Previous studies have shown that prognostically valuable molecules can be identified through bioinformatics analysis of high-throughput data of functional genes22. In the present study, based on WGCNA, we found that PRC1 and DLGAP5 play critical roles in tumor proliferation and development in HCC. Then we explored these two key genes using multicenter transcriptome sequencing data, including single-cell transcriptome sequencing, microarray data and clinicopathological data from online public databases and our in-house database. Our analysis revealed further insights into their clinical diagnostic and prognostic value, their potential functions in HCC, and their relationship with the immunosuppressive T cell.
PRC1 cross-links microtubules to stabilize their specific orientation, and it participates in cytokinesis23. PRC1 is overexpressed in a variety of different cancers and plays a major role in the malignant progression of tumors, such as lung adenocarcinoma13 and prostate cancer24. Knocking down PRC1 markedly inhibits the proliferation of breast cancer and bladder cancer cells25,26. We demonstrated overexpression of PRC1 in HCC, based on 1704 HCC tissues and 1104 non-cancerous cases at 14 centers in our in-house RNA sequencing data, GEO database and TCGA database. The overexpression of PRC1 protein in HCC tissues was confirmed by immunohistochemistry of the HPA database. In fact, PRC1 gave an AUC of 0.95 and a specificity of 0.93, indicating strong diagnostic value for HCC. In this way, PRC1 shows the potential to outperform the diagnostic combination of serum AFP and ultrasound, since the specificity of this combination is only 68.3%2. A previous small study also reported that PRC1 was up-regulated in HCC, suggesting diagnostic value for early-stage liver cancer 27.
DLGAP5 is a microtubule-associated protein28 that may specifically stabilize short or unstable spindles during mitosis or meiosis, helping ensure normal cell division16. Tsou et al. have demonstrated that levels of DLGAP5 mRNA fluctuate periodically during the cell cycle and peak in the M phase of liver cancer cells15. DLGAP5 has been reported to be up-regulated in pancreatic cancer and glioma 29,30. We analyzed the expression of DLGAP5 in the same HCC and control samples as PRC1, and the results indicated overexpression in HCC tissues. This result was confirmed through comprehensive meta-analysis across various databases and based on immunohistochemistry in the HPA database. These results are based on a large sample and so are likely to be highly reliable. A previous study also reported DLGAP5 overexpression in HCC1. In our study, DLGAP5 gave an AUC of 0.95 and specificity of 0.92, suggesting high diagnostic value for HCC. Future studies should explore whether serum levels of DLGAP5, perhaps together with serum levels of PRC1, can provide accurate HCC diagnosis.
When we compared expression of PRC1 and DLGAP5 with clinicopathological characteristics of patients at our center, we found that expression of both proteins was significantly associated with microvascular invasion and tumor recurrence. In uni- and multivariate analyses, high expression of PRC1 and DLGAP5 and microvascular invasion were independent predictors of the survival of HCC patients. We conclude that PRC1, DLGAP5 and microvascular invasion contribute to HCC in different ways, while PRC1 and DLGAP5 may also contribute to microvascular invasion. Previous studies have shown microvascular invasion to be a key determinant of early recurrence and survival in HCC31 and an independent predictor of tumor grade in patients with solitary small HCC32. High expression of PRC1 and DLGAP5 in our study was associated with shorter OS and DFS, based on our in-house data and Kaplan-Meier plotter data. PRC1 promotes tumor formation, metastasis, and stemness of early HCC through the Wnt/-catenin signaling pathway 33. Overexpression of PRC1 and DLGAP5 has previously been strongly linked to poor prognosis in HCC34.
Analysis of functional enrichment implicated both PRC1 and DLGAP5 were in the BPs of cell division and G1/S transition in mitosis, as well as cell division-related KEGG pathways such as cell cycle regulation and DNA replication. Based on single-cell sequencing data, these two genes were found to be mainly involved in cell proliferation and development. Numerous studies suggest that genes with key roles in cell cycle regulation may play an important role in tumorigenesis35,36. PRC1 enhances the chemoresistance of HCC cells to 5-FU by preventing the drug from arresting cells in G2/M phase, which leads to poor prognosis for HCC patients undergoing chemotherapy11,12. Like PRC1, DLGAP5 also makes HCC cells resistant to chemotherapeutics, and knockdown of DLGAP5 sensitizes SK-Hep-1 cells to cisplatin37. The high expression of DLGAP5 in fetal liver implicates the gene in regulation of cell cycle progression in hematopoietic stem cells38, and in enhancement of the invasiveness of hepatocellular carcinoma39. These results strongly suggest that PRC1 and DLGAP5 promote tumor cell proliferation via mechanisms involving the cell cycle and cell division, thereby contributing to the poor prognosis of tumor patients.
Another novel finding of the present study is that expression of PRC1 and DLGAP5 is positively related to levels of CD8 + T cells, CD4 + T cells, B cells, macrophages, neutrophils, and dendritic cells in the immune microenvironment. Similar results were reported in previous work40, and another study linked DLGAP5 expression to levels of B cells, CD8 + T cells, CD4 + T cells and PDZ-binding kinase (PBK), which are involved in HCC progression41. DLGAP5 upregulation might be involved in the progression of virus-infected CD4 + T cells and B cells toward malignancy in hematologic cancers42. In HCC, CD4 + T cells can promote tumor progression by interacting with different types of inflammatory/immune cells and are linked to poor prognosis after hepatectomy43. DLGAP5 expression has already been implicated in CD8 + T cell infiltration status in renal clear cell carcinoma, uveal melanoma and thymoma17. Further our studies using single-cell transcriptome sequencing showed that PRC1 and DLGAP5 were co-expressed in the Tprolif_C12 subtype in the immune microenvironment, and that this cell subtype shows strong proliferation through activation of E2F and its downstream target genes. Multiple immune suppressor genes, such as PDCD1, CTLA4, HAVCR2, LAG3 and TIGIT, were also expressed, suggesting that this subtype may be highly immunosuppressive. In the landscape of individual immune cells in HCC, proliferative T cells, also known as CD8-c1-MKI67 cells, were significantly enriched in tumor tissue44. These T cells are thought to be able to proliferate directionally into exhausted CD8 + T (Tex) cells44,45, and to participate in tumor metastasis into the ascites. Tex cells are a dysfunctional CD8 + T cell type thought to be related to tumor immunosuppression44. Tex cells contain a higher proportion of proliferating cells than other subtypes of CD8 + T cells do46, so they may share a close relationship with proliferative T cells because they are tumor-related. Our discovery of Tprolif_C12 cells and their functions is consistent with these reports. Taken together, our analyses suggest that HCC patients who express high levels of PRC1 and DLGAP5 are more likely to have poor prognosis, and this relationship may be mediated by Tprolif_C12 cells involved in the immune escape of tumor cells, which allows cancer cells to avoid being killed, and then rapidly proliferate and progress. This may also explain why shorter tumor doubling times were positively associated with upregulation of these two genes. More importantly, this subtype of immunosuppressive T cells co-expressing PRC1 and DLGAP5 also co-expressed multiple immunosuppressive markers. This suggests that a combination of ICI therapy targeting HCC patients enriched for PRC1 or/and DLGAP5-overexpressing T cells may benefit these patients. Therefore, we believe that PRC1 or DLGAP5 combined with CD8 T cell markers may serve as predictive biomarkers for the efficacy of ICI combination therapy. This result still needs to be confirmed by further clinical studies.
Our study shows that PRC1 and DLGAP5 are overexpressed in HCC tissues relative to adjacent non-cancerous tissues, and that both genes show strong potential as diagnostic markers of HCC. In addition, high expression of PRC1 and DLGAP5 is significantly associated with poor prognosis, so the two genes may also be useful as prognostic markers. Our analyses suggest that PRC1 and DLGAP5 are co-expressed in proliferative T cells in HCC that proliferate strongly as a result of activation of E2F and its target genes, facilitating immune escape by cancer cells and thereby contributing to poor prognosis. Most importantly, our results imply that a combination of ICI therapy may be more effective for HCC patients who strongly express PRC1 and DLGAP5. PRC1 or DLGAP5 combined with CD8 T cell markers may serve as predictive biomarkers for the efficacy of ICI combination therapy.