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

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.


INTRODUCTION
Thyroid cancer causes significant human mortalities globally [1,2]. The primary thyroid cancer form is papillary thyroid carcinoma [1,2]. Radiation and/or genetic susceptibility are key contributors for the initiation and progression of human papillary thyroid carcinoma [1,2]. The prognosis and five-year overall survival for the advanced and/or metastatic thyroid carcinoma are extremely poor [1,2]. Molecularlytargeted therapies are vital for the better treatment of this malignancy [3,4].
We have previously shown that BRD4 expression is elevated in thyroid carcinoma cells [13]. Importantly, AZD5153, a novel and specific BRD4 inhibitor, potently inhibited thyroid carcinoma cell growth in vitro and in vivo [13]. Wang et al., found that gambogic acid downregulated BRD4 to inhibit proliferation of anaplastic thyroid cancer cells [14]. Li et al., demonstrated that long non-coding RNA (LncRNA) UCA1 induced BRD4 upregulation, therefore promoting papillary thyroid cancer cell proliferation [15]. These results imply that BRD4 inhibition might AGING represent as an important therapeutic advance for the treatment of thyroid carcinoma [13].
The small molecule BRD4 inhibitors will, however, lead to feed-back BRD4 protein elevation in cancer cells, resulting in only modest anti-proliferative activity [16]. Lu et al., have recently developed ARV-825 as a heterobifunctional PROTAC (Proteolysis Targeting Chimera) compound. It recruits BRD4 directly to the E3 ubiquitin ligase cereblon [16], leading to fast, efficient and sustained BRD4 protein degradation [16]. Studies have shown that ARV-825 is far more efficient than the small molecule BRD4 inhibitors in suppressing BRD4 signaling, causing potent and sustained cancer cell inhibition and profound apoptosis induction [16][17][18][19][20]. Its potential efficacy in human thyroid carcinoma cells is tested in the present study.

ARV-825 oral administration inhibits TPC-1 xenograft tumor growth in SCID mice
At last experiments were carried out to test the potential anti-thyroid carcinoma activity of ARV-825 in vivo. As described previously [13] TPC-1 cells were s.c. injected to the flanks of SCID mice, forming tumor xenografts within 16-18 days ("Day-0"). By measuring tumor volumes, we demonstrated that oral administration of ARV-825 (daily, at 5 or 25 mg/kg body weight) potently inhibited TPC-1 xenograft growth in SCID mice ( Figure 4A). ARV-825 administration decreased estimated daily tumor growth, which was calculated by (tumor volume at Day-35-tumor volume at Day-0)/35 ( Figure 4B). At Day-35 all xenograft tumors were isolated. Tumors from ARV-825-treated mice weighted significantly lighter than those from vehicle control mice ( Figure  4C). ARV-825 at 25 mg/kg dose was more potent in suppressing TPC-1 tumor growth than it at 5 mg/kg dose ( Figure 4A-4C). The mice body weights were not significantly different among the three groups ( Figure   4D), neither did we notice any signs of apparent toxicities in the experimental mice.
To test BRD4 signaling proteins, at treatment Day-7 and Day-14 one tumor of each group (total six tumors) was isolated, and fresh tumor lysates subjected to Western blotting assays. As demonstrated, BRD4 protein was depleted in ARV-825-treated xenograft tumors ( Figure 4E and 4F). BRD4-dependent proteins, c-Myc, Bcl-xL and cyclin D1, were downregulated ( Figure 4E and 4F). Cleavages of caspase-3 and PARP were detected in ARV-825-treated tumor tissues ( Figure  4E and 4F), indicating apoptosis activation in vivo. These results suggest that ARV-825 oral administration in SCID mice inhibited BRD4 signaling and activated apoptosis in TPC-1 xenografts.

DISCUSSION
BRD4 is the most abundant and important BET family protein in cancer cells [9][10][11]28]. It is a key epigenetic regulator binding to acetylated-histones [9][10][11][12]. BRD4 is also required for the chromatin structure formation in the daughter cells [9][10][11]. By recruiting P-TEFb (the positive transcription elongation factor b) and phosphorylating RNA polymerase II, BRD4 is essential for transcription elongation and expression of multiple key oncogenic genes [11], including Bcl-xL, c-Myc, cyclin D1, among others [5,[8][9][10][11][12]. Our previous studies have demonstrated that BRD4 expression is significantly elevated in established and primary human thyroid carcinoma cells [13]. BRD4 inhibition, by the small molecule inhibitor AZD5153, potently inhibited thyroid carcinoma cell growth in vitro and in vivo [13]. These studies supported that BRD4 is a promising and valuable therapeutic target of thyroid carcinoma.
ARV-825 is a molecule designed by the PROTAC technology, causing sustained BRD4 protein degradation [16]. The results of this study suggest that ARV-825 exerted potent anti-thyroid carcinoma cell activity [16,18,19,22,29]. In TPC-1 cells and primary human thyroid carcinoma cells ARV-825 induced BRD4 protein degradation, causing downregulation of BRD4-dependent oncogenic proteins, including c-Myc, Bcl-xL and cyclin D1. ARV-825 potently inhibited thyroid carcinoma cell viability, proliferation and migration, and inducing robust cell apoptosis. The BRD4 PROTAC compound was, however, noncytotoxic nor pro-apoptotic to primary thyroid epithelial cells. In vivo, oral administration of ARV-825, at welltolerated doses, efficiently inhibited TPC-1 tumor xenograft growth in SCID mice. BRD4 protein degradation as well as c-Myc, Bcl-xL and cyclin D1 downregulation were detected in ARV-825-treated AGING TPC-1 tumor tissues. Therefore, BRD4 degradation by its PROTAC compound ARV-825 could be novel therapeutic advance of thyroid carcinoma.
It has been shown that BRD4 inhibitors could have reversible binding and incomplete inhibition of BRD4, which largely compromise the anti-cancer cell activity [16]. ARV-825, unlike BRD4 inhibitors, directly recruits BRD4 to the E3-ubiquitin ligase cereblon, causing robust and sustained BRD4 protein degradation [16]. In the present study, ARV-825-induced antithyroid carcinoma cell activity was significantly more potent than the small molecule BRD4 inhibitors, including JQ1, CPI203, and GSK1210151A. Our results suggest that BRD4 protein degradation should be the main reason to explain ARV-825-induced superior antithyroid carcinoma cell activity. As BRD4 KO, by the CRSIPR/Cas9 strategy, mimicked ARV-825-induced cytotoxicity in TPC-1 cells. More importantly, ARV-825 was completely ineffective in the BRD4-KO cells.

CONCLUSION
Taken together, we conclude that ARV-825 induced BRD4 protein degradation and inhibited thyroid. carcinoma cell growth in vitro and in vivo. It could be (100 mm 3 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 mm 3 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. an important therapeutic advance for the treatment of thyroid carcinoma.

Cell culture
The established TPC-1 human thyroid carcinoma cells were cultured as previously described [13]. TPC-1 cells were routinely checked for possible mycoplasma and microbial contamination. To confirm the the genotype STR profiling, population doubling time, and morphology were verified. The primary human thyroid carcinoma cells ("C1/C2") as well as the thyroid follicular epithelial cells ("E1/E2"), derived from two primary papillary thyroid carcinoma patients, were reported early [13], and cultured using the described protocol [13]. The protocols of using human cells were in accordance with the Declaration of Helsinki, approved by the Institutional Ethics Review Board of authors institutions.

Cell viability
As previously described [13], at 5,000 cells per well cells were initially plated into 96-well plates. Following the applied treatment, cell viability was tested by the MTT assay, with its optical density (OD) values measured at the wavelength of 550 nm.

Colony formation
After the applied ARV-825 treatment THP-1 cells were placed onto 10-cm dishes (20,000 cells/dish) [13]. Cells were maintained in ARV-825-containing complete medium for a total of 10 days (with medium renewed every two days). The number of viable THP-1 cell colonies remained was manually counted.

Caspase activity
Following the applied ARV-825 treatment, 30 μg of cytosolic protein extracts were mixed with the caspase assay buffer and the caspase (-3/-8/-9) substrate [13]. An Infinite 200-PRO reader was utilized to quantify the release of fluorogenic AFC at 400 nm excitation and 505 nm emission. AFC fluorescence value was always normalized to that of vehicle control.

TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining
Cells were initially seeded into the 12-well plates (5 x 10 4 cells/cm 2 ). With the applied treatment, a TUNEL In Situ Cell Death Detection Kit (Roche) was applied to test cell apoptosis. Briefly, cells were stained with TUNEL and DAPI, visualized under a fluorescent microscope (Leica). In each condition 600 cell nuclei in six random views were counted to calculate TUNELpositive nuclei ratio (% vs. DAPI).

Mitochondrial depolarization
With mitochondrial depolarization JC-1 dye shall aggregate in the mitochondria to form green monomers in the stressed cells [32]. After the applied ARV-825 treatment, cells were incubated with JC-1 (5 μg/mL, Sigma), tested under the fluorescence spectrofluorometer at the test-wavelength of 545 nm (green). The JC-1 fluorescence images, integrating the green (at 550 nm) and red (at 635 nm) channels, were presented as well.

"Transwell" assay
The thyroid carcinoma cells (10, 000 cells in 250 μL serum-free medium) with the applied ARV-825 treatment were seeded on the upper surface of the "Transwell" chambers (BD Biosciences). FBS-containing complete medium was added to the lower "Transwell" chambers. After incubation for 24h the migrated cells were stained and counted.

Western blotting
The detailed protocols of Western blotting were described previously [33,34]. In brief, total cellular lysates (30-40 μg proteins per treatment in each lane) were separated by 10-12% SDS-PAGE gels, transferred to a PVDF blot. After blocking, the blot was incubated with the applied primary and secondary antibodies, with the antibodyantigen binding detected by an ECL kit. For data quantification, the ImageJ software (NIH) was utilized.

RNA extraction and qPCR
Total RNA was extracted via the TRIzol reagents (Invitrogen Thermo-Fisher). Quantitative real-time reverse transcription-PCR ("qPCR") assay was carried using the previously-described protocol [23]. GAPDH was always examined as the reference gene for data quantification, using the 2 −∆∆Ct method. mRNA primers utilized in this study were provided by Dr. Zhao at Soochow University [23].

Xenograft assay
The detailed protocols were reported in our previous study [13]. Briefly, from the Experimental Animal Center of Soochow University (Suzhou, China) the severe combined immunodeficient (SCID) mice were obtained. To establish tumor xenografts TPC-1 cells were subcutaneously (s.c.) injected to the right flanks [13]. Tumor-bearing SICD mice were randomly assigned into three groups (10 mice per group), treated with ARV-825 or the vehicle control. Tumor volumes and the mice body weights were recorded every seven days. The animal studies were conducted with the standards of ethical treatment approved by the Institutional Animal Care and Use Committee (IACUC) of authors' institutions.

Statistical analyses
Data were presented as mean ± standard deviation (SD). Statistics were analyzed by one-way ANOVA followed by the Scheffe' and Tukey Test using SPSS software (21.0, SPSS Co. Chicago, CA). Significance was chosen as p < 0.05. To determine significance between two treatment groups, the two-tailed unpaired t tests (Excel 2007) were carried out.