Gene expression analysis
AK4 has been reported to be aberrantly expressed in cancer24,25. In this study, we observed differential expression of AK4 in 33 human cancers using TIMER 2.0 and GEPIA 2.0 databases. Specifically, AK4 was overexpressed in HNSC (head and neck squamous cell carcinoma), LUAD, LUSC, THCA (thyroid carcinoma), and UCEC (uterine corpus endometrial carcinoma) according to the TIMER 2.0 database (Fig. 1a). AK4 has been implicated in BLCA (bladder urothelial carcinoma), BRCA (breast invasive carcinoma), CHOL (cholangiocarcinoma), COAD (colon adenocarcinoma), KICH (kidney chromophobe), KIRC (kidney renal clear cell carcinoma), KIRP (kidney renal papillary cell carcinoma), STAD (stomach adenocarcinoma), PRAD (pancreatic adenocarcinoma) and SKCM (skin cutaneous melanoma) were decreased in 10 cancers (Fig. 1a) (p < 0.05).
After analyzing the GTEx and TCGA datasets in the GEPIA 2.0 database, we observed that AK4 is highly expressed in eight types of cancers, including ACC (adrenocortical carcinoma), CESC (cervical squamous cell carcinoma), DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), OV (ovarian serous cystadenocarcinoma), PAAD (pancreatic adenocarcinoma), PCPG (pheochromocytoma and paraganglioma), THYM (thymoma), and TGCT (testicular germ cell tumors) (Fig. 1b). Conversely, AK4 expression was low in SARC (sarcoma) and LAML (acute myeloid leukemia) (Fig. 1c). All these differences were statistically significant (p < 0.05).
Based on the HPA database, AK4 protein expression was highest in the kidneys (Fig. S1a) and testicular cancer (Fig. S1b). When comparing the immunohistochemical results from HPA with the differential expression data from previous databases, weak or absent AK4 staining was observed in normal brain, lung, cervix, pancreas, and lung tissues. Moderate staining was observed in LUAD, CESC, PADD, OV, and HNSC tissues. Intense staining was observed in normal breast, kidney, and gastric tissues, while weak or no staining was observed in BRCA, PRAD, and STAD tissues (Fig.S1 c-h).
We used the GEPIA 2.0 database to evaluate the relationship between AK4 expression and pathological staging in different types of cancer. The results showed that AK4 expression was significantly associated with pathological stages in ACC(p = 0.011), CHOL(p = 0.0433), KICH(p = 0.025), LUAD(p = 0.00539), PAAD(p = 0.0106), and THCA (p = 0.00297) (Fig. 2).
Prognostic value of AK4
To evaluate the prognostic value of AK4 in different cancers, we utilized the GEPIA 2.0 database and observed that AK4 expression was associated with overall survival (OS) and disease-free survival (DFS) in several types of cancer. In particular, upregulation of AK4 was associated with lower OS in UVM (p = 0.0013), STAD (p = 0.014), PADD (p = 0.016), LUAD (p = 0.001), LGG (p = 0.0062), KICH (p = 0.031), HNSC (p = 0.0054) and CESC (p = 0.0048) (Fig. 3a), while low AK4 expression was associated with lower OS in READ (p = 0.037) and KIRC(p = 9.6e-06). Additionally, high AK4 expression was associated with low DFS in THCA(p = 0.031), UVM(p = 0.032), HNSC(p = 0.013), LGG(p = 0.00089), and KICH(p = 0.013), while low AK4 expression was associated with poor DFS in PRAD(p = 0.039) (Fig. 3b). These results were statistically significant (p < 0.05) and suggest that AK4 may be a prognostic marker for these types of cancer.
We further assessed the prognostic value of AK4 using the Kaplan-Meier plotter. Our findings indicate that elevated AK4 expression is correlated with unfavorable overall survival (OS) in BRCA(p = 0.026), UCEC(p = 0.003), LIHC(p = 0.0012), HNSC(p = 0.02), PDAC (p = 7.4e-05), STAD(p = 0.00026), THYM(p = 0.021), LUAD(p = 0.0011), and CESC(p = 0.00031) (Fig. S2a), whereas low AK4 expression is associated with poor OS in KIRC(p = 0.0029) (Fig. S2b). Moreover, high AK4 expression is linked to reduced recurrence-free survival (RFS) in UCEC(p = 0.014), THCA(p = 0.0019), PAAD(p = 8e-04), LUAD(p = 0.038), LIHC(p = 0.033), and KIRC(p = 0.047) (Fig.S2c), while low AK4 expression is associated with poor RFS in OV(p = 0.045) and ESCA(p = 0.013) (Fig.S2d). All these differences are statistically significant (p < 0.05). In conclusion, high expression of AK4 is strongly associated with poor prognosis in various types of cancer.
Correlation between AK4 and immune infiltration in a variety of cancers
The TIMER 2.0 database was utilized to examine the relationship between AK4 expression and immune cell infiltration in several types of cancer. The findings revealed a negative correlation between AK4 expression and the infiltration of CD8 + T cells, CD4 + T cells, B cells, and dendritic cells in more than four types of cancer (Fig. 4a, b, c, and e). Conversely, AK4 expression was positively correlated with macrophages and neutrophils in more than four types of cancer (Fig. 4d and f). Furthermore, using the TISDB database, we confirmed that AK4 expression is negatively correlated with CD8 cells, B lymphocytes, and CD4 cells in most cancer types but positively correlated with macrophages and neutrophils (Fig. 4g and h). Thus, AK4 could potentially impact immune infiltration in tumors. As immune checkpoint genes are crucial targets for tumor immunotherapy, we used the TISDB database to analyze the correlation between AK4 expression and immune activation, immune checkpoints, chemokine, and chemokine receptor genes. AK4 was significantly associated with most immune-stimulating genes, such as poliovirus receptor (PVR) and 5'-nucleotidase (NT5E), in more than four cancers (Fig. 4i). AK4 expression was negatively correlated with various immune checkpoint inhibitors, such as CD160 (Fig. 4j). There was also a correlation between the expression level of AK4 and chemokine genes, being negatively correlated with CCL14, CXCL19, and CXC22 (Fig. 4k) and positively correlated with the chemokine receptor gene CCR1.
In summary, these findings suggest that AK4 may serve as a tumor immune checkpoint molecule; however, further experiments and clinical trials are necessary to validate this concept.
Functional analysis of AK4 in LUAD
We utilized the CancerSEA database to investigate the potential involvement of AK4 in tumor-associated malignant phenotypes at the single-cell level in LUAD and other cancer types. Our analysis revealed that AK4 was positively associated with cell cycle, interstitial epithelial transformation (EMT), hypoxia, invasion, and metastasis in more than 4 cancer types, including LUAD, BRCA, and RCC (Fig. 5a). In LUAD, we observed a significant positive correlation between AK4 expression and EMT and metastasis (r = 0.23, 0.32, respectively; p < 0.05, Fig. 5b). Further exploration of the potential functionality of AK4 was performed using the LinkedOmics database in LUAD. As shown in Fig. 5c, positive and negative genes associated with AK4 were identified. The top 50 ranked genes are presented in Fig. 5d and e. Furthermore, Gene Ontology (GO) analysis revealed that AK4 could potentially participate in the regulation of cysteine-type endopeptidase activities in LUAD, such as apoptosis, phagocytic cups, endocytic vesicles, early endosomes, autophagosomes, endolysin, DNA replication, organ growth, DNA conformational changes, necrotic cell death, lipid homeostasis, and processes that utilize autophagy mechanism (Fig. 5f, g and h). KEGG analysis showed that AK4 was related to LUAD autophagy, toll-like receptor signaling pathway, DNA replication, protein digestion, absorption, and adhesion (Fig. 5i).
Taken together, these results suggest that AK4 may play a role in promoting malignant phenotypes in LUAD and other cancer types, and further studies are needed to explore it as a potential therapeutic target.
AK4 knockdown
We selected HCC827 cells to perform AK4 knockout and assessed the knockdown efficiency of three knockout sequences. WB and qRT-PCR results showed that siRNA-3 had the best knockdown effect (p = 0.0008) (Fig. 6a and b), with all differences being statistically significant (p < 0.05).
AK4 knockdown inhibited HCC827 cell proliferation, invasion, and migration
AK4 knockdown and its effect on LUAD cell proliferation were investigated in the HCC827 cell line. CCK8 results showed that the knockdown of AK4 significantly inhibited the proliferation of HCC827 cells(p < 0.001) (Fig. 7a). The effect of AK4 knockdown on LUAD cell viability was quantified through wound healing experiments, which showed that AK4 knockdown significantly reduced the motility of HCC827 cells(p = 0.001) (Fig. 7b). Additionally, Transwell experiments demonstrated that knocking down AK4 significantly reduced the invasiveness of HCC827 cells(p = 0.0001) (Fig. 7c). These results were statistically significant (p < 0.05).
In summary, these results suggest that AK4 knockdown inhibits the proliferation, migration, and invasion of HCC827 cells in vitro.
The effect of AK4 knockdown on autophagy, apoptosis, and EMT in HCC827 cells
Through single-cell function, GO, and KEGG enrichment analyses, we found that AK4 is related to autophagy, apoptosis, and EMT in LUAD, which we verified experimentally in this study.
After AK4 knockdown, we observed increased autophagy, as indicated by the significant increase in the autophagy marker LC3A/B (p = 0.0369) and a decrease in p62(p = 0.0002), negatively correlated with autophagy activity. Additionally, Beclin1 expression, which promotes the formation of autophagy membranes and directs the localization of autophagy-associated proteins, was increased (p = 0.0153) (Fig. 8a, b), suggesting increased autophagy of HCC827 cells after AK4 knockdown.
Moreover, the expression of caspase 3(p = 0.0104), the most crucial terminal lyase associated with apoptosis, decreased after AK4 knockdown, while the expression of BCL2(p = 0.0227), negatively correlated with apoptotic activity, was significantly increased. The expression of Bax (p = 0.0104), which is positively correlated with apoptotic processes, was decreased (Fig. 8c, d), indicating a reduction in apoptosis of HCC827 cells after AK4 knockout.
Furthermore, when we detected EMT-related proteins, we observed an increase in the expression of the epithelial marker E-cadherin(p = 0.0137) and a decrease in the expression of vimentin(p = 0.0109) after AK4 knockdown (Fig. 8e, f), suggesting that AK4 inhibits EMT.