High expression of NRP2 is positively associated with worse prognosis in human HNC
To identify the value of NRP2 as a potential biomarker involved in tumorigenesis, we first analyzed the expression levels of NRP2 in normal tissues and nine kinds of human cancer tissues from the UALCAN database. The expression levels of NRP2 were highly expressed in all cancer types, including head and neck squamous cell carcinoma, compared to normal tissues (Fig. 1a). Correlation analysis, based on the cBioPortal dataset, demonstrated that the copy number of NRP2 is correlated with its mRNA levels in HNC samples (Fig. 1b). We further analyzed the mRNA levels of NRP2 based on GEO or TCGA datasets, which demonstrated that NRP2 mRNA levels in HNC tissues were significantly higher than those in normal or margin tissues (Figs. 1c-1e and S1). Further analysis of the CPTAC database comparing normal and tumor tissues showed that the NRP2 protein was present at higher levels in HNC tissues than in normal tissues (Fig. 1f). In contrast, there were no differences in NRP1 mRNA levels between the normal and HNC tissues (Figs. S2a-S2d). These results indicate that NRP2 can be a potential diagnostic marker for patients with HNC. To validate the results, using in silico analyses, we performed IHC staining of tissue samples from 53 OSCC cases, then classified them into low and high NRP2 expression groups. The intensity of NRP2 staining was remarkably higher in OSCC tissues than in the adjacent non-tumor epithelium (Fig. S3). Through multivariable analysis of NRP2 expression and clinicopathological characteristics, we observed that NRP2 expression positively associated with tumor stage, advanced tumor size, and lymph node (LN) metastasis in patients with OSCC (Table 1 and Fig. 1g). Notably, NRP2 was found to be more intense in cases with LN metastasis than in those without a history of LN metastasis (Fig. 1h). Furthermore, the expression pattern image between NRP2 expression and the final scores indicated that high expression of NRP2 significantly associated with LN metastasis in patients with OSCC (Figs. 1i and 1j). Consistently, NRP2 mRNA levels in LN-positive HNC samples, extracted from the GEO dataset, were statistically significant, compared with those in the LN-negative HNC samples (Fig. 1k). KM plotter analysis revealed that HNC patients with high NRP2 expression had a significantly adverse outcome (Fig. 1l). These results suggest that high NRP2 expression in HNC is positively associated with worse prognosis.
Table 1
Association between NRP2 expression and clinicopathological factors of OSCC patients
Variable | No. of cases (N = 53) | NRP2 | P value | Risk ratio | 95% CI |
Low (N = 30) | High (N = 23) |
Age | | | | | | |
< 58 | 27 | 15 | 12 | 0.875 | 1.05 | 0.57–1.94 |
≥ 58 | 26 | 15 | 11 | | 0.95 | 0.51–1.76 |
Gender | | | | | | |
Male | 36 | 21 | 15 | 0.712 | 0.89 | 0.47–1.67 |
Female | 17 | 9 | 8 | | 1.13 | 0.60–2.13 |
Differentiation status | | | | | | |
Well | 36 | 22 | 14 | 0.335 | 0.73 | 0.40–1.35 |
Moderately | 17 | 8 | 9 | | 1.36 | 0.74–2.50 |
Tumor size | | | | | | |
T1 + T2 | 32 | 24 | 8 | 0.001* | 0.35 | 0.18–0.68 |
T3 + T4 | 21 | 6 | 15 | | 2.86 | 1.48–5.52 |
LN metastasis | | | | | | |
Negative | 32 | 23 | 9 | 0.006* | 0.42 | 0.22–0.79 |
Positive | 21 | 7 | 14 | | 2.37 | 1.26–4.46 |
Distant metastasis | | | | | | |
Negative | 52 | 29 | 23 | 1.000 | 1.77 | 0.16–19.93 |
Positive | 1 | 1 | 0 | | 0.56 | 0.05–6.34 |
Stage | | | | | | |
I + II | 24 | 20 | 4 | < 0.001* | 0.25 | 0.10–0.65 |
III + IV | 29 | 10 | 19 | | 3.93 | 1.55–9.99 |
Recurrence | | | | | | |
No | 41 | 21 | 20 | 0.144 | 1.95 | 0.70–5.46 |
Yes | 12 | 9 | 3 | | 0.51 | 0.18–1.43 |
Nrp2 Promotes Proliferation, Motility, And Invasiveness Of Human Hnc Cells
We used NRP2-silencing HNC cells to obtain in vitro evidence supporting the relationship between NRP2 expression and aggressive behaviors in human HNC. First, we examined the expression levels of NRP2 in nine HNC cell lines and in a normal oral keratinocyte (HOK) cell line. The results demonstrated that NRP2 was remarkably overexpressed in all HNC cell lines compared to that in HOK cells (Fig. S4). We chose HSC-4, FaDu, and HN22 from among the HNC cell lines with high NRP2 expression levels to create NRP2-silencing cells. Real-time PCR and immunoblotting analyses verified that both mRNA and protein levels of NRP2 were significantly downregulated in the established NRP2-silencing cells (Figs. 2a and 2b). To define the functional role of NRP2 in the progression and tumorigenic potential of HNC in vitro, we conducted MTS, soft agar, and clonogenic assays using three NRP2-silencing cells. According to the findings, NRP2-silencing cells' proliferation and colony-forming capacities were slower or smaller than those of the respective control group (Fig. 2c-2e). Interestingly, with respect to morphological features, NRP2-silencing cells exhibited an epithelial-like phenotype with a polygonal shape, compared with the control groups, representing a mesenchymal-like phenotype with a spindle shape (Fig. 2f). To examine the role of NRP2 in the aggressive behavior of human HNC in vitro, we evaluated the motility and invasive abilities of NRP2-silencing cells using transwell migration and invasion assays. As shown in Fig. 2g, a significant reduction in migratory or invasive cells crossing the membranes was observed in the NRP2-silencing groups, compared to that in the control groups. These results demonstrated that NRP2 is required for the proliferation, motility, and invasiveness of human HNC.
Nrp2 Regulates Sox2 Expression To Promote Aggressiveness And Csc Properties Of Human Hnc Cells
To identify the molecular mechanism by which NRP2 exhibits aggressive behavior in human HNC in vitro, we performed a signaling explorer antibody array, consisting of 1,358 antibodies. Among them, 32 proteins with a fold shift greater than 1.2 in NRP2-silencing cells were selected (Fig. 3a). A previous study reported that mRNA levels of CSC markers such as Sox2, BMI-1, and Oct4 were upregulated during NRP2 overexpression in breast cancer cells [4]. Indeed, in our hierarchical clustering analysis, we observed a reduction in Sox2 enrichment in the NRP2-silencing group compared to the control group. This result was verified by immunoblotting (Fig. 3b). Next, we analyzed the CPTAC dataset to evaluate the relationship between NRP2 and Sox2 proteins in HNC samples, and the results showed a positive correlation between NRP2 and Sox2 proteins (Fig. 3c). To explore the biological function of Sox2 in human HNC in vitro, the endogenous levels of Sox2 in the two HNC cell lines were knocked down using siRNA. After confirming the reduced endogenous levels of Sox2 in both cell lines (Fig. 3d), we examined the effect of Sox2 knockdown on motility and invasiveness. As per the results, knocking down endogenous Sox2 in human HNC cells resulted in much weaker migratory or invasive capacities than those in the control group (Figs. 3e and 3f). To clarify the contribution of NRP2 expression to CSC stemness in human HNC cells, we performed a sphere-formation assay in NRP2-silencing cells. As shown in Fig. 3g, the sphere-forming abilities of NRP2-silencing cells were reduced compared to those of control cells. In addition, the expression levels of CSC markers, such as Nanog, were commonly reduced in the two NRP2-silencing cells compared with another CSC marker, Oct4 (Fig. S5a). These results demonstrated that Sox2 may be a critical factor in the NRP2 signaling pathway that is involved in the aggressiveness and CSC properties of HNC cells.
Rsk1 Mediates Between Nrp2 And Sox2 During Aggressive Behaviors Of Human Hnc Cells
To determine which protein kinase is an important determinant between NRP2 and Sox2 during the aggressive behaviors of human HNC cells, we examined the data from the signaling explorer antibody array. As shown in Fig. S6, five ribosomal protein kinases, RPS27 (MPS1), RPS6KA1 (RSK1), RPS6KA2 (RSK3), RPS6KA6 (RSK4), and RPS6KB1 (p70S6Kα), were chosen for validation. Of which, only RPS6KA1 (hereafter referred to as RSK1) exhibited a statistically significant decrease in NRP2-silencing cells compared to that in control cells (fold change = 0.56, p = 0.020). Furthermore, immunoblotting analysis verified that NRP2 silencing markedly reduced the expression levels of RSK1 and p-p90RSK (Fig. 4a). To clarify whether RSK1 mediates the relationship between NRP2 and Sox2, we evaluated the effect of the RSK inhibitor BI-D1870 on Sox2 expression. The results showed that BI-D1870 treatment suppressed the expression levels of both p-p90RSK and RSK1 in the two human HNC cell lines, which was accompanied by a significant reduction in Sox2 expression (Fig. 4b). We further examined whether RSK1 is necessary for the aggressive behavior of human HNC cells. After confirming the minimal effect of BI-D1870 on cell viability (Fig. 4c), we observed that BI-D1870 strongly inhibited the migratory and invasive capacities of human HNC cells (Figs. 4d and 4e). Furthermore, BI-D1870 treatment significantly reduced the ability of human HNC cells to form spheres when compared to control cells (Fig. 4f). Consistent with the results from NRP2-silencing cells, only Nanog was significantly decreased in BI-D1870-treated cells (Fig. S5b). These results demonstrate that RSK1 promotes the aggressiveness and CSC properties of human HNC cells, possibly by mediating the interaction between NRP2 and Sox2.
NRP2 is required for proliferation and aggressive behaviors of human HNC cells by activating the RSK1/Sox2 signaling pathway
To determine whether NRP2 is an important molecule for the RSK1/Sox2 signaling pathway, NRP2-overexpressing cells were established using the HSC-2 cell line, which expresses NRP2 at a low level. Immunoblotting analysis showed that the expression levels of p-p90RSK, RSK1, and Sox2 in NRP2-overexpressing cells were significantly higher than those in the control cells (Fig. 5a). We next investigated the tumorigenic potential of NRP2 in human HNC cells using soft agar and clonogenic assays, which revealed that the colony-forming capacity of NRP2-overexpressing cells increased (Figs. 5b and 5c). Furthermore, NRP2 overexpression markedly increased the migratory and invasive abilities of HSC-2 cells (Figs. 5d and 5e). The sphere-forming ability of NRP2-overexpressing cells was greater than that of the control cells (Fig. 5f). Consistently, the protein levels of Nanog were higher in NRP2-overexpressing cells than in the control cells (Fig. S5c). These results indicate that NRP2 promotes proliferation and aggressive behavior of human HNC cells by activating the RSK1/Sox2 signaling pathway.
Zeb1 Is Involved In Emt Progression Of Human Hnc Cells During The Nrp2/rsk1/sox2 Signaling Pathway
Since NRP2-silencing cells exhibited an epithelial-like phenotype, we further investigated the relationship between the NRP2/RSK1/Sox2 signaling pathway and EMT-related proteins. Among the seven EMT-related proteins, including mesenchymal and epithelial markers, the expression of mesenchymal marker Zeb1 was lower in two NRP2-silencing cells than in control cells, whereas the expression of the epithelial marker E-cadherin was higher in NRP2-silencing cells (Fig. 6a). In addition, the expression levels of Zeb1 were effectively reduced in both siSox2-transfected and BI-D1870-treated cells (Figs. 6b and 6c). However, NRP2 overexpression had the opposite effect (Fig. 6d). These results provide evidence that Zeb1 may be a crucial determinant of the NRP2/RSK1/Sox2 signaling pathway for EMT progression in human HNC cells.
Mecf Inhibits The Aggressiveness Of Human Hnc Cells By Suppressing Nrp2/rsk1/sox2/zeb1 Signaling Pathway
To discover the novel NRP2 inhibitor that inhibits aggressive behaviors of human HNC cells, the HNC cells were treated with 20 kinds of algal methanol extracts at the same dose for 48 h. Even though the viability of cells treated with algal methanol extracts did not change, only extract No. 17 remarkably suppressed the expression of NRP2, compared with other extracts (Figs. S7a and S7b). We further verified that the No. 17 extract (hereafter called MECF) decreased the expression levels of NRP2 in the three human HNC cell lines (Fig. 7a). To evaluate whether MECF regulates the motility and invasiveness of human HNC cells, we performed Transwell migration and invasion assays. The results revealed that the migratory and invasive abilities of HSC-4 and HN22 cells following MECF treatment were lower than those of the respective control cells, whereas MECF treatment only decreased the invasive ability of FaDu cells without affecting their migratory capacity (Figs. 7b and 7c). Nonetheless, MECF did not exhibit any noticeable cytotoxicity in the three human HNC cell lines (Fig. 7d). To determine whether MECF treatment regulated the RSK1/Sox2/Zeb1 signaling pathway, we performed immunoblotting analysis. As shown in Fig. 7e, the expression levels of p-p90RSK, RSK1, Sox2, and Zeb1 were significantly reduced in MECF-treated cells compared to the respective control cells. Furthermore, we found that MECF strongly inhibited the sphere-forming ability of human HNC cells (Fig. 7f). In addition, MECF repressed the expression of Nanog and Oct4 in both HNC cell lines (Fig. S5d). These results indicate that MECF can be a potential NRP2 inhibitor that reduces the aggressive behavior of human HNC cells, possibly by inhibiting the RSK1/Sox2/Zeb1 signaling pathway.