Low levels of SorCS3 expression in human glioma cancer tissues are associated with poor clinical outcomes.
To explore the potential roles of SorCS3 in glioma, we analysed RNA sequencing results from The Cancer Genome Atlas (TCGA) database and found that the mRNA level of SorCS3 was decreased in glioma (Fig. 1A). Further, we found that the mRNA level of SorCS3 in high-grade glioma was much lower than that in low-grade glioma (Fig. 1B). Using two independent datasets, we demonstrated that a low level of SorCS3 transcripts in low-grade glioma and high-grade glioma tissues was associated with shorter overall survival times in patients (Fig. 1C and 1D). In contrast to low expression, high expression of SorCS3 was significantly correlated with better survival. Taken together, these data demonstrated that SorCS3 is significantly downregulated in glioma, suggesting that it may function as a tumour suppressor.
SorCS3 inhibits glioma cell migration, invasion and proliferation in vitro.
To investigate the biological effects of SorCS3 in glioma progression, and considering that SorCS3 has not previously been studied in glioma cells, we first examined cellular SorCS3 levels in a quantitative manner. As shown in Fig. 2A, western blot analysis showed that SorCS3 expression was significantly decreased in the U251 cell line compared to the other glioma cell lines tested (U87, A172, and T98). Next, we performed cell proliferation, invasion and migration, apoptosis, and cell cycle assays in two glioma cell lines (U87 and U251) after transfection with the pCMV3-SorCS3-Flag plasmid (Fig. 2B). Further, we examined the effect of SorCS3 on the invasion and migration abilities of glioma cells. In vitro wound healing and Transwell assays demonstrated that overexpression of SorCS3 reduced the migratory and invasive potential of tumour cells (Fig. 2C-F). In addition, SorCS3 overexpression reduced the proliferation of glioma cells, as determined by an EdU incorporation assay (Fig. 2G) and a plate colony formation assay (Fig. 2H). Given that SorCS3 regulates cell proliferation, we examined whether SorCS3 regulates cell cycle progression or cellular apoptosis. We found that SorCS3 expression in U251 cells decreased the number of cells in S phase but increased the number of cells in G0/G1 phase (Supplementary Fig. S1A). However, overexpression of SorCS3 had no effect on apoptosis (Supplementary Fig. S1B). These findings showed that SorCS3 significantly attenuates the growth of glioma cells in vitro.
Hence, we examined the effects of SorCS3 on the expression of EMT and proliferation markers. Western blot analysis showed that in SorCS3-overexpressing glioma cells, the protein expression of the epithelial marker E-cadherin was upregulated, while the expression of the mesenchymal marker vimentin as well as the transcriptional repressors Snail and PCNA was markedly decreased (Fig. 2I). These results suggested that SorCS3 suppresses the proliferation and invasion of glioma cancer cells, functioning as an inhibitor of glioma development.
To verify the tumour suppressor roles of SorCS3, we first silenced SorCS3 expression using two siRNA constructs (si-SorCS3-1# and 2#) in U251 and U87 cells (Fig. 3A-B). Knockdown (KD) of SorCS3 significantly increased the invasion and migration of glioma cells compared to that of control cells in the Transwell assay (Fig. 3C-D and Supplementary Fig. S2A-B). Accordingly, we observed similar patterns in the wound healing assay (Fig. 3E-F and Supplementary Fig. S2C-D). Similarly, we found that knockdown of SorCS3 significantly enhanced the proliferation of glioma cells (Fig. 3G-H and Supplementary Fig. S2E). Upregulation of SorCS3 resulted in a reduction in glioma cell proliferation, which was blocked by si-SorCS3. In contrast, silencing SorCS3 resulted in obvious promotion of cell proliferation. Further, western blot analysis showed that in SorCS3-KD glioma cells, the protein levels of the EMT markers E-cadherin and Vimentin were significantly changed, while those of the transcriptional repressors Snail and PCNA were markedly increased (Fig. 3I). In summary, we concluded that SorCS3 functions as a tumour suppressor gene in glioma cells in vitro.
SorCS3 and p75NTR have a negative regulatory relationship.
The VPS10p domain receptor SorCS3 has been implicated in several pathways of protein internalization and sorting between the plasma membrane and endosomes. Although findings from an earlier report indicate that SorCS3 binds NGF and that NGF is the driving force of glioma cell progression [9], the functional implications of these interactions are poorly understood [11]. In addition, NGFR (TrkA and p75NTR) has been verified to play an important role in glioma progression. We speculated that SorCS3 is involved in the impact of increased tumour-promoting behaviour upon NGF-induced NGFR internalization. First, we explored the specific ligand-induced internalization ability of SorCS3 in glioma cell lines. Fluorescence microscopy revealed that the overexpression of SorCS3 on the cell membrane surface increased the internalization of albumin. Moreover, silencing SorCS3 suppressed the specific ligand-induced internalization ability and decreased the appearance of bright fluorescent puncta on the cell membrane surface (Fig. 4A-B). Next, we validated the correlation between the SorCS3 and NGFR levels after transfection with the SorCS3 overexpression plasmid or SorCS3 siRNA. Our findings showed that the SorCS3 and p75NTR levels were negatively related but that the SorCS3 and TrkA levels were not significantly related (Fig. 4C). Therefore, we analysed the correlation between the mRNA levels of SorCS3 and p75NTR. Interestingly, the results showed a weak negative correlation between these levels (Fig. 4D). Therefore, there might be another stronger regulatory relationship between SorCS3 and p75NTR. Previous studies have shown that p75NTR dramatically enhances the migration and invasion of genetically distinct glioma cells and frequently exhibits robust expression in specimens from patients with highly invasive glioblastoma [12]. We verified the biological significance of p75NTR in glioma. Kaplan-Meier analysis indicated that high expression of p75NTR was negatively correlated with the overall survival time of glioma patients (Fig. 4E). Further, we examined the expression of p75NTR in glioma samples of different grades by tissue microarray analysis (Fig. 4F). Taken together, these data indicated that SorCS3 regulates the expression of p75NTR in glioma.
SorCS3 interacts with p75NTR on the plasma membrane.
Intrigued by the co-internalization of SorCS3 and p75NTR, we generated a SorCS3-Flag fusion protein and then performed a set of Co-IP assays to investigate whether SorCS3 and p75NTR interact. First, we used an anti-Flag antibody to pull down p75NTR, and as shown in Fig. 4G, SorCS3 and p75NTR may interact. However, there seemed to be no interaction between SorCS3 and TrkA. Second, we found that endogenous p75NTR and SorCS3 co-precipitated in U87 cells, indicating that SorCS3 and p75NTR may exist in the same protein complex (Fig. 4H). Next, we sought to examine the subcellular localization of endogenous p75NTR and SorCS3 in U87 cells with high levels of SorCS3 expression. In U87 cells, endogenous SorCS3 colocalized with p75NTR (Fig. 4I). In addition, a portion of SorCS3 was demonstrated to have a distict overlap with EEA1 and Rab5, indicating its localization in early endosomes. Previous studies have indicated that p75NTR is mainly confined to the plasma membrane and overlaps with endosomes during internalization and sorting [13]. We speculated that p75NTR may be involved in the internalization process regulated by SorCS3.
Taken together, these findings indicated that SorCS3 co-precipitates with p75NTR. However, at this point, we do not know whether SorCS3 interacts directly with p75NTR or whether they are coupled through a molecular complex or an intermediary partner.
NGF stimulation promotes the interaction between SorCS3 and p75NTR.
We then sought to investigate the mechanism underlying the SorCS3-p75NTR interaction. We stimulated U87 human glioma cells with NGF under normal serum conditions, investigated whether NGF stimulation promoted the interaction between p75NTR and SorCS3, and analysed the canonical internalization pathways of SorCS3 in whole-cell lysates (WCLs). In addition, we explored whether the interaction between p75NTR and SorCS3 increases with NGF treatment (Fig. 5A). We speculated that NGF may act as a bridge or linker between P75NTR and SorCS3, forming a complex with these proteins to complete the internalization process. Subsequently, we measured the expression level of p75NTR in SorCS3-overexpressing glioma cells at different time points after NGF treatment. Unexpectedly, in cells with SorCS3 overexpression, the expression of p75NTR gradually decreased with increasing NGF treatment time. Interestingly, the expression level of the lysosomal marker Lamp2 was increased by stress, but the expression of the late endosome marker Rab7 was not changed (Fig. 5B). These results showed that the decreased level of p75NTR may result from its degradation through the lysosomal pathway. As NGF treatment triggered decreased intracellular retention of p75NTR in cells with SorCS3 overexpression, we investigated the subcellular localization of SorCS3 in U87 cells. Under NGF treatment conditions, immunofluorescence experiments showed that the overlap of SorCS3 with p75NTR and RAB5 was increased (Fig. 5C). Subsequently, we investigated whether overexpression of SorCS3 can impair the canonical p75NTR signalling pathway in cells treated with NGF. Overexpression of SorCS3 impaired p75NTR signalling, as evidenced by the decreased phosphorylation of both AKT and ERK1/2. After treatment with NGF, the inhibitory effect on the p75NTR signal was enhanced to some extent. In addition, SorCS3-depleted cells exhibited sustained p75NTR signalling (Fig. 5D).
Previous studies have shown that NGF is required for glioma cell invasion and proliferation [8]. To verify the role of NGF in SorCS3-mediated inhibition of glioma, we analysed the biological functions of metastasis and proliferation in SorCS3-overexpressing glioma cells treated with NGF (Fig. 6A-B). Under normal conditions, NGF treatment promoted the invasion and metastasis of glioma cells. However, when SorCS3 was overexpressed, the effect of NGF was blocked in two glioma cell lines (U87 and U251). Taken together, these results suggested that SorCS3 can weaken the cancer-promoting effect of NGF and that NGF can enhance the SorCS3-regulated internalization of p75NTR.
Inhibition of internalization abolishes the tumour-suppressive ability of SorCS3.
We then sought to investigate the underlying mechanism by which SorCS3 inhibits glioma. We inhibited endocytosis using Dynasore, a cell internalization inhibitor. To prevent the inhibition of Dynasore internalization, we also added NGF to promote internalization mediated by SorCS3. In Dynasore-treated cells, the inhibitory effect of SorCS3 on migration and invasion was abolished (Fig. 7A). As expected, in the EdU incorporation assay, the number of proliferating cells did not change significantly (Fig. 7B). Subsequently, we investigated whether suppression of internalization impairs the SorCS3-p75NTR signalling pathway. Interestingly, in cells with SorCS3 overexpression, the decrease in the p75NTR level was alleviated by the inhibition of internalization. In addition, blockade of internalization suppressed the change in p75NTR signalling, as evidenced by the unchanged phosphorylation of both AKT and ERK1/2 (Fig. 7C). To further verify that the biological function of SorCS3 in suppressing cancer is achieved through the NGF/p75NTR signalling pathway, we selected the competitive NGF inhibitor Ro 08-2750 to inhibit binding between endogenous NGF and p75NTR. As a result, we found that Ro 08-2750 restored the influence of SorCS3 on the downstream p75NTR signalling pathway. Taken together, these findings indicated that inhibiting the internalization of or the binding between NGF and SorCS3 interfered with the tumour suppressor function of SorCS3.
SorCS3 downregulation is associated with poorer prognosis.
Our results obtained from animal models revealed that overexpression of SorCS3 is associated with inhibited cellular proliferation and retarded tumour growth (Fig. 8A). To determine whether this phenomenon is also relevant to human glioma, we performed immunohistochemical analysis on glioma tissue microarrays containing samples from 108 patients. We analysed SorCS3 expression according to grade, with grades I–IV corresponding to well-differentiated to moderately differentiated tumours. Low levels of SorCS3 protein were found to positively correlate with greater tumour differentiation in the tissue microarray analysis (Fig. 8B). These results demonstrate that SorCS3 acts as a novel tumour suppressor and is negatively correlated with the degree of glioma malignancy.