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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Transcriptional repressors such as nuclear receptor corepressors (NCORs) and class I histone deacetylases (HDACs) are considered potential therapeutic targets in various human malignancies. In astrocytic gliomas, however, there is still a need to understand the role of these transcriptional repressors in tumor proliferation, tumor differentiation, and patient survival. We immunohistochemically analyzed the expression of NCOR1 and 2 as well as HDAC1, 2, and 3 on a tissue microarray comprising tumor samples from 283 astrocytic gliomas and correlated the expression levels with tumor differentiation, tumor proliferation, and patient survival. Strong nuclear expression was found in glioma cells for HDAC1, HDAC2, and NCOR2. In contrast, weak expression of NCOR1 and HDAC3 was detected in the cytoplasm and nuclei of tumor cells. HDAC3 expression was inversely associated with tumor grade. Consequently, increased HDAC3 expression was associated with better patient survival in univariate regression. Expression of HDAC1 and HDAC2 increased during tumor recurrence and malignant tumor progression, respectively, whereas expression of the remaining antigens did not seem to depend on tumor grade and was comparable to expression levels found in non-neoplastic brain tissues. Finally, we detected a positive association between HDAC2 expression and tumor proliferation as well as between NCOR1 and expression of the stem cell-associated intermediate filament protein nestin. Our findings suggest that “classical” transcriptional repressors are expressed in astrocytic tumors and that the roles of HDAC2 and HDAC3 in these tumors deserve further investigation. (Cancer Sci 2011; 102: 387–392)

There is increasing evidence suggesting that both increased proliferation and incomplete cellular differentiation are involved in the pathogenesis of astrocytic gliomas.(1,2) Various reports account for the existence of undifferentiated glioma cells and link their occurrence with an unfavorable patient prognosis.(2,3) As such, investigation of molecules involved in physiological differentiation processes of neural cells may be a promising approach to elucidate potential pathogenetic mechanisms and new molecular targets that contribute to gliomagenesis. Nuclear receptor corepressors (NCORs) mediate transcriptional repression in various differentiation-related pathways, such as vitamin D, retinoic acid, and peroxisome proliferator-activated receptor (PPAR) signaling.(4–9) In murine neural progenitors, nuclear localization of NCOR1 is required to maintain an undifferentiated, proliferative state, which can be reversed by Akt1 kinase-dependent phosphorylation and subsequent cytoplasmic relocation of NCOR1.(10) Similarly, nuclear NCOR2 (also known as SMRT) is critical for preventing retinoic acid- and NOTCH-dependent neurogenesis in the murine forebrain and maintain neural progenitors in an undifferentiated state.(11) Transcriptional repression through NCORs in turn requires interaction with histone deacetylases (HDACs), which hinder transcriptional accessibility of target genes through local chromatin condensation.(12) Although HDACs comprise a family of 18 different enzymes with several nuclear and cytoplasmic protein substrates, association with NCORs and subsequent target gene repression preferentially involves HDAC1, HDAC2, and HDAC3.(13) Based on their similarity with yeast homologues, these three enzymes constitute the so-called class I HDACs, along with HDAC8, which is still poorly investigated. Overexpression of HDAC1, 2, and 3 has been reported in various human malignancies, such as ovarian, endometrial, and colorectal cancer (reviewed in (12)). To date, knowledge on protein expression of class I HDACs and NCORs in astrocytic tumors is scarce,(14,15) and the prognostic influence of these molecules remains unknown. To address these questions we immunohistochemically analyzed the expression of HDAC1, 2, and 3 as well as NCOR1 and 2 in a patient sample comprising astrocytic gliomas from 283 patients and examined the relationship between expression levels and World Health Organization (WHO) grade, proliferative activity, as well as patient survival. Our findings show that “classical” HDACs and nuclear corepressors are expressed in astrocytic gliomas and that HDAC2 and NCOR1 in particular might play a role in tumor proliferation and tumor differentiation, respectively. In addition, HDAC3 inversely correlated with tumor malignancy. The present data might guide future studies to elucidate the pathological role of HDAC2 and HDAC3, as well as NCOR1, in astrocytic gliomas.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Tumor samples.  Study samples consisted of histologically representative, paraffin-embedded tumor biopsies from 283 patients with astrocytic gliomas of WHO grades II, III or IV, arranged on a tissue microarray (TMA) as described elsewhere.(3) Four additional samples of non-neoplastic brain tissue served as reference tissues for the expression of individual antigens in this study. Informed consent was obtained from each patient according to the research proposals approved by the Institutional Review Board at Heidelberg Medical Faculty (University of Heidelberg, Heidelberg, Germany). Patient characteristics are summarized in Table 1.

Table 1.   Characteristics of study sample
Histological diagnosisGradePatients n = 283 (%)Recurrent tumors n = 52 (%)
  1. †Therapy at primary tumor diagnosis. –, not applicable; WHO, World Health Organization.

Astrocytic gliomaWHO IV221 (78.1)29 (55.8)
WHO III17 (6.0)9 (17.3)
WHO II45 (15.9)14 (26.9)
AgeMean (years) ± SD 
Astrocytic gliomaWHO IV53.8 ± 12.8
WHO III36.1 ± 14.2
WHO II33.6 ± 18.7
GenderMale:Female 
Astrocytic gliomaWHO IV133:88
WHO III11:6
WHO II30:15
Extent of resectionTotal n (%)Subtotal/biopsy n (%)
Astrocytic gliomaWHO IV151 (68.3)70 (31.7)
WHO III11 (64.7)6 (35.3)
WHO II33 (73.3)12 (26.7)
Non-surgical treatments†Radiotherapy n (%)Chemotherapy n (%)
Astrocytic gliomaWHO IV186 (84.2)87 (39.4)
WHO III16 (94.1)1 (5.9)
WHO II14 (31.1)3 (6.7)

Immunohistochemistry.  Primary antibodies used in our study were: rabbit polyclonal anti-HDAC1 (1:200); rabbit polyclonal anti-HDAC2 (1:200); rabbit polyclonal anti-HDAC3 (1:200); rabbit polyclonal anti-NCOR1 (1:100; all Abcam, Cambridge, UK); and rabbit polyclonal anti-NCOR2 (1:50; Sigma-Aldrich, Schnelldorf, Germany). Prior to TMA staining, specificity of primary antibodies was verified using corresponding isotype controls (all Acris, Herford, Germany) on glioma control tissues in equal concentrations as primary antibodies and as indicated by the manufacturer. Antigen retrieval, incubation with primary and secondary antibodies, as well as detection with Vectastain Laboratories ELITE ABC KIT (Vector Laboratories, Burlingame, CA, USA) was carried out as previously described.(3) Additional antibodies used for double immunofluorescence staining were: mouse monoclonal anti-human Ki-67 (1:50) and mouse monoclonal anti-human CD31 (1:100; both BD Pharmingen, Hamburg, Germany); mouse monoclonal anti-human GFAP (ready to use; PROGEN, Heidelberg, Germany); mouse monoclonal anti-human CD68 (1:100; DAKO, Hamburg, Germany); as well as secondary antibodies anti-mouse ALEXA488 and anti-rabbit ALEXA555 (1:500; both Invitrogen, Karlsruhe, Germany).

Evaluation of TMA staining.  Staining of different antigens on subsequent TMA slides was carried out as previously described.(3) Briefly, each tumor biopsy was evaluated at 20× magnification by two independent investigators blinded to all clinical data. Staining of TMA biopsies was semiquantitatively graded in an antigen-dependent manner according to the estimated percentage of positive cells covering the whole tissue spot. In case of inter-observer variability, staining frequency on individual biopsies was counted manually. Average staining patterns from all biopsies of an individual tumor were taken as final staining result.

Statistical analysis.  Overall survival (OS) was calculated from date of first diagnosis to death and it was censored for patients alive at the end of study (November 30, 2009). In case of impossible patient contact (n = 11), the date of last visit was taken as the censored end-point. Recurrent tumors, patients with incomplete clinical follow-up, and patients enrolled in experimental therapies were excluded from survival analyses. Potential associations between OS and average staining results were investigated using the log–rank method and represented by Kaplan–Meier curves. In multivariate Cox regression analyses, hazard ratios (HR) were adjusted for the influence of WHO grade and established prognostic factors on OS, that is, patient age at diagnosis and extent of tumor resection. Average time of follow-up was 10.7 years (±4.3 years), assuming a 20:80 proportion of patients with increased : decreased expression and a type I error rate of 0.05 results in 0.95 statistical power to detect a HR higher than 1.3. The relationship between antigen expression and WHO grade, expression of the tumor proliferation marker Ki-67 and stem cell-associated filament protein nestin was quantified by Spearman’s rank correlation rho. Calculations were carried out using the statistical software environment R, version 2.4.1 (http://www.r-project.org). Probability values smaller than 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

NCOR1 weakly expressed in cytoplasm and nucleus of glioma cells; NCOR2 shows a strong nuclear staining pattern.  Immunohistochemical analyses of nuclear corepressors on our TMA revealed weak NCOR1 expression, discernable as cytoplasmic and nuclear staining in little more than half of the tumor samples (56.2%) (Fig. 1). Most tumors (80.4%) showed NCOR1 staining restricted to 25% or less of the tumor cells. Further, NCOR1 expression was not associated with tumor grade (ρ = 0.13; P = 0.11), and was comparable to non-neoplastic brain tissues in terms of staining, pattern, and percentage of NCOR1-postive cells (Fig. 1).

image

Figure 1.  Expression of nuclear receptor corepressor (NCOR)1 and 2 in astrocytic gliomas. Immunoreactivity of individual antigens in non-neoplastic brain tissue (column 1), diffuse astrocytomas World Health Organization (WHO) grade II (column 2), anaplastic astrocytomas WHO grade III (column 3), and glioblastomas WHO grade IV (column 4), as well as distribution of staining frequencies in the distinct WHO grades (lower panels). P-values indicate associations of immunoreactivity scores with WHO grade. Scale bar = 100 μm.

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In contrast, NCOR2 immunoreactivity was detectable as strong nuclear staining in most of the tumor samples (93.1%). Almost half of the tumors (49.1%) showed staining in more than 50% of the tumor cells (Fig. 1). NCOR2 expression did not increase with tumor grade (ρ = −0.06; P = 0.42) and was similar in non-neoplastic brain tissues (Fig. 1). Noteworthy, in addition to their expression in tumor cells, NCOR1 and NCOR2 expression was also clearly discernable in some non-neoplastic cells such as endothelial cells and microglia, both of which constitute major cell populations within the tumor bulk (Fig. S1).

Expression of HDAC1 and HDAC2 strong in gliomas but independent of grade; HDAC3 expression inversely correlates with grade.  Immunohistochemical analysis of HDAC protein expression revealed a strong and clearly discernable nuclear staining for HDAC1 and HDAC2 in most tumor samples (58.0% and 84.4%, respectively) (Fig. 2). More than a quarter of tumor biopsies showed staining in more than 25% of the tumor cells for HDAC1. Similarly, more than half of the tumors displayed staining of more than 50% of tumor cells for HDAC2 (Fig. 2). HDAC1 and HDAC2 expression was independent of WHO grade (ρ = −0.05; P = 0.45/ρ = 0.07; P = 0.31) and comparable to the expression in non-neoplastic brain tissues (Fig. 2).

image

Figure 2.  Expression of class I histone deacetylase (HDAC)1, 2, and 3 in astrocytic gliomas. Immunoreactivity of individual antigens in non-neoplastic brain tissues (column 1), diffuse astrocytomas World Health Organization (WHO) grade II (column 2), anaplastic astrocytomas WHO grade III (column 3), and glioblastomas WHO grade IV (column 4), as well as distribution of staining frequencies among the different WHO grades (lower panels). P-values summarize associations between immunoreactivity scores and WHO grade. Scale bars = 100 μm.

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HDAC3 expression was mainly found in the cytoplasm but was also distinguishable as nuclear staining (Fig. 2). Altogether, HDAC3 immunoreactivity was detectable in nearly half of the tumor samples (49.4%), although the expression was weak and the fractions of labeled cells were low compared to HDAC1 and HDAC2 (Fig. 2). Accordingly, most tumors (95.7%) showed HDAC3 staining restricted to 25% or less of the tumor cells. However, HDAC3 expression was inversely correlated with WHO grade (ρ = −0.29; P < 0.001), with strongest staining seen in WHO grade II diffuse astrocytomas (Fig. 2). In these tumors HDAC3 staining was comparable to non-neoplastic brain tissues (Fig. 2). As in the case of NCORs, it is worth mentioning that HDAC1, 2, and 3 were not exclusively expressed in tumor cells but were also detectable in non-neoplastic cells including endothelial and microglial cells (Fig. S2).

Expression of HDAC1 increased in recurrent tumors; HDAC2 expression augments with malignant tumor progression.  In addition to primary tumor biopsies, the TMA included tissue samples from recurrent tumors and tumors that had undergone malignant progression upon recurrence. Interestingly, in most patients suffering from glioblastomas, HDAC1 expression was higher in the recurrent compared to the primary tumor (11/14 cases). HDAC1 expression levels decreased upon recurrence in only a single case (1/14), and was unchanged in the remaining patients (2/14). Remarkably, this pattern of expression was exclusively seen for HDAC1, whereas all other antigens analyzed in this study showed no obvious differences between corresponding primary and recurrent tumors (data not shown).

In contrast, tumors that had undergone malignant progression upon recurrence showed increased expression of HDAC2 in the recurrences (7/10 cases). Decreased HDAC2 expression was only detectable in a single recurrence (1/10), and the remaining tumors retained their expression levels upon progression (2/14). There were no obvious differences between corresponding primary and recurrent tumors for all other antigens analyzed in this study (data not shown).

Expression of HDAC2 associated with tumor proliferation.  To investigate possible relationships between NCOR/HDAC expression and tumor proliferation as well as tumor differentiation, we compared the expression of each antigen with antigens previously evaluated on the same TMA. These included expression data of the proliferation marker Ki-67 (MIB1) and the stem cell-associated intermediate filament protein nestin.(3) Interestingly, HDAC2 expression in glioblastoma was positively correlated with proliferative activity as distinguished by MIB1 staining (ρ = 0.34; P < 0.001), confirmed by double immunofluorescent staining (Fig. S3). We did not find any correlation with tumor proliferation or nestin expression, with the exception of a positive association between NCOR1 and nestin expression in glioblastomas, which did not reach statistical significance (ρ = 0.15; P = 0.08).

Association of HDAC and NCOR expression with patient survival.  Finally, we explored potential associations between protein expression of NCORs, HDACs, and patient survival. Among all candidate antigens, only HDAC3 expression negatively associated with overall patient survival (Fig. 3; HR 0.71, 95% confidence interval 0.58–0.87; P = 0.001). However, taking into account the prognostic influence of WHO grade and known prognostic confounders, we found that the association was largely attributable to the correlation of WHO grade and HDAC3 expression. P-values and hazard ratios are summarized in Table 2.

image

Figure 3.  Association of antigen expression levels with patient survival. Kaplan–Meier plot illustrating patient survival according to antigen expression. P-value represents the association of expression levels with overall survival (OS).

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Table 2.   Results from (a) univariate and (b) multivariate survival analysis†
AntigenOverall survival
HR (95% CI)P
  1. †Hazard ratios were adjusted for age at diagnosis and extent of tumor resection. For analyses involving the whole study sample, HR were also adjusted for World Health Organization (WHO) grade. CI, confidence interval; HDAC, histone deacetylase; HR, hazard ratio; NCOR, nuclear receptor corepressor; n.d., not determined.

(a)
Whole study sample
 NCOR10.95 (0.80–1.13)0.540
 NCOR20.97 (0.85–1.10)0.590
 HDAC10.95 (0.83–1.09)0.450
 HDAC20.99 (0.88–1.10)0.830
 HDAC30.71 (0.58–0.87)0.001
(b)
Whole study sample
 NCOR10.91 (0.76–1.09)0.32
 NCOR20.95 (0.83–1.09)0.46
 HDAC11.01 (0.88–1.14)0.94
 HDAC20.94 (0.84–1.06)0.31
 HDAC30.88 (0.71–1.10)0.27
WHO grade II
 NCOR10.86 (0.34–2.19)0.76
 NCOR21.36 (0.81–2.28)0.25
 HDAC10.87 (0.47–1.59)0.64
 HDAC20.88 (0.58–1.32)0.53
 HDAC30.75 (0.42–1.35)0.34
WHO grade III
 NCOR1n.d.n.d.
 NCOR20.80 (0.42–1.53)0.50
 HDAC10.06 (0.00–13.8)0.31
 HDAC20.68 (0.37–1.24)0.20
 HDAC3n.d.n.d
WHO grade IV
 NCOR10.95 (0.79–1.15)0.63
 NCOR20.95 (0.83–1.10)0.52
 HDAC11.03 (0.90–1.17)0.68
 HDAC20.97 (0.86–1.10)0.66
 HDAC30.93 (0.74–1.19)0.57

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Transcriptional repressors such as NCORs and class I HDACs are receiving growing attention as potential therapeutic targets in human malignancies.(16–20) The relevance of NCORs in cancer is still a recent field of investigation, but several HDAC-specific inhibitors are currently being tested in preclinical and clinical trials,(17–19) including first trials with patients suffering from astrocytic gliomas.(21) However, the expression and functional role of NCORs and HDACs in gliomas is still a matter of ongoing investigation and current knowledge on the topic is scarce. Thus, there is a need to understand the influence of these transcriptional repressors on tumor proliferation, tumor differentiation, and most importantly, patient survival. Unlike previous reports on aberrant expression of NCORs and class I HDACs in extracranial tumors,(12) our present results suggest a heterogeneous expression of these transcriptional repressors in astrocytic tumors without obvious differences among tumor grades or between tumors and non-neoplastic brain tissues, except for HDAC3. In our study, HDAC3 expression was inversely correlated with tumor grade and was highest in low-grade tumors as well as non-neoplastic brain tissue. These results are in contrast to previous results reported by Lucio-Eterovic et al.,(14) who did not detect any differential expression of HDAC3 transcripts in a study involving mRNA from 43 astrocytic tumors and 10 brain tissues. It is conceivable, however, that these discrepant observations reflect methodological differences, such as mRNA and protein analysis, or might be due to the different case numbers in the study samples. Nevertheless, protein data on HDAC1 and HDAC2 expression in our study cohort are in good agreement with corresponding mRNA data in the above mentioned study, as both reports document comparable expression levels among different tumor grades as well as between tumorous and non-tumorous tissues. However, our data suggest that HDAC1 and HDAC2 expression increase during tumor recurrence and malignant tumor progression, respectively. As these expression patterns were exclusively related to HDAC1 and HDAC2 it might be worth investigating these findings in future studies. Another interesting finding in the present study involves the positive correlation between HDAC2 expression and tumor proliferation in glioblastomas. Despite this association, however, HDAC2 expression was not associated with patient survival.

To date, knowledge on the expression of NCORs in astrocytic tumors is based on a single study analyzing NCOR1 in seven glioblastoma tissues (WHO grade IV).(15) In this study, NCOR1 was identified as differentially expressed protein between normal white matter and tumor tissues. Using primary and established glioma cell lines, the authors could show a correlation between NCOR1 expression and an undifferentiated tumor phenotype in vitro. Although we were unable to confirm the differential expression of NCOR1 in our study sample, it is worth mentioning that NCOR1 expression seems to be linked to the expression of nestin in glioblastomas, although this association did not reach statistical significance. Expression of the remaining antigens studied in this report was not associated with nestin expression, suggesting that these proteins are not necessarily involved in the maintenance of an undifferentiated tumor phenotype in astrocytic gliomas. It is thus conceivable that “traditional” transcriptional repressors may not play the same pathogenetic role in astrocytic tumors as in extra-cranial cancers. Our present characterization of three prominent HDACs and two NCORs in a large cohort of astrocytic tumors could help to broaden the focus of future glioma studies on additional members of these protein families.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

This work was supported by the Verein zur Förderung der Krebsforschung (CHM); the Bundesministerium für Bildung und Forschung (01GS0886 to CHM; 01GS0884 to GR); the Tumorzentrum Heidelberg/Mannheim (to CHM); and the Deutsche Krebshilfe (109202 to CHM). The authors would like to thank Thora Pommerencke for undertaking microscope scans of the TMA slides as well as Prof. Peter Lichter and Dr Stefan Joos for help during the assembly of the tissue microarray. The authors would also like to thank D. Zito, H. Discher, F. Kashfi, I. Hearn and M. Greibich for excellent technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Fig. S1. Expression of nuclear receptor corepressors (NCORs) and class I histone deacetylases (HDACs) on normal and tumor cells.

Fig. S2. Expression of of nuclear receptor corepressors (NCORs) and class I histone deacetylases (HDACs) on normal and tumor cells.

Fig. S3. Co-expression of Ki-67 and class I histone deacetylase (HDAC)2.

FilenameFormatSizeDescription
CAS_1792_sm_figS1.jpg1186KSupporting info item
CAS_1792_sm_figS2.jpg1313KSupporting info item
CAS_1792_sm_figS3.jpg412KSupporting info item

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