The aurora kinase A inhibitor MLN8237 enhances cisplatin-induced cell death in esophageal adenocarcinoma cells

Abstract

Esophageal adenocarcinomas are poorly responsive to chemotherapeutics. This study aimed to determine the levels of Aurora kinase A (AURKA) and the therapeutic potential of MLN8237, an investigational AURKA inhibitor, alone and in combination with cisplatin. Using quantitative real-time PCR, we detected frequent AURKA gene amplification (15 of 34, 44%) and mRNA overexpression (37 of 44, 84%) in esophageal adenocarcinomas (P < 0.01). Immunohistochemical analysis showed overexpression of AURKA in more than two-thirds of esophageal adenocarcinoma tissue samples (92 of 132, 70%; P < 0.001). Using FLO-1, OE19, and OE33 esophageal adenocarinoma cell lines, with constitutive AURKA overexpression and mutant p53, we observed inhibition of colony formation with a single treatment of 0.5 μmol/L MLN8237 (P < 0.05). This effect was further enhanced in combination with 2.5 μmol/L cisplatin (P < 0.001). Twenty-four hours after treatment with the MLN8237 or MLN8237 and cisplatin, cell-cycle analyses showed a sharp increase in the percentage of polyploid cells (P < 0.001). This was followed by an increase in the percentage of cells in the sub-G(1) phase at 72 hours, concordant with the occurrence of cell death (P < 0.001). Western blot analysis showed higher induction of TAp73β, PUMA, NOXA, cleaved caspase-3, and cleaved PARP with the combined treatment, as compared with a single-agent treatment. Using xenograft models, we showed an enhanced antitumor role for the MLN8237 and cisplatin combination, as compared with single-agent treatments (P < 0.001). In conclusion, this study shows frequent overexpression of AURKA and suggests that MLN8237 could be an effective antitumor agent, which can be combined with cisplatin for a better therapeutic outcome in esophageal adenocarcinomas.

Figure 1. AURKA is significantly amplified and overexpressed in esophageal adenocarcinoma tissues

(A) Representative images of AURKA immunostaining in tumor (EAC) and histologically normal non tumor (NT) tissue samples are shown at x20 and x40. The Immunohistochemical analysis of AURKA protein expression demonstrated significant overexpression of AURKA in 70% of EACs (Average CES: 6.7±0.28) (**p<.001). (B) qPCR analysis of AURKA DNA in normal esophageal (n=14) and EAC (n=34) tissue samples demonstrates frequent DNA amplification (≥1.5 fold) in EAC (44.1%) tissues. (C) qRT-PCR analysis of AURKA mRNA in normal esophageal (n=23) and EAC (n=44) tissue samples show frequent mRNA overexpression (≥2.0 fold) in EAC (84.1%) tissues samples. (D) Chemical structure of MLN8237 (Millennium Pharmaceuticals, Inc.,).

Figure 2. MLN8237 and CDDP combination treatment promotes polyploidy and alters cell cycle progression

(A) OE33 EAC cells were treated with MLN8237 (0.5μM) and/or CDDP (2.5μM) for 24hr and cell cycle progression was analyzed with flow cytometry. MLN8237 (0.5μM) treatment alone and in combination with CDDP (2.5μM) induces G2-M arrest and polyploidy. (B) After 24hr of treatment, OE33 cells were incubated for 48hr in drug free medium and cell cycle progression was analyzed. MLN8237 (0.5μM) and CDDP (2.5μM) combination treatment significantly enhanced the percentage of sub-G1-phase cells. CV - Control Vehicle; MLN - MLN8237; CDDP - Cisplatin.

Figure 3. MLN8237 and CDDP combination treatment significantly inhibits cell survival and enhances apoptosis

(A & B) The cell survival assay data indicates significant inhibition of FLO-1 and OE33 EAC cell survival after treatment with MLN8237 and CDDP combination. (C & D) FLO-1 and OE33 EAC cells were treated with MLN8237 (0.5μM) and/or CDDP (2.5μM) for 24hr and incubated in drug free medium for 48hr. Subsequently, expression of apoptotic proteins and TAp73β activity was evaluated. MLN8237 (0.5μM) and CDDP (2.5μM) combination treatment significantly enhanced expression of apoptotic proteins and mRNA levels of TAp73β pro-apoptotic transcriptional targets, PUMA and NOXA, in EAC cells. CV - Control Vehicle; MLN 0.5 - MLN8237 0.5μM; CDDP 2.5 - Cisplatin 2.5μM & CDDP 5.0 - Cisplatin 5.0μM, * p<0.05 and ** p<0.01.

Figure 4. MLN8237 and CDDP combination treatment exhibits enhanced anti-tumor activity in vivo

FLO-1 and OE33 tumor xenografts were treated with MLN8237 (30mg/kg) and/or CDDP (2mg/kg) for 21 days and tumor size was measured every alternate day. (A & B) The data indicates that MLN8237 (30mg/kg) and CDDP (2mg/kg) combination treatment has significantly enhanced anti-tumor activity against FLO-1 and OE33 tumor xenografts. MLN - MLN8237; CDDP – Cisplatin, * p<0.05 and ** p<0.01.

Figure 5. MLN8237 and CDDP combination treatment inhibits tumor proliferation and enhances apoptotic marker expression in tumor xenografts

OE33 tumor xenografts were treated with MLN8237 (30mg/kg) and/or CDDP (2mg/kg) for 21 days. Subsequently, tumors were isolated and immunohistochemical analyses were done to measure Ki-67 and p73 expression. (A) The data indicates that MLN8237 (30mg/kg) and CDDP (2mg/kg) combination treatment significantly inhibits Ki-67 expression in OE33 tumor xenografts. (B) Combination treatment with MLN8237 (30mg/kg) and CDDP (2mg/kg) exhibits enhanced nuclear p73 protein expression in OE33 tumor xenografts. MLN - MLN8237; CDDP - Cisplatin, * p<0.05 and ** p<0.01.

Figure 6. MLN8237 and CDDP combination treatment induces TAp73β transcriptional activity and cleaved caspase 3 expression in tumor xenografts

OE33 tumor xenografts were treated with MLN8237 (30mg/kg) and/or CDDP (2mg/kg) for 21 days. Subsequently, tumors were extracted and immunohistochemical analyses were done to measure cleaved caspase 3 expression. (A) The data indicates that MLN8237 (30mg/kg) and CDDP (2mg/kg) combination treatment exhibits enhanced cleaved caspase 3 protein expression in OE33 tumor xenografts. Additionally, RNA was isolated and qRT-PCR was done to analyze mRNA levels of TAp73β pro-apoptotic transcriptional targets, PUMA and NOXA. (B & C) The data indicates that MLN8237 (30mg/kg) and CDDP (2mg/kg) combination treatment significantly enhances PUMA and NOXA mRNA levels in OE33 tumor xenografts. MLN - MLN8237; CDDP - Cisplatin, * p<0.05 and ** p<0.01.