PKD3 promotes metastasis and growth of oral squamous cell carcinoma through positive feedback regulation with PD-L1 and activation of ERK-STAT1/3-EMT signalling

Oral squamous cell carcinoma (OSCC) has a high incidence of metastasis. Tumour immunotherapy targeting PD-L1 or PD-1 has been revolutionary; however, only a few patients with OSCC respond to this treatment. Therefore, it is essential to gain insights into the molecular mechanisms underlying the growth and metastasis of OSCC. In this study, we analysed the expression levels of protein kinase D3 (PKD3) and PD-L1 and their correlation with the expression of mesenchymal and epithelial markers. We found that the expression of PKD3 and PD-L1 in OSCC cells and tissues was significantly increased, which correlated positively with that of mesenchymal markers but negatively with that of epithelial markers. Silencing PKD3 significantly inhibited the growth, metastasis and invasion of OSCC cells, while its overexpression promoted these processes. Our further analyses revealed that there was positive feedback regulation between PKD3 and PD-L1, which could drive EMT of OSCC cells via the ERK/STAT1/3 pathway, thereby promoting tumour growth and metastasis. Furthermore, silencing PKD3 significantly inhibited the expression of PD-L1, and lymph node metastasis of OSCC was investigated with a mouse footpad xenograft model. Thus, our findings provide a theoretical basis for targeting PKD3 as an alternative method to block EMT for regulating PD-L1 expression and inhibiting OSCC growth and metastasis.


INTRODUCTION
Head and neck squamous cell carcinoma (HNSCC) is the most common malignant tumour of the head and neck. Oral squamous cell carcinoma (OSCC), the most prevalent subtype of HNSCC, is characterised by aggressive invasion, early cervical lymph node metastasis, a high metastasis rate and fast growth. 1,2 Treatment of patients with metastatic OSCC remains a major clinical challenge. Despite recent breakthroughs in tumour therapy research, the 5-year survival rate of patients with OSCC remains~50%. 3 Many tumour patients currently receive immunotherapy, which has been approved for the treatment of recurrent or metastatic HNSCC. Notwithstanding some success, only a few patients respond to this treatment. Therefore, understanding the mechanisms underlying the growth and metastasis of OSCC is crucial to develop new treatment strategies.
Epithelial-mesenchymal transition (EMT) is one of the critical processes that promotes metastasis. 4,5 In the course of EMT, epithelial cells lose polarity, reorganise the actin cytoskeleton and acquire more mesenchymal characteristics, all of which enhance the cells' metastatic ability. EMT is characterised by reduced expression of adherens junction proteins (such as E-cadherin) and increased expression of mesenchymal markers (such as N-cadherin, Snail and Vimentin), which occur via transcriptional and post-transcriptional mechanisms. [5][6][7] Moreover, Snail can regulate the transcriptional repression of the epithelial marker E-cadherin (CDH1) during EMT. 8 Hence, identifying potential EMT blockers in patients with OSCC may pave the way for promising new therapies.
Protein kinase D (PKD) is a member of a class of serine/ threonine protein kinases that modulate numerous biological activities, such as protein transport, cell migration, differentiation, proliferation, apoptosis, EMT and immune regulation. 9-15 PKD3 expression is increased in highly invasive breast and prostate cancers. [9][10][11][12][13][14][15] The concomitant absence of PKD1 in highly invasive breast cancer indicates that while this protein plays an antitumour role, PKD3 may instead promote cancer. Unlike PKD1 and PKD3, PKD2 shows relatively unchanged expression during breast cancer progression. 13,15,16 Moreover, downregulation of PKD3 expression has a greater effect on cell migration than that of PKD2 expression. 12 Our research group obtained similar results in OSCC and found a significant correlation between the nuclear localisation of PKD3 and advanced tumour grade. 14 Increasing evidence shows that PKD3 is involved in the signalling pathways of multiple oncogenes, including extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (AKT), nuclear factor-kappa B (NF-κB), and signal transducer and activator of transcription 1/3 (STAT1/3). 12,13,17 These pathways can regulate EMT as well as tumour cell growth and metastasis. [18][19][20][21][22] EMT may also contribute to the immune escape of tumour cells. 23 ERK1/2, NF-κB and STAT1/3 are also important regulators of PD-L1 expression. 24 Previously, we showed that PKD3 can regulate the expression of PD-L1. In this study, we investigated whether PKD3 participates in EMT of OSCC and examined the contribution of PD-L1 and related signalling pathways to the induction of the EMT phenotype.

RESULTS
PKD3 is overexpressed in OSCC and is closely related to EMT Our previous studies determined that PKD3 can regulate PD-L1 expression. 14 Moreover, PD-L1 is closely related to EMT in HNSCC. 25 To verify whether PKD3 is involved in the regulation of EMT in OSCC, we first analysed the protein levels of PKD3, PD-L1 and EMT markers in OSCC cell lines and tissues using western blot and IHC. As shown in Fig. 1a, b, PKD3 was not only highly expressed in OSCC cell lines but also positively correlated with mesenchymal markers and negatively correlated with the epithelial marker E-cadherin. Moreover, the levels of PKD3, PD-L1 and Vimentin were higher in OSCC tissues than in normal tissues. With advanced tumour grade, the nuclear distribution of PKD3 as well as the levels of PKD3 and Vimentin gradually increased (Figs. 1c and S1a). However, the expression of PKD3 did not correlate with age, sex or race (Fig. S1b). Subsequently, we analysed the expression levels of PKD3 and PD-L1 in normal and HNSCC tissues using the UALCAN database. As shown in Fig. S1c, the levels of PKD3 and PD-L1 were significantly increased in tumour tissues compared to non-tumour tissues. We downloaded the RNA-seq data of the query genes from the UCSC Xena database and obtained the gene expression data of 564 HNSCC specimens to analyse the correlation of PKD3, PD-L1, and EMTrelated markers. In this large dataset, the expression of PKD3 and PD-L1 was positively correlated with that of mesenchymal markers (such as SNAI1, SNAI2, ZEB1, ZEB2 and VIM) and negatively correlated with that of epithelial markers (such as CDH1, CLDN4 and CLDN7; Fig. S1d). In summary, our data indicate that PKD3 may play an important role in the occurrence and development of OSCC and is closely related to its EMT.   Fig. 1a. We employed sh-PKD3-1 and sh-PKD3-2 because they were previously determined to effectively knockdown PKD3. First, we analysed the effect of PKD3 on cell proliferation using colony formation and CCK-8 assays. The CCK-8 assay showed that the growth of PKD3-knockdown cells was much slower than that of control cells. Additionally, statistical analysis showed significant differences from the third day after knockdown (Fig. 2a). After 2 weeks, the colony formation rate of PKD3-knockdown cells was significantly reduced (Fig. S2a). Next, we evaluated the effect of PKD3 on the invasion and metastasis of OSCC cells. Transwell migration assays showed that PKD3-knockdown cells migrated more slowly than did control cells (Fig. 2b). PKD3 silencing also significantly reduced the invasive ability of OSCC cells through a Matrigel-coated membrane (Fig. 2c). Furthermore, we examined the effects of PKD3 knockdown on the expression of epithelial and mesenchymal markers using western blotting. As shown in Fig. S2b, the expression of E-cadherin was increased, while that of mesenchymal markers and PD-L1 was decreased in PKD3knockdown cells. Together, these results indicate that knockdown of PKD3 inhibits the migration, invasion and EMT phenotype of OSCC cells.
Overexpression of PKD3 promotes the growth, migration and invasion of OSCC cells According to the results shown in Fig. 1a, we selected two cell lines with the lowest PKD3 expression, namely, DOKs and Cal-27 cells. After screening, we obtained cells stably overexpressing PKD3 and evaluated their proliferation, invasion and migration.
Growth curve analysis showed that PKD3-overexpressing cells also proliferated much faster than did control cells. DOKs started showing significant differences from the 4th day, while Cal-27 cells started showing significant differences from the 3rd day c Transwell invasion assay for the indicated cell lines. The number of cells passing through the Matrigel per field is shown in the bar graphs; scale bar size = 50 µm. Nuclei were stained with DAPI (blue). Red represents the cytoskeleton labelled with 555 phalloidin. Data are presented as the means ± SD (n = 3). **P < 0.01; ***P < 0.001. PKD3, protein kinase D3; CCK-8, Cell Counting Kit-8; OSCC, oral squamous cell carcinoma ( Fig. 3a). We found that the number of clones formed by PKD3-overexpressing cells increased significantly (Fig. S2c). Transwell assays showed that PKD3 overexpression significantly promoted the migration and invasion of DOKs and Cal-27 cells (Fig. 3b, c). Western blot analysis revealed that in PKD3overexpressing cells, the expression of PD-L1 slightly increased, whereas the expression of epithelial and mesenchymal markers decreased and increased, respectively (Fig. S2d). Collectively, these data demonstrate that PKD3 contributes to regulating EMT of OSCC and that its overexpression promotes the EMT phenotype and metastasis of OSCC.
PKD3 plays an important role in the EMT of OSCC induced by the PD-1/PD-L1 pathway Programmed death ligand-1 (PD-L1) can interact with programmed cell death protein 1 (PD-1) expressed on activated B cells, T cells and natural killer (NK) cells, thus activating the PD-1/ PD-L1 pathway, leading to T cell apoptosis or loss of function and downregulating the anti-tumour response of T cells. 26 However, how activation of the PD-1/PD-L1 pathway affects tumour cells is largely unknown. To answer this question, we stimulated Cal-27 cells with different concentrations of the PD-1 fusion protein for 24 h. As shown in Fig. 4a, b, E-cadherin expression was slightly lower while PKD3 and PD-L1 expression was significantly higher in cells treated with 1, 2 and 5 μg·mL −1 PD-1 fusion protein than those in cells treated with a low concentration (0.5 μg·mL −1 ) of PD-1 or untreated cells. We then examined the activation of PKD3 using Phos-tag SDS-PAGE immunoblots. PKD3 phosphorylation began to significantly increase after treatment with 1 μg·mL −1 PD-1. Intriguingly, stimulation of cells with all four tested concentrations of PD-1 significantly upregulated the expression of N-cadherin, Snail and Vimentin. Moreover, the levels of Snail and Vimentin gradually increased with increasing concentrations of the PD-1 fusion protein. These data suggest that the binding of the PD-1 fusion protein to PD-L1 promotes EMT in OSCC.
Subsequently, we investigated whether PD-1/PD-L1 pathwayinduced EMT in OSCC was dependent on PKD3. For this purpose, we examined the expression of epithelial and mesenchymal markers after 24 h of culture in the presence or absence of 1 μg·mL −1 PD-1 fusion protein. The expression of PD-L1, N-cadherin, Snail and Vimentin was increased after treatment with PD-1; however, knockdown of PKD3 significantly reduced the expression of PD-L1 and mesenchymal markers but increased the expression of E-cadherin (Fig. 4c, d). Taken together, these results show that knockdown of PKD3 can effectively block EMT induced by the PD-1/PD-L1 pathway.
PKD3 regulates EMT and PD-L1 through ERK1/2 and STAT1/ 3 signalling, establishing a positive feedback loop with PD-L1 To further explore the mechanism of PKD3-induced OSCC migration and tumour growth, we silenced PKD3 and PD-L1 in Cal-27 cells using siRNAs. As shown in Fig. 5a phalloidin. Data are presented as the means ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. PKD3, protein kinase D3; OSCC, oral squamous cell carcinoma of PD-L1 was significantly reduced in PKD3-knockdown cells. In these cells, the levels of EMT markers showed the same changes previously observed in sh-PKD3-transfected cells. Additionally, knockdown of PKD3 significantly inhibited the phosphorylation of STAT1/3 at Ser727 but not the phosphorylation of tyrosine residues. Phosphorylation of ERK1/2 was also significantly reduced after PKD3 knockdown. Considering that PKD3 could regulate the expression of PD-L1, which is closely associated with EMT, we speculated that the regulation of EMT by PD-L1 might also involve the ERK and STAT signalling pathways. Interestingly, knockdown of PD-L1 also reduced PKD3 expression. Following PD-L1 knockdown, the expression of mesenchymal markers was significantly reduced, whereas the expression of E-cadherin was slightly increased. Furthermore, knockdown of PD-L1 significantly reduced the phosphorylation of STAT1/3 at serine residues, as well as the phosphorylation, but not expression, of ERK1/2. Double knockdown of PKD3 and PD-L1 inhibited the phosphorylation of ERK1/2 and STAT1/3 on serine residues more effectively than single knockdown of either gene (Fig. 5c). In addition, double knockdown of PKD3 and PD-L1 significantly inhibited the EMT of OSCC, + similar to the single knockdown of PKD3. However, knockdown of neither PKD3 nor PD-L1 could significantly alter NF-κB pathway activity. As ERK1/2 and STAT1/3 play a crucial role in EMT, we analysed their effect on EMT of OSCC cells. Following knockdown of ERK1/ 2 and STAT1/3, the expression of PKD3 and PD-L1 was effectively suppressed in Cal-27 cells. Moreover, after silencing ERK1/2 and STAT1/3, the expression of mesenchymal markers in OSCC cell lines was still significantly inhibited, even upon stimulation with PD-1 fusion protein, whereas E-cadherin expression was only slightly affected ( Fig. 6a-c). Furthermore, the PD-1/PD-L1 interaction significantly increased the phosphorylation of ERK1/2 and STAT1/3; however, the phosphorylation of STAT1/3 at Ser727 was significantly inhibited after ERK1/2 knockdown (Fig. 6d). In conclusion, PKD3 and PD-L1 regulate the phosphorylation levels of ERK1/2 and STAT1/3. Interestingly, ERK1/2 and STAT1/3 in turn can also affect the expression of PKD3 and PD-L1 and thereby regulate EMT in OSCC. These data indicate that PKD3 regulates PD-L1 expression and EMT in OSCC through ERK1/2 and STAT1/3, suggesting a positive feedback mechanism between them.
PKD3 promotes the growth and lymph node metastasis of OSCC in vivo To study the effects of PKD3 on the growth and lymph node metastasis of OSCC in vivo, we injected Cal-27-sh-Con, Cal-27-sh-PKD3-1, or Cal-27-sh-PKD3-2 cells into the footpads of mice to establish a xenograft model. After 4 weeks, the mice were sacrificed, the size of the footpad tumours was measured, and the popliteal lymph nodes were removed and measured. IHC staining revealed that the expression of PD-L1 decreased with knockdown of PKD3 (Fig. 7a). Moreover, tumour size and lymph node metastasis were most pronounced in the Cal-27-sh-Con group and were smaller in the Cal-27-sh-PKD3-1 and Cal-27-sh-PKD3-2 groups (Fig. 7b, d). Since the lentiviral plasmids were labelled with green fluorescent protein (GFP), transfected cancer cells that metastasised into lymph nodes could be identified by IHC staining of GFP (Fig. 7c). Subsequently, we calculated the lymph node metastasis rates in different groups. This rate was highest in the Cal-27-sh-Con group (78%), followed by the Cal-27-sh-PKD3-1 (11%) and Cal-27-sh-PKD3-2 (22%) groups (Table 1). Importantly, the observed difference in metastasis between the control group and the PKD3-knockdown groups was significant, as determined by Fisher's exact test (P = 0.004). Our experiment thus proved that PKD3 can promote the growth and metastasis of OSCC in vivo.

DISCUSSION
OSCC is a common malignant tumour of the head and neck characterised by rapid growth and a high metastasis rate. Increasing evidence suggests that EMT is crucial for tumour progression and metastasis. In the initial stages of metastasis, epithelial tumour cells undergo EMT, leading to loss of intercellular adhesions, enhanced mobility and invasion of surrounding tissues. 5,27 Currently, the treatment of patients with metastatic OSCC remains a major clinical challenge. Therefore, it is urgent to explore the potential mechanism of growth and metastasis of OSCC to determine novel and more effective therapeutic targets.
In this study, we investigated the relevance of PKD3, a poorly understood member of the PKD family, to OSCC. Our research revealed that PKD3 regulates EMT and PD-L1 expression in OSCC through the ERK1/2 and STAT1/3 pathways and establishes a positive feedback loop with PD-L1 to promote tumour growth and metastasis. These findings indicate that PKD3 plays a pivotal role in the progression of OSCC, suggesting PKDs as potential therapeutic targets for the treatment of this type of cancer.
Based on data from the UALCAN database, UCSC Xena, and western blot, PKD3 and PD-L1 are significantly upregulated in OSCC. Previous studies have shown that PD-L1 regulates EMT and promotes tumour growth and metastasis. 23,25,[28][29][30][31][32] Moreover, PD-L1 expression can be modulated by PKD3. 14 Although this evidence implies that PKD3 may be closely related to EMT, no molecular mechanism has previously been reported between the two. Here, we found that PKD3 was positively correlated with mesenchymal markers and negatively correlated with epithelial markers. Downregulation of PKD3 inhibited the invasion, migration and proliferation of OSCC in vitro; reduced tumour growth and metastasis in vivo; and decreased the expression of mesenchymal markers while increasing that of E-cadherin. PKD3 overexpression instead significantly enhanced the ability of OSCC cells to invade, migrate and proliferate; promoted the expression of mesenchymal markers; and inhibited the expression of E-cadherin. IHC and TCGA analysis showed that the expression of PKD3, the frequency and intensity of PDK3 nuclear staining, and the expression of Vimentin gradually increased with advanced tumour grade. These findings support our view that PKD3 can regulate EMT in OSCC and promote tumour growth and metastasis.
PD-L1 interacts with PD-1 expressed by activated T cells, B cells, NK cells, some dendritic cells (DCs) and tumour-associated macrophages, activating the PD-1/PD-L1 pathway. In turn, activation of this pathway can inhibit the anti-tumour function of the same immune cells, thereby reducing anti-tumour immunity. 26,[33][34][35][36] However, the effect of the PD-1/PD-L1 interaction on tumour cells has rarely been reported. Here, we found that the expression of PKD3 and PD-L1 significantly increased after treatment of OSCC cell lines with the PD-1 fusion protein; in parallel, the expression of mesenchymal markers increased, whereas that of epithelial markers decreased, suggesting that the PD-1/PD-L1 pathway can induce EMT in tumour cells. EMT is key to tumour cell remodelling for metastasis. 5 The interaction between tumour cells and tumour-infiltrating lymphocytes induces the expression of PD-L1 in tumour cells through the STAT pathway; in HNSCC, the expression of PD-L1 is closely related to EMT. 25,37,38 These results are consistent with ours, which indicate that, in vivo, PD-1-expressing T cells, B cells, and NK cells interact with OSCC cells, promoting EMT and immune escape. This may also be the cause of early lymph node metastasis in OSCC.

Patients and clinical samples
In this study, a total of 34 patients with OSCC who did not receive radiotherapy or chemotherapy before surgery were enroled, and 26 normal oral epithelial tissues were collected from the West China Hospital of Stomatology, Sichuan University. The patients' clinical information is shown in Supplementary Table S3. All clinical samples were confirmed by pathological examination. This study was approved by the Ethics Committee of West China Hospital of Stomatology, Sichuan University, and all patients gave informed consent.
Western blot Total proteins were extracted for western blot detection according to the method described previously. 14 Protein concentration was measured using the BCA protein assay kit (Beyotime Institute of Biotechnology, China). Approximately 20 μg of protein was separated by SDS-PAGE through 6% or 10% gels and then transferred to PVDF membranes (Bio-Rad, USA). After the PVDF membrane was blocked in 5% skim milk at room temperature for 1 h, the primary antibody was added and incubated overnight on a shaker at 4°C. Membranes were washed with TBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (cat. no. 7076 or 7074; 1:2 000; Cell Signaling Technology, Inc.) for 1-2 h at room temperature. An enhanced chemiluminescence (ECL) substrate kit (Millipore, Inc.) and an automatic gel imaging system (Bio-Rad Laboratories, Inc.) were used to detect the luminescent signal of protein bands. Primary antibodies used for western blotting targeted the following proteins:  1:20 000) from Abcam. Phos-tag SDS-PAGE (Wako, Japan) was performed according to the protocol described previously. 51 The densitometry of the protein bands was quantified using Gel-Pro 32 software (version 3.1, Media Cybernetics, Bethesda, MD, USA).
Immunohistochemical staining All excised OSCC tissues and lymph nodes of mice were fixed with 10% neutral formalin, embedded in paraffin, and cut into 5-μm-thick sections. Immunohistochemical staining was performed as previously described. 14  Cell Counting Kit-8 assay After serial dilution of the cell suspension, the cells were seeded at a density of 3 × 10 3 cells per well in 96-well plates (100 μL per well), and at least three replicate wells were set per group. After 24 h, Cell Counting Kit-8 (CCK-8) was used to detect cell proliferation (DOJINDO, Japan). The test was continued for 6 days; the cells were incubated with CCK-8 solution for 1 h before absorbance measurements. The growth curve was made according to the absorbance at 450 nm.
Cell invasion and migration assay Cell invasion assays were performed using 24-well Transwell chambers (8-μm pore size; BD Bioscience, USA). Cells (1 × 10 5 ) were seeded in the upper chamber, which was coated with 50 μl of Matrigel (5 mg·mL −1 in cold medium, BD Bioscience, USA) and cultured with 1% FBS. Then, 600 μL of high-glucose DMEM supplemented with 10% FBS was added to the lower chamber. After incubation at 37°C for 24 h, the cells were fixed with 4% paraformaldehyde for 15 min. The cells in the upper chamber were removed, which was then washed with PBS three times, and the invading cells were stained with Acti-stain TM 555 phalloidin (Cytoskeleton, Inc. USA) according to the manufacturer's protocol. The chamber membrane was placed on a glass slide, and 10 fields of view were randomly selected under the microscope to take pictures and count stained cells. ImageJ 1.48v software was used to count the number of cells. Each group had three replicates.
Cell migration assays were performed similarly to cell invasion assays but without Matrigel in the upper chamber. The same methods described above were used to fix, stain, photograph and count the cells.
Popliteal lymph node metastasis model The animal experiments were approved by the Ethics Review Committee of the West China Hospital of Stomatology, Sichuan University (permit number: WCHSIRB-D-2017-211). Female BALB/c-nu mice (4-5 weeks old) were purchased from the Transgenic Engineering Mouse Centre of the West China Hospital of Sichuan University. The mice were randomly divided into three groups (Con, sh-PKD3-1 and sh-PKD3-2), with nine mice per group. Cal-27 cells (4 × 10 6 per mouse) were injected subcutaneously into the footpad of the left hind leg. All transfected cells stably expressed GFP. Four weeks later, the nude mice were euthanized. Primary tumours and popliteal lymph nodes were  Statistical analysis Correlation analysis was performed using Origin 2018 software (Origin Lab). Other analyses were performed using GraphPad Prism software (version 6; GraphPad Software, Inc.) using oneway ANOVA followed by Tukey's multiple comparison test. Fisher's exact test was used to analyse the relationship between PKD3 expression and lymph node metastasis. P < 0.05 was considered statistically significant.

CONCLUSION
In conclusion, we determined that the PD-1/PD-L1 interaction could promote EMT in OSCC. Furthermore, we demonstrated that PKD3 regulates PD-L1 expression through the ERK/STAT1/3 pathway and that there is a positive feedback loop between PKD3 and PD-L1 related to driving EMT and promoting the growth and metastasis of OSCC. Our study thus suggests that targeting PKD3 could be an effective strategy to block EMT, providing a new potential avenue for the treatment of OSCC.