Omipalisib Inhibits Esophageal Squamous Cell Carcinoma Growth Through Inactivation of Phosphoinositide 3-Kinase (PI3K)/AKT/Mammalian Target of Rapamycin (mTOR) and ERK Signaling

Abstract

BACKGROUND Esophageal squamous cell carcinoma (ESCC) is a life-threatening digestive tract malignancy with no known curative treatment. This study aimed to investigate the antineoplastic effects of omipalisib and its underlying molecular mechanisms in ESCC using a high throughput screen. MATERIAL AND METHODS MTT assay and clone formation were used to determine cell viability and proliferation. Flow cytometry was conducted to detect cell cycle distribution and apoptosis. Global gene expression and mRNA expression levels were determined by RNA sequencing and real-time PCR, respectively. Protein expression was evaluated in the 4 ESCC cell lines by Western blot analysis. Finally, a xenograft nude mouse model was used to evaluate the effect of omipalisib on tumor growth in vivo. RESULTS In the pilot screening of a 1404-compound library, we demonstrated that omipalisib markedly inhibited cell proliferation in a panel of ESCC cell lines. Mechanistically, omipalisib induced G₀/G₁ cell cycle arrest and apoptosis. RNA-seq, KEGG, and GSEA analyses revealed that the PI3K/AKT/mTOR pathway is the prominent target of omipalisib in ESCC cells. Treatment with omipalisib decreased expression of p-AKT, p-4EBP1, p-p70S6K, p-S6, and p-ERK, therefore disrupting the activation of PI3K/AKT/mTOR and ERK signaling. In the nude mouse xenograft model, omipalisib significantly suppressed the tumor growth in ESCC tumor-bearing mice without obvious adverse effects. CONCLUSIONS Omipalisib inhibited the proliferation and growth of ESCC by disrupting PI3K/AKT/mTOR and ERK signaling. The present study supports the rationale for using omipalisib as a therapeutic approach in ESCC patients. Further clinical studies are needed.

Figure 1

Omipalisib was identified as a potential agent against Esophageal squamous cell carcinoma (ESCC). (A) MTT assay was used to screen compounds in a 1404-compound library. (B) In total, 13 compounds showed inhibitory effects on 4 ESCC cell lines. (C) Chemical structure of omipalisib.

Figure 2

PIK3CA and mTOR were highly expressed in Esophageal squamous cell carcinoma (ESCC) patients. (A, B) The PIK3CA and mTOR expression levels in all tumor samples. (C, D) The PIK3CA and mTOR mRNA expression in esophageal cancer samples and normal tissues (* P<0.05).

Figure 3

Omipalisib inhibited the proliferation of Esophageal squamous cell carcinoma (ESCC) cells. (A) Representative data of 3 independent experiments in cell viability of ESCC exposed to a series of concentrations of omipalisib for 72 hours. (B, C) Representative image of clone formation assay for ESCC cells treated with omipalisib for 72 hours. Clone numbers were calculated. (D) ESCC cells were treated with a series of omipalisib and cisplatin concentrations for 72 hours, and cell viability was measured by MTT assay. (* P<0.05, ** P<0.01, *** P<0.001).

Figure 4

Omipalisib induced cell cycle arrest of Esophageal squamous cell carcinoma (ESCC) cells. (A) Representative data of 3 independent experiments in cell cycle distribution determined by flow cytometry in ESCC cells exposed to omipalisib for 24 hours. (B) Representative image of protein expression of 3 independent experiments in 4 ESCC cells exposed to omipalisib for 24 hours. (C) Density analysis of protein bands performed with ImageJ software. Plot is the average of 3 independent experiments. (* P<0.05, ** P<0.01, *** P<0.001).

Figure 5

Omipalisib induced apoptosis in Esophageal squamous cell carcinoma (ESCC) cells. (A) Representative image of apoptosis in ESCC cells. (B) Apoptotic cells in ESCC cells was quantified. (* P<0.05, ** P<0.01, *** P<0.001).

Figure 6

Identification of potential signaling pathways of omipalisib with RNA-seq assays. (A, B) DEGs indicated that the mTOR signaling pathway is the major pathway affected by omipalisib treatment according to KEGG pathway enrichment analysis (A) and GSEA (B). (C) qRT-PCR assays showed obvious alterations in DDIT4 and CCND1 gene expression, and the tendency was consistent with the RNA-seq results. (*** P<0.001).

Figure 7

Omipalisib decreased phosphorylation of proteins in mTOR and ERK signaling pathway. (A) Esophageal squamous cell carcinoma (ESCC) cells were treated with the various concentrations of omipalisib for 24 hours. The protein expression levels of 4EBP1, p-4EBP1, p70S6K, p-p70S6k, S6, p-S6, AKT, and p-AKT were identified by Western blot analysis. (B) ERK and p-ERK protein levels in the 4 ESCC cell lines treated with omipalisib at the indicated concentrations for 24 hours. (C) Density analysis of protein bands performed with ImageJ software. Plot is the average of 3 independent experiments. (* P<0.05, ** P<0.01, *** P<0.001).

Figure 8

Omipalisib inhibited the growth of Esophageal squamous cell carcinoma (ESCC) in vivo. (A) Representative image of mice administered different doses of omipalisib at the end of the experiment. (B) Tumor volume was calculated by the following equation: volume=long diameter×(short diameter)²/2. (C) Body weight was recorded every 2 days during the course of omipalisib administration. (D) Protein expression in tumor tissue from KYSE150-bearing mice by Western blot. (E) Density analysis of protein bands were performed with ImageJ software. (* P<0.05, ** P<0.01, *** P<0.001).