Partial Hepatectomy Promotes the Development of KRASG12V-Induced Hepatocellular Carcinoma in Zebrafish

Simple Summary

Partial hepatectomy (PH) is a common clinical surgery for managing hepatocellular carcinoma (HCC). A common concern about PH is the high tumor recurrence rate following PH. Previous studies have reported that PH can promote the growth of transplanted HCC in rodents. In the current study, we have used an inducible kras^G12V-driven zebrafish HCC model to investigate the effects of PH on the oncogene-induced de novo HCC development. We found that PH can significantly promote the development of kras^G12V-induced HCC in zebrafish. This enhancing effect could be attributed to the increased oxidative stress and the enhanced deregulation of molecular factors. Our findings may provide references for the future development of novel therapeutic strategies.

Abstract

The purpose of this study was to investigate the effects of PH on the development of oncogenic kras^G12V-induced HCC in zebrafish. The inducible HCC model in Tg(fabp10a:rtTA2s-M2; TRE2:EGFP-kras^G12V) zebrafish was used. PH or sham surgery was performed before the induction of oncogenic kras^G12V expression in the livers of transgenic zebrafish. Histological analysis was carried out to determine the progression of HCC and other HCC-associated features including hepatocyte proliferation, extracellular matrix production, and local oxidative stress. The similarity between the process of PH-induced liver regeneration and that of kras^G12V-induced HCC development was further compared by RNA-Seq analysis. The results show that PH promotes the development of kras^G12V-induced HCC in zebrafish possibly through enhancing neutrophil-mediated oxidative stress and promoting the upregulation of s100a1, and the downregulation of ribosome biogenesis.

1. Introduction

Partial hepatectomy (PH) is a major method for treating early- and mid-stage hepatocellular carcinoma (HCC), especially in regions with a shortage of liver donors and limited medical resources [1,2,3]. It is a well-established procedure to improve HCC patients’ survival, as the 5-year overall survival rate after the surgery reaches 40–75% [1,3,4,5]. Nevertheless, up to 80% of patients who received PH have HCC recurrence within 5 years, which prevents patients from having better prognoses [6].

The process of liver regeneration following PH has been suggested to activate a similar set of regulators with the process of HCC development [7,8]. Previous studies have suggested that PH can create a microenvironment in favor of HCC growth and metastasis with enhanced angiogenesis, cell dedifferentiation, and expression of growth factors [9,10,11]. This idea has been supported by studies using the rodent PH model with transplanted HCC [12,13,14,15,16]. According to these studies, the growth and metastasis of transplanted HCC cells and xenografts were facilitated by PH in rodents. Nevertheless, these studies only simulate the spread of a pre-existing HCC population but not the de novo development of HCC following PH.

Zebrafish have been widely used in cancer research due to their high capacity for forward genetics and phenotype-based, high-throughput screening [17]. Chemical carcinogen-induced zebrafish HCC has shown histology and gene signatures that resemble human HCC, which demonstrate the modeling potential of zebrafish for HCC [17,18]. The EGFR downstream effector RAS is a major oncogenic factor for human cancer, and KRAS mutation is the most frequent isoform among RAS mutations in liver cancer patients [19,20]. KRAS^G12V is a hyperactive mutation of KRAS due to its reduced activity of GTPase. To model KRAS mutation-induced HCC with zebrafish, Tg(fabp10a:rtTA2s-M2; TRE2:EGFP-kras^G12V) (gz32Tg) (abbreviated as kras+), which has the liver-specific expression of kras^G12V under the control of the Tet-on system, has been generated by our lab previously [21]. This inducible HCC model provides an excellent platform for studying the de novo development of HCC because of its rapid and robust tumorigenesis and the reversible induction of HCC.

Tumor recurrence following PH can occur in two forms: spread of remaining tumor cells and the de novo development of new tumors [2]. How PH affects the de novo development of HCC has not been investigated before. In the current study, we have applied the kras+ zebrafish to investigate the effects of PH on the de novo development of oncogene-induced HCC. We have found that kras^G12V-induced HCC developed faster in the PH-treated livers than in the control livers. Furthermore, phenotypes associated with HCC development such as hepatocyte proliferation, production of extracellular matrix, recruitment of neutrophils, and local oxidative stresses were enhanced by PH. Transcriptomic analysis revealed the overlap of several deregulated genes between the PH livers and the kras+ livers. Both PH-induced liver regeneration and kras^G12V-induced HCC development caused significant inhibition of ribosome biogenesis in the zebrafish livers. Together, PH can promote the development of kras^G12V-induced HCC in zebrafish, probably through the enhanced oxidative stresses and inhibited ribosome biogenesis in livers.

2. Materials and Methods

2.1. Zebrafish Husbandry

The maintenance of the zebrafish used in the current study followed the Institutional Animal Care and Use Committee guidelines from the Southern University of Science and Technology (SUSTech), China, and the National University of Singapore (NUS). The transgenic zebrafish used in the current study include Tg(fabp10a:DsRed; ela3l:EGFP) (gz15Tg) [22], Tg(lyz:DsRed) (nz50Tg) with DsRed-labelled neutrophils [23]; Tg(fabp10a:rtTA2s-M2; TRE2:EGFP-kras^G12V) (gz32Tg) with the inducible liver-specific expression of oncogenic kras^G12V in a Tet-On system [21]; and double-transgenic zebrafish Tg(fabp10a: rtTA2s-M2; TRE2: EGFP-kras^G12V; lyz: DsRed) generated by crossing Tg(fabp10a:rtTA2s-M2; TRE2:EGFP-kras^G12V) with Tg(lyz:DsRed), which were designated as Lipan, lyz+, kras+, and kras/lyz+, respectively.

2.2. Zebrafish Partial Hepatectomy

Adult male zebrafish (>12 weeks old) were used in the experiments of partial hepatectomy (PH). The procedure of performing PH on zebrafish was derived from the published protocol [24]. In general, after anesthesia with 150 mg/L of buffered tricaine (MS-222), a 2–3 mm incision was made on the zebrafish abdomen with a micro-stab knife and precise scissors to expose the liver. Subsequently, the ventral lobe of the liver was removed with tweezers, resulting in approximately 30% of the liver being resected. Sham surgery was performed as a control by making an incision on the abdomen without removing the liver tissue.

2.3. Induction of the Transgenic Oncogene Expression

To induce the expression of transgenic kras^G12V in the liver of kras+ zebrafish, adult and larvae kras+ zebrafish were exposed to 20 mg/L of doxycycline (Dox) (D9891; Sigma-Aldrich, St. Louis, MO, USA). For larvae exposure, Dox treatment began at 3 dpf for 48 h. For adult exposure, Dox treatment was continued for up to 7 days. During Dox treatment, the zebrafish were kept in dim light to prevent light-sensitive degradation of Dox.

2.4. RNA Extraction and RT-qPCR

The total RNA extraction from tissues and embryos was performed using TRIzol reagent (Invitrogen, Waltham, MA, USA), followed by reverse transcription using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). The synthesized cDNA was used for real-time quantitative PCR (RT-qPCR) with SsoAdvanced Universal Supermixes (Bio-Rad Laboratories, Hercules, CA, USA), GoTaq qPCR Master Mix (Promega, Madison, WI, USA) in CFX96 Touch Real-Time PCR System (Bio-Rad Laboratories), and ABI 7500 Fast Dx Real-Time PCR Instrument (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Genes of interest were amplified by 40 cycles using a standard three-step protocol (95 °C, 20 s; 65 °C, 15 s; 72 °C, 30 s). All primers used in RT-qPCR are listed in Table S1.

2.5. RNA-Seq and Bioinformatic Analysis

The RNAs used for RNA-Seq were extracted from the liver tissues with TRIzol reagent. For the RNAs extracted from PH/sham surgery-treated WT zebrafish, library preparation and Illumina sequencing were conducted by Novogen, Hong Kong. Each sample used for RNA-Seq contained RNAs from three individual zebrafish livers, and each biological group contained two pooled samples. For the RNAs extracted from kras+ and WT zebrafish after Dox treatment, library preparation and Illumina sequencing were conducted by Metware Biotechnology, New Zealand. Each sample used for RNA-Seq contained RNAs from two individual zebrafish livers, and each biological group contained three pooled samples. After sequencing, the reads were mapped to zebrafish reference genome assembly GRCz11 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000002035.6/, accessed on 5 March 2023) with TopHat2 (http://ccb.jhu.edu/software/tophat/index.shtml, accessed on 4 January 2021). The raw counts of sequences mapped to each gene were quantified using StringTie (https://ccb.jhu.edu/software/stringtie/, accessed on 4 January 2021), followed by differential expression analysis using DESeq2 [25]. The criteria of significantly deregulated genes were adjusted p-value (padj) ≤ 0.05 and fold change (FC) ≥ 2. To identify enriched pathways, pre-ranked gene set enrichment analysis (GSEA) was performed using the FGSEA package and clusterProfiler package [26,27]. The Hallmark pathway gene sets and KEGG pathway gene sets used for GSEA were downloaded from the Molecular Signature Database and the KEGG database, respectively [28,29]. The differentially expressed genes were ranked by their values of Wald statistic generated from DESeq2.

2.6. Morpholino Knockdown

For s100a1 knockdown, MO-s100a1-ATG (5′-GTGACGACAGCTTGAAAAGATGTT-3′) (Gene Tools, Philomath, OR, USA) was synthesized and applied. Aliquots of morpholino (50 nM) were injected into zebrafish embryos at the one-cell stage. The effect of MO-s100a1-ATG on S100A1 expression was validated in 1-dpf zebrafish embryos with western blotting using the Rabbit-anti-βactin antibody (bs-0061R, Bioss Antibodies, Woburn, MA, USA) (1:5000) and the Rabbit-anti-S100A1 antibody (50266-RP02; Sino Biological, Beijing, China) (1:1000). The total protein extraction from zebrafish embryos and western blotting were conducted as previously described [30].

2.7. Histological Analyses

Adult zebrafish liver and gut tissues were harvested together and fixed in 10% formalin and 4% paraformaldehyde (PFA) in PBS overnight for subsequent paraffin embedding and cryo-embedding, respectively. Paraffin-embedded samples were sectioned at 5 μm with a microtome, and frozen samples were sectioned at 8 μm with a cryostat. All frozen samples were used for immunofluorescence (IF) staining, while paraffin-embedded samples were primarily used for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) staining. For IHC staining, sections were incubated with diluted primary antibodies overnight at 4 °C, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody and color development with the DAKO Real Envision Detection System (K500711; Agilent, Santa Clara, CA, USA). For IF staining, the sections were incubated with diluted primary antibodies overnight at 4 °C, followed by incubation with diluted fluorescent-dye-conjugated secondary antibodies for 2 h at room temperature. The antibodies used in this study and their dilution ratio are Mouse anti-PCNA (sc-56; Santa Cruz) (1:200), Rabbit anti-HNF4α (ab201460; Abcam, Cambridge, UK) (1:2000), Rabbit anti-HIF1α (ab114977; Abcam) (1:200), Goat anti-rabbit IgG Alexa Fluor™ 488 (A11034; Invitrogen) (1:500), and Goat anti-mouse IgG Alexa Fluor™ 568 (A11031; Invitrogen) (1:500). During immunostaining, the tissue sections received heat-mediated antigen-retrieval by 95 °C water baths in citrate buffer (C9999; Sigma-Aldrich). For H&E staining, the rehydrated sections were stained with Mayer’s hematoxylin (Vector Laboratories, Newark, CA, USA) and eosin (Sigma-Aldrich). The classification of liver histology was conducted as previously described [31].

2.8. Photography and Image Analysis

Side views of dissected adult zebrafish were photographed individually with a stereo fluorescence microscope (SZX16; Olympus, Tokyo, Japan). Larvae photography was performed with the ZEISS Axio Imager 2 light microscope (Carl Zeiss AG, Oberkochen, Germany) after immobilizing the larvae in 3% methylcellulose. For IHC and H&E staining, theimaging was conducted using the ZEISS Axio Imager 2 light microscope and the NanoZoomer S60 digital slide scanner (Hamamatsu Photonics, Shizuoka, Japan). For imaging IHC and H&E staining, at least two high-power fields of view per sample were captured and analyzed. Tissue sections of IF staining were imaged with the ZEISS LSM 900 confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany). For imaging IF staining, at least three high-power fields of view per sample were captured and analyzed. All image analyses were performed with ImageJ [32].

2.9. Statistical Analysis

For statistical analysis, Prism 8.0 (GraphPad, San Diego, CA, USA) was applied. All statistical significances in the current study were determined by a two-tailed unpaired student t-test unless stated otherwise.

3. Results

3.1. PH Promotes kras^G12V-Induced HCC Development in Zebrafish

To investigate the effect of PH on the kras^G12V-induced development of HCC in zebrafish, we performed PH and sham surgery on kras+ zebrafish before inducing the liver-specific expression of kras^G12V with continuous Dox treatment (Figure 1A). Only male zebrafish were used in the current study because previous studies have demonstrated that HCC development in zebrafish is male-biased [33,34]. After 3 days of Dox induction, the PH zebrafish developed significantly larger livers than the sham and uncut zebrafish (Figure 1B,C). By Day 7, all zebrafish had developed excessive livers, which overshadowed the effect of PH on liver growth. These results indicate that PH facilitated kras^G12V-induced hepatomegaly. Subsequently, we evaluated the histology of the kras+ liver sections after H&E staining. Based on liver histology, we found more HCC fish in the PH group than in the two control groups on Day 3 (Figure 1D,E). Normal liver histology was not observed in the PH group on Day 3, indicating that all zebrafish in that group had either developed HCC or had been at the pre-stage of HCC development (liver hyperplasia). The sham group had more zebrafish with liver hyperplasia than the uncut group, which could be attributed to the inflammatory effects caused by the wound. Generally, PH-treated zebrafish have more advanced HCC progressions than the control zebrafish. By Day 7, all kras+ zebrafish had the liver histology of HCC due to the robust effect of oncogene overexpression across the whole liver (Figure 1F). Therefore, the oncogene kras^G12V-induced development of HCC in zebrafish can be facilitated by PH.

3.2. PH Enhanced the HCC-Associated Features in Kras+ Zebrafish

Because PH facilitated the histopathological transformation of the kras+ zebrafish livers, we speculated that PH may also promote other HCC-associated features. The development of kras^G12V-induced HCC in zebrafish is associated with robust cell proliferation, which has been demonstrated by previous studies [33,34]. To examine the effect of PH on the proliferation of hepatocytes during the kras^G12V-induced HCC development, IF staining of the proliferation marker PCNA and the hepatocyte marker HNF4α was conducted. Based on IF imaging, the PH-treated kras+ zebrafish had significantly higher levels of hepatocyte proliferation than the control groups after 3 days of Dox induction, which explained the enhanced liver expansion in the PH group (Figure 2A,B). Thus, PH can promote hepatocyte proliferation during the kras^G12V-induced zebrafish’s HCC development.

We then examined the effects of PH on the expression of HCC-associated genes by RT-qPCR in the kras+ zebrafish livers from different treatment groups after 3 days of Dox induction. Consistent with the enhanced cell proliferation, the liver expression of cell cycle-related genes, including ccnb1 and cdk1, was promoted significantly by PH (Figure 2C). Extracellular matrix (ECM) accumulation in the liver can contribute to tumor metastasis during HCC development [35]. In our experiments, the expression of genes encoding ECM, including col1a1b and lama5, was significantly increased by PH in the kras+ livers (Figure 2D). Furthermore, the genes that contribute to ECM production and liver fibrosis, including myofibroblast marker acta2 and cytokine tgfb1b, were also significantly upregulated by PH in the kras+ livers (Figure 2E). To rule out the possibility of discrepancy in the oncogene expression levels among the different groups, we examined the expression levels of kras^G12V in the kras+ livers and found no significant differences among the three groups (Figure 2F).

3.3. PH Enhances the Oxidative Stress and Neutrophil Recruitment in the Kras+ Zebrafish Liver

The reactive oxygen species (ROS)-induced oxidative stress has been reported to correlate with the development of HCC [36]. Based on the results of the IHC staining, we found that the protein level of hypoxia-inducible factor 1-alpha (HIF1α) in the kras+ zebrafish liver was significantly higher in the PH group than in the sham group on Day 5 (Figure 3A,B). In addition to that, the expression of the hif1aa gene, which encodes HIF1α, was upregulated significantly in the PH-treated kras+ livers compared with the control kras+ livers on Day 3 (Figure 3C). To confirm whether PH alone can induce HIF1α upregulation, we examined the expression of hif1aa in WT male zebrafish livers following PH/sham surgery. As shown in Figure 3D, the expression of hif1aa was not changed by 24 h post-PH but upregulated significantly by 72 h post-PH. These results show that PH could enhance oxidative stress in the kras+ liver. As a main source of ROS, tumor-associated neutrophils (TANs) have been reported to play essential roles in liver regeneration and HCC development [37,38]. To investigate the effect of PH on the recruitment of neutrophils during HCC development, we employed the kras/lyz+ zebrafish. Based on IF imaging results, PH significantly increased the number of neutrophils in the kras/lyz+ liver after 5 days of Dox induction (Figure 3E,F). Neutrophils produce ROS mainly through activating NADPH oxidase 2 (NOX2) [39]. In our experiments, the expression of cybb, which encodes NOX2, in the kras/lyz+ liver was increased significantly by PH 3 days after Dox induction (Figure 3G). Therefore, the enhanced recruitment of neutrophils in the PH kras/lyz+ liver may promote ROS production, which contributes to the development of HCC in zebrafish.

3.4. Comparison of Liver Transcriptomic Regulation between PH-Induced Liver Regeneration and Kras^G12V-Induced Hepatocellular Carcinogenesis

To investigate the mechanism underlying the promoting effect of PH on the development of kras^G12V-induced zebrafish HCC, we performed RNA-Seq with the kras+ and WT male zebrafish livers on Day 5 after Dox induction and determined the differential expressed genes (DEGs) of the kras+ livers versus the WT livers (Figure 4A). Similarly, RNA-Seq was also performed using the WT male zebrafish livers on Day 1 after PH/sham surgery, and the DEGs in the PH livers compared with the sham livers were determined. In total, 4029 DEGs were found in the kras+ livers, while 197 DEGs were found in the PH livers (Figure 4B). We then compared the DEGs induced by HCC development with those induced by PH. Sixteen common upregulated and 42 common downregulated genes were found in the kras+ livers and PH livers (Figure 4C,D). Because previous studies have suggested that PH could promote HCC development through additive effects, we focused on the overlap of DEGs between the PH livers and the kras+ livers (Figure 4E,F) (Table S2) [7,8]. fgf13b and s100a1 were significantly upregulated in both the kras+ livers and the PH livers. By RT-qPCR, we confirmed that PH could further upregulate the expression of fgf13b and s100a1 during kras^G12V-induced HCC development at 24 h post-PH (Figure 4G,H). Both FGF13 and S100A1 have been suggested to promote cancer cell growth and survival by previous studies [40,41]. Thus, the enhanced expression of fgf13b and s100a1 in the PH-treated livers may contribute to the promoting effects of PH on HCC development. Curiously, the kras+ livers and the PH livers showed an overlap in the significant downregulation of rsl24d1, which is crucial to the biogenesis of the 60S large ribosome subunit [42]. Apart from some genes of interest found in the common DEGs between the PH livers and the kras+ livers, the majority of DEGs in the PH livers were not found among the DEGs in the kras+ livres (Table S3). Therefore, it is possible that PH can also affect HCC progression through mechanisms completely different from kras^G12V-induced tumorigenesis.

By gene set enrichment analysis (GSEA) of the Hallmark gene sets and KEGG pathways, we found that the deregulation of biological functions and pathways caused by kras^G12V overexpression shared little similarity with those caused by PH-induced liver regeneration (Figure 5A–C). These results were consistent with the small overlaps of DEGs between the kras+ liver and the PH liver. The GSEA of Hallmark gene sets indicated an overlap in the activation of epithelial to mesenchymal transition between the kras+ liver and the PH liver, which explained the enhanced production of ECM in the kras+ livers after PH. Apart from that, based on the GSEA of KEGG pathways, both the kras+ liver and the PH liver had a significant downregulation in the ribosome pathway. As shown in Figure 6A,B, the expression of several ribosomal proteins such as L5e, L24e, S26e, and S27e was reduced in both the kras+ liver and the PH liver, suggesting that both PH and kras^G12V overexpression caused a general inhibition on ribosome biogenesis in the zebrafish liver. FGF13-mediated repression on ribosome biogenesis can reduce cell death caused by acute oncogene overexpression and promote cancer cell growth in vitro [40]. Thus, it is possible that PH promoted kras^G12V-induced HCC development in zebrafish by an additive effect on the downregulation of ribosome biogenesis.

With regard to S100A1, it has been suggested that S100A1regulates the Hippo-YAP-signaling pathway, which is an oncogene suppressor, in HCC cells [41]. To test whether S100A1plays a role in kras^G12V-induced zebrafish’s HCC development, we completed knockdown of s100a1 in kras+ larvae and LiPan larvae with the morpholino (MO) before Dox treatment (Figure 7A). The effectiveness of the morpholino targeting the start codon of s100a1 (s100a1-ATG) was verified with western blot analysis (Figure 7B). As shown in Figure 7C,D, s100a1 knockdown abolished the hepatomegaly caused by kras^G12V overexpression after 48 h of Dox induction in kras+ larvae. Thus, s100a1 was required for the kras^G12V-induced tumor growth, and the enhanced s100a1 expression in the PH livers may contribute to HCC development in kras+ zebrafish. Notably, the s100a1-ATG MO-treated LiPan larvae showed significantly smaller liver sizes than the control MO-treated larvae, indicating that s100a1 knockdown can also compromise normal liver growth.

Sox family protein Sox9 has been reported to activate the expression of s100a1, and this regulatory function of Sox9 can be enhanced by Sox5 [43]. To determine the mechanism underlying the upregulation of s100a1 in the PH-treated kras+ livers, we examined the expression of sox5, sox9a, and sox9b in the kras+ male zebrafish livers at 24 h post-PH by RT-qPCR. As shown in Figure 7E, PH significantly enhanced the expression of sox5, sox9a, and sox9b in the kras+ livers. Thus, the enhanced s100a1 expression in the PH-treated kras+ livers could be due to the upregulation of sox5 and sox9 induced by PH.

4. Discussion

As mentioned earlier, the recurrence of HCC following PH can be classified into two types: the expansion of pre-existing tumors and the de novo development of new tumors [2]. According to previous studies, transplanted HCC cells and xenografts in rodents have shown enhanced growth and metastasis after PH [12,13,14,15,16]. With the inducible HCC zebrafish model driven by kras^G12V, the current study was the first one to investigate the effects of PH on the oncogene-induced de novo development of HCC. We found that PH promoted HCC development indicated by the enhanced liver expansion and facilitated the pathological transformation of liver histology 3 days after the induction of oncogene overexpression. The recruitment of neutrophils and the local oxidative stress in the kras+ livers were also enhanced by PH. Neutrophils-derived ROS has been suggested to promote tumor cell growth in vitro and hepatocarcinogenesis in rodents [44,45]. Thus, the enhanced HCC development in the PH-treated kras+ zebrafish could be attributed to the enhanced neutrophil recruitment and local oxidative stresses in their livers.

In addition to the facilitated development of HCC-associated histopathology, PH enhanced hepatocyte proliferation and ECM production in the kras^G12V-induced zebrafish HCC. Sustaining cell proliferation is the fundamental feature of cancers because it drives the growth of tumors [46]. Thus, the enhanced expansion of livers in the PH-treated kras+ zebrafish can be attributed to their enhanced hepatocyte proliferation. Disorganized ECM contributes to the formation of the tumor microenvironment. Excessive ECM deposition in the liver could lead to liver fibrosis and cirrhosis, which predispose to HCC and promote tumor growth and metastasis by inducing inflammatory factors, promoting abnormal vasculature, and increasing tissue rigidity [35]. In the current study, the PH-treated kras+ zebrafish livers have shown enhanced ECM gene expressions, which could contribute to the speed of tumorigenesis. Consistent with our results, the promoting effects of PH on hepatocyte proliferation and ECM production have also been reported in rodent HCC models [13,15,16].

Previous studies have summarized a sizable set of genes and signaling pathways that are shared between HCC development and liver regeneration [7,8]. Unexpectedly, our RNA-Seq results showed a very limited pool of genes that were deregulated in the same direction by both kras^G12V overexpression and PH-induced liver regeneration. This could be due to the fact that the time points of sampling the PH/sham zebrafish were too late to catch more significant changes. In addition, because the comparison was made between two independent experiments conducted with different batches of zebrafish, the differences in their genetic backgrounds may interfere with the results. Among the several genes that were significantly upregulated in both kras+ livers and PH livers, fgf13b has the potential to contribute to the enhanced HCC development in kras+ zebrafish after PH. FGF13 plays dual roles in cell proliferation depending on the types of cells and tissues, as reported by previous studies [47,48]. In tumorigenesis, fgf13 was found to be upregulated in lung adenocarcinoma patients and to be essential to the survival of cancer cells in vitro [40]. Another candidate gene that was significantly upregulated by both kras^G12V-induced HCC development and PH-induced liver regeneration is s100a1. The upregulation of s100a1 has been found in various types of tumors including HCC in the clinic, and a high S100A1level is correlated with poor outcomes for patients [41,49,50]. It was demonstrated that S100A1promoted HCC cell proliferation in vitro by promoting YAP activation [41]. Thus, it is possible that PH enhances YAP activity during HCC development by further enhancing the expression of s100a1.

Based on the GSEA of the KEGG pathway, we found that the ribosome pathway was downregulated in both the kras+ livers and the PH livers. The downregulation of the ribosome pathway in the kras+ livers is consistent with the previous data our lab obtained from the kras^G12V-driven HCC and the xmrk-driven HCC in zebrafish [51]. These observations seem counterintuitive because the intense cell proliferation during HCC development should require ribosome-mediated protein synthesis. Several ribosomal proteins have been reported to be overexpressed in HCC and contribute to tumorigenesis [52]. However, they are usually involved in specific oncogenic pathways rather than merely mediating protein synthesis universally. Moreover, a previous study analyzing RNA-Seq data from 372 human HCC tissues has shown that most genes in the KEGG ribosome pathway were downregulated, which is similar to our data from the current study [53]. In the C-myc-induced mouse HCC model, the downregulation of ribosomal proteins and rRNAs was observed in the livers during the early formation of HCC but not in the advanced HCC, suggesting that ribosome biogenesis was not regulated uniformly across different stages of tumorigenesis [54]. The downregulation of the ribosome pathway in the kras+ livers and PH livers occurred simultaneously with the upregulation of fgf13b. FGF13 has been found to promote cancer cell survival and growth by repressing ribosome biogenesis and restricting protein synthesis to reduce endoplasmic reticulum stress [40]. It was not directly upregulated by acute overexpression of the oncogene but was increased gradually during the oncogene-induced neoplastic transformation of cells. Similarly, the downregulation of rRNAs and ribosomal proteins was observed in C-myc-induced mouse HCC only after 7 days of overexpression of the oncogene [54]. Thus, the upregulation of fgf13 and the downregulation of ribosome biogenesis could occur temporally in response to endoplasmic reticulum stress during the formation of tumors to enhance the survivability of cancer cells. Enhanced fgf13b expression in the PH-treated kras+ livers may contribute to the facilitated HCC development by mediating the downregulation of ribosome biogenesis. In the clinic, the protein synthesis inhibitor sorafenib is commonly applied to manage HCC. The possibility of repressed protein synthesis promoting HCC development at certain stages shows a potential need to further optimize the clinical application of protein synthesis inhibitors.

As for the limitations of the current study, although the EGFR downstream effector RAS is a major proto-oncogene for human cancer, the RAS mutation is not very prevalent in patients with liver cancers, with 7% of patients having KRAS mutations and 4% of patients having HRAS mutations [20]. It is unclear whether the findings from the current study can be applied to HCC induced by other types of oncogenes. However, up to 50% of HCC patients have been reported to have RAS activation, and RAS is a common therapeutic target for HCC in the clinic [55]. Thus, the kras+ zebrafish HCC model used in our study has the potential to represent the situation of a substantial category of HCC. In addition, the kras+ zebrafish has the ectopic expression of kras^G12V across the whole liver upon Dox induction, while HCC in the clinic usually develops from a few spots and spreads during progression. This discrepancy in the approach of tumor expansion could cause problems in the translatability of the current study.

5. Conclusions

In conclusion, the current study confirmed that PH can promote kras^G12V-induced HCC development in zebrafish and enhance neutrophil-mediated oxidative stresses in their livers. In addition, PH could enhance the deregulation of molecular factors that potentially contribute to HCC development including the Hippo-YAP regulator S100A1 and the ribosome biogenesis repressor FGF13 in zebrafish livers during kras^G12V-induced HCC development. These factors could serve as therapeutic targets for developing intervention methods that are intended to apply to patients following PH to reduce HCC recurrence. Overall, the findings from the current study could improve the understanding of the relationship between PH-induced liver regeneration and HCC development, which may provide clues for developing novel therapeutic strategies.