Background
Glioblastomas are the most common histological subtype among all the malignant brain tumours [
1,
2]. With the distinct molecular subtypes of glioblastoma recently characterised, the hope of new glioblastoma therapeutics is imminent [
3‐
5]. Paired box-containing (PAX) transcription factors are largely expressed during development and at low levels in adult tissue [
6]. Aberrant
PAX gene expression is present in multiple cancer types, including cancers of the lymphoid tissue, thyroid, kidney, breast, and endometrium [
7‐
10]. Paired box-containing proteins also possess many tumour-promoting functions, such as the promotion of cell survival and anti-apoptotic properties, because a reduction in
PAX gene expression induces apoptosis in normal and tumour cells [
11‐
15].
PAX8 is expressed at the midbrain-hindbrain junction during brain development and is virtually absent in the adult brain [
16]. In earlier studies involving PAX8 and glioblastomas, we found increased
PAX8 expression in tumours using a small panel of 14 telomerase-positive tumours and cell lines [
14,
17]. The tumour-promoting functions of
PAX8 include the ability to transform cells and to form tumours in mice [
18], an increased telomerase activity [
17], and the promotion of cell cycle progression [
19]. The genes upregulated by PAX8 include
b cell lymphoma 2 (BCL2) and
Wilms tumour 1 (WT1). High-grade gliomas have a higher
WT1 expression level compared with low-grade gliomas [
20], and
BCL2 is associated with the higher tumour grades, poorer patient survival, and the conferring of treatment resistance through its own action and the action of other gene family members [
21‐
25].
The prevalence of increased PAX8 expression has not been extensively explored in glioma, especially with regard to the effect of increased PAX8 expression in telomerase-negative gliomas. Here, we surveyed the PAX8 expression in a range of brain tumours, including different grades of gliomas and varieties of telomere maintenance mechanisms.
Methods
Tumour samples
Brain tumours were procured during surgery from patients admitted to New Zealand hospitals. The Multi-region Ethics Committee, New Zealand, approved this study, and all patients provided written informed consent. Each hospital made the original histological diagnoses, which were subsequently reviewed by consultant neuropathologists at the referral centres, and confirmed by the study consultant neuropathologist who was blind to the original diagnoses. The glioblastomas used in this already had the telomere maintenance mechanism established as part of previous studies or had the telomere maintenance mechanism typed in the current study by methods outlined elsewhere [
26,
27]. Briefly, ALT positive tumours had heterogeneous telomere lengths by terminal restriction fragment (TRF) analysis and were positive for ALT associated promyelocytic leukaemia nuclear bodies (APB), but were negative for telomerase activity using the telomere repeat amplification protocol (TRAP) assay. Telomerase positive tumours were positive for telomerase activity using the TRAP assay and did not have APBs or heterogeneous telomere lengths by TRF analysis. NDTMM tumours did not have heterogeneous telomeres by TRF analysis and were negative for telomerase activity by TRAP analysis.
Immunohistochemistry (IHC)
Paraffin-embedded brain tissues were mounted on microscope slides and were subjected to heat-mediated antigen retrieval. Primary antibodies raised against PAX8 (MRQ-50 and PAX8 [polyclonal] antibodies, Cell Marque, Rocklin, CA) and PAX5 (clone 24, Cell Marque, Rocklin, CA) were used and detected using the EDL (Dako, Glostrup, Denmark) and DAB methods. PAX5- or PAX8-positive cells were detected with light microscopy, and the percentage of positive cells per 1000 tumours cells was calculated (DM 2000 microscope, DFC 295 camera and Application Suite software, version 3.5.0, Leica, Solms, Germany). The slides were assessed by three authors (AS, NH and TS) independently. A tumour was considered positive for PAX8 or PAX5 when 10% or more of the tumour nuclei were moderately or faintly stained by IHC.
Quantitative PCR
Total RNA was extracted from glioma specimens using the RNeasy Lipid Tissue Mini Kit (Qiagen, GmbH, Germany) following the manufacturer’s instructions. For quantitative PCR (QPCR), the first-strand cDNA from 50 ng RNA was used. Relative quantification of the PAX8 transcripts and the two housekeeping genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) by real time PCR was determined utilising the SYBR-green detection protocol and the ABI PRISM 7000 or 7300 Sequence Detection System (Life Technologies, Carlsbad, CA). The primer sequences used were as follows:
PAX8 forward primer: 5′-TTTGCTTGGCTCTTTCTACACCTC-3′
PAX8 reverse primer: 5′-GAATGTCTGTTTTAAGCTCCCTGG-3′
GAPDH forward primer: 5′-TGCACCACCAACTGCTTAGC-3′
GAPDH reverse primer: 5′-GGCATGGACTGTGGTCATGAG-3′
HPRT1 forward primer: 5′-TGACACTGGCAAAACAATGCA-3′
HPRT1 reverse primer: 5′-GGTCCTTTTCACCAGCAAGCT-3′
The cycling conditions were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s, 60°C for 1 min, and then from 60°C to 95°C for 20 min. The relative expression levels were calculated using the ΔΔCt method with the
GAPDH and
HPRT1 genes used as internal controls. The tumours that expressed
PAX8 at a level at least 3 times higher than the HEK-293 cell (no or low expression of
PAX8) levels were considered positive [
28,
29].
Construction and transfection of siRNAs
PAX8 siRNAs were designed following previously developed and described guidelines [
30].
The sequences targeting PAX8 were as follows:
PAX8-1: 5′-AGACAAAATTGAAGAAGAA-3′
PAX8-2: 5′-CGCCAGAACCCTACCATGT-3′
PAX8-3: 5′-TCTTTATTTATTACATGAA-3′
The other controls included:
Non-targeting 1 (NT1) to GFP: 5′-ACTACCAGCAGAACACCCC-3′
Scrambled sequence for PAX8-1 (sc8-1): 5′-AAGTTAGAAAAAAACGAAAAG-3′
Scrambled sequence for PAX8-2 (sc8-2): 5′-AACACCGGGAAACACCUTCCU-3′
All siRNAs were synthesised using the Ambion Silencer™ siRNA construction Kit (Life Technologies, Carlsbad, CA) following the manufacturer’s instructions. The control
GAPDH siRNA template was provided with the kit. The siRNA for p53 and the non-targeting 2 (NT2) control siRNA were purchased from Qiagen (GmbH, Hilden, Germany). Two additional siRNA for p53 sc29435 (siTP53 2 in this study) and sc44218 (siTP53 3 in this study) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Three
BCL2 siRNA were used (sc-61899) along with the control siRNA (sc-37007, NT3), and were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Two additional siRNA for BCL2 214532 (siBCL2 2 in this study) and 214533 (siBCL2 3 in this study) purchased from Life Technologies (Carlsbad, CA). All siRNAs were handled and prepared according to manufacturers’ instructions. The transfection experiments utilised A172 cells because of their endogenous
PAX8 expression, as previously described [
17]. Briefly, cells were plated at densities ranging from 2 × 10
4 to 1 × 10
5 cells/well 24 hours prior to transfection. The siRNAs were diluted with serum-free medium to a final concentration of 10 nM and transiently transfected into cells using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) or Ambion siPORT NeoFX (Life Technologies, Carlsbad, CA). The medium was replaced after 4 hours, and the cells were harvested 24–96 hours after siRNA transfection. The viable cells were counted using the trypan blue exclusion assay. Apoptotic nuclei were detected in paraffin-embedded cell clot sections using the Klenow FragEL DNA Fragmentation Kit (Merck, Darmstadt, Germany) and light microscopy. The percentage of apoptotic cells per 500 cells was measured.
Western blot analysis
A172 protein lysates were prepared in the presence of protease inhibitors, and 100 μg protein were separated on NuPAGE 4-12% Bis-Tris Gels (Life Technologies, Carlsbad, CA). Blots were probed with primary antibodies raised against PAX8 (MRQ-50, Cell Marque), Bcl-2 (Clone 124, Dako), p53 (1C12, Cell Signaling Technology, Beverly, MA), WT1 (6FH2, Dako, Glostrup, Denmark) and β-actin (AC-15, Abcam, UK) according to the manufacturers’ instruction, or that optimised in the current study (1:200 dilution for WT1). Alkaline phosphatase conjugated antibodies were detected using the Western Breeze Immunodetection kit (Life Technologies, Carlsbad, CA).
Statistical analysis
To analyse the PAX8-positive tumours, a comparison between the experimental groups was made using the Fisher’s exact test. For cell transfection experiments, the data are expressed as the mean ± SD, and the statistical significance was determined between the experimental groups using the Student t test. P < 0.05 was considered statistically significant and the GraphPad Prism software, version 6.00 for Macintosh (GraphPad Software, San Diego, CA) to perform all statistical tests.
Discussion
The current study represents the first extensive analysis of the PAX8 expression levels in gliomas. Our data showed that PAX8 is increased in most high-grade gliomas and is a pro-survival factor for glioma cells. In another study with a large tumour panel, PAX8-positive tumours were frequently detected in renal cell carcinomas (90%), thyroid cancers (90%), endometrial cancers (84%), cervical adenocarcinomas (83%), and ovarian cancers (79%) [
33]. Our data include glioblastoma and malignant meningioma amongst the cancers with a high incidence of PAX8-positive tumours. PAX8 transactivates the promoters of the telomerase catalytic subunit (hTERT) and the telomerase RNA component (hTR) to increase telomerase activity [
17], and as might be predicted, the majority of the telomerase-positive tumours were also PAX8-positive. Therefore, in telomerase-positive glioblastomas, the
PAX8 expression may play an important part in the immortalisation process by regulating telomerase activity. But
PAX8 expression was not restricted to telomerase-positive glioblastomas. The frequency of PAX8-positive tumours was similar between the telomerase- and NDTMM-positive tumours and was lower in the ALT-positive glioblastomas (44% of tumours, of which only half showed strong PAX8 immunostaining).
In cancer, the over-expression of the
PAX genes is often attributed to chromosomal rearrangements that result in fusion proteins [
7,
10,
34,
35]. In thyroid adenocarcinomas the PAX8/PPAR-γ (peroxisome proliferator-activator receptor gamma 1) fusion protein confers many oncogenic properties, including increased proliferation, decreased apoptosis and the inhibition of wild-type PPAR-γ [
7,
36‐
38]. The cause for the increased
PAX8 expression in glioblastomas is unknown. In gliomas, the chromosome 2q13 locus, where the
PAX8 gene is located, is not a glioma susceptibility locus (OMIM #137800), but other mechanisms for the increased
PAX expression in cancer have been described. Hypomethylation, for example, produces an increase in
PAX2 expression in endometrioid carcinoma [
9]. In adult tissues, the
PAX genes are proposed to be important for maintaining stem cells; therefore, the increased PAX8 expression in glioblastomas may be indicative of an early cell lineage [
39]. Additionally, co-activators of PAX8 are increased in gliomas. Increased TAZ (transcriptional co-activator with PDZ-binding motif) is observed in the mesenchymal subtype of glioblastoma [
40,
41]. Furthermore, TAZ is reduced in proneural glioblastomas, which are usually ALT-positive tumours and those with reduced PAX8 positivity in the current study [
41].
In low-grade gliomas, PAX8 was not detected in the majority of tumours. A reduction of the PAX8 expression levels in low-grade tumours is consistent with the association of PAX8 expression with more aggressive tumours. Our results are also consistent with another study in which the transcriptional target of PAX8,
WT1, was decreased in low-grade compared with high-grade gliomas [
20]. A larger cohort of the low-grade PAX8-positive tumours might show an association with poorer outcomes because in our cohort, the single PAX8-positive grade I astrocytoma was a recurrent tumour.
In non-malignant cells, PAX8 expression was detected in a minimal number of cerebellar cells. Otherwise, all other non-malignant cells examined were PAX8-negative. The virtual absence of
PAX8 in the adult brain is consistent with studies in mice in which the adult murine brain expressed
PAX8 at levels no higher than the background signal [
16]. PAX8 expression in the adult human brain has not been previously studied, and our results suggest that the residual PAX8 expression does occur in a small minority of cells. The predominance of high-grade gliomas expressing high levels of PAX8 and the virtual absence of PAX8 expression in normal brain makes PAX8 signalling an appealing therapeutic target pathway.
We found that PAX8 acted as a pro-survival factor for glioblastomas. The silencing
PAX8 in several glioma cell lines caused a marked reduction in cell number, which is partly explained by an increase in apoptosis. Reduced PAX8 expression produced a reduction in the BCL2 expression levels, and
BCL2 inhibition by siRNA-knockdown reduced the glioma cell growth rate. These findings are consistent with previous reports that demonstrate
PAX expression enhances cell growth and survival [
15,
42], and upregulated BCL2 is found in gliomagenesis [
21,
31]. In other studies
BCL2 silencing induced cell death
in vitro[
43,
44], led to an arrest of cell cycle progression [
19], and was associated with the downregulation of multiple developmental genes [
45].
Competing interests
The authors have no competing interests.
Authors’ contributions
NH, Histo-pathological assessment, interpretation of the results and writing of the manuscript; YJC, quantitative PCR and knockdown experiments, and interpretation of the results; AT, MO, RB, BB, and MM the collection and selection of tumours, patient consenting, and collecting and interpreting clinical data, interpretation of the results and writing of the manuscript; TW, quantitative PCR and interpretation of the results; AW, AS, and RE telomere maintenance mechanism typing, immunohistochemistry staining, and interpretation of slides; ME and AB, in vitro experimental design and interpretation of results; JR, project conception, experimental design, interpretation of the results and writing of the manuscript; TS, Histo-pathological assessment, knockdown experiments, project conception, experimental design, interpretation of the results and writing of the manuscript. All authors read and approved the final manuscript.