3.1 CENPW is abnormally upregulated in ccRCC.
First, using the TIMER database, the author investigated the changes in CENPW expression levels in cancer tissues compared to normal samples. The results showed that CENPW is significantly upregulated in most cancer types, including ccRCC (Fig.1. a-b). CENPW expression profiles from the GSE36895 and GSE53757 datasets also confirmed our conclusion (Fig.1. c-d). To further validate the results based on high-throughput sequencing and gene microarray, samples collected from clear cell renal cell carcinoma patients were subjected to an RT-qPCR experiment. There was a significant upregulation of CENPW mRNA expression in clear cell renal cell carcinoma tissues (Fig.1. e). Perform a statistical analysis of the sample information used in the experiments (Table. 1)
3.2 High expression of CENPW is associated with clinical characteristics.
The objective of this work is to explore the role of CENPW in ccRCC pathogenesis by analyzing the correlation between CENPW expression and clinical features of ccRCC. In the TCGA-KIRC dataset, the author found that the expression level of CENPW was significantly associated with T-stage, N-stage, M-stage, clinical stage, and patient’s vital status but had no correlation with gender and age (Table. 2). Besides, the expression level of CENPW was higher in high levels of T-stage (Fig.1.f), N-stage (Fig.1. g), and M-stage (Fig.1. h). Also, high expression of CENPW was correlated to worse grade and stage (Fig.1. i-j).
3.3 High expression of CENPW predicts poor clinical outcomes.
From the above analysis, the author has shown the elevating expression of CENPW in ccRCC and the association between CENPW expression levels and clinical characteristics. However, whether CENPW is positively associated with ccRCC malignancy is still largely unknown. To address this question, the author stratified patients into high and low CENPW expression groups based on the median CENPW expression level in the TCGA-KIRC dataset. Then KM survival analysis was conducted, and the findings indicated that the high expression group had a notably lower overall survival rate compared to the low expression group (Fig.2. a), and the disease-free survival of the high expression group was markedly lower than that of the low expression group (Fig.2. b). The author performed a ROC curve analysis to confirm the ability of CENPW to predict survival rates. The results demonstrated that CENPW was an accurate diagnostic indicator for patients with ccRCC (Fig.2. a-b). In order to confirm the reported findings, Cox regression analysis was employed to ascertain if CENPW was a risk factor for ccRCC patient outcomes. The results showed that in addition to Age, Grade, Stage, T-stage, and M-stage, CENPW was a significant risk factor for the prognosis of ccRCC patients (Fig.2. c). Furthermore, the multivariate analysis also identified CENPW as an adverse factor (Fig.2. d). It turned out that Age, Grade, and Stage were also deleterious factors in the TCGA-KIRC dataset (Fig.2. d). Furthermore, three independent datasets validated that CENPW influenced prognosis for patients with ccRCC (Fig.2. e).
3.4 Investigate the function of CENPW in ccRCC.
Knowing that CENPW is a deleterious factor for ccRCC patients, this study further analyzed the possible signal pathways in which CENPW may be involved. The author first classified the data from the TCGA-KIRC dataset into high and low expression groups according to the median CENPW expression value. Gene expression profiles from these two groups showed significant differences (Fig.3. a). Then, KEGG and Gene Ontology analysis were performed to identify the significant pathways enriched in these two groups. The results showed that several pathways often abnormally upregulated in malignancy, such as the PI3K-Akt and Wnt signaling pathways, were enriched (Fig.3. b). Surprisingly, cholesterol metabolism, high-density lipoprotein particle pathways and fat digestion and absorption were also enriched (Fig.3. b-c). High-density lipoprotein is the main carrier involved in cholesterol transport, which indicates that CENPW may be involved in cholesterol metabolism in ccRCC. The author also found enriched cytokine-cytokine receptor interaction (Fig.3. b), B cell-mediated immunity, antigen binding, and immunoglobulin receptor binding (Fig.3. c). These results indicated that CENPW may be involved in immune cell infiltration and tumor microenvironment changes in ccRCC. GSEA was also performed to complete GO and KEGG enrichment results. After analyzing the results, the author found that CENPW may promote ccRCC development by activating the P53 signal pathway and participating in DNA replication. Significant enrichment of the cytokine-cytokine receptor interaction pathway was seen in the group with high CENPW expression. (Fig.3. d), which was consistent with KEGG enrichment results (Fig.3. b).
3.5 PPI network and co-expression analysis of CENPW.
The PPI network was established by using the STRING database (Fig.4. a). Then, Degree and Maximum Neighborhood Component (MNC) algorithms were carried out to recognize the hub genes from this network. The top 10 genes calculated by these two methods showed significant consistency (Fig.4. b). The GO enrichment of these hub genes showed that these genes were related to protein-DNA complex, chromosome, and kinetochore (Fig.4. c). The results of this study indicate that CENPW mainly associates with DNA replication and cell division-related genes to promote proliferation of ccRCCs. The co-expressed genes with CENPW were determined based on Spearman correlation analysis across the whole transcriptome sequences of TCGA-KIRC. These are the top five positively correlated genes based on correlation coefficients (UBE2C, PTTG1, AURKB, CDC20, BIRC5) and the top 5 negatively correlated genes (HOOK1, PRKAA2, OSBPL1A, SPATA18, EMX2OS) were selected under the criterion of P-value less than 0.01.
3.6 CENPW is involved in immune cell infiltration of ccRCC.
GO enrichment analysis data pointed out that CENPW may be involved in tumor immunology. Given that tumor immune cell infiltration is essential in tumor development, CENPW and immune infiltration need to be investigated.
First, the author used the TIMER database to analyze the association between diverse immune cell infiltration and CENPW expression levels. A negative association was seen between the expression of CENPW and tumor purity, whereas a positive correlation was found between CENPW expression with the quantification of CD8+ cells, CD4+ T cells, B cells, Neutrophils, Macrophages, and Dendritic cells infiltrating the tumor (Fig.5. a).
Then, the tumor-associated stroma cell and Infiltration of immune cells level differences between the two groups were assessed by the ESTIMATE algorithm based on TCGA-KIRC expression profiles. According to (Fig.5. b), as reported by TIMER, there was a significant decrease in tumor purity in the CENPW-overexpressed group. The CENPW high-expressed group exhibited higher immune cell and tumor-associated stroma cell abundance, there was a higher Immune Score and Stroma Score in the group with high CENPW expression (Fig.5. b). ESTIMATE also generated an ESTIMATE Score, which comprehensively investigates immune and stroma cell infiltration levels. The ESTIMATE Score was higher in the CENPW high-expressed group contrasts to the CENPW low-expressed group (Fig.5. b).
Next, to compensate for the data from the TIMER database, CIBERSORT algorithms were used to analyze the difference in numbers of immune cells infiltrated between groups with high and low CENPW expression, respectively. In agreement with (Fig.5. a), CD8+ T cells and macrophages M0 infiltrated more heavily in the group with high CENPW expression (Fig.5. c). The author also found that activated, follicular helper T cells, CD4 memory T cells, NK cells, γδ T cells and Dendritic cells were differentially elevated in the high CENPW expression group (Fig.5. c).
At last, as immune checkpoint therapy has become increasingly important in cancer therapy, an exploration of the co-relation between immune checkpoints and CENPW expression is necessary. It founded out that 27 immune checkpoints were elevated in the CENPW high-expressed group and were positively associated with CENPW expression level to varying degrees (Fig.5. d-e). CTLA4, a target of Ipilimumab for immunotherapy, was associated with the expression level of CENPW. Another gene, PDCD1 encoding PD1, was also positively correlated with CENPW. While for CD274 and IDO1, CENPW was negatively correlated with (Fig.5. f).
3.7 Immunotherapy and chemotherapy responsibility of CENPW.
To better evaluate the value of immune checkpoint inhibitors targeting CTLA4 and PD1 and find potential chemotherapy molecules for ccRCC, the author performed immunophenoscore (IPS) analysis and IC50 analysis based on the TCIA database and the ‘pRRophetic’ R package. The results revealed that the CENPW high-expressed group showed greater IPS-CTLA4-pos-PD1-neg and IPS-CTLA4-pos-PD1-pos values but Statistical analysis did not reveal a significant difference between the IPS_CTLA4_neg_PD1_pos and IPS_CTLA4_neg_PD1_neg values (Fig.6. a), which indicates that high CENPW group responded better to CTLA4 inhibitor alone or CTLA4 inhibitor combined with PD-1 inhibitor. High CENPW expression patients may be more responsible for CTLA4 inhibitor treatment. As for chemotherapy, patients with high CENPW expression were more susceptible to first-line chemotherapy drugs, such as 5-Fluorouracil, Cisplatin, and Doxorubicin. In addition, the author also found other candidate drugs for high CENPW expression patients (Fig.6. b).
3.8 Verification of the function of CENPW in ccRCC.
The author also identified the expression level of CENPW in several renal cell carcinoma cell lines and selected 786-O and CAKI-1 cell lines, which were highly expressed CENPW compared to other cancer cells, for further experimental verification (Fig.7. a). After knocking down the expression of CENPW by siRNA (Fig.7. b), the cell viability (Fig.7. c), migration, and invasion abilities (Fig.7. d) were significantly reduced.
3.9 CENPW Regulates Lipid Metabolism in ccRCC
Through GO pathway enrichment analysis of the CENPW dataset, we found that CENPW might be associated with lipoprotein activity, which can enhance the uptake of lipid synthesis precursors through the cell membrane, thereby promoting the accumulation of lipid droplets in ccRCC (Fig.8. a). GSEA enrichment analysis revealed that when CENPW is highly expressed, the adipocytokine signaling pathway and fatty acids metabolism are activated, thus regulating lipid metabolism in ccRCC (Fig. 8b-c). To investigate the impact of CENPW on lipid metabolism in ccRCC, we performed an Oil Red O staining experiment. In this experiment, we found that knocking down CENPW significantly reduced the amount of lipid droplets in the 786-O cell line. Then, we conducted BODIPY 493/503 staining on 786-O cells and observed that neutral lipid droplets in renal cell carcinoma were markedly reduced after CENPW knockdown. Therefore, CENPW can influence the content of lipid droplets in ccRCC.