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Human Cancer Biology |
Authors' Affiliations: 1 Institut für Pathologie, Charité Universitätsmedizin, Berlin, Germany and 2 Therapeutic Area Oncology, Altana Pharma AG, Konstanz, Germany
Requests for reprints: Wilko Weichert, Institut für Pathologie, Charité Universitätsmedizin, Schumannstraße 20/21, 10117 Berlin, Germany. Phone: 49-304-50536006; Fax: 49-304-50536922; E-mail: wilko.weichert{at}charite.de.
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Abstract |
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Experimental Design: We characterized class I HDAC isoform expression patterns in a cohort of 140 colorectal carcinomas by immunohistochemistry. In addition, effects of HDAC inhibition by valproic acid and suberoylanilide hydroxamic acid, and specific HDAC isoform knockdown by short interfering RNA, were investigated in a cell culture model.
Results: We found class I HDACs highly expressed in a subset of colorectal carcinomas with positivity for HDAC1 in 36.4%, HDAC2 in 57.9%, and HDAC3 in 72.9% of cases. Expression was significantly enhanced in strongly proliferating (P = 0.002), dedifferentiated (P = 0.022) tumors. High HDAC expression levels implicated significantly reduced patient survival (P = 0.001), with HDAC2 expression being an independent survival prognosticator (hazard ratio, 2.6; P = 0.03). Short interfering RNA–based inhibition of HDAC1 and HDAC2 but not HDAC3 suppressed growth of colon cancer cells in vitro, although to a lesser extent than chemical HDAC inhibitors did.
Conclusions: The strong prognostic impact of HDAC isoforms in colorectal cancer, the interactions of HDACs with tumor cell proliferation and differentiation in vivo, and our finding that HDACs are differentially expressed in colorectal tumors suggest that the evaluation of HDAC expression in clinical trials for HDAC inhibitors might help to identify a patient subgroup who will exceptionally profit from such a treatment.
To date, four classes of HDACs comprising >18 isoenzymes have been identified in humans (3). In our study, we focus on NAD+-independent class I isoforms HDAC1, HDAC2, and HDAC3, which are known to represent a target for a growing class of new therapeutics, the HDAC inhibitors (HDI). HDIs have been shown to suppress tumor growth in vitro and in vivo and some of these substances, including hydroxamic acids such as suberoylanilide hydroxamic acid (SAHA) and short-chain fatty acids such as valproic acid (VPA), have entered late-phase clinical trials for the treatment of a variety of solid and hematologic malignancies including colorectal cancer (1). SAHA was recently approved for the treatment of cutaneous T-cell lymphoma in second- or third-line therapy.
Novel treatment options for colorectal cancer, the third most common cancer in the Western world (7), are still urgently needed. Although this disease is surgically curable at early stages, the tumor frequently does not become symptomatic before the metastatic stage, which is associated with high mortality rates. Although changes in the chemotherapeutic regimens used and combination with radiotherapy have considerably improved survival of patients with advanced neoplasms (8), the current therapeutic concepts, especially for late-stage tumors, are far from being optimal. Based on the fact that synergistic effects of HDIs and conventional chemotherapeutics have been proposed (9–11), and that HDIs have been shown to act as radiosensitizers in a variety of cancer cell lines, including colon cancer cells (12, 13), HDIs might be extremely useful for radiochemotherapeutic combination therapies.
The role of HDACs in colorectal carcinogenesis is further emphasized by the fact that short-chain fatty acids such as butyrates, physiologically produced as a consequence of high-fiber diet, are known HDIs (14). This finding provides an interesting possible functional explanation for the long known interlink between nutrition and colorectal cancer formation. Finally, the detection of antibodies directed against HDAC3 has been suggested as a serologic antigen/biomarker for colorectal cancer (15).
Although, lately, several studies reported a functional link between specific class I HDACs and the development of tumors of the large intestine (16–18), it is surprising how little is known on the expression patterns and specific functions of single HDAC isoforms in primary human tumors. However, this information is of utmost importance because pan-HDIs will most likely be used for the treatment of malignant solid human tumors in the near future and it is not yet clear whether selectivity of inhibitors for specific HDAC isoforms might reduce unwanted side effects. Even more important is the fact that selection strategies to prospectively identify patients who might benefit from such a treatment have not been established thus far.
In our study, we addressed this apparent lack of translational information by characterizing specific class I HDAC isoform expression patterns and functions in a large number of colorectal tumor samples and in colon cancer cell culture models. Our goal was to provide information that might help to identify specific HDAC isoforms important in the tumorigenesis of colorectal cancer. In addition, we wanted to test the hypothesis that a stratification of patients according to their HDAC expression patterns might allow for prediction of individual patient prognosis. Our data might be the basis for studies on HDACs as biomarkers for the prediction of response to HDI treatment in future clinical trials.
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Patients, Materials, and Methods |
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TopAbstractPatients, Materials, and MethodsResultsDiscussionReferences |
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Immunohistochemistry. For immunohistochemical detection of HDAC isoforms on tissue samples, prediluted polyclonal rabbit IgG antibody directed against HDAC1 (1:11, Abcam), monoclonal mouse IgG antibody directed against HDAC2 (1:5,000, Abcam), and monoclonal mouse IgG antibody directed against HDAC3 (1:500, Becton Dickinson) were used on 5-µm paraffin sections. As there are known structural homologies between the three proteins, antibody specificity was ascertained by selective short interfering RNA (siRNA) inhibition and subsequent Western blotting for all three HDAC isoforms (see below). Immunohistochemistry was done, as previously described (20). Treatment of slides under omission of the primary antibodies served as negative control.
Ki-67 (MIB-1) staining was done with a Ventana Discovery autostainer (Ventana) using a monoclonal mouse IgG antibody (1:50, Dako) under standard conditions.
Evaluation of staining of tissue slides. Nuclear staining of HDAC isoforms was scored by applying a semiquantitative immunoreactivity scoring (IRS) system, as previously described (20). Briefly, intensity of staining (score 0-3) as well as percentage of cells stained (score 0-4) was evaluated separately (for examples of staining, see Supplementary Fig. S1). The IRS for each individual case ranging from 0 to 12 was calculated by multiplication of the intensity and frequency scores. Cases exhibiting an IRS from 0 to 6 were combined in one group (HDAC negative); cases with an IRS higher than 6 were combined in a HDAC-positive group. Staining of tissue slides was evaluated independently by two pathologists (W.W. and C.D.) who were blinded toward patient characteristics and outcome. To test whether our grouping system (negative/positive) gives robust results, we additionally did all statistical analyses (if applicable) with the raw expression scores and with a three-tied grouping system (weak/moderate/strong), which essentially gave the same results as the two-tied system used (data not shown).
Ki-67 index was determined by counting Ki-67–positive tumor cell nuclei per 100 tumor cells in a representative carefully selected tumor area. The invasive front, the tumor surface, and the transitional zone to normal epithelium were avoided, because in these areas proliferative activity was extremely high in almost all tumors. To ascertain the representativeness of the data, overall proliferative activity on whole tumor sections was additionally estimated by view (data not shown). Correlation of both variables resulted in a correlation coefficient of r = 0.93 (P < 0.001, Spearman's rank order correlation), thus proving the reliability of the proliferation data obtained. The mean proliferative activity of colorectal carcinomas was 36.3% (SD, 12.3%; Supplementary Table S1).
Statistical analysis. Statistical analyses were done with SPSS 13.0 and GraphPad Prism 4.0. The respective statistical tests used are given in Results, and in the table and figure legends.
Cell culture and treatment with HDIs and 5-FU. The CX-2 colon adenocarcinoma cell line was obtained from the German Cancer Research Center. HT-29 and HCT-116 were from the American Type Culture Collection. Cells were cultured in RPMI (CX-2) or DMEM (HT-29, HCT-116) supplemented with 10% fetal bovine serum.
For chemical HDAC inhibition and treatment with 5-FU, cells were seeded into 96-well plates at a density of 4 x 104/mL, grown for 24 h, and then treated with given concentrations of SAHA (Alexis Biochemicals) and VPA (Sigma) dissolved in methanol and DMSO, respectively. Control experiments confirmed that the respective inhibitor solvents had no effect on cell number (data not shown). For combined treatment with 5-FU (GRY Pharma), the respective concentrations of the drugs were added at the same time point. Predesigned siRNA duplexes (for sequences, see Supplementary Table S2) were purchased from Qiagen. A nonsilencing siRNA was used as negative control. The day before transfection, cells were seeded into 24-well plates at a density of 4 x 104/mL. Cells were transfected with 30 pmol siRNA using Oligofectamine transfection reagent (Invitrogen) according to the manufacturer's instructions. After 72 h, the efficacy of transfection was checked by immunoblotting.
Immunoblotting and immunofluorescence. Immunoblotting of cell extracts was done as previously described (20). The previously mentioned antibodies directed against HDAC1 (1:30), HDAC2 (1:800), and HDAC3 (1:200) were used. All blots were probed for actin (monoclonal mouse IgG, 1:5,000, Chemicon) to ensure equal protein loading.
For immunofluorescence, 3 x 104 cells/mL were plated onto Lab-Tek four-well chamber slides (Nalge Nunc). After treatment with SAHA or VPA at the indicated concentrations, slides were washed twice with PBS and fixed with ice-cold methanol for 20 min at –20°C. Fixed cells were blocked with 10% bovine serum albumin and 1% normal goat serum in PBS and incubated with polyclonal goat IgG directed against acetyl H3/H4 (1:100, Santa Cruz Biotechnology) for 90 min. Then, slides were washed thrice with PBS and incubated for 30 min with a biotinylated secondary antibody (rabbit anti goat IgG, 1:5,000, Dako). After treatment with rhodamine (TRITC)–conjugated streptavidin (1:250, Jackson Immunoresearch Laboratories) and 4',6-diamidino-2-phenylindole (1:1,000, Invitrogen), cells were visualized by confocal microscopy.
2,3-Bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay and cell cycle analysis. Cell viability was measured after 48 and 72 h of siRNA or drug treatment using a 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt cell proliferation kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. Cells were incubated at 37°C for 4 h. Formazan formation was measured at 490 nm. All experiments were done in triplicates.
For cell cycle analysis, cells were treated for 72 h with either HDAC1 or HDAC2 siRNA, VPA, or SAHA as described above. Then, cells were harvested and fixed overnight in 70% ice-cold ethanol at –20°C. The percentage of cells in G0-G1, S, and G2-M phase was quantified by using a propidium iodide–based cellular DNA flow cytometry analysis method. Briefly, cells were rinsed in PBS and 500 µL of diluent buffer (50 µL of Triton X-100 and 250 mg of bovine serum albumin in 50 mL PBS) were added. RNase digestion was carried out by adding 4 µL RNase (10 mg/mL) for 1 h at 37°C. Then, cells were centrifuged and diluted in 500 µL diluent buffer containing 20 µL propidium iodide. Flow cytometry was done with FACSCalibur according to the manufacturer's instructions (BD Biosciences). All experiments were done in triplicates. Analyses of cell cycle data were done with Cylchred and WinMDI (Freeware).
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Results |
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TopAbstractPatients, Materials, and MethodsResultsDiscussionReferences |
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Correlation of HDAC expression with clinicopathologic variables, tumor differentiation, and cell proliferation. To gain insight on the biological role of HDACs in colorectal cancer, we investigated the association of HDAC isoform expression with clinicopathologic variables that are used to describe the progression and aggressiveness of a tumor. The expression of all three isoforms correlated statistically significantly with WHO tumor stage, with locally more advanced tumors showing higher expression levels of all three proteins (HDAC1, P = 0.008; HDAC2, P = 0.017; HDAC3, P = 0.005; Supplementary Table S1). The expression of all HDAC isoforms was higher in tumors with distant metastases (HDAC1, P = 0.037; HDAC2, P = 0.045; HDAC3, P = 0.062; Supplementary Table S1). Interestingly, when the patients were grouped according to their overall class I HDAC expression pattern (all three isoforms negative versus one or two isoforms positive versus all three isoforms positive), high HDAC expression levels were associated with enhanced tumor cell proliferation (P = 0.022, one-way ANOVA) and with poor tumor differentiation as indicated by a high (G3) tumor grade (P = 0.002, 
2 test for trends; Fig. 2
). Correlation of the raw IRS scores with the number of cycling cells revealed a significant positive correlation of weak to moderate strength for HDAC1 (r = 0.23, P = 0.025) and HDAC2 (r = 0.29, P = 0.005) but not HDAC3 (r = 0.03, P = 0.808), indicating that some but not all of the variance in the expression of these isoforms is associated with altered proliferative capacity. This finding is supported by the fact that HDAC isoform expression was not restricted to cycling cells alone (Supplementary Fig. S2). These in vivo data from human tumor tissue fits well with the observation that HDI treatment leads to differentiation and reduced proliferation of tumor cells in vitro (21–24).
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Inhibition of colon carcinoma cell proliferation by treatment with HDI and isoform-specific siRNA. To test whether the effects of chemical HDI inhibition could be contributed to an inhibitory effect on one specific class I HDAC isoform, we compared the effects of HDI treatment with specific class I HDAC isoform knockdown by siRNA. Treatment of colon cancer cells with SAHA (maximum dose 4 µmol/L) revealed a significant dose-dependent reduction in cell number between 69.9% (CX-2) and 39.2% (HT-29) after 72 hours. Treatment with the maximum dose of VPA (4 mmol/L) resulted in a reduction of cell numbers between 77.3% (CX-2) and 35.6% (HT-29) after 72 hours (Fig. 4 ). After 48 hours, attenuated reductions of cell number were observed in CX-2 (Supplementary Fig. S4).
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Combined treatment of CX-2 with 5-FU and HDAC inhibitors. CX-2 cells were rather resistant to treatment with 5-FU. Low and moderately high doses of 5-FU resulted only in a weak to moderate, albeit significant, reduction of CX-2 cell numbers of up to 26.5% (5-FU, 2 mg/mL) after 72 hours (data not shown). In the low-dose treatment groups (5-FU, 50-200 µg/mL), the reduction of cell numbers could be slightly enhanced by adding 2 mmol/L VPA or 2 µmol/L SAHA, respectively. However, these differences did not reach statistical significance when compared with treatment with the respective doses of 5-FU alone or treatment with SAHA and VPA alone. In the treatment groups with higher doses of 5-FU (500 µg/mL to 2 mg/mL), no additional reductions in cell number were observed after adding SAHA (2 µmol/L) and VPA (2 mmol/L).
Effects of HDI treatment on class I HDAC isoform expression in CX-2. To assess whether the effects of SAHA and VPA on CX-2 are due to a degradation of specific HDAC isoforms as observed in other cell lines (25), we did Western blots for all three HDAC isoforms after treatment with SAHA (2 µmol/L/4 µmol/L) and VPA (2 mmol/L/4 mmol/L). HDAC1, HDAC2, or HDAC3 expression did not seem to be influenced by treatment with the respective inhibitors after 24 hours, or even after 48 or 72 hours (Supplementary Fig. S4).
Cell cycle alterations in CX-2 cells after treatment with HDI and isoform-specific siRNA. Treatment of CX-2 for 72 hours with 2 mmol/L VPA resulted in an accumulation of cells in G0-G1 phase of the cell cycle, whereas cells in the S and G2-M phases were reduced (Supplementary Table S3; Supplementary Fig. S4). In contrast, SAHA treatment (2 µmol/L, 72 hours) led to an accumulation of cells in G2-M. Specific knockdown of HDAC1 with siRNA produced no discernible effects on the cell cycle after 72 hours. HDAC2 knockdown by siRNA resulted in a weak accumulation of cells in G2-M (Supplementary Table S3; Supplementary Fig. S4). Induction of apoptosis, as indicated by the emergence of a sub-G1 peak, was observed neither after chemical HDAC inhibition (SAHA, 2 µmol/L; VPA, 2 mmol/L) nor after treatment with isoform-specific siRNA in our experimental setting (data not shown). This was confirmed by the absence of a relevant number of apoptotic cells when similarly treated cells were analyzed by fluorescence-activated cell sorting with a modified terminal deoxyribonucleotide transferase–mediated nick-end labeling assay (APO-direct, data not shown).
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Discussion |
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TopAbstractPatients, Materials, and MethodsResultsDiscussionReferences |
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Our findings are in line with previous studies reporting an elevation of both selected class I HDAC isoform RNA (18) and protein (17). In contrast, some studies have reported a loss (26) of class I HDAC isoforms in small sets of colorectal tumors when compared with normal mucosa. However, such an analysis of HDAC mRNA as well as protein expression in extracts of tumor tissue must be interpreted with caution because not only tumor epithelium but also normal and tumor stroma as well as inflammatory cells express relevant amounts of all three isoforms, as we were able to show. HDAC1 (18) and HDAC2 (16, 18) were confirmed to be overexpressed in colorectal cancer by exploratory tissue-based expression analysis on small sets of tumors. In contrast, in one study, Ropero and coworkers (27) reported truncating mutations of the HDAC2 gene in 20.4% of microsatellite unstable (MSI-H) sporadic colorectal cancers, which led to a loss of expression of the protein. However, as 
20% of sporadic colorectal cancers are MSI-H (28), this affects only 4% of sporadic tumors. Nevertheless, because MSI-H tumors are said to have slight survival advantages, this may contribute marginally to the survival differences seen in our cohort. Occasional mutational inactivation of histone acetylases has also been described for colorectal cancer (29, 30). Functional data on the regulation of HDAC expression are very sparse. There is some evidence that the expression of class I HDAC isoforms is under shared control of specific growth factors. In addition, an autoregulatory feedback loop controlling expression of class I HDACs has been suggested (31, 32). This fits well with our observation of a coordinate regulation of class I HDAC isoform expression in colorectal cancer tissue.
Based on the results of cell culture studies, it has been proposed that HDI treatment leads to reduced tumor cell proliferation and initiates differentiation. Our finding of a direct correlation of elevated class I HDAC expression levels with tumor dedifferentiation and enhanced tumor proliferation is the first report strongly suggesting that this interaction is also relevant for progression of primary human tumors.
There are few reports on the expression patterns of distinct HDAC isoforms in other tumor entities; however, most of the cohorts investigated thus far were of very small sample size. Moreover, most of the authors did not test for correlations with clinicopathologic data and patient prognosis. Our study, to the best of our knowledge, is the first report on an adverse independent prognostic impact of HDAC isoform expression in human cancer.
Suppressive effects of various HDIs on colon cancer cell growth in vitro and in vivo have been proven by several groups (33–38). The results of inhibitor-specific cell cycle alterations and cell reduction observed in colon cancer cells fits well with the results of these previous studies on other human colon cancer cell lines. However, degradation of class I HDAC isoforms, which was observed in other cancer cell lines as a consequence of HDI treatment (25), could not be seen in CX-2.
Combined treatment of CX-2 cells with 5-FU and VPA or SAHA did not result in additive reductions in cell number in our experimental setting, which is in line with findings of other authors in other cancer cell lines (10). However, synergistic effects of HDI other than the ones we used and 5-FU have also been described for colorectal cancer cell lines (11). Effects may be largely dependent on the cell lines used and the inhibitor concentrations applied.
Data on the cellular effects of a specific HDAC isoform knockdown are sparse. In one study, Wilson and colleagues (17) reported a reduction in cell number of up to 20% after knockdown of each HDAC1, HDAC2, and HDAC3 by siRNA treatment in the colon carcinoma cell line HCT-116 with an especially prominent effect for HDAC3. In addition, they observed induction of apoptosis after HDAC2 and HDAC3 but not after HDAC1 knockdown. In another study on HeLa cells, specific knockdown of HDAC1 and HDAC3 by siRNA led to a concentration-dependent inhibition of proliferation of up to 50% (39). In our study on CX-2, the effects of HDAC isoform inhibition on cell number were especially pronounced for HDAC2, whereas no effect was observed for HDAC3. The inhibitory effects of specific HDAC2 knockdown on cell number could be confirmed in HT-29 but not in HCT-116 colon cancer cells, which is in line with previous findings (16, 17). These differences may be explained by the fact that different cancer cell lines may need different HDAC isoforms for maintaining cellular proliferation and survival.
However, when compared with the effects of either nonselective (SAHA) or putative semiselective class I (VPA) synthetic small-molecule HDIs, selective HDAC inhibition by siRNA, albeit effectively reducing protein expression, only produced modest alterations with respect to the mentioned cellular readouts, which indicates that a comparably broad therapeutic strategy targeting different HDACs with one therapeutic substance may be more promising than a specific inhibition of single HDAC isoforms. This is in line with the results of Wilson et al. (17) in HCT-116.
An outstanding role of HDAC2 in colon carcinogenesis has been suggested lately in a comprehensive work by the group of Martin Göttlicher, in which the authors showed that loss of the adenomatosis polyposis coli tumor suppressor induced HDAC2 expression depending on the Wnt pathway and c-Myc in the colon carcinoma cell line HT-29 (16). In addition, they showed that HDAC2 expression is required for and sufficient on its own to prevent apoptosis in this cell line. These findings fit well with our in vitro results and are also perfectly in line with our clinical findings. Our observation that HDAC2 expression is an independent prognosticator in colorectal cancer further emphasizes the role of this isoform in colorectal carcinogenesis and tumor progression. However, based on our data, it is likely that some but not all of the effects of HDI can be contributed to an inhibition of HDAC2, as HDAC2 knockdown alone only shows moderate cellular effects in vitro when compared with HDI treatment.
In conclusion, our results emphasize the important role of class I HDACs, especially HDAC2, in colon cancer biology. Patients showing an overexpression of class I isoforms had dramatically reduced survival times, which hint on a potential use of these markers to clarify individual patient prognosis. Even more important, the prognostic impact of HDAC isoforms, together with our confirmation of the proposed interactions of HDACs with tumor cell proliferation and differentiation in vivo, strongly suggests that the evaluation of HDAC expression profiles before HDI treatment might help to identify patient populations who will exceptionally benefit from such a treatment. This should be considered when clinical trials for HDIs are planned.
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Acknowledgments |
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Footnotes |
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Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
W. Weichert and A. Röske contributed equally to this work.
Received 4/25/07; revised 10/ 4/07; accepted 1/ 9/08.
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References |
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TopAbstractPatients, Materials, and MethodsResultsDiscussionReferences |
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and β messenger RNAs by histone deacetylase inhibitors trichostatin A (TSA) and butyrate in SW620 human colon carcinoma cells. Cancer Lett 2002;188:127–40.[CrossRef][Medline]This article has been cited by other articles:
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