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. 2017 Nov;153(5):1429-1443.e5.
doi: 10.1053/j.gastro.2017.07.036. Epub 2017 Jul 29.

Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

Yangchun Xie  1 , Shan Zhu  2 , Meizuo Zhong  3 , Manhua Yang  4 , Xiaofan Sun  2 , Jinbao Liu  2 , Guido Kroemer  5 , Michael Lotze  6 , Herbert J Zeh 3rd  6 , Rui Kang  6 , Daolin Tang  7
Affiliations

Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

Yangchun Xie et al. Gastroenterology. 2017 Nov.

Abstract

Background & aims: Induction of nonapoptotic cell death could be an approach to eliminate apoptosis-resistant tumors. We investigated necroptosis-based therapies in mouse models of pancreatic ductal adenocarcinoma cancer (PDAC).

Methods: We screened 273 commercially available kinase inhibitors for cytotoxicity against a human PDAC cell line (PANC1). We evaluated the ability of the aurora kinase inhibitor CCT137690 to stimulate necroptosis in PDAC cell lines (PANC1, PANC2.03, CFPAC1, MiaPaCa2, BxPc3, and PANC02) and the HEK293 cell line, measuring loss of plasma membrane integrity, gain in cell volume, swollen organelles, and cytoplasmic vacuoles. We tested the effects of CCT137690 in colon formation assays, and the effects of the necroptosis (necrostatin-1 and necrosulfonamide), apoptosis, autophagy, and ferroptosis inhibitors. We derived cells from tumors that developed in Pdx1-Cre;K-RasG12D/+;p53R172H/+ (KPC) mice. Genes encoding proteins in cell death pathways were knocked out, knocked down, or expressed from transgenes in PDAC cell lines. Athymic nude or B6 mice were given subcutaneous injections of PDAC cells or tail-vein injections of KPC tumor cells. Mice were given CCT137690 (80 mg/kg) or vehicle and tumor growth was monitored; tumor tissues were collected and analyzed by immunohistochemistry. We compared gene expression levels between human pancreatic cancer tissues (n = 130) with patient survival times using the online R2 genomics analysis and visualization platform.

Results: CCT137690 induced necrosis-like death in PDAC cell lines and reduced colony formation; these effects required RIPK1, RIPK3, and MLKL, as well as inhibition of aurora kinase A (AURKA). AURKA interacted directly with RIPK1 and RIPK3 to reduce necrosome activation. AURKA-mediated phosphorylation of glycogen synthase kinase 3 beta (GSK3β) at serine 9 inhibited activation of the RIPK3 and MLKL necrosome. Mutations in AURKA (D274A) or GSK3β (S9A), or pharmacologic inhibitors of RIPK1 signaling via RIPK3 and MLKL, reduced the cytotoxic activity of CCT137690 in PDAC cells. Oral administration of CCT137690 induced necroptosis and immunogenic cell death in subcutaneous and orthotopic tumors in mice, and reduced tumor growth and tumor cell phosphorylation of AURKA and GSK3β. CCT137690 increased survival times of mice with orthotopic KPC PDACs and reduced tumor growth, stroma, and metastasis. Increased expression of AURKA and GSK3β mRNAs associated with shorter survival times of patients with pancreatic cancer.

Conclusions: We identified the aurora kinase inhibitor CCT137690 as an agent that induces necrosis-like death in PDAC cells, via RIPK1, RIPK3, and MLKL. CCT137690 slowed growth of orthotopic tumors from PDAC cells in mice, and expression of AURKA and GSK3β associate with patient survival times. AURKA might be targeted for treatment of pancreatic cancer.

Keywords: ATP; Antitumor Immunity; HMGB1; Regulated Cell Death.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. Identification of CCT137690 as a novel anticancer agent limiting PDAC cells
(A, B) PANC1 cells were treated with a kinase inhibitor (10 μM) for 24 hours and then cell viability was assayed. Ranking of the anticancer activity of 273 kinase inhibitors is shown by the heat map; one block represents a kinase inhibitor (A). The top five anti-cancer kinase inhibitors are shown in panel B (n=3, *p < 0.05 versus untreated group). (C) Indicated PDAC or normal HPDE cells were treated with CCT137690 (2.5–40 μM) for 24 hours, and then cell viability was assayed. (D, E) Clonogenic cell survival assay determined the reproductive ability of a cell after treatment with CCT137690 (10 μM) (n=3, *p < 0.05 versus untreated group).
Figure 2
Figure 2. Induction of necroptosis contributes to the anticancer activity of CCT137690
(A) Necrotic-like morphology was observed in PANC1 cells following treatment with CCT137690 (10 μM) for 24 hours. (B) Western blot analysis of indicated proteins in PANC1 and PANC2.03 cells following treatment with CCT137690 (2.5–10 μM) for 24 hours (n=3, *p < 0.05 versus untreated group). (C, D) PANC1 and PANC2.03 cells were treated with CCT137690 (C) or staurosporine (D) or erastin (D) in the absence or presence of indicated cell death inhibitors for 24 hours. Cell viability was assayed (n=3, *p < 0.05). (E) Indicated MEFs were treated with CCT137690 (10 μM) for 24 hours, and then cell viability was assayed (n=3, *p < 0.05). (F–I) Knockdown of RIPK1, RIPK3, and MLKL by shRNAs inhibited CCT137690 (10 μM, 24 hours)-induced cell death and HMGB1 and ATP release (n=3, *p < 0.05 versus control shRNA group).
Figure 3
Figure 3. AURKA inhibits necroptosis in a cell cycle-independent manner
(A) Western blot analysis of indicated proteins in PDAC cells following treatment with CCT137690 (2.5–10 μM) for 24 hours (n=3, *p < 0.05 versus untreated group). AU=arbitrary unit. (B) Q-PCR analysis AURKA mRNA in stable AURKA-knockdown cells (n=3, *p < 0.05 versus control shRNA group). (C, D) Clonogenic cell survival assay determines the reproductive ability of AURKA-knockdown cells in the absence or presence of indicated cell death inhibitors (n=3, *p < 0.05). (E, F) ELISA analysis of HWGB1 and ATP levels in culture supernatants (n=3, *p < 0.05 versus AURKA shRNA group). (G) Cell cycle analysis in AURKA-knockdown cells. (H) Cell cycle analysis in PANC1 and PANC2.03 cells following CCT137690 (10 μM), genistein (50 μM), and doxorubicin (0.2 μg/ml) for 24 hours. (I) Indicated MEFs were treated with CCT137690 (10 μM), genistein (50 μM), and doxorubicin (0.2 μg/ml) for 24 hours, and cell viability was assayed (n=3, *p < 0.05 versus WT group). (J) PANC1 cells were treated with CCT137690 (10 μM), genistein (50 μM), and doxorubicin (0.2 μg/l) in the absence or presence of indicated cell death inhibitors for 24 hours, and then cell viability was assayed (n=3, *p < 0.05 versus control group).
Figure 4
Figure 4. AURKA inhibits necroptosis by binding the RIPK1/RIPK3 necrosome
(A) Western blot analysis of indicated proteins in PDAC cells following treatment with CCT137690 (10 μM) for three to 24 hours (n=3, *p < 0.05 versus untreated group). (B) Western blot analysis of indicated proteins in control and stable AURKA-knockdown PDAC cells (n=3, *p < 0.05 versus control shRNA group). (C) Immunoprecipitation (IP) analysis of the levels of RIPK3 binding to RIPK1 and MLKL in PANC1 and PANC2.03 cells following treatment with CCT137690 (10 μM) for 24 hours. (D) IP analysis of the levels of RIPK3 binding to RIPK1 and MLKL in control and stable AURKA-knockdown PANC1 and PANC2.03 cells. (E) IP analysis of the levels of AURKA binding to RIPK1, RIPK3, and MLKL in PANC1 and PANC2.03 cells following treatment with CCT137690 (10 μM) for 24 hours. (F) IP analysis of the levels of AURKA binding to RIPK1, RIPK3, and MLKL in HEK293 cells after expressed AURKA wild type and D274A mutant. (G) PANC1 cells were treated with CCT137690 (10 μM) or TNF (50 ng/ml) in the absence or presence of anti-TNFR1 antibody (1 mg/ml) for 24 hours and cell death was analyzed (n=3, *p < 0.05).
Figure 5
Figure 5. GSK3β contributes to AURKA-mediated necroptosis inhibition
(A) Western blot analysis of indicated proteins in PDAC cells following treatment with CCT137690 (10 μM) for three to 24 hours (n=3, *p < 0.05 versus untreated group). (B) Western blot analysis of indicated proteins in control and stable AURKA-knockdown PANC1 and PANC2.03 cells (n=3, *p < 0.05 versus control shRNA group). (C, D) PANC1 and PANC2.03 cells were treated with XXVI (C) or AR-A014418 (D) in the absence or presence of indicated cell death inhibitors for 24 hours. Cell viability was assayed (n=3, *p < 0.05). (E) Indicated MEFs were treated with XXVI or AR-A014418 for 24 hours, and then cell viability was assayed (n=3, *p < 0.05). (F) Knockdown of RIPK3 and MLKL, but not RIPK1, inhibited XXVI- or AR-A014418-induced cell death (n=3, *p < 0.05 versus control shRNA group). (G–I) HEK293 cells were expressed with GSK3β WT and S9A mutant and then treated with CCT137690 (10 μM) for 24 hours. Protein level (G), cell viability (H), caspase-3 activity (H), and complex formation (I) were assayed.
Figure 6
Figure 6. Anticancer activity of CCT137690 in subcutaneous tumor models
(A) Athymic nude mice were injected subcutaneously with PANC1 cells (5 × 106 cells/mouse) and treated with CCT137690 (80 mg/kg/p.o., twice every other day) at day seven for two weeks. Tumor volume was calculated weekly (n=10 mice/group, * p < 0.05). (B) Representative photographs of isolated tumors at day 34 after treatment. (C) ELISA analysis of serum HMGB1 levels in mice at day 34 after treatment (n=10 mice/group, * p < 0.05). (D) Western blot analysis of indicated protein expressions in isolated tumor at day 34 after treatment. (E–G) Average body weight (E) and serum ALT (F) and BUN (G) levels in mice at day 34 after treatment (n=10 mice/group, * p < 0.05). (H–L) B6 mice were injected subcutaneously with PANC02 cells (1 × 106 cells/mouse) or KPC cells (1 × 106 cells/mouse) and treated with CCT137690 (80 mg/kg/p.o., twice every other day) at day seven for two weeks. Tumor volume was calculated weekly (H, I). The serum HMGB1 (J) and ATP (K) levels and frequency of IFN-γ-producing tumor-infiltrating CD8+ T cells (L) were assayed in isolated tumor at day 34 after treatment (n=10 mice/group, * p < 0.05). (M) Athymic nude mice were injected subcutaneously with PANC1 cells (5 × 106 cells/mouse) and treated with CCT137690 (40 mg/kg/p.o., twice every other day) and/or gemcitabine (20 mg/kg/i.p., once every other day) at day 14 for two weeks. Tumor volume was calculated weekly (n=8 mice/group, * p < 0.05).
Figure 7
Figure 7. Anticancer activity of CCT137690 in orthotopic tumor and genetically engineered mouse models
(A) Injection of CCT137690 (80 mg/kg/p.o., twice every other day, at day seven for two weeks) prolonged animal survival of orthotopic KPC (1 × 106 cells/mouse) tumor models (n=10 mice/group, * p < 0.05). (B) Representative photomicrographs of isolated tumors at day 21 after treatment. (C) Average pancreas weight at day 21 following treatment (n=10 mice/group, * p < 0.05). (D, E) Histological analysis of pancreas at day 21 after treatment (bar=400 03BCm, n=5 mice/group). (F, G) Histological analysis of liver at day 21 after treatment (bar=400 03BCm, n=5 mice/group). (H–J) Analysis of serum HMGB1 (H), serum ATP (I), and frequency of IFN-γ-producing tumor-infiltrating CD8+ T cells (H) in mice at day 21 after treatment (n=10 mice/group, * p < 0.05). (K) Western blot analysis of indicated protein expression in isolated tumor at day 21. (L–N) Injection of CCT137690 (80 mg/kg/p.o., twice every other day, for one month) reduced pancreatic weight and ductal/stromal response in KC mice at the age of 10 months (bar=400 03BCm). (O–Q) Analysis of serum HMGB1 (O), serum ATP (P), and frequency of IFN-γ-producing tumor-infiltrating CD8+ T cells (Q) in mice at day 30 after treatment (n=10 mice/group, * p < 0.05). (R) Western blot analysis of indicated protein expression in isolated tumor at day 30 after treatment.
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