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Hepatocellular Carcinoma (HCC) is the most common primary liver malignancy and a leading cause of cancer-related deaths worldwide [1]. Understanding the molecular mechanisms underlying HCC progression is crucial for developing effective therapeutic strategies [2]. The combination of atezolizumab and bevacizumab [3]. Significantly increased the overall survival of patients with advanced HCC compared to sorafenib from 13.4 months to 19.2 months [4]. However, there are still unsolved clinical problems, including approximately 26% of nonresponders to atezolizumab plus bevacizumab and intolerance to immune-oncologic drugs, such as underlying autoimmune diseases (e.g., ulcerative colitis) [5]. Also, 26–30% of non-responders to atezolizumab plus bevacizumab often have poorly differentiated tumor biology and progress very rapidly [6]. Therefore, there is an urgent need to identify novel biomarkers and therapeutic targets for poorly differentiated HCCs.

Recent studies have highlighted the significant role of the SenescenceAssociated Secretory Phenotype (SASP) in hepatocarcinogenesis, especially in aspect of progression to poor biology [7]. SASP is characterized by the secretion of various pro-inflammatory cytokines, chemokines, and growth factors by senescent cells, contributing to chronic inflammation and tumor promotion. However, the specific involvement of the SASP in HCC pathogenesis and its relationship with Hepatic Stellate Cell (HSC) activation remain poorly elucidated. Although previous studies have explored the pathogenic role of the SASP in various cancer types, including liver cancer, its specific impact on hepatocarcinogenesis remains largely unexplored. The current literature lacks detailed investigations of the interplay between bile acid-mediated HSC activation and the expression of SASP components in HCC cells.

In previous studies, bile acids induced Cyclooxygenase-2 (COX-2) and Myeloid cell leukemia-1 (Mcl-1) protein expression in HSCs and cholangiocytes, mediating their survival against bile acid-induced apoptosis [8]. Unlike hepatocytes undergoing apoptosis [9]. COX-2 is an enzyme that is involved in prostanoid metabolism and may promote cellular growth, inhibit apoptosis, and support angiogenesis. Moreover, a growing body of evidence shows that COX-2 expression is correlated to cancer invasion and metastasis by reducing the activity of E-cadherin, inhibiting the separation of cancer cells from tissues via matrix metalloproteinase upregulation. Mcl-1 is a potent anti-apoptotic Bcl-2 family protein induced by various stimuli. Specifically, it blocks the release of cytochrome c, which is required for the intrinsic apoptosis pathway. During liver damage and secondary inflammatory reactions, DNA damage of a significant magnitude might induce cellular senescence.

In the present study, we investigated the mechanism underlying the bile acid-mediated induction of HSC activation via SASP expression in HCC cells. The mechanisms underlying these signaling cascades may provide new targets for preventing HCC progression by inhibiting HSC activation. This study aimed to fill this knowledge gap by elucidating the mechanisms underlying the bile acid-mediated induction of HSC activation and its relationship with the expression of SASP components in hepatocellular carcinoma cells.

Cell lines and co-culture

The following human HCC cell lines and an HSC line were obtained from the Korean cell-line bank (Seoul, Korea) for this study: Huh-7, HepG2, which are well-differentiated HCC cell lines; SNU-761, a poorly differentiated HCC cell line; SNU-3058, a hypovascular HCC cell line; and LX-2, an activated human HSC line. We also established Huh-SR cells in vitro by culturing Huh-7 cells in a culture medium containing sorafenib for three months. The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Huh-7, HepG2, and LX-2) or RPMI 1640 (SNU-761) supplemented with 10%Fetal Bovine Serum (FBS), 100,000 U/l penicillin, and 100 mg/l streptomycin, with or without 100 nM insulin. Co-culture experiments were performed in serum-free DMEM or RPMI 1640 using 1 μm pore size Transwell inserts (Corning, Lowell, MA, USA), which permitted media diffusion but prevented cell migration. HCC and LX-2 cell lines were incubated alone or together using Transwell inserts under standard culture conditions (20% O2 and 5% CO2, 37°C).

Cell proliferation analysis (MTT assay)

Using the CellTiter 96 aqueous one solution cell proliferation assay (Promega, Madison, WI, USA), cell proliferation was measured based on the cellular conversion of the colorimetric reagent 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into soluble formazan by the dehydrogenase enzyme found in metabolically proliferating cells. Following each treatment, 20 μl of dye solution was added into each well of a 96-well plate and incubated for 2 hours. Subsequently, the absorbance was recorded at 490 nm using an Enzyme-Linked Immunosorbent Assay (ELISA) plate reader (Molecular Devices, Sunnyvale, CA, USA).

Small interfering RNA (siRNA) transfection

Cells were seeded in a 6-well culture plate (2 × 105 cells/well) in 2 ml antibiotic-free medium supplemented with 10% FBS. At 60–80% confluence, the cells were transfected with siRNAs using a siRNA

Transfection Reagent (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) according to the manufacturer’s instructions. The cells were incubated with the siRNAs for 6 hours at 37°C before adding a growth medium containing 20% FBS and antibiotics. After 18 h, the spent medium was replaced with fresh medium containing 10% FBS and antibiotics. At 24 hours after transfection, the cells were used for further experiments.

In vivo subcutaneous xenograft model

Briefly, control short hairpin RNA (shRNA)-transfected Huh-SR cells (5 × 10⁷ cells/mouse) were subcutaneously transplanted into the flanks of nude mice (male, 5 weeks old) in the control group (n=10). Mcl-1 shRNA-transfected Huh-SR cells (5 × 10⁷ cells/mouse) were subcutaneously transplanted into mice in the study group (n=10). Control shRNA-transfected Huh-SR cells (5 × 10⁷ cells/mouse) were subcutaneously injected into another group (n=10), which received celecoxib treatment daily at a dose of 50 mg/kg after tumor budding. Tumor volume was measured using a Vernier caliper and calculated as follows: (length × (width)²)/2. The maximal diameter and volume of each tumor were measured three times per week for three weeks.

Human blood sample collection and cytokine measurement

All human blood samples were provided by the NCC BioBank of the National Cancer Center, Korea. The serum was stored without preservative at –70℃ and then thawed just prior to testing. IL-6 levels were determined using commercially available human IL-6 ELISA kit supplied by Abcam (ab178013) from serum samples of 296 patients with HCC. All samples were assayed in duplicate.

Immunoblot analysis

Cells were lysed for 20 minutes on ice with lysis buffer and centrifuged at 14,000 ×g for 10 minutes at 4°C. Samples were resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, blotted with the appropriate primary antibodies (1:1000 dilution), and treated with the corresponding peroxidase-conjugated secondary antibodies (Biosource International, Camarillo, CA, USA). The bound antibodies were visualized using a chemiluminescent substrate (ECL; Amersham, Arlington Heights, IL) and exposed to Kodak X-OMAT film (Kodak, New Haven, CT). Rabbit anti-REG3A antibodies were obtained from Abcam (Cambridge, UK; ab95316). Primary antibodies, including rabbit anti-phospho-p42/44 MitogenActivated Protein Kinase (MAPK), anti-phospho-Akt, rabbit anti-cMyc, rabbit anti-caspase 8, anti-caspase 9, and anti-caspase 7 (cleaved) were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat anti-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Densitometric analyses were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Real-time Polymerase Chain Reaction (qPCR) analysis

Total RNA was extracted from HSC or HCC cells using the Trizol Reagent (Invitrogen, Carlsbad, CA, USA). cDNA templates were prepared using random oligo (dT) primers and Moloney Murine Leukemia Virus reverse transcriptase. After reverse transcription, the cDNA template was amplified via PCR using Taq polymerase (Invitrogen). COX-2 and Mcl-1 mRNA was quantitated using realtime PCR and the following primers: COX-2 forward, 5′- TGAAACCCACTCCAAAACA-3′, reverse, 5′- CCCATGGGCATTCAATAAAC-3′; Mcl-1 forward, 5′- ATGCTTCGGAAACTGGACAT-3′, reverse, 5′- TCCTGATGCCACCTTCTAGG-3′. For gene expression quantification, we used a real-time PCR (LightCycler; Roche Molecular Biochemicals, Mannheim, Germany) with SYBR green as a fluorophore (Molecular Probes, Eugene, OR, USA). Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene expression was used as a control. The relative intensity of the COX-2 and Mcl-1 bands were compared to that of GAPDH using the 2–ΔΔCt method. All PCR experiments were performed in triplicate.

Statistical analyses

Statistical analyses were performed using PASW version 21.0 (SPSS Inc., Chicago, IL, USA). All experimental results were obtained from three independent experiments using cells from three separate isolations and presented as the mean ± Standard Deviation (SD). The Mann–Whitney U test was used for non-parametric measures. For comparisons between groups, data were analyzed using the Mann–Whitney U test or one-way ANOVA.

The Kaplan–Meier method was used to estimate overall survival. A multivariate Cox proportional hazards regression analysis was performed to derive a model. If there was positive collinearity between the covariates, the most objective and easily applicable variable was selected as the representative variable for creating the model. The output of the model was expressed as a coefficient that was used to compute the Hazard Ratio (HR). In addition, the coefficients were used to calculate the risk score, which was used to predict the tumor recurrence rate. The discrimination function was evaluated using Harrell’s concordance index (c). For all tests, p<0.05 was considered statistically significant.

Ethics statement

Ethics approval was obtained from the Ethics Committee of the National Cancer Center, Korea. This study was conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. The in vivo study protocol was approved by the Institutional Animal Care and Use Committee (NCC-22-771) of the National Cancer Center of Korea. The study was reported in accordance with ARRIVE guidelines. All in vivo surgical procedures were performed under anesthesia with 2,2,2-tribromoethanol, and efforts were made to minimize suffering.

All experiments involving human tissues were approved by the Institutional Review Board of the National Cancer Center (NCC2022-0200). The period for serum samples of the study subjects was from May, 2002, to December, 2013, and the period for accessing medical records was from May, 2002, to December, 2021. Participant consent was waived by the Institutional Review Board of National Cancer Center, Korea.

Bile acid significantly increased the invasion of HSC’s

Using an invasion assay, we evaluated whether bile acid treatment increased the invasion of HSCs and examined how SASP contributes to this phenomenon. Treatment with 50 μM Deoxycholic Acid (DC) increased the invasion of HSCs (Figure 1A). Treatment of LX-2 cells with DC also significantly increased the levels of IL-6 or IL-8 as determined using real-time PCR (Figure 1B). These findings indicate that bile acids significantly increased the invasion of HSCs via IL-6 or IL-8, which are some of the most important SASP factors. The treatment of LX-2 cells with DC also increased Mcl-1 and COX-2 protein expression (Figure 1C).

 

Figure 1: Bile acid significantly increased the invasion of HSCs.

Note: (A) The invasion of HSCs significantly increased after treatment with 50 μM Deoxycholic acid (DC). The experiment was repeated three times. The data are expressed as the mean ± SD. (B) The treatment of LX-2 cells with DC significantly increased the levels of IL-6 or IL-8, as determined via real-time PCR analysis. (C) The treatment of LX-2 cells with DC also increased the protein expression levels of Mcl-1 and COX-2. The experiment was repeated three times.

The selective inhibition of Mcl-1 or COX-2 attenuates HSC invasion and IL-8 production

LX-2 cells were transfected with a Mcl-1-specific siRNA to selectively inhibit Mcl-1 induction. While the treatment of LX-2 cells with DC in the control group significantly increased HSC invasion, Mcl-1 siRNA-transfected LX-2 cells treated with DC showed an attenuated degree of HSC invasion (Figure 2A). Similarly, COX-2 inhibition by celecoxib attenuated bile acid-mediated HSC invasion, while increased invasion was observed in cells not treated with 20 μM celecoxib (Figure 2B). We found that inhibitors of either Mcl-1 (via siRNA transfection) or COX-2 (using celecoxib) attenuated bile acidmediated HSC invasion.

We also tested whether the selective inhibition of Mcl-1 or COX-2 decreased the bile acid-mediated increase in IL-8 levels. We found that the siRNA-mediated inhibition of Mcl-1 significantly diminished IL-8 levels regardless of DC treatment (Figure 2C). The celecoxib mediated selective inhibition of COX-2 resulted in a robust decrease in the levels of IL-8 without DC treatment; however, no significant change in the expression of IL-8 was detected with DC treatment (Figure 2C). These findings further suggest that SASP (i.e., IL-8) induction is regulated by Mcl-1 and COX-2, with Mcl-1 having a greater impact than COX-2.

 

Figure 2: The selective inhibition of Mcl-1 or COX-2 attenuated HSC invasion and IL-8 production.

Note: Mcl-1 siRNA transfection of LX-2 cells (A) or celecoxib treatment (20 μM) (B) with DC treatment (50 μM) reduced HSC invasion. The experiment was repeated three times. The data are expressed as the mean ± SD. (C) Celecoxib treatment or Mcl-1 siRNA transfection significantly diminished the mRNA levels of IL-8 regardless of DC treatment. The experiment was repeated three times. The data are expressed as the mean ± SD.

Crosstalk of HCCs and HSCs increased SASP, COX2 , and Mcl-1 levels

We then examined whether the co-culture of HCC cells with HSCs increased the levels of IL-6 or IL-8. Compared to HCC cells (Huh-SR or SNU-761) cultured alone, co-culture with LX-2 cells significantly increased the mRNA levels of IL-6 or IL-8 (Figure 3A). In addition, co-culturing HCC cells with LX-2 cells significantly increased Mcl-1 and COX-2 protein expression levels compared to their respective monocultures (Figure 3B).

To selectively inhibit Mcl-1 induction, HCC cells were transfected with a Mcl-1 specific siRNA. Mcl-1 siRNA-transfected HCC cells treated with DC showed reduced expression of IL-6 or IL-8 mRNA (Figure 3C). Similarly, COX-2 inhibition by celecoxib attenuated the bile acid-induced IL-6 or IL-8 mRNA expression in HCC cells (Figure 3C).

 

Figure 3: Crosstalk of HCCs and HSCs increased SASP, COX2, and Mcl-1 levels.

Note: (A) Co-culturing Huh-SR or SNU-761 cells with LX-2 cells significantly increased the mRNA levels of IL-6 and IL-8. The experiment was repeated three times. The data are expressed as mean ± SD. (B) Co-culturing HCC cells with LX-2 cells significantly increased the Mcl-1 and COX-2 protein expression levels compared to HCC cell monocultures. The experiment was repeated three times. (C) Mcl-1 siRNA transfection or celecoxib treatment of HCC cells with DC treatment attenuated the mRNA expressions of IL-6 or IL-8. The experiment was repeated three times. The data are expressed as the mean ± SD.

Upregulated mRNA expression of IL-6 in patients with HCC was correlated with poor overall survival

Next, 168 serum samples from patients with HCC from the National Cancer Center Biobank were used to determine serum ELISA levels. Supplementary Table 1 presents the baseline patient characteristics. The median age was 56 years, and the median level of IL-6 was 5.62 pg/mL (interquartile range, 3.58–14.833). Cox proportional hazard analyses were performed to identify the significant factors for overall survival. Among these variables, we used the square root values of the serum Alpha-Fetoprotein (AFP) and Protein Induced by Vitamin K Antagonist-II (PIVKA-II) levels to fit normal distributions. In the univariate analysis, the number of tumors, tumor size, Child-Pugh score, √Serum AFP, √Serum PIVKA −II, IL-6, and the MoRAL score (11 × √PIVKA−II + 2 × √AFP) were significantly associated with the overall survival of study subjects (Supplementary Table 2). There were significant confounding effects between the tumor factors and serum tumor markers; thus, we used serum AFP and PIVKA-II levels in the multivariate analysis, which were objective and reproducible. Multivariate Cox regression analysis showed that IL-6 (HR=1.011, p<0.0001) and the MoRAL score (HR=1.0004, p<0.0001) were independently associated with the overall survival of patients with HCC. At a cut-off of 5.973, serum IL-6 levels significantly discriminated the OS of patients with HCC with a c-index of 0.741 (Figure 4A). Based on the Cox proportional hazards model, the risk score was calculated as the IL-6+MoRAL score. The c-index of the MoRAL score on overall survival was 0.765 (95% Confidence Interval (CI): 0.719–0.808) with a cut-off of 172.22, which showed higher performance in predicting overall survival compared to IL-6 or the MoRAL score alone (Supplementary Table 3, Figure 4B).

 

Figure 4: IL-6 upregulation in patients with HCC was correlated with poor overall survival.

Note: (A) Overall survival according to serum IL-6 (pg/ml) levels with a cut-off of 5.973. (B) Overall survival according to serum IL-6 (pg/ml) levels+MoRAL score, with a cut-off of 172.22.

Bile acid or IL-6 treatment significantly increased the invasion of HCC cells through the COX2 and Mcl-1 pathways

Next, we evaluated whether bile acids or human IL-6 treatment increased the invasion of HCC cells and examined how the SASP contributes to this phenomenon. The invasion of HCC cells (Huh-SR or SNU-3058) significantly increased after treatment with 50 μM DC (Figure 5A) or 200 ng/ml human IL-6 (Figure 5B). Co-culturing HCC cells with HSCs also increased the levels of Epithelial-Mesenchymal Transition (EMT) markers, including fibronectin, N-cadherin, Ecadherin, vimentin, α-SMA, and CK19 compared to HCC cell monocultures (Figure 5C). In addition, Mcl-1 siRNA-transfected HCC cells treated with DC showed an attenuated expression of EMT markers (Figure 5D). The downregulation of the Mcl-1 pathway significantly suppressed Huh-SR cell invasion (Figure 5E).

 

Figure 5: Bile acid or IL-6 treatment significantly increased the invasion of HCC cells through the COX2 and Mcl-1 pathways.

Note: The invasion of Huh-SR or SNU-3058 cells significantly increased after treatment with 50 μM 1 Deoxycholic acid (DC) (A) or 200 ng/ml human IL-6 (B). The experiment was repeated three 2 times. The data are expressed as the mean ± SD. (C) Co-culturing HCC cells with HSCs increased the protein levels of Epithelial-Mesenchymal Transition (EMT) markers. The experiment was repeated three times. (D) Mcl-1 siRNA-transfected HCC and DC treatment attenuated the protein expressions of EMT markers. The experiment was repeated three times.

(E) Downregulation of the Mcl-1 pathway significantly suppressed the invasion of Huh-SR cells. The experiment was repeated three times. The data are expressed as the mean ± SD.

The selective inhibition of Mcl-1 or COX-2 attenuates the proliferation of HCC cells, which had been enhanced by crosstalk with HSCs

Initially, we examined whether co-culture with HSCs and bile acid treatment could modulate the proliferation of HCC cells. Co-culture with LX-2 cells significantly increased the proliferation of HCC cells compared to HCC cell monocultures. Treatment with a low concentration of DC (50 μM) slightly increased HCC cell proliferation, especially in SNU-3058 cells (Figure 6A). These findings indicate that the proliferation of HCC cells was enhanced by crosstalk between HCC cells and HSCs under SASP activation.

Mcl-1 siRNA transfection and DC treatment attenuated proliferation in HCC cells (Figure 6B). Similarly, COX-2 inhibition by celecoxib resulted in the attenuation of bile acid-mediated HCC cell proliferation. In addition, co-treatment of HCC cells (Huh-SR, SNU-3058, and SNU-761) with celecoxib and Mcl-1 siRNA significantly suppressed their proliferation.

Interestingly, the co-culture of HCC cells with LX-2 cells under DC treatment significantly increased the protein expression of the survival pathways, including the mTOR, PARP, Akt, ERK, and YAP pathways (Figure 6C). In addition, Mcl-1 siRNA-transfected HCC cells treated with DC attenuated the expression of these proteins (Figure 6D).

 

Figure 6: The selective inhibition of Mcl-1 or COX-2 attenuated the proliferation of HCC cells, which had been enhanced by crosstalk with HSCs.

Note: (A) Treatment with a low concentration of Deoxycholic acid (DC) (50 μM) slightly increased HCC cell proliferation. The experiment was repeated three times. The data are expressed as the mean ± SD. (B) Mcl-1 siRNA transfection or celecoxib treatment and DC treatment attenuated the proliferation of HCC cells. Co-treatment with celecoxib and Mcl-1 siRNA transfection under DC treatment significantly suppressed the proliferation of HCC cells. The experiment was repeated three times. The data are expressed as the mean ± SD. (C) Co-culturing HCC cells with LX-2 cells under DC treatment significantly increased the protein expressions of survival pathways. The experiment was repeated three times. (D) Mcl-1 siRNA-transfected HCC cells treated with DC showed attenuated expressions of survival pathways. The experiment was repeated three times.

The selective inhibition of TGR-5 suppressed bile acidmediated HSC and HCC cell invasion and IL-6/-8 expression

To further elucidate the pathway regulating the bile acid-induced SASP, LX-2, and HCC cells were treated with MDL-12,330A, a Takeda G Protein-Coupled Receptor 5 (TGR5) inhibitor (10 μM). TGR5, also known as G Protein-coupled Bile Acid Receptor 1 (GPBAR1) or Membrane-type Receptor for Bile Acids (M-BAR), functions as a cell surface receptor for bile acids and induces the production of intracellular cAMP and the activation of the MAPK pathway. To demonstrate the TGR-5-dependent induction of the SASP (i.e., IL-6 or IL-8), LX-2 or HCC cells were treated with a TGR-5 inhibitor in the absence or presence of DC. As shown in Figure 7A, while cells treated with the TGR-5 inhibitor in the absence of DC showed increased expression levels of IL-6 or IL-8, cells treated with the TGR-5 inhibitor and DC showed the opposite. These findings collectively indicate that SASP induction (i.e., IL-6 or IL-8), as well as Mcl-1 and COX-2, is due to transcriptional enhancement dependent on TGR-5 activation. As shown in Figure 7B, cells treated with a TGR-5 inhibitor also showed reduced bile acid-mediated HSC or HCC cell invasion under DC treatment.

 

Figure 7: The selective inhibition of TGR-5 suppressed the bile acid-mediated HSC and HCC cell invasion and the expressions of IL-6 or IL-8.

Note: (A) HCC cells or HSCs treated with a 24 TGR-5 inhibitor in the presence of DC showed a significant attenuation in IL-6 or IL-8 expression levels. In contrast, cells treated with a TGR-5 inhibitor in the absence of DC showed increased expression levels of IL-6 or IL-8. The experiment was repeated three times. The data are expressed as the mean ± SD. (B) Treatment with a TGR-5 inhibitor also reduced the degree of bile acid-mediated HSC or HCC cell invasion under DC treatment. The experiment was repeated three times. The data are expressed as the mean ± SD.

Modulation of Mcl-1 expression in HCC cells showed antitumor effects in an in vivo xenograft mouse model

The potential anti-proliferative effects of Mcl-1 downregulation were investigated using shRNA transfection in Huh-SR cells and an in vivo xenograft mouse model. The growth of liver tumors was significantly inhibited in the Mcl-1 shRNA-transfected group (n=10) compared to that in the control group (n=10) during the 19 days after tumor budding (Figure 8A; p<0.05). Oral treatment with celecoxib (50 mg/kg per day, n=10) for 19 days after tumor budding did not significantly suppress tumor growth compared with the control group (data not shown). We also performed immunohistochemical staining for Mcl-1 in the HCC tissues at the end of the in vivo study (Figure 8B). Mcl-1 protein expression was significantly suppressed in Mcl-1 shRNA-transfected HCC cells compared to control shRNA-transfected HCC cells. Mcl-1 shRNA transfection also attenuated the protein expressions of β-catenin and YAP (Figure 8C), which are common and novel markers of hepatocarcinogenesis, respectively.

 

Figure 8: Modulating Mcl-1 expression in HCC cells showed antitumor effects in an in vivo xenograft mouse model.

Note: (A) The growth of liver tumors was significantly inhibited in the Mcl-1 shRNA-transfected group compared with the control group during the 19 days after tumor budding (p<0.05). (B) Immunohistochemistry staining revealed significant suppression of Mcl-1 protein expression in Mcl-1 shRNA-transfected HCC cells compared to control shRNA-transfected HCC cells. (C) Mcl-1 shRNA transfection also attenuated the protein expressions of β-catenin and YAP on the HCC tissue of mice. The experiment was repeated three times.

In this study, we identified a novel target for Hepatocellular Carcinoma (HCC) treatment, Mcl-1, which was modulated through a TGR-5-dependent mechanism in response to elevated IL-6 levels induced by SASP in the tumor microenvironment. This effect originates from the communication between HCC cells and HSCs. This study revealed that modulating Mcl-1 expression within HCC cells led to notable anti-tumor effects.

Identifying novel biomarkers or target molecules for poorly differentiated HCC is an urgent clinical need. Poorly differentiated HCC is associated with aggressive tumor behavior, increased metastatic potential, and resistance to conventional therapies. Therefore, developing a deeper understanding of the underlying molecular mechanisms and identifying specific biomarkers involved in the progression of poorly differentiated HCC could lead to improved prognosis and tailored therapeutic approaches. However, to date, there is a lack of comprehensive studies investigating the role of SASP and its potential as a biomarker or therapeutic target in poorly differentiated HCC. In this study, the bile acid-mediated induction of HSC activation enhanced the invasion of HCC cells via the expression of SASP proteins, including IL-6.

The role of IL-6 in hepatocarcinogenesis has garnered significant attention owing to its multifaceted impact on tumorigenesis and tumor progression. As a pleiotropic cytokine, IL-6 is recognized for its involvement in chronic inflammation, immune regulation, and cell proliferation. It has been shown that the serum IL-6 level is elevated in patients with HCC, and its increased expression is associated with poor prognosis and advanced disease stages. IL-6 has also been implicated in promoting the activation of HSCs, which are key players in the progression of liver fibrosis to HCC. Furthermore, IL-6 has been linked to the acquisition of SASP in cancer cells, contributing to a protumorigenic microenvironment by promoting inflammation, angiogenesis, and immunosuppression. A translational study on the bile acid- mediated induction of HSC activation and SASP in HCC sheds new light on the intricate mechanisms of hepatocarcinogenesis, suggesting a potential link between bile acid signaling, HSC activation, IL-6 expression, and the development of HCC. These findings underscore the critical role of IL-6 in orchestrating the molecular pathways that contribute to HCC progression and suggest that it is a potential target for therapeutic interventions to disrupt the hepatocarcinogenic process.

Mcl-1 has emerged as a pivotal factor in hepatocarcinogenesis, drawing attention from previous studies because of its significant impact on tumorigenesis and disease progression. Mcl-1, an antiapoptotic member of the Bcl-2 protein family, has been implicated in regulating cell survival, resistance to apoptosis, and maintaining mitochondrial integrity. Furthermore, previous studies have suggested that Mcl-1 contributes to the evasion of apoptosis in HCC cells, enabling their survival and uncontrolled proliferation. A translational study investigating the bile acid-mediated induction of HSC activation and SASP in HCC provided novel insights into the intricate mechanisms of hepatocarcinogenesis, offering potential connections between bile acid signaling, Mcl-1 expression, and the development of HCC. These findings underscore the crucial role of Mcl-1 in orchestrating the cellular pathways that fuel HCC progression and highlight its potential as a therapeutic target for interventions aimed at disrupting hepatocarcinogenesis.

There is an intriguing disparity between the significant effect of COX-2 observed in in vitro studies and its lack of a substantial impact on in vivo mouse models. Previous studies have indicated that in vitro systems often lack the complex microenvironment and interactions present in vivo, which can lead to differences in cellular behavior and its associated signaling pathways. COX-2, an enzyme involved in prostaglandin synthesis and inflammation, has been linked to tumor promotion and progression by modulating the tumor microenvironment. While an in vitro model might have provided an isolated view of the direct impact of COX-2 on HSC activation and Mcl-1 expression, the in vivo environment incorporates additional factors such as immune responses, systemic inflammation, and crosstalk between different cell types. To fully elucidate this discrepancy, future investigations should determine the intricate interplay between COX-2, HSC activation, and Mcl-1 expression within the dynamic in vivo milieu, providing a more comprehensive understanding of the role of COX-2 in HCC progression.

One limitation of this study is that the role of Mcl-1 in hepatocarcinogenesis has not yet been elucidated in patients with poorly differentiated HCC. Future studies may prove that the inhibition of Mcl-1 could be a therapeutic target for patients with HCC. Nonetheless, the results of this study provide critical knowledge regarding the mechanistic link between bile acid signaling, HSC activation, and the SASP in HCC. Addressing these findings is essential for identifying novel therapeutic targets, such as Mcl-1, and developing innovative strategies to combat HCC progression.

In conclusion, Mcl-1 expression was modulated through a TGR-5- dependent mechanism in response to elevated IL-6 levels induced by SASP in the tumor microenvironment of HCC. This effect originates from the communication between HCC cells and HSCs. These findings may contribute to our understanding of the role of the SASP in hepatocarcinogenesis and provide novel insights into identifying the role of Mcl-1 in poorly differentiated HCC and sorafenib-resistant HCC cells. Ultimately, these findings have the potential to pave the way for the development of innovative therapeutic strategies to improve patient outcomes following HCC treatment.

Key messages

• Bile acid-mediated induction of hepatic stellate cell activation aggravated HCC cell progression.
• Bile acid significantly increased IL-6 levels as a result of HSC and HCC crosstalk.
• SASP induced the increased expressions of Mcl-1 and COX in HCC cells and HSCs.

Ethical Statement

Ethics approval was obtained from the Ethics Committee of the National Cancer Center, Korea. This study was conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. The in vivo study protocol was approved by the Institutional Animal Care and Use Committee (NCC-22-771) of the National Cancer Center of Korea. All experiments involving human tissues were approved by the Institutional Review Board of the National Cancer Center (NCC2022-0200). Participant consent was waived by the Institutional Review Board of National Cancer Center, Korea.

Conceived and designed the analysis: YC, ML, BHK, JWP

Collected the data: YC, ML, MJP, NJ

Contributed data or analysis tools: YC, ML, MJP, NJ, BHK, JWP

Performed the analysis: YC, ML MJP, NJ

Wrote the paper: YC, ML All authors have read and agreed to the published version of the manuscript.

Authors disclose no conflicts of interest.

This work was supported by the Genomics Core Facility in National Cancer Center Korea. This work has supported by the National Research Foundation of Korea grant funded by the Korea government (2021R1A2C4001401), National Cancer Center, Korea (2210420), the Korea Health Technology R and D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health and Welfare, Republic of Korea (HI22C1948, HI22C0828).

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