Targeting thiol isomerase activity with zafirlukast to treat ovarian cancer from the bench to clinic

Abstract

Thiol isomerases, including PDI, ERp57, ERp5, and ERp72, play important and distinct roles in cancer progression, cancer cell signaling, and metastasis. We recently discovered that zafirlukast, an FDA-approved medication for asthma, is a pan-thiol isomerase inhibitor. Zafirlukast inhibited the growth of multiple cancer cell lines with an IC₅₀ in the low micromolar range, while also inhibiting cellular thiol isomerase activity, EGFR activation, and downstream phosphorylation of Gab1. Zafirlukast also blocked the procoagulant activity of OVCAR8 cells by inhibiting tissue factor-dependent Factor Xa generation. In an ovarian cancer xenograft model, statistically significant differences in tumor size between control vs treated groups were observed by Day 18. Zafirlukast also significantly reduced the number and size of metastatic tumors found within the lungs of the mock-treated controls. When added to a chemotherapeutic regimen, zafirlukast significantly reduced growth, by 38% compared with the mice receiving only the chemotherapeutic treatment, and by 83% over untreated controls. Finally, we conducted a pilot clinical trial in women with tumor marker-only (CA-125) relapsed ovarian cancer, where the rate of rise of CA-125 was significantly reduced following treatment with zafirlukast, while no severe adverse events were reported. Thiol isomerase inhibition with zafirlukast represents a novel, well-tolerated therapeutic in the treatment of ovarian cancer.

Keywords: CA-125; ERP72; ERp5; ERp57; PDI; drug repurposing; montelukast.

Figure 1 –

Zafirlukast and montelukast are broad-spectrum thiol isomerase inhibitors. The structures of zafirlukast (A) and montelukast (B). Both zafirlukast (C) and montelukast (D) inhibit the thiol isomerases PDI, ERp5, ERp57, and ERp72 in a concentration-dependent manner when examined via the insulin turbidity assay. (E) The structure of a zafirlukast analogue missing the cyclopentyl moiety, decreasing or losing its affinity for the leukotriene receptor. (F) The analogue inhibits PDI, ERp5, ERp57, and ERp72 similarly to zafirlukast.

Figure 2 –

Cellular thiol isomerase activity is inhibited by zafirlukast. (A) Cellular thiol isomerase activity is inhibited by zafirlukast treatment in a concentration dependent manner as measured by di-eosin-GSSG fluorescence (n=4) Data is presented as mean ±SD. One-way ANOVA and a post-hoc Dunnett’s test where *p=0.0217 for 3 µM zafirlukast, **p=0.0020 for 10 µM zafirlukast and ****p<0.0001 for 30 µM zafirlukast compared to the control. (B) Levels of PDI, ERp5, ERp57, and ERp72 remain similar with increasing concentrations of zafirlukast in OVCAR8 cells (n=3). Data is presented as mean ±SD. A student’s t-test was used for comparison to control. No significant changes were present. (C & D) Cellular thiol isomerase activity is also inhibited by (C) montelukast (n=3) and (D) the zafirlukast analogue (n=4). Data is presented as mean ±SD. One-way ANOVA and a post-hoc Dunnett’s test where *p=0.0219 for 10 µM montelukast, **p=0.0018 for 30 µM montelukast and ***p=0.0002 for 100 µM montelukast compared to the control, while *p=0.0383 for 1 µM zafirlukast analog, **p=0.0017 for 3 µM zafirlukast analog, ***p= 0.0004 for 10 µM zafirlukast analog and ****p<0.0001 for 30 µM zafirlukast analog compared to the control.

Figure 3 –

Zafirlukast and montelukast selectively cause cancer cell cytotoxicity. (A) Zafirlukast is about 5x more cytotoxic than montelukast and 1.5× more cytotoxic than the analogue (n=3). (B) Comparison of the relative potency of each compound in the experiments from Figure 1, Figure 2 and Figure 3A. (C) Zafirlukast also induced cytotoxicity to HCT116 colon tumor cells, PC3 prostate cancer cells, and A549 lung cancer cells selectively over the HEK293 non-cancerous cell line (n=3).

Figure 4 –

Zafirlukast effects downstream measures of thiol isomerase inhibition. (A) Zafirlukast treatment inhibits the activation of the EGFR receptor (n=3). Data is presented as mean ±SD. A student’s t-test where *p=0.0112 for 1 hour 30 µM treated zafirlukast and ***p= 0.0002 for 4 hour 30 µM treated zafirlukast compared to the EGF control. (B) The known ERp57 inhibitor RL90 and PACMA 31 also inhibit EGFR activation (n=3). Data is presented as mean ±SD. A student’s t-test where ***p=0.0007 for 30 µM zafirlukast, **p=0.004 for 5 µg RL90, *p=0.0433 for 10 µg RL90, **p=0.0059 for 15 µg RL90 and **p=0.0092 for 10 µM PACMA 31 compared to the EGF control. (C) A time-course study of zafirlukast RL-90, and PACMA 31’s effect on OVCAR8 cell viability (n=4). Data is presented as mean ±SD. A two-way ANOVA and a post-hoc Sidak’s test demonstrate no significant changes between the control and any of the test groups. (D) Phosphorylation of Gab1, an immediate downstream target of EGFR phosphorylation (n=3) Data is presented as mean ±SD. A student’s t-test where *p=0.0153 for 1 hour 10 µM zafirlukast and ****p<0.0001 for 4 hour 10 µM and 1 and 4 hour 30 µM zafirlukast compared to the EGF control. (E) Zafirlukast inhibits the tissue factor dependent generation of Factor Xa (n=4). Data is presented as mean ±SD. A one-way ANOVA and post-hoc Dunnett’s test where **p=0.0041 for 10 µM zafirlukast and ****p<0.0001 for 30 µM zafirlukast.

Figure 5 –

Zafirlukast inhibits the growth of OVCAR-8 tumors on xenograft mice. (A) NOG mice were SC injected with OVCAR8 cells at 4 weeks old (n=13/group). Tumors were allowed to grow to an average size of 30 mm³ before daily treatment with 30 mg/kg of zafirlukast (red bars) or vehicle control (black bars). Tumor size was measured twice weekly. Data is presented as mean ±SD. A student’s t-test where *p=0.0149 at 18 days, ***p=0.0002 at 21 and 25 days, ****p<0.0001 at 28 days and ***p=0.0001 at 28 days. (B) PDI and ERp57 levels in tumors were measured by immunoblotting after day 32 of treatment in control vs zafirlukast treated mice with minimal difference in expression. (C. & D) Lung metastasis (n=4/group) were graded on a scale of 0–4. A significant difference was seen between the control and zafirlukast treated groups. Data is presented as mean ±SD. A student’s t-test where ***p=0.0004 for the treated group compared to the control. (E) Similar to A, except mice were treated with 5 mg/kg cisplatin and 120 mg/kg gemcitabine once weekly in the presence (red bars) or absence (black bars) of 30 mg/kg zafirlukast daily (n=12/group). Data is presented as mean ±SD. A student’s t-test where *p=0.023, 0.012, 0.018 and 0.027 for days 14, 18, 21 and 25 respectively, and **p=0.004 for day 28. (F) A summary of the three different treatment groups, normalized to the control group by percentage.

Figure 6 –

Zafirlukast inhibits CA-125 doubling time. The rate of CA-125 doubling time (A) prior to and (B) after zafirlukast treatment. The mean change in CA-125 was 0.036 U/mL per day prior to treatment compared with 0.015 U/mL per day following zafirlukast (paired t-test p=0.026) (C) Comparison of the average pre and post treatment doubling times of CA-125. (D) The relative thiol isomerase activity of each patient 28 days after treatment, the reduction of which was well correlated (E) to the decrease in CA-125 doubling time.