HDAC 1 and 6 modulate cell invasion and migration in clear cell renal cell carcinoma
© The Author(s). 2016
Received: 18 February 2016
Accepted: 22 July 2016
Published: 9 August 2016
Class I histone deacetylases (HDACs) have been reported to be overexpressed in clear cell renal cell carcinoma (ccRCC), whereas the expression of class II HDACs is unknown.
Four isogenic cell lines C2/C2VHL and 786-O/786-OVHL with differential VHL expression are used in our studies. Cobalt chloride is used to mimic hypoxia in vitro. HIF-2α knockdowns in C2 and 786-O cells is used to evaluate the effect on HDAC 1 expression and activity. Invasion and migration assays are used to investigate the role of HDAC 1 and HDAC 6 expression in ccRCC cells. Comparisons are made between experimental groups using the paired T-test, the two-sample Student’s T-test or one-way ANOVA, as appropriate. ccRCC and the TCGA dataset are used to observe the clinical correlation between HDAC 1 and HDAC 6 overexpression and overall and progression free survival.
Our analysis of tumor and matched non-tumor tissues from radical nephrectomies showed overexpression of class I and II HDACs (HDAC6 only in a subset of patients). In vitro, both HDAC1 and HDAC6 over-expression increased cell invasion and motility, respectively, in ccRCC cells. HDAC1 regulated invasiveness by increasing matrix metalloproteinase (MMP) expression. Furthermore, hypoxia stimulation in VHL-reconstituted cell lines increased HIF isoforms and HDAC1 expression. Presence of hypoxia response elements in the HDAC1 promoter along with chromatin immunoprecipitation data suggests that HIF-2α is a transcriptional regulator of HDAC1 gene. Conversely, HDAC6 and estrogen receptor alpha (ERα) were co-localized in cytoplasm of ccRCC cells and HDAC6 enhanced cell motility by decreasing acetylated α-tubulin expression, and this biological effect was attenuated by either biochemical or pharmacological inhibition. Finally, analysis of human ccRCC specimens revealed positive correlation between HIF isoforms and HDAC. HDAC1 mRNA upregulation was associated with worse overall survival in the TCGA dataset.
Taking together, these results suggest that HDAC1 and HDAC6 may play a role in ccRCC biology and could represent rational therapeutic targets.
Inactivation of the tumor suppressor gene von Hippel Lindau, VHL, is a common alteration in sporadic clear cell renal cell carcinomas (ccRCCs) . VHL protein is responsible for the proteasomal degradation of hypoxia inducible factors (HIF) by binding to the oxygen dependent domain on HIF, thus inhibiting downstream target genes involved in angiogenesis, glycolysis and cell cycle [2–4].
Histone deacetylases (HDACs), enzymes that regulate chromatin status and gene expression, are subdivided into four classes (I, II, III and IV), based on their structure . In ccRCC, class I HDACs (i.e. HDAC 1 and HDAC 2) have been reported to be overexpressed, as compared to adjacent non-tumor tissues . Our lab has shown that class II HDACs, HDAC 4 and HDAC 6, stabilize HIF-1α in renal and prostate tumor cells [7, 8]. However, studies related to the regulation of HDAC expression and the role of HDACs in ccRCC tumor biology remain limited.
Class I HDACs, specifically HDAC 1, is upregulated at both the mRNA and protein level under hypoxic conditions, which corresponds to increased HDAC activity that can be blocked by the HDAC inhibitor (HDACi) trichostatin A (TSA) . Class I HDACs further regulate HIF-1α stability, and TSA abrogates this effect in HeLa cells . The pan HDACi panobinostat downregulates HIF-1α protein in HUVECs as well as in prostate cancer cell lines under normoxic and hypoxic conditions . The class II HDAC 6 increases invasiveness and motility in kidney epithelial cells through deacetylation of α-tubulin, which is counteracted by a specific HDAC 6 inhibitor (tubacin) and TSA . HDAC 6 translocation to the plasma membrane is associated with membrane estrogen receptor alpha (ERα), and deacetylation of α-tubulin increases motility of breast tumor cells in vitro . HDAC 6 upregulation in MCF7 cells changed the morphological features as well as the migration capacity of these cells . In addition, estrogen receptor (ER)-positive tumors with concomitant HDAC 6 overexpression showed significant increase in overall and cancer specific survival after tamoxifen treatment . Early evidence for the expression of ERα in kidney tumors has been demonstrated in an estradiol-induced hamster kidney tumor model that showed the presence of ERα in epithelial tumor cells and stromal cells in both female and male hamsters .
There are several studies involving the combination of HDAC inhibitors and ERα antagonists in breast cancer. In ER-positive tumors, panobinostat increases cell death in synergy with hydroxytamoxifen , whereas valproic acid in combination with tamoxifen augmented the inhibition of cell proliferation and apoptosis . TSA also enhanced the effectiveness of hormonal therapy in ER-negative breast tumors through ERβ activity . Additionally, RCC cells when treated with estrogen showed decreased proliferation, migration and invasion of cells, primarily through ERβ effects .
In this study, we investigated the role of class I and II HDACs in ccRCC tumor biology by utilizing in vitro models and human samples.
Cell lines, treatments and antibodies
Renal cell lines C2, C2VHL and 786–0 were kindly provided by Drs. Jennifer Isaacs and Len Neckers (National Cancer Center). Cells were cultured in DMEM media supplemented with 10 % FBS at 37 °C and 5 % CO2 concentration. 5x105 cells in duplicate 12-well plates were serum-starved for 24 h followed by treatment with media/10 % FBS with or without the hypoxia. Cobalt chloride (100 μM) (Sigma Aldrich, Cat.no. 232696) addition for 24 h was used as hypoxia mimic in these studies. At the designated time point, cells were harvested in RIPA buffer (Sigma Aldrich, Cat. no. R0278) with protease and phosphatase inhibitors (Roche) for western blot. For short term effects on the levels of acetylated alpha tubulin, 3000 cells were plated on coverslips overnight, followed by treatment with hydroxytamoxifen (Sigma Aldrich, Cat. no. T176) and/or panobinostat (Novartis) for 4 h. Antibodies against HIF-1α (Cayman chemical, Cat.no. 10006421), HIF-2α (Abcam, Cat.no. ab199), HDAC 1 (Cell signaling, Cat.no. 5356), acetylated H3 (Millipore, Cat.no. 06–599), HDAC 6 (Santacruz Cat. no. sc-11420), ER-alpha (Santacruz, Cat. no. sc-543), acetylated α-tubulin (Life technologies, Cat. no. 32–2700), total histone H3 (Cell signaling, Cat.no. 9715), GAPDH (Cell signaling, Cat. No. 2118), and HRP-conjugated rabbit (BioRad, Cat.no. 170–6515) and mouse (Dako, Cat.no. P0260) secondary antibodies were used at the recommended dilutions.
Western blot analysis and flow cytometry
Cells were harvested using RIPA buffer for Western blot, and 40 μg of total protein were run on 12 % gels followed by wet transfer at 25 V overnight at room temperature. The blots were then blocked with 10 % milk, followed by incubation with primary antibody and HRP-conjugated secondary antibody. Protein bands were detected with ECL (Perkin Elmer, Cat.no. NEL105001EA). 8x105 cells were plated for flow cytometry, treated and harvested for fixation and permeabilization (BD Pharmingen, Cat. no. 560409). Cells were blocked with blocking serum, incubated with HDAC 1 antibody, washed, incubated with secondary FITC-conjugated anti-mouse antibody (BD bioscience, Cat.no. 554001) and finally stained with propidium iodide for cell cycle analysis. Cells were run on a LSR Fortessa, and results were analyzed using FCS Express software.
The wt-VHL plasmid was kindly provided Dr. Michael Ohh (University of Toronto) and transfected into 786–0 cells with Lipofectamine 2000 (Life technologies, Cat.no. 11668–019) and OptiMEM media (Life Technologies, Cat. no. 31985070). The following day, cells were incubated with media containing neomycin and selected for two weeks for stable transfection. The HDAC 6 plasmid (kindly provided by Dr. Tso Pang Yao at Duke University) and the HDAC 1 shRNA were transfected and packaged in retroviral cells at the RPCI genomics core facility. Retroviral supernatants were added to C2 and 786–0 cells, spun for 45 min at 1800 rpm and incubated for 4 h at 37 °C. Regular medium was then added to the cells, and puromycin (for HDAC 1 knockdown selection) or neomycin (for HDAC 6 selection) was added for selection the next day. Cells that were infected were selected for a period of two weeks. HDAC 1 and HDAC 6 knockdown was observed by Western blot and immunofluorescent analysis, respectively. For HIF-2α knockdown, shRNA against HIF-2α was purchased from Addgene (Plasmid 22131) and transfected using retroviral supernatants generated at the RPCI genomics core facility. The next day, cells were incubated with regular media and selected with neomycin for a period of two weeks. The cells were tested for HIF-2α knockdown efficiency by Western blot analysis. For ERα knockdown, siRNA against ERα was transfected using Lipofectamine 2000 in OptiMEM media. The cells were tested for ERα knockdown efficiency by Western blot, and acetylated α-tubulin levels were measured by immunofluorescence.
Proliferation and Invasion assay
For proliferation assays, 8x103 cells were plated in 24 well plates with regular media and harvested after 24, 48 and 72 h for measurement of proliferation by staining the wells with crystal violet. This was followed by dissolution of the stain in methanol for 2–3 h, and the plates were read at 590 nm. Proliferation at different time points was compared to 24 h for growth rate calculations. For invasion assays, 5x105 cells were plated on top of Matrigel-coated chambers (BD bioscience, Cat.no. 354480) in regular medium with serum overnight. The medium on top was replaced with serum free media the next day, and media with serum was added to the bottom of the well as a chemoattractant. The non-invading cells at the top of the chamber were removed with cotton swabs (after 4 h for 786–0 cells and 24 h for C2 cells), and cells on the lower surface were stained with crystal violet (Sigma Aldrich, Cat. no. HT 90132) for 30 min. The inserts were washed thrice with distilled water, and the number of invading cells were counted by observation under the microscope. The HDAC 1 knockdown cells were compared to the parental cell lines for measuring invasion capability. In addition, a gelatin zymography assay was performed to analyze the matrix metalloprotease (MMP) activity in the cell lysate as well as in the supernatant. Briefly, 5x104 cells were plated in a 24-well plate in regular DMEM with serum for 24 h. This was followed by media change to DMEM without serum (to analyze MMP activity in the cell supernatant) for 16 h. Cell supernatants and cell lysates (harvested by RIPA Buffer) were collected at the end of the experiment. The lysates and supernatants were then run on 7.5 % acrylamide gels with 1 % gelatin (substrate for MMP); followed by incubation with renaturing buffer for 30 min, developing buffer overnight at 37 °C, stained with commassie blue for 30 min, and finally destained with destaining solution until the bands on the gel were strong and clear.
After treatments, cells were fixed in 4 % formaldehyde, followed by permeabilization with Triton-X 100 (Sigma Aldrich; Cat. no. T8787) and blocking with 1 % bovine serum albumin for one hour. The cells were stained for acetylated α-tubulin, HDAC 6 and ERα and detected by secondary FITC or Alexa-fluor tagged secondary antibody. Zeiss AxioImager and the axiovision software were used to capture immunofluorescent images at 20X magnification. Immunofluorescent images were analyzed using the NIH software Image J. Integrated density of images was calculated using Image J and plotted as bar graphs for comparison of intensity of fluorescence in images.
Motility and migration assay
5x105 cells were plated in a 12-well plate overnight to develop a monolayer, followed by creating a horizontal scratch in the plate using a sterile pipette tip. The cells were then placed in fresh media and observed over a period of 24 h. A grid was developed for the 12-well plate to maintain the same field of observation for cell motility.
Analysis of HDAC 1 promoter and non-promoter region by Chromatin immunoprecipitation assay
HDAC 1 promoter region: Forward primer: 5’-GACCGACTGACGGTAGGGA-3’, Reverse primer: 5’-GGTGCTCACCGTCGTAGTAG-3’
HDAC 1 non-promoter region: Forward primer: 5’-GAGTGTGCAGGTTCTGCTCT-3’, Reverse primer: 5’-CACACCCAGCCAGACTGAAT-3’
VEGF promoter region: Forward primer: 5’-GATCTGTGTGTCCCTCTCCC-3’, Reverse primer: 5’-AAAGTGAGGTTACGTGCGGA-3’
Immunohistochemistry and Immunofluorescence of TMA
A small cohort of TMAs was established in the lab with ccRCC that were collected immediately after nephrectomy, deidentified by the tissue procurement core at RPCI and received by the lab. The tissues were fixed in formalin, placed in a multi-tissue cassette, paraffin embedded and used for HDAC 1 and HDAC6/ERα staining. A larger cohort of patients obtained from the pathology core facility containing 120 ccRCC (GuCa2) and 88 metastatic ccRCC (GuCa4), were additionally analyzed for HDAC 1 expression. For TMAs and paraffin embedded formalin fixed tissue, the slides were first deparaffinized in xylene and decreasing concentrations of ethanol; antigen retrieval was carried out by boiling the slides in 10 mM sodium citrate in a microwave. The slides were washed, incubated with 3 % hydrogen peroxidase to inhibit peroxidase activity, blocked with horse serum (Vector Laboratories, Cat. no. S1000) and incubated with HDAC 1 or HDAC 6/ERα primary antibody overnight. For immunohistochemistry, slides were washed the next day, incubated with secondary HRP-conjugated antibody (Vector Laboratories, Cat. no. MP7401) followed by incubation with DAB (Dako, Cat. no. 3467), hematoxylin counterstaining, dehydration and mounting on coverslips using cytoseal. For immunofluorescence, slides were washed the next day, incubated with secondary antibodies conjugated to fluorochromes, followed by incubation with DAPI for nuclear staining and mounted on coverslips using Vectashield. Brightfield and fluorescent images were taken on Zeiss microscope with Axiovision software. HIF-1α and HIF-2α status in the TMA was obtained from Chintala et al.  and correlated with HDAC 1 status in these tumors.
The outcomes measures (HDAC expression, cell counts, acetylated α-tubulin levels, and qPCR data) of the in vitro studies are reported by experimental group using the mean and standard deviation; and graphically using dot- or mean-plots. Comparisons are made between experimental groups using the paired T-test (when comparing expression between tumor and adjacent non-tumor tissue) and the two-sample Student’s T-test or one-way ANOVA, as appropriate. In the TMA samples, the association between the HDAC expression and HIF-1α/HIF-2α status was assessed using one-way ANOVA; and reported graphically using box-plots. The association between HDAC expression and tumor stage (T1-2 versus T3-4) or grade (I/II versus III/IV) was assessed using the Student’s T-test. The survival outcomes (overall, disease-specific, and progression-free survival) were summarized by HDAC mRNA status (unaltered versus upregulated) using standard Kaplan-Meier methods, with comparisons made using the log-rank test. Analyses were completed in SAS v9.4 (Cary, NC) at a significance level of 0.05; therefore a p-value less than 0.05 is considered statistically significant.
TCGA data analysis
All patients gave written informed consent for the collection of biomaterials. The study was approved by the Genitourinary Disease Site Research Group at Roswell Park Cancer Institute (number: BDR 036713).
Class I and II HDACs are overexpressed in a subset of ccRCC tumors
HDAC 1 and HDAC 6 increase invasion and motility of renal tumor cell lines in vitro
HDAC 1 and HDAC 6 increase invasiveness and motility through increased MMP2/9 activity and decreased acetylated α-tubulin, respectively
HIF-α regulates HDAC 1 expression in renal cell lines in vitro
ER-α regulates acetylated α-tubulin levels in renal tumor cells in vitro through its interaction with HDAC 6
HDAC 1 expression correlates with HIF expression in ccRCC and is associated with poorer survival
HDAC 6 is associated with worse overall survival and progression free survival in TCGA data set
Although not statistically significant, HDAC 6 mRNA upregulation (in 9 % of tumors) was associated with worse overall survival and progression free survival in ccRCC patients (Additional file 1: Figure S5a-d) [20, 21].
The VHL tumor suppressor gene is deregulated in approximately 70 % of the sporadic cases of ccRCC, and its major role is to decrease the stability of HIF isoforms under normal oxygen levels [3, 26–28]. The VHL-HIF axis deregulation has been implicated in the activation of several oncogenic pathways in ccRCC [29–31]. HIF isoforms are stabilized by histone deacetylases and can be antagonized by the use of HDAC inhibitors [7, 10, 32]. However, there have been only a few studies assessing the role of HIF isoforms in regulating HDAC expression and activity. A subset of ccRCC tumors overexpresses class I and II HDACs when compared to adjacent non-tumor tissues. Class I HDACs, HDAC 1 and HDAC 2, have been shown to increase invasion through the upregulation of MMP-2 and MMP-9 in different cancer cell lines [22, 24, 33]. TSA exhibits anti-invasive properties through upregulation of RECK, which further inhibits MMP-2 and MMP-9 in a variety of cell lines, including breast cancer cell lines and hepatocytes, amongst other cancer types [23, 24]. Our study demonstrates that HDAC 1 knockdown reduces the invasive capacity of renal tumor models through decreased MMP activity in these cells. On the other hand, HDAC 6 is known to be overexpressed in different tumor types, to have oncogenic functions and to interact with other pathways to enhance tumorigenesis [34–37]. 786–0 cells were associated with lower acetylated α-tubulin, suggesting increased HDAC 6 activity as compared to C2 cells. The clinical relevance of this is not yet fully understood; however, the 786–0 cells are thought to represent a more aggressive type of ccRCC as compared to C2 cells. Upon enhancing HDAC 6 activity in the C2 cells by using an overexpression system, we observed that HDAC 6 was associated with not only decreased acetylated α-tubulin levels but also with functional consequences in terms of enhanced cell motility. Thus, it can be speculated that those tumors with higher HDAC 1 or HDAC 6 expression have a potential to be more aggressive as well as more metastatic in the clinical setting.
Further investigation of the regulation of HDAC 1 revealed a possible role of HIF isoforms in ccRCC cell lines. When comparing VHL-null cell lines with wild type VHL cell lines of renal tumor models, HDAC 1 expression was found to be upregulated in VHL-null cell lines. In addition, hypoxia stimulation of wt-VHL cells led to both HIF and HDAC 1 upregulation. Chromatin immunoprecipitation assay findings showed that the HDAC 1 promoter region is enriched upon pull down of both HIF isoforms and knockdown of HIF-2α in renal tumor cell lines reduced HDAC 1 expression in these cells. HDAC 1 activity as measured by acetylated histone H3 is also affected in these cell lines. Therefore, the above results indicate that HIF is involved in the regulation of HDAC 1 protein expression and activity. To date, anti-angiogenic drugs and agents inhibiting the mTOR pathway have been extensively studied in ccRCC treatment. Targeting HDACs in ccRCC, therefore, has the potential to reduce not only HDAC activity but also reduce HIF stability. This may further decrease HDAC expression and activity by HIFs.
The examination of ccRCC tumor samples obtained by nephrectomies demonstrated overexpression of HDAC 6 in a subset of tumors along with ERα expression. This phenomenon occurs regardless of the gender of the patient, indicating that ERα may have a potential role in the biology of ccRCC tumors. By immunofluorescent microscopy analysis of the expression of these proteins in the tumor, both ERα and HDAC 6 were present exclusively in the cytoplasm. In breast cancer, the colocalization of HDAC 6 and ERα in the cytoplasm has been associated with better clinical outcome in tamoxifen-treated patients as well as increased deacetylation of α-tubulin, which led to enhanced cell motility in vitro [13, 14]. Thus, we carried out ERα knockdown assays to examine the putative role of ERα in renal tumor cell lines, in particular the role on deacetylation of α-tubulin. Knockdown of ERα in renal tumor cell lines drastically increased the levels of acetylated α-tubulin, confirming that both HDAC 6 and ERα regulate the levels of acetylated α-tubulin and, thus, play roles in regulating the motility of these cells. Thus, the HDAC 6/ER-α interaction represents a potential therapeutic target, for reducing the metastatic potential of ccRCC. Pharmacological inhibition of HDAC 6 and ERα in renal tumor cell lines showed similar effects as ERα knockdown in these cells. Tamoxifen showed enhanced effects on α-tubulin acetylation in 786–0 cells, and this effect was greater with the addition of panobinostat. Single agent and combination treatments increased HDAC 6 and ER-α expression in C2H6 and 786-O cells; however this did not result in increased HDAC 6 activity. The mechanisms of increased ERα and 786-O expression by treatments are not entirely known. These results indicate that in ccRCC, tamoxifen treatment may reduce metastatic potential. Furthermore, when combined with an HDAC inhibitor, such as panobinostat (that has cytotoxic effects), can lead to both anti-tumor effects and reduced metastasis. Moreover, in 100 ccRCC tumors, HIF-1/2α and HDAC 1 positively correlated with one another; however, HDAC 1 expression did not correlate with overall or disease free survival. TCGA data further revealed that HDAC 1 overexpression is associated with worse overall survival and higher tumor stage (not statistically significant). Hence, targeting HDAC 1 by using class I specific HDAC inhibitors may not only reduce the invasiveness of the disease, but the level of HDAC 1 itself can be used as a prognostic indicator in ccRCC. Similarly, HDAC 6 mRNA upregulation, although not statistically significant, showed a trend towards worse overall survival as well as higher tumor stage in ccRCC patients. Therefore, targeting HDAC 6 using a class II specific HDAC inhibitor may reduce the motility and aggressiveness of ccRCC tumors.
Although HDAC inhibitors have shown potential anti-tumor activities in preclinical models, the clinical development of this class of drugs has only achieved moderate success in hematological malignancies and not in solid tumors. However, these agents have been tested primarily as single agents in solid tumors and combination with already-approved therapies have not been extensively studied. In ccRCC, our lab has previously shown that the class I HDAC inhibitor, Entinostat, enhances immunotherapy  and another study demonstrated synergistic anti-proliferative effects of the combination of sorafenib with HDAC inhibitors . The subset of ccRCCs that express high levels of HDAC 1 and HDAC 6 may be the most suitable patient population for rational combination strategies using HDAC inhibitors with other agents.
Our study indicates a potential use for HDAC 1 and HDAC 6 expression status in identifying a subset of ccRCC patients who are suitable for treatment with HDAC inhibitors. Clinical testing of histone remodeling drugs in rational combination strategies will shed additional light on the potential therapeutic value of this class of agents in ccRCC.
We would like to thank Drs. Jennifer Isaacs and Len Neckers (National Cancer Institute) for providing the C2, C2-VHL and 786–0 cell lines, Dr. Michael Ohh (University of Toronto) for providing the VHL expressing vector, and Dr. Tso Pang Yao (Duke University) for providing the HDAC 6 expressing vector. Clinical data delivery and Honest Broker services for this study were provided by the Clinical Data Network, which is funded by the National Cancer Institute and is a Roswell Park Cancer Institute Cancer Center Support Grant shared resource.
This study was in part supported by the National Institutes of Health-NCI (R01CA135321) and a donation from Dr. Richard Turner and Mrs. Deidre Turner.
Availability of data and materials
The dataset analyzed in this manuscript are publicly available through The Cancer Genome Atlas and cbioportal.
SR, SK, EC, KMM, SC, LS, LE, PS, RH, DC and AO carried out the experimental studies. KA performed statistical analysis. WS and GD provided experimental materials. SR and RP analyzed data, designed experiments and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All patients gave written informed consent for the collection of biomaterials. The study was approved by the Genitourinary Disease Site Research Group at Roswell Park Cancer Institute (number: BDR 036713).
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