Polycystin‐1 and hydrostatic pressure are implicated in glioblastoma pathogenesis in vitro

Abstract The mechanobiological aspects of glioblastoma (GBM) pathogenesis are largely unknown. Polycystin‐1 (PC1) is a key mechanosensitive protein which perceives extracellular mechanical cues and transforms them into intracellular biochemical signals that elicit a change in cell behaviour. The aim of the present study was to investigate if and how PC1 participates in GBM pathogenesis under a mechanically induced microenvironment. Therefore, we subjected T98G GBM cells to continuous hydrostatic pressure (HP) and/or PC1 blockade and evaluated their effect on cell behaviour, the activity of signalling pathways and the expression of mechano‐induced transcriptional regulators and markers associated with properties of cancer cells. According to our data, PC1 and HP affect GBM cell proliferation, clonogenicity and migration; the diameter of GBM spheroids; the phosphorylation of mechanistic target of rapamycin (mTOR), extracellular signal‐regulated kinase (ERK) and focal adhesion kinase (FAK); the protein expression of transcription cofactors YES‐associated protein (YAP) and transcriptional coactivator with PDZ‐binding motif (TAZ); and the mRNA expression of markers related to anti‐apoptosis, apoptosis, angiogenesis, epithelial to mesenchymal transition (EMT) and proliferation. Together, our in vitro results suggest that PC1 plays an important role in GBM mechanobiology.


| INTRODUC TI ON
Mechanotransduction is a key biological process whereby cells convert extracellular mechanical cues into intracellular biochemical signals, resulting in the regulation of cell phenotype and behaviour. In the context of cancer, mechanical forces are continuously applied within the tumour microenvironment. Mechanical changes in the microenvironment of brain tumours include increased pressure due to oedema, cellular compression, stiffening of the extracellular matrix (ECM), increased cellular contractility and pressure applied to the cell membrane, all of which can favour gliomagenesis by triggering the activation of mechano-induced oncogenic signalling pathways. [1][2][3][4][5][6][7] In gliomas, it appears that the stiffness of the ECM progressively increases from low-grade to high-grade tumours. Glioblastoma (GBM), which is the most aggressive glioma, exhibits the greatest stiffness despite its heterogeneous nature. Importantly, this increased stiffness is associated with a worse prognosis. 4 Progressive transformation from low-grade glioma to GBM is also accompanied by enhanced mechanotransduction, as evidenced by the phosphorylation of mechanically activated proteins, such as focal adhesion kinase (FAK) and myosin regulatory light chain 2 (MLC2). 4 Gliomas with isocitrate dehydrogenase (IDH) mutations, which have a better prognosis, are characterized by less stiffness than tumours with wild-type IDH, which are more rigid and present with a worse prognosis. In the latter, increased expression of structural components that increase the hardness of ECM is detected, including hyaluronic acid and tenascin-C protein (TNC). 4 The median pressure, generated by the pressure difference that exists between normal tissue and tumour, has also been shown to increase the shear forces of the extracellular fluid, promoting the local invasion of glioma cells. 3,5 Polycystins have emerged as major mechanosensitive molecules 2) genes, located on chromosomes 16p13.3 and 4q21-23, respectively. 8 Mutations in these genes cause autosomal dominant polycystic kidney disease (ADPKD), the most common inherited kidney disease. 9 PC1 is a transmembrane protein with a large and flexible amino (N)-terminus, and a carboxy (C)-terminus that produces transcriptionally active fragments. It functions as a mechanosensory molecule that detects extracellular mechanical stimuli. As an atypical G protein-coupled receptor it can modulate cellular responses that include proliferation, differentiation and apoptosis. 10 PC1 is located in multiple focal adhesion structures, the primary cellular structures that mediate cell communication with ECM. 11 PC2 is a non-selective cation channel permeable to calcium ions, which belongs to the family of transient receptor potential (TRP) channels. 12 In terms of their expression in the human brain, high levels of PC1 are found in the cerebral cortex. 13 Specifically, PC1 presents high expression in astrocytes relative to precursor nerve cells (20-fold higher). 14 PC2 is diffusely expressed in several neural tissues, particularly in the neural tube and nerve ganglia, while in the sixteenth week of organogenesis its expression is more pronounced in the anterior roots of the spinal cord. 15 As far as the role of polycystins in cancer is concerned, they have already been identified as mechanosensitive proteins involved in the biology of various types of cancer, such as colorectal cancer (CRC), renal cell carcinoma, prostate cancer, breast cancer, lung cancer and GBM. [16][17][18][19][20][21] In GOS-3 GBM cells, PC1 has been reported to regulate cancer cell behaviour and to interact with mechanistic target of rapamycin (mTOR) and Janus kinase (JAK) signalling pathways. 19 All the above data led us to the hypothesis that polycystins play a significant role in the development and progression of gliomas.
Therefore, the aim of the present study was to probe the potential role of mechanosensitive PC1 in GBM pathogenesis in relation to mechanical stimulation.

| Human tissue
A sample of adult normal brain (cerebellum) tissue (both as preserved specimen and in paraffin blocks) was used in the present study and was obtained from the archives of the First Department of Pathology, "Laikon" General Hospital, Medical School, National and Kapodistrian University of Athens.

| Hydrostatic pressure (HP) apparatus
T98G cells were cultured in cell plates or dishes depending on the assay applied. When cells reached the appropriate confluency, they were placed into the chamber of the "Continuous Flow Constant Pressure" hydrostatic pressure system ( "Continuous flow constant pressure for cell culture" apparatus, designed and developed by Inspiration Technology Innovation, ITI, Athens, Greece, http://iti. com.gr/) 22 and 100 g/cm 2 of continuous hydrostatic pressure (HP) was delivered to the cell monolayer. Following application of HP, cells were harvested immediately.

| PKD1 knockdown
Prior to transfection, cells were starved for 6 h in order to achieve

| Antibodies
The following primary antibodies were used for Western blot analy-

| Semi-quantitative PCR and quantitative realtime PCR
Total RNA was extracted from T98G cells using RNeasy Mini Kit

| Cell proliferation assay
Cells were seeded in a 96-well plate at a density of 10 3 -10 5 cells/

| Clonogenic assay
Cancer cells were seeded in 6-well plates, at an appropriate seeding density (~10 3 cells/well). Cells were allowed to attach to the wells and then were treated with IgPC1 and/or HP. Plates were placed in a CO 2 incubator at 37°C for 10-15 days, until control cells formed sufficiently large colonies. Cells were then fixed with a solution containing 1 acetic acid: 7 methanol and stained with 0.5% crystal violet in methanol for 15 min. Plates were carefully immersed in a tank with tap water and left to dry. Next, they were scanned, and the relative capacity to produce colonies was evaluated by a densitometry analysis using ImageJ software.

| Hanging drop cell culture for generation of spheroids
Using a 20μl pipettor, 10 μl of 2.5 × 10 6 T98G cells/ml was deposited onto the bottom of the lid of a 10-cm tissue culture dish. The lid was placed back to the dish filled with 15 ml sterile PBS, and cells were incubated at 37°C, 5% CO 2 -95% humidity. The drops were incubated for 2-3 days until aggregates formed. Following, spheroids were transferred to low-adherence plates and treated with IgPC1 and/or HP for 4 days. Each spheroid was photographed in a computer-connected microscope at days 1, 2, 3 and 4. Spheroid diameter (μm) was measured using ZEN 2 software.

| Statistical and image analysis
All experiments were performed at least three times. Data are presented as mean ± SD and were analysed by one-way ANOVA.
GraphPad Prism 6 software was employed for these statistical analyses. All statistical tests were two-sided. p < 0.05 was considered statistically significant.

| PC1 and PC2 expression in GBM cell lines and normal brain tissue
Initially, the RNA and protein expression of both polycystins, PC1 and PC2, were identified in GBM cell lines GOS-3 and T98G, as well as in normal brain tissue ( Figure 1A,B). While PC1 and PC2 present marginal protein expression in normal brain, they are firmly expressed in GBM cells (Figure 1B), indicating a potential implication of these two proteins in GBM pathogenesis. Of note, the presence of polycystins RNA in combination with the absence of protein expression in normal brain implies strong post-transcriptional regulation.

| Impact of HP and/or PC1 blockade on proliferation, clone formation, and migration of GBM cells
In promotes these oncogenic processes.

| Impact of HP and/or PC1 blockade on GBM spheroid diameter
Next, we generated T98G spheroid cultures in order to overcome Surprisingly, treatment of GBM spheroids with both IgPC1 and HP enhanced this effect. alone and the combination of IgPC1 with HP ( Figure 4A  except for the expression of TAZ at 48 h which was upregulated.

| Impact of HP and/or PC1 blockade on the expression of mechano-induced transcription cofactors in GBM cells
According to these findings, PC1 and HP can regulate the protein expression of transcription cofactors YAP and TAZ in GBM cells and this regulation is time-dependent.

| Impact of HP and/or PC1 blockade on the expression of apoptotic, angiogenic, EMT and proliferation markers
The observed effect of PC1 and HP on cell behaviour, the activation

| DISCUSS ION
Gliomas represent the most common primary tumour of the central nervous system and are associated with a particularly high mortality and morbidity. 37 Several factors have been shown to play a role in GBM pathogenesis, with cancer tissue mechanics emerging as a key contributor to its development and progression. Abnormal ECM stiffness and aberrant mechanotransduction can promote glioma growth. In addition, HP has been shown to increase GBM invasiveness. 6 The response of glioma cells to increased mechanical pressure involves mechanosensitive protein molecules such as Piezo1, talin-1, caveola-forming proteins, tenascin-c and Rac1. 1,2,4,6,7,38,39 Furthermore, characterization of the nano-mechanical properties of GBM provides a useful tool for distinguishing normal from malignant brain tissue. 40 Polycystins are mechanosensitive proteins that have been as- In this study, T98G GBM cells were subjected to HP in order to simulate the increased interstitial pressure that develops in GBM   Overall, our in vitro findings highlight the role of PC1 in the mechanobiological mechanisms of GBM pathogenesis. Based on our work, GBM cells use PC1 to sense their mechanical microenvironment and respond to it by translating mechanical forces into biochemical signals that govern their oncogenic behaviour. Therefore, PC1 emerges as an additional mechanosensitive protein that participates in GBM development and progression, suggesting that it may represent a potential novel therapeutic target for this lethal brain cancer. 42,43 Further studies will provide a better understanding of the molecular underpinnings of the effects of mechano-induced PC1 on GBM cells and will validate its role in the mechanobiology of GBM.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest. Writing -review & editing (lead).