Wnt3a promotes radioresistance via autophagy in squamous cell carcinoma of the head and neck

Abstract The canonical Wnt/β‐catenin signalling pathway and autophagy play critical roles in cancer progression. However, the role of Wnt‐mediated autophagy in cancer radioresistance remains unclear. In this study, we found that irradiation activated the Wnt/β‐catenin and autophagic signalling pathways in squamous cell carcinoma of the head and neck (SCCHN). Wnt3a is a classical ligand that activated the Wnt/β‐catenin signalling pathway, induced autophagy and decreased the sensitivity of SCCHN to irradiation both in vitro and in vivo. Further mechanistic analysis revealed that Wnt3a promoted SCCHN radioresistance via protective autophagy. Finally, expression of the Wnt3a protein was elevated in both SCCHN tissues and patients' serum. Patients showing high expression of Wnt3a displayed a worse prognosis. Taken together, our study indicates that both the canonical Wnt and autophagic signalling pathways are valuable targets for sensitizing SCCHN to irradiation.

which is important for the discovery and development of novel radiation sensitizers for cancer radiotherapy, are urgently needed.
Wnt signalling is essential for stem cell regulation during development and tissue homoeostasis. 3 Mutated components in the Wnt pathway cause multiple growth-related pathologies and cancer. 4 In the canonical Wnt/β-catenin signalling pathway (hereafter referred to as the Wnt signalling pathway), diverse Wnt ligands bind to Frizzled receptors and low-density lipoprotein-related protein 5/6 co-receptors, stabilizing β-catenin protein by inhibiting the function of the protein destruction complex constituted by adenomatous polyposis coli, Axin, casein kinase 1 and glycogen synthase kinase 3. Stabilized β-catenin enters the nucleus and replaces T cell factor-associated co-repressors Groucho with coactivators such as TCF/LEF, leading to the transcriptional activation of β-catenin target genes such as the Cyclin D1, c-Myc and Survivin genes. 3,4 The activated Wnt signalling pathway up-regulates multiple genes such as aldehyde dehydrogenase, 5 DNA ligase 4, 6 high mobility group box 1 7 and functions critically in cancer malignant behaviours including radioresistance. However, the underlying molecular mechanisms of Wnt signalling-mediated radioresistance remain unclear.
Autophagy is a dynamic cellular metabolism process that depends on the lysosome to reutilize proteins and damaged organelles. 8 Autophagy plays a protective role in cells and tissues under stress conditions. Irradiation induces autophagy to sustain the survival of cancer cells, thereby contributing to treatment resistance and recurrence. 9 Inhibiting the autophagic response using specific inhibitors or by targeting autophagy-associated genes has shown efficacy for sensitizing cancer cells to radiotherapy. 10 Multiple clinical trials targeting autophagy as part of a combination therapy strategy in diverse cancers are undergoing and have shown encouraging results, highlighting the importance of clarifying the regulatory mechanisms of autophagy and accelerating the discovery of molecular targets for selective and specific inhibition aimed at autophagy. 11 However, the role of Wnt-mediated autophagy in cancer radioresistance is unclear. In this study, we demonstrated that irradiation induced the activation of both the Wnt/β-catenin and autophagic signalling pathways in SCCHN cells. Wnt3a-mediated activation of the Wnt/β-catenin signalling pathway and autophagy decreased the sensitivity of SCCHN cells to irradiation both in vitro and in vivo.
Further analysis of the mechanism revealed that Wnt3a promoted SCCHN radioresistance by promoting protective autophagy. Finally, the expression of Wnt3a protein was elevated in both SCCHN tissues and patients' serum. Patients with high expression of Wnt3a displayed a worse prognosis. Taken together, our results indicate that both the canonical Wnt and autophagic signalling pathways are valuable targets that sensitize SCCHN to irradiation.

| Cell culture
The human SCCHN cell line Tu686 was kindly provided by Dr Zhuo Georgia Chen (Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA). 12 The nasopharyngeal carcinoma cell line 6-10B was purchased from the Cell Center of Central South University (Changsha, China). As we previously described, gradually increased doses of irradiation were administered to nasopharyngeal carcinoma 6-10B cells to screen and establish 6-10B cells with enhanced radioresistant capacity (abbreviated as 6-10B-Rs). 13,14 Tu686 cells were maintained in DMEM/F12 (1:1). 6-10B and 6-10B-Rs cells were cultured in RPMI1640 medium. All media were supplemented with 10% foetal bovine serum, 100 IU/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified atmosphere with 5% CO 2 . Cell lines were routinely excluded for mycoplasma contamination and cells in the exponential phase were used in the following experiments.

| Wnt3a overexpression or Beclin1 knockdown in SCCHN cells
To overexpress Wnt3a, full-length human Wnt3a cDNA was amplified by PCR and then cloned into the pLV lentiviral plasmid (Addgene, Watertown, MA, USA). An empty vector was used as a control. For

| Irradiation
Irradiation (IR) was delivered at room temperature using a 6-MeV electron beam generated by a linear accelerator (2100EX, Varian Medical Systems, Palo Alto, CA, USA) at a dose rate of 300 cGy/min. Compensation glue (1.5-cm thick) was used to cover the cell culture containers. The source-to-skin distance was 100 cm.

| Immunoblotting assays
Protein from tumour cells, xenograft samples and human SCCHN samples was extracted using RIPA buffer (10 mmol L −1 Tris-Cl (pH 8.0), 1 mmol L −1 EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mmol L −1 NaCl, 1 mmol L −1 PMSF) supplemented with inhibitors of protease and phosphatase. Western blotting assays were then performed as we described previously. [14][15][16] Briefly, protein samples were separated in 8%-12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). Blotted membranes were incubated with primary antibodies at 4°C overnight or for 2 hours at room temperature and then secondary antibodies for 1 hours at room temperature. GAPDH was used as a loading control. Protein bands were visualized using enhanced chemiluminescence reagents and images were captured with Image Lab 4.1 (Bio-Rad, Hercules, CA, USA). Each experiment was performed in duplicate or triplicate. The antibody working concentration, catalogue number, company source and reaction species are listed in Table S1.

| Plate clonogenic survival assays
Radioresistance was measured by clonogenic survival assay following exposure to different doses of irradiation. Briefly, 300-600 cells were seeded into 6-well plates and exposed to specific doses of irradiation. After irradiation, the cells were cultured for another 12-14 days and the number of surviving colonies (defined as a colony with > 50 cells) was counted. The survival fraction was calculated as we previously described. 14,17 Each experiment was performed in duplicate or triplicate.

| Treatment with small molecular compounds
The small synthetic molecules 3-methyladenine (3MA) (TargetMol Corp., Boston, MA, USA) and recombinant human Wnt3a protein (rhWnt3a) (Abnova, CA, USA) were used to inhibit autophagy or activate the Wnt/β-catenin signalling pathway. 3MA blocks autophagy via inhibiting the PI3K class III. SCCHN Tu686 and 6-10B cells from different groups were treated with 800 µM 3MA and 20 ng/mL rh-Wnt3a for the indicated times and then subjected to a clonogenic survival assay, Western blotting assay and immunofluorescence staining.  using the modified ellipse formula (volume = length × width 2 /2).

| Xenograft tumour model
Two weeks after the first irradiation exposure, all mice were killed.
Xenograft tumours in all groups were removed and the final individual weight of each tumour was measured. Each tumour sample was cut into two parts and fixed with formaldehyde or stored in nitrogen. Tumour samples were used for haematoxylin and eosin staining, Western blotting assay and immunostaining.

| SCCHN patient information and tissue preparation
One hundred thirty-eight paraffin-embedded SCCHN patient samples from the Department of Otolaryngology in Xiangya Hospital

| Haematoxylin and eosin staining, immunohistochemistry and quantification
Four-micrometre thick paraffin-imbedded tumour sections were initially stained with haematoxylin and eosin to confirm the tumourigenesis of SCCHN cells in vivo. All immunohistochemistry staining was performed as described in our previous studies. [19][20][21][22] Briefly, human SCCHN and xenograft sections were stained with the indicated primary antibodies, followed by sequential incubation of secondary antibody and diaminobenzidine. Slides were incubated with immunoglobulin G rather than primary antibodies as negative controls.

| Irradiation activates Wnt signalling pathway and induces autophagy in SCCHN
Our previous RNA-sequencing data revealed that radioresistant 6-10B-Rs cells displayed differentially expressed genes compared to the parent 6-10B cells. 14  We also investigated the alterations in the autophagic cascades.
As shown in Figure 1B, autophagic signalling proteins including   Figure 2D and 2), indicating an enhanced capacity to repair DNA double-strand breaks, which is in accordance with the enhanced radioresistance in Tu686 and 6-10B cells ( Figure 2B and 2). Taken together, these data indicate that Wnt3a induces autophagy and enhances SCCHN radioresistance in vitro.

| Autophagy confers radioresistance in SCCHN
Autophagy functions in a dual manner in cancer malignant progression. 8,9 To confirm the precise protective role of autophagy in SCCHN radioresistance, the expression of Beclin1 was inhibited in 6-10B-Rs and Tu686 cells ( Figure 3A  Immunofluorescence staining was conducted to quantify γH2AX foci in 6-10B-Rs (C) and Tu686 (F) cells. SCCHN 6-10B (G-I) and Tu686 (J-L) cells were forced to express Beclin1 and then proteins associated with the Wnt and autophagy signalling pathways were examined by Western blotting assays (G, J). Clonogenic assays were conducted to determine the survival fraction of 6-10B (H) and Tu686 (K) cells. Immunofluorescence staining was used to quantify γH2AX foci in 6-10B-Rs (I) and Tu686 (L) cells. *P < 0.05; **P < 0.01; ***P < 0.001 and LC3B ( Figure 3A and 3), but showed no effect on the expression of β-catenin, c-Myc and Survivin ( Figure S3A), indicating that Beclin1-mediated autophagy did not regulate the Wnt signalling pathway. More importantly, Beclin1 knockdown re-sensitized Tu686 and 6-10B-Rs cells to irradiation ( Figure 3B and 3; Figure   S3B), which was accompanied by a significantly increased number of γH2AX ( Figure 3C and 3; Figure S3C). In contrast, without the influence on the Wnt signalling pathway, Beclin1 overexpression in Tu686 and 6-10B cells activated the autophagic proteins Atg3, Atg5, Atg7, Atg12 and LC3B, inhibited the formation of γH2AX and led to SCCHN radioresistance ( Figure 3G-L; Figure S3B and C). Consistent with the data obtained from Beclin1 modulation, the autophagy inhibitor 3MA also restored the radiosensitivity of 6-10B-Rs ( Figure S4A and 4B) and Tu686 ( Figure S4D and E) in vitro, while 3MA did not regulate the expression of Beclin1 or proteins associated with the Wnt signalling pathway ( Figure S4C and F).
To further confirm the above results in vivo, 6-10B-Rs cells infected with control and Beclin1 shRNA were subcutaneously injected into the flanks of nude mice. Mice were consecutively subjected to 4 Gy irradiation exposure twice and killed at 28 days after cancer cell injection ( Figure 4A). Without radiotherapy, Beclin1 knockdown had no effect on the proliferation of 6-10B-Rs cells in vivo ( Figure 4B-D). However, Beclin1 knockdown sensitized 6-10B-Rs cells to a total of 8 Gy radiotherapy ( Figure 4B-D). Western blotting assays and immunohistochemistry (IHC) staining showed that Beclin1 and LC3B were efficiently inhibited in xenograft tumours ( Figure 4E and 4). Taken together, our data indicate that inhibition of Beclin1-mediated autophagy improves the therapeutic efficiency of radiotherapy in SCCHN.

| Wnt3a enhances radioresistance via autophagy
The above data clearly reveal that both the Wnt and autophagic signalling pathways contribute to SCCHN radioresistance. Therefore, we examined whether the Wnt pathway promotes radioresistance by modulating autophagy. To evaluate this, 6-10B and Tu686 cells transfected with Wnt3a cDNA were treated with 3MA or left untreated and then the changes in radioresistance were evaluated. As shown in Figure 5A and D, 3MA blocked Wnt3a-induced up-regulation of LC3B. More importantly, 3MA also reversed the decreased γH2AX and radioresistance caused by rhWnt3a ( Figure 5B, C, E, and F;  Figure 6A). We found that Wnt3a improved the survival advantage radiotherapy-treated mice compared to in mice without irradiation exposure ( Figure 6B-D). Similar to the in vitro results, 3-MA effectively impeded the up-regulation of Beclin1 ( Figure 6E) and significantly restored radiosensitivity in Wnt3a-overexpressing 6-10B cells ( Figure 6B-D). Collectively, these data reveal that Wnt3a-mediated activation of the canonical Wnt signalling pathway promotes SCCHN radioresistance via protective autophagy.

| Clinical relevance of Wnt3a and Beclin1 in SCCHN patients
Finally, IHC was conducted to evaluate the expression of Wnt3a and then correlated with clinicopathological parameters in 138 patients with SCCHN. As shown in Figure 7A and B, Wnt3a and Beclin1 expression levels were positively correlated in SCCHN tissues. Wnt3a expression was positively correlated with lymph node metastasis and clinical stages and negatively associated with poor prognosis ( Figure 7C; Table 1), indicating their potential value in the surveillance of cancer progression and prognosis. Wnt3a, a secretory protein, can be detected in patient serum. Therefore, serum Wnt3a was also quantified in patients with SCCHN. Although we found that serum Wnt3a levels in patients with SCCHN were much higher than those in healthy donors ( Figure 7D) and its expression level was not correlated with multiple clinical parameters (Table S3). Beclin1 was mildly increased in patients with SCCHN at late clinical stages, however, we did not determine the clinical significance of Beclin1 in patients with SCCHN ( Figure 7A and B; Table S2). Univariate Cox regression analyses determined that T classifications, clinical stages and Wnt3a protein levels were significantly associated with overall survival status in patients with SCCHN. However, only Wnt3a expression was an independent prognostic factor via multivariate Cox regression analyses (Table 2). Thus, our clinical data suggest that Wnt3a not only impacts SCCHN radioresistance, but also has potential value for the surveillance of SCCHN progression and prognosis.

| D ISCUSS I ON
In the current study, SCCHN cells with high activity of the Wnt signal- Irradiation exposure enriches cancer stem cell-like progenitor cells when the Wnt signalling pathway is highly activated. 24 The Wnt pathway can induce both epithelial-mesenchymal transition and cancer stem cell-like properties, playing a key role in cancer malignant proliferation and therapy resistance, 25,26 which also occurs in patients with SCCHN. 27 Thus, the Wnt pathway may contribute to cancer radioresistance by inducing the epithelial-mesenchymal transition and maintaining cancer stem cell-like properties, which requires future verification in SCCHN radioresistance. In contrast, autophagy inhibition has been shown to improve cancer radiosensitivity in preclinical investigations. 11 However, whether the mutual interaction between the Wnt and autophagic signalling pathways and whether this interaction can cooperate in cancer radioresistance remains unknown.
The Wnt signalling pathway was recently reported to suppress the autophagic pathway in cancer cells. 28,29 In osteosarcoma cells, the Wnt/β-catenin pathway inhibits autophagy and then rescues gemcitabine resistance in vitro. 29 Similarly, β-catenin inhibition using siRNA blocks the Wnt signalling pathway and represses stress-induced autophagy in colorectal carcinoma. 28    vessel-and tumour cell-derived Wnt3a, which easily enters the circulating blood system. This is not the case in SCCHN, which is relatively absent of tumour angiogenesis. 42 Clinically, Beclin1 has been reported to be positively or negatively correlated with a poor or better survival status in distinct solid malignancies. [43][44][45] In this study, Beclin1 was found to act as an oncogene to promote radioresistance in patients with SCCHN. However, although expression of Beclin1 was mildly increased in patients with SCCHN at advanced clinical stages, no significant clinical associations were observed in our patient cohort.
Probability prediction of successful radiotherapy is very important for personalized treatment of patients with SCCHN. The current risk stratification system for patients with SCCHN is mainly based on tumour-node-metastasis stage, which is important for making clinical treatment strategy decisions, but this system must be improved.
Reliable indicators of the response of patients with SCCHN to radiotherapy are urgently needed. Retrospective clinical investigations of numerous cancers demonstrated that potential biomarkers in SCCHN samples are important tools for predicting radiotherapy outcome and appropriate treatment selection. 46 Particularly, combined with functional and molecular investigations of these biomarkers, these studies provide a foundation for the discovery of molecular targets for managing SCCHN radioresistance. Our data clearly indicate that Wnt3a is a useful biomarker for the surveillance of SCCHN progression and a potential target for resensitizing SCCHN cells to irradiation. Therefore, evaluating Wnt3a protein may provide valuable information for predicting the prognosis of patients with SCCHN.
Additionally, such information will improve strategies for irradiation sensitization of patients with SCCHN.
In summary, our data indicate that Wnt3a, as a canonical ligand for the Wnt signalling pathway, binds to its receptor on the membrane of SCCHN cells, activates the canonical Wnt signalling pathway, accelerates the nuclear translocation of β-catenin and then enhances the expression of Beclin1. Thus, increased Beclin1 promotes autophagy, which prevents DNA damage and the formation of γH2AX foci following exposure to irradiation, finally inducing radioresistance. This study not only revealed the signalling interaction by which Wnt3a-mediated activation of the canonical Wnt signalling pathway induces protective autophagy and contributes to SCCHN radioresistance, but also provides a clinical opportunity involving a combinational target of the Wnt signalling pathway and autophagy for treating patients with SCCHN with radioresistance. Further studies are needed to determine whether these results can be extended to the management of other solid human cancers. All authors discussed the results and commented on the manuscript.