mTORC1/S6K1 signaling promotes sustained oncogenic translation through modulating CRL3IBTK-mediated ubiquitination of eIF4A1 in cancer cells.

Enhanced protein synthesis is a crucial molecular mechanism that allows cancer cells to survive, proliferate, metastasize, and develop resistance to anti-cancer treatments, and often arises as a consequence of increased signaling flux channeled to mRNA-bearing eukaryotic initiation factor 4F (eIF4F). However, the post-translational regulation of eIF4A1, an ATP-dependent RNA helicase and subunit of the eIF4F complex, is still poorly understood. Here, we demonstrate that IBTK, a substrate-binding adaptor of the Cullin 3-RING ubiquitin ligase (CRL3) complex, interacts with eIF4A1. The non-degradative ubiquitination of eIF4A1 catalyzed by the CRL3IBTK complex promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and cervical tumor cell growth both in vivo and in vitro. Moreover, we show that mTORC1 and S6K1, two key regulators of protein synthesis, directly phosphorylate IBTK to augment eIF4A1 ubiquitination and sustained oncogenic translation. This link between the CRL3IBTK complex and the mTORC1/S6K1 signaling pathway, which is frequently dysregulated in cancer, represents a promising target for anti-cancer therapies.

YANK2 activated by Fyn promotes glioma tumorigenesis via the mTOR-independent p70S6K activation pathway.

Glioma, particularly glioblastomas (GBM), is incurable brain tumor. The most targeted receptor tyrosine kinase (RTKs) drugs did not bring benefit to GBM patients. The mechanism of glioma growth continues to be explored to find more effective treatment. Here, we reported that Ser/Thr protein kinase YANK2 (yet another kinase 2) is upregulated in glioma tissues and promotes the growth and proliferation of glioma in vitro and in vivo. Further, we confirmed that oncogene Fyn directly activated YANK2 through phosphorylation its Y110, and Fyn-mediated YANK2 phosphorylation at Y110 site promotes glioma growth by increasing its stability. Finally, YANK2 was proved to be a novel upstream kinase of p70S6K and promotes glioma growth by directly phosphorylating p70S6K at T389. Taken together, we found a new mTOR-independent p70S6K activation pathway, Fyn-YANK2-p70S6K, which promotes glioma growth, and YANK2 is a potential oncogene and serves as a novel therapeutic target for glioma.

LOXL3 Inhibits Autophagy of Chondrocytes by Activating Rheb in Osteoarthritis.

OBJECTIVE: This study aimed to investigate the potential mechanisms by which lysyl oxidase like 3 (LOXL3) affects the autophagy in chondrocytes in osteoarthritis (OA), specifically through the activation of mammalian target of rapamycin complex 1 (mTORC1). METHODS: To establish an OA model, rats underwent anterior cruciate ligament transection (ACLT). Chondrocytes were isolated from cartilage tissues and cultured. Western blotting was performed to assess the expression of LOXL3, Rheb, phosphorylation of p70S6K (p-p70S6K, a downstream marker of mTORC1), and autophagy markers. The autophagy of chondrocytes was observed using an immunofluorescence assay. RESULTS: The expression levels of both LOXL3 and Rheb proteins were upregulated in chondrocytes isolated from the OA model cartilage, in comparison to those from the normal cartilage. The silencing of LOXL3 resulted in a decrease in the protein levels of Rheb and p-p70S6K, as well as an increase in the expression of autophagy-related proteins. Additionally, the effect of LOXL3 could be reversed through the silencing of Rheb. The results of the immunofluorescence assay confirmed the impact of LOXL3 and Rheb on chondrocyte autophagy. CONCLUSION: LOXL3 inhibits chondrocyte autophagy by activating the Rheb and mTORC1 signaling pathways.

EIF4A3 Acts on the PI3K-AKT-ERK1/2-P70S6K Pathway through FLOT1 to Influence the Development of Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) is a major lung cancer subtype. In this study, we discovered that the eukaryotic translation initiation factor EIF4A3 expression was significantly higher in LUAD tissues and that this higher expression was closely linked to a poor prognosis for LUAD. In addition, we demonstrated that the knockdown of EIF4A3 significantly inhibited the proliferation, invasion, and migration of LUAD cells in vitro and in vivo. The findings of mass spectrometry analysis revealed that EIF4A3 could interact with Flotillin-1 in LUAD cells and that EIF4A3 could positively regulate the expression of FLOT1 at the protein level. Meanwhile, transcriptome sequencing showed that EIF4A3 could influence the development of LUAD by affecting PI3K-AKT-ERK1/2-P70S6K and PI3K class III-mediated autophagy in the Apelin pathway. In addition, we confirmed that Flotillin-1 expression was upregulated in LUAD based on the existing literature, and knockdown of FLOT1 could inhibit the proliferation and migration of LUAD cells. In addition, the knockdown of Flotillin-1 reversed the increase of cell proliferation and migration caused by EIF4A3 overexpression. Furthermore, we found that the activation of PI3K-AKT-ERK1/2-P70S6K signaling pathway and PI3K class III-mediated autophagy caused by EIF4A3 overexpression was rescued by the knockdown of FLOT1. In a word, we proved that EIF4A3 positively regulates the expression of FLOT1 and plays a procancer role in LUAD. IMPLICATIONS: Our study revealed the role of EIF4A3 in prognosis and tumor progression in LUAD, indicating that EIF4A3 could be used as the molecular diagnostic and prognostic therapeutic target.

AMPK/mTOR/p70S6K axis prevents apoptosis of Porphyromonas gingivalis-infected gingival epithelial cells via BadSer136 phosphorylation.

Epithelial disruption is the initiation of most infectious disease. Regulation of epithelium apoptosis may play a key role in balance the survival competition between resident bacteria and host cells. The role of the mTOR/p70S6K pathway in preventing apoptosis of human gingival epithelial cells (hGECs) infected with Porphyromonas gingivalis (Pg) was investigated in order to further understand the survival strategy of the epithelial cells in during Pg infecting. hGECs was challenged with Pg for 4, 12, and 24 h. Additionally, hGECs was pretreated with LY294002 (PI3K signaling inhibitor) or Compound C (AMPK inhibitor) for 12 h and exposed them to Pg for 24 h. Subsequently, apoptosis was detected using flow cytometry, and expression and activity of Bcl-2, Bad, Bax, PI3K, AKT, AMPK, mTOR, and p70S6K proteins were analyzed using western blotting. Pg-infecting did not increase apoptosis of hGECs; but the expression ratio of Bad to Bcl-2 was increased after infecting. In contrast, BadSer136 phosphorylation was promoted, accompanied by a significant reduction of mTOR/p70S6K and PI3K/AKT signaling, along with the upregulation of AMPKThr172 signaling. Morrover, the PI3K inhibitor LY294002 promoted Pg-mediated reduction of mTOR/p70S6K expression, and the increase of AMPK signaling and BadSer136 phosphorylation rate, eventually decreasing apoptosis. While Compound C inhibited Pg-mediated activation of AMPK and downregulation of mTOR/p70S6K signaling, significantly reduced the BadSer136 phosphorylation rate, thereby increasing apoptosis. Thus, hGECs prevent apoptosis via an inherent cellular-homeostasis, pro-survival mechanism during Pg infection, the AMPK/mTOR/p70S6K pathway helps prevent apoptosis in hGECs infected with Pg by regulating BadSer136 phosphorylation.

Neuregulin-1/ErbB4 upregulates acetylcholine receptors via Akt/mTOR/p70S6K: a study in a rat model of obstetric brachial plexus palsy and in vitro.

In obstetric brachial plexus palsy (OBPP), the operative time window for nerve reconstruction of the intrinsic muscles of the hand (IMH) is much shorter than that of biceps. The reason is that the atrophy of IMH becomes irreversible more quickly than that of biceps. A previous study confirmed that the motor endplates of denervated intrinsic muscles of the forepaw (IMF) were destabilized, while those of denervated biceps remained intact. However, the specific molecular mechanism of regulating the self-repair of motor endplates is still unknown. In this study, we use a rat model of OBPP with right C5-C6 rupture plus C7-C8-T1 avulsion and left side as a control. Bilateral IMF and biceps are harvested at 5 weeks postinjury to assess relative protein and mRNA expression. We also use L6 skeletal myoblasts to verify the effects of signaling pathways regulating acetylcholine receptor (AChR) protein synthesis in vitro. The results show that in the OBPP rat model, the protein and mRNA expression levels of NRG-1/ErbB4 and phosphorylation of Akt/mTOR/p70S6K are lower in denervated IMF than in denervated biceps. In L6 myoblasts stimulated with NRG-1, overexpression and knockdown of ErbB4 lead to upregulation and downregulation of AChR subunit protein synthesis and Akt/mTOR/p70S6K phosphorylation, respectively. Inhibition of mTOR abolishes protein synthesis of AChR subunits elevated by NRG-1/ErbB4. Our findings suggest that in the OBPP rat model, lower expression of AChR subunits in the motor endplates of denervated IMF is associated with downregulation of NRG-1/ErbB4 and phosphorylation of Akt/mTOR/p70S6K. NRG-1/ErbB4 can promote protein synthesis of the AChR subunits in L6 myoblasts via phosphorylation of Akt/mTOR/p70S6K.

S6K1 phosphorylates Cdk1 and MSH6 to regulate DNA repair.

The mTORC1 substrate, S6 Kinase 1 (S6K1), is involved in the regulation of cell growth, ribosome biogenesis, glucose homeostasis, and adipogenesis. Accumulating evidence has suggested a role for mTORC1 signaling in the DNA damage response. This is mostly based on the findings that mTORC1 inhibitors sensitized cells to DNA damage. However, a direct role of the mTORC1-S6K1 signaling pathway in DNA repair and the mechanism by which this signaling pathway regulates DNA repair is unknown. In this study, we discovered a novel role for S6K1 in regulating DNA repair through the coordinated regulation of the cell cycle, homologous recombination (HR) DNA repair (HRR) and mismatch DNA repair (MMR) mechanisms. Here, we show that S6K1 orchestrates DNA repair by phosphorylation of Cdk1 at serine 39, causing G2/M cell cycle arrest enabling homologous recombination and by phosphorylation of MSH6 at serine 309, enhancing MMR. Moreover, breast cancer cells harboring RPS6KB1 gene amplification show increased resistance to several DNA damaging agents and S6K1 expression is associated with poor survival of breast cancer patients treated with chemotherapy. Our findings reveal an unexpected function of S6K1 in the DNA repair pathway, serving as a tumorigenic barrier by safeguarding genomic stability.

Damage to the DNA in our cells can cause harmful changes that, if unchecked, can lead to the development of cancer. To help prevent this, cellular mechanisms are in place to repair defects in the DNA. A particular process, known as the mTORC1-S6K1 pathway is suspected to be important for repair because when this pathway is blocked, cells become more sensitive to DNA damage. It is still unknown how the various proteins involved in the mTORC1-S6K1 pathway contribute to repairing DNA. One of these proteins, S6K1, is an enzyme involved in coordinating cell growth and survival. The tumor cells in some forms of breast cancer produce more of this protein than normal, suggesting that S6K1 benefits these cells' survival. However, it is unclear exactly how the enzyme does this. Amar-Schwartz, Ben-Hur, Jbara et al. studied the role of S6K1 using genetically manipulated mouse cells and human cancer cells. These experiments showed that the protein interacts with two other proteins involved in DNA repair and activates them, regulating two different repair mechanisms and protecting cells against damage. These results might explain why some breast cancer tumors are resistant to radiotherapy and chemotherapy treatments, which aim to kill tumor cells by damaging their DNA. If this is the case, these findings could help clinicians choose more effective treatment options for people with cancers that produce additional S6K1. In the future, drugs that block the activity of the enzyme could make cancer cells more susceptible to chemotherapy.

[Effect of moxibustion on autophagy in mice with Alzheimer's disease based on mTOR/p70S6K signaling pathway].

OBJECTIVE: To investigate the effect of moxibustion on autophagy and amyloid ß-peptide1-42 (Aß1-42) protein expression in amyloid precursor protein/presenilin 1 (APP/PS1) double-transgenic mice with Alzheimer's disease (AD). METHODS: After 2-month adaptive feeding, fifty-six 6-month-old APP/PS1 double transgenic AD mice were randomly divided into a model group, a moxibustion group, a rapamycin group and an inhibitor group, 14 mice in each group. Another 14 C57BL/6J mice with the same age were used as a normal group. The mice in the moxibustion group were treated with monkshood cake-separated moxibustion at "Baihui"(GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) for 20 min; the mice in the rapamycin group were intraperitoneally injected with rapamycin (2 mg/kg); the mice in the inhibitor group were treated with moxibustion and injection of 1.5 mg/kg 3-methyladenine (3-MA). All the treatments were given once a day for consecutive 2 weeks. The morphology of hippocampal tissue was observed by HE staining; the ultrastructure of hippocampal tissue was observed by transmission electron microscopy; the expression of Aß1-42 protein in frontal cortex and hippocampal tissue was detected by immunohistochemistry; the expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), p70 ribosomal protein S6 kinase (p70S6K) and phosphorylated p70S6K (p-p70S6K) protein in hippocampus were detected by Western blot method. RESULTS: Compared with the normal group, the number of neuron cells was decreased, cells were necrotic and deformed, and autophagy vesicle and lysosome were decreased in the model group. Compared with the model group, the number of neuron cells was increased, cell necrosis was decreased, and autophagy vesicle and lysosome were increased in the moxibustion group and the rapamycin group. Compared with the normal group, the protein expressions of Aß1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the model group were increased (P<0.05); compared with the model group, the protein expressions of Aß1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group, rapamycin group and inhibitor group were decreased (P<0.05); compared with the inhibitor group, the protein expressions of Aß1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group and rapamycin group were decreased (P<0.05); compared with the rapamycin group, the protein expressions of mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group were decreased (P<0.05). CONCLUSION: Moxibustion could enhance autophagy in hippocampal tissue of APP/PS1 double transgenic AD mice and reduce abnormal Aß aggregation in brain tissue, the mechanism may be related to the inhibition of mTOR/p70S6K signaling pathway.

Upregulation of KIF18B facilitates malignant phenotype of esophageal squamous cell carcinoma by activating CDCA8/mTORC1 pathway.

BACKGROUND: Kinesin family member 18B (KIF18B) has been regarded as an oncogene that is abnormally overexpressed in some cancers, but its mechanism in esophageal squamous cell carcinoma (ESCC) remains unclear, which is thereby investigated in this study. METHODS: Bioinformatics analysis was performed to analyze the expression of KIF18B in esophageal carcinoma (ESCA). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect KIF18B expression in ESCC cells. After KIF18B overexpression or cell division cycle associated 8 (CDCA8) deficiency, ESCC cells were subjected to determination of qRT-PCR, Western blot, cell counting kit-8 assay, flow cytometry, wound healing, and Transwell assay. The mechanism of KIF18B in the mechanistic target of rapamycin complex 1 (mTORC1) pathway was detected by Western blot. RESULTS: KIF18B was overexpressed in ESCA samples and ESCC cells. Upregulation of KIF18B enhanced the viability, accelerated cell cycle by elevating CDK4 and Cyclin D3 levels as well as promoted the migration and invasion by decreasing E-cadherin level and increasing Vimentin and N-cadherin levels in ESCC cells, which was counteracted by CDCA8 silencing. The expression of CDCA8 in ESCC cells was upregulated by KIF18B overexpression. KIF18B overexpression activated the mTORC1 pathway by upregulating phosphorylated (p)-/p70S6K and p-/mTOR levels in the ESCC cells, which was reversed by CDCA8 silencing. CONCLUSION: KIF18B overexpression promotes the proliferation, migration, and invasion of ESCC cells via CDCA8-mediated mTORC1 signaling pathway in vitro.

[Effect of eIF4B knockout on apoptosis of mouse fetal liver cells].

Eukaryotic translation initiation factor 4B (eIF4B) plays an important role in mRNA translation initiation, cell survival and proliferation in vitro, but the in vivo function is poorly understood. In this study, via various experimental techniques such as hematoxylin-eosin (HE) staining, flow cytometry, Western blotting, and immunohistochemistry, we investigated the role of eIF4B in mouse embryo development using an eIF4B knockout (KO) mouse model and explored the mechanism. We found that the livers, but not lungs, brain, stomach, or pancreas, derived from eIF4B KO mouse embryos displayed severe pathological changes characterized by enhanced apoptosis and necrosis. Accordingly, high expression of cleaved-caspase 3, and excessive activation of mTOR signaling as evidenced by increased expression and phosphorylation of p70S6K and enhanced phosphorylation of 4EBP1, were observed in mouse embryonic fibroblasts and fetal livers from eIF4B KO mice. These results uncover a critical role of eIF4B in mouse embryo development and provide important insights into the biological functions of eIF4B in vivo.

S6K1 amplification confers innate resistance to CDK4/6 inhibitors through activating c-Myc pathway in patients with estrogen receptor-positive breast cancer.

BACKGROUND: CDK4/6 inhibitors combined with endocrine therapy has become the preferred treatment approach for patients with estrogen receptor-positive metastatic breast cancer. However, the predictive biomarkers and mechanisms of innate resistance to CDK4/6 inhibitors remain largely unknown. We sought to elucidate the molecular hallmarks and therapeutically actionable features of patients with resistance to CDK4/6 inhibitors. METHODS: A total of 36 patients received palbociclib and endocrine therapy were included in this study as the discovery cohort. Next-generation sequencing of circulating tumour DNA in these patients was performed to evaluate somatic alterations associated with innate resistance to palbociclib. Then the candidate biomarker was validated in another independent cohort of 104 patients and publicly available datasets. The resistance was verified in parental MCF-7 and T47D cells, as well as their derivatives with small interfering RNA transfection and lentivirus infection. The relevant mechanism was examined by RNA sequencing, chromatin immunoprecipitation and luciferase assay. Patient-derived organoid and patient-derived xenografts studies were utilized to evaluated the antitumor activity of rational combinations. RESULTS: In the discovery cohort, S6K1 amplification (3/35, 9%) was identified as an important reason for innate resistance to CDK4/6 inhibitors. In the independent cohort, S6K1 was overexpressed in 15/104 (14%) patients. In those who had received palbociclib treatment, patients with high-expressed S6K1 had significantly worse progression free survival than those with low S6K1 expression (hazard ratio = 3.0, P = 0.0072). Meta-analysis of public data revealed that patients with S6K1 amplification accounted for 12% of breast cancers. Breast cancer patients with high S6K1 expression had significantly worse relapse-free survival (hazard ratio = 1.31, P < 0.0001). In breast cancer cells, S6K1 overexpression, caused by gene amplification, was sufficient to promote resistance to palbociclib. Mechanistically, S6K1 overexpression increased the expression levels of G1/S transition-related proteins and the phosphorylation of Rb, mainly through the activation of c-Myc pathway. Notably, this resistance could be abrogated by the addition of mTOR inhibitor, which blocked the upstream of S6K1, in vitro and in vivo. CONCLUSIONS: S6K1 amplification is an important mechanism of innate resistance to palbociclib in breast cancers. Breast cancers with S6K1 amplification could be considered for combinations of CDK4/6 and S6K1 antagonists.

[Protective effect of salidroside on high fat-induced apoptosis in H9c2 cardiomyocytes through AMPK/mTOR/p70S6K pathway].

The study explored the effect of salidroside(SAL) on high fat-induced apoptosis in H9 c2 cardiomyocytes based on AMPK/mTOR/p70 S6 K pathway.H9 c2 cardiomyocytes were cultured in vitro and the lipotoxicity model of H9 c2 cardiomyocytes was constructed by 0.2 mmol·L~(-1) palmitic acid(PA) treatment for 24 hours.The cells were divided into control group, PA group, and SAL group(20 µmol·L~(-1)).Cell proliferation was detected with cell proliferation kit I(MTT) assay after SAL and PA treatment.Dihydroethidium(DHE) probe, Annexin V-FITC/PI kit, and JC-1 probe were used to estimate reactive oxygen species(ROS) level, cell apoptosis, and mitochondrial membrane potential(MMP) change, respectively.The expression levels of p-AMPK/AMPK, p-mTOR/mTOR, p-p70 S6 K/p70 S6 K and apoptosis-related proteins Bax, Bcl-2, and cleaved caspase-3 were investigated with Western blot.The mRNA levels of AMPK, mTOR and p70 S6 K were determined by quantitative reverse transcription-polymerase chain reaction(qRT-PCR).RESULTS:: showed that compared with control group, PA group had decreased cell proliferation ability, MMP, Bcl-2 protein expression and AMPK protein and mRNA expression, while increased ROS level, Bax and cleaved caspase-3 protein expression, and mTOR and p70 S6 K mRNA and protein expression, and the difference was statistically significant(P<0.05, P<0.01).Compared with PA group, SAL improved cell proliferation ability, MMP level, Bcl-2 protein expression, and AMPK mRNA and protein expression, while down-regulated ROS level, cell apoptosis, Bax and cleaved caspase-3 protein expression, and mTOR and p70 S6 K mRNA and protein expression, and the difference was statistically significant(P<0.05, P<0.01).In conclusion, SAL exerted protective effects on high fat-induced lipotoxicity of H9 c2 cardiomyocytes, alleviated the oxidative stress injury and reduced cell apoptosis via regulating AMPK/mTOR/p70 S6 K signaling pathway.

BDK knockout skeletal muscle satellite cells exhibit enhanced protein translation initiation signal in response to BCAA in vitro.

We examined the effects of branched-chain amino acids (BCAAs) and electrical pulse stimulation (EPS) on the mTORC1 pathway in muscle satellite cells (MSCs) isolated from branched-chain α-keto acid dehydrogenase kinase (BDK) knockout (KO) mice in vitro. MSCs were isolated from BDK KO and wild-type (WT) mice, proliferated, and differentiated into myotubes. BCAA stimulation increased the phosphorylation of p70 S6 kinase (p70S6K), a marker of protein translation initiation, in MSCs from WT and BDK KO mice, but the rate of the increase was higher in MSCs isolated from BDK KO mice. Contrarily, there was no difference in the increase in p70S6K phosphorylation by EPS. Acute BDK knockdown in MSCs from WT mice using shRNA decreased p70S6K phosphorylation in response to BCAA stimulation. Collectively, the susceptibility of mTORC1 to BCAA stimulation was elevated by chronic, but not acute, enhancement of BCAA catabolism.

p70 S6 kinase as a therapeutic target in cancers: More than just an mTOR effector.

p70 S6 kinase (p70S6K) is best known for its regulatory roles in protein synthesis and cell growth by phosphorylating its primary substrate, ribosomal protein S6, upon mitogen stimulation. The enhanced expression/activation of p70S6K has been correlated with poor prognosis in some cancer types, suggesting that it may serve as a biomarker for disease monitoring. p70S6K is a critical downstream effector of the oncogenic PI3K/Akt/mTOR pathway and its activation is tightly regulated by an ordered cascade of Ser/Thr phosphorylation events. Nonetheless, it should be noted that other upstream mechanisms regulating p70S6K at both the post-translational and post-transcriptional levels also exist. Activated p70S6K could promote various aspects of cancer progression such as epithelial-mesenchymal transition, cancer stemness and drug resistance. Importantly, novel evidence showing that p70S6K may also regulate different cellular components in the tumor microenvironment will be discussed. Therapeutic targeting of p70S6K alone or in combination with traditional chemotherapies or other microenvironmental-based drugs such as immunotherapy may represent promising approaches against cancers with aberrant p70S6K signaling. Currently, the only clinically available p70S6K inhibitors are rapamycin analogs (rapalogs) which target mTOR. However, there are emerging p70S6K-selective drugs which are going through active preclinical or clinical trial phases. Moreover, various screening strategies have been used for the discovery of novel p70S6K inhibitors, hence bringing new insights for p70S6K-targeted therapy.

Circulating tumor DNA predicts efficacy of a dual AKT/p70S6K inhibitor (LY2780301) plus paclitaxel in metastatic breast cancer: plasma analysis of the TAKTIC phase IB/II study.

The phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is frequently activated in HER2-negative breast cancer and may play a role in taxane resistance. The phase IB/II TAKTIC trial (NCT01980277) has shown that combining a dual AKT and p70 ribosomal protein S6 kinase (p70S6K) inhibitor (LY2780301) taken orally with weekly paclitaxel in HER2-negative advanced breast cancer is feasible, with preliminary evidence of efficacy. We wanted to explore whether circulating tumor DNA (ctDNA) may be a surrogate marker of treatment efficacy in this setting. Serial plasma samples were collected and cell-free DNA was sequenced using low-coverage whole-genome sequencing, and analysis was completed with droplet digital polymerase chain reaction (PCR) for some patients with driver mutations. Baseline tumor fraction (TF) and TF after 7 weeks on treatment were compared to progression-free survival (PFS) and the overall response rate. We also explored circulating copy number alterations associated with treatment failure. Of the 51 patients enrolled in the TAKTIC trial, at least one plasma sample was available for 44 cases (96 timepoints). All patients with tumor TP53, PI3KCA, or AKT1 mutations harbored at least one of these alterations in plasma. TF at inclusion was correlated with PFS (6m-PFS was 92% for ctDNAneg patients vs 68% for ctDNApos cases; hazard ratio [HR] = 3.45, 95% confidence interval [CI] [1.34-8.90], P = 0.007). ctDNA status at week 7 was not correlated with prognosis. Even though most circulating copy number alterations were conserved at disease progression, some genomic regions of interest were altered in post-progression samples. In conclusion, ctDNA detection at baseline was associated with shorter PFS in patients included in the TAKTIC trial. Plasma-based copy number analysis may help to identify alterations involved in resistance to treatment.

Low-frequency electroacupuncture improves disordered hepatic energy metabolism in insulin-resistant Zucker diabetic fatty rats via the AMPK/mTORC1/p70S6K signaling pathway.

BACKGROUND AND AIM: Disordered hepatic energy metabolism is found in obese rats with insulin resistance (IR). There are insufficient experimental studies of electroacupuncture (EA) for IR and type 2 diabetes mellitus (T2DM). The aim of this study was to probe the effect of EA on disordered hepatic energy metabolism and the adenosine monophosphate (AMP)-activated protein kinase (AMPK)/mammalian target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase, 70-kDa (p70S6K) signaling pathway. METHODS: Zucker Diabetic Fatty (ZDF) rats were randomly divided into three groups: EA group receiving EA treatment; Pi group receiving pioglitazone gavage; and ZF group remaining untreated (n = 8 per group). Inbred non-insulin-resistant Zucker lean rats formed an (untreated) healthy control group (ZL, n = 8). Fasting plasma glucose (FPG), fasting insulin (FINS), C-peptide, C-reactive protein (CRP) and homeostatic model assessment of insulin resistance (HOMA-IR) indices were measured. Hematoxylin-eosin (H&E) staining was used to investigate the liver morphologically. The mitochondrial structure of hepatocytes was observed by transmission electron microscopy (TEM). Western blotting was adopted to determine protein expression of insulin receptor substrate 1 (IRS-1), mTOR, mTORC1, AMPK, tuberous sclerosis 2 (TSC2) and p70S6K, and their phosphorylation. RT-PCR was used to quantify IRS-1, mTOR, mTORC1, AMPK and p70S6K mRNA levels. RESULTS: Compared with the ZF group, FPG, FINS, C-peptide, CRP and HOMA-IR levels were significantly reduced in the EA group (p < 0.05, p < 0.01). Evaluation of histopathology showed improvement in liver appearances following EA. Phosphorylation levels of AMPK, mTOR and TSC2 decreased, and IRS-1 and p70S6K increased, in hepatocytes of the ZF group, while these negative effects appeared to be alleviated by EA. CONCLUSIONS: EA can effectively ameliorate IR and regulate energy metabolism in the ZDF rat model. AMPK/mTORC1/p70S6K and related molecules may represent a potential mechanism of action underlying these effects.

PP2A-mTOR-p70S6K/4E-BP1 axis regulates M1 polarization of pulmonary macrophages and promotes ambient particulate matter induced mouse lung injury.

To identify key signaling pathways involved in ambient particulate matter (PM)-induced pulmonary injury, we generated a mouse model with myeloid-specific deletion of Ppp2r1a gene (encoding protein phosphatase 2 A (PP2A) A subunit), and conducted experiments in a real-ambient PM exposure system. PP2A Aα-/- homozygote (Aα HO) mice and matched wild-type (WT) littermates were exposed to PM over 3-week and 6-week. The effects of PM exposure on pulmonary inflammation, oxidative stress, and apoptosis were significantly enhanced in Aα HO compared to WT mice. The number of pulmonary macrophages increased by 74.8~88.0% and enhanced M1 polarization appeared in Aα HO mice upon PM exposure. Secretion of M1 macrophage-related inflammatory cytokines was significantly increased in Aα HO vs. WT mice following PM exposure. Moreover, we demonstrated that PP2A-B56α holoenzyme regulated M1 polarization and that the mTOR signaling pathway mediated the persistent M1 polarization upon PM2.5 exposure. Importantly, PP2A-B56α holoenzyme was shown to complex with mTOR/p70S6K/4E-BP1, and suppression of B56α led to enhanced phosphorylation of mTOR, p70S6K, and 4E-BP1. These observations demonstrate that the PP2A-mTOR-p70S6K/4E-BP1 signaling is a critical pathway in mediating macrophage M1 polarization, which contributes to PM-induced pulmonary injury.

Inhibition of TRIM32 by ibr-7 treatment sensitizes pancreatic cancer cells to gemcitabine via mTOR/p70S6K pathway.

Pancreatic cancer is one of the most notorious diseases for being asymptomatic at early stage and high mortality rate thereafter. However, either chemotherapy or targeted therapy has rarely achieved success in recent clinical trials for pancreatic cancer. Novel therapeutic regimens or agents are urgently in need. Ibr-7 is a novel derivative of ibrutinib, displaying superior antitumour activity in pancreatic cancer cells than ibrutinib. In vitro studies showed that ibr-7 greatly inhibited the proliferation of BxPC-3, SW1990, CFPAC-1 and AsPC-1 cells via the induction of mitochondrial-mediated apoptosis and substantial suppression of mTOR/p70S6K pathway. Moreover, ibr-7 was able to sensitize pancreatic cancer cells to gemcitabine through the efficient repression of TRIM32, which was positively correlated with the proliferation and invasiveness of pancreatic cancer cells. Additionally, knockdown of TRIM32 diminished mTOR/p70S6K activity in pancreatic cancer cells, indicating a positive feedback loop between TRIM32 and mTOR/p70S6K pathway. To conclude, this work preliminarily explored the role of TRIM32 in the malignant properties of pancreatic cancer cells and evaluated the possibility of targeting TRIM32 to enhance effectiveness of gemcitabine, thereby providing a novel therapeutic target for pancreatic cancer.

Ribosomal protein S6 kinase beta-1 gene variants cause hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a genetic heart muscle disease with preserved or increased ejection fraction in the absence of secondary causes. Mutations in the sarcomeric protein-encoding genes predominantly cause HCM. However, relatively little is known about the genetic impact of signalling proteins on HCM. METHODS AND RESULTS: Here, using exome and targeted sequencing methods, we analysed two independent cohorts comprising 401 Indian patients with HCM and 3521 Indian controls. We identified novel variants in ribosomal protein S6 kinase beta-1 (RPS6KB1 or S6K1) gene in two unrelated Indian families as a potential candidate gene for HCM. The two unrelated HCM families had the same heterozygous missense S6K1 variant (p.G47W). In a replication association study, we identified two S6K1 heterozygotes variants (p.Q49K and p.Y62H) in the UK Biobank cardiomyopathy cohort (n=190) compared with matched controls (n=16 479). These variants are neither detected in region-specific controls nor in the human population genome data. Additionally, we observed an S6K1 variant (p.P445S) in an Arab patient with HCM. Functional consequences were evaluated using representative S6K1 mutated proteins compared with wild type in cellular models. The mutated proteins activated the S6K1 and hyperphosphorylated the rpS6 and ERK1/2 signalling cascades, suggesting a gain-of-function effect. CONCLUSIONS: Our study demonstrates for the first time that the variants in the S6K1 gene are associated with HCM, and early detection of the S6K1 variant carriers can help to identify family members at risk and subsequent preventive measures. Further screening in patients with HCM with different ethnic populations will establish the specificity and frequency of S6K1 gene variants.

MiRNA-30e downregulation increases cancer cell proliferation, invasion and tumor growth through targeting RPS6KB1.

Human esophagus carcinoma (EC) is one of the most common malignant tumors, especially in Africa and Asia including China. In EC initiation and progression, genetic and epigenetic aberrations have been reported to play a major role, but the underlying molecular mechanisms are largely unknown. In this study, the miR-30e levels were analyzed in human EC tissues and TCGA databases, and the results demonstrated that miR-30e expression in EC tissues was significantly decreased compared to adjacent normal tissues. To further investigate the role of miR-30e in cancer cells, we found that forced expression of miR-30e dramatically inhibited cell proliferation, invasion, tube formation, and colony formation of cancer cells. To determine the underlying mechanism of miR-30e, we found that RPS6KB1 was a direct target of miR-30e by binding to its 3'-UTR, which was verified by luciferase activity assay using reporters with wild-type miR-30e and its seed sequence mutant constructs and Western blotting assay. In vivo experiment showed that miR-30e overexpression significantly inhibited tumor growth and decreased RPS6KB1 expression in xenografts. In EC, high expression of RPS6KB1 in tumor tissues indicated poor prognosis of patients with less survival rate. High levels of RPS6KB1 and low levels of miR-30e closely correlated poor survival of patients with several other types of cancer. These findings show that miR-30e and its target RPS6KB1 are important in cancer development and clinical outcomes, and miR-30e/RPS6KB1 is a potential future therapeutic pathway for EC intervention.