meningioma - carcinogenesis - apoptosis - gene expression
meningioma - carcinogênese - apoptose - expressão gênica
Meningioma is the most frequent primary intracranial tumor in adults[1]. They arise from the meninges and are composed of neoplastic arachnoid (meningothelial)
cells. They generally grow slowly and rarely show an aggressive behavior[2],[3],[4],[5].
According to the current World Health Organization histological grade system, meningiomas
are classified into three grades: grade I or benign (90%), generally following a benign
clinical course; grade II or atypical (5–7%) are associated with increased risk of
recurrence; and grade III, or anaplastic (1–2%), defined as meningiomas that have
obvious malignant histology or high mitotic rates that frequently present with local
and brain invasion, recurrence and even metastases[3],[4]. Currently, the curative treatment option is complete surgical resection[6], and there is no established chemotherapy regimen for unresectable meningiomas[7]. Despite being benign, meningiomas have a significant rate of recurrence after surgery.
While recurrence rates of benign meningiomas range from 7% to 20%, recurrence of the
atypical and anaplastic tumors occur at approximately 29% to 40% and 50% to 78% of
the cases, respectively[8]. Many advances have been made in the knowledge of the different molecular mechanisms
that drive tumorigenesis[9].
Physiological apoptosis is a form of cell death that eliminates unwanted cells through
the activation of a coordinated series of events synchronized by a unique set of gene
products[10]. Caspases constitute a large family of homologous proteins, whose enzymatic activity
is activated in cells marked for apoptosis death. They are the central components
of the apoptotic response and are generally divided into two classes: initiators,
which include caspases 2, 8, 9 and 10; and the effectors, which include caspases 3,
6 and 7[11]. The protein inhibitors of apoptosis (IAPs) are known as endogenous inhibitors of
caspases that effectively eliminate the cell death program. There are currently several
human IAPs including: X chromosome linked IAP (XIAP), c-IPA1, c-IAP2, NAIP, Survivin,
BRUCE and NL-IAP[12]. They block the apoptotic pathway mediated by mitochondria directly inhibiting initiator
and effector caspases. The IAPs are regulated by cellular proteins such as the second
mitochondria-derived activator of caspases/direct inhibitor of apoptosis (Smac/DIABLO),
a mitochondrial protein released along with cytochrome c from the mitochondria to the cytoplasm responsible for apoptotic stimulation[13]. During apoptosis, the inhibition of caspase, mediated by the IAP, is removed by
the Smac/DIABLO proteins. When there is the stimulation of apoptosis, Smac/DIABLO
proteins are released from the intermembrane space of mitochondria to the cytoplasm
with cytochrome c. While the cytochrome c directly activates the APAF1 and caspase 9, the Smac/DIABLO removes the IAP (XIAP,
c-IAP1, c-IAP2, and ML-IAP), which means the inhibition of caspases initiators and
effectors, empowering the apoptosis[11],[14]. The Smac/DIABLO inhibits the interaction between the field BIR3 of XIAP with caspase
9 and field BIR 2 with caspase 3 and caspase 7, and mitigates the inhibiting effects
of XIAP on these caspases[15].
Several studies have been conducted to better clarify the different genetic mechanisms
involved in the carcinogenesis of meningiomas. The sensitivity of tumor cells to apoptosis
is probably one of these mechanisms and is influenced, at least partly, by the interactions
and the relative amount of several proteins inducing and inhibiting this process.
However, the correlation of events on gene expression related to cellular apoptosis,
and also the possible association with the tumor progression, are not completely clear
in meningiomas[11].
The purpose of this study was to examine, in meningiomas, the pattern of gene expression
of the anti-apoptotic genes BCL2, XIAP, cellular FLICE-inhibitory protein (c-FLIP),
and pro-apoptotic genes Smac/DIABLO, complex apoptosome, caspase 3 and caspase 8,
related to extrinsic and intrinsic pathways of apoptosis using a polymerase chain
reaction – real-time (PCR-RT) technique.
The characterization of these processes is paramount for a better understanding of
these events and it may also lead to establishing more effective diagnostic tests
and treatment[11].
METHODS
For this study, 20 grade I and 10 grade II meningioma samples from adult patients
were used. The selected patients had not undergone previous treatment. For the control
group, we used 10 arachnoid mater samples from patients who had vascular surgery without
a tumor diagnosis. The histopathologic diagnoses were based on 2016 World Health Organization
criteria[16]. This study was approved by the Research Ethics Committee of the University Hospital
of the Medical School of Ribeirão Preto of the University São Paulo.
RNA extraction and cDNA synthesis
Total cellular RNA was extracted using Trizol® Reagent (Invitrogen, Carlsbad, CA,
USA) and RNA was reverse transcribed to single-stranded cDNA using a High Capacity
Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol.
Analysis of Gene Expression Patterns by RQ-PCR
For the quantitative analysis of the genes under study, caspase 9 (assay ID Hs00154260_m1),
cytochrome c (assay ID Hs01588973_m1), APAF-1 (assay ID Hs00559441_m1), Smac/DIABLO (assay ID
00219876_m1), XIAP (assay AI 00236913_m1), BCL2 (assay ID 00608023_m1), caspase 3
(assay ID Hs00234385_m1), caspase 8 (assay ID Hs00236278_m1) and c-FLIP (assay ID
Hs0235998_m1), we used the commercially available TaqMan Gene Expression Assay-on-demand,
which consists of oligonucleotides and probes (Applied Biosystems, Foster City, CA,
USA). Reverse transcription was performed using 1 mg total RNA for each sample in
20 mL of the total reaction mixture. The cDNA obtained was diluted 1:10 and 4.5 mL
was used for each 10 mL of the RQ-PCR mixture using the TaqMan Master Mix (Applied
Biosystems).
All reactions were carried out in duplicate and analyzed with the 7500 Sequence Detection
System apparatus (Applied Biosystems). Data were analyzed using the ABI-7500 SDS software.
The maximum standard deviation between duplicates was 10%. The total RNA absorbed
was normalized on the basis of the Ct value for β-Actin (ACT-β) gene. The variation
of expression among samples was calculated by the 2-ΔΔCt method, with the mean delta
Ct value of the control group being used as a calibrator. To obtain the Ct (cycle)
values, we established a threshold of 0.1 and the PCR conditions were: pre-heating
at 50° for 2 min, denaturation at 95° for 10 min and 50 cycles of amplification and
quantification (15 sec at 95° and 1 min. at 60°).
Statistical analysis
Comparisons of gene expression of grade I, grade II meningiomas and control samples
were performed by One-way ANOVA and post-test Newman-Keuls Multiple Comparison Test.
The level of significance was set at p < 0.05 in all analyses. The tests were carried
out using the Graphpad Prism - version 4.0 program (Graph Pad Software Inc, San Diego,
CA, USA).
RESULTS
Expression of pro-apoptotic genes: cytochrome c, APAF1, caspase 9, Smac/DIABLO, caspase
8 and caspase 3
The mRNA levels of cytochrome C, APAF1, caspase 9, Smac/DIABLO, caspase 8 and caspase
3 in grade I and II meningiomas and control arachnoid tissue were compared. No significant
difference in gene expression was found: cytochrome c (p = 0.3991), caspase 9 (p = 0.7807), APAF1 (p = 0.6486), Smac/DIABLO (p = 0.5011),
caspase 8 (p = 0.1381), and caspase 3 (p = 0.0507) ([Figure 1]).
Figure 1 Representation of pro-apoptotic genes: cytochrome c (p = 0.3991); caspase 9 (p = 0.7807); APAF1 (p = 0.6486), Smac (p = 0.5011), caspase
8 (p = 0.1381), and caspase 3 (p = 0.0507) among the groups.
Expression of anti-apoptotic genes: XIAP, Bcl-2 and c-FLIP
The mRNA levels of the anti-apoptotic genes XIAP, Bcl-2 and c-FLIP in grade I and
II meningiomas and control arachnoid tissue were compared. We observed overexpression
of all genes studied in grade II meningioma tumors when compared to control arachnoid
cells and grade I meningiomas: XIAP (p = 0.0012), Bcl-2 (p = 0.0020) and c-FLIP (p
= 0.0025) ([Figure 2]).
Figure 2 Representation of anti-apoptotic genes: XIAP (p = 0.0012), Bcl-2 (p = 0.0020) and
c-FLIP (p = 0.0025) among the groups.
DISCUSSION
A series of genetic alterations lead to neoplastic transformation and development,
and tumor growth is considerably influenced by the balance between cell proliferation
and cell death. In meningiomas, the understanding of growth regulation is currently
limited. The ratio between anti- and pro-apoptotic proteins appears to influence the
sensitivity of cells for apoptosis[17]. Abnormal apoptosis is a hallmark of human tumors and the deregulation of the genes
that control the apoptotic cascade is closely related to tumor development, progression
and recurrence. Among the known regulators and effectors of apoptosis, the family
of caspases plays an important role in the execution phases of apoptosis[10],[18].
Bcl2 is a member of the Bcl2 proto-oncogene family known to be important regulators
of susceptibility to apoptotic cell death and proliferation, and its immunoreactivity
in meningiomas has been described by several authors[19]. The Bcl2 protein itself is an anti-apoptotic protein[20]. Karamitopoulou et al.[21] observed a good correlation between histological grading of meningiomas (benign,
atypical and malignant), MIB1 and TP53 protein expressions by immunohistochemistry.
Their results also indicate that the presence of Bcl2 protein expression together
with high MIB1 labeling index is associated with an unfavorable prognosis of the disease
in benign meningiomas. Sixteen (26.6%) of the 60 meningiomas expressed Bcl2 protein
(21.6% of the benign, 20% of the atypical and 46.1% of the anaplastic meningiomas)
and the intensity of the Bcl2 protein expression was higher in malignant meningiomas
than in atypical and benign samples. A higher rate of Bcl2 positivity was observed
in recurrent meningiomas when compared to non-recurrent benign meningiomas[19]. Similar results were found in this study, where the analysis of gene expression
of Bcl2 by PCR-RT, demonstrated higher expression of this gene in atypical grade II
meningiomas when compared to grade I.
The IAPs are known as caspase inhibiting proteins that have been reported to bind
and to inhibit specific members of the caspase family. The IAPs are responsible for
blocking the pathway of intrinsic apoptosis, directly inhibiting the initiator and
effector caspases. They are regulated by cellular proteins like Smac/DIABLO, a mitochondrial
protein released along with cytochrome c of mitochondria to the cytosol, and responsible for stimulating apoptosis. Among
the several IAPs, we can also highlight the XIAP and Survivin, important proteins
that act as suppressors of apoptosis[13]. Our results showed a significant increase in the expression of XIAP in grade I
and II meningiomas when compared to samples of arachnoid tissue used as a control.
The increase in the expression of XIAP was two-fold higher when compared to the expression
of Bcl2. In another study, Gogineni and colleagues also found an overexpression of
XIAP protein in meningiomas when compared to control tissue; moreover, they saw that
the Transforming Growth Factor β (TGFβ1), involved in physiological processes such
as cancer, is one of the factors responsible for the increase of XIAP expression[22].
Few studies involving the expression of complex apoptosome (cytochrome c, caspase 9 and APAF-1) are reported in meningiomas. Our results showed a low expression
of the genes that constitute the apoptosome. The same results were observed in the
expression of Smac/DIABLO, with low expression in grade I and II meningiomas. This
demonstrates that apoptosis mediated by intrinsic pathway is low in this type of tumor.
Rajaraman et al.[23] evaluated three brain tumor types (glioma, meningioma and acoustic neuronoma) with
respect to 12 single nucleotide polymorphisms from 10 genes involved in apoptosis
and cell cycle control, including two caspase 8 polymorphisms (location 2q33-q34)
selected for their common occurrence in the population. The haplotype analyses further
implicating the chromosomal region indicated by the two caspase 8 single nucleotide
polymorphisms were evaluated. The significantly increased risk of meningioma observed
with the CT (haplotype) suggests that this chromosomal region either harbors or is
in linkage disequilibrium with a functional polymorphism that affects meningioma risk
and may be a promising candidate for brain tumor susceptibility genes[23]. In Koschny et al.[7], caspase 8 expression showed a high variability between the tumor samples (grade
I, II and III meningiomas), and there was no significant difference in caspase 8 expression
between the grades of malignancy. Our results of the expression of this gene by PCR-RT
showed that its expression is very low in meningiomas, and very similar in grades
I, II and control samples.
The activation of caspase 3 is a central and specific event in the process of apoptosis
and represents the point of convergence of multiple “executioner” apoptotic pathways
and the experimental evidence on animal models the known caspases in the brain with
important implications in brain development. Konstantinidou et al.[18] studied the caspase 3 immunohistochemical expression in 58 primary intracranial
meningiomas by using one monoclonal antibody to detect both precursor and cleaved
caspase 3 (CPP32) and a second to recognize only the cleaved activated form (ASP175).
Caspase 3 expression was analyzed mostly in relation to the anti-apoptotic protein
BCL2, proliferation indices (KI67 and PCNA), standard clinic pathological parameters
and patient disease-free survival. Caspase 3 immunostaining was observed in 62% of
cases for CPP32 and in 24% for ASP175. The CPP32, but not the ASP175, labeling indices
positively correlated with the mitotic index and PCNA labeling indices. Both CPP32
and ASP175 labeling indices were increased in nonbenign meningiomas. Caspase 3 predicted
meningioma recurrence, regardless of how this affected disease-free survival. The
weak correlation between active caspase 3 expression and the ss-DNA (recognizing exposed
single-stranded–ss regions in the DNA of apoptotic cells), as well as the broader
range of ASP175 values when compared to ss-DNA range, indicate that caspase 3 activation
may occur in the absence of cell death and apoptosis. Endogenous inhibitors of caspases
exist and are known as inhibitors of apoptosis or IAPs[24]. The authors concluded that the present findings indicate that apoptosis in meningiomas
has a prognostic value[18]. These results are higher than those obtained in our studies, which evaluated the
gene expression of caspase 3 through the reaction of PCR-RT, observing low expression
in grade I and II meningiomas, as well as in control samples. The high expression
of the clone CPP32 of caspase 3, obtained by Konstantinidou et al.[18] using immunohistochemistry, can be explained by representing both the protein expression
of its precursor form and the form cleaved. Despite observing a low expression of
caspase 3, it was still higher when compared to the expression of caspase 8, perhaps
explained by the fact that it was an effector caspase in apoptotic cascade.
The c-FLIP blocks death receptor-induced apoptosis and has been implicated in tumor
progression. The c-FLIP interacts with caspase 8, thereby preventing activation of
the caspase cascade. Jönsson et al.[25] investigated the endogenous expression of c-FLIP and caspase 8 in bladder carcinoma
cells in relation to their sensitivity to death receptor ligation. Stable expression
of c-FLIP long (c-FLIPL) was obtained by retroviral transduction and the relative
ratio of c-FLIP and caspase 8 was directly correlated to resistance to anti-CD95 or
tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis.
Overexpression of c-FLIPL shifted the responsiveness towards resistant status and,
in bladder carcinomas showing c-FLIP, is an important determinant of susceptibility
to death receptor-induced apoptosis and could function as a prognostic marker for
death receptor sensitivity in future immune therapy[24]. Also, in Koschnya et al.[7], it was observed that the pattern expression of c-FLIP was evenly distributed in
all tumor samples and does not depend on the grade of malignancy. We observed an increase
in gene expression of meningioma c-FLIP in grades I and II when compared to normal
arachnoid tissue, and it is statistically significant in grade II meningiomas. Consistent
with the results of Jönsson et al.[25], this increase in the expression of c-FLIP also suggests a resistance to apoptosis
induced by receptors of death through its extrinsic pathway, once the action of this
protein is to inhibit the activation of caspase 8[25]. However, no data was found in the literature related to the expression of c-FLIP
in meningiomas.
The TRAIL is a cytokine of the TNF super family that induces apoptosis in tumor cells
but not in most normal tissues[26],[27]. Activation of agonistic TRAIL-R2 enables the Fas-associated death domain (FADD)
adapter protein and the caspase 8 (FLICE, MACH) to bind to the receptor. Recruitment
of caspase 8 leads to its proteolytic activation, which initiates a cascade of caspases,
leading to apoptosis[28]. The c-FLIP is a recently-identified protein homolog to caspase 8 that lacks catalytic
activity. Expression of c-FLIP has been suggested to play a significant role in controlling
the susceptibility of tumor cells to the apoptosis-inducing effects of TRAIL[29].
The TRAIL preferentially triggers apoptosis in tumor cells versus normal cells, thus
providing a therapeutic potential. Hao et al.[29]examined a large panel of glioma cell lines for their susceptibility to death ligands
and demonstrated that, of four well-characterized soluble death ligands (TNFα, FasL,
Apo3L, and TRAIL), TRAIL killed the majority (54%) of human malignant glioma cells
with medium to high efficiency[29]. Fas-mediated apoptosis of glioma cells using anti-Fas antibodies or membrane bound
FasL has been reported previously. Soluble FasL apoptotic-inducing capacity is reduced
when compared with membrane-bound FasL[30]. The use of soluble FasL in this study and the high constitutive levels of c-FLIP
in the cells might explain why little Fas-mediated cell death was observed. Hao et
al.[30] showed that the TRAIL-resistant phenotype of some glioma cells can be converted
to sensitive by treatment with protein synthesis inhibitors. This suggests the presence
of short-lived apoptosis inhibitors in the cells. The demonstration of caspase 8 activation
in these cells indicates that the regulation of apoptosis may occur between the death
receptors and caspase 8. Apoptosis inhibitor c-FLIP is a prime candidate for this
intracellular regulation because it possesses a DED through which it interrupts FADD
and caspase 8 binding, preventing death-inducing signaling complex formation and apoptosis[29]. The c-FLIP expression in meningioma cells correlates with sensitivity to TRAIL
in our study. Furthermore, protein synthesis inhibitors did not decrease c-FLIP expression
at concentrations enhancing TRAIL-induced apoptosis. These results suggest that c-FLIP
is unlikely to be involved directly in regulating TRAIL-induced apoptosis in meningioma
cells.
In conclusion, this study showed that the gene expression of caspase 8 and caspase
3 is low in grade I and II meningiomas, suggesting that apoptosis through extrinsic
pathway is small in this type of tumor. On the other hand, the gene expression of
c-FLIP was investigated in meningiomas and its high expression may explain the high
competition of this protein with caspase 8, thereby inhibiting their activation by
the lack of connection with the receiver and blocking the Fas activation of the cascade
of apoptosis by its extrinsic pathway. It was also demonstrated that the up regulation
of anti-apoptotic genes Bcl-2 and XIAP blocks the activation of intrinsic caspase
cascades for apoptosis in grade I and II meningiomas. Prospective studies are needed
to better understand the action of these genes in the anti-apoptotic mechanisms. These
studies may represent attractive novel markers for the diagnosis and follow-up of
brain tumors, especially in meningiomas.
