POU2F1 induces the immune escape in lung cancer by up-regulating PD-L1

Abstract

Purpose: The aim was to research the POU2F1 related genes and mechanism during the progress of immune escape of lung cancer.

Methods: Lung cancer cell lines (H1993, HCC827, A549, H2228, H3122 and H1975) and Human normal lung epithelial cell line (BEAS-2B) were involved in this study. Overexpression or knockdown of POU2F1 was processed in lung cancer cells. POU2F1, PD-L1 and CRK expression in cells were detected by WB and RT-PCR. Flow cytometry and immunofluorescence was used to detect PD-L1 expression on the cell surface. Luciferase reporter detected the promoter activity of CRK. C57BL/6 mice models with knocked down of of POU2F1 were constructed. After tumor formation, anti-PD-1 was administered to detect tumor suppressing ability. IHC assay showed the number of intratumoral CD3+, CD8+, GranzB+ T cells.

Results: POU2F1 and PD-L1 were positively correlated in lung cancer cell lines. Overexpression of POU2F1 promoted the expression level of PD-L1 in lung cancer cells. POU2F1 transcription activated the expression of CRK, and further promoted the expression of PD-L1. Knockdown of POU2F1 promoted the efficacy of Anti-PD-1. In addition, tumor growth ability decreased after POU2F1 was knocked down. Cytotoxic effector cytokines levels, tumor suppressive chemokines and interleukin increased, while IL17a level decreased when POU2F1 was knocked down.

Conclusion: POU2F1 activates the expression of CRK, further promotes the expression of PD-L1, and finally improves the immune escape in lung cancer.

Keywords: Lung cancer; PD-L1; POU2F1; immune escape.

Figure 1

The levels of POU2F1 and PD-L1 were positively correlated in lung cancer cell lines. A. The expression of POU2F1 and PD-L1 were significant higher in most lung cancer cell lines (H1993, HCC827, H2228, H3122, H1975) than that in BEAS-2B cells. B. The protein levels of POU2F1 and PD-L1 were significant higher in most lung cancer cell lines (H1993, HCC827, H2228, H3122, H1975) than that in BEAS-2B cells. C. PD-L1 was higher expressed in the surface of HCC827 and H1975 cells, while they were low expressed on the surface of A549 cells. *P < 0.05, **P < 0.01.

Figure 2

Overexpression of POU2F1 promoted the expression level of PD-L1 in lung cancer cell lines. A. POU2F1 was overexpressed in lung cancer cells (A549 and H1993). B. The protein level of POU2F1 was higher in lung cancer cells (A549 and H1993). C. PD-L1 expressions significantly rise in mRNA levels. D. PD-L1 expressions significantly rise in protein levels. E. After POU2F1 was overexpressed, the POU2F1 level was up-regulated in both A549 and H1993 cells. F. Immunofluorescence assay was used to detect PD-L1 levels. The POU2F1 level was up-regulated in both cells **P < 0.01.

Figure 3

Knockdown of POU2F1 inhibited the expression level of PD-L1 in lung cancer cell lines. A. POU2F1 level was decreased after shPOU2F1 transfection. B. POU2F1 protein level was decreased after shPOU2F1 transfection. C. PD-L1 expressions were significantly decreased in mRNA levels after knockdown of POU2F1. D. PD-L1 expressions were significantly decreased in protein levels after knockdown of POU2F1. E. The PD-L1 level was decreased in both HCC827 and H1975 cells after POU2F1 was knockdown. F. Immunofluorescence assay confirmed that POU2F1 positively regulated the expression of PD-L1. **P < 0.01.

Figure 4

POU2F1 transcription activated the expression of CRK. A. POU2F1 positive regulated CRK expression. B. Western blot assay also confirmed that POU2F1 positive regulated CRK expression. C. POU2F1 was predicted to combine with the promoter of CRK. D. CRK promoter activity declined after POU2F1 knockdown. E. POU2F1 was overexpressed in lung cancer cells, and luciferase reporter was used to detect the promoter activity of CRK. F. The promoter fragment content increased in the anti-POU2F1 sample. *P < 0.05, **P < 0.01.

Figure 5

POU2F1 promoted the expression of PD-L1 by promoting the expression of CRK. A. CRK was knocked down in lung cancer cells. B. shCRK down-regulated the expression of PD-L1. C. Flow cytometry also confirmed that PD-L1 positively regulated PD-L1 Level. D. Rescue assay. **Compared with control+shNC group, P < 0.05; ^##Compared with POUF21+shNC group, P < 0.01.

Figure 6

POU2F1 knockdown promoted the efficacy of Anti-PD-1. A. Knockdown of POU2F1 and/ or anti-PD-1 treatment was processed in C57BL/6 mice. The mice were divided into four groups, including shNC+IgG, shPOU2F1+IgG, shNC+Anti-PD-1 and shPOU2F1+Anti-PD-1 groups. The tumor volume decreased in shPOU2F1 group and shPOU2F1+Anti-PD-1 groups. B. The tumor weight decreased in shPOU2F1 group and shPOU2F1+Anti-PD-1 groups. C. Body weight was lower decreased in shPOU2F1 group and shPOU2F1+Anti-PD-1 groups. D. The number of CD3+, CD8+, GranzB+ T cells in the tumor increased, indicating that T cell infiltration was promoted in shPOU2F1 group and shPOU2F1+Anti-PD-1 groups. E. Cytotoxic effector cytokines (granzyme B, TNF-a, IFN-g) levels increased, and tumor suppressive chemokines (CCL5, CXCL9, CXCL10) levels increased. The level of interleukin (IL1b, IL12b, IL15) increased, while IL17a level decreased. *P < 0.05, **P < 0.01, ns, the different with no significance.