysis. Our findings indicate that RICTOR was overexpressed in twelve cancer types, and a high RICTOR expression was linked to poor overall survival. Moreover, the CRISPR Achilles' knockout analysis revealed that RICTOR was a critical gene for the survival of many tumor cells. Function analysis revealed that RICTOR-related genes were mainly involved in TOR signaling and cell growth. We further demonstrated that the RICTOR expression was significantly influenced by genetic alteration and DNA-methylation in multiple cancer types. Additionally, we found a positive relationship between RICTOR expression and the immune infiltration of macrophages and cancer-associated fibroblasts in Colon adenocarcinoma and Head and Neck squamous cell carcinoma. Finally, we validated the ability of RICTOR in sustaining tumor growth and invasion in the Hela cell line using cell-cycle analysis, the cell proliferation assay, and wound-healing assay. Our pan-cancer analysis highlights the critical role of RICTOR in tumor progression and its potential as a prognostic marker for various cancer types.

Molecular mechanisms underlying equine endometrial cup regression: a transcriptomic analysis

The gonadotropin-secreting equine endometrial cups (EC) have a major role in pregnancy maintenance, but an intriguingly short lifespan. First seen in the maternal endometrium at days (d) 38-40 post-ovulation, the EC undergo degeneration via poorly understood mechanisms starting around d 60-70, with complete dehiscence at approximately 120-150 d of gestation. Pregnancy losses during the EC physiological lifespan and EC pathological persistence after abortion or uncomplicated parturition usually lead to irregular estrous cyclicity and subfertility, with only anecdotal and inconsistent therapeutic options currently available. A holistic understanding of the cellular signaling leading to EC regression may provide insight for development of effective therapies for retained or persistent EC. We therefore hypothesized that comparing the transcriptome of EC during development and regression could elucidate key regulators and molecular pathways leading to EC trophoblast cell demise. Samples of EC were dissected from the adjacent endometrium of pregnant pony mares during early development (DEC, 42-47 d post-ovulation, n = 5), and regression (REC, 96 – 120 d, n = 5). Next generation RNA sequencing was performed with Illumina NovaSeq6000 and reads mapped to EquCab3.0 (STAR-2.5.2b). Differentially expressed genes (DEGs) were identified with Cuffdiff-2.2.1 (FDR < 0.05). A total of 2,504 DEG were identified, being 1,648 upregulated and 856 downregulated in REC when compared to DEC. Pathway Analysis of DEGs was also performed (IPA, Qiagen). Interestingly, the main pathways affected in REC included oxidative phosphorylation (P = 1.08e-38);mitochondrial dysfunction (P = 2e-25); estrogen receptor signaling (P = 2.2e-10); Sirtuin signaling pathway (P = 9.64e-16), which has central regulatory roles in cell senescence and apoptosis; and Granzyme A Signaling (3.29e-15), associated with Cytotoxic T Lymphocyte and Natural Killer-mediated apoptosis via caspase-independent pathways. Upstream regulator analysis highlighted the roles of mitochondrial protein ubiquinol-cytochrome c reductase complex-3; rapamycin-insensitive companion of mTOR, an essential molecule in cellular homeostasis; Torin-1, a potent mTOR inhibitor; dead box protein 5, which may act as a co-activator of estrogen receptor signaling; and transducing-like enhancer protein-3, a member of a family of proteins that regulate cell fate through the Notch signaling pathway. Importantly, the main upstream regulators and downstream molecules are also involved in cancer cell biology (P = 1.32e-07 – 1.71e-122), including the downstream effectors of neoplastic and epithelial cell death secreted phosphoprotein 1 (Spp1), and secreted protein acidic and cysteine rich (Sparc). Along with aromatase, Spp1 and Sparc were upregulated in REC in comparison to DEC. In summary, we have highlighted key molecular pathways and regulators of EC regression, which merit further molecular and functional investigations. Since multiple identified molecules have also been targeted in human oncological treatments, further investigation of their effectiveness in hastening EC demise is warranted.

MTORC2-AKT-LAT1 signalling participates in methionine-induced β-CASEIN expression in mammary epithelial cells of dairy cows.

This study investigated the role of the mammalian target of rapamycin complex 2 (mTORC2)-protein kinase B (AKT) signalling in methionine (Met)-induced L-type amino acid transporter 1 (LAT1) expression and milk protein production. Primary mammary epithelial cells (MECs) from mammary parenchymal tissues of three lactating cows and MAC-T bovine MECs were cultured with or without 0.6 mM Met. Rapamycin-insensitive companion of mTOR (RICTOR) siRNA, the mTORC1 inhibitor rapamycin and the AKT activator SC79 were used to evaluate the effects of mTORC2-AKT signalling on Met-induced LAT1 expression and function. Each experiment was performed three times. Data were analysed with a two-sided unpaired t test or ANOVA with the Bonferroni multiple-comparison test. Western blotting showed that Met stimulation increased RICTOR expression (~244.67%; p < 0.05; control, 0.15 ± 0.026; Met, 0.517 ± 0.109) and AKT-S473 levels (~281.42%; p < 0.01; control, 0.253 ± 0.067; Met, 0.965 ± 0.019) in both primary MECs and MAC-T cells. Rapamycin-induced mTORC1 signalling inhibition decreased only Met-induced β-CASEIN expression by ~21.24% (p < 0.01; Met, 0.777 ± 0.01; Met and rapamycin, 0.612 ± 0.04) and did not affect Met-stimulated AKT-S473 levels, suggesting that mTORC2-AKT activation upon Met stimulation also contributes to milk protein synthesis. LAT1 participates in Met-induced β-CASEIN expression. In dairy cow MECs, mTORC2 inhibition by RICTOR siRNA decreased LAT1 levels on the plasma membrane by ~45.13% (p < 0.01; control, 0.359 ± 0.006; siRICTOR, 0.197 ± 0.004). However, SC79-induced AKT activation had the opposite effect (p < 0.01). In primary MECs and MAC-T cells, Met stimulation increased cytosolic and plasma membrane LAT1 expression respectively (MECs, 113.98% and 58.43%; MAC-T, 165.85% and 396.39%; p < 0.05). However, RICTOR siRNA significantly reduced Met-induced plasma membrane LAT1 expression (~76.48%; Met, 0.539 ± 0.05; Met and siRICTOR, 0.127 ± 0.012; p < 0.05). Thus, Met increased LAT1 expression and function via mTORC2-AKT signalling, upregulating milk protein synthesis in dairy cow MECs.

Differential protein expression in endothelial cells exposed to serum from patients with acute graft-vs-host disease, depending on steroid response.

Graft-versus-host disease (GVHD) is a complication of allogeneic haematopoietic cell transplantation. Endothelial injury is crucial as pathophysiological substrate for GVHD. GVHD first-line treatment is high-dose corticosteroids, although some patients are steroid-refractory. Through the present study, we compared the endothelial proteomic profiles in response to serum from steroid-refractory acute GVHD (SR-aGVHD) and steroid-sensitive acute GVHD (SS-aGVHD) patients. Blood samples from SR-aGVHD (n= 4) and SS-aGVHD (n= 8) patients were collected at aGVHD diagnosis. Endothelial cell cultures were exposed (48 h) to patients' serum. Protein extraction and proteomic analysis were performed. Differences were statistically evaluated by multivariate analysis. Forty-four proteins contributed to separate all samples into the two study groups, among which 15 participated significantly (p< 0.05), 10 exhibiting a fold change >1.2. Differentially expressed proteins were mainly associated with oxidative phosphorylation (Cytochrome C oxidase subunit 6B1, CX6B1), inflammation and angiogenesis (Apolipoprotein D, APOD), cell survival (Rapamycin-insensitive companion of mTOR, RICTR), and oxidative stress (Riboflavin kinase, RIFK). This pilot study used a novel approach to distinguish the aGVHD response to steroid treatment. The proteins differentially expressed could constitute potential biomarkers for steroid-treatment response. These findings signify a step forward to identify the mechanisms of response to steroids, of high clinical relevance considering the SR-aGVHD elevated mortality.

Maternal hyperglycemia inhibits pulmonary vasculogenesis during mouse fetal lung development by promoting GβL Ubiquitination-dependent mammalian target of Rapamycin assembly

BackgroundGestational diabetes mellitus (GDM) is associated with retarded lung development and poor lung health in offspring. Mammalian target of rapamycin (mTOR) is a key regulator of vasculogenesis and angiogenesis. The aim of this study was to investigate the role mTOR plays in pulmonary vasculogenesis during fetal lung development under maternal hyperglycemia.MethodsFirst, GDM was induced via streptozotocin injection in pregnant C57BL/6 mice before the radial alveolar count (RAC) in the fetal lungs was assessed using hematoxylin and eosin staining. The angiogenic ability of the cultured primary mouse fetal lung endothelial cells (MFLECs) was then assessed using the tube formation assay technique, while western blot and real-time polymerase chain reaction were performed to determine the expression of mTOR, regulatory-associated protein of mTOR (Raptor), rapamycin-insensitive companion of mTOR (Rictor), stress-activated protein kinase interacting protein 1 (Sin1), G protein beta subunit-like protein (GβL), Akt, tumor necrosis receptor associated factor-2 (TRAF2), and OTU deubiquitinase 7B (OTUD7B) in both the fetal lung tissues and the cultured MFLECs. Immunoprecipitation assays were conducted to evaluate the status of GβL-ubiquitination and the association between GβL and mTOR, Raptor, Rictor, and Sin1 in the cultured MFLECs.ResultsThe GDM fetal lungs exhibited a decreased RAC and reduced expression of von Willebrand factor, CD31, and microvessel density. The high glucose level reduced the tube formation ability in the MFLECs, with the mTOR, p-mTOR, p-Raptor, and TRAF2 expression upregulated and the p-Rictor, p-Sin1, p-Akt, and OTUD7B expression downregulated in both the GDM fetal lungs and the high-glucose-treated MFLECs. Meanwhile, GβL-ubiquitination was upregulated in the high-glucose-treated MFLECs along with an increased GβL/Raptor association and decreased GβL/Rictor and GβL/Sin1 association. Furthermore, TRAF2 knockdown inhibited the high-glucose-induced GβL-ubiquitination and GβL/Raptor association and restored the tube formation ability of the MFLECs.ConclusionMaternal hyperglycemia inhibits pulmonary vasculogenesis during fetal lung development by promoting GβL-ubiquitination-dependent mTORC1 assembly.

Mammalian Target of Rapamycin Inhibition Decreases Angiotensin II-Induced Steroidogenesis in HAC15 Human Adrenocortical Carcinoma Cells.

Mammalian target of rapamycin (mTOR) inhibitors suppress adrenal cortical carcinoma cell proliferation and cortisol production; the relationship between mTOR and aldosterone production has not been examined. HAC15 cells were incubated with an mTOR activator and several inhibitors including AZD8055 (AZD) in the presence and absence of angiotensin II (AngII). The expression of rapamycin-sensitive adapter protein of mTOR (Raptor) and rapamycin-insensitive companion of mTOR (Rictor), adaptor proteins of mTOR complex 1 and 2, respectively, were studied in the HAC15 cells and deleted by CRISPR/gRNA. The mTOR inhibitors decreased aldosterone induced by AngII. Inhibition of mTOR by AZD significantly suppressed AngII-induced aldosterone and cortisol formation in a dose-dependent manner, whereas the mTOR activator MHY had no effect. AZD did not alter forskolin-induced aldosterone production showing that it is specific to the AngII signaling pathway. AngII-mediated ERK and mTOR activation were suppressed by AZD, along with a concomitant dose-dependent reduction of AngII-induced steroidogenic enzymes including steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase-type 2, CYP17A1, and aldosterone synthase protein. Furthermore, mTOR components ribosomal protein S6 kinase (P70S6K) and protein kinase B phosphorylation levels were decreased by AZD. As mTOR exerts its main effects by forming complexes with adaptor proteins Raptor and Rictor, the roles of these individual complexes were studied. We found an increase in the phosphorylation of Raptor and Rictor by AngII and that their CRISPR/gRNA-mediated knockdown significantly attenuated AngII-induced aldosterone and cortisol production. mTOR signaling has a critical role in transducing the AngII signal initiating aldosterone and cortisol synthesis in HAC15 cells and that inhibition of mTOR could be a therapeutic option for conditions associated with excessive renin-angiotensin system-mediated steroid synthesis.

In Situ Analysis of mTORC1/C2 and Metabolism-Related Proteins in Pediatric Osteosarcoma.

Activation of the mTOR pathway has been observed in osteosarcoma, however the inhibition of mammalian target of rapamycin (mTOR) complex 1 has had limited results in osteosarcoma treatment. Certain metabolic pathways can be altered by mTOR activation, which can affect survival. Our aim was to characterize the mTOR profile and certain metabolic alterations in pediatric osteosarcoma to determine the interactions between the mTOR pathway and metabolic pathways. We performed immunohistochemistry on 28 samples to analyze the expression of mTOR complexes such as phospho-mTOR (pmTOR), phosphorylated ribosomal S6 (pS6), and rapamycin-insensitive companion of mTOR (rictor). To characterize metabolic pathway markers, we investigated the expression of phosphofructokinase (PFK), lactate dehydrogenase-A (LDHA), β-F1-ATPase (ATPB), glucose-6-phosphate dehydrogenase (G6PDH), glutaminase (GLS), fatty acid synthetase (FASN), and carnitin-O-palmitoyltransferase-1 (CPT1A). In total, 61% of the cases showed low mTOR activity, but higher pmTOR expression was associated with poor histological response to chemotherapy and osteoblastic subtype. Rictor expression was higher in metastatic disease and older age at the time of diagnosis. Our findings suggest the importance of the Warburg-effect, pentose-phosphate pathway, glutamine demand, and fatty-acid beta oxidation in osteosarcoma cells. mTOR activation is linked to several metabolic pathways. We suggest performing a detailed investigation of the mTOR profile before considering mTORC1 inhibitor therapy. Our findings highlight that targeting certain metabolic pathways could be an alternative therapeutic approach.

RICTOR Affects Melanoma Tumorigenesis and Its Resistance to Targeted Therapy.

The network defined by phosphatidylinositol-3-kinase (PI3K), AKT, and mammalian target of rapamycin (mTOR) plays a major role in melanoma oncogenesis and has been implicated in BRAF inhibitor resistance. The central role of RICTOR (rapamycin-insensitive companion of mTOR) in this pathway has only recently begun to be unraveled. In the present study, we assessed the role of mTORC2/RICTOR in BRAF-mutated melanomas and their resistance to BRAF inhibition. We showed that RICTOR was significantly overexpressed in melanoma and associated with bad prognoses. RICTOR overexpression stimulated melanoma-initiating cells (MICs) with ‘stemness’ properties. We also showed that RICTOR contributed to melanoma resistance to BRAF inhibitors and rendered the cells very sensitive to mTORC2 inhibition. We highlighted a connection between mTORC2/RICTOR and STAT3 in resistant cells and revealed an interaction between RAS and RICTOR in resistant melanoma, which, when disrupted, impeded the proliferation of resistant cells. Therefore, as a key signaling node, RICTOR contributes to BRAF-dependent melanoma development and resistance to therapy and, as such, is a valuable therapeutic target in melanoma.

Increases in Integrin-ILK-RICTOR-Akt Proteins, Muscle Mass, and Strength after Eccentric Cycling Training.

Recently, it has been suggested that a cellular pathway composed of integrin, integrin-linked kinase (ILK), rapamycin-insensitive companion of mTOR (RICTOR), and Akt may facilitate long-term structural and functional adaptations associated with exercise, independent of the mTORC1 pathway. Therefore, we examined changes in integrin-ILK-RICTOR-Akt protein in vastus lateralis (VL) before and after 8 wk of eccentric cycling training (ECC), which was expected to increase muscle function and VL cross-sectional area (CSA). Eleven men (23 ± 4 yr) completed 24 sessions of ECC with progressive increases in intensity and duration, resulting in a twofold increase in work from the first three (75.4 ± 14.1 kJ) to the last three sessions (150.7 ± 28.4 kJ). Outcome measures included lower limb lean mass, VL CSA, static strength, and peak and average cycling power output. These measures and VL samples were taken before and 4-5 d after the last training session. Significant (P < 0.05) increases in integrin-β1 (1.64-fold) and RICTOR (2.99-fold) protein as well as the phosphorylated-to-total ILK ratio (1.70-fold) were found, but integrin-α7 and Akt did not change. Increases in lower limb, thigh, and trunk lean mass (2.8%-5.3%, P < 0.05) and CSA (13.3% ± 9.0%, P < 0.001) were observed. Static strength (18.1% ± 10.8%) and both peak (8.6% ± 10.5%) and average power output (7.4% ± 8.3%) also increased (P < 0.05). However, no significant correlations were found between the magnitude of increases in protein and the magnitude of increases in CSA, static strength, or power output. In addition to increased muscle mass, strength, and power, we demonstrate that ECC increases integrin-β1 and RICTOR total protein and p-ILK/t-ILK, which may play a role in protection against muscle damage as well as anabolic signaling to induce muscle adaptations.

Rictor, an essential component of mTOR complex 2, undergoes caspase-mediated cleavage during apoptosis induced by multiple stimuli.

Caspase-mediated cleavage of proteins ensures the irreversible commitment of cells to undergo apoptosis, and is thus a hallmark of apoptosis. Rapamycin-insensitive companion of mTOR (rictor) functions primarily as a core and essential component of mTOR complex 2 (mTORC2) to critically regulate cellular homeostasis. However, its role in the regulation of apoptosis is largely unknown. In the current study, we found that rictor was cleaved to generate two small fragments at ~ 50 kD and ~ 130 kD in cells undergoing apoptosis upon treatment with different stimuli such as the death ligand, TRAIL, and the small molecule, AZD9291. This cleavage was abolished when caspases were inhibited and could be reproduced when directly incubating rictor protein and caspase-3 in vitro. Furthermore, the cleavage site of caspase-3 on rictor was mapped at D1244 (VGVD). These findings together robustly demonstrate that rictor is a substrate of caspase-3 and undergoes cleavage during apoptosis. These results add new information for understanding the biology of rictor in the regulation of cell survival and growth.

WAVE2 suppresses mTOR activation to maintain T cell homeostasis and prevent autoimmunity.

Cytoskeletal regulatory protein dysfunction has been etiologically linked to inherited diseases associated with immunodeficiency and autoimmunity, but the mechanisms involved are incompletely understood. Here, we show that conditional Wave2 ablation in T cells causes severe autoimmunity associated with increased mammalian target of rapamycin (mTOR) activation and metabolic reprogramming that engender spontaneous activation and accelerated differentiation of peripheral T cells. These mice also manifest diminished antigen-specific T cell responses associated with increased inhibitory receptor expression, dysregulated mitochondrial function, and reduced cell survival upon activation. Mechanistically, WAVE2 directly bound mTOR and inhibited its activation by impeding mTOR interactions with RAPTOR (regulatory-associated protein of mTOR) and RICTOR (rapamycin-insensitive companion of mTOR). Both the T cell defects and immunodysregulatory disease were ameliorated by pharmacological mTOR inhibitors. Thus, WAVE2 restraint of mTOR activation is an absolute requirement for maintaining the T cell homeostasis supporting adaptive immune responses and preventing autoimmunity.

An Immunohistochemical Study of the PTEN/AKT Pathway Involvement in Canine and Feline Mammary Tumors.

Simple SummaryThe PTEN/AKT pathway is involved in several human and animal tumors’ pathogenesis. This study investigates the PTEN/AKT pathway’s biological and prognostic values in canine and feline mammary tumors. PTEN, phospho-AKT (p-AKT) and Rictor expression was determined by immunohistochemistry in canine mammary adenomas and carcinomas and feline mammary carcinomas. In mammary tumors of both species p-Akt was inversely correlated with PTEN expression and positively with Rictor expression; p-Akt and Rictor expression correlated with poorer prognosis. This data could provide a rationale for further studies of this pathway in veterinary oncology due to prognostic and therapeutic implications.Phosphatase and tensin homolog deleted on chromosome10 (PTEN), phospho-v-Akt murine thymoma viral oncogene homolog (AKT), and the Rapamycin-Insensitive Companion of mTOR (Rictor) expression was investigated by immunohistochemistry in 10 canine mammary adenomas (CMAs), 40 canine mammary carcinomas (CMCs), and 30 feline mammary carcinomas (FMCs). All the CMAs, 25 of 40 CMCs (63%) and 7 of 30 FMCs (23%), were PTEN-positive. In dogs, no CMAs and 15 of 25 CMCs (37%) expressed phospho-AKT (p-AKT), while 24 of 30 FMCs (82%) were p-AKT-positive. One of 10 CMAs (10%), 24 of 40 CMCs (60%) and 20 of 30 FMCs (67%) were Rictor-positive. In the dog, PTEN expression correlated with less aggressive tumors, absence of lymphatic invasion, and longer survival. P-AKT expression correlated with more aggressive subtype, lymphatic invasion, and poorer survival and Rictor expression with lymphatic invasion. In cats, PTEN correlated with less aggressive carcinomas, absence of lymphatic invasion, and better survival. P-AKT and Rictor expression correlated with poorer survival. PTEN expression was inversely correlated with p-AKT and Rictor in both species, while p-AKT positively correlated with Rictor expression. A strong PTEN/AKT pathway involvement in behavior worsening of CMT and FMTs is demonstrated, providing a rationale for further studies of this pathway in veterinary oncology.

Proteomic Dissection of the Impact of Environmental Exposures on Mouse Seminal Vesicle Function

Seminal vesicles are an integral part of the male reproductive accessory gland system. They produce a complex array of secretions containing bioactive constituents that support gamete function and promote reproductive success, with emerging evidence suggesting these secretions are influenced by our environment. Despite their significance, the biology of seminal vesicles remains poorly defined. Here, we complete the first proteomic assessment of mouse seminal vesicles and assess the impact of the reproductive toxicant acrylamide. Mice were administered acrylamide (25 mg/kg bw/day) or control daily for five consecutive days prior to collecting seminal vesicle tissue. A total of 5013 proteins were identified in the seminal vesicle proteome with bioinformatic analyses identifying cell proliferation, protein synthesis, cellular death, and survival pathways as prominent biological processes. Secreted proteins were among the most abundant, and several proteins are linked with seminal vesicle phenotypes. Analysis of the effect of acrylamide on the seminal vesicle proteome revealed 311 differentially regulated (FC ± 1.5, p ≤ 0.05, 205 up-regulated, 106 downregulated) proteins, orthogonally validated via immunoblotting and immunohistochemistry. Pathways that initiate protein synthesis to promote cellular survival were prominent among the dysregulated pathways, and rapamycin-insensitive companion of mTOR (RICTOR, p = 6.69E-07) was a top-ranked upstream driver. Oxidative stress was implicated as contributing to protein changes, with acrylamide causing an increase in 8-OHdG in seminal vesicle epithelial cells (fivefold increase, p = 0.016) and the surrounding smooth muscle layer (twofold increase, p = 0.043). Additionally, acrylamide treatment caused a reduction in seminal vesicle secretion weight (36% reduction, p = 0.009) and total protein content (25% reduction, p = 0.017). Together these findings support the interpretation that toxicant exposure influences male accessory gland physiology and highlights the need to consider the response of all male reproductive tract tissues when interpreting the impact of environmental stressors on male reproductive function.

TELO2 induced progression of colorectal cancer by binding with RICTOR through mTORC2.

Colorectal cancer (CRC) is a common cancer worldwide, and its treatment strategies are limited. The underlying mechanism of CRC progression remains to be determined. Telomere maintenance 2 (TELO2) is a mTOR-interacting protein. Both the role and molecular mechanism of TELO2 in cancer progression remain unknown. In this study, the gene expression database of normal and tumor tissue, in addition to western blot analysis, and immunohistochemistry (IHC) were used to determine the expression and location of TELO2 in CRC and normal tissues. Clinical features of a tissue array were collected and analyzed. WST-1, soft agar, flow cytometry, wound healing, and invasion assays were employed to verify the role of TELO2 in the growth, cell cycle, migration, and invasion of CRC cells. The correlation between TELO2 and RICTOR (rapamycin-insensitive companion of mTOR) was analyzed by bioinformatics, IHC, and immunoprecipitation. Normal and serum-deprived cells were collected to detect the protein level of TELO2 and its downstream effectors. The results revealed that TELO2 was significantly upregulated in CRC, and TELO2 inhibition significantly restrained the growth, cell cycle, and metastasis of CRC cells. TELO2 overexpression correlated with age, lymph node metastasis, and TNM stage of CRC patients. In addition, TELO2 was positively correlated with RICTOR in CRC and induced tumor progression mainly via RICTOR with serum in culture. RICTOR induced the degradation of TELO2 upon serum deprivation in an mTOR-independent manner. These findings indicate that TELO2 promotes tumor progression via RICTOR in a serum-dependent manner, which may be a potential therapeutic target for CRC.

MTORC2 negatively controls the maturation process of medullary thymic epithelial cells by inhibiting the LTβR/RANK-NF-κB axis.

The differentiation of mature medullary thymic epithelial cells (mTECs) is critical for the induction of central immune tolerance. Although the critical effect of mechanistic target of rapamycin complex 1 (mTORC1) in shaping mTEC differentiation has been studied, the regulatory role of mTORC2 in the differentiation and maturation of mTECs is poorly understood. We herein reported that TEC-specific ablation of a rapamycin-insensitive companion of mTOR (RICTOR), a key component of mTORC2, significantly decreased the thymus size and weight, the total cell number of TECs, and the cell number of mTECs with a smallerdegree of reduced cortical thymic epithelial cells. Interestingly, RICTOR deficiency significantly accelerated the mTEC maturation process, as indicated by the increased ratios of mature mTECs (MHCIIhi , CD80+ , and Aire+ ) to immature mTECs (MHCIIlo , CD80- , and Aire- ) in Rictor-deficient mice. The RNA-sequencing assays showed that the upregulated nuclear factor-κB(NF-κB) signaling pathway in Rictor-deficient mTECs was one of the obviously altered pathways compared with wild-type mTECs. Our studies further showed that Rictor-deficient mTECs exhibited upregulated expression of receptor activator of NF-κB (RANK) and lymphotoxin β receptor (LTβR), as well as increased activity of canonical and noncanonical NF-κB signaling pathways as determined by ImageStream and Simple Western. Finally, our results showed that inhibition of NF-κB signaling pathways could partially reverse the accelerated maturation of mTECs in Rictor conditional KO mice. Thus, mTORC2 negatively controls the kinetics of the mTEC maturation process by inhibiting the LTβR/RANK-NF-κB signal axis.