HHLA2 deficiency inhibits pancreatic cancer progression and THP-1 macrophage M2 polarization via EGFR/MAPK/ERK and mTOR/AKT pathway

Abstract

Background: Human endogenous retrovirus subfamily H long terminal repeat associating protein 2, (HHLA2), a member of B7 family, exhibits heightened expression in various malignant tumors. However, the exact functions of HHLA2 in pancreatic cancer (PC) remain incompletely elucidated.

Methods: We initially conducted an analysis of the B7 family members' expression pattern in pancreatic tumor samples and adjacent normal tissues using The Cancer Genome Atlas (TCGA) database. Subsequently, immunohistochemistry, RT-qPCR and western blot methods were used to assess HHLA2 expression levels in PC tissues and cell lines. Furthermore, after silencing HHLA2 in PC cell lines, cell migration and proliferation of PC cells were detected by wound healing and CCK-8 assays, and cell invasion of PC cells was detected by transwell assays. We also investigated the regulation of epithelial-mesenchymal transition (EMT) markers and levels of EGFR, MEK, ERK1/2, mTOR and AKT via western blot analysis. Finally, the correlation between HHLA2 expression and immune infiltration was further explored.

Results: Silencing of HHLA2 resulted in the inhibition of PC cell proliferation, migration and invasion, potentially through the suppression of the EGFR/MAPK/ERK and mTOR/AKT signaling pathway. Additionally, silencing HHLA2 led to the inhibition of M2-type polarization of tumor associated macrophages (TAMs).

Conclusion: The knockdown of HHLA2 was observed to inhibit the migration and invasion of PC cells through the regulation of the EMT process and EGFR/MAPK/ERK and mTOR/AKT pathway. Furthermore, silencing HHLA2 was found to modulate M2 polarization of TAMs. These finding suggest that HHLA2 could be a promising therapeutic target for Pancreatic cancer.

Keywords: HHLA2; Invasion; Migration; Pancreatic cancer; Tumor-associated macrophages.

Fig. 1

The expressions of B7 family members between PC tissues and normal tissues. A Expression of B7 family members in pancreatic cancer tissues and normal tissues. B Heatmap of B7 family member in normal and cancer tissues (green: low-expression; red: high-expression). C Spearman correlation analysis for the 10 types of B7 family members (red: positive correlation; blue: negative correlation). D Forest plot of B7 family members by univariate Cox regression analysis. E&F Univariate and multivariate Cox regression analysis of clinical parameters and risk score

Fig. 2

The B7 family members were correlated with the survival of PC patients. A-J Survival curves of B7 family members and prognosis of PC; K The distribution of risk scores, survival status and heatmap of the selected genes expression levels in datasets of TCGA-PC (left of the dotted line: low-risk population; right of the dotted line: high-risk population; distribution of patients based on the median risk score.) L Correlation heatmap of B7 family members and clinicopathological features in datasets of TCGA-PC

Fig. 3

The expression level of HHLA2 in pancreatic cancer tissues and cells. A The expression of HHLA2 in pancreatic cancer tissue was detected by WB. B The expression of HHLA2 in pancreatic cancer tissue was detected by RT-qPCR. C&D The expression of HHLA2 in 62 paired adjacent normal tissues and tumor tissues was detected by IHC. Scale bar: 50 μm. E&F The expression of HHLA2 in pancreatic cancer cells was detected by WB and RT-qPCR. G Differential expression of HHLA2 in PC tissues and normal tissues and its survival curve with patients were analyzed. ^*p<0.05, ^**p<0.01, ^***p<0.001, ^****p<0.0001

Fig. 4

Effects of siHHLA2 on viability and proliferation of PC cells. A The relative expression level of protein of siHHLA2 in AsPC-1 and Capan-2. B The relative expression level of mRNA of siHHLA2 in AsPC-1 and Capan-2. The effect of siHHLA2-2 and siHHLA2-3 on the growth of PC cells was detected by colony formation assay (C), CCK-8 (D) and immunofluorescence staining. Scale bar: 100 μm (E&F). ^*p<0.05, ^**p<0.01, ^***p<0.001, ^****p<0.0001

Fig. 5

Function enrichment analysis of HHLA2-related gene sets in PC. A Differentially expressed genes (DEGs) for high expression of HHLA2 vs. low expression of HHLA2 in PC were shown in the volcano plot, with red dots representing significantly up-regulated genes and blue dots representing significantly down-regulated genes in PC with high expression of HHLA2. B The heatmap exhibits the expression level. C Enrichment analysis for KEGG pathway of up-regulated genes. D Enrichment analysis for Hallmark pathway of up-regulated genes. E Enrichment analysis for GO term of up-regulated genes. F Enrichment analysis for KEGG pathway of down-regulated genes. G Enrichment analysis for Hallmark pathway of down-regulated genes. H Enrichment analysis for GO term of down-regulated genes

Fig. 6

Effects of siHHLA2-2 and siHHLA2-3 on the invasion and migration. A&B, migration and invasion (C&D) of AsPC-1 and Capan-2 cells. Scale bar: 100 μm. ^*p<0.05, ^**p<0.01, ^***p<0.001, ^****p<0.0001

Fig. 7

Effects of siHHLA2-2 and siHHLA2-3 on EGFR/MEK/ERK1/2/AKT/mTOR pathway in AsPC-1 and Capan-2 cells. A&B WB and RT-qPCRwere performed to evaluate the protein and mRNA levels of E-Cadherin and Vimentin expression after HHLA2 knockdown. C Knockdown of HHLA2 inhibited the activity of EGFR/MAPK/ERK signaling pathway in AsPC-1 and Capan-2 cells. D Knockdown of HHLA2 inhibited the activity of AKT/mTOR signaling pathway in AsPC-1 and Capan-2 cells. ^*p<0.05, ^**p<0.01, ^***p<0.001, ^****p<0.0001

Fig. 8

Silencing HHLA2 in AsPC-1 and Capan-2 inhibited M2 polarization of TAMs. A-C TIMER2.0 and TCGA database suggested that M2 macrophage infiltration in pancreatic tissue was positively correlated with HHLA2 expression; D M0 macrophages and co-cultured TAMs expressed changes in the expression levels of M2-type macrophage markers. ^*p<0.05, ^**p<0.01, ^***p<0.001, ^****p<0.0001