Disruption of Autophagic Degradation with ROC-325 Antagonizes Renal Cell Carcinoma Pathogenesis

Abstract

Purpose: Although autophagy plays important roles in malignant pathogenesis and drug resistance, there are few clinical agents that disrupt this pathway, and the potential therapeutic benefit of autophagy inhibition remains undetermined. We used medicinal chemistry approaches to generate a series of novel agents that inhibit autophagic degradation.Experimental Design: ROC-325 was selected as a lead compound for further evaluation. Comprehensive in vitro and in vivo studies were conducted to evaluate the selectivity, tolerability, and efficacy of ROC-325 in preclinical models of renal cell carcinoma (RCC) with HCQ serving as a comparator. Markers of autophagy inhibition and cell death were evaluated in tumor specimens.Results: ROC-325 exhibited superior in vitro anticancer effects compared with the existing autophagy inhibitor hydroxychloroquine (HCQ) in 12 different cancer cell lines with diverse genetic backgrounds. Focused studies of the mechanism of action and efficacy of ROC-325 in RCC cells showed that drug treatment induced hallmark characteristics of autophagy inhibition, including accumulation of autophagosomes with undegraded cargo, lysosomal deacidification, p62 stabilization, and disruption of autophagic flux. Subsequent experiments showed that ROC-325 antagonized RCC growth and survival in an ATG5/7-dependent manner, induced apoptosis, and exhibited favorable selectivity. Oral administration of ROC-325 to mice bearing 786-0 RCC xenografts was well tolerated, was significantly more effective at inhibiting tumor progression than HCQ, and inhibited autophagy in vivoConclusions: Our findings demonstrate that ROC-325 has superior preclinical anticancer activity compared with HCQ and support the clinical investigation of its safety and preliminary efficacy in patients with RCC and other autophagy-dependent malignancies. Clin Cancer Res; 23(11); 2869-79. ©2016 AACR.

Figure 1

ROC-325 induced vacuolization and lysosome membrane permeability. (A) Chemical structures of ROC-325 and related compounds. (B) ROC-325 induces vacuolization. 786-O and A498 cells were treated for 24 h with 5 μM ROC-325. Cell morphology and vacuolization were visualized by Giemsa staining. Scale bars indicate 20 microns. (C) Electron microscopy demonstrates vacuolization and electron dense particle accumulation. Cells were treated with 5 μM ROC-325 for 24 h. Cells were fixed and prepared for electron microscopy. Arrows indicate vacuoles with undegraded cargo in the cytosol of imaged cells. Scale bars for images with 8000X magnification indicate 2 microns and scale bars for images with 20000X magnification indicate 500 nm. (D) Measurement of lysosome membrane permeability by loss of acridine orange fluorescence. Red acridine orange staining was measured in 786-O and A498 cells by immunofluorescence and quantified using ImageJ software. Mean ± SD, n = 5. *Indicates a significant difference from the controls. P < 0.05.

Figure 2

ROC-325 inhibits autophagy. (A) ROC-325 induces LC3B accumulation. RCC cells were treated with 5 μM ROC-325 for 24 h. LC3B accumulation was visualized by immunocytochemistry. (B) ROC-325 increases LC3B, p62, and cathepsin D expression. 786-O and A498 cells were treated with the indicated concentrations of ROC-325 for 24 h. Protein levels were determined by immunoblotting. The lower band on all LC3 blots in the manuscript depicts the lipidated (LC3-II) form of LC3B, which is an established autophagosome marker. (C–D) Bafilomycin A1 does not augment ROC-325-mediated LC3B or p62 accumulation or apoptosis. 786-O cells were treated with 5 μM ROC-325, 100 nM bafilomycin A1, or both agents for 48 h. Protein expression was determined by immunoblotting and apoptosis by PI-FACS analysis. Mean ± SD, n = 3. *Indicates a significant difference from control, P<0.05. There is no significant difference between bafilomycin A1, ROC-325, and ROC-325 + bafilomycin A1 treatments.

Figure 3

ROC-325 has selective anticancer effects that are dependent upon autophagy function. (A) The essential autophagy genes ATG5 and ATG7 were knocked down in 786-0 RCC cells using lentiviral shRNA. Knockdown efficiency was assessed by immunoblotting. Tubulin documented equal protein loading. (B) Cells infected with non-targeted control, ATG5- or ATG7-directed lentiviral shRNAs were treated with the indicated concentrations of ROC-325 for 72h. The effects of drug treatment on cell viability were quantified for each experimental condition by MTT assay. Mean ± SD, n = 3. (C) ROC-325 selectively decreases RCC cell line viability more effectively than HCQ. RCC cell lines and normal RPTEC cells were treated with varying concentrations of ROC-325 or HCQ for 72 h. Cell viability was measured using the MTT assay. Mean ± SD, n = 3. (D) ROC-325 stimulates apoptosis. RCC cell lines were treated with the indicated concentrations of ROC-325 for 48 h. Active caspase-3 (left) was measured using a FITC-labeled active caspase-3 antibody followed by flow cytometric analysis. Mean ± SD, n = 3. DNA fragmentation (right) was measured by PI-FACS analysis. Mean ± SD, n = 3.

Figure 4

ROC-325 reduces tumor burden more effectively than HCQ in RCC xenografts. (A) 786-O cells were injected into the flanks of nude mice. Mice were pair-matched and randomized into groups when mean tumor burden reached approximately 100 mm³. Mice were treated with 25, 40, or 50 mg/kg ROC-325 PO and 60 mg/kg HCQ IP QDx5 throughout the course of the study. Tumor volumes were measured twice weekly. Mean ± SEM, n = 5. *Indicates a significant difference in tumor burden compared to vehicle control based on 2-way ANOVA analyses. P < 0.05. (B) ROC-325 is well tolerated in mice. Body weight was determined at the end of the study (Day 46) to quantify drug-induced weight loss. Mean ± SD, n = 5.

Figure 5

ROC-325 significantly increases LC3B and p62 expression and induces apoptosis in RCC xenografts. (A) Tumors were harvested 6 h following the last dose of each drug was administered. LC3B immunohistochemistry. Tumors were stained with an anti-LC3B antibody and the relative intensity of expression was quantified by densitometry. Mean ± SD, n = 5. (B) p62 immunohistochemistry. Tumors were stained with an anti-p62 antibody and the relative intensity of expression was quantified by densitometry. Mean ± SD, n = 5. (C) Apoptosis was determined by active caspase-3 immunohistochemistry. Tumors were stained with an antibody to cleaved caspase-3. The percentage of positive stained cells was determined manually under 20X magnification. Mean ± SD, n = 5. *Indicates a significant difference from vehicle, P<0.05.

Scheme I

a) Pd(II)acetate, BINAP, K₃PO₄, dioxane, 100 °C, 17 h; b) 4N HCl in dioxane; c) Pd(II)acetate, BINAP, K₃PO₄, dioxane, 100 °C, 15 h; d) Methanol, 4N HCl in dioxane