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. 2020 Mar 12;12(5):4547-4557.
doi: 10.18632/aging.102910. Epub 2020 Mar 12.

ARV-825-induced BRD4 protein degradation as a therapy for thyroid carcinoma

Ling He  1 , Chen Chen  1 , Guoyu Gao  1 , Kun Xu  1 , Zhaoqun Ma  1
Affiliations

ARV-825-induced BRD4 protein degradation as a therapy for thyroid carcinoma

Ling He et al. Aging (Albany NY). .

Abstract

Bromodomain-containing protein 4 (BRD4) is overexpressed in thyroid carcinoma, represents as an important therapeutic target. ARV-825 is a novel cereblon-based PROTAC (Proteolysis Targeting Chsimera) compound. It can induce fast and sustained BRD4 protein degradation. Its potential effect in human thyroid carcinoma cells was studied here. In TPC-1 cells and primary human thyroid carcinoma cells, ARV-825 potently inhibited cell viability, proliferation and migration. Furthermore, ARV-825 induced robust apoptosis activation in the thyroid carcinoma cells. ARV-825 induced BRD4 protein degradation and downregulation of its targets, including c-Myc, Bcl-xL and cyclin D1 in thyroid carcinoma cells. It was significantly more potent in inhibiting thyroid carcinoma cells than the known small molecule BRD4 inhibitors. In vivo studies demonstrated that ARV-825 oral administration potently suppressed TPC-1 xenograft tumor growth in severe combined immunodeficient mice. BRD4 protein degradation as well as c-Myc, Bcl-xL and cyclin D1 downregulation were detected in ARV-825-treated TPC-1 tumor tissues. Taken together, ARV-825 induces BRD4 protein degradation and inhibits thyroid carcinoma cell growth in vitro and in vivo.

Keywords: ARV-825; BRD4; c-Myc; proteolysis targeting chimera; thyroid carcinoma.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
ARV-825 inhibits human thyroid carcinoma cell viability, proliferation and migration. TPC-1 cells (A–D), the primary human thyroid carcinoma cells (“C1”/“C2”, E–G) or the primary human thyroid epithelial cells (“E1”/“E2”, E–G) were left untreated (“Ctrl”, same for all Figures) or treated with ARV-825 (5-250 nM). Cells were further cultured in complete medium for indicated time periods, cell viability (MTT OD, A and E), colony formation (B), cell proliferation (EdU incorporation, C and F) and migration (“Transwell” assays, D and G) were tested. Data were presented as mean ± standard deviation (SD, n=5) (same for all Figures). *p < 0.05 vs. “Ctrl” group. ***p < 0.001 vs. “Ctrl” group. The experiments were repeated three times, with similar results obtained. Bar= 100 μm (C and D).
Figure 2
Figure 2
ARV-825 induces apoptosis activation in human thyroid carcinoma cells. TPC-1 cells (A–D), the primary human thyroid carcinoma cell (“C1”/“C2”, G and H) or the primary human thyroid epithelial cells (“E1”/“E2”, G and H) were treated with ARV-825 (5-250 nM) and cultured in for indicated time periods, the caspase activity (A), expression of apoptosis-associated proteins (B) as well as nuclear TUNEL staining (C and G) and mitochondrial depolarization (JC-1 green fluorescence intensity, D and H) were tested to examine cell apoptosis. TPC-1 cells were pre-treated for 30 min with 50 μM of z-DEVD-fmk, z-LEHD-fmk or z-VAD-fmk, following by ARV-825 (100 nM) treatment for 48-72h, cell apoptosis and cell viability were tested by TUNEL staining (E) and MTT assay (F), respectively. Expression of the listed proteins was quantified and normalized to loading control (B). “DMSO” stands for vehicle control (0.1% DMSO, E and F). **p < 0.01 vs. “Ctrl” group. ***p < 0.001 vs. “Ctrl” group. # p < 0.001 vs. “DMSO” group (E and F). The experiments were repeated three times, with similar results obtained. Bar=100 μm (D).
Figure 3
Figure 3
ARV-825 inhibits BRD4 signaling in human thyroid carcinoma cells. TPC-1 cells (A and B) or the primary human thyroid carcinoma cells (“C1”/“C2”) (F) were treated with applied concentration of ARV-825 for 24h, tested by Western blotting and qPCR assays of listed genes. TPC-1 cells were pre-treated with MG-132 (25 μM) for 2h, followed by ARV-825 (100 nM) treated for 24h, expression of listed proteins was shown (C). The stable TPC-1 cells with CRISPR/Cas9 BRD4-KO construct (“BRD4-KO”) cells were treated with or without ARV-825 (100 nM, for indicated time periods), control TPC-1 cells with empty vector (“Cas9-C”) were left untreated; expression of listed proteins was shown (D); Cell viability and proliferation were tested by MTT and EdU staining assays (E), respectively. TPC-1 cells or “C1” human thyroid carcinoma cells were treated with ARV-825 (100 nM), JQ1 (500 nM), CPI203 (500 nM) or GSK1210151A (500 nM) for indicated time periods, cell viability (MTT assay, 96h) (G and J), proliferation (testing nuclear EdU/DAPI ratio, 48h) (H and K) and apoptosis (nuclear TUNEL ratio, 48h) (I and L) were tested, and results were quantified. Expression of listed proteins was quantified and normalized to the corresponding loading control (A, C, D and F). # p < 0.001 vs. “ARV-825” treatment group (G–L). The experiments were repeated three times, and similar results obtained.
Figure 4
Figure 4
ARV-825 oral administration inhibits TPC-1 xenograft tumor growth in SCID mice. SCID mice bearing TPC-1 xenografts (100 mm3 at “Day-0”) were treated with ARV-825 (daily gavage, 5 or 25 mg/kg body weight, for 21 consecutive days), the tumor volumes (A) and the mice body weights (D) were recorded every seven days (recording five rounds). The estimated daily tumor growth (in mm3 per day) was calculated (B). At “Day-35” tumors of all three groups were isolated and weighted (C). At treatment “Day-7” and “Day-14”, one tumor of each group was isolated, with fresh tumor tissue lysates analyzed by Western blotting for listed proteins (E and F). Expression of listed proteins was quantified and normalized to Erk1/2 (E and F). Vehicle stands for 10% DMSO, 40% PEG300, 5% Tween-80 plus 45% saline. For each group, n= 10 mice. ***p < 0.001 vs. “Vehicle” group.
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