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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

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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 International Journal of Oral Science www.nature.com/ijos ARTICLE OPEN 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 Bomiao Cui1, Jiao Chen1, Min Luo 1, Yiying Liu1, Hongli Chen1, Die Lü1, Liwei Wang1, Yingzhu Kang1, Yun Feng1, Libin Huang2 and Ping Zhang1 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 1234567890();,: theoretical basis for targeting PKD3 as an alternative method to block EMT for regulating PD-L1 expression and inhibiting OSCC growth and metastasis. International Journal of Oral Science (2021) ;13:8 https://doi.org/10.1038/s41368-021-00112-w INTRODUCTION regulate the transcriptional repression of the epithelial marker Head and neck squamous cell carcinoma (HNSCC) is the most E-cadherin (CDH1) during EMT.8 Hence, identifying potential EMT common malignant tumour of the head and neck. Oral squamous blockers in patients with OSCC may pave the way for promising cell carcinoma (OSCC), the most prevalent subtype of HNSCC, is new therapies. characterised by aggressive invasion, early cervical lymph node Protein kinase D (PKD) is a member of a class of serine/ metastasis, a high metastasis rate and fast growth.1,2 Treatment of threonine protein kinases that modulate numerous biological patients with metastatic OSCC remains a major clinical challenge. activities, such as protein transport, cell migration, differentiation, Despite recent breakthroughs in tumour therapy research, the proliferation, apoptosis, EMT and immune regulation.9–15 PKD3 5-year survival rate of patients with OSCC remains ~50%.3 Many expression is increased in highly invasive breast and prostate tumour patients currently receive immunotherapy, which has cancers.9–15 The concomitant absence of PKD1 in highly invasive been approved for the treatment of recurrent or metastatic breast cancer indicates that while this protein plays an anti- HNSCC. Notwithstanding some success, only a few patients tumour role, PKD3 may instead promote cancer. Unlike PKD1 and respond to this treatment. Therefore, understanding the mechan- PKD3, PKD2 shows relatively unchanged expression during breast isms underlying the growth and metastasis of OSCC is crucial to cancer progression.13,15,16 Moreover, downregulation of PKD3 develop new treatment strategies. expression has a greater effect on cell migration than that of Epithelial–mesenchymal transition (EMT) is one of the critical PKD2 expression.12 Our research group obtained similar results in processes that promotes metastasis.4,5 In the course of EMT, OSCC and found a significant correlation between the nuclear epithelial cells lose polarity, reorganise the actin cytoskeleton and localisation of PKD3 and advanced tumour grade.14 Increasing acquire more mesenchymal characteristics, all of which enhance evidence shows that PKD3 is involved in the signalling pathways the cells’ metastatic ability. EMT is characterised by reduced of multiple oncogenes, including extracellular signal-regulated expression of adherens junction proteins (such as E-cadherin) kinase 1/2 (ERK1/2), protein kinase B (AKT), nuclear factor-kappa B and increased expression of mesenchymal markers (such as (NF-κB), and signal transducer and activator of transcription 1/3 N-cadherin, Snail and Vimentin), which occur via transcriptional (STAT1/3).12,13,17 These pathways can regulate EMT as well as and post-transcriptional mechanisms.5–7 Moreover, Snail can tumour cell growth and metastasis.18–22 EMT may also contribute 1State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & West China Hospital of Stomatology, Sichuan University, Chengdu, China and 2Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu, China Correspondence: Libin Huang ([email protected]) or Ping Zhang ([email protected]) These authors contributed equally: Bomiao Cui, Jiao Chen, Min Luo. Received: 2 September 2020 Revised: 16 November 2020 Accepted: 5 January 2021 PKD3 promotes metastasis and growth of OSCC Cui et al. 2 to the immune escape of tumour cells.23 ERK1/2, NF-κB and and Vimentin were higher in OSCC tissues than in normal tissues. STAT1/3 are also important regulators of PD-L1 expression.24 With advanced tumour grade, the nuclear distribution of PKD3 as Previously, we showed that PKD3 can regulate the expression of well as the levels of PKD3 and Vimentin gradually increased PD-L1. In this study, we investigated whether PKD3 participates in (Figs. 1c and S1a). However, the expression of PKD3 did not EMT of OSCC and examined the contribution of PD-L1 and related correlate with age, sex or race (Fig. S1b). Subsequently, we signalling pathways to the induction of the EMT phenotype. 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 RESULTS tumour tissues compared to non-tumour tissues. We downloaded PKD3 is overexpressed in OSCC and is closely related to EMT the RNA-seq data of the query genes from the UCSC Xena Our previous studies determined that PKD3 can regulate PD-L1 database and obtained the gene expression data of 564 HNSCC expression.14 Moreover, PD-L1 is closely related to EMT in specimens to analyse the correlation of PKD3, PD-L1, and EMT- HNSCC.25 To verify whether PKD3 is involved in the regulation related markers. In this large dataset, the expression of PKD3 of EMT in OSCC, we first analysed the protein levels of PKD3, and PD-L1 was positively correlated with that of mesenchymal PD-L1 and EMT markers in OSCC cell lines and tissues using markers (such as SNAI1, SNAI2, ZEB1, ZEB2 and VIM) and western blot and IHC. As shown in Fig. 1a, b, PKD3 was not only negatively correlated with that of epithelial markers (such as highly expressed in OSCC cell lines but also positively correlated CDH1, CLDN4 and CLDN7; Fig. S1d). In summary, our data indicate with mesenchymal markers and negatively correlated with the that PKD3 may play an important role in the occurrence and epithelial marker E-cadherin. Moreover, the levels of PKD3, PD-L1 development of OSCC and is closely related to its EMT. ab 1.000 Vimentin E-cadherin 0.680 Snail Vimentin N-cadherin Snail 0.360 E-cadherin N-cadherin PD-L1 0.040 PD-L1 PKD3 -0.280 GAPDH PKD3 -0.600 HOK DOK Snail Cal-27HSC-4HSC-3 PKD3 SCC25 PD-L1 Vimentin E-cadherin N-cadherin c Normal Grade l Grade Grade ll Grade Grade lll Grade PKD3 PD-L1 E-cadherin Vimentin Fig. 1 Expression of PKD3 in OSCC and its relationship with EMT. a, b The expression of PKD3, PD-L1 and EMT markers was detected by western blot in OSCC cells and non-tumour cells of the oral epithelium. Densitometry was analysed, and the correlation between PKD3, PD-L1, and EMT marker proteins was determined. c Expression of PKD3, PD-L1, and EMT markers in OSCC tissues detected by immunohistochemistry. Black scale bars are 100 µm. PKD3, protein kinase D3; OSCC, oral squamous cell carcinoma; PD-L1, programmed death ligand-1; EMT, epithelial-mesenchymal transition International Journal of Oral Science (2021) 13:8 PKD3 promotes metastasis and growth of OSCC Cui et al. 3 Knockdown of PKD3 significantly inhibits the growth, migration Matrigel-coated membrane (Fig. 2c). Furthermore, we examined and invasion of OSCC cells the effects of PKD3 knockdown on the expression of epithelial To determine the role of PKD3 in EMT of OSCC, we silenced the and mesenchymal markers using western blotting. As shown in expression of PKD3 in the OSCC cell lines Cal-27 (without obvious Fig. S2b, the expression of E-cadherin was increased, while that of mesenchymal characteristics) and HSC-4 (with obvious mesench- mesenchymal markers and PD-L1 was decreased in PKD3- ymal characteristics), the results of which are shown in Fig. 1a. We knockdown cells. Together, these results indicate that knockdown employed sh-PKD3-1 and sh-PKD3-2 because they were pre- of PKD3 inhibits the migration, invasion and EMT phenotype of viously determined to effectively knockdown PKD3. First, we OSCC cells. analysed the effect of PKD3 on cell proliferation using colony formation and CCK-8 assays. The CCK-8 assay showed that the Overexpression of PKD3 promotes the growth, migration and growth of PKD3-knockdown cells was much slower than that of invasion of OSCC cells control cells. Additionally, statistical analysis showed significant According to the results shown in Fig. 1a, we selected two cell differences from the third day after knockdown (Fig. 2a). After lines with the lowest PKD3 expression, namely, DOKs and Cal-27 2 weeks, the colony formation rate of PKD3-knockdown cells was cells.
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