The sialidase NEU1 directly interacts with the juxtamembranous segment of the cytoplasmic domain of mucin-1 to inhibit downstream PI3K-Akt signaling

The extracellular domain (ED) of the membrane-spanning sialoglycoprotein, mucin-1 (MUC1), is an in vivo substrate for the lysosomal sialidase, neuraminidase-1 (NEU1). Engagement of the MUC1-ED by its cognate ligand, Pseudomonas aeruginosa-expressed flagellin, increases NEU1-MUC1 association and NEU1-mediated MUC1-ED desialylation to unmask cryptic binding sites for its ligand. However, the mechanism(s) through which intracellular NEU1 might physically interact with its surface-expressed MUC1-ED substrate are unclear. Using reciprocal coimmunoprecipitation and in vitro binding assays in a human airway epithelial cell system, we show here that NEU1 associates with the MUC1-cytoplasmic domain (CD) but not with the MUC1-ED. Prior pharmacologic inhibition of the NEU1 catalytic activity using the NEU1-selective sialidase inhibitor, C9-butyl amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid, did not diminish NEU1-MUC1-CD association. In addition, glutathione-S-transferase (GST) pull-down assays using the deletion mutants of the MUC1-CD mapped the NEU1-binding site to the membrane-proximal 36 aa of the MUC1-CD. In a cell-free system, we found that the purified NEU1 interacted with the immobilized GST-MUC1-CD and the purified MUC1-CD associated with the immobilized 6XHis-NEU1, indicating that the NEU1-MUC1-CD interaction was direct and independent of its chaperone protein, protective protein/cathepsin A. However, the NEU1-MUC1-CD interaction was not required for the NEU1-mediated MUC1-ED desialylation. Finally, we demonstrated that overexpression of either WT NEU1 or a catalytically dead NEU1 G68V mutant diminished the association of the established MUC1-CD binding partner, PI3K, to MUC1-CD and reduced downstream Akt kinase phosphorylation. These results indicate that NEU1 associates with the juxtamembranous region of the MUC1-CD to inhibit PI3K-Akt signaling independent of NEU1 catalytic activity.     

PAK4 confers cisplatin resistance in gastric cancer cells via PI3K/Akt- and MEK/ERK-dependent pathways

CDDP [cisplatin or cis-diamminedichloroplatinum(II)] and CDDP-based combination chemotherapy have been confirmed effective against gastric cancer. However, CDDP efficiency is limited because of development of drug resistance. In this study, we found that PAK4 (p21-activated kinase 4) expression and activity were elevated in gastric cancer cells with acquired CDDP resistance (AGS/CDDP and MKN-45/CDDP) compared with their parental cells. Inhibition of PAK4 or knockdown of PAK4 expression by specific siRNA (small interfering RNA)-sensitized CDDP-resistant cells to CDDP and overcome CDDP resistance. Combination treatment of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 [the inhibitor of PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B or PKB) pathway] or PD98509 {the inhibitor of MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] pathway} with PF-3758309 (the PAK4 inhibitor) resulted in increased CDDP efficacy compared with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or PD98509 alone. However, after the concomitant treatment of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and PD98509, PF-3758309 administration exerted no additional enhancement of CDDP cytotoxicity in CDDP-resistant cells. Inhibition of PAK4 by PF-3758309 could significantly suppress MEK/ERK and PI3K/Akt signalling in CDDP-resistant cells. Furthermore, inhibition of PI3K/Akt pathway while not MEK/ERK pathway could inhibit PAK4 activity in these cells. The in vivo results were similar with those of in vitro. In conclusion, these results indicate that PAK4 confers CDDP resistance via the activation of MEK/ERK and PI3K/Akt pathways. PAK4 and PI3K/Akt pathways can reciprocally activate each other. Therefore, PAK4 may be a potential target for overcoming CDDP resistance in gastric cancer.     

p21-activated Kinases (PAKs) Mediate the Phosphorylation of PREX2 Protein to Initiate Feedback Inhibition of Rac1 GTPase

Phosphatidylinositol 3,4,5-trisphosphate (PIP₃)-dependent Rac exchanger 2 (PREX2) is a guanine nucleotide exchange factor (GEF) for the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase, facilitating the exchange of GDP for GTP on Rac1. GTP-bound Rac1 then activates its downstream effectors, including p21-activated kinases (PAKs). PREX2 and Rac1 are frequently mutated in cancer and have key roles within the insulin-signaling pathway. Rac1 can be inactivated by multiple mechanisms; however, negative regulation by insulin is not well understood. Here, we show that in response to being activated after insulin stimulation, Rac1 initiates its own inactivation by decreasing PREX2 GEF activity. Following PREX2-mediated activation of Rac1 by the second messengers PIP₃ or Gβγ, we found that PREX2 was phosphorylated through a PAK-dependent mechanism. PAK-mediated phosphorylation of PREX2 reduced GEF activity toward Rac1 by inhibiting PREX2 binding to PIP₃ and Gβγ. Cell fractionation experiments also revealed that phosphorylation prevented PREX2 from localizing to the cellular membrane. Furthermore, the onset of insulin-induced phosphorylation of PREX2 was delayed compared with AKT. Altogether, we propose that second messengers activate the Rac1 signal, which sets in motion a cascade whereby PAKs phosphorylate and negatively regulate PREX2 to decrease Rac1 activation. This type of regulation would allow for transient activation of the PREX2-Rac1 signal and may be relevant in multiple physiological processes, including diseases such as diabetes and cancer when insulin signaling is chronically activated.     

Transient Activation of the PI3K-AKT Pathway by Hepatitis C Virus to Enhance Viral Entry

The PI3K-AKT signaling pathway plays an important role in cell growth and metabolism. Here we report that hepatitis C virus (HCV) transiently activates the PI3K-AKT pathway. This activation was observed as early as 15 min postinfection, peaked by 30 min, and became undetectable at 24 h postinfection. The activation of AKT could also be mediated by UV-inactivated HCV, HCV pseudoparticle, and the ectodomain of the HCV E2 envelope protein. Because antibodies directed against CD81 and claudin-1, but not antibodies directed against scavenger receptor class B type I or occludin, could also activate AKT, the interaction between HCV E2 and its two co-receptors CD81 and claudin-1 probably triggered the activation of AKT. This activation of AKT by HCV was important for HCV infectivity, because the silencing of AKT by siRNA or the treatment of cells with its inhibitors or with the inhibitor of its upstream regulator PI3K significantly inhibited HCV infection, whereas the expression of constitutively active AKT enhanced HCV infection. The PI3K-AKT pathway is probably involved in HCV entry, because the inhibition of this pathway could inhibit the entry of HCV pseudoparticle but not the VSV pseudoparticle into cells. Furthermore, the treatment of cells with the AKT inhibitor AKT-V prior to HCV infection inhibited HCV infection, whereas the treatment after HCV infection had no obvious effect. Taken together, our studies indicated that HCV transiently activates the PI3K-AKT pathway to facilitate its entry. These results provide important information for understanding HCV replication and pathogenesis and raised the possibility of targeting this cellular pathway to treat HCV patients.     

DNA methylation and miR-92a-3p-mediated repression of HIP1R promotes pancreatic cancer progression by activating the PI3K/AKT pathway

Pancreatic cancer (PAAD) is a highly malignant tumour characterized of high mortality and poor prognosis. Huntingtin-interacting protein 1-related (HIP1R) has been recognized as a tumour suppressor in gastric cancer, while its biological function in PAAD remains to be elucidated. In this study, we reported the downregulation of HIP1R in PAAD tissues and cell lines, and the overexpression of HIP1R suppressed the proliferation, migration and invasion of PAAD cells, while silencing HIP1R showed the opposite effects. DNA methylation analysis revealed that the promoter region of HIP1R was heavily methylated in PAAD cell lines when compared to the normal pancreatic duct epithelial cells. A DNA methylation inhibitor 5-AZA increased the expression of HIP1R in PAAD cells. 5-AZA treatment also inhibited the proliferation, migration and invasion, and induced apoptosis in PAAD cell lines, which could be attenuated by HIP1R silencing. We further demonstrated that HIP1R was negatively regulated by miR-92a-3p, which modulates the malignant phenotype of PAAD cells in vitro and the tumorigenesis in vivo. The miR-92a-3p/HIP1R axis could regulate PI3K/AKT pathway in PAAD cells. Taken together, our data suggest that targeting DNA methylation and miR-92a-3p-mediated repression of HIP1R could serve as novel therapeutic strategies for PAAD treatment.     

miR‐126 reduces trastuzumab resistance by targeting PIK3R2 and regulating AKT/mTOR pathway in breast cancer cells

MicroRNAs (miRNAs) have been found to play a key role in drug resistance. In the current study, we aimed to explore the potential role of miR‐126 in trastuzumab resistance in breast cancer cells. We found that the trastuzumab‐resistant cell lines SKBR3/TR and BT474/TR had low expression of miR‐126 and increased ability to migrate and invade. The resistance, invasion and mobilization abilities of the cells resistant to trastuzumab were reduced by ectopic expression of miR‐126 mimics. In comparison, inhibition of miR‐126 in SKBR3 parental cells had the opposite effect of an increased resistance to trastuzumab as well as invasion and migration. It was also found that miR‐126 directly targets PIK3R2 in breast cancer cells. PIK3R2‐knockdown cells showed decreased resistance to trastuzumab, while overexpression of PIK3R2 increased trastuzumab resistance. In addition, our finding showed that overexpression of miR‐126 reduced resistance to trastuzumab in the trastuzumab‐resistant cells and that inhibition of the PIK3R2/PI3K/AKT/mTOR signalling pathway was involved in this effect. SKBR3/TR cells also showed increased sensitivity to trastuzumab mediated by miR‐126 in vivo. In conclusion, the above findings demonstrated that overexpression of miR‐126 or down‐regulation of its target gene may be a potential approach to overcome trastuzumab resistance in breast cancer cells.     

Downregulation of NEAT1 reverses the radioactive iodine resistance of papillary thyroid carcinoma cell via miR-101-3p/FN1/PI3K-AKT signaling pathway

Considering the resistance of papillary thyroid cancer (PTC) ¹³¹I therapy, this study was designed to find a solution at molecular respect. By probing into lncRNA-NEAT1/miR-101-3p/FN1 axis and PI3K/AKT signaling pathway, this study provided a potential target for PTC therapy. ¹³¹I-resistant cell lines were established by continuous treatment with median-lethal ¹³¹I. Bioinformatic analysis was applied to filtrate possible lncRNA/miRNA/mRNA and related signaling pathway. Luciferase reporter assay was employed in the verification of the targeting relationship between lncRNA and miRNA as well as miRNA and mRNA. MTT assay and flow cytometry assay were performed to observe the impact of NEAT1/miR-101-3p/FN1 on cell viability and apoptosis in radioactivity iodine (RAI)-resistant PTC cell lines, respectively. Western blot and qRT-PCR were conducted to measure the expression of proteins and mRNAs in RAI-resistant PTC tissues and cells. Meanwhile, endogenous PTC mice model were constructed, in order to verify the relation between NEAT1 and RAI-resistance in vivo. NEAT1 was over-expressed in RAI-resistant PTC tissues and cell lines and could resist RAI by accelerating proliferation accompanied by suppressing apoptosis. It indicated that overexpressed NEAT1 restrained the damage of RAI to tumor in both macroscopic and microcosmic. Besides, NEAT1/miR-101-3p exhibited a negative correlation by directly targeting each other. The expression of FN1, an overexpressed downstream protein in RAI-resistance PTC tissues, could be tuned down by miR-101-3p, while the decrease could be restored by NEAT1. In conclusion, both in vitro and in vivo, NEAT1 suppression could inhibit ¹³¹I resistance of PTC by upregulating miR-101-3p/FN1 expression and inactivated PI3K/AKT signaling pathway both in vitro and in vivo.     

ANXA2 Facilitates Enterovirus 71 Infection by Interacting with 3D Polymerase and PI4KB to Assist the Assembly of Replication Organelles

Virologica Sinica - Similar to that of other enteroviruses, the replication of enterovirus 71 (EV71) occurs on rearranged membranous structures called replication organelles (ROs)....     

Genipin up-regulates heme oxygenase-1 via PI3-kinase-JNK1/2-Nrf2 signaling pathway to enhance the anti-inflammatory capacity in RAW264.7 macrophages

Genipin, an aglycon of geniposide, has been reported to exhibit diverse pharmacological functions such as antitumor and anti-inflammatory effects. This study aimed to elucidate the anti-inflammatory mechanism of genipin, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. In RAW264.7 cells, genipin increased HO-1 expression and its enzyme activity via a NF-E2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway. These effects were significantly inhibited by exposure to the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, or by expression of a dominant negative mutant of PI 3-kinase. Additional experiments showed that the activation of c-Jun NH₂-terminal kinase 1/2 (JNK1/2) is required for genipin-induced phosphorylation and nuclear translocation of Nrf2 and antioxidant response element (ARE)-driven induction of HO-1, and acts as a downstream effector of PI 3-kinase. Furthermore, functional significance of HO-1 induction was revealed by genipin-mediated inhibition of lipopolysaccharide-stimulated inducible nitric oxide synthase expression or cyclooxygenase-2 promoter activity, the response was reversed by the blocking of HO-1 protein synthesis or HO-1 enzyme activity. Therefore, identification of PI 3-kinase-JNK1/2-Nrf2-linked signaling cascade in genipin-mediated HO-1 expression defines the signaling event that could participate in genipin-mediated anti-inflammatory response.     

Down-regulation of Mcl-1 by inhibition of the PI3-K/Akt pathway is required for cell shrinkage-dependent cell death.

The anti-apoptotic effect of a chloride-bicarbonate exchange blocker has been previously examined in endothelial cells and cardiomyocytes. However, the anti-apoptotic effects of this blocker on epithelial cells and the mechanism of the anti-apoptotic effect remain unknown. We examined the anti-apoptotic effects of a chloride-bicarbonate exchange blocker in a renal epithelial cell line (MDCK cells). Changes in the expression of bcl-2 family proteins, which are known to have anti-apoptotic effects, were also examined. Staurosporine was used to induce apoptotic cell death in the MDCK cells. Staurosporine treatment was sufficient to induce apoptotic cell death, detected by propidium iodide and DNA ladder formation. A chloride-bicarbonate exchange blocker was added 24 h before the staurosporine treatment and during treatment. The chloride-bicarbonate exchange blocker inhibited the staurosporine-induced apoptosis in the MDCK cells in a dose-dependent manner. The expression of bcl-2 family gene products was detected by RT-PCR and Western blotting. No changes in the expression of Bax, Bid and Bik (pro-apoptotic proteins), or Bcl-2 (an anti-apoptotic protein) were detected. However, Mcl-1 expression was reduced by the staurosporine treatment, and this reduction was recovered when the chloride-bicarbonate exchange blocker was added. LY294002, a PI 3-kinase inhibitor, partially inhibited this anti-apoptotic effect. In conclusion, chloride-bicarbonate exchange blockers appear to offer cell-protective effects via Mcl-1 up-regulation.     

BMP2 accelerates the motility and invasiveness of gastric cancer cells via activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway

Up-regulation of bone morphogenetic proteins (BMPs) and their receptors by tumor is an important hallmark in cancer progression, as it contributes through autocrine and paracrine mechanisms to tumor development, invasion, and metastasis. Generally, increased motility and invasion are positively correlated with the epithelial-mesenchymal transition (EMT). The purpose of the present study was to determine whether BMP-2 signaling to induce gastric cancer cells to undergo EMT-mediated invasion might pass through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Herein we showed that gastric cancer cell lines express all the components of BMP-2 signaling, albeit to different extents. Moreover, an increased concentration of BMP-2 strongly enhanced motility and invasiveness in gastric cancer cells, whereas no increase was observed in cells treated with either Noggin (a BMP-2 inhibitor) or BMP-2 blocking antibodies. The stimulation of BMP-2 in gastric cancer cells induces a full EMT characterized by Snail induction, E-cadherin delocalization and down-regulation, and up-regulation of mesenchymal and invasiveness markers. Furthermore, blockade of BMP-2 signaling by Noggin or BMP-2 blocking antibodies also restored these changes in EMT markers. In addition, phosphorylation of Akt was also enhanced by treatment with BMP-2, but not Noggin or BMP-2 blocking antibodies. Pretreatment of gastric cancer cells with PI-3 kinase/Akt kinase inhibitor (kinase-dead Akt [DN-Akt], Akt siRNA, or LY294002) significantly inhibited BMP-2-induced EMT and invasiveness. Overall, our studies suggest that BMP-2 promotes motility and invasion of gastric cancer cells by activating PI-3 kinase/Akt and that targeting of this signaling pathway may provide therapeutic opportunities in preventing metastasis mediated by BMP-2.     

Regulation of PI(3)K/Akt signalling and cellular transformation by inositol polyphosphate 4‐phosphatase‐1

Akt is a crucial phosphoinositide 3-kinase (PI(3)K) effector that regulates cell proliferation and survival. PI(3)K-generated signals, PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂, direct Akt plasma membrane engagement. Pathological Akt plasma membrane association promotes oncogenesis. PtdIns(3,4)P₂ is degraded by inositol polyphosphate 4-phosphatase-1 (4-ptase-1) forming PtdIns(3)P; however, the role of 4-ptase-1 in regulating the activation and function of Akt is unclear. In mouse embryonic fibroblasts lacking 4-ptase-1 (^−/−MEFs), the Akt-pleckstrin homology (PH) domain was constitutively membrane-associated both in serum-starved and agonist-stimulated cells, in contrast to ^+/+MEFs, in which it was detected only at the plasma membrane following serum stimulation. Epidermal growth factor (EGF) stimulation resulted in increased Ser⁴⁷³ and Thr³⁰⁸-Akt phosphorylation and activation of Akt-dependent signalling in ^−/−MEFs, relative to ^+/+MEFs. Significantly, loss of 4-ptase-1 resulted in increased cell proliferation and decreased apoptosis. SV40-transformed ^−/−MEFs showed increased anchorage-independent cell growth and formed tumours in nude mice. This study provides the first evidence, to our knowledge, that 4-ptase-1 controls the activation of Akt and thereby cell proliferation, survival and tumorigenesis.     

MiR‐3116 sensitizes glioma cells to temozolomide by targeting FGFR1 and regulating the FGFR1/PI3K/AKT pathway

Glioma is a brain tumour that is often diagnosed, and temozolomide (TMZ) is a common chemotherapeutic drug used in glioma. Yet, resistance to TMZ is a chief hurdle towards curing the malignancy. The current work explores the pathways and involvement of miR‐3116 in the TMZ resistance. miR‐3116 and FGFR1 mRNA were quantified by real‐time PCR in malignant samples and cell lines. Appropriate assays were designed for apoptosis, viability, the ability to form colonies and reporter assays to study the effects of the miR‐3116 or FGFR1. The involvement of PI3K/AKT signalling was assessed using Western blotting. Tumorigenesis was evaluated in an appropriate xenograft mouse model in vivo. This work revealed that the levels of miR‐3116 dipped in samples resistant to TMZ, while increased miR‐3116 caused an inhibition of the tumour features mentioned above to hence augment TMZ sensitivity. miR‐3116 was found to target FGFR1. When FGFR1 was overexpressed, resistance to TMZ was augmented and reversed the sensitivity caused by miR‐3116. Our findings further confirmed PI3K/AKT signalling pathway is involved in this action. In conclusion, miR‐3116 sensitizes glioma cells to TMZ through FGFR1 downregulation and the PI3K/AKT pathway inactivation. Our results provide a strategy to overcome TMZ resistance in glioma treatment.     

Growth hormone modulation of EGF-induced PI3K-Akt pathway in mice liver

The epidermal growth factor (EGF) activates the phosphatidylinositol 3-kinase (PI3K)-Akt cascade among other signaling pathways. This route is involved in cell proliferation and survival, therefore, its dysregulation can promote cancer. Considering the relevance of the PI3K-Akt signaling in cell survival and in the pathogenesis of cancer, and that GH was reported to modulate EGFR expression and signaling, the objective of this study was to analyze the effects of increased GH levels on EGF-induced PI3K-Akt signaling.EGF-induced signaling was evaluated in the liver of GH-overexpressing transgenic mice and in their normal siblings. While Akt expression was increased in GH-overexpressing mice, EGF-induced phosphorylation of Akt, relative to its protein content, was diminished at Ser473 and inhibited at Thr308; consequently, mTOR, which is a substrate of Akt, was not activated by EGF. However, the activation of PDK1, a kinase involved in Akt phosphorylation at Thr308, was not reduced in transgenic mice. Kinetics studies of EGF-induced Akt phosphorylation showed that it is rapidly and transiently induced in GH-overexpressing mice compared with normal siblings. Thus, the expression and activity of phosphatases involved in the termination of the PI3K-Akt signaling were studied. In transgenic mice, neither PTEN nor PP2A were hyperactivated; however, EGF induced the rapid and transient association of SHP-2 to Gab1, which mediates association to EGFR and activation of PI3K. Rapid recruitment of SHP2, which would accelerate the termination of the proliferative signal induced, could be therefore contributing to the diminished EGF-induced activity of Akt in GH-overexpressing mice.     

MAPK and PI3K pathways regulate hypoxia-induced atrial natriuretic peptide secretion by controlling HIF-1 alpha expression in beating rabbit atria

Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways are pivotal and intensively studied signaling pathways in hypoxic conditions. However, the roles of MAPK and PI3K in the regulation of hypoxia-induced atrial natriuretic peptide (ANP) secretion are not well understood. The purpose of the present study was to investigate the mechanism by which the MAPK/ERK (extracellular signal-regulated kinase) and PI3K signaling pathways regulate the acute hypoxia-induced ANP secretion in isolated beating rabbit atria. An acute hypoxic perfused beating rabbit atrial model was used. The ANP levels in the atrial perfusates were measured by radioimmunoassay, and the hypoxia-inducible factor-1α (HIF-1α) mRNA and protein levels in the atrial tissue were determined by RT-PCR and Western blot. Acute hypoxia significantly increased ANP secretion and HIF-1α mRNA and protein levels. Hypoxia-induced ANP secretion was markedly attenuated by the HIF-1α inhibitors, rotenone (0.5 μmol/L) and CAY10585 (10 μmol/L), concomitantly with downregulation of the hypoxia-induced HIF-1α mRNA and protein levels. PD098059 (30 μmol/L) and LY294002 (30 μmol/L), inhibitors of MAPK and PI3K, markedly abolished the hypoxia-induced ANP secretion and atrial HIF-1α mRNA and protein levels. The hypoxia-suppressed atrial dynamics were significantly attenuated by PD098059 and LY294002. Acute hypoxia in isolated perfused beating rabbit atria, markedly increased ANP secretion through HIF-1α upregulation, which was regulated by the MAPK/ERK and PI3K pathways. ANP appears to be part of the protective program regulated by HIF-1α in the response to acute hypoxic conditions.     

GRP94 promotes muscle differentiation by inhibiting the PI3K/AKT/mTOR signaling pathway

The glucose-regulated endoplasmic reticulum chaperone protein 94 (GRP94) is required for many biological processes, such as secretion of immune factors and mesoderm induction. Here, we demonstrated that GRP94 promotes muscle differentiation in vitro and in vivo. Moreover, GRP94 inhibited the PI3K/AKT/mTOR signaling pathway. Using both in vitro and in vivo approaches, in myoblasts, we found that this inhibition resulted in reduced proliferation and increased differentiation. To further investigate the mechanism of GRP94-induced muscle differentiation, we used co-immunoprecipitation and proximity ligation assays and found that GRP94 interacted with PI3K-interacting protein 1 (Pik3ip1). The latter protein promoted muscle differentiation by inhibiting the PI3K/AKT/mTOR pathway. Furthermore, GRP94 was found to regulate Pik3ip1 expression. Finally, when Pik3ip1 expression was inhibited, GRP94-induced promotion of muscle differentiation was diminished. Taken together, our data demonstrated that GRP94 promoted muscle differentiation, mediated by Pik3ip1-dependent inhibition of the PI3K/AKT/mTOR signaling pathway.