1.Differential HK2 expression among tumor diseases
To explore the difference of HK2 expression among tumor diseases, the differential analysis of the mRNA expression in pan-cancer was performed in accordance with the TCGA databases. As indicated by the result, HK2 displayed a higher expression level in LUSC, HNSC and COAD, and it showed a relatively lower expression level in PCPG, KICH and LIHC (Fig. 1a). Next, the difference of HK2 expression of tumoral and normal tissues was further explored. HK2 was highly expressed within most tumour diseases, as indicated by the results presented in Fig. 1b. HK2 expression in nine types of cancer was significantly higher than in normal tissues (e.g., UCEC, THCA, STAD, PRAD, LUSC, KIRP, KIRC, GBM, and CHOL). Furthermore, in COAD, KICH, LUAD and READ, HK2 expression in normal tissues was up-regulated. The above results conform to the existing study that HK2 is highly expressed in various tumor diseases and may facilitate the tumor progression[9].
2. Prognostic value and clinicopathologic significance of HK2 across cancers
Survival analysis was conducted in different tumor diseases, which involved progression free interval (PFI), disease-specific survival (DSS) and overall survival (OS), with the aim of finding the relationship of survival and HK2 expression. The Kaplan–Meier (KM) curves were plotted. According to the KM curves of OS, HK2 expression displayed a relationship to poor prognosis in CESC, ESCA, LGG and LIHC (Fig. 2a-d). While, for ACC and LAML patients, KM expression displayed a correlation to better prognosis (Fig. 2e-f). Next, the KM curves of DSS showed that HK2 expression displayed a negative relationship to DSS in patients subjected to CESC, LGG and PRAD (Fig. 3a-c). However, ACC patients subjected to higher HK2 expression showed better DSS (Fig. 3d). What’s more, HK2 expression displayed a relationship to poor PFS in LGG and THYM (Fig. 4a-b), and higher HK2 expression was unrelated to better PFS across cancers. In addition, we explored the relationship of HK2 expression and clinicopathologic features. As indicated by the result, HK2 was highly expressed in > 65 years old group among COAD, SARC, STAD and TGCT (Fig. 5). In contrast, HK2 showed relatively higher expression in < 65 years old group in LAML (Fig. 5). HK2 expression also displayed positive association with tumor stage in LIHC, TGCT and THCA (Fig. 6). In contrast, the stage of COAD patients showed negative association with HK2 expression (Fig. 6).
3. Relationship between HK2 expression and tumor immunity
The tumour microenvironment (TME) covers vascular cells, immune cells, stromal cells, epithelial cells, and tumor cells. To be specific, the major components include immune cells and stromal cells[25]. Immune cells’ infiltration in TME displays a close relationship to clinical outcome and therapeutic effect. Thus, the relationship of HK2 expression and stromal score in the TME, immune infiltration score among cancers was studied. In general, immune score displays a positive relationship to stromal score and a negative relationship to tumour purity[26]. The results here suggested that HK2 expression displayed a negative relationship to the immune score and stromal score in numerous tumour diseases (e.g., CESC, ESCA, LUSC, PAAD, SKCM and UCS) (Fig. 7a-l). While in LGG, LIHC, KICH and PCPG, HK2 expression may promote the immune and stromal cells infiltration (Fig. 8a-h).
To investigate the potential role played by HK2 in specific immune cells in depth, TIMER 2.0 database was adopted for displaying the relationship of infiltrating immune cells among cancers and HK2 expression. CD8+ T cells serve as the major anti-tumor immune effector cells by generating perforin (PFN) and other cytotoxin[27]. As indicated by the results of this study, HK2 expression displayed a negative relationship to the CD8+ T cells infiltration in CESC, ESCA, LUSC, PAAD and SKCM (Fig. 9). The above phenomena were consistent with the previous analysis regarding the infiltration of immune cells (Fig. 7a-l). Moreover, the similar results can be reported in GBM and OV (Fig. 9), suggesting that HK2 was also likely to enhance the immune resistance in the above-mentioned two tumour diseases. In contrast, the HK2 expression displayed a positive relationship to CD8+ T cells in UVM. CD4+ T cells displayed a negative relationship to HK2 expression level in CHOL and TGCT (Fig. 9). NK cells have been reported as a member of innate immune cells and the first line of defense against tumors, while the functions of NK cells can be often downmodulated in the TME[28]. As indicated by the result of this study, the infiltration of NK cells may be relatively up-regulated in HK2 highly expressed patients subjected to COAD and UVM (Fig. 9). B cells are vital parts of the TME, whereas their antitumor effects remain unclear[29]. As indicated by the result of this study, HK2 expression displayed a negative relationship to B cells infiltration in STAD and TGCT, whereas it showed a positive relationship to CHOL, CESC, and KICH (Fig. 9). Macrophages are potent immune effector cells and can be classified into the M1 and M2 phenotypes with distinct roles in the TME[30]. The results of this study suggested that HK2 expression showed a positive relationship to the infiltration of anti-tumoral M1 phenotype in DLBC, KICH, and LGG (Fig. 10), while the opposite result occurred in GBM. For the pro-tumoral M2 phenotype, HK2 expression displayed a negative relationship to infiltration in CESC, UCS and UCEC (Fig. 10). Regulatory T cells (Tregs) are subsets of T cells with significant immunosuppressive effect, such that the immune response can be inhibited, and immune escape in the TME can be mediated[31]. Based on the results of this study, HK2 expression displayed a negative relationship to Tregs infiltration in DLBC and STAD (Fig. 10). Cancer associated fibroblasts (CAFs), one of the critical components of the TME, are capable of secreting a variety of cytokines to promote the tumour angiogenesis, induce the epithelial interstitial transformation of tumour cells, break the homeostasis between tissues and cells, and shape the TME more conducive to tumour progression[32, 33]. This study suggested that the HK2 expression displayed a significant negative relationship to CAFs in STAD. However, HK2 may promote the infiltration of CAFs in patients subjected to KICH, LGG, LIHC, CHOL and PCPG (Fig. 10). Myeloid-Derived Suppressor Cells (MDSCs) refer to a heterogeneous group of cells that can inhibit the immune responses mediated by NK cells, CD4+ and CD8+ T cells[34]. Interestingly, the results of this study revealed that HK2 expression displayed a positive relationship to the MDSCs in most tumour diseases except for STAD (Fig. 10).
The relationship of HK2 expression and the levels of immunomodulators was investigated based on the TISIDB database, with the aim of uncovering the potential immunomodulatory effect of HK2. First, we determined the relative coefficient between HK2 expression and immunosuppressive factors. Most immunosuppressive factors displayed a negative relationship to HK2 expression in LUSC, OV and UCS (Fig. 11). While, in patients subjected to LGG, HK2 expression displayed a notable positive relationship to almost all immunosuppressive factors (Fig. 12a-e). Second, the relationship of immunostimulatory factors and HK2 expression was investigated (Fig. 13). We found that in patients subjected to KICH, HK2 expression showed a positive relationship to various immunostimulatory factors (Fig. 14a-i). While in patients subjected to LUSC, HK2 may facilitate the expression of immunostimulatory factors (Fig. 14j-p). Third, we explored the relationship of HK2 expression and chemokines and chemokine receptors across cancers. The results showed that most chemokines especially CXCL8 displayed a positive relationship to HK2 expression in KICH (Fig. 15a). Besides, HK2 expression showed a relatively negative relationship to most chemokines especially CCL14 in PAAD (Fig. 15a). For chemokine receptors, patients subjected to LGG showed significantly positive relationship to HK2 expression (Fig. 15b). In contrast, patients subjected to LUSC displayed a significant negative relationship to HK2 expression (Fig. 15b). Lastly, the relationship of HK2 expression and MHC molecules was investigated. Interestingly, all the MHC molecules showed a significant positive relationship to HK2 expression in ACC, KICH, and LGG (Fig. 16). However, the distinct outcomes occurred in LUSC and UCS (Fig. 16).
4. GSEA of HK2 in GO and KEGG pathways
We further performed GSEA in GO and KEGG pathways, with the aim of exploring HK2’s potential role in the specific immune-related pathways. The results revealed that HK2 expression showed a relationship to various immune response-related pathways, and it was likely to affect immune cells’ functions. GO pathways were analyzed. The result suggested that in patients subjected to LGG, HK2 showed significantly enrichment in adaptive immune response, cytokine-mediated signaling pathways, and leukocyte mediated immunity (Fig. 17). In patients subjected to GBM, HK2 expression showed a positive relationship to leukocyte chemotaxis, mononuclear cell migration, receptor signaling pathway via STAT, cytokine activity (Fig. 17). In patients subjected to ACC, HK2 expression showed a positive relationship to myeloid leukocyte migration, leukocyte migration, immune response regulating signaling pathway and immune response regulating cell surface receptor signaling (Fig. 17). In patients subjected to KICH, HK2 expression showed a positive relationship to the activation of immune response, adaptive immune response and cell chemotaxis (Fig. 17). In patients subjected to SARC, HK2 expression showed a positive relationship to the adaptive immune response, cytokine-mediated signaling pathways, leukocyte proliferation and mononuclear cell differentiation (Fig. 17). Similar outcomes occurred in the KEGG analysis as the patients subjected to LGG indicated a positive relationship of HK2 expression and cytokine/cytokine receptor interaction, JAK/STAT signaling pathway, and chemokine signaling pathway (Fig. 18). In patients subjected to GBM, HK2 expression showed a positive relationship to toll-like receptor signaling pathway, JAK/STAT signaling pathway and cytokine/cytokine receptor interaction (Fig. 18). While in patients subjected to KICH, HK2 expression showed a positive relationship to chemokine signaling pathway and cytokine/cytokine receptor interaction (Fig. 18). In patients subjected to LUAD, HK2 showed significantly enrichment in natural killer cell mediated cytotoxicity, JAK/STAT signaling pathway, as well as antigen processing and presentation (Fig. 18). Lastly, in patients subjected to SARC, HK2 expression showed a positive relationship to natural killer cell mediated cytotoxicity, JAK/STAT signaling pathway and chemokine signaling pathway (Fig. 18). The above results reveal that HK2 acts as a traditional metabolic enzyme while exerting diverse effects on various immune-related pathways.
5. Relationship of HK2 and TMB, MSI and the effect of immunotherapy.
TMB acts as an emerging prediction biomarker for tumor diseases. It has aroused wide attention in predicting the efficacy of tumor immunotherapy[35]. As indicated by existing research, patients subjected to high TMB may achieve better efficacy of immune checkpoint inhibitor therapy. Thus, the relationship of HK2 expression and TMB across cancers was studied. The result suggested that HK2 expression displayed a notable positive relationship to TMB in UCEC, STAD, PAAD, HNSC, COAD, CESC and BRCA (Fig. 19a). In contrast, patients subjected to ACC indicated a negative relationship of HK2 and TMB (Fig. 19a). MSI serves as a vital indicator of immunotherapy. As indicated by the result of the analysis, HK2 expression showed a positive relationship to MSI in UCEC, STAD and LUSC (Fig. 19b). While in patients subjected to DLBC, HK2 expression displayed a negative relationship to MSI (Fig. 19b). For the in-depth investigation of the relationship of HK2 and the effect of immunotherapy, the relationship of HK2 expression and the response to anti-PD1, anti-CTLA4 therapy across cancers was determined and analyzed. As indicated by the result, patients subjected to low HK2 expression levels may benefit more from anti-PD1 and anti-CTLA4 therapy in LUSC (Fig. 20a) and THCA (Fig. 20b).
6. Immunohistochemical analysis of HK2 protein expression level across cancers.
We employed the HPA database to explore the differential protein expression level of HK2 across cancers. As indicated by the result, HK2 protein showed weak staining in normal breast (Fig. 21a), colon (Fig. 21c), ovary (Fig. 21e) and liver (Fig. 21g) pathological sections. In contrast, HK2 protein showed moderate-to-strong staining in breast cancer (Fig. 21b), colorectal cancer (Fig. 21d), ovarian cancer (Fig. 21f), as well as liver cancer (Fig. 21h) pathological sections. As revealed by the above-mentioned results, HK2 may affect the tumour progression at the protein level.