WNT5A promotes stemness characteristics in nasopharyngeal carcinoma cells leading to metastasis and tumorigenesis

Abstract

Nasopharyngeal carcinoma (NPC) has the highest metastasis rate among head and neck cancers with unclear mechanism. WNT5A belongs to the WNT family of cysteine-rich secreted glycoproteins. Our previous high-throughput gene expression profiling revealed that WNT5A was up-regulated in highly metastatic cells. In the present study, we first confirmed the elevated expression of WNT5A in metastatic NPC tissues at both the mRNA and protein levels. We then found that WNT5A promoted epithelial-mesenchymal transition (EMT) in NPC cells, induced the accumulation of CD24-CD44+ cells and side population, which are believed to be cancer stem cell characteristics. Moreover, WNT5A promoted the migration and invasion of NPC cells in vitro, while in vivo treatment with recombinant WNT5A promoted lung metastasis. Knocking down WNT5A diminished NPC tumorigenesis in vivo. When elevated expression of WNT5A coincided with the elevated expression of vimentin in the primary NPC, the patients had a poorer prognosis. Among major signaling pathways, protein kinase C (PKC) signaling was activated by WNT5A in NPC cells. A positive feedback loop between WNT5A and phospho-PKC to promote EMT was also revealed. Taken together, these data suggest that WNT5A is an important molecule in promoting stem cell characteristics in NPC, leading to tumorigenesis and metastasis.

Keywords: PKC; WNT5A; metastasis; nasopharyngeal carcinoma; tumorigenesis.

Figure 1. Elevated WNT5A expression in metastatic NPC tissues

A, IHC staining of WNT5A in primary NPC tissues as well as in pulmonary metastatic NPC tissues. B, WNT5A mRNA expression in the pulmonary metastatic NPC tissues. C, The relative levels of WNT5A mRNA expression in different human tissues measured using quantitative real time-PCR. Fold change (y-axis) represents the relative expression of the gene in different cancerous tissues compared with the level of WNT5A mRNA expression in nasopharyngeal (NP) mucosa, normalized to GAPDH gene expression. The highest WNT5A mRNA level was found in the hepatic metastasis, followed by lymph node metastasis.

Figure 2. WNT5A promotes migration, invasion, and metastasis of NPC cells

A and B, Overexpression of both WNT5A mRNA (A) and protein (B) was achieved in low-metastasis S26 cells by transfection of a WNT5A expression vector. C, The migration and invasion of S26 cells was significantly promoted by WNT5A overexpression. D and E, Knocking down WNT5A in S18 cells resulted in reduced WNT5A mRNA (D) and protein (E). F, The migration and invasion of S18 cells was significantly inhibited by knocking down WNT5A. G, Histological image of a lung metastasis in a nude mouse after tail vein injection of S26 cells. H, The wet lung weights of the mice treated with rWNT5A were significantly higher, suggesting more metastases into the lung. I, The largest diameters of the pulmonary metastatic nodules in the mice treated with rWNT5A were also significantly larger, suggesting that rWNT5A promoted the metastasis of NPC cells.

Figure 3. WNT5A regulates stem-like cell markers in NPC cells

A, Flow cytometry analyses showed that stable knocking down WNT5A in S18 cells could significantly decrease the percentage of CD24-/CD44+ cells, which are believed to possess stemness characteristics. Over-expression of WNT5A in S26 cells by transfecting expression vector could significantly increase the percentage of CD24-/CD44+ cells. B and C, Comparisons of the detected values from the experiments shown in A. D, Side population analyses showed that over expression of WNT5A in S26 cells resulted in significant increase of the side population. E, the images of flow cytometry for side population analysis. FTC, fumitremorgin C, an inhibitor of ABCG2 protein. *, P < 0.01.

Figure 4. WNT5A promotes EMT in NPC cells, and this procedure associates with poor patient survival

A, Knocking down WNT5A in high-metastasis S18 and 5-8F cells resulted in reduction of mesenchymal proteins vimentin, N-cadherin, and fibronectin, as well as increment of epithelial protein desmoplakin and E-cadherin, whereas over-expression of WNT5A in the low-metastasis S26 and SUNE-1 cells resulted in accumulation of mesenchymal proteins and reduction of epithelial proteins. B, IHC staining of vimentin in NPC tissue. C, By combining the IHC scores of vimentin shown in B with the WNT5A IHC score shown in Figure 1A, survival analyses were performed in a cohort of 220 patients. Shorter disease-free survival (DFS) and relapse-free survival (RFS) were observed in the patients with primary NPC expressing high levels of both WNT5A and vimentin proteins.

Figure 5. WNT5A activates PKC signaling

A positive loop between WNT5A and phospho-PKC promotes EMT in NPC cells. A, By using SUNE-1 cells with WNT5A over-expression as well as 5-8F cells with WNT5A knocked-down, several major signaling pathways were examined, including PKC, ERK, AKT, and JNK pathways. Only phosphorylation of PKC was altered by WNT5A expression in the cells. B, Over-expression or knocking down of WNT5A did not alter the protein level of β-catenin in the nucleus, indicating that the classic β-catenin signaling pathway could not be activated by WNT5A in NPC cells. C, Over-expression of WNT5A increased phospho-PKC level, while knocking down WNT5A resulted in reduction of phosphorylated PKC. D, PKC activator PMA could successfully induce phosphorylation of PKC in S26 cells. E, Activation of PKC by PMA in S26 cells could further up-regulate WNT5A level and subsequently increase mesenchymal marker Snail and decrease epithelial marker E-cadherin. F, PKC inhibitor GF10923X could decrease WNT5A level in S18 cells and subsequently reduce Snail and gain E-cadherin.

Figure 6. A schematic model to summarize WNT5A signaling in NPC

WNT5A activates PKC signaling, subsequently promotes EMT and the presentation of other stem cell properties, resulting in NPC metastasis and tumorigenesis. A positive loop between WNT5A and phospho-PKC is an ideal therapeutic target.