Potent antitumor activity of HSP90 inhibitor AUY922 in adrenocortical carcinoma
Junchao Huang . Chengchao Sun . Ting Zhang . Lei Pan . Suqing Wang . Qiqiang He . Dejia Li
Received: 16 April 2014 / Accepted: 6 May 2014
C International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The objective of this study is to investigate the expression of HSP90 and the effect of HSP90 inhibitor AUY922 in ACC. The expression of HSP90 was measured in tissue samples from 36 human sporadic adrenocortical tumors by immunohistochemistry, Western blotting, and real-time PCR. The effect of AUY922 was tested on SW13 and H295R cells by evaluating cell viability and apoptosis in vitro. Transwell assay was performed to evaluate the mi- gration of SW13 cells after different concentrations of AUY922. Western blot, real-time PCR, and immunohisto- chemistry revealed that both HSP90 mRNA and protein were obviously expressed in a higher degree in ACC tissues than ACA tissues and normal adrenal tissues (P<0.01). Positive staining for HSP90 was found in 15 of 20 ACCs (75.00 %) and in 3 of 16 (18.75 %) ACAs. There existed the significant statistical difference (P<0.001). AUY922 inhibited the prolif- eration of ACC cells in a time- and concentration-dependent manner, and increasing apoptosis was observed in tumor cells treated with the HSP90 inhibitor. Finally, migration of SW13 cells was distinctly suppressed after undergoing treatment with AUY922. Our data suggest that the specific HSP90 inhibitor AUY922 can play a therapeutic role in treatment of ACC and, thus, HSP90 could qualify as a promising new target in ACC.
J. Huang
Hubei Province Key Laboratory on Cardiovascular, Cerebrovascular, and Metabolic Disorders, School of Nuclear Technology and
Chemistry & Biology, Hubei University of Science and Technology, Xianning 437100, China
C. Sun . T. Zhang . L. Pan . S. Wang . Q. He . D. Li () Department of Occupational and Environmental Health, School of Public Health, Wuhan University, 115 Donghu Road, Wuhan 430071, China
e-mail: lidjwuhan@163.com
Keywords Adrenocortical carcinoma . Heat shock protein 90 . AUY922 · Targeted therapy
Introduction
Adrenocortical carcinoma (ACC) is a rarely but typically aggressive malignancy characterized by dismal prognosis and lacking efficacious therapeutic regimens [1]. Radical sur- gery remains the solely potentially curative option; however, about one third of patients present initially with distant metas- tases and some other patients develop local or metastatic recurrence even after apparently complete resection [2]. The poor prognosis may be attributable to the fact that many ACCs are not detected until they are at advanced stage [3]. For patients without disseminated disease, aggressive surgical excision should be the mainstay of treatment of ACC. For patients with metastasized ACC and not amenable to complete resection, mitotane in combination with cytotoxic drugs are promising treatment regimens. Results have been published for the phase III clinical of etoposide, doxorubicin, cisplatin, and mitotane (EDP/M) versus streptozocin and mitotane (Sz/ M) in advanced ACC. Preliminary results released that EDP/ M is superior to Sz/M in terms of progression-free survival and thus should be considered the standard first-line treatment for patients presenting with stage III or IV ACC [4]. The 2nd Annual International Adrenal Cancer Symposium unanimous- ly stated that patients with potential residual disease (R1 or Rx resection) and/or Ki67 more than 10 % should be offered adjuvant mitotane, whereas adjuvant therapy is not considered mandatory in patients fulfilling all of the following criteria: stage I or II disease (based on the ENSAT Stage), histologi- cally proven R0 resection, and Ki67 expressed in ≤10 % of neoplastic cells [5]. Despite of the treatment, the 5-year sur- vival rate is less than 15 % among patients with metastatic disease. No effective treatment has been developed yet. The
overall prognosis is limited, indicating the need for improved therapies directed at potential molecular targets.
Small numbers of patients with ACC have been evaluated for response to targeted therapy in individual case reports or as part of clinical trials of endocrine tumors [6, 7]. As yet, no clinical study demonstrating positive impact of therapy on progression-free survival or overall survival has been reported in patients with ACC, highlighting the importance of explor- ing additional molecular targets. Heat shock protein 90 (HSP90) is a molecular chaperone that comprises 1-2 % of total cellular protein content and regulates the correct confor- mation, activity, function, and stability over 200 client pro- teins [8]. Many client proteins of HSP90 are involved in cell cycle control and proliferative/antiapoptotic signalling whose dysregulation may drive cancer. Targeting HSP90 may repre- sent a therapeutic strategy that could potentially interfere with multiple oncogenic pathways.
The classic HSP90 inhibitors were the benzoquinone and ansamycins, including geldanamycin and its derivatives 17- allylamino-17-demethoxygeldanamycin (17-AAG). These in- hibitors have been extensively studied in phase I and phase II trials [8, 9]. Recently, HSP90 inhibition was performed with novel low molecular weight ATP-competitive non- geldamycin HSP90 inhibitors AUY922. The compound has been speculated to offer advantages over ansamycin benzo- quinone HSP90 inhibitors such as 17-AAG based on the independence from NAD (P) H: quinone oxidoreductase 1 (NQO1) metabolism, P-glycoprotein expression, and favor- able aqueous solubility. In this article, we investigate the expression of HSP90 in ACCs and evaluate the antitumor efficacy of HSP90 inhibitor AUY922 in vitro.
Materials and methods
Ethical statement
For the analyzed tissue specimens, all patients gave informed consent to use excess pathological specimens for research purposes. The protocols employed in this Subjects Commit- tee. The use of human tissues was approved by the institu- tional review board of the Wuhan University and conformed to the Helsinki Declaration and to the local legislation. Pa- tients offering samples for the study signed informed consent forms.
Patients and tissue samples
Approved by the Institutional Ethics Review Board, patholo- gy specimens and medical records were reviewed from the database. The preoperative diagnosis was based on the clinical history, symptoms, signs, endocrine evaluation, and image examination (e.g., MRI and CT). Histological features,
including high mitotic rate, atypical mitoses, high nuclear grade, low percentage of clear cells, necrosis, diffuse archi- tecture of tumor, capsular invasion, sinusoidal invasion, and venous invasion, are carefully analyzed according to the method of Weiss and Medeiros [10]. The pathological diag- nosis of ACC was based on Weiss’s criteria with its score ≥3 [11]. Thirty-six adrenocortical tumor tissues, collected from surgical specimens of 36 patients, were divided into two groups: 16 benign adrenal cortical tumors and 20 ACCs. Besides clinical diagnosis, histopathologic slides were classi- fied by two pathologists independently and no discrepancy exists between them.
Immunohistochemical analysis
To evaluate the HSP90 expression in ACC, the immunohis- tochemical analyses were performed using the En Vision method (DAKO, Glostrup, Denmark). Antigen retrieval was achieved by microwave at 750 W for 15 min, and the sections were incubated with 10 % normal goat serum at room tem- perature for 10 min to block non-specific reactions. This was followed by a PBS wash and incubation with polyclonal mouse antihuman HSP90 antibody (Abcam, USA) diluted to 1:100 for 12 h at 4 ℃. Localization of immunostaining was demonstrated by incubation with EnVision-peroxidase sys- tem. The staining results of pancreatic carcinoma tissue sec- tions which HSP90-positive had already known were regarded as positive control, PBS instead of primary antibod- ies was as negative control.
Evaluation of immunohistochemical results
Positive HSP90 staining was characterized by purple brown granules located diffusely in the cell cytoplasm. Lack of any obvious purple brown or brown red pigmentation in the cyto- plasm of tumor cell was considered negative. For quantitative analyses of expression, five visual fields were randomized selected per section under high power microscope (×400), and 200 cells were counted in each high power field. Staining was scored according to the percent of positive staining tumor cells, including 0 to <5 %, 1-5 to 29 %, 2-30 to 50 %, and 3 to >50 %, and as intensity, including 0, no; 1, weak; 2, moderate; and 3, strong staining. Positive or negative expression was determined according to the combination of these two vari- ables. A total score of greater than 3 was considered positive and a total score of 3 or less was considered negative. The results were scored by two independent pathologists who were blinded to the diagnosis.
Total RNA extraction and cDNA synthesis
Upon collection, the tissues were snap frozen in liquid nitro- gen and subsequently kept at -80 ℃ until required. The
tissues were pulverized and total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The purity and concentration of RNA were determined by spectrophotometric methods. Three micrograms of total RNA were reverse-transcribed into first- strand cDNA using reverse transcription system kit (Promega, Madison, WI, USA) according to the following protocol with the reaction kit. Briefly, samples were preincubated at 70 ℃ for 10 min, cooled on ice, then added to a reaction mixture of 10 mmol/L dNTP mixture, 25 mmol/L MgCl2, 15 U of AMV reverse transcriptase, reverse transcription 10× buffer, 0.5 U of Rnasin and 0.5 µg oligo-(dT)15 primer, and scaled up to a final volume of 20 L. The reaction mixture was sequentially incubated at 44 ℃ for 15 min, 99 ℃ for 5 min, and 4 ℃ for 5 min. The cDNA was stored at 20 ℃ before use.
Quantitative real-time polymerase chain reaction
Quantitative RT-PCR was performed using SYBR Master Mix (Takara) on an ABI Prism 7900HT (Applied Biosystems). A human GAPDH gene was used as an endog- enous control for sample normalization. Results were present- ed as the fold expression relative to that of GAPDH. PCR primers were as follows: for human GAPDH, forward 5’- GAGTCAACGGATTTGGTCGT-3’ and reverse 5’- GACA AGCTTCCCGTTCTCAG-3’; for human HSP90 forward 5’- TTGCTTCAGTGTCCCGGTGCG-3’ and reverse 5’-TGGT TGGTCTTGGGTCTGGGT-3’.
Western blot
Western immunoblot analyses were performed with protein lysates obtained from snap-frozen tissue samples. Protein levels were determined using the BCA Protein Assay Kit (Pierce, USA). Thirty micrograms of the respective tissue protein were separated by SDS-PAGE (using 10 % gels) and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). The membranes were blocked with 5 % non-fat milk and then incubated with mouse antihuman HSP90 polyclonal antibody (1:100; Abcam, USA) and actin (1:10,000; MP Biomedicals). Each membrane was washed three times for 10 min with Tris-buffered saline (50 mM Tris, pH 7.4, 0.9 % NaCl) containing 0.05 % Tween-20 (TBS-T) and incubated with horseradish peroxidase-conjugated sec- ondary antibodies. Each membrane was then washed again three times for 10 min with TBS-T. Target protein bands were visualized using the enhanced chemiluminescence method. All western immunoblot analyses were performed three times.
Cell culture and reagents
H295R and SW13 cell lines were supplied from the American Type Culture Collection (ATCC). The H295R cells were
cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 Nutrient mixture (DMEM/F12; Sigma) supplemented with 1 %L-glutamine (Sigma) and 2.5 % of Nu-Serum (Becton Dickinson) and enriched with 1 % ofITS + Premix (Becton Dickinson), whereas SW13 cells were cultured in DMEM (Sigma) supplemented with 10 % FBS (ATCC). The HSP90 inhibitor AUY922 was purchased from Selleck (Houston, USA). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) at stock solutions and stored at-20 ℃. Mouse antihuman HSP90 antibody (Abcam, USA) was obtained.
Cell viability measurement
Cell viability was analyzed by Thiazolyl Blue (MTT, Sigma- Aldrich, USA). The cells were divided into three groups: negative controls (the solvent DMSO-treated cells served as control), blank controls, and experiment groups. Cells of the experiment group that were grown to 70-80 % confluency in 96 well plates were treated with AUY922 at a final concen- tration of 1, 2.5, 5, 10, 25, 50, and 100 nM for 24, 48, or 72 h, respectively. The blank group was only added with culture fluid. Cells treated with 100 µL cell suspension and DMSO was used as negative control. After the reaction with the drugs for 24, 48, 72 h, cells were then treated with MTT for 4 h at 37 °℃. Cells were subjected to absorbance reading at 570 nm using a 96-well microplate reader. The OD values were nor- malized to cells treated with 0 nM of AUY922. Percentage of residual cell viability was determined as [(OD of experiment group - OD of blank group)//(OD of negative group - OD of blank group)]×100 %. Assays were performed three times.
Flow cytometry assay of cell apoptosis
After treating cells with AUY922, the cells were washed twice in cold PBS and labeled with Annexin V-FITC and propidium iodide (PI) and analyzed immediately after staining with a FACScan flow cytometer (BD Biosciences) and FlowJo soft- ware according to the manufacturer’s recommendations. Quantification of Annexin V-FITC and PI binding was con- ducted with a FACScan flow cytometer. Cell flow cytometric analysis was used to differentiate between living, early apo- ptotic, late apoptotic/necrotic, and necrotic cells by staining with Annexin V-FITC and PI.
Cell migration assay
Motility and invasion capabilities in vitro were measured with Transwell chambers (Corning, Corning, NY, USA). Four groups of cells (5×10) were seeded on the upper wells with serum-free medium. Medium with 20 % FBS was plated in the bottom wells as chemoattractants. After 48 h incubation, cells were fixed with methanol and stained with 1 % crystal
| Clinical characteristic | ACCs | ACAs |
|---|---|---|
| Total number of tumors/ patients | 20/20 | 16/16 |
| Gender | ||
| Male | 7 | 10 |
| Female | 13 | 6 |
| Age at presentation (year) mean (range) | 59.31 (43-70) | 47.45 (41-75) |
| Tumor location | ||
| Left adrenal | 11 | 7 |
| Right adrenal | 9 | 9 |
| Bilateral | 0 | 0 |
| Primary tumor size (cm) | ||
| <5.0 | 2 | 13 |
| 5.0-10.0 | 15 | 3 |
| >10.0 | 3 | 0 |
| Mean diameter (cm) | 7.7±2.1 | 3.9±2.4(P<0.001) |
| ENSAT stage | ||
| I | 4 | |
| II | 11 | |
| III | 5 | |
| IV | 0 | |
| The Weiss score | ||
| 0-3 | 0 | 16 |
| 3-6 | 18 | 0 |
| 6-9 | 2 | 0 |
violet for 30 min at 37 ℃. Cells staying on the upper side of the membranes were wiped, while those on the lower side were counted and photographed with microscope.
Statistical analysis
All statistical analyses were conducted using the SPSS soft- ware, version 15.0 (Chicago, USA). Patient characteristics are
shown as the mean ± SD for continuous variables and as the count and percent for discrete variables. Phenotypic differ- ences in quantitative traits were assessed by genotype using the t test or ANOVA. Differences in the distribution of qual- itative traits by genotype were assessed by standard chi-square analysis and Fisher’s exact test. A P value less than 0.05 was considered significant.
Results
HSP90 was overexpressed in ACC tissues compared with ACA tissues and normal adrenal tissues
HSP90 was previously detected to be overexpressed in vari- ous cancers; we, thus, wondered whether it is also overexpressed in ACC tissues compared with adrenocortical adenoma (ACA) tissues and the normal ones, so the expres- sion of HSP90 was measured in a series of tissue samples from 36 human sporadic adrenocortical tumors by immuno- histochemistry, Western blotting, and real-time PCR. These specimens were classified as adenomas (n=16) and carcino- mas (n=20) according to the histological criteria defined by Weiss. Surgical specimens of adrenal gland were from 20 patients undergoing nephrectomy; histopathologic slides of adrenal gland were confirmed to normal tissue by patho- logical diagnosed. All of these characteristics were ob- tained from patient medical records and summarized in Table 1.
As shown in Fig. 1a, b, positive staining for HSP90 was observed in 75.00 % (15/20) of the ACC group and 18.75 % (3/16) of the ACA group. The difference of HSP90 expression between ACA and ACC was statistically significant (P<0.001) (magnification×40). The expression of HSP90 mRNA normalized to GAPDH mRNA was detected by quan- titative real-time PCR (Fig. 2a). The expression level of HSP90 mRNA was significantly increased in ACC tissues compared with that in ACA tissues and normal prostate tissue (P<0.01). The results were confirmed by Western blot analy- ses, and we also found the protein expression levels of HSP90
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was higher in ACCs than in ACA tissues and normal adrenal tissues, and the difference between these groups had statistic significance (P<0.01) (Fig. 2b).
AUY922 plays an inhibiting role in proliferation of ACC cells and induces apoptosis in ACC cells
As shown in Fig. 3, MTT assay shows that AUY922 signif- icantly inhibited the proliferation of SW13 (Fig. 3a) and H295R (Fig. 3b) cells in a time- and dose-dependent manner (P<0.05). The half maximal growth inhibitory concentration in SW13 and H295R cells after 72 h (IC50) was 9.2 and 4.1 nM, respectively. In SW13 and H295R cells, apoptosis significantly increased after inhibition of HSP90. We test the survival rates after action of AUY922 in 24 and 48 h in the experiment group. The results show that the AUY922 induced apoptosis in SW13 (Fig. 4a) and H295R cells (Fig. 4b).
Inhibition of migration of SW13 cells by AUY922
We used the Transwell assay to verify the AUY922’s effect on migration of ACC cell in vitro. The results of SW13 showed that in migration assay, the number of SW13 cells that pene- trated through the membrane in the AUY922-treated groups was significantly less than the negative control group, and SW13 cells in the 100-nM group penetrated less cells through the polycarbonate membrane than the 10-nM group (P<0.05) (Fig. 5). These results show a critical role of AUY922 in the inhibition on ACC migration.
Discussion
Unlike benign ACA that can be cured by surgical resection, ACC is a rare malignancy characterized by dismal prognosis and lacking efficacious therapeutic regimens [12]. Veytsman et al. [13] point out that evidence on the efficacy of adjuvant mitotane in patients with ACC is based only on retrospective studies; however, no randomized prospective trials have so far been published, and no data from randomized clinical trials on adjuvant treatment in ACC will be available in the near future. The option of long-term monotherapy is restricted to patients who tolerate mitotane and either experiences a clinical re- sponse or is at high risk for recurrence. A preferable adminis- tration schedule is to start with 1 to 2 g/day and to increase the daily dose by 1 to at most 2 g every 1 to 2 weeks to the maximum-tolerated dose (never >6 to 10 g/day). Four to six grams is usually sufficient. The molecular pathogenesis of ACC, particularly in various signaling pathways, has attracted growing attention during the last decade. Preclinical investi- gations and clinical trials of emerging targeted therapy (e.g., small-molecule tyrosine kinase inhibitors and antiangiogenic compounds) have been initiated to seek the more rational treatment of choice in advanced ACCs; however, the first preliminary results of these new drugs have been largely disappointing [14].
HSP90 is the most abundant cytosolic HSP and regulates the maturation and stability of various proteins crucial for multiple cell signaling processes. In a variety of cancers, including hepatocellular carcinoma, breast cancer, non-
Fig. 3 AUY922 inhibits proliferation of ACC cells. Cell number was measured by MTT assay, AUY922 concentration dependently inhibited cell proliferation at 24, 48, and 72 h. a Cell proliferation curve of SW13; b cell proliferation curve of H295R
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small-cell lung cancer, and prostate cancer, HSP90 is overexpressed and may contribute to tumor cell survival by mediating the maturation and stability of a variety of client proteins, including the IGF1 receptor and elements of the PI3/ Akt, STAT3, and MAPK signalling pathways [15]. Therefore, HSP90 is recognized as a crucial facilitator of oncogene addiction and cancer cell survival [8, 16]. Although HSP90 was identified to have oncogenic abilities in several cancers, the oncogenic contribution of HSP90 in ACC has not been clarified until recently.
The aim of our study was to examine the impact on the oncogenetic process through investigating the expression and function of HSP90 in ACC. In the present study, immunohis- tochemistry, real-time PCR, and Western immunoblot analysis were performed for comparison of expression of HSP90 be- tween ACCs and ACAs; our findings on ACCs are in line with the previous reported overexpression of HSP90 in different tumor entities [17]. The expression level of HSP90 mRNA and protein were significantly increased in ACC tissues com- pared with that in ACA tissues and normal adrenal tissue (P<0.01). The HSP90 chaperone machine, consisting of HSP90, the chaperone HSP70, and additional proteins termed co-chaperones, can be used by cancer cells to contribute to resistance to drugs, proliferation, tissue invasion, metastasis, and angiogenesis, all critical components for tumor progres- sion and survival.
Recently, inhibition of HSP90 has become one of the most popular research focuses [18]. HSP90 inhibitors have been
considerably improved; AUY922 is part of the isoxazole HSP90 inhibitor family and inhibits ATPase activity with an IC50 of 30 nM [19]. This molecule is a non-geldanamycin analog, which have the promise of more prolonged target inhibition and which so far have not been associated with the same degree of hepatotoxicity as their geldanamycin counterparts. AUY922 exerts its effects by binding to the ATPase domain of the HSP90 N-terminal, preventing HSP90 from its chaperone functions. This leads to the proteasomal degradation of many relevant client proteins. Single-agent AUY922 has shown potent preclinical antican- cer activity in vitro and in vivo against a range of histologic cell types including head and neck squamous cell carcinomas (HNSCC); pancreatic, prostatic, lung, cervical, colorectal, and breast carcinomas; myelomas; and melanomas [20-24]. In our study, we treated SW13 and H295R cells with different con- centrations of AUY922, cell survival rate was measured by MTT assay, and cell apoptosis was detected by flow cytometry in order to test the inhibiting effect of AUY922 on ACC cells. Our study found that AUY922 significantly inhibited cell proliferation and induced apoptosis of SW13 and H295R cells in a time- and dose-dependent manner. AUY922 can inhibit various tumor cells from proliferation and induce apopto- sis of the tumor cell according to previous research re- ports; therefore, our results validate HSP90 as an impor- tant target in ACC.
The presumptive tumor suppressor function of HSP90 inhibitor AUY922 in human ACC was further investigated
A
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by in Transwell assay. Results showed that cell migration ability was significantly suppressed by AUY922. The migra- tion assay revealed that SW13 cells in AUY922-treated groups penetrated less cells through the polycarbonate mem- brane than the negative control group, and the data were dose- dependent.
In conclusion, our results validate HSP90 as an important target in ACC and provide rationale for the testing of HSP90 inhibitors as a promising therapeutic agent in clinical trials. The AUY922 treatment may provide a promising strategy for the antitumor therapy of ACC, and HSP90 qualifies as a promising new target in ACC.
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