Keywords:
brain tumor - angiogenesis - genetics
Palavras-chave:
neoplasias encefálicas - angiogênese - genética
Central nervous system cancers are rare, but present high mortality and morbidity
rates worldwide. Gliomas are the second most common brain tumors, accounting for almost
one fourth of all brain tumors in adults[1]. Based on histopathological analysis, gliomas are classified into 4 grades (I-IV).
Grade I: gliomas are usually benign and easily curable. Lower grade gliomas (LGG)
are grade II, which are often encountered in young adults (mean age of 35), presented
with lesions in the temporal, insular or frontal lobes, as well as seizure disorders[2]. Glioblastomas, which constitute half of all glioma cases, are grade IV gliomas[3]. In adults, glioblastoma multiforme (GBM) is the most common and most aggressive
type of primary brain tumor, which usually occurs in elderly people (mean age of 65).
Accordingly, 5-year post-diagnosis survival rates are as low as 5.1% in GBM patients,
whereas 90% of LGG patients survive 10 years post-diagnosis[1].
Angiogenesis, which is the formation of new blood vessels, is a typical feature of
solid tumors. In order to compensate the nutrition and oxygen need of highly proliferating
tumor cells, a new network of blood vessels needs to be established within the tumor
microenvironment. This is often achieved with an increased secretion of growth factors,
including vascular endothelial growth factor (VEGF) family proteins, causing abnormal
endothelial proliferation[4],[5]. VEGFA and VEGFB are important drivers of vasculogenesis, cell migration and permeabilization
of blood vessels, all of which are hallmarks of malignant cancers[6]. KDR, which is also known as VEGFR2, is a tyrosine kinase with a weak kinase activity
and preferentially acts as a cell-surface receptor for VEGF family proteins. The inflammatory
response is also frequently adjusted in favor of tumor angiogenesis[7]. Studies show that interleukin IL-8 (CXCL8) is secreted to the GBM tumor microenvironment
in high levels[8]. Receptors for CXCR8 signalling are CXCL1 and CXCL2, which play critical roles in
microvascular endothelial cells[9].
Vascularization is one of the major pathological distinctions between GBM and LGG.
Studies have suggested that the high mortality of GBM is strongly influenced by tumor
angiogenesis, which provided the tumor with the ability to infiltrate throughout the
brain tissue and persist through drug therapies[10],[11]. Therefore, this study aims to comparatively analyze the angiogenesis-related genes,
namely VEGFA, VEGFB, KDR, CXCL8, CXCR1 and CXCR2 in LGG vs. GBM using DNA sequencing
and mRNA expression data available on The Cancer Genome Atlas (TCGA) to identify molecular
distinctions that could explain the progress from LGG to GBM.
METHODS
Mutation analysis
The cBio Cancer Genomics Portal (http://cbioportal.org) is an open-access tool that
provides mutation data, copy number alterations, microarray-based and RNA sequencing-based
mRNA expression changes, DNA methylation values, protein and phosphoprotein levels
based on the TCGA-derived data[12],[13]. Seeking to comparatively study the mutations in VEGFA, VEGFB, KDR, CXCL8, CXCR1
and CXCR2 genes in brain lower grade glioma (LGG) and glioblastoma multiforme (GBM),
we first selected our cancer studies of interest on the web interface. The TCGA LGG
and GBM data sets we selected comprised of genome wide sequencing and mRNA expression
data for a cohort of 514 and 592 cancer patients respectively, among which we carried
on analyzing complete tumor samples that had mRNA, copy number alteration and sequencing
data[14]. We then selected VEGFA, VEGFB, KDR, CXCL8, CXCR1 and CXCR2 genes and determined
their mutational status using the OncoPrint feature.
mRNA expression analysis
GEPIA (http://gepia.cancer-pku.cn/index.html) is an online tool that provides differential
expression analysis between tumor vs. normal samples based on RNA expression data
obtained from 9,736 tumor and 8,587 normal samples from TCGA and the Genotype-Tissue
Expression (GTEx) projects[15]. Dot plots and box plots of gene expression profiles of the selected genes across
all tumor samples and paired normal tissues were generated on GEPIA. Finally, p-values
were automatically calculated by the tool, and p-values below 0.05 (%5) were considered
significant.
RESULTS
Aiming to identify genetic alterations within angiogenesis-related genes in LGG and
GBM patients, we analyzed the DNA sequencing and mRNA expression data for 507 glioma
and 360 hepatocellular carcinoma patients, available on The Cancer Genome Atlas (TCGA)
through the cBioPortal interface. We found that 7.2% of all LGG patients and 14.5%
of all GBM patients were subjected to at least one genetic alteration (amplifications,
deletions and point mutations) in VEGFA, VEGFB, KDR, CXCL8, CXCR1 and CXCR2 genes
([Figure 1]). Among the selected genes we analyzed, we identified KDR (VEGFR2) as the most commonly
altered gene within the patient groups, with 4 and 11% alteration percentages in LGG
and GBM, respectively. Most of these alterations were gene amplifications.
Figure 1 Genetic alterations within the angiogenesis-related genes VEGFA, VEGFB, KDR, CXCL8,
CXCR1 and CXCR2 in the LGG (upper panel) and GBM (lower panel) patient groups. Each
bar represents a patient. LGG: Brain Lower Grade Glioma; GBM: Glioblastoma Multiforme.
Considering KDR was the most frequently altered gene, we investigated the point mutations
within the KDR gene in more detailed and compared way, whether LGG and GBM patients
shared some common point mutations. There were six distinct KDR mutations in the LGG
patients, five of which resulted in amino acid substitutions (missense mutations:
E207G, R347H, V398A, T723I and K981N), as well as a nonsense mutation at position
R944*, that resulted in a truncated protein ([Figure 2], upper panel). On the other hand, we detected a total of 18 distinct KDR mutations
in the GBM patients, including missense (I36M, R347H, A352V, A505T, S620N, A632T,
D636N, E759K, R961W, R962C, V1012M, V1093I, S1104F, P1243L, S1290R) and nonsense (W63*,
R1032*) mutations, frame shift deletion (D703Ifs*6) and altered splice region (G23=)
([Figure 2], lower panel). Of all the mutations that we detected, only R347H amino acid substitution
was common in both patient groups.
Figure 2 KDR domain structures and the point mutations detected within the LGG and GBM patient
cohorts.
Cancer is a disease characterized by aberrant protein expression. In order to determine
whether the gene expression profiles of angiogenesis-related genes display alterations
in LGG and GBM patients, we investigated the expression levels of VEGFA, VEGFB, KDR,
CXCL8, CXCR1 and CXCR2 genes in the GBM or LGG tumor samples in comparison to the
paired normal tissues ([Figure 3]). Results showed that KDR was significantly upregulated in both tumors when compared
to the normal tissues. Furthermore, the expression levels of KDR were similar for
both LGG and GBM. We did not detect any significant changes in VEGFB, CXCR1 and CXCR2
between LGG and GBM or tumor and normal tissue samples. Interestingly, however, VEGFA
and CXCL8 were significantly upregulated in GBM tumors in comparison to the paired
normal tissue, as well as the LGG tumor tissue, which did not show any significant
alterations at the expression level when compared to the normal tissue.
Figure 3 Box plots showing gene expression profiling of VEGFA, VEGFB, KDR, CXCL8, CXCR1 and
CXCR2 genes in the GBM or LGG tumor samples (red) in comparison to the paired normal
tissues (grey). (*indicates p<0.05).
DISCUSSION
It is highly suspected in the literature that angiogenesis is a key feature of GBM,
thus explaining its aggressiveness and higher mortality over LGG. Although GBM is
characterized as a genetically heterogeneous disease with patients carrying one or
more genetic alterations at their EGFR, PTEN, RB1 and NF1 genes[16], there are no current studies that comprehensively investigate the genetic alterations
in angiogenesis-related genes in GBM in comparison to LGG, which is a less aggressive
brain tumor type. Therefore, in the present study, we aimed to identify molecular
distinctions within the angiogenesis-related genes between GBM and LGG, and provide
evidence for the role of angiogenesis in brain cancer progression from LGG to GBM.
We found that the alterations within all the selected genes, except for KDR, were
rare changes (<2%). The most frequently altered gene within both patient cohorts,
KDR, was subjected to several point mutations. R347H amino acid substitution, which
was the only common mutation type between the LGG and GBM patients, is a mutation
that has been linked with several cancer types, including leukemia, colorectal carcinoma,
brain lower grade glioma and lung cancer[14],[17],[18],[19], whereas its function is yet to be characterized. Similarly, although the other
KDR point mutations were identified in several genome-wide cancer studies, they have
not been linked with any function or pathology so far. Therefore, at this point, we
can only speculate that these mutations have cancer-driving roles.
Our analysis on the expression profiles of the angiogenesis-related genes revealed
that KDR was upregulated in both gliomas when compared to the normal tissue counterparts.
This could be directly reflected by the fact that KDR gene was amplified in several
LGG and GBM patients, resulting in an overexpressed protein. We did not detect significant
changes in VEGFB, CXCR1 and CXCR2 expression levels. VEGFA was suggested as a prime
candidate driving angiogenesis in GBM by several studies[20]. In line with this, we found that its expression levels were significantly higher
in the GBM tumor tissues than both its paired healthy tissue and LGG tumor and normal
samples; suggesting that it could be an important inducer of angiogenesis in GBM.
A similar trend was observed for the chemokine CXCL8 as well; because its expression
levels were significantly upregulated specifically in the GBM tumor tissue. Previous
studies attributed important roles for CXCL8 in the proliferation, survival, invasion,
and angiogenesis in breast cancer[21], melanoma[22] and glioblastoma[23] and suggested it as a pro-angiogenic factor during carcinogenesis[24]. These results are consistent with our findings, showing that angiogenesis-related
genes, VEGFA and CXCL8, are significantly overexpressed within the GBM patients. Bearing
in mind the proposed role of angiogenesis in its progression to GBM, this study provides
further evidence that VEGFA and CXCL8 could induce angiogenesis and promote LGG to
progress to GBM. Identifying the molecular distinctions between LGG and GBM could
prove to be useful in developing novel targeted therapeutics approaches in the future.
