HPCAL1 promotes glioblastoma proliferation via activation of Wnt/β‐catenin signalling pathway

Abstract Glioblastoma (GBM) is the most prevalent primary malignancy of the central nervous system with obvious aggressiveness, and is associated with poor clinical outcome. Studies have indicated that calcium ion (Ca2+) can positively regulate the initiation of malignancy with regard to GBM by modulating quiescence, proliferation, migration and maintenance. Hippocalcin like‐1 protein (HPCAL1) serves as a sensor of Ca2+. However, the understanding of HPCAL1 activity in GBM is limited. The present study revealed that the gene HPCAL1 was up‐regulated by Ca2+ in the tissues and cells of GBM. Ectopic expression of HPCAL1 promoted proliferation of cells. Exhaustion of HPCAL1 inhibited cell growth not only in vivo, but also in vitro. In addition, HPCAL1 enhanced the Wnt pathway by stimulating β‐catenin accumulation and nuclear translocation in GBM cells, while β‐catenin silencing significantly inhibited the proliferation and growth of the GBM cells. Our results showed that Ser9 phosphorylation of GSK3β was significantly decreased after HPCAL1 knockdown in GBM cells, and knockdown of the gene GSK3β in GBM cells enhanced cell proliferation and promoted transcription of the genes CCND1 and c‐Myc. Furthermore, the phosphorylation of ERK was decreased in the cells with HPCAL1 knockdown, while it was promoted via overexpression of HPCAL1. The suppression or depletion of the gene ERK decreased proliferation triggered by overexpression of HPCAL1 and impaired transcription of the genes c‐Myc and CCND1. These studies elucidate the tumour‐promoting activity of HPCAL1. They also offer an innovative therapeutic strategy focusing on the HPCAL1‐Wnt/β‐catenin axis to regulate proliferation and development of GBM.

36.5%. 3 Even with numerous clinical trials conducted with different agents, there has been no proven success in terms of efficacy. 4 Furthermore, the understanding of the biology of glioma is limited. Calcium ion (Ca 2+ ) has been regarded as a dominant secondary messenger in eukaryotic cells. Emerging reports indicate that Ca 2+ could serve as an essential positive modulator, as it influences the progression of GBM via enhanced quiescence, proliferation, migration and maintenance of malignant cells. 5 Considering treatment approaches that block structures relying on Ca 2+ , such as channels and pumps, can never cure GBM. 5 However, various approaches relying on Ca 2+ utilization by cells in order to conquer checkpoints can be exploited to reprogram malignant stem cells to a different destiny.
The visinin-like protein (VILIP) superfamily, includes VILIP1, VILIP2, VILIP3 (also called HPCAL1), neurocalcin-δ and hippocalcin. [6][7][8] Biological activities of the proteins belonging to this family are atypical. It has been reported that VILIP1 suppresses the invasiveness and proliferation of squamous cell carcinoma cells via inhibiting the function of matrix metalloproteinase-9 and RhoA. Furthermore, VILIP1 also inhibits cancer progression via down-regulation of α5 and αV integrins. 9 Some studies have proved that the expression of HPCAL1 mainly occurred in the Purkinje cells of brain, and the protein HPCAL1 might participate in the regulation of neuron types. 6 Studies have also indicated that reinforced expression of HPCAL1 stimulated ERK2 and its expression. 10 It has been recognized that HPCAL1 is an innovative inhibitor of liver cancer, which was downregulated in the hepatocellular carcinoma (HCC) tissues and cells.
Suppressed HPCAL1 expression worsened clinical outcome in patients with HCC. 11 On the contrary, interaction between HPCAL1 and wild-type paired-like homeobox 2b (WT PHOX2B) influenced the outgrowth of neurites in human neuroblastoma cells with PHOX2B expression. Elimination of the interaction by HPCAL1 knockdown with small hairpin RNA (shRNA) in the neuroblastoma cells with PHOX2B expression reduced the outgrowth of neurites.
Furthermore, the transcriptional profile predicted suppressed differentiation of sympathetic neurons. 12 The understanding of the influence of HPCAL1 on the development of GBM cells is limited.
The present study evaluated the effect of: (a) overexpression of HPCAL1 in the tissues and cells obtained from patients with GBM, and (b) abnormally stimulated Wnt/β-catenin axis in order to enhance cell growth. Stimulation of HPCAL1 was regulated via Ca 2+ concentration within the cells, which enhances ERK stimulation and inhibits the enzyme glycogen synthase kinase 3 beta (GSK3β). The findings of the present study will elucidate the innovative effects of HPCAL1 on progression of GBM, and also offer a promising strategy to treat GBM.

| Analysis of β-catenin nuclear localization
Immunofluorescence was used to evaluate nuclear β-catenin in the cells LN-18 and A172. 14 Nikon fluorescence microscope (type TS800) supplemented with a SPOT camera and imaging software was used for observation.

| RNA separation and real-time quantitative polymerase chain reaction analysis
Ribonucleic acid was separated from tissues and cells using Trizol reagent (Invitrogen). Subsequently, 2 μg of RNA was supplemented ZHANG ET AL.
| 3109 with RQ1 DNase (Promega, Madison, WI, USA) to produce RNA without DNase. Complementary DNA (cDNA) was generated via avian myeloblastosis virus reverse transcriptase (AMV-RT) (Promega) using 1 μg of RNA after treatment with DNase. RT-qPCR was carried out. Fold alteration in the expression of gene was evaluated according to 2 −ΔΔCt method with GAPDH transcripts as reference.

| Western blotting
Ten percent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to isolate protein lysates, which were then moved to PVDF membrane (GE Healthcare, Buckinghamshire, Great Britain). Subsequently, immunoblotting and detection were car-   The mice were examined for the initiation and progression of malignancy. The volume of cancer was calculated according to the formula: 4/3π (major axis/2 × minor axis/2). The cancer tissue was fixed in formalin before haematoxylin and eosin staining and marker analysis. The tissue was then cut into pieces and fixed in 10% formalin and then embedded with paraffin. Immunostaining was carried out with antibodies, such as c-Myc, p-ERK (Cell Signaling Technology, USA), β-catenin (BD Biosciences) and Brdu (Abcam).

| Statistical analysis
GraphPad Prism V software was used for statistical analysis. The data were regarded as significant at P < 0.05. The results are presented as means ± SD.

| HPCAL1 was up-regulated via Ca 2+ in GBM in vivo and in vitro
To investigate the influence of HPCAL1 on GBM, we firstly detected the protein and messenger RNA (mRNA) levels of HPCAL1 in various GBM cells. The results revealed that the expression of the gene HPCAL1 was elevated in the majority of GBM cells in comparison with that of the normal cells, HCN-2 and HCN-1A ( Figure 1A,B).
However, the expression of HPCAL1 has no correlation with the TMZ response in GBM cells, as both TMZ-resistant cells (U-118MG) and TMZ-sensitive cells (U-87MG and A172) have higher HPCAL1 expression. 13 The expression of the gene HPCAL1 in 19 pairs of cancer tissues and the surrounding non-cancer tissues was evaluated.
Similar to the findings in cells, HPCAL1 transcription and translation were significantly promoted in the cancer tissues in comparison with those in non-cancer tissues ( Figure 1C,D). As the markers in circulation have been most frequently used for the diagnosis of cancer and diagnosis before surgery, 15 the presence of HPCAL1 in blood was examined. The concentration of HPCAL1 was significantly enhanced in GBM patients in comparison with that of the healthy counterparts according to ELISA ( Figure 1E). As HPCAL1 serves as a sensor of Ca 2+ , the present study verified if the up-regulation of HPCAL1 was associated with the concentration of Ca 2+ in GBM patients. The concentration of Ca 2+ was significantly promoted in patients with GBM, as expected ( Figure 1E). Furthermore, activation via CaCl 2 promoted the expression of HPCAL1, while supplementation of Ca 2+ scavenger (BAPTA-AM) exhibited opposite effects ( Figure 1F). These findings suggest that HPCAL1 expression was enhanced in GBM specimens, and it relied on Ca 2+ concentration.

| HPCAL1 enhanced GBM proliferation
To investigate the influence of HPCAL1 on biological activities of

catenin expression
As HPCAL1 can regulate β-catenin pathway, the present study veri-

GSK3β phosphorylation
The present study also investigated the mechanisms of regulation of Wnt/β-catenin axis via HPCAL1 to enhance the progression of GBM.
The concentration of β-catenin was modulated via protein degeneration mediated by ubiquitin/proteasome after its phosphorylation via GSK3β, 16 and its kinase function was determined via suppressed Ser9 phosphorylation. 17 It was found that Ser9 phosphorylation of GSK3β was significantly decreased after HPCAL1 knockdown in the cells A172, U-118MG and U-87MG ( Figure 5A; Figure S1A Figure 6A,B). The immunochemical staining of HPCAL1 confirmed the knockdown effect of shRNA in vivo ( Figure 6C). Simultaneously, proliferation of cells was retarded in HPCAL1-knockdown cancers according to WB of Ki67 ( Figure 6D) and immunostaining of Brdu ( Figure 6E). Furthermore, the expression of the genes c-Myc and βcatenin was reduced in the HPCAL1-knockdown cancer cells in comparison with that of the parent cancer cells ( Figure 6F). This indicates that HPCAL1 enhanced proliferation of GBM not only in vivo, but also in vitro.

| DISCUSSION
Glioblastoma is the most prevalent cancer with noticeable aggressiveness in brain. Relationship between cancer growth and proliferation indicates that various cancer stages preserve molecular features for a specific period of the development of autonomic system. 18 Studies on regulation of proliferation and differentiation of malignant cells throw light on GBM and also an innovative therapeutic strategy. Emerging reports indicate that Ca 2+ could play a crucial part in positive regulation of initiation of GBM by affecting the maintenance, migration, quiescence and proliferation of malignant cells. 5 As calcium pathway has been recognized to regulate diverse cellular reactions, it can be presumed that the pathway influences the progression of malignancy. 19 Up-regulation of HPCAL1 has been demonstrated in patients with GBM. 12 In the present study, there was an up-regulation of HPCAL1 in GBM cells, which enhanced proliferation of GBM cells worsening clinical outcome in participants with GBM. Further, HPCAL1 stimulated phosphorylation of not only GSK3β, but also ERK, simultaneously with the stimulation of Wnt/βcatenin axis, which influenced the proliferation of GBM cells.
Hippocalcin like-1 protein (HPCAL1), a member of visinin-like (VSNL) subfamily protein, is characterized by a strict pattern of expression in brain cells. It has been found in the granule cells, Purkinje cells 6 and sympathetic ganglia during the developmental period.
The VSNL family has been reported to exhibit specific activities with regard to signal transduction, membrane trafficking and differentiation among specific subgroups of neuronal cells. Each neuronal calcium sensor (NCS) protein was specific to several kinds of cells, receptors and pathways. The translocation capability of VSNL proteins from the cytoplasm to the subcellular membrane compartments, especially after an increase in the level of Ca 2+ in cytoplasm, is due to the EF-hand calcium-binding motifs and the consensus Nterminal myristoylation sequence. 6,20 Despite the dependence of HPCAL1 on Ca 2+ in brain homogenates, few studies have demonstrated its influence on brain tumour. Several studies have also indicated that HPCAL1 enhanced neuroblastoma differentiation, which was impaired via interaction with PHOXB. 12 However, the interaction between PHOXB and HPCAL1 does not rely on calcium. Consequently, the understanding of calcium sensor activity of HPCAL1 In our study, we found that HPCAL1 expression was positively associated with Ca 2+ concentration within the cells. Escalated cellular Ca 2+ usually results in mRNA level changes of Ca 2+ binding proteins, such as regucalcin, 21 VILIP-1, Calbindin-D28K. 22 Besides, under disturbed Ca 2+ -homeostasis condition, reduced expression of HPCAL1 lead to enhanced oxidative stress, and increased the apoptosis in neuron, which will result in Alzheimer's disease. 22 Thus, increased expression of HPCAL1 may be protective for neuron against oxidative stress, and promotes neuron cell growth. Consistently, we also found that the increased Ca 2+ level in GBM promote the expression of HPCAL1, and enhanced the GBM cell proliferation. Silence of HPCAL1 abolished the proliferation effect of Ca 2+ in GBM cells (Figure 2E), but enhanced expression of HPCAL1 reversed the suppression effect of Ca 2+ scavenger on GBM proliferation ( Figure 2F).
These results suggested that HPCAL1 should be the Ca 2+ sensor in the GBM cell proliferation. However, the detail mechanism for HPCAL1 induction by Ca 2+ level still remain unclear, and worth more efforts to be further investigated.
The up-regulation of HPCAL1 stimulated the Erk-Wnt/β-catenin axis, which led to the proliferation of GBM cells. Further, the present study highlighted the influence of HPCAL1 on enhancing the progression of GBM. Extracellular signal-regulated protein kinases 1 and 2 are a part of mitogen-stimulated protein kinase family, which regulates proliferation of cells. Several studies have suggested that stimulation of Ras-Raf-MEK-ERK axis [23][24][25] and Raf-MEK-ERK axis can result in glioma. 26 In the present study, our results showed that the increased intracellular Ca 2+ enhanced the expression of HPCAL1 followed by enhanced ERK activity. Consequently, the wnt/β-catenin pathway was activated and promotes the proliferation ability of glioma cells. Furthermore, ERK and MEK were abnormally stimulated in gliomas and other malignancies, 27 and could serve as an upstream event, and is necessary to deactivate GSK3β. 17 Previous studies have demonstrated the interaction between ERK2 and HPCAL1. 11 Accordingly, our results provided also suggested that the expression of HPCAL1 lead to the activation of ERK, which would deactivate GSK3β, and therefore constrain the activity of β-catenin. However, the precise mechanism of interaction between ERK and HPCAL1 in GBM requires further exploration.
It has been demonstrated that the Wnt/β-catenin axis participates in GBM 28 The expression of β-catenin was enhanced in several GBM cells, thus triggering Wnt target genes and promoting proliferation, 29 invasion and migration. 30 Findings of the present study suggest that HPCAL1 is an upstream effector of the Wnt/β-catenin axis of GBM. This is implied by a close relationship between the pro-  29 The function of Wnt and malignant stemness was modulated via the micro-environment, where agents, such as growth factor of liver cells generated via myofibroblasts, stimulated transcription relying on β-catenin and promoted cancer stem cell clonogenicity. 32 No mutation specific to β-catenin in GBM has been reported. Promoter hypermethylation of Wnt pathway suppressors, such as NKD2 and sFRP2, has been frequently reported in numerous GBMs. 33 Findings of the present study indicate that HPCAL1 stimulates the Wnt pathway by enhancing ERK and indirectly inactivating GSK3β, thus enhancing β-catenin nuclear translocation. As β-catenin is also involved in GBM metastasis, 30 we believed that HPCAL1 might also have function to promote GBM cell invasion and migration. Therefore, further studies focusing on the expression of HPCAL1 that propels proliferation and migration of GBM are necessary.
The results of the present study proved the influence of HPCAL1 on the stimulation of Wnt/β-catenin axis and proliferation of GBM.
Future research on the functional effect of HPCAL1 can offer innovative strategies to identify pharmacological or biological targets of GBM.