J Pathol Transl Med.  2025 Jan;59(1):68-83. 10.4132/jptm.2024.11.27.

PLUNC downregulates the expression of PD-L1 by inhibiting the interaction of DDX17/β-catenin in nasopharyngeal carcinoma

• Feng R
• Guo Y
• Chen M
• Tian Z
• Liu Y
• Jiang S
• Zhou J
• Liu Q
• Li X
• Xiong W
• Shi L
• Fan S
• Li G
• Zhang W

• KMID: 2563886
• DOI: http://doi.org/10.4132/jptm.2024.11.27

Abstract

Background
Nasopharyngeal carcinoma (NPC) is characterized by high programmed death-ligand 1 (PD-L1) expression and abundant infiltration of non-malignant lymphocytes, which renders patients potentially suitable candidates for immune checkpoint blockade therapies. Palate, lung, and nasal epithelium clone (PLUNC) inhibit the growth of NPC cells and enhance cellular apoptosis and differentiation. Currently, the relationship between PLUNC (as a tumor-suppressor) and PD-L1 in NPC is unclear.
Methods
We collected clinical samples of NPC to verify the relationship between PLUNC and PD-L1. PLUNC plasmid was transfected into NPC cells, and the variation of PD-L1 was verified by western blot and immunofluorescence. In NPC cells, we verified the relationship of PD-L1, activating transcription factor 3 (ATF3), and β-catenin by western blot and immunofluorescence. Later, we further verified that PLUNC regulates PD-L1 through β-catenin. Finally, the effect of PLUNC on β-catenin was verified by co-immunoprecipitation (Co-IP).
Results
We found that PLUNC expression was lower in NPC tissues than in paracancer tissues. PD-L1 expression was opposite to that of PLUNC. Western blot and immunofluorescence showed that β-catenin could upregulate ATF3 and PD-L1, while PLUNC could downregulate ATF3/PD-L1 by inhibiting the expression of β-catenin. PLUNC inhibits the entry of β-catenin into the nucleus. Co-IP experiments demonstrated that PLUNC inhibited the interaction of DEAD-box helicase 17 (DDX17) and β-catenin.
Conclusions
PLUNC downregulates the expression of PD-L1 by inhibiting the interaction of DDX17/β-catenin in NPC.

Keyword

Nasopharyngeal carcinoma; PLUNC; PD-L1; DDX17; β-catenin

Figure

• 

  Fig. 1. Palatal, lung, and nasal epithelial clone (PLUNC) negatively correlates with programmed death-ligand 1 (PD-L1) expression in nasopharyngeal carcinoma (NPC). (A) PLUNC and PD-L1 gene expressions in the head and neck squamous cell carcinoma database. T represents tumor and N represents normal. (B) The forest map of risk factor analysis for NPC tissue chips, *p < .05, ***p < .001, with significant differences. (C) Detection of PD-L1 expression levels in NPC tissue and paracancer tissues using tissue chips. (D) Immunohistochemical staining was performed on paraffin sections of NPC and adjacent tissues to detect the expression levels of PLUNC and PD-L1. (E) Verification of PD-L1 expression in 5-8F and HNE2 cell lines after overexpression of PLUNC through western blot. (F) After transfection of PLUNC-shRNA in 5-8F and HNE2 cells, western blot was used to detect the expression of PD-L1. NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; WBC, white blood cell; AIC, Akaike information criterion.

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  Fig. 2. β-catenin upregulates activating transcription factor 3 (ATF3)/programmed death-ligand 1 (PD-L1) expression. (A) Verification of PD-L1 and ATF3 expression in S18 and HNE2 cell lines by western blot after overexpression of β-catenin. (B) Verification of PD-L1 and ATF3 expression in 5-8F and HONE1 cell lines by western blot after transfection with β-catenin–shRNA. (C, D) Semi-quantitative analysis of the protein expressions of β-catenin, PD-L1, and ATF3 after overexpression and knockdown with β-catenin. *p < .05. (E, F) After overexpressing β-catenin, the expression of PD-L1 was verified by immunofluorescence. (G, H) After knocking down β-catenin, the expression of PD-L1 was verified by immunofluorescence. NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Fig. 3. Palatal, lung, and nasal epithelial clone (PLUNC) suppresses the β-catenin pathway. (A) Detection of the β-catenin pathway expression in 5-8F and HNE2 cell lines by western blot after overexpression of PLUNC. (B, C) Semi-quantitative analysis of the protein expressions of β-catenin, glycogen synthase kinase 3β (GSK-3β), p–GSK-3β, T-cell factor 4 (TCF4), and activating transcription factor 3 (ATF3) after overexpression with PLUNC. *p < .05. (D) β-catenin expression in 5-8F and HNE2 cell lines by immunofluorescence after overexpression of PLUNC. (E) Detection of β-catenin pathway expression in 5-8F and HNE2 cell lines by western blot, and the semi-quantitative analysis of the protein expression after knocking down PLUNC. (F) β-catenin expression in 5-8F and HNE2 cell lines by immunofluorescence after knocking down PLUNC. NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 4. Palatal, lung, and nasal epithelial clone (PLUNC) downregulates the expression of activating transcription factor 3 (ATF3)/programmed death-ligand 1 (PD-L1) by inhibiting the β-catenin pathway. (A, B) After overexpression of PLUNC in 5-8F cells, decreased ATF3 and PD-L1 expression was confirmed by western blot and immunofluorescence. (C, D) After overexpression of PLUNC in HNE2 cells, decreased ATF3 and PD-L1 expression was confirmed by western blot and immunofluorescence. (E–H) After knocking down of PLUNC in 5-8F and HNE2 cells, upregulated ATF3 and PD-L1 expression was confirmed by western blot and immunofluorescence. (I, J) After the cells were treated with PLUNC-shRNA and/or XAV-939, the expressions of β-catenin, phospho–glycogen synthase kinase 3β (p–GSK-3β), ATF3, and PD-L1 were verified by western blot. NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Fig. 5. DEAD-box helicase 17 (DDX17) interacts with β-catenin in nasopharyngeal carcinoma cells. (A–D) Co-immunoprecipitation (Co-IP) of DDX17 and β-catenin in 5-8F and HNE2 cells. (E) Co-localization of DDX17 and β-catenin by immunofluorescence (β-catenin: red, DDX17: green). NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 6. Palatal, lung, and nasal epithelial clone (PLUNC) inhibits the interaction of DEAD-box helicase 17 (DDX17) and β-catenin. (A, B) Co-immunoprecipitation (Co-IP) of DDX17 and PLUNC in 5-8F and HNE2 cells. (C, D) Co-localization of DDX17 and PLUNC by immunofluorescence (PLUNC: red, DDX17: green). (E) Successful co-transfection of PLUNC, β-catenin, and DDX17 plasmids in 293T cells was verified through western blot. (F) Co-IP confirmed that PLUNC inhibited the interaction of DDX17 and β-catenin. (G, H) After overexpression of PLUNC in 5-8F and HNE2 cells, the co-localization of DDX17 and β-catenin was reduced (β-catenin: red, DDX17: green). NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 7. Palatal, lung, and nasal epithelial clone (PLUNC) negatively correlates with DEAD-box helicase 17 (DDX17) and β-catenin expression in nasopharyngeal carcinoma (NPC). (A) Immunohistochemical staining results of PLUNC, DDX17, and β-catenin in NPC and paracancer. (B) Research model diagram of PLUNC inhibiting programmed death-ligand 1 (PD-L1) expression. GSK-3β, glycogen synthase kinase 3β.

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Reference

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