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. Author manuscript; available in PMC: 2020 Jan 14.
Published in final edited form as: Clin Cancer Res. 2017 Jun 9;23(17):5044–5054. doi: 10.1158/1078-0432.CCR-17-1043

Dual Inhibition of HDAC and Tyrosine Kinase Signaling Pathways with CUDC-907 Inhibits Thyroid Cancer Growth and Metastases

Shweta Kotian 1, Lisa Zhang 1, Myriem Boufraqech 1, Kelli Gaskins 1, Sudheer Kumar Gara 1, Martha Quezado 2, Naris Nilubol 1, Electron Kebebew 1,3
PMCID: PMC6959516  NIHMSID: NIHMS1066218  PMID: 28600475

Abstract

Purpose:

There is currently no standard therapy for anaplastic thyroid cancer (ATC) and poorly differentiated thyroid cancer (PDTC), which account for two-thirds of thyroid cancer-related deaths. Driver mutations in the PI3K/AKT and RAF/RAS/MEK/ERK pathways are common in ATC and PDTC. Histone deacetylases (HDAC) regulate cancer initiation and progression. Our aim was to determine the therapeutic efficacy of simultaneously targeting these pathways in thyroid cancer with a single agent and to evaluate biomarkers of treatment response.

Experimental Design:

CUDC-907 is a first-in-class compound, functioning as a dual inhibitor of HDACs and the PI3K/AKT pathway. We investigated its antiproliferative effect in vitro and in vivo.

Results:

CUDC-907 significantly inhibited cellular proliferation in thyroid cancer cell lines, induced G2-M arrest with decreased levels of the checkpoint regulators cyclin B1, AURKA, AURKB, PLK1, and increased p21 and p27. Treatment induced apoptosis with increased caspase-3/7 activity and decreased survivin levels and decreased cellular migration and invasion. CUDC-907 treatment caused H3 hyperacetylation and decreased HDAC2 expression. HDAC2 was upregulated in ATC and other thyroid cancer histologic subtypes. CUDC-907 treatment reduced both p-AKT and p-ERK1/2 levels. Finally, CUDC-907 treatment, in a metastatic mouse model of thyroid cancer, showed significant inhibition of growth and metastases, and tumors from treated mice had decreased HDAC2 expression, suggesting that this may be a useful biomarker of response.

Conclusions:

Dual inhibition of HDAC and the tyrosine kinase signaling pathways with CUDC-907 is a promising treatment strategy for advanced, metastatic thyroid cancer.

Introduction

Thyroid cancer is the most common endocrine malignancy, and the incidence and mortality rates differ significantly based on histologic subtype (1). Differentiated thyroid cancer is generally associated with a good prognosis unless there is distant metastasis or the cancer does not take up radioiodine, rendering 131I therapy ineffective. There has been progress in the treatment of radioiodine-refractory differentiated thyroid cancer with two drugs, sorafenib and lenvantinib, showing improved progression-free survival (2, 3). Both drugs have recently been approved by the FDA for the treatment of patients with differentiated thyroid cancer who fail standard therapy [surgical resection, thyroid hormone for thyroid-stimulating hormone (TSH) suppression, and radioiodine ablation]. Unfortunately, most thyroid cancer-related deaths are due to poorly differentiated thyroid cancer (PDTC) and undifferentiated (anaplastic) thyroid cancer (ATC), and less common aggressive variants of differentiated thyroid cancer such as Hürthle cell carcinoma, tall-cell variant of papillary thyroid cancer (PTC), and sclerosing variant (4). ATC is one of the most fatal solid malignancies with a median survival of just 4.9 months, and a 1-year survival rate of less than 10% (5). Patients with locally advanced PDTC have a 5-year overall survival rate of only 47% (6). Patients with Hürthle cell carcinoma and aggressive variant (tall-cell, sclerosing, and insular) of differentiated thyroid cancer have a 5-year disease-specific mortality rate of 4% to 15% and 18% to 29%, respectively (7–10). Thus, the need for effective therapeutics for PDTC and ATC, rare and neglected malignancies, is enormous, as well as for the less common aggressive variant of differentiated thyroid cancer. Furthermore, survival in patients with PDTC and ATC has not changed in more than six decades primarily due to uncontrolled systemic metastases (5, 11).

Our understanding of the molecular and genetic events that are involved in thyroid cancer initiation and progression has improved. For example, TERT promoter mutations and genetic alterations causing activation of the PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways are common in PDTC, aggressive variants of differentiated thyroid cancer (Hürthle cell carcinoma, tall-cell variant, sclerosing variant, and insular variant), andATC (11–13). Multiple driver mutations are commonly present in PDTC and ATC as compared with being mutually exclusive events in differentiated thyroid cancer, and have important implications for treatment selection (11–15). Targeted monotherapy with compounds that target these driver genetic events in patients with ATC has shown a remarkable response in case reports, but has not been durable with disease-progression being common (16–18).

Epigenetic changes in cancer are common and have been associated with dedifferentiation and driver genetic events in thyroid cancer (14, 19, 20). Aberrant expression of histone deacetylaces (HDACs) is frequent in human cancers (21). A recent study demonstrated that HDAC1, HDAC2, HDAC4, and HDAC6 are overexpressed in thyroid cancer as compared with benign lesions (22). HDAC inhibitors have been evaluated in thyroid cancer clinical trials (23). HDAC inhibitors such as vorinostat, belinostat, panobinostat, and romidepsin have been approved by the FDA, and about 20 others are in various phases of clinical trials (24). Although monotherapy with HDAC inhibitors has shown good efficacy in treating hematological malignancies, the clinical outcomes in treating solid tumors have been discouraging.

ATC, PDTC, and aggressive variants of differentiated thyroid cancer have multiple driver genetic and epigenetic events, and there has been no or limited durable response to most monotherapy. Thus, we hypothesized that targeting multiple altered pathways simultaneously could result in a durable response, and that mediators of these pathways could be biomarkers of treatment response. To test this hypothesis, we evaluated CUDC-907, a novel compound that is an HDAC and PI3K signaling pathway inhibitor, in preclinical studies that recapitulate the cancer phenotype of the most aggressive forms of thyroid cancer (25).

Materials and Methods

Cell lines, cell culture, reagents, and tissue samples

The thyroid cancer cell line TPC-1 (derived from a patient with papillary thyroid cancer) was provided by Dr. Nabuo Satoh (Japan), FTC-133 (derived from a follicular thyroid cancer lymph node metastasis) was provided by Dr. Peter Goretzki (University Dusseldorf, Germany), XTC-1 (derived from a breast Hürthle cell carcinoma metastasis) was provided by Dr. Orlo H. Clark (University of California, San Francisco, CA),THJ-16T andTHJ-29T cell lines derived from patients with ATC were a kind gift from Dr. John A. Copland (Mayo Clinic, Jacksonville, FL), and 8505C, an ATC cell line, was purchased from European Collection of Cultures (United Kingdom). All the thyroid cancer cell lines were authenticated by short tandem repeat profiling and had testing for mycoplasma contamination (NCI’s Frederick National Laboratory for Cancer Research, Frederick, MD). Supplementary Table S1 is a summary of the genetic alterations present in these cell lines and differentiation markers in follicular thyroid cells which commonly occur and that are absent or decrease in ATC and PDTC, respectively (Supplementary Table S1). CUDC-907 (Curis) was dissolved in dimethyl sulfoxide DMSO for in vitro experiments.

The cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 4,500 mg/L of D-glucose, 2 mmol/L of l-glutamine, and 110 mg/L of sodium pyruvate, supplemented with 10% fetal bovine serum (FBS), thyroid stimulating hormone (TSH; 10 mU/Ml), penicillin (10,000 U/mL), streptomycin (10,000 U/mL), fungizone (250 ng/mL), and insulin (10 μg/mL) in a standard humidified incubator at 37°C in 5% CO2 and 95% O2 atmosphere.

Human thyroid tissue samples were collected in a clinical protocol at the National Institutes of Health Clinical Center (NCT01005654). This protocol was approved by the National Cancer Institute Institutional Review Board. All patients were enrolled after written informed consent. Tissue samples were stored at −80°C until their use for experiments.

Cell proliferation assay

Cell proliferation experiments were performed using the CyQUANT (Thermo Fisher Scientific) assay kit. Cells were seeded at a density of 2,000 cells/100 μL in quadruplicate in 96-well opaque plates with clear bottoms (Greiner Bio-One). The next day, cells were supplemented with 100 μL of fresh medium containing the indicated concentrations of CUDC-907 and dimethyl sulfoxide (DMSO). Medium with drug/vehicle was replenished every 48 hours. Cell numbers were determined using a 96-well fluorescence microplate reader (Molecular Devices).

Western blot analysis and antibodies

Cell lysates were prepared using a buffer containing 10 mmol/L Tris-HCl at pH 7.4 and 1% sodium dodecyl sulfate (SDS). Protein concentration was determined using the Pierce Bicinchoninic Acid (BCA) Assay Kit (Thermo Fisher Scientific). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was then performed on the cell lysates, transferred to polyvinylidene fluoride membranes, and immunostained overnight using antibodies as described below. The following antibodies were purchased from Cell Signaling Technology: p21 (dilution at 1:1,000), p27 (1:1,000), cyclinB1 (1:1,000), aurora kinase A (AURKA; 1:1,000), polo-like kinase 1 (PLK1; 1:1,000), cyclin-dependent kinase 1 (CDK1; 1:1,000), E-cadherin (1:1,000), Vimentin (1:1,000), acetyl-histone H3 (1:1,000), histone H3 (1:1,000), p44/p42 MAPK (ERK1/2; 1:1,000), p-p44/p42 MAPK (ERK1/2; 1:1,000), p-AKT1 (1:1,000), and AKT1 (1:1,000). Aurora kinase B (AURKB; 1:1,000) and HDAC2 (1:1,000) antibodies were purchased from Abcam. Survivin (1:1,000) was obtained from Novus Biologicals, N-cadherin (1:1,000) from EMD Millipore, HDAC1 (1:1,000) from Thermo Fisher Scientific, and HDAC10 from BioVision. The following antibodies were purchased from Santa Cruz Biotechnology: p27(1:1,000), HDAC4(1:1,000), HDAC6 (1:1,000), and GAPDH (1:5,000). Band densitometry analysis was performed using ImageJ software (NCI).

Cell cycle assay

Cells were plated at a density of 2 × 105 cells in a 9-cm2 dish with 2 mL of culture medium. The next day, CUDC-907 or DMSO was added, and after 12 and 24 hours, cells were trypsinized, washed with phosphate-buffered saline (PBS), and fixed with ice cold 70% ethanol. Cells were then washed with PBS and resuspended in a solution consisting of PBS, ribonuclease A (0.1 mg/mL), and propidium iodide (0.05 mg/mL). The DNA content of cells was then measured by FACS analysis using Canto II (Becton Dickinson). Cell cycle analysis of the gated propidium iodide (PI) distribution was done using ModFit software (Verity Software House, Inc.).

Apoptosis assay and screening of regulatory protein using an apoptosis protein array

Cells were seeded at a density of 2,000 cells per well in a 96-well plate in triplicate. Next, cells were treated with drug versus vehicle at specified concentrations. After 24 and 48 hours, caspase-3/7 activity was measured using the Caspase-Glo 3/7 Assay from Promega Corp. per the manufacturer’s instructions. Relative luminescence was calculated by normalizing to the total cell number using a SpectraMax microplate reader (Molecular Devices).

A human antibody apoptosis array kit from R&D Systems (Catalog # ARY009) was used to detect the expression of 35 apoptosis-related proteins in samples treated with CUDC-907 versus vehicle. Protein levels that changed with CUDC-907 treatment were validated by Western blot analysis.

Cellular migration and invasion assay

The ability of the cells to migrate and invade was determined using a Transwell Chamber assay (BD Biosciences) per the manufacturer’s instructions. Cells were treated with CUDC-907 versus vehicle at specified concentrations for 24 hours, and then plated in the upper transwell chamber at a density of 50,000 cells/mL in 500 mL total volume of medium without FBS. In the bottom well, 750 μL of DMEM supplemented with 10% FBS was added to act as a chemoattractant. Cells were incubated at 37°C for 22 hours, at which point the cells that migrated/invaded were fixed and stained using the Diff Quik staining kit (Dade Behring). Cells were then photographed and analyzed using ImageJ software.

Immunohistochemistry

Tissues were fixed in 10% formalin and then embedded in paraffin. Sections were deparaffinized, serially rehydrated, treated with 1 × citrate buffer at 120° C for antigen retrieval, blocked, and then immunostained with primary antibody (anti-HDAC2, 1:100, rabbit, Abcam, Y461-ab32117 and anti-Survivin, 1:100, rabbit, Novus Biologicals, NBS-500–201) overnight at 4°C. The Vectastain ABC and DAB kits (Vector Laboratories) were used to detect immunoreactivity of the primary antibody per the manufacturer’s instructions. Slides were scanned at 20× magnification using an Aperio ScanScope XT digital pathology slide scanner (Leica Biosystems), and analyzed using ImageScope software (Leica Biosystems). A thyroid tissue microarray (US Biomax #TH641)—consisting of six cores in duplicate each of follicular, papillary, and anaplastic thyroid carcinoma tissue, and 16 cores from normal lung, thyroid, and testes tissue—was immunostained using anti-HDAC2 antibody. Immunohistochemistry was also performed in 16 tall-cell variant of papillary thyroid cancer samples from our institution.

Animal studies

An in vivo mouse model of metastatic thyroid cancer was used to determine the anticancer activity of CUDC-907 (25). The Animal Care and Use Committee (ACUC) at the NCI, NIH approved the animal protocol. FTC133-Luc2 cells were injected into the tail vein of 6-month-old NOD Cg-Prkdcscid Il2rgtmlWjl/SzJ mice. Luminescence as an estimate of tumor burden was measured using a Xenogen IVIS in vivo imaging system. A week after, in vivo imaging was performed to confirm lung metastases and treatment started. CUDC-907 (Curis) was reconstituted in 30% captisol. Mice were randomized into two groups—a control treated with 30% captisol and a treatment group, which was administered 75 mg/kg CUDC-907 by oral gavage, on a 5-day on/2-day off dosing regimen. Mice were imaged weekly, and body weight was measured weekly. Treatment was continued until the first mouse reached humane endpoint criteria, upon which all mice were euthanized using C02 inhalation.

Statistical analyses

Data were analyzed using paired and unpaired (Mann-Whitney) Student t test and ANOVA. A two-tailed P value of less than 0.05 was considered to be statistically significant. The values are represented as mean ± standard deviation or mean ± standard error. GraphPad Prism software (version 6; Graphpad Software Inc.) was used to perform all statistical analyses.

Results

CUDC-907 inhibits cellular proliferation and causes G2/M arrest and apoptosis

We first investigated the effect of CUDC-907 treatment on cellular proliferation using six thyroid cancer cell lines with various driver mutations and differentiation marker expression levels consistent with PDTC and ATC (Supplementary Table S1). We found that CUDC-907 treatment significantly inhibited cellular proliferation in a time- and dose-dependent manner and caused cell death at higher concentrations regardless of driver mutation status (Fig. 1).

Figure 1.

Figure 1.

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CUDC-907 inhibits cellular proliferation. The antiproliferative effect of CUDC-907 was tested in six thyroid cancer cell lines. Cells were treated with CUDC-907 at doses ranging from 5 to 80 nmol/L. CUDC-907 treatment significantly inhibited cellular proliferation that was dose- and time-dependent in all six cell lines tested. Error bars are ± SD. *, P < 0.05; **, P < 0.01.

To understand the mechanism by which CUDC-907 treatment inhibited cellular proliferation, we tested its effect on cell cycle progression and apoptosis. We treated thyroid cancer cells with CUDC-907 for varying time intervals, and observed G2-M arrest at 12 hours post drug treatment (Fig. 2A and B). We evaluated the effect of CUDC-907 treatment on cell-cycle-regulatory proteins (p21, cyclin B1, AURKA, AURKB, and PLK1) involved in G2-M transition and that are dysregulated in thyroid cancer (26–28). CUDC-907 treatment increased p21 protein expression and decreased cyclin B1, AURKA, AURKB, and PLK1 protein levels (Fig. 2C and D).

Figure 2.

Figure 2.

Figure 2.

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CUDC-907 treatment causes G2-M arrest and induces apoptosis. A, Thyroid cancer cell lines were treated with 20 and 50 nmol/L of CUDC-907 for 12 hours. Cells were then stained with PI, and analyzed by FACS. CUDC-907 caused a significant increase in the G2-M peak in all cell lines tested at the 12-hour time-point after drug treatment. B, Histogram representation of the percentage of cells in different cell-cycle phases. C, Western blot analysis of cell-cycle regulatory proteins. Thyroid cancer cells were treated with 20 and 50 nmol/L of CUDC-907 for 24 hours. Top, blot for cell-cycle-regulatory proteins. D, Band densitometry of Western blot protein bands. E, Caspase-3/7 activity assay. Thyroid cancer cell lines were treated with vehicle versus 20 and 50 nmol/L doses of CUDC-907 for 24hours and 48 hours. Treatment increased caspase-3/7 activity. Error bars are ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. F, Apoptosis-regulatory proteins altered with CUDC-907 treatment. A human apoptosis array was used to screen for apoptosis-regulatory proteins altered with CUDC-907 treatment in TPC-1 and THJ-16T cell lines. Cells were treated with 50 nmol/L CUDC-907 for 24 hours, and lysates were hybridized to the array. Apoptosis array hybridization signals are shown. Red box shows proteins altered with CUDC-907 treatment. G, Western blot validation of p27 and Survivin in thyroid cancer cell lines treated with 20 nmol/L and 50 nmol/L of CUDC-907 for 24 hours. H, Band densitometry of reference controls, p27, and Survivin.

We next evaluated the effect of CUDC-907 treatment on apoptosis using the caspase-3/7 activity assay and found significantly increased activity at 24 hours that was dose dependent (Fig. 2E). To determine the apoptosis-regulatory proteins that could mediate this effect, we used an apoptosis array to screen for altered protein levels with CUDC-907 treatment. We found that CUDC-907 treatment resulted in increased p27 protein levels and decreased survivin protein levels (Fig. 2F). p27 is a cyclin-dependent kinase inhibitor (29) and thus regulates apoptosis. Survivin represses apoptosis by preventing caspase activation; therefore, survivin inhibition increases levels of caspases 3 and 9 (30). These results were validated by Western blot analysis (Fig. 2G and H).

CUDC-907 inhibits cell migration and invasion

One of the hallmarks of ATC and PDTC is that these cancers undergo epithelial-to-mesenchymal transition (EMT; refs. 31–33). Furthermore, driver mutations that are common in these cancers activate the RAF/RAS/ERK/MEK and PI3K/AKT signaling pathways and regulate cancer progression through their effect on cellular migration and invasion, and EMT (34). Therefore, we evaluated whether CUDC-907 treatment influenced thyroid cancer cell migration and invasion. CUDC-907 treatment significantly decreased cellular migration and invasion in all six thyroid cancer cell lines tested (Fig. 3A). We next evaluated the effect of CUDC-907 treatment on proteins known to regulate cancer cell migration and invasion, and EMT. We found that the protein expression of E-cadherin, an important regulator of EMT, was induced with CUDC-907 treatment, but there was a modest compensatory increase in N-cadherin and vimentin protein levels in some cell lines (Fig. 3B and C). To understand the mechanism by which E-cadherin protein expression was increased with CUDC-907 treatment, we evaluated TWIST1 protein expression as it transcriptionally represses E-cadherin expression (35). We found that CUDC-907 treatment downregulated TWIST1 protein expression in all of the thyroid cancer cell lines tested, suggesting that TWIST1 may mediate the upregulation of E-cadherin (Fig. 3B).

Figure 3.

Figure 3.

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CUDC-907 inhibits cellular migration and invasion and upregulates E-cadherin. A, CUDC-907 inhibited cellular migration and invasion in all six thyroid cancer cell lines tested. Thyroid cancer cell lines were treated with 50 nmol/L of CUDC-907 for 24 hours and then plated into transwells. After 22 hours, cells were fixed and stained. Matrigel-coated inserts in transwell chambers were used to study invasion under the same treatment conditions. CUDC-907 treatment resulted in decreased cellular migration and invasion. Left, representative images; right, quantification of the number of cells migrated and invaded. Error bars are mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. B, Effect of CUDC-907 treatment on E-cadherin, vimentin, N-cadherin, and TWIST1 protein expression. Thyroid cancer cell lines were treated with 20 and 50 nmol/L of CUDC-907 for 24 hours. Western blot analysis was performed for each protein (top; Note: E-cadherin blot is depicted with proteins that were run on the same gel, but exposed at different times, i.e., lanes 7–9 in gel 1 and lanes 1–3 in gel 2) with band densitometry measurements (C).

CUDC-907 inhibits HDAC2 expression

We evaluated the effect of CUDC-907 as a histone deacetylase inhibitor in thyroid cancer cells and the specific HDAC targeted by it. As expected, CUDC-907 treatment in thyroid cancer cells resulted in increased acetylation of histone 3, consistent with its effect as a histone deacetylase inhibitor (Fig. 4A and B). However, the effect of CUDC-907 on specific HDAC protein levels is unknown. Because HDAC1, HDAC2, HDAC4, and HDAC6 have been reported to be overexpressed in thyroid cancer as compared with benign thyroid tumors, and HDAC1 and HDAC2 are overexpressed in ATC as compared with normal tissue, we evaluated the effect of CUDC-907 treatment on these specific HDAC proteins (22, 36). We found that CUDC-907 treatment specifically reduced HDAC2 protein levels in thyroid cancer cells (Fig. 4A and B).

Figure 4.

Figure 4.

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CUDC-907 inhibits RAF/RAS/MEK/ERK and PI3K-AKT signaling and HDAC2 protein levels. A, CUDC-907 treatment increased acetyl-H3 levels in thyroid cancer cell lines. The effect of CUDC-907 was also tested on protein levels of HDACs 1, 2, 4, 6, and 10. CUDC-907 treatment only reduced HDAC2 protein levels. Western blot analysis of proteins was performed. B, Band densitometry for proteins in blots. C, CUDC-907 treatment reduced p-AKT1 and p-ERK1/2 levels, which are downstream effectors of the RAF/RAS/MEK/ERK and PI3K-AKT pathways. Bottom left, representative Western blot; bottom right (D), band densitometry of blots. Thyroid cancer cell lines were treated with 20 nmol/L and 50 nmol/L of CUDC-907 for 24 hours.

We next evaluated the effect of CUDC-907 treatment on its other target, the PI3K/AKT pathway, in thyroid cancer cells. As expected, we found that CUDC-907 treatment effectively reduced phospho-AKT levels (Fig. 4C and D). As driver mutations activating the RAF/RAS/ERK/MEK pathway are common in ATC and PDTC, we also investigated the effect of CUDC-907 treatment on the RAF/RAS/MEK/ERK pathway. We found that phospho-ERK protein levels were reduced with CUDC-907 treatment in all cell lines except for FTC133 (Fig. 4C and D). This might be due to the presence of a PTEN mutation in FTC133 cells. Although CUDC-907 is a PI3K inhibitor, several studies have shown that inhibition of this pathway may result in inhibition of the RAF/RAS/ERK/MEK pathway, and our results are consistent with these observations (37, 38).

HDAC2 is overexpressed in thyroid cancer

We found that CUDC-907 treatment specifically reduced HDAC2 protein levels, and therefore, we decided to characterize its expression in a broad range of thyroid neoplasms. In a thyroid tissue microarray (consisting of 6 cores each of benign, PTC, FTC, and ATC tissue and 8 cores of normal tissue), we found that HDAC2 immunostaining was highest in ATC followed by papillary thyroid cancer; follicular thyroid cancer and most benign thyroid neoplasms and normal thyroid tissue showed weak or no staining (Fig. 5A). In an independent sample set of tall-cell variant of papillary thyroid cancer, we also observed high HDAC2 protein staining (Fig. 5B). These findings suggest that the target of CUDC-907, HDAC2, is overexpressed in thyroid cancer and highest in ATC and aggressive thyroid cancer.

Figure 5.

Figure 5.

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HDAC2 is overexpressed in thyroid cancer. A, IHC for HDAC2 in TMA. Top left, shows representative staining for HDAC2; right, H score for HDAC2 by tissue category using tissue microarray. HDAC2 expression, which is significantly higher in ATC, followed by PTC and then FTC, in comparison with normal/benign tissue. B, Bottom left, representative sample of tall-cell variant of papillary thyroid cancer (TC-PTC, n = 16) and adjacent normal thyroid tissue staining for HDAC2. Bottom right, H score for comparison of TC-PTC and adjacent normal thyroid tissue. Tissues displaying HDAC2 staining. *, P < 0.05; **, P < 0.01; ****, P < 0.001.

CUDC-907 inhibits tumor growth and metastases in a mouse model of metastatic thyroid cancer

We evaluated the in vivo effect of clinical-grade CUDC-907 in a metastatic mouse model of thyroid cancer that recapitulates the clinical behavior of ATC and PDTC (25). Mice were injected with FTC-133 Luc2 or 8505C thyroid cancer cells via tail vein. A week later, once metastases were established, drug treatment was commenced (Fig. 6A). During treatment, mice developed mild diarrhea, which was treated with a trans-gel diet supplement. There was no significant difference in body weight between control and treated mice (Fig. 6B). CUDC-907 treatment resulted in significantly less overall tumor burden and liver metastases (Fig. 6C and D). We next evaluated HDAC2 expression based on treatment group and response to treatment by IHC. CUDC-907 treatment and response was associated with decreased HDAC2 expression (Fig. 6E). This suggests that HDAC2 protein levels could be used as a biomarker of CUDC-907 treatment response.

Figure 6.

Figure 6.

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CUDC-907 treatment inhibits tumor growth and metastases. A, Treatment schema. Mice were divided into 2 groups: control and treatment (n = 10 in each group), and injected with FTC133-Luc2 cells via tail vein injection. Treatment with CUDC-907 was started on day 7. CUDC-907 was administered by oral gavage (75 mg/kg). B, There was no significant change in body weight between the control and treatment groups. C, CUDC-907 treatment significantly decreased tumor growth as measured by whole-body luciferase activity. D, Number of metastatic lesions in the liver per mouse as determined by H&E. E, Representative image and scoring of HDAC2 IHC of tumor tissue in treated and control group mice. Mice treated and responding to CUDC-907 had no HDAC2 immunoreactivity. Error bars are mean ± SD. *, P < 0.05; **, P < 0.01; ****, P < 0.001, ns: not significant.

Discussion

In this study, we evaluated the anticancer activity of CUDC-907, a first-in-class dual PI3K and HDAC inhibitor, in in vitro and in vivo studies. We found that CUDC-907 inhibits growth and metastases, effectively inhibits commonly activated signaling pathways in thyroid cancer, and induces G2-M arrest and apoptosis through key regulator proteins involved in these processes and that are dysregulated in thyroid cancer. We also found that HDAC2 is overexpressed in thyroid cancer. Thus, our preclinical data show that CUDC-907 is a promising candidate for thyroid cancer therapy, and HDAC2 could be utilized as a biomarker of treatment response.

HDACs maintain a dynamic equilibrium in the cell in conjunction with histone acetyl transferases, and deacetylate lysine residues on histones to bring about transcriptional repression (39). In this manner, HDAC2 regulates the activity of many biologically important proteins. It functions as part of the NCoR/SMRT complex along with HDAC1 and HDAC3 to repress the transcriptional activity of cell-cycle checkpoint regulators and differentiation genes (40,41). HDAC2 is overexpressed in several cancers, including thyroid cancer, and HDAC2 overexpression may be associated with a less- or undifferentiated state (21, 22, 36).

The RAF/RAS/MEK/ERK and PI3K/AKT/mTOR signaling pathways are commonly activated in thyroid cancer due to frequent driver mutations involved in these pathways occurring and leading to cancer initiation and/or progression (42, 43). Several targeted therapies are in various phases of clinical trials, and a few isolated case reports of response in patients with ATC have been reported, but these patients usually demonstrate resistance, and often there is disease progression (16, 17). Thus, monotherapy with agents that target a driver event is not likely to yield durable responses, especially in patients with ATC and PDTC that have multiple genetic alterations (15). Thus, the effective inhibition of multiple pathways active in thyroid cancer with CUDC-907 treatment and its effect as an HDAC inhibitor suggest that it may be more effective than monotherapy.

In our studies to understand the mechanism of action of CUDC-907 on cellular proliferation, migration, and invasion, and target HDACs, we have identified important mediators of these cellular processes that are frequently dysregulated in aggressive thyroid cancer. CUDC-907 inhibited cellular proliferation by inducing G2-M arrest and apoptosis. We found CUDC-907 causes cell-cycle arrest at G2-M, followed by activation of apoptosis posttreatment. The effect of CUDC-907 on caspase-3/7 activation was more pronounced at 24 hours. We believe that the decrease of apoptosis observed at 48 hours is because the caspase activation window has passed, and cell death starts to occur. Our cell proliferation data are consistent with this.

Regulatory proteins involved in G2-M transition and dysregulated in thyroid cancer were downregulated (cyclin B1, AURKA, AURKB, and PLK1) and upregulated (p21) with CUDC-907 treatment (44–46). In addition to increasing apoptosis through the caspase pathway, we found CUDC-907 treatment reduced survivin protein levels and induced p27 protein expression, which is also a G2-M checkpoint regulatory protein. Survivin is an inhibitor of apoptosis, wherein survivin inhibition amplifies caspase activation (30), and survivin is upregulated in thyroid cancer, its expression being the highest in ATC (47). p27 is absent in most thyroid cancers (29, 48). E-cadherin is an epithelial adhesion molecule, and its loss is an important step during the EMT process, primarily due to loss of cell-cell contact (49). This results in the induction of multiple transcription factors that regulate E-cadherin expression, such as TWIST1, which is important for E-cadherin loss-induced metastasis (35, 49). Importantly, expression of E-cadherin is reduced in ATC (50). Although CUDC-907 treatment inhibited cellular migration and invasion with induction of E-cadherin protein expression, which was associated with increased TWIST1 expression, a known regulator of E-cadherin, there was reciprocal increase in N-cadherin and vimentin protein levels. Thus, the effective targeting of multiple downstream effectors dysregulated in thyroid cancer, in addition to inhibiting multiple pathways, suggests that CUDC-907 maybe an effective anticancer agent for thyroid cancer, especially ATC and PDTC.

In the current study, we used clinical grade CUDC-907 at clinically achievable concentrations and a dosing regimen used in a recent phase I clinical trial (51). In this study, it was demonstrated that CUDC-907 had an HDAC inhibitory effect by using skin biopsy, measuring acetylated histone H3 levels in peripheral blood mononuclear cell samples. On the basis of this and our promising preclinical study results, we have opened a phase II clinical trial focused on ATC and PDTC, and aggressive histologic variants of differentiated thyroid cancer.

In summary, we determined that CUDC-907 treatment results in significant inhibition of growth and distant metastases, effectively targets commonly altered pathways in thyroid cancer that mediate cellular proliferation, migration, and invasion, and represents a therapeutic alternative strategy for PDTC and ATC that should be investigated in a clinical trial.

Supplementary Material

suppl

Translational Relevance.

There is currently no standard or effective therapy for anaplastic thyroid cancer (ATC) and poorly differentiated thyroid cancer (PDTC), which account for two-thirds of thyroid cancer-related deaths. Driver mutations in the PI3K/AKT and RAF/RAS/MEK/ERK pathways are common in ATC and PDTC. Histone deacetylases (HDAC) regulate cancer initiation and progression. CUDC-907, a first-in-class compound, significantly inhibited cellular proliferation in thyroid cancer cell lines, induced G2-M arrest and apoptosis, and decreased cellular migration and invasion. CUDC-907 treatment caused H3 hyperacetylation and decreased HDAC2 expression. HDAC2 was upregulated in ATC and other thyroid cancer histologic subtypes. CUDC-907 treatment reduced both p-AKT and p-ERK1/2 levels. CUDC-907 treatment showed significant inhibition of growth and metastases, and tumors from treated mice had decreased HDAC2 expression, suggesting that this may be a useful biomarker of response. These findings support that CUDC-907 should be tested in a clinical trial for ATC and PDTC.

Acknowledgments

The authors would like to express gratitude to Curis, Inc. for providing them with clinical grade CUDC-907 for the animal studies.

Grant Support

This research was supported by the intramural research program of the Center for Cancer Research, NCI, NIH (grant number 1ZIABC011275-06 to E. Kebebew).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

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