ESCO2 promotes the proliferation of hepatocellular carcinoma through PI3K/AKT/ mTOR signaling pathway

doi:10.21203/rs.3.rs-3901584/v1

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ESCO2 promotes the proliferation of hepatocellular carcinoma through PI3K/AKT/ mTOR signaling pathway

https://doi.org/10.21203/rs.3.rs-3901584/v1

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Background: Establishment of sister chromatid cohesion N-Acetyltransferase 2(ESCO2), a gene reported to involved in the establishment of sister chromatid cohesion (SCC) and cell proliferation. We aim to explore how ESCO2 affects the proliferation of hepatocellular carcinoma (HCC).

Methods: ESCO2’s expression value and its relationship with clinical prognosis were explored based on TCGA, HCCDB and ICGC databases. We then utilized bioinformatics method analysis to investigate the potential regulatory pathways in which ESCO2 may be implicated. CCK-8, clone assay, and flow cytometry were utilized to examine the impact of ESCO2 knockdown on the malignant biological activity of HCC cells. Finally, we identified the specific regulatory mechanism of ESCO2 using Western blotting.

Results: We determined ESCO2 was significantly upregulated in HCC tissues and high ESCO2 expression was linked to a worse prognosis. Bioinformatic analysis revealed that ESCO2 regulated pathways related to the cell cycle and cell proliferation. Furthermore, knockdown of ESCO2 significantly inhibited HCC cell proliferation in vivo and in vitro. Most significantly, ESCO2 stimulates PI3K/AKT/mTOR pathway, which ultimately accelerates up the cell cycle and inhibits apoptosis, promoting the progression of HCC.

Conclusion: We revealed the mechanism by which ESCO2 regulates HCC proliferation: ESCO2 promotes HCC proliferation by accelerating the cell cycle and inhibiting apoptosis via the PI3K/AKT/mTOR signaling pathway.

Cancer Biology

Cell Cycle & Proliferation

Oncology

Hepatocellular carcinoma

ESCO2

Cell cycle

Apoptosis

PI3K/AKT/mTOR signaling pathway

Liver cancer, more specifically hepatocellular carcinoma (HCC), is one of the most prevalent solid cancers globally and represents an enormous health care burden[1, 2]. With a relative 5-year survival rate of only about 18%, HCC remains one of the deadliest malignancies[3]. The majority of HCC patients receive only local and systemic treatments because they are diagnosed at an advanced stage, which precludes them from surgery or even liver transplantation. Presently, systemic therapy is the standard strategy for treating advanced HCC in clinic[4], ranging from single-agent targeted therapies (sorafenib or levatinib) into combinations of immune checkpoint inhibitors and targeted therapies (atezolizumab plus bevacizumab therapy). Advanced HCC continues to be one of the most challenging and unpromising cancers because, despite substantial advancements in systemic therapy, only a minority of patients achieve durable clinical benefit[5]. The carcinogenesis of HCC is quite complicated, and researches has demonstrated that abnormal proliferation is a key component in the HCC development[6]. Investigating the molecular processes that control HCC proliferation will aid in understanding how it develops as well as in identifying crucial therapeutic targets, which may improve the patients’ prognosis in the future.

Establishment of sister chromatid cohesion N-Acetyltransferase 2 (ESCO2), a novel gene reported to have a functional role in cell proliferation. It belongs to the family of histone acetyltransferases (HAT) that regulate numerous key biological processes[7]. To establish sister chromatid cohesion (SCC) in S phase, the ESCO2 protein is required.[8]. Sister chromatid cohesion facilitates effective chromosome segregation that is vital for cell cycle progression [9]. Earlier studies on ESCO2 discovered that human ESCO2 mutations result in the inherited developmental disorder known as Roberts syndrome, which is brought on by SCC defects and incorrect transcription of acetylation[10]. ESCO2 is linked to the development of several malignancies according to previous researches, making it a reliable biomarker and prospective therapeutic target. ESCO2 is significantly upregulated in kidney cancer tissues, and ESCO2 knockdown prevented the growth, invasion, and migration of cancer cells via controlling the AKT/mTOR pathway[11]. Zhu et al. also demonstrated that ESCO2 promotes Lung adenocarcinoma (LUAD) cell proliferation and metabolic reprogramming of metastasis in vitro and in vivo[12]. ESCO2, however, was capable of inhibiting cancer metastasis in colorectal cancer via reducing MMP2 expression[13]. As of right now, no research has explored the expression of ESCO2 and its effector function in HCC.

In this study, multi-source high-throughput data enabled us to identify that ESCO2 was significantly elevated in HCC tissues and HCC patients with high ESCO2 expression possessed poorer prognosis. In addition, the knockdown of ESCO2 markedly suppressed HCC cell proliferation in vivo and in vitro. Most notably, ESCO2 may promote PI3K/AKT/mTOR pathway, accelerating the cell cycle and inhibiting apoptosis and thus increasing HCC growth. Hence, ESCO2 is a viable molecular target for HCC.

2.1 Data preparation and Differential Expression Analysis

The bulk mRNA sequencing profiles of Liver hepatocellular carcinoma (LIHC) and normal liver tissue samples were acquired from the international cancer genome consortium (ICGC) and the Cancer Genome Atlas (TCGA) database. Simultaneously, we obtained the single-cell RNA sequencing matrix from GEO149614[14], which included 10 tumor tissues and seven normal ones. Spatial transcriptome sequencing data (HRA000437)[15] was accessed from Genome Sequence Archive (GSA). We selected the spatial transcriptomics data of HCC leading-edge sections.

The TCGA database was first utilized to assess the difference in ESCO2 mRNA value between tumor and normal samples. HCCDB database[16]-a powerful platform that incorporated all HCC mRNA sequencing data-was also used to examine the ESCO2 expression level. In addition, we utilized the Seurat R package to process spatial transcriptomics data and performed log-normalization to standardize data. We represented the expression level of ESCO2 and its distribution on the leading-edge sections utilizing Seurat's SpatialFeaturePlot function.

2.2 Survival Analysis and Functional Enrichment Analysis

We carried out survival analysis using the survival R program. The Kaplan-Meier survival curve was modeled using the survfit function. The "surv cutpoint" function of the survminer R package was implemented to determine the optimal cutoff point. Using the optimal cutoff point for gene expression, we then split patients into two groups and modeled the Kaplan-Meier survival curves. We further calculated the correlation between clinical data (tumor size, stage, grade, and survival status) and the ESCO2 expression value.

To explore the biological functions of ESCO2 in HCC, individuals in LIHC cohort were split into ESCO2-high and ESCO2-low subsets on the basis of ESCO2’s median value. Gene Set Enrichment Analysis (GSEA) was performed to ascertain whether priori defined gene sets exhibit significant differences between two subgroups. Two HCC RNA-seq datasets, TCGA-LIHC and ICGC datasets, were then utilized to identify ESCO2-related regulatory genes. The individuals were divided into ESCO2-high and ESCO2-low subsets relied on ESCO2’s median value in each dataset. Subsequently, a differential expression analysis between ESCO2-high and ESCO2-low subgroup was conducted to detect the differentially expressed genes (DEGs) (p < 0.05, Log FC ≥ 1). The association between ESCO2 and these DEGs was later determined using Spearman's correlation analysis in the two datasets (p < 0.05, Cor ≥ 0.3). Intersecting genes for these closely related genes were identified as ESCO2-related regulatory genes. Finally, the effector function of these regulatory genes was ascertained using the Metascape online platform.

2.3 Single-Cell Analysis

All cells were filtered through quality control (RNA counts were ranged from 200 to 7000; and mitochondrial gene expression percentages were less than 5%). Data were normalized using the “LogNormalize” approach with 10,000 scale factor. The Seutat's CellCycle Scoring function determines each cell's cell cycle score. Using the Seutat’s ScaleData function, influence of UMI’s and percent mitochondrial content was regressed out. Next, the batch effect was eliminated through the harmony R package. The top 30 principal components and top 2000 variable genes were picked for cell clustering and the uniform manifold approximation and projection (UMAP) visualization. The canonical marker genes identified in previous studies were applied to mark the cell type. DEGs were identified utilizing FindMarkers function. Then the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for DEGs were implemented using clusterProfiler R packages[17]. In addition to that, we used Gene Set Variation Analysis (GSVA) to detect the expression score of 50 hallmaker pathways among cell clusters.

2.4 Cell culture and transfection

Huh7 and MHCC97H cell lines were acquired from ICell Bioscience Inc (Shanghai, China). At 37°C, DMEM medium (Gibco BRL Life Technologies Inc., USA) 10% fetal bovine serum (FBS) (Gibco BRL Life Technologies Inc., USA) and 1% penicillin-streptomycin (Hy-clone, CA, USA) was applied to cultivate all of the hepatoma cell lines. Two independent small interfering RNAs (siRNA) were assigned to assure the effectiveness of siRNA in suppressing ESCO2 expression. siRNA was ordered from GenePharma, and the siRNA sequences were listed in Table S1. According to the manufacturer's protocol, we transfected siRNA into the hepatoma cell line when the cells filled 70–80% of the culture plate with transfection reagent (Thermo Fisher Scientific, United States).

ShRNA knockdown for ESCO2 and negative controls were orders from GenePharma. Huh7 and MHCC97H cell lines were infected with lentiviral particles targeting ESCO2 followed by manufacturer's protocol. The stable transfectants were acquired after following 10 µg/mL puromycin selection (Sigma-Aldrich, St. Louis, MO, USA). The sequence of the shRNA for ESCO2 was 5'-GCAAATCAAGGCTCACCAT-3'.

2.5 RT-qPCR and Western blotting

The cells were harvested for RNA and protein extraction after 48 and 72 hours of siRNA transfection, respectively. Total RNA was extracted employing TRIzol reagent (Invitrogen). We then quantified RNA utilizing the One-Step SYBR Prime Script RT-PCR kit (Takara, Japan) and synthesized cDNA with the cDNA Reverse Transcription Kit. The ESCO2 expression was standardized to GAPDH and calculated through the 2^−△△CT approach. Table S1 lists the primers that were applied. (Sangon biotech).

Cells were harvested and incubated with RIPA lysis buffer (Thermo Fisher Scientific, USA) supplemented with a protease inhibitor cocktail (Roche, Switzerland) on ice for 30 minutes to extract cellular proteins. Tumor tissue proteins were isolated using the Tissue Protein Extraction Reagent (Thermo Fisher Scientific, USA) following the protocol. Utilizing the BCA assay (Thermo Fisher Scientific, USA), the protein concentration was measured. Equivalent amounts of total proteins from each group were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a nitrocellulose membrane. Following a 1-hour blocking step in 5% non-fat milk, primary and secondary antibodies (Table S1) were applied according to standard procedures. Ultimately, protein bands were visualized using SuperKine™ West Femto Maximum Sensitivity Substrate (Abbkine) and detected utilizing an imaging system (Tanon, Shanghai, China). The grayscale of the protein bands was quantified using Image J software. All antibodies used are listed in Table S1.

2.6 Cell proliferation and colony formation assay

For the CCK-8 experiment, 2000 cells were planted into 96-well plate, transfected, and cultured for a set period of time. Each well received 10 µL CCK8 reagent. After 2h incubation, the absorbance (450 Nm) was measured. To evaluate colony formation capacity,1000 cells were inoculated into 6-well plates and cultured for 12 days. Cells are fixed and stained when visible colonies appear. The colonies in each well are then counted.

2.7 Ethynyl-2-Deoxyuridine (EdU) Incorporation Assay

Using an EdU imaging analysis kit (Abbkine, Wuhan, China), cell proliferation was found. Following the inoculation of 50,000 cells per 15 mm glass-bottom culture dish, the cells were transfected. In accordance with the manufacturer's instructions, A suitable volume of Edu (ApexBio Technology LLC) was added after removing the entire medium, and the cells were then incubated for 2 hours. Nuclei were counterstained with DAPI (Sigma-Aldrich, USA) for 10 minutes. A fluorescence microscope (Nikon, Tokyo, Japan) was then used to fix and stain the cells such that the percentage of EdU-positive cells (red) could be seen and determined.

2.8 Flow cytometry related to cell cycle and apoptosis

Collect and wash both normal and transfected cells. Employ fluorescent labeling dyes, Annexin V and PI (Beyotime), for apoptosis labeling and incubate for 30 minutes. Analyze individual cells for distinct fluorescence intensities using a flow cytometer (BD Biosciences). Process the data with FlowJo software to obtain the percentage of apoptotic cells.

Cells were trypsinized and then resuspended in complete growth medium. Following PBS washing, cells were subsequently fixed overnight in 70% cold ethanol.Stain the collected cells using the Cell Cycle and Apoptosis Analysis Kit (Beyotime). Employ a flow cytometer (BD Biosciences) to determine the distribution of cells with different DNA content. Analyze and process the data using FlowJo.

2.9 In vivo proliferation experiment

All animal procedures were authorized by the Animal Ethics Committee of Tianjin Medical University. We purchased four-week-old female BALB/c-nude mice from SPF (Beijing) Biotechnology Co., Ltd. 1 × 10⁷ MHCC97H cells with stable ESCO2 knockdown or control RNA were injected subcutaneously into the right flank of each nude mice (n = 5 per group). On day 21, mice were executed, and the tumors were removed. Tumor tissues were then fixed, paraffin-embedded, sectioned, and stained with anti-Ki67 antibody (dilution 1:200, Affinity BioSciences, Cincinnati, OH, USA). The slide images were examined and quantified by a pathologist.

2.10 Statistical analysis

Analyses between the two groups were performed using the Wilcoxon test, while the one-way analysis of variance (ANOVA) test was utilized for three or more groups. R studio and GraphPad software were employed for all statistical calculations.

3.1 ESCO2 is overexpressed in HCC tissues and associated with poor prognosis

Previous researches have demonstrated that ESCO2 is markedly upregulated in lung, kidney, and colorectal malignancies[11–13]; however, no studies have specifically focused on the ESCO2 expression in HCC. Using the TCGA database, we first identified that HCC samples had considerably greater ESCO2 mRNA levels than normal tissues (Fig. 1A). We also discovered ESCO2 was significantly overexpressed in the majority of HCC tissues using RNA-seq data from 50 matched cohorts of HCC and paracancerous tissues (Fig. 1B). The HCCDB database analysis of eight independent HCC study cohorts indicated ESCO2 expression was considerably higher in HCC tissues than normal ones (Fig. 1C). Finally, we employed spatial transcriptomic data from two HCC leading-edge sections to further confirm whether ESCO2 is overexpressed in HCC tissues (Fig. 1D). The findings demonstrated that HCC tissues displayed a considerable overexpression of ESCO2 compared to nearby non-tumor tissues.

The potential of ESCO2 as a prognostic biomarker was then explored as ESCO2 was overexpressed in HCC tissues. Kaplan-Meier survival analyses for HCC patients were performed using the TCGA database to determine the relationship between ESCO2 expression and prognosis of HCC patients, concentrating on OS, DFI, DSS, and PFI. Upregulated ESCO2 expression was significantly related to poor outcomes (Fig. 2A). Next, we evaluated the connection between survival outcomes and ESCO2 expression in the ICGC cohort. Similarly, HCC patients with increased ESCO2 expression demonstrated poor OS in ICGC dataset (Fig. 2A). To substantiate the involvement of ESCO2 in cancer progression, the association between ESCO2 expression and clinical data was evaluated. ESCO2 expression value positively correlated with tumor stage, size, and grade and survival status (Fig. 2B). Furthermore, univariate and multivariate Cox regression algorithms revealed the ESCO2 expression value was a prognostic indicator independent of certain other clinical indicators (Fig. 2C and 2D). In summary, the current analysis identified ESCO2 as a potential promoter of HCC initiation and progression.

3.2 ESCO2 regulates the cell cycle

Our study demonstrated that ESCO2 is a trustworthy biomarker for HCC and further investigated the effector function of ESCO2 in the formation of tumor. HCC patients were first split into the ESCO2-high and -low subsets based on ESCO2 median value to explore the oncogenic role of this protein. The gene sets enriched in both groups was identified via GSEA. The top-10 NSE-ranked enriched pathways were visualized in this study. For ESCO2-high subset, upregulated genes demonstrated the enrichment of G2M checkpoint, PI3K ATK MTOR signaling, and mTORC1 signaling, which were connected to cell proliferation and cell cycle. In addition, the apoptosis and P53 pathway was also significantly enriched in the ESCO2-high subset (Fig. 3A). The TCGA and ICGC databases were then utilized to explore ESCO2-regulated genes so that we could investigate the regulatory pathways of ESCO2 in more detail as described in methods. We identified 128 ESCO2-related regulatory genes (Fig. 3B). These genes showed enrichment of cell cycle, mitotic cell cycle process, cell cycle phase transition and mitotic cytokinesis (Fig. 3C). We further create a PPI network of ESCO2-related regulatory genes using the String database[18] in order to identify the core genes. The top 5 core genes are CDK1, BUB1, BUB1B, CCNB1, and CCNB2, all of which encode essential proteins for controlling the cell cycle (Fig. 3D). Cyclin-dependent kinase 1 (CDK1) is essential to facilitate the G2M transition, control the G1 process, and govern the G1-S transition[19]. Numerous studies have demonstrated that CDK1 dysregulation causes chromosomal instability, aggressive tumor growth, and accelerated cell proliferation[20]. CDK1 inhibitors are potential effective anti-cancer small-molecule drug. For instance, in HCC PDX tumor model, treatment with the CDK1 inhibitor RO3306 in conjunction with sorafenib dramatically decreased tumor growth[21]. Similarly, the BUB1 and BUB1B are crucial for preserving correct chromosomal segregation and reducing aneuploidy formation during mitosis[22]. Experiments have showed that BUB1 and BUB1B can booster cancer cell proliferation and are promising therapeutic targets[23–25]. The appropriate regulation of the G2/M transition phase depends on the presence of CCNB1 (Cyclin B1), which is located on 5q13.2. CCNB1 is aberrantly expressed in a number of malignancies and highly related with patient prognosis[26, 27]. HCC tissues displayed considerably higher levels of CCNB1, and HCC cell proliferation may be suppressed by CCNB1 knockdown. [28]. In conclusion, our bioinformatic analysis suggests ESCO2 regulates pathways connected to the cell cycle.

3.3 single-cell analysis of ESCO2

Cancers develop in complex organic environments that they rely on for continued growth, invasion, and metastasis. The tumor microenvironment (TME) is critical for tumor development and immune escape of tumor cells[29]. Stromal and immune cells in the TME are genetically stable, in contrast to tumor cells. Modifying TME components has promise for overcoming the pervasive issue of immunotherapy resistance. As a result, current cancer research has shifted from cancer cell-centric to non-tumor cell-centric[30]. Owing to the TME's high levels of heterogeneity and flexibility, oncogenes may play unique functions in each cell type. The functional study for oncogenes is not accurate enough using traditional bulk RNA sequencing data. We then employed single-cell analysis to explore the effector function of ESCO2 in malignant cells at the single cell solution. It was discovered myeloid cells, B cells, T/NK cells, and malignant cells all expressed ESCO2 (Fig. 4A and 4B). The hepatoma cells were then extracted and divided into ESCO2-positive and ESCO2-negative subgroups according to ESCO2 expression level. The GSVA results showed that E2F targets, G2M checkpoint, and MYC targets signaling were enriched in ESCO2-positive cells, which is in line with our previous bioinformatics results (Fig. 4C). DEG procedure was performed between ESCO2-positive and ESCO2-negative subgroups (Fig. 4D). GO enrichment analysis identified the DEGs were mainly enriched in terms associated to proliferation, including regulation of mitotic cell cycle, nuclear division, and sister chromatid segregation. Furthermore, DEGs showed the enrichment of DNA replication and Cell cycle in KEGG analysis (Fig. 4E). After that, when the cell cycles of the two aforementioned groups were compared, more ESCO2-positive group cells were observed to be in the G2/M phase, suggesting a more robust capacity for proliferation (Fig. 4F).

3.4 ESCO2 knockdown inhibits the growth of HCC cells in vitro and in vivo

We first validated the knockdown efficiency of ESCO2-siRNA in hepatoma cell lines.

Western blotting and PCR results confirmed that two siRNAs were effective in downregulating ESCO2 in the mRNA and protein levels (Fig. 5A,5B,6A,6B). After confirming ESCO2's knockdown efficiency, we carried out several in vitro experiments to examine how ESCO2’s knockdown affected the biological activity of HCC cells. First, the CCK8 assay revealed in Huh7 and MHCC97 cell lines, the absorbance of the siRNA1# and siRNA2# groups were considerably lower than the si-NC group at 24 h, 48 h, and 72 h (Fig. 5C and 6C). Similarly, EDU incorporation Assay demonstrated that the proliferation of HCC cells drastically halted by ESCO2 knockdown (Fig. 5E and 6E). Intriguingly, the clone formation experiment found that at day 11, a significantly smaller number of cell clones were formed following the knockdown of ESCO2 (Fig. 6D and 6D). The results presented above all indicated that ESCO2 knockdown suppressed the ability of hepatoma cell lines to proliferate.

We then constructed a subcutaneous xenograft mouse model to examine the effects of regulating ESCO2 expression on HCC cell proliferation in vivo. MHCC97H cells stably transfected with sh-ESCO2 or sh-Control were injected into the flanks of nude mice, and then tumor volumes were recorded periodically. Tumor volume and weight were considerably reduced in the sh-ESCO2 group than in the sh-Control animals (Fig. 7A and 7B). We then removed the tumors for IHC analysis. The results exhibited the Ki-67 level was substantially lower in mice injected with ESCO2-knockdown MHCC97H cells than in the corresponding control mice (Fig. 7C and 7D). Taken together, these findings demonstrate ESCO2 could promote the growth of HCC cells in vivo and in vitro.

3.5 Knockdown of ESCO2 inhibits HCC cell proliferation by inducing G1-S cell cycle arrest and stimulating apoptosis in vitro

According to our bioinformatics investigation, ESCO2 primarily participates in cell cycle regulation. Therefore, flow cytometry was performed to study the cell cycle of HCC cells after ESCO2 knockdown. In comparison to sh-NC group, the sh-ESCO2 group exhibited a considerably larger percentage of the G1 stage cells (Fig. 8A). This finding indicates that ESCO2 knockdown could trigger S- phase arrest in HCC cells. In addition, apoptosis-related flow cytometry results showed the proportion of apoptotic cells in the MHCC97H and Huh7 cell lines was considerably greater in the sh-ESCO2 group than in the sh-NC (Fig. 9A). As a result, ESCO2 knockdown could stimulate apoptosis in HCC cells.

3.6 Investigation of the mechanism by which ESCO2 regulates the development of HCC cells

We subsequently investigated the specific oncogenic mechanism of ESCO2 after confirming that ESCO2 knockdown prevents HCC cell growth by stimulating G1-S phase cell cycle arrest and apoptosis. First, in Huh7 and MHCC97H cell lines, western blotting of proteins associated to the cell cycle revealed the sh-NC group exhibited increased levels of CDK1 and cyclin B1 expression compared to the sh-ESCO2 group. In addition, CDK2 and cyclin A2 protein expression was significantly decreased after ESCO2 knockdown (Fig. 8B). These findings imply that ESCO2 knockdown causes cell cycle arrest via controlling the CDK1/CDK2 signaling pathways. Subsequently, western blotting of apoptosis-related proteins demonstrated that the inhibition of ESCO2 significantly increased the expression value of apoptosis-positive regulators (BAX and Caspase-3). However, compared to the sh-NC group, the sh-ESCO2 group displayed significantly decreased expression of the apoptotic negative regulatory protein (BCL-2) (Fig. 9B). Hence, ESCO2 knockdown can trigger apoptosis in HCC cells by controlling these apoptosis-specific proteins. Meanwhile, considering the GSEA results showed that ESCO2 might be implicated in the control of PI3K/AKT/mTOR signaling. Furthermore, the PI3K/AKT/mTOR pathway is frequently activated on cancer and is crucial for controlling the cell cycle and apoptosis. We then investigated how ESCO2 knockdown affected the PI3K/AKT/mTOR pathway. The bands of P-PI3K, P-AKT, and P-mTOR were significantly weaker in sh-ESCO2 cells compared to control cells, indicating that knockdown of ESCO2 dramatically inhibited the PI3K/AKT/mTOR pathway (Fig. 9C). To sum up, ESCO2 could influence the expression of proteins involved in the cell cycle and apoptosis by enhancing the PI3K/AKT/mTOR signaling, which supports the growth of HCC cells.

Advanced HCC is characterized by a poor prognosis. Despite numerous significant advancements in systemic therapy have increased survival time in this population, the median overall survival is still extremely short. As the current first-line treatment for advanced HCC, atezolizumab/bevacizumab therapy shown superior efficacy and tolerability over sorafenib[31]. Other drug combination treatment options are being tested right now. Therefore, the effective combination of immunotherapy and targeted medicine is research highlight to further improve the prognosis of advanced HCC patients. Sequencing technology advancements present greater possibilities to identify diagnostic and therapeutic targets that could benefit HCC patients through early detection, precise treatment, and prognostic monitoring. Cell division and cell death are the basic processes that govern organism growth and development. Both processes are abnormally regulated in cancer, which causes uncontrolled proliferation[32]. Uncontrolled cancer growth and metastasis are the main causes of liver cancer's high mortality and recurrence rates. Understanding the HCC proliferation mechanism would therefore aid in the discovery of innovative and better therapeutic strategies to raise the survival rate. In previous studies, ESCO2 was identified as an effective target for cancer therapy, which is a pivotal protein in the cell division process. Acetylation of the SMC3 subunit of the adhesive protein via ESCO2 acetyltransferase facilitates SCC. The cohesiveness of the cohesin protein complex between sister chromatids ensures correct chromosomal segregation[33, 34]. ESCO2 was noticeably elevated in tumor tissues in several malignancies and ESCO2 knockdown could prevent cancer cell proliferation, invasion, and migration. However, the mechanism underlying ESCO2 upregulation for cancer development is still not fully elucidated. ESCO2's potential as a therapeutic target for HCC remains unknown. Our research identifies the mechanism by which ESCO2 supports HCC proliferation. We believe that ESCO2 may be a valuable molecular diagnostic marker and potential target for HCC patients.

Our work first identified ESCO2 expression was significantly upregulated in HCC tissues using extensive HCC sequencing data. Thanks to the public database, our data contains 1446 HCC tissues and 943 normal ones, making our results more robust. Using spatial transcriptome sequencing data, we additionally analyze the expression level of ESCO2 and its distribution on the leading-edge sections. Because ESCO2 is highly expressed in HCC tissues, it may be an oncogene that promotes the hepatocarcinogenesis. Moreover, upregulated ESCO2 expression had a strong association with worse outcomes in HCC patients. ESCO2 expression has a positive relationship with tumor grade, size, and stage. To some extent, tumor stage and grade represent the progression of liver cancer. Hence, ESCO2 may significantly contribute to the HCC progression. Finally, we verified that ESCO2 was an independent prognostic indicator for HCC patients. According to the aforementioned findings, ESCO2 is a trustworthy biomarker for HCC patients. Oncogenes are known to have an impact on cancer cells' biological behavior (migration, invasion, and proliferation), thereby assisting the progression of cancer. Bioinformatic analysis implied that ESCO2 expression was closely related to hepatocarcinogenesis and development, so we employed siRNA to inhibit ESCO2 expression in HCC cell lines. Our cellular experiments supported the conclusion that ESCO2 knockdown significantly suppressed the ability of HCC cell lines to proliferate. Meanwhile, the subcutaneous xenograft nude mouse model also confirmed that ESCO2 could promote the growth of HCC cells in vivo. As a result, we were able to confirm that ESCO2, a potential oncogene, may encourage the growth of HCC and result in a bad prognosis by encouraging the biologically malignant behavior of HCC cells. In previous studies, lung, kidney, and stomach cancer cells' capacity for proliferating and migrating could be enhanced by ESCO2. So our findings concur with earlier research.

Oncogenes typically regulate cancer cell biology through modifying cellular signaling pathways. Previous research found that ESCO2 affects the AKT/mTOR pathway in kidney cancer[11]. Furthermore, ESCO2 knockdown inhibited mTOR/RPS6K1 activation and upregulated AMPKα and p53 phosphorylation in gastric cancer cells[35]. In lung cancer, ESCO2 acetylates hnRNPA1, maintaining it in the nucleus and ultimately enhancing aerobic glycolysis by increasing PKM2 expression and lowering PKM1 expression[12]. We first explored the effector function of ESCO2 in HCC via bioinformatic methods. GSEA result revealed that ESCO2 participants in E2F targets, G2M checkpoint, PI3K ATK MTOR signaling, and mTORC1 signaling, which were related to cell cycle and proliferation. We also identified 128 ESCO2-related regulatory genes. These genes showed enrichment of cell cycle, mitotic cell cycle process, cell cycle phase transition and mitotic cytokinesis. In addition, our study explored the ESCO2’s biological function at the single-cell level, which avoids the interference of other non-tumor cells. Consistent with the results of Bulk RNA sequencing data analysis, cell proliferation-associated pathways, such as E2F targets, G2M checkpoint, and MYC targets signaling, were significantly enriched in ESCO2-positive hepatoma cells. In conclusion, our bioinformatic analysis suggests ESCO2 possesses significance in regulating cell cycle and cell proliferation-related pathways.

We delved into the ESCO2's role in controlling the cell cycle and cell growth through a series of experiments. Flow cytometry demonstrated a significantly larger percentage of G1 phase HCC cells after ESCO2 knockdown. In addition, ESCO2 knockdown regulated the expression levels of HCC cell-cycle proteins (CDK1, cyclinB1, CDK2, and cyclin A2), resulting in cell cycle arrest in G1 phase. CyclinB1 is a crucial cyclin that regulates the G2/M phase. During the S and G2 phases in cell cycle, cyclin B is synthesized in significant amounts, it then enters the nucleus, binds to CDK1, and activates the G2 to M phase transition[26]. Similarly, CyclinA2 (CCNA2) is a regulator of essential CDK protein kinases. CyclinA2 binds and activates CDK2, thereby promoting the transition through G1/S and G2/M[36]. Hence, ESCO2 knockdown causes cell cycle arrest via controlling the CDK1/CDK2 singling pathway. Human malignancies frequently exhibit cell cycle dysregulation, and targeting cell cycle proteins is one of the most promising areas for cancer therapy. Scientists have demonstrated that it is effective to target proteins associated with the cell cycle in order to limit tumor growth. For instance, researchers identified that homoharringtonine (HHT) binds to the PPI site of CDK2, interrupting the interaction between CDK2 and Cyclin A, resulting in a loss of CDK2 activity and protein degradation, and preventing tumor progression[37]. In addition, all three CDK4/6 inhibitors were authorized by the United States Food and Drug Administration (FDA) for breast cancer treatment[38]. Therefore, ESCO2 may be a promising target for cell cycle-related protein targeting. Furthermore, we discovered that ESCO2 knockdown stimulated apoptosis in HCC cells. Western blotting results displayed knockdown of ESCO2 increased the level of apoptosis signature proteins (BAX, Caspase-3), thus promoting apoptosis in HCC cells. A crucial step in the death of tumor cells is apoptosis. Evasion of apoptosis contributes to cancer cell escape and resistance to treatment. As a result, a lot of cancer therapies depend on successful apoptotic induction[39]. Numerous drugs targeting apoptosis-related proteins have been developed with exciting outcomes[40]. Furthermore, our research discovered that ESCO2 controls cell cycle and apoptosis via promoting the PI3K/AKT/mTOR pathway. The PI3K/Akt/mTOR signaling pathway is indispensable in many cellular biological processes, including cell proliferation, survival, metabolism, motility, angiogenesis, and response to stress and therapy. Its critical function in controlling tumor growth, metabolism, metastasis, and treatment resistance is supported by extensive researches[41, 42]. Small molecule inhibitors that target the key kinases in the PI3K/Akt/mTOR pathway have therefore been created and assessed in preclinical models and human clinical trials. For instance, the FDA authorized idelalisib, a PI3K inhibitor, to treat follicular b-cell non-Hodgkin's lymphoma and relapsed chronic lymphocytic leukemia[43]. There are now over 35 clinical trials targeting on the PI3K/Akt/mTOR pathway in HCC. However, outcomes show that the clinical benefit of monotherapy with these inhibitors remains limited[44]. This could be as a result of the PI3K/Akt/mTOR pathway interacting with multiple signaling pathways, which increases the likelihood of adverse events and treatment resistance. combination therapy may a better strategy for advanced HCC. Understanding crosstalk and feedback with other pathways will therefore be key challenges in targeting PI3K/Akt/mTOR pathway. Future studies should proceed to explore the upstream and downstream regulatory mechanisms of PI3K/Akt/mTOR pathway, which will offer crucial insights for creating new therapeutic targets or proposing novel combination therapeutic regimens. Our study will provide valuable suggestions for the drug development targeting PI3K/Akt/mTOR.

In our work, we determined ESCO2 was significantly up-regulated in HCC tissues and increased ESCO2 expression was linked to a poorer prognosis.

Knockdown of ESCO2 significantly inhibited HCC cell proliferation in vivo and in vitro. Most notably, ESCO2 may promote PI3K/AKT/mTOR pathway, accelerating the cell cycle and inhibiting apoptosis and thus increasing HCC growth. These findings offer innovative and valuable ideas for HCC targeted therapy.

However, this study contains some limitations. First, investigations on the specific mechanism of ESCO2's involvement in HCC are still insufficient, which is the future study focus. Further, our study lacks a real-world clinical cohort to judge the predictive power of ESCO2 for prognosis in HCC.

In a nutshell, our team reveals the molecular mechanism by which ESCO2 promotes HCC proliferation for the first time. ESCO2 may promote PI3K/AKT/mTOR, accelerating the cell cycle and inhibiting apoptosis and thus increasing HCC growth. Overall, this work provides a novel HCC target gene, which offer valuable information for HCC prognostic assessment and molecular therapy.

Funding

This work was supported by the Tianjin Natural Science Foundation (21JCYBJC00320), the Tianjin Science and technology project (19ZXDBSY00010), and the Tianjin Health Science and technology project (TJWJ2021ZD002 and ZC20218).

Author Contributions

The research scheme was developed by DPC and YH, who also carried out the bioinformatics analysis. YH and DPC performed the experiments. YB and YCZ collected and organized the gene expression matrix. DPC and YH wrote the manuscript. JQZ and YMZ provided administrative assistance while critically examining the article for crucial intellectual content. JQZ, ZHZ and YMZ were the guarantors for this study.

Data Availability

The datasets involved in our work are available in the TCGA, GEO and GSA.

Competing Interests

The authors have declared that no competing interests exists.

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The authors declare no competing interests.

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The antibodies, primers, oligonucleotides and shRNAs used in this study are shown.

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ESCO2 promotes the proliferation of hepatocellular carcinoma through PI3K/AKT/ mTOR signaling pathway

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Abstract

Figures

1. Background

2. Methods and Materials

2.1 Data preparation and Differential Expression Analysis

2.2 Survival Analysis and Functional Enrichment Analysis

2.3 Single-Cell Analysis

2.4 Cell culture and transfection

2.5 RT-qPCR and Western blotting

2.6 Cell proliferation and colony formation assay

2.7 Ethynyl-2-Deoxyuridine (EdU) Incorporation Assay

2.8 Flow cytometry related to cell cycle and apoptosis

2.9 In vivo proliferation experiment

2.10 Statistical analysis

3. Results

3.1 ESCO2 is overexpressed in HCC tissues and associated with poor prognosis

3.2 ESCO2 regulates the cell cycle

3.3 single-cell analysis of ESCO2

3.4 ESCO2 knockdown inhibits the growth of HCC cells in vitro and in vivo

3.6 Investigation of the mechanism by which ESCO2 regulates the development of HCC cells

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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