Gliomas are the most common central nervous system tumor type accounting for approximately 80% of all malignant intracranial tumors with high episodes of recurrence (Ostrom et al., 2014).

Depending on the cell type of origin, they can be classified or subdivided into astrocytomas, ependymomas, oligodendrogliomas, oligoastrocytomas, and glioblastoma multiforme (GBM) (Ostrom et al., 2014).

Glioblastoma is the most aggressive brain tumor owing to its high invasiveness. Despite advances in therapeutic modalities (tumor resection combined with chemotherapy and radiotherapy), the overall patient survival rate is 12–15 months from the initial diagnosis, with only 5% of patients reaching a 5-year survival rate (Fedele et al., 2019).

Likewise, temozolomide (TMZ), a DNA alkylating agent, is the standard first-line treatment for GBM, has a significant tumor recurrence rate and confers drug resistance (Strobel et al., 2019) due to its low specificity and limitations in crossing the blood-brain barrier (BBB).

Persistent headaches, neurological and behavioral deficits, seizures, cognitive deficits, drowsiness, dysphagia, confusion, aphasia, motor deficits, fatigue, dyspnea, and mood changes are common symptoms in patients diagnosed with glioblastoma (Maugeri et al., 2016; IJzerman-Korevaar et al., 2018). Usually, its localization, size, and grade make it inoperable and difficult to treat, drastically decreasing the quality of life of patients with glioma (Fedele et al., 2019).

Many symptoms are derived from neurological findings such as increased intracranial pressure and brain herniation. Edema around the tumor causes hypoxia (lack of oxygen supply) (Solar et al., 2022), resulting in long-term disability, psychiatric disorders, substance abuse, or self-harm (Huang et al., 2021).

A clinical study carried out by Wu et al. (2015) showed that within the identification of peritumoral edema during the preoperative period in patients with glioblastoma, factors such as necrosis and edema extent were independent factors for poor outcome and overall survival (OS).

Aquaporins (AQPs), a family of transmembrane water channel proteins, play a critical role in responding to changes in the osmotic environment. Eight members of the water channel family are expressed in the central nervous system (Saadoun et al., 2002; Maugeri et al., 2016). AQP1 and AQP4 are the major water channels in the brain and play a pivotal role in water homeostasis and the maintenance of BBB integrity (Galán-Cobo et al., 2016).

Brain AQPs are distributed primarily in astroglial membranes, especially in perivascular astrocyte foot processes and glia limitans, near the brain fluid compartments (Hubbard et al., 2018; Li et al., 2019) and ependymal cells, providing an efficient system to promote water and solute transport between the perivascular space and glial cells, and to support brain parenchymal clearance through the glymphatic system (Smith et al., 2017; Mestre et al., 2018; Lohela et al., 2022).

AQP4, an orthodox AQP type, is upregulated in gliomas and is involved in the tumorigenesis process, that is, cell migration, invasion, and functional changes in the surrounding tissue. AQP4 distribution has been hypothesized to underlie mechanisms related to the degree of malignancy (Lan et al., 2017), edema formation in the peritumoral region, increased intracranial pressure, and seizure episodes (Dubois et al., 2014; Maugeri et al., 2016).

Tumor cell migration is a complex mechanism not well understood. However, angiogenesis is a critical feature differentiating high-grade and low-grade glioma. Glioblastoma as the most aggressive brain tumor shows morphological and vascular changes characterized by a high invasiveness and migration, which are associated with the expression of AQP1 and AQP4 and mesenchymal transformation as previously described (Hardee and Zagzag, 2012; Brennan et al., 2013).

Aquaporins have been described to have a vital role in enhanced invasion and tumor cell migration as reviewed by De Ieso and Yool (2018) and Moon et al. (2022). Of special importance is the role of AQP1 and -4 in the proposed mechanism of invasiveness and migration of glioma cells. During tumor cells metastasis modifications of blood supply, cytoskeleton and extracellular matrix structure are needed to promote angiogenesis for tissue demands of oxygen and nutrient delivery. AQP1 was found to boost endothelial cell migration and, in association with overexpression of AQP4 to regulate water influx and extracellular matrix interaction leading to tumor cell membrane filopodia formation, mechanisms linked to metastasis progression.

Several in vitro and in vivo studies have reported reduced invasive capacity and migration in tumors after AQP4 depletion or downregulation (Ding et al., 2010, 2011; Cheng et al., 2017) by decreasing water permeability, which in turn upregulates transmembrane water fluxes during tumor or healthy astroglial cell movement (Huang et al., 2021).

In contrast, AQP1 (also considered a classic aquaporin) is predominantly expressed in the circumventricular brain and areas associated with cerebrospinal fluid production. Its expression is altered in the human brain under pathological conditions (Lima et al., 2012) such as cancer. In brain tumors, AQP1 and AQP4 expression is upregulated with the malignancy grade (Saadoun et al., 2002).

Inhibition of tumor growth and suppression of vasculogenic mimicry formation were observed following AQP1 silencing in vivo, which could be related to reduced tumor aggressiveness, malignancy, and metastasis (Perry and Wesseling, 2016).

More recently, the newly updated World Health Organization (WHO) Classification of Tumors of the Central Nervous System classifies brain tumors based on their histological appearance and molecular markers (genotypic classifications). Namely: isocitrate dehydrogenase (IDH) mutation, 1p/19q codeletion, and O-6-methylguanine-DNA methyltransferase (MGMT) methylation, avoiding imprecise diagnosis, thereby significantly influencing in the selection of treatment options for patients (Martinez-Lage and Sahm, 2018; Huang et al., 2020).

Owing to the role of molecular markers in the early diagnosis of gliomas, we hypothesized that AQP1 and AQP4, as homeostatic brain proteins, could act as potential anticancer therapeutic targets.

In this study, we aimed to characterize the expression patterns of genes encoding AQP1 and AQP4 proteins and to identify the genes present in the cancerous microenvironment that potentially regulate or correlate with their expression in human gliomas. Our findings, from a bioinformatic approach, allowed us to conduct a wide and diversified scale analysis and provided evidence for the role of these proteins and other related genes, thus paving the way for the development of potential biomarker candidates for the diagnosis and therapeutic targets of glioma for future research.

In this study, we aimed to identify the expression pattern of AQP1 and AQP4 genes in human gliomas, as well as to highlight their regulatory potential within the cancer microenvironment.

We used RNA-seq as a search strategy to identify the differential expression of AQP1 and AQP4 transcripts in glioma tissues data compared to normal brain tissues data. Indeed, AQPs genes are overexpressed in patients with glioma. Among the glioma subtypes, AQP1 and AQP4 are overexpressed in astrocytoma (LGG) and classical glioma (GBM). OS analysis showed that both AQP genes can be used as prognostic factors for patients with LGG, confirming the results of previous studies and reinforcing their clinical value. We also observed a correlation between the expression of genes involved in the tyrosine and thyroid hormone pathways and AQPs.

Emerging evidence suggests a positive relationship between AQP1 and AQP4 expression and histological tumor grade and brain edema volume. Poor neurological prognosis has also been reported (Suzuki et al., 2018; Chow et al., 2020). Our results are supported by these findings, as aquaporin expression patterns increased when compared between molecular subtypes of gliomas: AQP1 is increased in other subtypes of LGG classification (astrocytoma); in contrast, AQP4 is increased in only the classical subtype GBM.

Some current studies have highlighted the regulatory function of AQPs on brain volume in many other neurological diseases besides tumors (Sun et al., 2022; Ohmura et al., 2023). In gliomas, both AQP1 and AQP4, when suppressed, offer a better prognosis for patients affected by these types of tumors by edema reduction (Faraj et al., 2022; Ohmura et al., 2023), facilitating the management of neurological deficits in the pre-and postoperative periods, with peritumoral edema being more relevant, in terms of concerns the aggressiveness of this cancer, rather than its extent, per se (Faraj et al., 2022). AQP1, for example, is upregulated in the peritumoral area, especially in the vessels surrounding reactive astrocytes in high-grade gliomas. In LGG, it showed an intense pattern of intratumoral expression (Zoccarato et al., 2021). The discovery of the glymphatic system was also important for the consolidation of AQPs as targets therapeutic in brain edema regulation (Ding et al., 2023).

From these findings it is possible to suggest that AQPs are perhaps more relevant in the tumor progression process, that is, the transformation of LGG into GBM through the formation of expansive edema, facilitating the invasion of tumor cells into healthy tissue. The determining factors in the survival rate of patients with GBM, in turn, are other and more diversified, in addition to the expression pattern of AQPs (Deb et al., 2012; Saito et al., 2022), which would explain our results from the survival analysis, where AQP genes only functioned as a risk factor for survival in low-grade gliomas.

AQP4 isoforms, known as M1 and M23, have been shown to change the aggregation/disaggregation state into orthogonal arrangements of particles (OAPs) and glioma cell survival. One study revealed that the isoform M23 reduced the invasion and proliferation of glioma cells to promote their apoptosis (Simone et al., 2019).

Some endogenous factors such as hormonal and metabolic changes also modulate AQP4 expression in human gliomas (Lan et al., 2017). Likewise, an increase in AQP1 expression was observed during central necrosis and hypoxia, which are characteristics of GBM tumors (Hayashi et al., 2007; Honasoge and Sontheimer, 2013).

Gene expression is orchestrated by several endogenous and exogenous factors, such as DNA-binding transcription factors, regulatory proteins, and profile hormones (Bhat et al., 2021).

A study conducted by Nauman et al. (2004) evaluated the cellular concentrations of thyroxine (T4) and triiodothyronine (T3) in non-thyroidal illness syndrome (NTIS) patients with gliomas, where the thyroid stimulating hormone (TSH) serum concentration was within the normal range and T4/T3 levels were lower than normal (Nauman et al., 2004). They demonstrated that thyroid hormone levels were significantly lower in glioma tissues than in healthy tissues. In addition, both iodothyronine deiodinases (types 2 and 3) were higher in tumor tissues than in non-glioma tissues.

Some studies have shown that hypothyroidism is an important factor in the prognosis and progression of metastatic cancer in patients with brain cancer. For example, a cohort study (Berghoff et al., 2020) identified a beneficial role of this clinical condition in overall survival in patients with brain metastases, almost doubling life expectancy from the diagnosis of primary and metastatic cancer. Furthermore, the results of a clinical survey showed that almost 40% of patients with brain tumors evaluated had overt or subclinical hypothyroidism and 31% required hormone replacement (Faghih-Jouybari et al., 2018).

In pediatric patients, an important probability of developing thyroid dysfunction was found post-treatment with surgery, chemotherapy, and radiotherapy (Jin et al., 2018; Cosnarovici et al., 2020); moreover, abnormalities in thyroid function were found in patients with AQP4 antibody–seropositive optic neuritis (Zhao et al., 2017).

We previously identified a negative modulator of AQP4 protein and demonstrated that triiodothyronine (T3) treatment of GBM-95-line cells resulted in a slight reduction in cell migration (Costa et al., 2019); other studies have also evaluated the modulatory action of this hormone on the expression of AQP4 in stroke animal models (Sadana et al., 2015).

In recent years, a specific AQP4 inhibitor has been tested in rodents and human brain glioma to directly measure water efflux rate during tumorigeneses (Harrison et al., 2020; Rosu et al., 2020). A promising study conducted by Jia et al. (2023) found a correlation with water efflux in glioma cell cultures, rat and human glioma and cell proliferation as measured by magnetic resonance imaging. This discovery is in agreement with our results showing overexpression of AQP4 genes in gliomas, placing aquaporin 4 as an important biomarker to be used both in bioinformatics analyzes and in brain imaging as a potential therapeutic treatment of gliomas. A summary of what is hypothesized/proven about the modulation of the expression of AQPs 1 and 4 in gliomas is briefly outlined in Figure 6.

Therefore, according to our results, we suggest two new hypotheses: (1) As all patient data are post-mortem (including data other than this one), it is possible that standard cancer treatment led to hypothyroidism and subsequently downregulated the thyroid hormone pathways. This could result in the overexpression of AQP1 and AQP4 in the more aggressive glioma subtypes [astrocytoma (LGG) and classical, (GBM)], as we present in the differential expression results, (2) hypothyroidism would already be a clinical risk condition for the development of glioma; patients diagnosed early with the tumor would present a sub-clinical pattern of thyroid gland dysfunction. This condition would be accentuated by the anticancer therapeutic approach, and these patients would have the same outcome and genetic, and molecular profiles as mentioned in hypothesis 1.

Additional studies to clarify the specific inhibitors or promoters of these pathways, as well as transcription factors and post-transcription/translation modification methods, will help to identify the most appropriate therapeutic targets for AQP1 and AQP4 modulation, relationships between genes, and signaling pathways in gliomas.

To the best of our knowledge, this is the first study to suggest a modulatory interaction between the gene encoding AQP1 and pathways related to thyroid hormones.

The brain tissue is substantially plastic and heterogeneous. Factors such as age and sex can influence their genetic constitution. Glial tumor characteristics may also change depending on the topographic region of the brain where the tumor occurs. The non-separation of categorical patient groups based on the aforementioned factors is a limitation of this study. Single-cell analysis from different glioma patient groups would help us to confirm the oncogenic role of the AQPs studied here, as well as their modulation by thyroid hormones.

To the best of our knowledge this study is the first to show a strong potential functional relationship between AQP1 and thyroid hormone pathways in brain tumors. The expression of AQP1 and AQP4 is significantly associated with a worse prognosis in patients with LGG. The molecular pathways and AQP1 and AQP4 genes identified here may be useful for the molecular diagnosis of gliomas and for screening new anti-tumor drugs for these malignant tumors.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

CM: Conceptualization, Formal Analysis, Investigation, Writing – original draft, Writing – review and editing. LR: Writing – review and editing. CC: Data curation, Writing – original draft. AX: Conceptualization, Data curation, Project administration, Supervision, Writing – original draft, Writing – review and editing.