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B cell development in bone marrow (BM) is a multi-staged process involving pro-B, pre-B, immature B, and mature B cells, among which pre-B cells undergo vigorous proliferation, differentiation, apoptosis, and gene rearrangement. While several signaling pathways participate in pre-B cell development have been clarified, detailed intrinsic mechanisms regulating pre-B cell proliferation and survival have not been fully understood. In the current study, we report that miR-582 regulates pre-B cell proliferation and survival. miR-582 is enriched in pre-B cells. Deletion of miR-582 in mice expanded the BM pre-B cell population in a cell-autonomous manner as shown by competitive BM transplantation. We show that forced miR-582 overexpression inhibited pre-B cell proliferation and survival, whereas downregulation of miR-582 by siRNA significantly promoted pre-B cell proliferation and survival in vitro. We identified that Hif1α and Rictor are authentic targets of miR-582 in pre-B cells as shown by reporter assays. Moreover, miR-582 overexpression reduced the expression of Hif1α and its downstream molecule Glut1, as well as Rictor and mTORC2 activity as shown by attenuated AKT and FoxO1 phosphorylation, while miR-582 knockdown showed opposite effects. miR-582 knockdown-induced increases in pre-B proliferation and survival was abrogated by Hif1α and Rictor inhibitors. Together, miR-582 functions as a negative regulator of pre-B cell proliferation and survival by simultaneously targeting Hif1α and mTORC2 signaling that regulates metabolism in early B cell development.

Non-small cell lung cancer (NSCLC) is one of the deadliest cancers in the world. Metastasis is considered one of the leading causes of treatment failure and death in NSCLC patients. A crucial factor of promoting metastasis in epithelium-derived carcinoma has been considered as epithelial-mesenchymal transition (EMT). Rictor, one of the components of mTORC2, has been reportedly involved in EMT and metastasis of human malignancies. However, the regulatory mechanisms of Rictor, Rictor-mediated EMT and metastasis in cancers remain unknown. Our present study indicates that Rictor is highly expressed in human NSCLC cell lines and tissues and is regulated, at least partially, at the transcriptional level. Knockdown of Rictor expression causes phenotype alterations through EMT, which is accompanied by the impairment of migration and invasion ability in NSCLC cells. Additionally, we have cloned and identified the human

The encoding of noxious stimuli into action potential firing is largely mediated by nociceptive free nerve endings. Tissue inflammation, by changing the intrinsic properties of the nociceptive endings, leads to nociceptive hyperexcitability and thus to the development of inflammatory pain. Here, we showed that tissue inflammation-induced activation of the mammalian target of rapamycin complex 2 (mTORC2) triggers changes in the architecture of nociceptive terminals and leads to inflammatory pain. Pharmacological activation of mTORC2 induced elongation and branching of nociceptor peripheral endings and caused long-lasting pain hypersensitivity. Conversely, nociceptor-specific deletion of the mTORC2 regulatory protein rapamycin-insensitive companion of mTOR (Rictor) prevented inflammation-induced elongation and branching of cutaneous nociceptive fibers and attenuated inflammatory pain hypersensitivity. Computational modeling demonstrated that mTORC2-mediated structural changes in the nociceptive terminal tree are sufficient to increase the excitability of nociceptors. Targeting mTORC2 using a single injection of antisense oligonucleotide against Rictor provided long-lasting alleviation of inflammatory pain hypersensitivity. Collectively, we showed that tissue inflammation-induced activation of mTORC2 causes structural plasticity of nociceptive free nerve endings in the epidermis and inflammatory hyperalgesia, representing a therapeutic target for inflammatory pain.

Purpose: To explore the effects of pelareorep on autophagy in multiple models of colorectal cancer, including patient-derived peripheral blood mononuclear cells (PBMCs). Experimental Design: HCT116 [KRAS mutant (mut)] and Hke3 [KRAS wild-type (WT)] cells were treated with pelareorep (multiplicity of infection, 5) and harvested at 6 and 9 hours. LC3 A/B expression was determined by immunofluorescence and flow cytometry; five autophagic proteins were analyzed by Western blotting. The expression of 88 autophagy genes was determined by qRT-PCR. Syngeneic mouse models, CT26/Balb-C (KRAS mut) and MC38/C57B6 (KRAS WT), were developed and treated with pelareorep (10 × 106 plaque-forming unit/day) intraperitoneally. Protein and RNA were extracted from harvested tumor tissues. PBMCs from five experimental and three control patients were sampled at 0 (pre) and 48 hours, and on days 8 and 15. The gene expression normalized to “pre” was determined using 2−ΔΔCt method. Results: Pelareorep induced significant upregulation of LC3 A/B in HCT116 as compared with Hke3 cells by immunofluorescence (3.24 × and 8.67 ×), flow cytometry (2.37 × and 2.58 ×), and autophagosome formation (2.02 × and 1.57 ×), at 6 and 9 hours, respectively; all P < 0.05. Western blot analysis showed an increase in LC3 A/B (2.38 × and 6.82 ×) and Beclin1 (1.17 × and 1.24 ×) at 6 and 9 hours, ATG5 (2.4 ×) and P-62 (1.52 ×) at 6 hours, and VPS-34 (1.39 ×) at 9 hours (all P < 0.05). Induction of 13 transcripts in cell lines (>4 ×; 6 and 9 hours; P < 0.05), 12 transcripts in CT26 (qRT-PCR), and 14 transcripts in human PBMCs (P < 0.05) was observed. LC3 A/B, RICTOR, and RASD1 expression was upregulated in all three model systems. Conclusions: Pelareorep hijacks host autophagic machinery in KRAS-mut conditions to augment its propagation and preferential oncolysis of the cancer cells.

Background Despite reports of a link between human papillomavirus (HPV) infection and mechanistic target of rapamycin (mTOR) signaling activation, the role of the mTOR pathway, especially raptor and rictor, in HPV-related head and neck cancer is still unclear. The aim of the present study was to elucidate the role of the mTOR pathway in HPV-related oropharyngeal squamous cell carcinoma (OPSCC). Methods The present study involved two strategies. The first was to investigate the activity of mTOR and mTOR-related complexes in high-risk HPV-positive (UM-SCC47 and CaSki) and HPV-negative (SCC-4 and SAS) cancer cell lines. The second was to elucidate mTOR complex expression in 80 oropharyngeal cancer tissues and to examine the relationship between mTOR complex expression and survival in patients with OPSCC. Results The UM-SCC47 and CaSki cell lines showed high gene and protein expression of raptor. They also exhibited G1/S and G2/M phase cell cycle arrest following 24 h incubation with 6 μM temsirolimus, a rapamycin analog, and temsirolimus administration inhibited their growth. HPV-related OPSCC samples showed high gene and protein expression of raptor and rictor compared with HPV-unrelated OPSCC. In addition, HPV-related OPSCC patients with high raptor and rictor expression tended to have a worse prognosis than those with low or medium expression. Conclusions These results suggest that raptor and rictor have important roles in HPV-related OPSCC and that temsirolimus is a potential therapeutic agent for patients with HPV-related OPSCC. This is the first report to reveal the overexpression of raptor and rictor in HPV-related OPSCC.

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 smaller degree 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.

Neuronal hyperactivation of the mTOR signaling pathway may play a role in driving the pathological sequelae that follow status epilepticus. Animal studies using pharmacological tools provide support for this hypothesis, however, systemic inhibition of mTOR – a growth pathway active in every mammalian cell – limits conclusions on cell type specificity. To circumvent the limitations of pharmacological approaches, we developed a viral/genetic strategy to delete Raptor or Rictor, inhibiting mTORC1 or mTORC2, respectively, from excitatory hippocampal neurons after status epilepticus in mice. Raptor or Rictor was deleted from roughly 25% of hippocampal granule cells, with variable involvement of other hippocampal neurons, after pilocarpine status epilepticus. Status epilepticus induced the expected loss of hilar neurons, sprouting of granule cell mossy fiber axons and reduced c-Fos activation. Gene deletion did not prevent these changes, although Raptor loss reduced the density of c-Fos positive granule cells overall relative to Rictor groups. Findings demonstrate that mTOR signaling can be effectively modulated with this approach and further reveal that blocking mTOR signaling in a minority (25%) of granule cells is not sufficient to alter key measures of status epilepticus-induced pathology. The approach is suitable for producing higher deletion rates, and altering the timing of deletion, which may lead to different outcomes.

Triple-negative breast cancer (TNBC), characterized by decreased expression of hormone receptors and human epidermal growth factor type 2 receptor, has poor prognosis and lacks effective therapeutics. Recently, the mTOR inhibitor rapamycin and its analogs have attracted growing interests and evaluated as therapeutic agents against TNBC, in which the PI3K/AKT/mTOR pathway is often activated. However, some TNBCs are less sensitive to these drugs. In this study, we found that the sensitivity of TNBC cells to rapamycin was highly dependent on the expression level of rapamycin-insensitive companion of mTOR (Rictor), a key component of the mTOR complex 2. Repression of the Rictor expression strongly suppressed the growth of rapamycin-insensitive tumor cells. Furthermore, we showed that the suppression of Rictor expression was also effective in rapamycin-insensitive cells that had acquired resistance to mTOR kinase inhibitors. These findings indicate that Rictor can be a predictive marker for the use of rapamycin analogs in TNBC and highlight the need to develop therapeutics targeting Rictor in the treatment of TNBC.

Obesity‐induced activation and proliferation of resident macrophages and infiltration of circulating monocytes in adipose tissues contribute to adipose tissue inflammation and insulin resistance. These effects further promote the development of metabolic syndromes, such as type 2 diabetes, which is one of the most prevalent health conditions severely threatening human health worldwide. Our study examined the potential molecular mechanism employed by fibroblast growth factor 1 (FGF1) to improve insulin sensitivity. The leptin receptor‐deficient obese mice (db/db) served as an insulin‐resistant model. Our results demonstrated that FGF1‐induced amelioration of insulin resistance in obese mice was related to the decreased levels of pro‐inflammatory adipose tissue macrophages (ATMs) and plasma inflammatory factors. We found that FGF1 enhanced the adipocyte mTORC2/Rictor signalling pathway to inhibit C‐C chemokine ligand 2 (CCL2) production, the major cause of circulating monocytes infiltration, activation and proliferation of resident macrophages in adipose tissues. Conversely, these alleviating effects of FGF1 were substantially abrogated in adipocytes with reduced expression of mTORC2/rictor. Furthermore, a model of adipocyte‐specific mTORC2/Rictor‐knockout (AdRiKO) obese mice was developed to further understand the in vitro result. Altogether, these results demonstrated adipocyte mTORC2/Rictor was a crucial target for FGF1 function on adipose tissue inflammation and insulin sensitivity.

Key points Muscle contractions increase protein synthesis in a mechanistic target of rapamycin (mTOR)-dependent manner, yet it is unclear which/how mTOR complexes regulate muscle protein synthesis. We investigated the requirement of mTOR Complex 2 (mTORC2) in contraction-stimulated muscle protein synthesis. mTORC2 inhibition by muscle-specific Rictor knockout (Rictor mKO) did not prevent contraction-induced muscle protein synthesis. Rapamycin prevented contraction-induced muscle protein synthesis in Rictor mKO but not wild-type mice. Abstract Protein synthesis increases following muscle contractions. Previous studies have shown that inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) suppresses the early but not late muscle protein synthesis response, while inhibition of both mTORC1 and mTORC2 abolishes the two effects. Therefore, we hypothesized that mTORC2 regulates muscle protein synthesis following muscle contractions. To test this, we investigated the effect of mTORC2 inhibition by mouse muscle-specific Rictor knockout (Rictor mKO) on muscle protein synthesis 3 h after contraction. The right gastrocnemius muscles of Rictor mKO and wild-type (WT) mice were isometrically contracted using percutaneous electrical stimulation, while the left gastrocnemius muscles served as controls. Vehicle or the mTORC1 inhibitor rapamycin (1.5 mg/kg) was injected intraperitoneally 1 h before contraction. Treatment of WT mice with rapamycin and Rictor mKO lowered protein synthesis in general, but the response to contractions was intact 3 h after contractions in both conditions. Rapamycin treatment in Rictor mKO mice prevented contraction-stimulated muscle protein synthesis. Notably, signalling traditionally associated with mTORC1 was increased by muscle contractions despite rapamycin treatment. In rapamycin-treated Rictor mKO mice, the same mTORC1 signalling was blocked following contractions. Our results indicate that although neither rapamycin-sensitive mTOR/mTORC1 nor mTORC2 is necessary for contraction-induced muscle protein synthesis, combined inhibition of rapamycin-sensitive mTOR/mTORC1 and mTORC2 synergistically inhibits contraction-induced muscle protein synthesis.

The role of β-estradiol (E2) in lipoprotein metabolism in mammary tumors is unclear, therefore, we investigated the effect of E2 on the secretion of lipoprotein lipase (LPL) from mouse mammary tumor FM3A cells. E2-treated cells increased the secretion of active LPL from FM3A cells in a time- and dose-dependent manner. Activity of mitogen-activated protein kinase (MAPK) was increased in the tumor cells treated with E2, and enhanced secretion of LPL was suppressed by MAPK kinase 1/2 inhibitor, PD98059, extracellular signal-regulated kinase (ERK) 1/2 inhibitor, FR180204, p38 MAPK inhibitor, SB202190, and phosphatidyl inositol 3-kinase (PI3K) inhibitor, LY294002. In addition, the effect of E2 on LPL secretion was markedly suppressed by an inhibitor of mammalian target of rapamycin complex (mTORC) 1 and 2, KU0063794, but were not by a mTORC1 inhibitor, rapamycin. Furthermore, a small interfering RNA (siRNA)-mediated decrease in the expression of rapamycin-insensitive companion of mTOR (Rictor), a pivotal component of mTORC2, suppressed secretion of LPL by E2. These results suggest that the stimulatory secretion of LPL by E2 from the tumor cells is closely associated with an activation of mTORC2 rather than mTORC1 possibly via the MAPK cascade.

Introduction. Growing evidence indicates that Rictor (Rapamycin-insensitive companion of mTOR) is overexpressed across several malignancies and associated with poor survival. However, only limited data indicate that Rictor plays a role in gastric cancer (GC). We sought to explore the prognostic value of Rictor in GC and present interaction analysis between Rictor expression and H. pylori status regarding their effects over the prognosis of GC patient. Materials and methods. 250 GC tissues and 124 lymph node metastases were collected for the detection of Rictor by immunohistochemistry. Cox regression model was used to assess the association between Rictor expression and patient prognosis. Functional experiments were examined in transfected cells using Rictor siRNA. Additive and multiplicative interactions of Rictor and H. pylori were evaluated. Results. In this study, the positive rate of Rictor was 51.6% (129/150) in GC tissues. Multivariate analyses showed that Rictor was independent unfavorable predictor for OS (HR = 1.554, 95% CI = 1.076–2.244, P = 0.019) and DFS (HR = 1.556, 95% CI = 1.081–2.240, P = 0.017). Patients with upregulated Rictor in the primary tumor and lymph node metastases had the worst prognosis. We observed significant additive and multiplicative interactions between Rictor expression and H. pylori status for OS and DFS (P < 0.05). Our in vitro experiment showed that knockdown of Rictor could suppress cell proliferation, induce apoptosis and inhibit tumor migration and invasion. Conclusion. Our results demonstrate that Rictor, acting as an oncogene, might be a potential prognostic biomarker and therapeutic target in GC. We suggest that Rictor expression and H. pylori status may be a prognostic marker in gastric cancer.

Recent characterization of spatiotemporal genomic architecture of IDH-wild-type multifocal glioblastomas (M-GBMs) suggests a clinically unobserved common-ancestor (CA) with a less aggressive phenotype, generating highly genetically divergent malignant gliomas/GBMs in distant brain regions. Using serial MRI/3D-reconstruction, whole-genome sequencing and spectral karyotyping-based single-cell phylogenetic tree building, we show two distinct types of tumor evolution in p53-mutant driven mouse models. Malignant gliomas/GBMs grow as a single mass (Type 1) and multifocal masses (Type 2), respectively, despite both exhibiting loss of Pten/chromosome 19 (chr19) and PI3K/Akt activation with sub-tetraploid/4N genomes. Analysis of early biopsied and multi-segment tumor tissues reveals no evidence of less proliferative diploid/2N lesions in Type 1 tumors. Strikingly, CA-derived relatively quiescent tumor precursors with ancestral diploid/2N genomes and normal Pten/chr19 are observed in the subventricular zone (SVZ), but are distantly segregated from multi focal Type 2 tumors. Importantly, PI3K/Akt inhibition by Rictor/mTORC2 deletion blocks distant dispersal, restricting glioma growth in the SVZ., Recent studies suggest spatial segregation of tumor initiation and manifestation in IDH-WT glioblastomas. Here, the authors use serial MRI/3D-reconstruction, whole-genome sequencing and spectral karyotyping-based single-cell phylogenetic tree building to establish this unique evolutionary mode in a murine model.

Background A high percentage of small cell lung cancer (SCLC) cases harbor cell cycle-related gene mutations and RICTOR amplification. Based on underlying somatic mutations, the authors have conducted a phase 2 biomarker-driven, multiarm umbrella study. Methods The SCLC Umbrella Korea StudiES (SUKSES) is an adaptive platform trial that undergoes continual modification according to the observed outcomes. This study included 286 patients with SCLC who failed platinum therapy and who had known genomic profiles based on a predesigned screening trial. Patients with MYC amplification or CDKN2A and TP53 co-alterations were allocated to adavosertib (SUKSES protocol C [SUKSES-C]; 7 patients) and those with RICTOR amplification were allocated to vistusertib (SUKSES-D; 4 patients). Alternatively, patients who were without any predefined biomarkers were assigned to a non-biomarker-selected arm: adavosertib (SUKSES-N1; 21 patients) or AZD2811NP (SUKSES-N3; 15 patients). Results Patients in the SUKSES-C and SUKSES-N1 arms demonstrated no objective response. Three patients presented with stable disease (SD) in SUKSES-C and 6 patients in SUKSES-N1. The median progression-free survival (PFS) was 1.3 months (95% confidence interval, 0.9 months to not available) for SUKSES-C and 1.2 months (95% CI, 1.1-1.4 months) for SUKSES-N1. Patients in the SUKSES-D arm demonstrated no objective response and no SD, with a PFS of 1.2 months (95% CI, 1.0 months to not available). The SUKSES-N3 arm had 5 patients with SD and a PFS of 1.6 months (95% CI, 0.9-1.7 months), without an objective response. Grade≥3 adverse events (graded according to National Cancer Institute Common Terminology Criteria for Adverse Events [version 4.03]) were observed as follows: 3.2% in the SUKSES-C and SUKSES-N1 arms and 50.0% in the SUKSES-D arm. Target-related neutropenia (grade≥3) was observed in approximately 60.0% of patients in the AZD2811NP arm using the current dosing schedule. Conclusions To the best of the authors' knowledge, the current study is the first biomarker-driven umbrella study conducted in patients with recurrent SCLC. Although the current study demonstrated the limited clinical efficacy of monotherapy, novel biomarker approaches using other cell cycle inhibitor(s) or combinations warrant further investigation.

Background and aims Hepatic macrophages can be activated by many factors such as gut-derived bacterial components and factors released from damaged hepatocytes. Macrophage polarization toward a proinflammatory phenotype (M1) represents an important event in the disease progression of nonalcoholic fatty liver disease (NAFLD). However, the underlying molecular mechanisms remain incompletely understood. Exosomes have been identified as important mediators for cell-cell communication by transferring various biological components such as microRNAs (miRs), proteins, and lipids. The role of exosomes in crosstalk between hepatocytes and macrophages in disease progression of NAFLD is yet to be explored. Approach and results In the present study, we reported that lipotoxic injury-induced release of hepatocyte exosomes enriched with miR-192-5p played a critical role in the activation of M1 macrophages and hepatic inflammation. Serum miR-192-5p levels in patients with NAFLD positively correlated with hepatic inflammatory activity score and disease progression. Similarly, the serum miR-192-5p level and the number of M1 macrophages, as well as the expression levels of the hepatic proinflammatory mediators, were correlated with disease progression in high-fat high-cholesterol diet-fed rat models. Lipotoxic hepatocytes released more miR-192-5p-enriched exosomes than controls, which induced M1 macrophage (cluster of differentiation 11b-positive [CD11b+ ]/CD86+ ) activation and increase of inducible nitric oxide synthase, interleukin 6, and tumor necrosis factor alpha expression. Furthermore, hepatocyte-derived exosomal miR-192-5p inhibited the protein expression of the rapamycin-insensitive companion of mammalian target of rapamycin (Rictor), which further inhibited the phosphorylation levels of Akt and forkhead box transcription factor O1 (FoxO1) and resulted in activation of FoxO1 and subsequent induction of the inflammatory response. Conclusions Hepatocyte-derived exosomal miR-192-5p plays a critical role in the activation of proinflammatory macrophages and disease progression of NAFLD through modulating Rictor/Akt/FoxO1 signaling. Serum exosomal miR-192-5p represents a potential noninvasive biomarker and therapeutic target for nonalcoholic steatohepatitis.

Background: Hepatocellular carcinoma (HCC) has high morbidity and mortality, but current therapeutic methods cannot effectively improve patient's prognosis. FOXD3-AS1, a new identified long noncoding RNA, is dysregulated in several cancers and functions as a carcinogenic or tumor-suppressor factor. However, the function of FOXD3-AS1 in HCC has not been reported. Materials and Methods: Quantitative real time-polymerase chain reaction was applied to evaluate the expression of FOXD3-AS1 in HCC tissues and cell lines. miRDB and TargetScan websites were utilized to predict the interaction network of FOXD3-AS1 as a competing endogenous RNA. The interaction was confirmed by luciferase reporter assay and RNA binding protein immunoprecipitation (RIP) assay. The effect of FOXD3-AS1 on HCC cells (Huh6) were measured by cell counting kit (CCK)-8, BrdU cell proliferation assay, Transwell invasion assay, and wound healing assay. Results: FOXD3-AS1 was overexpressed in HCC, and HCC patients with the high level of FOXD3-AS1 had a poor prognosis. In addition, FOXD3-AS1 knockdown considerably inhibited the proliferation, migration, and invasion of Huh6 cells. Besides, FOXD3-AS1 functioned as a sponge of miR-335, and RICTOR was a direct target gene of miR-335. Furthermore, FOXD3-AS1 could enhance the level of RICTOR through sponging miR-335. Moreover, the knockdown of FOXD3-AS1 could competitively bind with miR-335 to suppress RICTOR expression, thereby inhibiting the growth of Huh6 cells through the deactivation of AKT signaling pathway. Conclusions: FOXD3-AS1 is crucial for the tumorigenesis and progression of HCC. The interaction among FOXD3-AS1, miR-335, and RICTOR provides a novel insight for understanding the molecular mechanism of HCC, and FOXD3-AS1, miR-335, and RICTOR can be regarded as the potential targets for HCC treatment.

Rictor is an essential component that directly activates the mammalian target of rapamycin (mTOR) activity, which contributes to the intrinsic axon growth capacity of adult sensory neurons after injury. However, whether its action also applies to regeneration after spinal cord injury (SCI) remains unknown. In this study, rats were given spinal cord contusion at the T9-10 level to establish the SCI model and were subsequently treated with intraspinal cord injection of a Rictor overexpression lentiviral vector to locally upregulate the Rictor expression in the injured spinal cord. Thereafter, we investigated the therapeutic effects of Rictor overexpression in the injured spinal cords of SCI rats. Rictor overexpression not only significantly attenuated the acute inflammatory response and cell death after SCI but also markedly increased the shift in macrophages around the lesion from the M1 to M2 phenotype compared to those of the control lentiviral vector injection-treated group. Furthermore, Rictor overexpression dramatically increased neurogenesis in the lesion epicenter, subsequently promoting the tissue repair and functional recovery in SCI rats. Interestingly, the mechanism underlying the beneficial effects of Rictor overexpression on SCI may be associated with the Rictor overexpression playing a role in the anti-inflammatory response and driving macrophage polarization toward the M2 phenotype, which benefits resident neuronal and oligodendrocyte survival. Our findings demonstrate that Rictor is an effective target that affects the generation of molecules that inhibit spinal cord regeneration. In conclusion, localized Rictor overexpression represents a promising potential strategy for the repair of SCI.

Deciphering molecular targets to enhance sensitivity to chemotherapy is becoming a priority for effectively treating cancers. Loss of function mutations of SMAD4 in colon cancer are associated with metastatic progression and resistance to 5-fluorouracil (5-FU), the most extensively used drug of almost all chemotherapy combinations used in the treatment of metastatic colon cancer. Here, we report that SMAD4 deficiency also confers resistance to irinotecan, another common chemotherapeutic frequently used alone or in combination with 5-FU against colon cancer. Mechanistically, we find that SMAD4 interacts with and inhibits RICTOR, a component of the mTORC2 complex, resulting in suppression of downstream effector phosphorylation of AKT at Serine 473. In silico meta-analysis of publicly available gene expression datasets derived from tumors indicates that lower levels of SMAD4 or higher levels of RICTOR/AKT, irrespective of the SMAD4 status, correlate with poor survival, suggesting them as strong prognostic biomarkers and targets for therapeutic intervention. Moreover, we find that overexpression of SMAD4 or depletion of RICTOR suppresses AKT signaling and increases sensitivity to irinotecan in SMAD4-deficient colon cancer cells. Consistent with these observations, pharmacologic inhibition of AKT sensitizes SMAD4-negative colon cancer cells to irinotecan in vitro and in vivo. Overall, our study suggests that hyperactivation of the mTORC2 pathway is a therapeutic vulnerability that could be exploited to sensitize SMAD4-negative colon cancer to irinotecan. Implications: Hyperactivation of the mTORC2 pathway in SMAD4-negative colon cancer provides a mechanistic rationale for targeted inhibition of mTORC2 or AKT as a distinctive combinatorial therapeutic opportunity with chemotherapy for colon cancer.

PURPOSE 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 weeks of eccentric cycling training (ECC) which was expected to increase muscle function and VL cross-sectional area (CSA). METHODS Eleven men (23 ± 4 years) completed 24 sessions of ECC with progressive increases in intensity and duration, resulting in a 2-fold 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 days after the last training session. RESULTS 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. CONCLUSION In addition to increased muscle mass, strength, and power, we demonstrate that eccentric cycling training 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.

Monocyte migration to the sites of inflammation and maturation into macrophages are key steps for their immune effector function. Here, we show that mechanistic target of rapamycin complex 2 (mTORC2)-dependent Akt activation is instrumental for metabolic reprogramming at the early stages of macrophage-mediated immunity. Despite an increased production of proinflammatory mediators, monocytes lacking expression of the mTORC2 component Rictor fail to efficiently migrate to inflammatory sites and fully mature into macrophages, resulting in reduced inflammatory responses in vivo. The mTORC2-dependent phosphorylation of Akt is instrumental for the enhancement of glycolysis and mitochondrial respiration, required to sustain monocyte maturation and motility. These observations are discussed in the context of therapeutic strategies aimed at selective inhibition of mTORC2 activity.

Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2-interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain-of-function and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase.

Hepatocellular carcinoma (HCC) is the global leading cause of cancer-related deaths due to the deficiency of targets for precision therapy. A new modality of epigenetic regulation has emerged involving RNA–RNA crosstalk networks where two or more competing endogenous RNAs (ceRNAs) bind to the same microRNAs. However, the contribution of such mechanisms in HCC has not been well studied. Herein, potential HMGB1-driven RNA–RNA crosstalk networks were evaluated at different HCC stages, identifying the mTORC2 component RICTOR as a potential HMGB1 ceRNA in HBV+ early stage HCC. Indeed, elevated HMGB1 mRNA was found to promote the expression of RICTOR mRNA through competitively binding with the miR-200 family, especially miR-429. Functional assays employing overexpression or interference strategies demonstrated that the HMGB1 and RICTOR 3′untranslated regions (UTR) epigenetically promoted the malignant proliferation, self-renewal, and tumorigenesis in HCC cells. Intriguingly, interference against HMGB1 and RICTOR in HCC cells promoted a stronger anti-PD-L1 immunotherapy response, which appeared to associate with the production of PD-L1+ exosomes. Mechanistically, the HMGB1-driven RNA-RNA crosstalk network facilitated HCC cell glutamine metabolism via dual mechanisms, activating a positive feedback loop involving mTORC2-AKT-C-MYC to upregulate glutamine synthetase (GS) expression, and inducing mTORC1 signaling to derepress SIRT4 on glutamate dehydrogenase (GDH). Meanwhile, this crosstalk network could impede the efficacy of immunotherapy through mTORC1-P70S6K dependent PD-L1 production and PD-L1+ exosomes activity. In conclusion, our study highlights the non-coding regulatory role of HMGB1 with implications for RNA-based therapeutic targeting together with a prediction of anti-PD-L1 immunotherapy in HCC.

Long term glucagon receptor (GCGR) agonism is associated with hyperglycemia and glucose intolerance, while acute GCGR agonism enhances whole-body insulin sensitivity and hepatic AKTSer473 phosphorylation. These divergent effects establish a critical gap in knowledge surrounding GCGR action. mTOR Complex 2 (mTORC2) is composed of seven proteins, including RICTOR, which dictates substrate binding and allows for targeting of AKTSer473. We utilized a liver specific Rictor knockout mouse (RictorΔLiver) to investigate whether mTORC2 is necessary for insulin-receptor (INSR) and GCGR crosstalk. RictorΔLiver mice were characterized by impaired Akt signaling and glucose intolerance. Intriguingly, RictorΔLiver mice were also resistant to GCGR-stimulated hyperglycemia. Consistent with our prior report, GCGR agonism increased glucose infusion rate and suppressed hepatic glucose production during hyperinsulinemic-euglycemic clamp of control animals. However, these benefits to insulin sensitivity were ablated in RictorΔLiver mice. We observed diminished AKTSer473 and GSK3α/βSer21/9 phosphorylation in RictorΔLiver mice, whereas phosphorylation of AKTThr308 was unaltered in livers from clamped mice. These signaling effects were replicated in primary hepatocytes isolated from RictorΔLiver and littermate control mice, confirming cell autonomous crosstalk between GCGR and INSR pathways. In summary, our study reveals the necessity of RICTOR, and thus mTORC2, in GCGR-mediated enhancement of liver and whole-body insulin action.

International audience; The hyperfiltration theory has been used to explain the mechanism of low birth weight (LBW)-related nephropathy. However, the molecular changes in the kidney proteome have not been defined in this disease, and early biomarkers are lacking. We investigated the molecular pathogen-esis of LBW rats obtained by intraperitoneal injection of dexamethasone into pregnant animals. Nor-mal-birth-weight (NBW) rats were used as controls. When the rats were four weeks old, the left kidneys were removed and used for comprehensive label-free proteomic studies. Following uninephrectomy, all rats were fed a high-salt diet until 9 weeks of age. Differences in the molecular composition of the kidney cortex were observed at the early step of LBW nephropathy pathogenesis. Untargeted quantitative proteomics showed that proteins involved in energy metabolism, such as oxidative phosphorylation (OXPHOS), the TCA cycle, and glycolysis, were specifically downregu-lated in the kidneys of LBW rats at four weeks. No pathological changes were detected at this early stage. Pathway analysis identified NEFL2 (NRF2) and RICTOR as potential upstream regulators. The search for biomarkers identified components of the mitochondrial respiratory chain, namely, ubiquinol-cytochrome c reductase complex subunits (UQCR7/11) and ATP5I/L, two components of mitochondrial F1FO-ATP synthase. These findings were further validated by immunohistology. At later stages of the disease process, the right kidneys revealed an increased frequency of focal segmental glomerulosclerosis lesions, interstitial fibrosis and tubular atrophy. Our findings revealed proteome changes in LBW rat kidneys and revealed a strong downregulation of specific mitochon-drial respiratory chain proteins, such as UQCR7.

BACKGROUND/AIM Rictor is an adaptor protein essential for mTORC2, which regulates cell growth and survival. The aim of this study was to identify microRNAs (miR) that down-regulate Rictor and investigate their function on breast cancer cell survival. MATERIALS AND METHODS Trypan blue assay, MTT assay, polymerase chain reaction analysis, luciferase reporter assay and western blot analysis were carried out in breast cancer cell lines HCC1954, MDA-MB-231, SK-BR-3, and BT474. RESULTS miR-3188 overexpression suppressed the expression of Rictor and inhibited cell viability in HCC1954 and MDA-MB-231, highly Rictor-expressing breast cancer cells. In addition, miR-3188 overexpression decreased the protein level of p-AKT at Ser473, a substrate of mTORC2. Moreover, miRNA-3188 overexpression sensitized breast cancer cells to ionizing radiation (IR) by down-regulating Rictor and p-AKT. CONCLUSION miR-3188 enhances IR sensitivity by affecting the mTORC2/AKT signalling pathway by altering the expression of Rictor, which could be a promising therapeutic strategy for the future treatment of breast cancer.

Glucocorticoids (GC) are the mainstay treatment option for inflammatory conditions. Despite the broad usage of GC, the mechanisms by which GC exerts its effects remain elusive. Here, utilizing murine autoimmune and allergic inflammation models, we report that Foxp3

Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the related mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously; however, important questions remain, particularly regarding the structural determinants mediating substrate specificity and context-dependent activity. Here, we used cryo-EM to obtain high-resolution structures of the human mTORC2 apo-complex in the presence of substrates Akt and SGK1. Using functional assays, we then tested predictions suggested by substrate-induced structural changes in mTORC2. For the first time, we visualized in the apo-state the side chain interactions between Rictor and mTOR that sterically occlude recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin. Also in the apo-state, we observed that mSin1 formed extensive contacts with Rictor via a pair of short α-helices nestled between two Rictor helical repeat clusters, as well as by an extended strand that makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, we found that SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1 residue Arg-83, which then interacts with a patch of negatively charged residues within Rictor. Finally, we demonstrate mutation of Arg-83 to Ala selectively disrupts mTORC2-dependent phosphorylation of SGK1, but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activity.

Yun-Yun Yang,1 Wei Zhang,2 Hui Liu,2 Jun-Jie Jiang,2 Wen-Jie Wang,3 Zheng-Yan Jia4 1Outpatient Comprehensive Treatment, Cangzhou Central Hospital, Cangzhou, Hebei Province, People’s Republic of China; 2Department of Thyroid and Breast I, Cangzhou Central Hospital, Cangzhou, Hebei Province, People’s Republic of China; 3Department of General Surgery, Botou Hospital, Cangzhou, Hebei Province, People’s Republic of China; 4Department of General Surgery, Qingxian People’s Hospital, Cangzhou, Hebei Province, People’s Republic of ChinaCorrespondence: Wei ZhangDepartment of Thyroid and Breast I, Cangzhou Central Hospital, Cangzhou, Hebei Province, People’s Republic of ChinaEmail weiesiz685125@163.comIntroduction: Breast cancer is a malignant tumor that seriously threatens women’s life and health.Methods: In this study, we proposed to use graphene nanoparticles loaded with siRNA that can silence Rictor molecules essential for the mammalian target of rapamycin (mTOR) complex 2 (mTORC2) complex to enhance gene delivery to tumor cells through modification of cell-penetrating peptide (CPP) for the treatment of breast cancer.Results: Remarkably, we successfully synthesized graphene oxide (GO)/polyethyleneimine (PEI)/polyethylene glycol (PEG)/CPP/small interfering RNA (siRNA) system, and the results were observed by atomic force microscopy (AFM) and ultraviolet visible (UV-Vis) absorption spectra. The optimum mass ratio of siRNA to GO-PEI-PEG-CPP was 1:0.5. We screened out Rictor siRNA-2 from 9 candidates, which presented the highest inhibition rate, and this siRNA was selected for the subsequent experiments. We validated that Rictor siRNA-2 significantly reduced the Rictor expression in triple negative breast cancer (TNBC) cells. Confocal fluorescence microscope and flow cytometry analysis showed that GO-PEI-PEG-CPP/siRNA was able to be effectively uptake by TNBC cells. GO-PEI-PEG-CPP/siRNA improved the effect of siRNA on the inhibition of TNBC cell viability and the induction of TNBC cell apoptosis. The expression of Rictor and the phosphorylation of Akt and p70s6k were inhibited by GO-PEI-PEG-CPP/siRNA. Tumorigenicity analysis in nude mice showed that GO-PEI-PEG-CPP/siRNA significantly repressed the tumor growth of TNBC cells in vivo. The levels of ki-67 were repressed by GO-PEI-PEG-CPP/siRNA, and the apoptosis was induced by GO-PEI-PEG-CPP/siRNA in the system.Discussion: Therefore, we concluded that CPP-modified GO nanoparticles loaded with Rictor siRNA significantly repressed TNBC progression by the inhibition of PI3K/Akt/mTOR signaling. Our finding provides a promising therapeutic strategy for the treatment of TNBC.Keywords: triple-negative breast cancer, graphene oxide, cell-penetrating peptide, Rictor, PI3K/Akt/mTOR signaling

Innervation sustains cornea integrity. Pigment epithelium‐derived factor (PEDF) plus docosahexaenoic acid (DHA) regenerated damaged nerves by stimulating the synthesis of a new stereoisomer of Resolvin D6 (RvD6si). Here, we resolved the structure of this lipid isolated from mouse tears after injured corneas were treated with PEDF + DHA. RvD6si synthesis was inhibited by fluvoxamine, a cytochrome P450 inhibitor, but not by 15‐ or 5‐LOX inhibitors, suggesting that the 4‐ and 17‐hydroxy of DHA have an R R‐ or S R‐configuration. The two compounds were chemically synthesized. Using chiral phase HPLC, four peaks of RvD6si1‐4 from tears were resolved. The R R‐RvD6 standard eluted as a single peak with RvD61 while pure S R‐RvD6 eluted with RvD63. The addition of these pure mediators prompted a trigeminal ganglion transcriptome response in injured corneas and showed that R R‐RvD6 was the more potent, increasing cornea sensitivity and nerve regeneration. R R‐RvD6 stimulates Rictor and hepatocyte growth factor (hgf) genes specifically as upstream regulators and a gene network involved in axon growth and suppression of neuropathic pain, indicating a novel function of this lipid mediator to maintain cornea integrity and homeostasis after injury.

Cardioprotective medications are still unmet clinical needs. We have previously identified several cardioprotective microRNAs (termed ProtectomiRs), the mRNA targets of which may reveal new drug targets for cardioprotection. Here we aimed to identify key molecular targets of ProtectomiRs and confirm their association with cardioprotection in a translational pig model of acute myocardial infarction (AMI). By using a network theoretical approach, we identified 882 potential target genes of 18 previously identified protectomiRs. The Rictor gene was the most central and it was ranked first in the protectomiR-target mRNA molecular network with the highest node degree of 5. Therefore, Rictor and its targeting microRNAs were further validated in heart samples obtained from a translational pig model of AMI and cardioprotection induced by pre- or postconditioning. Three out of five Rictor-targeting pig homologue of rat ProtectomiRs showed significant upregulation in postconditioned but not in preconditioned pig hearts. Rictor was downregulated at the mRNA and protein level in ischemic postconditioning but not in ischemic preconditioning. This is the first demonstration that Rictor is the central molecular target of ProtectomiRs and that decreased Rictor expression may regulate ischemic postconditioning-, but not preconditioning-induced acute cardioprotection. We conclude that Rictor is a potential novel drug target for acute cardioprotection.

Successful embryo implantation requires well-functioning endometrial luminal epithelial cells to establish uterine receptivity. Inadequate uterine receptivity is responsible for approximately two thirds of implantation failures in humans. However, the regulatory mechanism governing this functional process remains largely unexplored. A previous study revealed that the expression of Rictor, the main member of mTORC2, in mouse epithelial cells is increased on the fourth day of gestation (D4). Here, we provide the first report of the involvement of Rictor in the regulation of endometrial receptivity. Rictor was conditionally ablated in the mouse endometrium using a progesterone receptor cre (PRcre ) mouse model. Loss of Rictor altered polarity remodeling and the Na+ channel protein of endometrial cells by mediating Rac-1/PAK1(pPAK1)/ERM(pERM) and Sgk1/pSgk1 signaling, respectively, ultimately resulting in impaired fertility. In the endometrium of women with infertility, the expression of Rictor was changed, along with the morphological transformation and Na+ channel protein of epithelial cells. Our findings demonstrate that Rictor is crucial for the establishment of uterine receptivity in both mice and humans. The present study may help improve the molecular regulatory network of endometrial receptivity and provide new diagnostic and treatment strategies for infertility.

Tumor necrosis factor receptor-associated factors (TRAFs) are crucial for receptor activator of nuclear factor-κB (RANK) activation in osteoclasts. However, the upstream mechanisms of TRAF members in the osteoclastic lineage remain largely unknown. Here, we demonstrated that Rictor, a key component of mechanistic target of rapamycin complex 2 (mTORC2), was crucial for TRAF6/TRAF3 expression in osteoclasts. Our ex vivo and in vivo studies showed that Rictor ablation from the osteoclastic lineage reduced osteoclast numbers and increased bone mass in mice. Mechanistically, we found that Rictor ablation restricted osteoclast formation, which disrupted TRAF6 stability and caused autophagy block in a manner distinct from mTORC1, resulting in reduced TRAF3 degradation. Boosting TRAF6 expression or knockdown of TRAF3 levels in Rictor-deficient cells could both overcome the defect. Moreover, Rictor could interact with TRAF6 upon RANK ligand (RANKL) stimulation and loss of Rictor impaired TRAF6 stability and promoted its ubiquitinated degradation. These findings established an innovative link between Rictor, TRAF protein levels, and autophagic block. More importantly, mTOR complexes in the osteoclastic lineage are likely switches for coordinating TRAF6 and TRAF3 protein levels, and Rictor may function as an essential upstream regulator of TRAF6/TRAF3 that is partially independent of mTORC1 activity. Inhibitors targeting Rictor may therefore be valuable for preventing or treating osteoclast-related diseases. © 2021 American Society for Bone and Mineral Research (ASBMR).

Small cell lung carcinoma (SCLC) accounts for approximately 15% of all lung cancers and remains a challenging disease, with no significant improvement in the field of targeted therapies. The RICTOR gene (rapamycin-insensitive companion of mTOR [mammalian target of rapamycin]), which encodes a key structural (scaffold) protein of mTOR complex 2), has recently been identified as one of the most frequently amplified genes and a potential therapeutic target in SCLC. The aim of this study was to compare immunohistochemical (IHC) expression of Rictor and phospho-Akt (a downstream target of mTOR complex 2) with RICTOR amplification as detected by fluorescence in situ hybridization (FISH) in SCLC. RICTOR FISH and Rictor and phospho-Akt IHC staining were performed on 100 formalin-fixed, paraffin-embedded SCLC samples. RICTOR amplification was detected in 15 samples (15%). IHC positivity for Rictor and phospho-Akt was observed in 37 (37%) and 42 (42%) samples, respectively. Considering FISH as the diagnostic standard, the sensitivity and specificity of Rictor IHC were 93% and 73%, whereas the sensitivity and specificity of phospho-Akt IHC were 80% and 65%, respectively. Rictor expression was higher in distant metastases than in primary tumor samples and lymph node metastases. There was no association between RICTOR amplification and clinical outcome. However, high expression of either Rictor or phospho-Akt was associated with significantly decreased overall survival. In conclusion, IHC expression of Rictor correlates highly with RICTOR amplification. Therefore, Rictor IHC can be used as a cost-effective method to select patients for RICTOR FISH and, potentially, for mTORC1/2 inhibitor therapy.

The mTOR complex 2 (mTORC2) is recognized as a promising target for breast cancer treatment. As mTORC2-specific inhibitors do not yet exist, studies into the role of mTORC2 in cancer are performed by deleting Rictor or by RNAi-mediated Rictor silencing. The purpose of this study was to explore the effects of Rictor ablation in bone mesenchymal stromal cells (BMSCs) on bone metastasis of breast cancer. First, female mice with the genotype of Prx1-Cre;Rictorf/f (hereafter RiCKO) or Rictorf/f (as control) were injected intratibially with cells of the breast cancer cell line (TM40D) at 4 months of age. Three weeks later, osteolytic bone destruction was detected in metastatic legs by X-ray and micro-CT. We found that Rictor ablation in BMSCs inhibited TM40D-induced osteolytic bone destruction and resulted in greater bone volume maintenance in vivo. Lower CTX-I serum level, a decreased number of TRAP+ osteoclasts and lower Cathepsin-K expression observed at the tumor-bone interface indicated that osteoclastogenesis was inhibited in RiCKO mice. Additionally, co-culture experiments confirmed that Rictor deletion in BMSCs diminished osteoclast differentiation partly via down regulation of RANKL expression. Furthermore, Rictor deficiency was found to reduce the transition of BMSCs to CAFs coupled with decreased secretion of cytokines (IL-6, RANKL, TGFβ), which resulted in lower chemotaxis and less proliferation in TM40D cells. These results suggest that Rictor ablation in BMSCs plays dual roles in breast cancer bone metastasis: (1) repression of osteolytic bone destruction; (2) inhibition of tumor growth.