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. 2019 Jul 3;1(2):56-63.
doi: 10.1016/j.sopen.2019.05.004. eCollection 2019 Oct.

Antineoplastic effects of auranofin in human pancreatic adenocarcinoma preclinical models

Mayrim V Rios Perez  1 , David Roife  2 , Bingbing Dai  3 , Michael Pratt  4 , Ryszard Dobrowolski  5 , Ya'an Kang  3 , Xinqun Li  3 , Jithesh J Augustine  3 , Rafal Zielinski  6 , Waldemar Priebe  6 , Jason B Fleming  2
Affiliations

Antineoplastic effects of auranofin in human pancreatic adenocarcinoma preclinical models

Mayrim V Rios Perez et al. Surg Open Sci. .

Abstract

Background: Auranofin, a Food and Drug Administration-approved anti-rheumatic agent with anticancer properties for lung and ovarian cancer, has never been studied for pancreatic cancer. We hypothesize that auranofin may prevent pancreatic ductal adenocarcinoma progression by inhibition of Txnrd1 and HIF-1α.

Methods: In vitro sensitivity of human pancreatic ductal adenocarcinoma cell lines was determined based on IC50. Western blot assays were used to interrogate mechanisms of apoptosis and resistance. Ex vivo live tissue slice assays of xenografts allowed for testing of a larger number of PDX samples with high efficiency. In vivo pancreatic ductal adenocarcinoma orthotopic mouse models using MiaPaCa-2 Luc + cells were designed to determine optimal dose and antitumor effect.

Results: We found that 10 of 15 tested pancreatic ductal adenocarcinoma cell lines were sensitive to auranofin based on IC50s below 5 μmol/L. Ex vivo tissue growth inhibition greater than 44% was observed for 13 PDX tissue cases treated with 10 μmol/L auranofin. High Txnrd1 expression was observed for resistant cell lines. In vivo studies showed 15 mg/kg IP as the optimal dose with absence of gross solid organ metastasis up to 13 weeks post-treatment (median survival 8 and 12 weeks, respectively; P = .0953).

Conclusions: We have demonstrated that auranofin prevents pancreatic ductal adenocarcinoma progression using multiple models. Our study suggests inhibition of Txnrd1 and HIF-1α as possible mechanisms of action, and Txnrd1 as a biomarker of resistance. Based on these data, an off-label Phase 0 clinical trial with this FDA-approved drug should be considered for patients with pancreatic cancer.

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Figures

Fig. 1
Fig. 1
Live tissue slice assay for chemosensitivities of pancreatic adenocarcinoma patient-derived xenografts. (A and B) Representative figures showing the testing of a panel of FDA-approved or clinical testing agents on patient-derived xenograft tissue slices of PDAC using a live tissue slice assay (LTSA). Auranofin was noticed to be more active by decreasing tissue viability at a higher degree, when compared to other agents [Gemcitabine, Irinotecan, AZD6244 (MEK inhibitor), MK2206 (AKT inhibitor), Sunitinib (receptor protein-tyrosine kinase inhibitor), Dual Antiplatelet Therapy (DAPT), Crizotinib (ALK/c-Met/HGFR inhibitor), AZD2281 (PARP inhibitor) and untreated tumor xenograft tissue slices. (C) Normalized tissue viability was calculated for 13 tested tissue samples, and growth inhibition (%Inhibition = 100 – %Normalized Viability) induced by auranofin was found to be 44–95%.
Fig. 2
Fig. 2
Mechanisms of action of auranofin in human pancreatic adenocarcinoma models. (A) MTT assay was performed to assess for cell viability 72 hours after auranofin treatment at different concentrations. (B) IC50 determination for 15 human PDAC cell lines, 10 of which were defined as auranofin-sensitive (in green) based on IC50 < 5 μmol/L. The remaining cell lines were defined as auranofin-resistant (in red) due to IC50 ≥ 5μmol/L. (C) Western blot for Txnrd1 and baseline expression was performed for a panel of 13 human PDAC cell lines. Dotted vertical line divides cell lines in 2 subgroups based on IC50. (D) Txnrd1 protein expression and IC50 from human PDAC cell lines were found with a statistically significant positive correlation. (E) Western blot analysis was performed following low-dose (0.5–1 μmol/L) auranofin treatment showing higher PARP cleavage for MiaPaCa-2 and MDA-PATC53 (auranofin-sensitive), unlike auranofin-resistant cell lines SW1990 and AsPc-1. β-Actin was used as a loading control, and 0.1% DMSO used as the control treatment.
Fig. 3
Fig. 3
Hypoxia enhances antiangiogenesis mechanism of auranofin in human PDAC. Inhibition of Hif1α was observed at 6 hours post-treatment for MDA-PATC53 under hypoxia (O2 < 1%), which was not present during normoxia. (B) Treatment of PDX79-F4 with auranofin 10 μmol/L induced a statistically significant inhibition of tissue viability under both normoxia and hypoxia, and trended towards a bigger difference under hypoxia. (C) Human VEGF ELISA of tissue culture media from tissue slices of PDX79 revealed inhibition of VEGF secretion in all auranofin-treated groups, but became statistically significant under hypoxia at 6 and 24 hours.
Fig. 4
Fig. 4
Auranofin delays progression and prolongs survival of nude mice with MiaPaCa-2 tumors. MiaPaCa-2-Luc + cells (2.5 × 105 cells in 1:1 PBS to Matrigel ratio) were orthotopically implanted in twenty 6-week-old female nude mice with 100% take rate confirmed by BLI (not shown) 2-weeks post-implantation. Mice were randomized (5 mice/group), and treated Monday to Friday once a day with: Vehicle, Auranofin 5, 10, or 15 mg/kg as intraperitoneal injections. Mice were euthanized when: abdominal distension or large tumor burden (> 2 cm) occurred. Mice treated with 15 mg/kg auranofin trended towards longer survival (P = .095). (B) BLI (in radiance × 106 p/sec/cm2/sr) was recorded twice a week over the first 20 days, until the first mortality occurred. Groups treated with 10 and 15 mg/kg at day 20 were found to have significantly less luminescence compared to the control group, implying lower tumor burdens (*). (C) Representative photos showing the lack of gross solid organ metastasis for mice in the 15 mg/kg group versus the control treatment group. Total metastatic event occurrence (lower left) and ascites (lower right) were recorded during necropsy, with proportions showing less of either as treatment dosage increased.
Fig. 5
Fig. 5
Auranofin biodistribution, mouse weight change, and MiaPaCa-2 tumor Hif1α expression. Gold content was quantified from different tissues (tumor, liver, and left kidney) retrieved at the time of death (8–9 weeks and 12–14 weeks) for one mouse (M) per group as shown in the figure, to demonstrate gold biodistribution. Vehicle group was omitted due to levels below level of detection. (B) Intratumoral gold (μg of gold per g of tissue) was analyzed from tissue harvested at the time of death. (C) Mice weights were recorded weekly and were statistically significant different than control over the first 4 weeks post-treatment versus control (*). (D) Representative figure of Hif1α tissue expression for MiaPaCa-2 tumors under different doses of auranofin treatment, indicating less expression of Hif1α with higher doses of auranofin.
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