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. Author manuscript; available in PMC: 2016 Aug 19.
Published in final edited form as: Endocr Relat Cancer. 2013 May 21;20(3):361–370. doi: 10.1530/ERC-12-0403

TOP2A is overexpressed in and a therapeutic target for adrenocortical carcinoma

Meenu Jain 1, Lisa Zhang 1, Mei He 1, Ya-Qin Zhang 2, Erin E Patterson 1, Naris Nilubol 1, Min Shen 2, Electron Kebebew 1
PMCID: PMC4990817  NIHMSID: NIHMS803967  PMID: 23533247

Abstract

Background

Adrenocortical carcinoma (ACC) is a rare but aggressive malignancy with no effective therapy for patients with locally advanced and metastatic disease. The current study aimed to evaluate expression and function of TOP2A in human adrenocortical neoplasm, and anticancer activity of agents that target TOP2A.

Methods

TOP2A mRNA and protein expression levels were evaluated in 112 adrenocortical tissue samples. In vitro siRNA knockdown of TOP2A in ACC cell lines (NCI-H295R and SW13) was used to determine its effect on cellular proliferation, cell cycle, anchorage-independent growth and cellular invasion. We screened 14 TOP2A inhibitors for their anticancer activity in ACC cell lines.

Results

TOP2A mRNA expression was significantly higher in ACC than in benign and normal adrenocortical tissue samples (p < 0.001); likewise, TOP2A protein expression was significantly higher in benign and malignant samples than in normal samples (p < 0.05). TOP2A mRNA expression was enhanced up to 2.5-fold in ACC cell lines after decitabine treatment. Knockdown of TOP2A gene expression decreased cell proliferation, anchorage-independent growth and invasion (p < 0.05). Aclarubicin, TOP2A inhibitor, had potent antiproliferative activity out of 11 active TOP2 inhibitors, which significantly decreased proliferation and tumor spheroid size in ACC cell lines (p< 0.05).

Conclusions

Our data indicate that most TOP2A inhibitors are effective compounds for ACC, with aclarubicin having best anticancer activity. Thus, TOP2A inhibitors should be tested in future clinical trials for patients with locally advanced and metastatic ACC and that expression of TOP2A may be enhanced by the use of demethylation agents.

Keywords: Adrenocortical carcinoma, TOP2A, TOP2 inhibitors, aclarubicin, invasion

Introduction

Adrenocortical carcinoma (ACC) is a rare but aggressive malignancy of the adrenal cortex1–4. Prognosis is poor in patients with locally advanced and metastatic ACC with five-year survival rate of less than 10%1,5,6. Therefore, there is a significant need for the identification of new therapeutic options that may be effective in patients with ACC.

Genome-wide gene expression profiling analysis has been used to identify dysregulated gene expression associated with ACC7–11. One of the genes consistently observed to be overexpressed in ACC is topoisomerase alpha 2 (TOP2A)10–12. TOP2A encodes a DNA topoisomerase that controls and alters the topologic states of DNA during transcription and thus is involved in processes such as chromosome condensation and chromatid separation. TOP2A has been shown to be a marker of proliferation, aggressive disease and chemotherapy resistance in a variety of human cancers13–18. More recently, targeting TOP2A with a variety of agents having TOP2A inhibitor activity, such as the anthracycline drugs, has emerged as an attractive strategy for cancer therapy and has led to clinical trials to test the efficacy of these compounds19–25.

In the present study, we examined the expression of TOP2A in human adrenocortical tissue samples and found that TOP2A was overexpressed in 100% of the ACC samples. Using siRNA knockdown of TOP2A in ACC cell lines, we characterized the function of TOP2A and found that it regulates not only cellular proliferation but also cellular invasion in ACC cells. Given the overexpression of TOP2A, we tested 14 TOP2A inhibitors for their antiproliferative effect and found 11 of the 14 had good efficacy. Aclarubicin had the most potent anticancer activity.

Materials and Methods

Tissue specimens

Adrenal tissue samples were collected at surgery and stored at −80°C. In this study, 112 human adrenocortical tissue specimens were analyzed (21 normal adrenal glands, 80 benign adrenocortical tumors and 11 primary ACC). The clinical protocol was approved by the Institutional Review Board and written informed consent was obtained. The inclusion criterion for diagnosis of ACC was made by the presence of local invasion and or regional or distant metastases.

Cell culture and reagents

The, NCI-H295R and SW13 ACC cell lines (ATCC, Rockville, MD) were grown and maintained in DMEM media supplemented with 1% insulin transferrin selenium (ITS) (BD Biosciences, San Jose, CA) and 2.5% Nu-Serum I (BD Biosciences) in a standard humidified incubator at 37°C in a 5% CO2 atmosphere.

Immunohistochemistry

Immunohistochemistry was performed as previously described26. Primary anti-TOP2A mouse monoclonal antibody was used (Enzo Life Sciences, Ann Arbor, MI; ADI-KAM-CC21) at a 2.5-μg/ml dilution overnight at 4°C. The biotinylated secondary antibody was used 1:1000 dilution (Vector Laboratories, Burlingame, CA) for 1 hr at room temperature.

RNA preparation

RNA was extracted using the TRIzol reagent according to the manufacturer’s instructions (Invitrogen Inc., Carlsbad, CA). RNA quantity and quality were assessed as previously described26.

Reverse transcription and real-time quantitative PCR

Total RNA (200–500 ng) was reverse-transcribed using a High Capacity Reverse Transcription cDNA kit and cDNA was amplified according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). The PCR primers and probes for TOP2A (Hs_010180383_m1) and GAPDH (Hs_99999905_m1) were obtained from Applied Biosystems.

Copy number evaluation

Total DNA (10 ng) was used to determine the copy number of TOP2A gene using real-time quantitative PCR. The PCR primers probes for TOP2A27 and RNAseP (reference gene) were obtained from Applied Biosystems. All reactions were performed according to the manufacturer protocol (Applied Biosystems). The initial step of the PCR reaction was set at 50°C for 2 min, and the denaturation step was performed at 95°C for 10 min. The amplification was performed for 40 cycles at 95°C for 15 sec and at 60°C for 1 min. Data were collected using the absolute quantification method in the SDS software (Version 2.3).

Copy number calculation was performed using the DDCt method with CopyCaller software (ABI, Version 1.0). The detection of RNaseP, known to exist only in two copies in a diploid genome, was used as the calibrator to estimate the copy number of TOP2A.

Decitabine Treatment

NCI-H295R and SW13 adrenocortical carcinoma cell lines were maintained as described previously28. The cell lines were treated with the demethylating agent, 5-aza-2-deoxycytidine (5-Aza-dC; Sigma, St. Louis, MO). The cells were exposed to 5-Aza-dC at 10 μM and vehicle (dimethyl sulfoxide [DMSO]) for up to 4 days. The media were replaced with 5-Aza-dC and vehicle every 24 hr.

Western Blot

The whole-cell lysate was prepared with 1% sodium dodecyl sulfate (SDS) plus 10 mM Tris [pH 7.5] buffer and western blot was performed on 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel as previously described26. Primary mouse monoclonal antibodies, anti-TOP2A (Enzo Life Sciences; ADI-KAM-CC21) was used at 5 μg/ml dilution and anti-GAPDH (sc-32233; Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 1:3000 dilution.

Cell culture and siRNA transfection

Cells were transfected as previously described previously28. A nonspecific negative control siRNA (AM4613) and TOP2A-specific siRNAs at a final concentration of 90 nM were used (si#1; s14308 and si#3; s14309, Applied Biosystems).

TOP2A inhibitors

A quantitative proliferation assay of drugs that inhibit TOP2A was performed in the NCI-H295R ACC cell line using the CellTiter-Glo® Luminescent Cell Viability Assay. The drugs were serially diluted 1:2.24 in DMSO (Thermo Fisher Scientific, Waltham, MA) in 384-well plates. The stock concentrations of the test compounds ranged from 10 mM to 0.13 μM. Fourteen TOP2A inhibitors were tested: aclarubicin, idarubicin, teniposide, daunorubicin, doxorubicin, mitoxantrone, amsacrine, topotecan, pirarubicin, rubitecan, etoposide, irinotecan, sarafloxacin and gatifloxacin. We further validated the antiproliferative effect of aclarubicin (InterBioscreen Ltd., Moscow, Russia) in monolayer and three-dimensional multicellular aggregate (MCA) culture of NCI-H295R and SW13 cell lines.

Cell proliferation

Cells were seeded at a concentration of 5000 cells (NCI-H295R) and 2000 cells (SW13) per 150 μL culture medium in a 96-well plate in six replicates. Cells were treated with different drug concentrations based on their half-maximal inhibitory concentration (IC50) obtained from our drug screening using six replicates. The CyQUANT™ assay kit (Invitrogen) was used to evaluate cell number according to the manufacturer’s instructions.

Three-dimensional MCAs

We used a three-dimensional MCA model to mimic an in vivo solid tumor and test the anticancer activity of TOP2A inhibitors. A total of 1 × 105 NCI-H295R cells/well and 6 × 104 SW13 cells/well (in 0.5 ml) were plated in 24-well ultra-low attachment plates (Corning® Costar®, Corning NY) to generate MCAs. The plates were cultured at 37°C in 5% CO2 for 1 week, and the medium was changed every 3 days. After 1 week of culture for NCI-H295R cells and 3 weeks of culture for SW13 cells, distinct MCAs were formed. At these time points, the MCAs were photographed and treated with different concentrations (0.05–1 μM) of aclarubicin or vehicle (DMSO). The MCAs were treated twice a week for 3 weeks and photographed. All experiments were repeated at least three times for each testing concentration.

Cell invasion assay

Cell invasion assay was performed as previously described26,28. Invaded cells were fixed, stained with Diff Quik Stain (Dade Behring, Newark, DE) and counted under a light microscope in four separate fields. The experiments were repeated three times.

Soft agar anchorage independent growth assay

The soft agar assay in NCI-H295R cells was performed as previously described26,28.

Statistical Analyses

Data were represented as mean ± standard deviation (SD) or standard error of mean (SEM). Student’s t-test or two-tailed analysis was used to assess variance for multiple comparisons among differences among groups. The statistical analysis was done using StatView 5.0 (SAS Institute, Cary, NC) and SPSS v 16.0 (SPSS Inc., Chicago, IL) statistical software. A p-value < 0.05 was considered as significant. For the quantitative proliferation assay of the TOP2A inhibitors, titration-response data for each sample were plotted and modeled by a four-parameter logistic fit to determine compound activity. Curve-fits were then classified by criteria previously described29. Class 1.1 and 1.2 were full curves containing upper and lower asymptotes with efficacy ≥ 80% and < 80%, respectively. Class 2.1 and 2.2 were incomplete curves, having only one asymptote with efficacy ≥ 80% and < 80%, respectively. Class 3 curves showed activity at only the highest concentration or were poorly fit. Class 4 curves were inactive having a curve-fit of insufficient efficacy or lacking a fit altogether. Only TOP2A inhibitors with Class −1.1, −1.2 and −2.1 curves were considered as high-confidence actives.

Results

TOP2A is overexpressed in ACC

TOP2A mRNA expression was significantly higher in ACC than in normal adrenocortical tissue and benign adrenocortical tumors (p < 0.001) (Fig. 1a). TOP2A mRNA expression was also high in both ACC cell lines (Fig. 1b). TOP2A protein expression was also higher in ACC and benign adrenocortical tumor samples than in normal adrenocortical tissue (Fig. 1c).

Figure 1.

Figure 1

Figure 1

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TOP2A mRNA expression in ACC. (a) Real time quantitative PCR was used to measure TOP2A mRNA expression in normal adrenal cortex, benign adrenocortical tumors, ACC. The relative expression was normalized to GAPDH. Mean ± SEM. **, (p < 0.005). (b) TOP2A mRNA expression in NCI-H295R and SW13 cell lines. (c) Representative images from TOP2A immunohistochemistry analysis in normal adrenocortical tissue, benign tumors and primary ACC at 20 × magnification.

Mechanism of regulation of TOP2A expression

To ascertain whether the TOP2A overexpression was a result of amplification, we evaluated its genomic status in DNA isolated from ACC, benign tumors and normal adrenocortical samples. However, we observed no significant change in copy number in malignant samples as compared to benign or normal samples (data not shown).

We previously found no difference in CpG methylation in TOP2A in our prior study evaluating benign and malignant adrenocortical tumors30, we wanted to determine if demethylation agents commonly used in cancer therapy to enhance molecular target expression could modulate TOP2A expression. Thus, we treated adrenocortical cell lines (NCI-H295R and SW13) with decitabine. Interestingly, we found that TOP2A mRNA expression was enhanced by 2.5- to 3.1-fold in the decitabine-treated group as compared to vehicle control group (Fig. 2).

Figure 2.

Figure 2

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Decitabine treatment enhances TOP2A expression in NCI-H295R and SW13 cell lines. Fold change in NCI-H295R and SW13 cells is shown at indicated time points. Mean± SEM. *, (p < 0.05; relative to vehicle control).

Effect of TOP2A knockdown on cellular proliferation and cell cycle in ACC cell lines

Given the high expression of TOP2A in ACC, we next determined whether TOP2A regulates cell proliferation in ACC cells by using siRNA to knockdown TOP2A expression in NCI-H295R and SW13 cells (Fig. 3a). In NCI-H295R, cell proliferation decreased by as much as 30% as compared to the negative control (p < 0.05) (Fig. 3b). In SW13 cells, cellular proliferation was decreased modestly with siRNA knockdown of TOP2A as compared to the negative control (p < 0.05) (Fig. 3c). TOP2A knockdown did not have a significant effect on the cell cycle (data not shown).

Figure 3.

Figure 3

Figure 3

Figure 3

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siRNA knockdown of TOP2A protein expression in ACC cell lines. (a) Total cell lysate was extracted after 7 days of knockdown in ACC cells (siRNA and negative control) and TOP2A protein expression was determined by western blot. (b,c) Cell proliferation in ACC cell lines. The number of NCI-H295R (b) and SW13 (c) cells for the TOP2A siRNA–treated and negative control–treated groups is shown at 24h, 72h, 120h, and 168 h of transfection. Mean ± SEM. **, (p < 0.005; relative to negative control). NC = negative control (d,e) Knockdown of TOP2A in ACC cell lines reduced (d) invasion and (e) soft agar anchorage-independent growth. Invading cells and soft agar colonies were stained and visualized as described in methods. The bars represent the number of invaded cells or colonies in each group. Mean ± SEM.*, (p < 0.05; relative to negative control).

TOP2A knockdown decreases cellular invasion and soft agar anchorage-independent growth in ACC cells

We were interested in determining whether TOP2A regulates hallmarks of malignant cell phenotype, cellular invasion and anchorage-independent growth, because this gene has been suggested to be only a marker of proliferation. Cell invasion decreased by 57–71% with TOP2A knockdown as compared to the negative control in both ACC cell lines (p < 0.05) (Fig. 3d). We also performed soft agar growth assays to assess whether TOP2A regulates anchorage-independent growth. We observed significantly fewer and smaller colonies (p = 0.006) (Fig. 3e).

TOP2A positive ACC cell lines are sensitive to TOP2A inhibitors

Given that TOP2A was highly expressed in ACC samples, we evaluated its potential as a therapeutic target for ACC in vitro. Eleven of 14 TOP2A inhibitors had an antiproliferative effect in the NCI-H295R ACC cell line. The dose–response titration curve of the 11 most active drugs showed that aclarubicin had the most potent activity among TOP2A inhibitors evaluated (Fig. 4). Aclarubicin is an antitumor anthracycline drug that has been used in patients with acute myelocytic leukemia. The antiproliferative effect of aclarubicin was further validated in monolayer culture of both NCI-H295R and SW13 cell lines at concentrations ranging from 0.05 μM to 1 μM (Fig. 5a and 5b). To further confirm the cytotoxic effect of aclarubicin in a three-dimensional model that better mimics solid tumors, it was also administered to NCI-H295R and SW13 MCAs. We observed a significant decrease in the size of the MCAs after aclarubicin treatment in both NCI-H295R and SW13 cell lines at 0.10-μM and 0.05-μM concentration, which was below achievable serum concentration of aclarubicin in humans (0.34 μM) (Figures 5c and 5d). However, in NCI-H295R cells, 0.05 μM concentration did not show as dramatic a decrease in MCAs as the other doses. To determine the differences in sensitivity to TOP2 inhibitors in the SW13 and NCI-H295R cell lines, we hypothesized that altered expression of other topoisomerases, TOP1 and TOP2B, accounts for the effect. The gene expression of TOP1 and TOP2B was evaluated in cell lines but no significant difference in gene expression level was observed between the cell lines (Fig. 6).

Figure 4.

Figure 4

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Dose-response titration curve of active drugs against toposiomerase. X axis indicates the log concentration tested, and Y axis indicates the growth inhibition.

Figure 5.

Figure 5

Figure 5

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Effect of aclarubicin on NCI-H295R and SW13 cell lines. (a) Cell proliferation assay of NCI-H295R at drug concentrations ranging from 0.05 μM to1 μM and their vehicles DMSO (dimethylsulfoxide). (b) Cell proliferation assay of SW13 cell line at drug concentrations of 0.05, 0.1, 1 μM and their vehicles. X axis indicates days of treatment, and Y axis indicates the cell number. Asterisks (**) indicate p value < 0.005 for the comparison of drug treatment vs. vehicle for each concentration tested. (c, d) Effect of aclarubicin on NCI-H295R and SW13 multicellular aggregates (MCAs). MCAs were treated with 0.05–0.1 μM concentration of aclarubicin and its vehicle (DMSO) in triplicates. Representative images are shown at 25×.

Figure 6.

Figure 6

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mRNA expression of TOP2A, TOP2B and TOP1 in NCI-H295R and SW13 cell lines. Y axis indicates relative mRNA expression (2ˆ−ΔCt)*100%, and X axis indicates the specific gene (TOP1, TOP2B, TOP2A).

Discussion

There is a significant need for the development of effective treatment for patients with locally advance and metastatic ACC. In this study, we analyzed TOP2A expression and function in ACC. We found TOP2A was overexpressed in ACC and regulates cellular proliferation and invasion. As a consequence of this finding, we screened the antiproliferative activity of TOP2A inhibitors in ACC cell lines and found that most of these agents showed a significant antiproliferative activity and aclarubicin had the most potent anti-cancer activity. Moreover, TOP2A expression was enhanced in ACC cell lines treated with the demethylating agent, decitabine.

Several investigators have demonstrated that TOP2A is overexpressed in a variety of human malignancies and that high levels may be associated with more aggressive disease and a worse prognosis15,31–35. Methylation in the DNA promoter regions (CpG islands) is a mechanism that leads to aberrant gene expression in cancer. Recently, a study has described the epigenetic regulation of the TOP2A gene in a panel of 45 breast cancer cell lines36. Our study also found that TOP2A was up-regulated twofold after decitabine treatment in adrenocortical cell lines (NCI-H295R and SW13). Thus, epigenetically increasing TOP2A expression with the use of demethylating agents may be an effective strategy for increasing TOP2A expression when using agents which target TOP2A37. However, we are not able to experimentally prove this as both of the widely used and authenticated ACC cell lines have high levels of basal TOP2A expression and demethylating agents such as decitabine have an antiproliferative effect in ACC cell lines38.

The function of TOP2A is not well characterized. TOP2A was highly expressed in NCI-H295R and SW13 cell lines. We used gene knockdown strategy to effectively silence its expression. Using this strategy, we observed a modest decrease in cellular proliferation but a dramatic inhibitory effect on anchorage-independent growth and invasion. This is the first study to demonstrate that TOP2A regulates invasion in ACC cells. Our findings are consistent with the association of TOP2A overexpression with the invasiveness of cancers such as liposarcoma and in hepatocellular carcinoma35,39.

Currently, targeting of TOP2A with anthracycline drugs is an appealing strategy for the development of an effective cancer therapy. Aclarubicin is an anthracycline anticancer agent that is a strong DNA intercalating agent that prevents the binding of TOP2 to DNA40. It also inhibits TOP1. Based on these studies, several phase I and phase II clinical trials have been performed to evaluate the safety, tolerability and efficacy of this agent20–25. As TOP2A is overexpressed in several malignancies and can be targeted, we studied its expression in ACC and found it to be overexpressed in 100% of the ACC samples analyzed. We thus hypothesized that TOP2A may be an excellent therapeutic target for ACC. Indeed, we found that 11 of 14 TOP2A inhibitors tested had a significant antiproliferative effect in NCI-H295R ACC cells. Given that aclarubicin exhibited the most potent activity, we further validated this finding in monolayer cultures of NCI-H295R and SW13 cell lines. Although monolayer cell cultures can provide cell-specific response to drugs, this model lacks the important features of a three-dimensional solid tumor observed in vivo, such as the hypoxic area of the tumor center, regional differences of tumor growth and cell cycling, as well as poor delivery of drugs into deeper tumor tissue layers. Thus, we used the MCA assay to confirm the anticancer activity of aclarubicin. The effect of aclarubicn was more dramatic in the SW13 cell line than in the NCI-H295R cell line. The difference in sensitivity is not likely due to the expression levels of TOP2A or other topoisomerases (TOP1, TOP2B); as the expression levels were similar in both cell lines.

Currently, mitotane is the only therapy approved by the U.S. Food and Drug Administration for advanced or metastatic ACC, but it has poor efficacy and a narrow therapeutic window41–44. As compared to mitotane, we found aclarubicin, idarubicin, teniposide, daunorubicin and doxorubicin had a lower IC50 and higher efficacy in our screening of the TOP2A inhibitors in the NCI-H295R cell line. Aclarubicin is approved as a second-line therapy for acute myelocytic leukemia in those with refractory disease20,23,45–47. Our findings suggest that aclarubicin may be an effective therapeutic alternative in patients with advanced ACC.

In summary, the data from our study demonstrates that TOP2A is overexpressed and epigenetically regulated in ACC and may influence tumor progression, as it regulates anchorage-independent growth and invasion of ACC cells. Furthermore, most of the TOP2A inhibitors screened had good antiproliferative activity in ACC cells. Of these, aclarubicin should be further evaluated as a potential therapeutic alternative for patients with locally advanced or metastatic ACC.

Acknowledgments

This research was supported by the Intramural Research Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health.

Footnotes

Disclosure: The authors have nothing to disclose.

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