Dataset Information

PI3K-Akt-mTOR inhibition by GNE-477 inhibits renal cell carcinoma cell growth in vitro and in vivo.

ABSTRACT: Sustained activation of PI3K-Akt-mTOR cascade is important for renal cell carcinoma (RCC) cell progression. GNE-477 is a novel and efficacious PI3K-mTOR dual inhibitor. The current study tested its anti-RCC cell activity. In the primary cultured human RCC cells, GNE-477 potently inhibited cell growth, viability and proliferation, as well as cell cycle progression, migration and invasion. Furthermore, it induced robust apoptosis activation in primary RCC cells, but being non-cytotoxic to HK-2 epithelial cells and primary human renal epithelial cells. In the primary RCC cells GNE-477 inactivated PI3K-Akt-mTOR cascade by blocking phosphorylation of p85, Akt1, p70S6K1 and S6. Restoring Akt-mTOR activation by a constitutively-active Akt1 reversed GNE-477-induced anti-RCC cell activity. In nude mice intraperitoneal injection of GNE-477 potently suppressed RCC xenograft tumor growth. Collectively, targeting PI3K-Akt-mTOR cascade by GNE-477 inhibits RCC cell growth in vitro and in vivo.

SUBMITTER: Ye X

PROVIDER: S-EPMC7288912 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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PI3K-Akt-mTOR inhibition by GNE-477 inhibits renal cell carcinoma cell growth <i>in vitro</i> and <i>in vivo</i>.

Ye Xueting X Ruan Jian-Wei JW Huang Hang H Huang Wei-Ping WP Zhang Yan Y Zhang Fangyi F

Aging 20200518 10

Sustained activation of PI3K-Akt-mTOR cascade is important for renal cell carcinoma (RCC) cell progression. GNE-477 is a novel and efficacious PI3K-mTOR dual inhibitor. The current study tested its anti-RCC cell activity. In the primary cultured human RCC cells, GNE-477 potently inhibited cell growth, viability and proliferation, as well as cell cycle progression, migration and invasion. Furthermore, it induced robust apoptosis activation in primary RCC cells, but being non-cytotoxic to HK-2 epi ...[more]

PMID: 32421688

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Project description:In spite of the promising in vitro and preclinical results, dual PI3K/Akt/mTOR inhibitor NVP-BEZ235, and ATP-competitive mTOR inhibitor PP242 both failed to confirm their inhibitory efficacy against renal cell carcinoma (RCC) in clinical settings. Therefore, a better understanding of the molecular mechanism is essential so as to provide possibilities for their use in combination with other agents. In present study, RCC cell lines (UMRC6, 786-0 and UOK121) were treated with NVP-BEZ235, PP242 or Rapamycin, an mTOR complex 1 (mTORC1)-specific inhibitor. They all suppressed cell proliferation and invasion, induced apoptosis and cell cycle arrest, and the effects were in the order of NVP-BEZ235 > PP242 > Rapamycin. Accordingly, the marked and sustained decrease in speckle-type POZ protein (SPOP) expression and phosphorylation of Akt and mTOR kinases was observed in RCC cells treated with NVP-BEZ235 and PP242, whereas only potent inhibition of mTOR activity was induced in Rapamycin-treated cells. In considering the overactivation of c-Jun and IκB-α in human renal tumor tissue, we next investigated the role of JNK and IKK pathways in the response of RCC cells to these compounds. First of all, transforming growth factor β activated kinase 1 (TAK1)-dependent activation of JNK/ (activator protein-1) AP-1 axis in RCC cells was proved by the repression of AP-1 activity with TAK1 or JNK inhibitor. Second, the profound inhibition of TAK1/JNK/AP-1 pathway was demonstrated in RCC cells treated with NVP-BEZ235 or PP242 but not Rapamycin, which is manifested as a reduction in activity of TAK1, c-Jun and AP-1. Meanwhile, subsequent to TAK1 inactivation, the activation of IκB-α was also reduced by NVP-BEZ235 and PP242. Likewise, in vivo, treatment with NVP-BEZ235 and PP242 suppressed the growth of xenografts generated from 786-0 and A498 cells, along with decreased expression of phospho-TAK1, phospho-c-Jun, and phospho-IκB-α. In contrast, Rapamycin elicited no significant inhibitory effects on tumor growth and phosphorylation of TAK1, c-Jun and IκB-α. We conclude that besides PI3K/Akt/mTOR signaling, NVP-BEZ235, and PP242 simultaneously target TAK1-dependent pathways in RCC cells. Notably, these effects were more marked in the presence of NVP-BEZ235 than PP242, indicating the potential application of NVP-BEZ235 in combination therapy for RCC.

Project description:PURPOSE:To provide rationale for using phosphoinositide 3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathway inhibitors to treat rhabdomyosarcomas, a major cause of pediatric and adolescent cancer deaths. EXPERIMENTAL DESIGN:The prevalence of PI3K/MAPK pathway activation in rhabdomyosarcoma clinical samples was assessed using immunohistochemistry. Compensatory signaling and cross-talk between PI3K/MAPK pathways was determined in rhabdomyosarcoma cell lines following p110? short hairpin RNA-mediated depletion. Pharmacologic inhibition of reprogrammed signaling in stable p110? knockdown lines was used to determine the target-inhibition profile inducing maximal growth inhibition. The in vitro and in vivo efficacy of inhibitors of TORC1/2 (AZD8055), MEK (AZD6244), and P13K/mTOR (NVP-BEZ235) was evaluated alone and in pairwise combinations. RESULTS:PI3K pathway activation was seen in 82.5% rhabdomyosarcomas with coactivated MAPK in 36% and 46% of alveolar and embryonal subtypes, respectively. p110? knockdown in cell lines over the short and long term was associated with compensatory expression of other p110 isoforms, activation of the MAPK pathway, and cross-talk to reactivate the PI3K pathway. Combinations of PI3K pathway and MAP-ERK kinase (MEK) inhibitors synergistically inhibited cell growth in vitro. Treatment of RD cells with AZD8055 plus AZD6244 blocked reciprocal pathway activation, as evidenced by reduced AKT/ERK/S6 phosphorylation. In vivo, the synergistic effect on growth and changes in pharmacodynamic biomarkers was recapitulated using the AZD8055/AZD6244 combination but not NVP-BEZ235/AZD6244. Pharmacokinetic analysis provided evidence of drug-drug interaction with both combinations. CONCLUSIONS:Dual PI3K/MAPK pathway activation and compensatory signaling in both rhabdomyosarcoma subtypes predict a lack of clinical efficacy for single agents targeting either pathway, supporting a therapeutic strategy combining a TORC1/2 with a MEK inhibitor.

Project description:BackgroundRenal cell carcinoma (RCC) is a fatal disease, in which the PI3K/AKT/mTOR signaling pathway serves an important role in the tumorigenesis. Previous studies have reported the prognostic significance of PI3K/AKT/mTOR signaling pathway members in RCC; however, there is insufficient evidence to date to confirm this. Thus, the present study aimed to systematically investigate the prognostic roles of multiple PI3K/AKT/mTOR signaling proteins in clear cell RCC (ccRCC) using online large-scale databases.MethodsThe mRNA expression profiles of PI3K/AKT/mTOR signaling pathway proteins PTEN, PIK3CA, PIK3CB, PIK3CD, PIK3CG, AKT1, AKT2, AKT3 and mTOR were investigated using the Gene Expression Profiling Interactive Analysis (GEPIA) and Oncomine databases, and the protein expression levels of PI3K, AKT and mTOR were detected using western blotting (WB) analysis. In addition, the correlation between mRNA or protein expression levels and the prognostic significance was analyzed using the Kaplan-Meier (K-M) plotter (n = 530), the Human Protein Atlas (HPA; n = 528) and The Cancer Protein Atlas (TCPA; n = 445) databases.ResultsThe GEPIA revealed that the mRNA expression of major PI3K/AKT/mTOR pathway members, including PTEN, PIK3CA, PIK3CB, AKT1, AKT2 and AKT3, were negatively correlated with ccRCC stages (P < 0.05), though most of their mRNA and protein expression levels were notsignificantly different between ccRCC and normal tissues using GEPIA, Oncomine and WB analyses (P < 0.05). Meanwhile, using the K-M plotter and HPA prognostic analysis, it was found that the mRNA expression levels of the majority of the PI3K/AKT/mTOR signaling pathway members, including PTEN, PIK3CA, PIK3CB, PIK3CG, AKT3 and mTOR were positively correlated with overall survival (OS), whereas PIK3CD mRNA expression was negatively correlated with OS (P < 0.05). Furthermore, TCPA prognostic analysis observed that several of the key molecules of the PI3K/AKT/mTOR signaling pathway [PTEN, p-AKT (S473) and p-mTOR (S2448)] were also positively correlated with OS in patients with ccRCC (P < 0.05). In conclusion, the present study suggested that several members of the PI3K/AKT/mTOR signaling pathway, especially PTEN, may be favorable prognostic factors in ccRCC, which indicated that the PI3K/AKT/mTOR signaling pathway may be implicated in ccRCC initiation and progression.

Project description:Glioblastoma is a high-grade glioma with poor prognosis even after surgery and standard therapy. Here, we asked whether carnosine (β-alanyl-L-histidine), a naturally occurring dipeptide, exert its anti-neoplastic effect on glioblastoma cells via PI3K/Akt/mTOR signaling. Therefore, glioblastoma cells from the lines U87 and T98G were exposed to carnosine, to the mTOR inhibitor rapamycin and to the PI3K inhibitor Ly-294,002. Pyruvate dehydrogenase kinase (PDK4) expression, known to be a target of PI3K/Akt/mTOR, and which is also affected by carnosine, was analyzed by RT-qPCR, and reporter gene assays with the human PDK4 promoter were performed. Cell viability was assessed by cell-based assays and mTOR and Akt phosphorylation by Western blotting. Rapamycin and Ly-294,002 increased PDK4 mRNA expression in both cell lines but significance was only reached in U87. Carnosine significantly increased expression in both lines. A significant combinatorial effect of carnosine was only detected in U87 when the dipeptide was combined with Ly-294,002. Reporter gene assays revealed no specific effect of carnosine on the human PDK4 promoter, whereas both inhibitors increased reporter gene expression. Rapamycin reduced phosphorylation of mTOR, and Ly-294,002 that of Akt. A significant reduction of Akt phosphorylation was observed in the presence of carnosine in U87 but not in T98G, and carnosine had no effect on mTOR phosphorylation. Cell viability as determined by ATP in cell lysates was reduced only in the presence of carnosine. We conclude that carnosine's anti-neoplastic effect is independent from PI3K/Akt/mTOR signaling. As the dipeptide reduced viability in tumor cells that do not respond to PI3K or mTOR inhibitors, it appears to be worth to further investigate the mechanisms by which carnosine exerts its anti-tumor effect and to consider it for therapy, especially as it is a naturally occurring compound that has already been used for the treatment of other diseases without indication of side-effects.

Dataset Information

PI3K-Akt-mTOR inhibition by GNE-477 inhibits renal cell carcinoma cell growth in vitro and in vivo.

Publications

PI3K-Akt-mTOR inhibition by GNE-477 inhibits renal cell carcinoma cell growth <i>in vitro</i> and <i>in vivo</i>.

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