Carbonic anhydrase related protein expression in astrocytomas and oligodendroglial tumors

Carbonic anhydrases (CAs) catalyze the reversible conversion of carbon dioxide and water to bicarbonate ion and proton. These enzymes are fundamental in normal physiological processes in organisms [1, 2]. The role of the CAs in diseases has been investigated extensively, and at least CA II, IX and XII have been associated to neoplastic growth [3–5].

Three inactive CA isoforms were discovered in 1990s and were named carbonic anhydrase related proteins (CARPs) VIII, X, and XI [1, 6]. They lack the one or more of the three zinc binding histidine residues which are required for the enzymatic activity of CAs [1]. The distribution pattern of CARPs in adult and fetal brain implies a role in brain development [7]. In the human enteric nervous system, CARP VIII and X are expressed in neural cell bodies, whereas CARP XI is absent [7]. In the human central nervous system (CNS), weak but significant CARP X mRNA expression has been detected in almost all parts of the system, whereas it is absent in the fetal brain and other developing tissues. In the fetal brain, CARP VIII and XI expression has been detected first at the 84th day of gestation in the neuroprogenitor cells. Later expression has been found in the migrating neural cells. CARP X expression has been observed in the cerebral cortical neurons at the 141st day of gestation [8].

Immunohistochemical analysis has shown strong expression of CARP VIII in neuron bodies of almost all parts of the adult brain. Weak reactivity was observed in the astrocytes, while the expression was completely absent in the oligodendrocytes. Choroid plexus and pia arachnoid have been strongly positive for CARP VIII. The CARP XI expression is weaker than CARP VIII, but the overall distribution among the neurons and glial cells is similar. CARP X expression differs from the other CARPs; it has been found mainly in the myelin sheath of the oligodendrocytes, and only weak expression has been detectable in the Purkinje cells and neurons of olivary nuclei [8].

CARP VIII is predominantly expressed in cerebellar Purkinje cells and appears to modulate IP3-induced calcium release in these cells [9, 10]. A homozygous missense mutation in human CA8 gene that codes for CARP VIII, leads to reduction in cerebellar volume causing mild mental retardation and cerebellar ataxia and some patients exhibit quadrupedal gait [11, 12]. In waddles mice, Car8 gene mutation and the consequential protein deficiency are associated with obtrusive gait disorder [13]. In zebrafish, knockdown of the CA8 gene leads to reduction in cerebellar volume and abnormal swim pattern, which are similar to the consequences of CA8 gene mutation in humans and mice [14]. Thus, CARP VIII plays an important role in motor control in humans, mice and zebrafish.

The function of CARP X and XI is unclear in humans. However, expression studies in humans show that CARP X is highly expressed in pineal gland at night suggesting its role in circadian rhythm (day-night cycle) [15]. The 5′-untranslated region of the CA10 gene contains a CCG trinucleotide repeat in normal humans. The presence of CCG repeats in the CA10 gene indicates that CARP X could be involved in neurologic disorders due to mutations expanding the number of repeats [16]. Studies in humans and cultured cells showed that CARP XI is associated with spinocerebellar ataxia 3/Machado Joseph’s disease [17]. The latest studies on CARP X and CARP XI in zebrafish showed that these genes are predominantly expressed in CNS, and knockout of these genes induces to apoptosis in the brain leading to ataxic swim pattern in the ca10a and ca10b mutant zebrafish [18].

Altered expression patterns of CARPs VIII and XI have been also linked to cancer. CARP VIII has been reported to promote colon cancer cell growth and invasiveness [19]. In colorectal adenocarcinomas, strong expression of CARP VIII has been found at the tumor infiltrative border [20]. In lung adenocarcinoma cells, CARP VIII increases cell growth in a laminin-rich environment, which led to a hypothesis that it may interrupt apoptosis signaling [21]. Strong CARP VIII expression has been observed in the developing fetal lung and at the infiltrative border of the non-small cell lung carcinoma, but only minor expression has been found in the adult lung [22]. Most of the gastrointestinal stromal tumors (GIST) overexpress both CARP VIII and XI, and especially CARP XI seems to enhance proliferation and invasion of these tumors [23]. Similar to the lung and colorectal cancers, the most intense expression of CARPs VIII and XI is present in the marginal areas rather than the center of the GISTs [23].

Recent study showed an increased expression of CA8 gene in more aggressive types of human osteosarcoma (HOS) cells and the CA8 expression correlated with the disease stages, showing intense expression in the late stages of the disease. Further experiments involving overexpression of CA8 gene in HOS cells showed significant increase in cell proliferation both in vitro and in vivo. Interestingly, downregulation of CA8 gene in HOS cells decreased cell invasion and colony formation ability in soft agar, and reduced the expression of metalloproteinase 9 (MMP9) and focal adhesion kinase (FAK), suggesting a role for CARP VIII in cancer cell invasion through the activation of FAK-MMP9 signaling [24]. Even though several tumors overexpress CARPs, the exact molecular mechanisms beneath their cancer promoting qualities have not yet been discovered [19, 23].

In the case of brain tumors, there have been no reports on CARPs in human astrocytomas and oligodendroglial tumors (oligodendrogliomas and oligoastrocytomas). These tumors derive from glial cells or glial progenitor cells and the majority of them are highly malignant. The surgical treatment is often inadequate and the prognosis of patients is still poor [25, 26]. Because the progenitor cells have been shown to express CARPs during fetal development, we wanted to assess the expression of different CARPs in astrocytomas, oligodendrogliomas and oligoastrocytomas, and to study their association with different clinicopathological features and patient survival. Since CAs II, IX and XII have shown high expression in gliomas, their association with CARPs was also evaluated.

The cellular origin of the gliomas has been a focus of intensive research for decades. Many recent studies have implied that the neural stem cells can initiate and maintain the growth of human brain tumors, and hence could act as considerable targets for therapy [25]. In the development of CNS, fetal neuroprogenitor cells express CARP VIII and XI [8]. Mature oligodendrocytes, excluding the myelin sheath, do not express CARPs at all. Astrocytes have shown weak expression of CARPs VIII and XI [8]. The presence of CARPs in both the fetal brain and some tumors suggests that they might represent oncofetal proteins and play a role in the development of gliomas.

In this study, we first evaluated the expression of CARPs VIII, X and XI in astrocytomas, oligodendrogliomas and oligoastrocytomas by immunohistochemistry. Because the expression of CARP X turned out to be extremely low in gliomas, it was excluded from the final analyses. Some representative images of CARP VIII and XI staining are shown in Fig. 1.

In further statistical analysis the grade II – IV gliomas with inconclusive genetic testing (according WHO 2016 CNS tumor classification) were also excluded. The excluded tumors are marked as NOS (not otherwise specified) tumors in the Table 1.

In diffuse astrocytic and oligodendroglial tumors tested for the appropriate molecular markers CARP XI expression differed significantly between astrocytomas and oligodendrogliomas; all oligodendrogliomas were negative (p = 0.040, chi-square test). In astrocytomas and oligodendrogliomas, there were no association between CARPs and proliferation by Ki-67 / MIB-1 (p = n.s, Kruskal-Wallis test).

In diffuse gliomas tested for the appropriate molecular markers CARP XI expression differed significantly between diffuse astrocytomas and oligodendrogliomas; all oligodendrogliomas were negative (p = 0.040, chi-square test). In diffusively infiltrating astrocytomas and oligodendrogliomas, there were no association between CARPs and proliferation by Ki-67 / MIB-1 (p = n.s, Kruskal-Wallis test).

In gliomas, hypoxia plays a key role in the regulation of tumor aggressiveness and treatment resistance. Hypoxia-inducible factors (HIF1 and 2) enhance tumor growth under hypoxic conditions by turning on several hypoxia-responsive genes, such as vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and, importantly, CA9 which has been shown to be strongly controlled by hypoxia also in brain tumors [32]. Both EGFR and VEGF gene amplifications are usually associated to higher tumor grade and thus poorer outcome of patients. In gliomas, VEGF is mainly found in perinecrotic tumor areas. EGFR gene is amplified in 40% of the glioblastomas [33].

In our study, the majority of the tumors expressing either CARP VIII or CARP XI were negative concerning VEGF. Almost all CARP VIII-positive astrocytomas inversely correlated to VEGF (p = 0.005, chi-square test, n = 197. These observations suggest that the CARPs are not related to hypoxic conditions.

In gastrointestinal, colorectal and lung cancers, the most intense expression of CARPs has been reported in the tumor infiltrative border rather than in the hypoxic center areas of the tumors [20, 22, 23]. These findings imply that CARPs might play a specific role in tumor invasion. In our study, we were not able to define the precise intratumoral location of CARPs, because each tissue microarray specimen contained only a single representative area of the tumor. On the other hand, diffusely infiltrating gliomas usually invade either individually or in small groups of cells [34]. Obviously, analyses of larger sections or multiple intratumor biopsies are warranted to evaluate further CARP expression separately in hypoxic and invasive areas. Similarly, the role of tumor cell invasion should be tested in patient derived in vitro analysis in the future studies. In light of our increasing understanding of genetic diversity with sub-clones of malignant tumors and stem cells, CAs II, VII, IX and XII have been shown to be present in several categories of brain tumors [28, 31, 35–37]. CA IX seems to be the most interesting CA in terms of its potential role in brain tumor diagnostics and drug therapy [35]. Its transcription is facilitated by HIF-1 alpha [32]. In a recent study, a negative correlation was found between CA IX and enzyme cytosolic isocitrate dehydrogenase 1 (IDH1) mutation in astrocytomas [38]. In our study, no correlation was found between CARP VIII expression and the other CAs (p = n.s., chi-square test) or IDH1 mutations (p = n.s., chi-square test). Similarly, there was no correlation between CARP XI and IDH1, nor did CARP XI correlate with the expression of CA VII, CA IX, and CA XII (p = n.s., chi-square test). Interestingly, the expression of CARP XI positively correlated with the cytoplasmic CA II in astrocytic tumors (p = 0.037, chi-square test). Endothelial expression of CA II has been associated with neoplastic growth and considered a potential molecule in diagnostics and treatment of gliomas [39]. CA II is regulated mainly by factors other than hypoxia [40], which might be the case with CARPs as well. When primary and recurrent tumors were analyzed in different tumor types, no differences in CARP VIII and XI was found (p = n.s., chi-square test). 1p/19q co-deletion is an independent prognostic and predictive marker in oligodendroglial tumors [40]. In our study, all oligodendrogliomas (with defined 1p/19q – co-deletion) were CARP XI negative.

From the FANTOM project, the samples where expression of the CARP VIII CAGE peak is found most strongly are: small-cell gastrointestinal carcinoma, acute myeloid leukemia, cerebellum, rectal cancer, and reticulocytes; whereas the expression was extremely low in astrocytoma, glioblastoma, and glioma samples (Additional file 1: Table S1). Similarly, the top five samples where expression of the CA11 CAGE peak is found most strongly are: middle temporal gyrus, medial temporal gyrus, amygdala, parietal lobe, and caudate nucleus; while also near zero in astrocytoma, glioblastoma, and glioma (Additional file 2: Table S2). In our study, CARP XI protein immunopositivity was observed only in one case (1%) of the group of oligodendrogliomas.

It is of interest that CARP VIII and XI are only weakly expressed in the mature astrocytes and they are absent in the oligodendrocytes [8]. In our study, the overall expression of the CARPs in the astrocytomas and oligodendroglial tumors. Was rather weak. The most benign tumors, pilocytic astrocytomas, did not express CARPs at all. When the grade II-IV astrocytomas and oligodendrogliomas were evaluated separately, no correlation to WHO tumor grade was found (p = n.s, chi-square test).

However, in a recent study the inhibition of CA8 gene in human osteosarcoma cells sensitized the tumor cells to cisplatin chemotherapy [24]. Since this chemotherapy is also used in glioma treatment, it would be of interest to analyze the effect of CA8 inhibition combined to cisplatin in gliomas.

Our analysis of the CA8 promoter has identified a number of putative TFBSs which may affect CA8 expression (Fig. 2). CAGE data from the FANTOM project shows a peak near the Ensembl defined TSS. This peak overlaps, and is supported by, an Ensembl predicted promoter region. Within this promoter region our analysis has predicted a cluster of overlapping TFBSs within an area that is − 59 to − 41 nt upstream of the TSS. Specifically, the TF proteins, which are predicted to bind here, are KLF14, SP4, and EGR1. Two additional binding clusters exist further upstream of the TSS, STAT5A:STAT5B and STAT3 at − 524 to − 512, and a site for CEBPA, HLF, CEBPE, CEBPD, and CEBPB at − 783 to − 771. A complete list of predictions is available as Additional file 3: Table S3.

Early growth response-1 (EGR1) has demonstrated both tumor-suppressor and oncogene properties depending on the type of tumor. In high-grade astrocytoma, EGR1 expression is associated with enhanced patient survival [41]. However, Sakakini et al. recently observed that EGR1 expression was much more consistently observed in glioblastoma than in pilocytic astrocytomas, that in proliferating cyclin A/OLIG2-positive cells EGR1 was localized to the nucleus, and that this produced increased aggressiveness and stemness [42]. Additional links are found between glioma and STAT3 and CEBPB TFs. Specifically, these two proteins correlate with mesenchymal differentiation and poor clinical outcome [43]. STAT5B inhibition reduces glioblastoma cell growth, cell cycle progression, invasion, and migration [44]. There are a number of candidate TFs, across three clusters of binding sites, that have established roles in astrocytoma and glioblastoma. Based on expression values from the extensive FANTOM project, we see that CA8 is expressed highly in cerebellum (95.76 TPM) but near zero in astrocytoma, glioblastoma, and glioma. With the significantly higher CA8 expression in normal brain tissues than in tumor tissues, these putative TFBSs would make interesting subjects for future study as possible repressors of CA8.

Our analysis of the CA11 promoter showed that the strongest predicted binding sites are in two distinct clusters (Fig. 2). The first cluster is comprised entirely of numerous ZNF263 binding sites on the positive strand overlapping the most highly expressed FANTOM CAGE peak, locations + 86 to + 132 nt relative to TSS. The second cluster of predicted TFBSs all overlap one another immediately upstream of the same most strongly expressed CAGE peak, and on both positive and negative DNA strands: LHX3, ARID3B, PHOX2A, PROP1, PHOX2B, FOXB1, ARID5A, HOXD8, and HOXD9 at locations + 64 to + 83 nt. A complete list of predictions is available as Additional file 4: Table S4.

Compared to the CA8 promoter analysis, these predictions for potential TFBSs were more limited in their correlation with astrocytoma. However, this makes sense as nearly all the best scoring predictions seemed to occupy one position. PHOX2B has been identified to have a hypermethylated promoter in glioma, thus resulting in its down-regulation [45]. Similarly, a CpG locus in the HOXD8 gene 5’ UTR is differentially hypermethylated in short-term survival gliomas vs. long-term survivors [46]. Increased expression of HOXD9, the other HOX family member predicted at this location, has been found associated with cell proliferation, and may be a marker of glioma stem cells [47]. The prolific set of potential ZNF263 binding sites overlapping the CAGE peak could be a locus for CA11 repression, as ZNF263 serves that function in some genes [48]. These two proximal sites represent potential regulatory sites worth investigating further to understand the decreased expression of CA11 in astrocytoma and oligodendroglioma.

Our studies showed no CARP expression in pilocytic astrocytomas. In higher grade gliomas, the CARP expression is mainly absent or weak. In this study, the CARP VIII and XI proteins were associated to non-hypoxic conditions and CARP XI also to the expression of cytoplasmic CA II staining. The expression of CARPs did not associate to survival or tumor WHO grade. Due to their presence in a subset of malignant gliomas, CARPs may have a role in tumorigenic processes. However, further investigations on the functions of CARPs are warranted.