MASH as an emerging cause of hepatocellular carcinoma: current knowledge and future perspectives.

Hepatocellular carcinoma is one of the deadliest and fastest-growing cancers. Among HCC etiologies, metabolic dysfunction-associated fatty liver disease (MAFLD) has served as a major HCC driver due to its great potential for increasing cirrhosis. The obesogenic environment fosters a positive energy balance and results in a continuous rise of obesity and metabolic syndrome. However, it is difficult to understand how metabolic complications lead to the poor prognosis of liver diseases and which molecular mechanisms are underpinning MAFLD-driven HCC development. Thus, suitable preclinical models that recapitulate human etiologies are essentially required. Numerous preclinical models have been created but not many mimicked anthropometric measures and the course of disease progression shown in the patients. Here we review the literature on adipose tissues, liver-related HCC etiologies and recently discovered genetic mutation signatures found in MAFLD-driven HCC patients. We also critically review current rodent models suggested for MAFLD-driven HCC study.

Triclosan administration to humanized UDP-glucuronosyltransferase 1 neonatal mice induces UGT1A1 through a dependence on PPARα and ATF4.

Triclosan (TCS) is an antimicrobial toxicant found in a myriad of consumer products and has been detected in human tissues, including breastmilk. We have evaluated the impact of lactational TCS on UDP-glucuronosyltransferase 1A1 (UGT1A1) expression and bilirubin metabolism in humanized UGT1 (hUGT1) neonatal mice. In hUGT1 mice, expression of the hepatic UGT1A1 gene is developmentally delayed resulting in elevated total serum bilirubin (TSB) levels. We found that newborn hUGT1 mice breastfed or orally treated with TCS presented lower TSB levels along with induction of hepatic UGT1A1. Lactational and oral treatment by gavage with TCS leads to the activation of hepatic nuclear receptors constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor alpha (PPARα), and stress sensor, activating transcription factor 4 (ATF4). When CAR-deficient hUGT1 mice (hUGT1/Car-/-) were treated with TCS, TSB levels were reduced with a robust induction of hepatic UGT1A1, leaving us to conclude that CAR is not tied to UGT1A1 induction. Alternatively, when PPARα-deficient hUGT1 mice (hUGT1/Pparα-/-) were treated with TCS, hepatic UGT1A1 was not induced. Additionally, we had previously demonstrated that TCS is a potent inducer of ATF4, a transcriptional factor linked to the integrated stress response. When ATF4 was deleted in liver of hUGT1 mice (hUGT1/Atf4ΔHep) and these mice treated with TCS, we observed superinduction of hepatic UGT1A1. Oxidative stress genes in livers of hUGT1/Atf4ΔHep treated with TCS were increased, suggesting that ATF4 protects liver from excessive oxidative stress. The increase oxidative stress may be associated with superinduction of UGT1A1. The expression of ATF4 in neonatal hUGT1 hepatic tissue may play a role in the developmental repression of UGT1A1.

The Past and Future of Inflammation as a Target to Cancer Prevention.

Inflammation is an essential defense mechanism in which innate immune cells are coordinately activated on encounter of harmful stimuli, including pathogens, tissue injury, and toxic compounds and metabolites to neutralize and eliminate the instigator and initiate healing and regeneration. Properly terminated inflammation is vital to health, but uncontrolled runaway inflammation that becomes chronic begets a variety of inflammatory and metabolic diseases and increases cancer risk. Making damaged tissues behave as "wounds that do not heal" and sustaining the production of growth factors whose physiologic function is tissue healing, chronic inflammation accelerates cancer emergence from premalignant lesions. In 1863, Rudolf Virchow, a leading German pathologist, suggested a possible association between inflammation and tumor formation, but it took another 140 years to fully elucidate and appreciate the tumorigenic role of inflammation. Key findings outlined molecular events in the inflammatory cascade that promote cancer onset and progression and enabled a better appreciation of when and where inflammation should be inhibited. These efforts triggered ongoing research work to discover and develop inflammation-reducing chemopreventive strategies for decreasing cancer risk and incidence.

Cholesterol esterification and p53-mediated tumor suppression.

Many human cancers carry missense mutations in or deletions of the tumor protein 53 (TP53) tumor suppressor gene. TP53's product, p53 regulates many biological processes, including cell metabolism. Cholesterol is a key lipid needed for the maintenance of membrane function and tissue homeostasis while also serving as a precursor for steroid hormone and bile acid synthesis. An over-abundance of cholesterol can lead to its esterification and storage as cholesterol esters. The recent study has shown that the loss of p53 leads to excessive cholesterol ester biosynthesis, which promotes hepatocellular carcinoma in mice. Blocking cholesterol esterification improves treatment outcomes, particularly for liver cancers with p53 deletions/mutations that originate in a background of non-alcoholic fatty liver disease.

IRF1 regulates self-renewal and stress responsiveness to support hematopoietic stem cell maintenance.

Hematopoietic stem cells (HSCs) are tightly controlled to maintain a balance between blood cell production and self-renewal. While inflammation-related signaling is a critical regulator of HSC activity, the underlying mechanisms and the precise functions of specific factors under steady-state and stress conditions remain incompletely understood. We investigated the role of interferon regulatory factor 1 (IRF1), a transcription factor that is affected by multiple inflammatory stimuli, in HSC regulation. Our findings demonstrate that the loss of IRF1 from mouse HSCs significantly impairs self-renewal, increases stress-induced proliferation, and confers resistance to apoptosis. In addition, given the frequent abnormal expression of IRF1 in leukemia, we explored the potential of IRF1 expression level as a stratification marker for human acute myeloid leukemia. We show that IRF1-based stratification identifies distinct cancer-related signatures in patient subgroups. These findings establish IRF1 as a pivotal HSC controller and provide previously unknown insights into HSC regulation, with potential implications to IRF1 functions in the context of leukemia.

Therapeutic blockade of ER stress and inflammation prevents NASH and progression to HCC.

The incidence of hepatocellular carcinoma (HCC) is rapidly rising largely because of increased obesity leading to nonalcoholic steatohepatitis (NASH), a known HCC risk factor. There are no approved treatments to treat NASH. Here, we first used single-nucleus RNA sequencing to characterize a mouse model that mimics human NASH-driven HCC, the MUP-uPA mouse fed a high-fat diet. Activation of endoplasmic reticulum (ER) stress and inflammation was observed in a subset of hepatocytes that was enriched in mice that progress to HCC. We next treated MUP-uPA mice with the ER stress inhibitor BGP-15 and soluble gp130Fc, a drug that blocks inflammation by preventing interleukin-6 trans-signaling. Both drugs have progressed to phase 2/3 human clinical trials for other indications. We show that this combined therapy reversed NASH and reduced NASH-driven HCC. Our data suggest that these drugs could provide a potential therapy for NASH progression to HCC.

Multifaceted collagen-DDR1 signaling in cancer.

In addition to immune cells and fibroblasts, the tumor microenvironment (TME) comprises an extracellular matrix (ECM) which contains collagens (COLs) whose architecture and remodeling dictate cancer development and progression. COL receptors expressed by cancer cells sense signals generated by microenvironmental alterations in COL state to regulate cell behavior and metabolism. Discoidin domain receptor 1 (DDR1) is a key sensor of COL fiber state and composition that controls tumor cell metabolism and growth, response to therapy, and patient survival. This review focuses on DDR1 to NRF2 signaling, its modulation of autophagy and macropinocytosis (MP), and its role in cancer and other diseases. Elucidating the regulation of DDR1 activity and expression under different pathophysiological conditions will facilitate the discovery of new therapeutics.

Collagen architecture and signaling orchestrate cancer development.

The tumor microenvironment (TME) controls tumor progression and maintenance. Accordingly, tumor-centric cancer treatment must adjust to being more holistic and TME-centric. Collagens are the most abundant TME proteins, and their dynamic remodeling profoundly affects both TME architecture and tumor development. Recent evidence shows that in addition to being structural elements, collagens are an important source of nutrients and decisive growth controlling and immunoregulatory signals. This review focuses on macropinocytosis-dependent collagen support of cancer cell metabolism and the role of collagen fiber remodeling and trimer heterogeneity in control of tumor bioenergetics, growth, progression, and response to therapy. If properly translated, these basic advances may alter the future of cancer treatment.

Signalling by senescent melanocytes hyperactivates hair growth.

Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.

Single-nucleus RNA sequencing of pre-malignant liver reveals disease-associated hepatocyte state with HCC prognostic potential.

Current approaches to staging chronic liver diseases have limited utility for predicting liver cancer risk. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and pre-malignant livers using two distinct mouse models. Downstream analyses unraveled a previously uncharacterized disease-associated hepatocyte (daHep) transcriptional state. These cells were absent in healthy livers but increasingly prevalent as chronic liver disease progressed. Copy number variation (CNV) analysis of microdissected tissue demonstrated that daHep-enriched regions are riddled with structural variants, suggesting these cells represent a pre-malignant intermediary. Integrated analysis of three recent human snRNA-seq datasets confirmed the presence of a similar phenotype in human chronic liver disease and further supported its enhanced mutational burden. Importantly, we show that high daHep levels precede carcinogenesis and predict a higher risk of hepatocellular carcinoma development. These findings may change the way chronic liver disease patients are staged, surveilled, and risk stratified.

Angiotensin II receptor inhibition ameliorates liver fibrosis and enhances hepatocellular carcinoma infiltration by effector T cells.

Although viral hepatocellular carcinoma (HCC) is declining, nonviral HCC, which often is the end stage of nonalcoholic or alcoholic steatohepatitis (NASH, ASH), is on an upward trajectory. Immune checkpoint inhibitors (ICIs) that block the T cell inhibitory receptor PD-1 were approved for treatment of all HCC types. However, only a minority of HCC patients show a robust and sustained response to PD-1 blockade, calling for improved understanding of factors that negatively impact response rate and duration and the discovery of new adjuvant treatments that enhance ICI responsiveness. Using a mouse model of NASH-driven HCC, we identified peritumoral fibrosis as a potential obstacle to T cell-mediated tumor regression and postulated that antifibrotic medications may increase ICI responsiveness. We now show that the angiotensin II receptor inhibitor losartan, a commonly prescribed and safe antihypertensive drug, reduced liver and peritumoral fibrosis and substantially enhanced anti-PD-1-induced tumor regression. Although losartan did not potentiate T cell reinvigoration, it substantially enhanced HCC infiltration by effector CD8+ T cells compared to PD-1 blockade alone. The beneficial effects of losartan correlated with blunted TGF-ß receptor signaling, reduced collagen deposition, and depletion of immunosuppressive fibroblasts.

Hepatic stellate cell stearoyl co-A desaturase activates leukotriene B4 receptor 2 - ß-catenin cascade to promote liver tumorigenesis.

Hepatocellular carcinoma (HCC) is the 3rd most deadly malignancy. Activated hepatic stellate cells (aHSC) give rise to cancer-associated fibroblasts in HCC and are considered a potential therapeutic target. Here we report that selective ablation of stearoyl CoA desaturase-2 (Scd2) in aHSC globally suppresses nuclear CTNNB1 and YAP1 in tumors and tumor microenvironment and prevents liver tumorigenesis in male mice. Tumor suppression is associated with reduced leukotriene B4 receptor 2 (LTB4R2) and its high affinity oxylipin ligand, 12-hydroxyheptadecatrienoic acid (12-HHTrE). Genetic or pharmacological inhibition of LTB4R2 recapitulates CTNNB1 and YAP1 inactivation and tumor suppression in culture and in vivo. Single cell RNA sequencing identifies a subset of tumor-associated aHSC expressing Cyp1b1 but no other 12-HHTrE biosynthetic genes. aHSC release 12-HHTrE in a manner dependent on SCD and CYP1B1 and their conditioned medium reproduces the LTB4R2-mediated tumor-promoting effects of 12-HHTrE in HCC cells. CYP1B1-expressing aHSC are detected in proximity of LTB4R2-positive HCC cells and the growth of patient HCC organoids is blunted by LTB4R2 antagonism or knockdown. Collectively, our findings suggest aHSC-initiated 12-HHTrE-LTB4R2-CTNNB1-YAP1 pathway as a potential HCC therapeutic target.

Fructose-1,6-bisphosphatase is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness.

Insulin inhibits gluconeogenesis and stimulates glucose conversion to glycogen and lipids. How these activities are coordinated to prevent hypoglycemia and hepatosteatosis is unclear. Fructose-1,6-bisphosphatase (FBP1) is rate controlling for gluconeogenesis. However, inborn human FBP1 deficiency does not cause hypoglycemia unless accompanied by fasting or starvation, which also trigger paradoxical hepatomegaly, hepatosteatosis, and hyperlipidemia. Hepatocyte FBP1-ablated mice exhibit identical fasting-conditional pathologies along with AKT hyperactivation, whose inhibition reversed hepatomegaly, hepatosteatosis, and hyperlipidemia but not hypoglycemia. Surprisingly, fasting-mediated AKT hyperactivation is insulin dependent. Independently of its catalytic activity, FBP1 prevents insulin hyperresponsiveness by forming a stable complex with AKT, PP2A-C, and aldolase B (ALDOB), which specifically accelerates AKT dephosphorylation. Enhanced by fasting and weakened by elevated insulin, FBP1:PP2A-C:ALDOB:AKT complex formation, which is disrupted by human FBP1 deficiency mutations or a C-terminal FBP1 truncation, prevents insulin-triggered liver pathologies and maintains lipid and glucose homeostasis. Conversely, an FBP1-derived complex disrupting peptide reverses diet-induced insulin resistance.

Angiotensin II receptor inhibition ameliorates liver fibrosis and enhances hepatocellular carcinoma infiltration by effector T cells.

Although viral hepatocellular carcinoma (HCC) is declining, non-viral HCC, which often is the end-stage of non-alcoholic or alcoholic steatohepatitis (NASH, ASH), is on an upward trajectory. Immune checkpoint inhibitors (ICI) that block the T cell inhibitory receptor PD-1 were approved for treatment of all HCC types. However, only a small portion of HCC patients show a robust and sustained response to PD-1 blockade, calling for improved understanding of factors that negatively impact response rate and duration and the discovery of new adjuvant treatments that enhance ICI responsiveness. Using a mouse model of NASH-driven HCC, we identified peritumoral fibrosis as a potential obstacle to T cell mediated tumor regression and postulated that anti-fibrotic medications may increase ICI responsiveness. We now show that the angiotensin II receptor inhibitor losartan, a commonly prescribed and safe antihypertensive drug, reduced liver and peritumoral fibrosis and substantially enhanced anti-PD-1 induced tumor regression. Although losartan did not potentiate T cell reinvigoration, it substantially enhanced HCC infiltration by effector CD8 + T cells compared to PD-1 blockade alone. The beneficial effects of losartan correlated with inhibition of TGF-ß receptor signaling, collagen deposition and depletion of immunosuppressive fibroblasts. Significance: Immune checkpoint inhibitors are used in HCC treatment but overall response rates for single agent PD-1/PD-L1 blockers have remained stubbornly low. Using a mouse model of NASH-driven HCC, we show that co-treatment with the safe and inexpensive angiotensin II receptor inhibitor losartan substantially enhanced anti-PD-1 triggered HCC regression. Although losartan did not influence the reinvigoration of exhausted CD8 + T cells it considerably enhanced their intratumoral invasion, which we postulated to be compromised by peritumoral fibrosis. Indeed, the beneficial effect of losartan correlated with inhibition of TGF-ß signaling and collagen deposition, and depletion of immunosuppressive fibroblasts. Losartan should be evaluated for its adjuvant activity in HCC patients undergoing PD-1/PD-L1 blocking therapy.

ATF4 suppresses hepatocarcinogenesis by inducing SLC7A11 (xCT) to block stress-related ferroptosis.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC), a leading cause of cancer-related death, is associated with viral hepatitis, non-alcoholic steatohepatitis (NASH), and alcohol-related steatohepatitis, all of which trigger endoplasmic reticulum (ER) stress, hepatocyte death, inflammation, and compensatory proliferation. Using ER stress-prone MUP-uPA mice, we established that ER stress and hypernutrition cooperate to cause NASH and HCC, but the contribution of individual stress effectors, such as activating transcription factor 4 (ATF4), to HCC and their underlying mechanisms of action remained unknown. METHODS: Hepatocyte-specific ATF4-deficient MUP-uPA mice (MUP-uPA/Atf4Δhep) and control MUP-uPA/Atf4F/F mice were fed a high-fat diet to induce NASH-related HCC, and Atf4F/F and Atf4Δhep mice were injected with diethylnitrosamine to model carcinogen-induced HCC. Histological, biochemical, and RNA-sequencing analyses were performed to identify and define the role of ATF4-induced solute carrier family 7a member 11 (SLC7A11) expression in hepatocarcinogenesis. Reconstitution of SLC7A11 in ATF4-deficient primary hepatocytes and mouse livers was used to study its effects on ferroptosis and HCC development. RESULTS: Hepatocyte ATF4 ablation inhibited hepatic steatosis, but increased susceptibility to ferroptosis, resulting in accelerated HCC development. Although ATF4 activates numerous genes, ferroptosis susceptibility and hepatocarcinogenesis were reversed by ectopic expression of a single ATF4 target, Slc7a11, coding for a subunit of the cystine/glutamate antiporter xCT, which is needed for glutathione synthesis. A ferroptosis inhibitor also reduced liver damage and inflammation. ATF4 and SLC7A11 amounts were positively correlated in human HCC and livers of patients with NASH. CONCLUSIONS: Despite ATF4 being upregulated in established HCC, it serves an important protective function in normal hepatocytes. By maintaining glutathione production, ATF4 inhibits ferroptosis-dependent inflammatory cell death, which is known to promote compensatory proliferation and hepatocarcinogenesis. Ferroptosis inhibitors or ATF4 activators may also blunt HCC onset. IMPACT AND IMPLICATIONS: Liver cancer or hepatocellular carcinoma (HCC) is associated with multiple aetiologies. Most HCC aetiologies cause hepatocyte stress and death, as well as subsequent inflammation, and compensatory proliferation, thereby accelerating HCCdevelopment. The contribution of individual stress effectors to HCC and their underlying mechanisms of action were heretofore unknown. This study shows that the stress-responsive transcription factor ATF4 blunts liver damage and cancer development by suppressing iron-dependent cell death (ferroptosis). Although ATF4 ablation prevents hepatic steatosis, it also increases susceptibility to ferroptosis, due to decreased expression of the cystine/glutamate antiporter SLC7A11, whose expression in human HCC and NASH correlates with ATF4. These findings reinforce the notion that benign steatosis may be protective and does not increase cancer risk unless accompanied by stress-induced liver damage. These results have important implications for prevention of liver damage and cancer.

IRF1 regulates self-renewal and stress-responsiveness to support hematopoietic stem cell maintenance.

Inflammatory mediators induce emergency myelopoiesis and cycling of adult hematopoietic stem cells (HSCs) through incompletely understood mechanisms. To suppress the unwanted effects of inflammation and preserve its beneficial outcomes, the mechanisms by which inflammation affects hematopoiesis need to be fully elucidated. Rather than focusing on specific inflammatory stimuli, we here investigated the role of transcription factor Interferon (IFN) regulatory factor 1 (IRF1), which receives input from several inflammatory signaling pathways. We identify IRF1 as a master HSC regulator. IRF1 loss impairs HSC self-renewal, increases stress-induced cell cycle activation, and confers apoptosis resistance. Transcriptomic analysis revealed an aged, inflammatory signature devoid of IFN signaling with reduced megakaryocytic/erythroid priming and antigen presentation in IRF1-deficient HSCs. Finally, we conducted IRF1-based AML patient stratification to identify groups with distinct proliferative, survival and differentiation features, overlapping with our murine HSC results. Our findings position IRF1 as a pivotal regulator of HSC preservation and stress-induced responses.

P53 deficiency affects cholesterol esterification to exacerbate hepatocarcinogenesis.

BACKGROUND AND AIMS: Cholesterol ester (CE) biosynthesis and homeostasis play critical roles in many cancers, including HCC, but their exact mechanistic contributions to HCC disease development require further study. APPROACH AND RESULTS: Here, we report on a proposed role of tumor suppressor P53 in its repressing ubiquitin-specific peptidase 19 (USP19) and sterol O-acyltransferase (SOAT) 1, which maintains CE homeostasis. USP19 enhances cholesterol esterification and contributes to hepatocarcinogenesis (HCG) by deubiquitinating and stabilizing SOAT1. Loss of either SOAT1 or USP19 dramatically attenuates cholesterol esterification and HCG in P53-deficient mice fed with either a normal chow diet or a high-cholesterol, high-fat diet (HCHFD). SOAT1 inhibitor avasimibe has more inhibitory effect on HCC progression in HCHFD-maintained P53-deficient mice when compared to the inhibitors of de novo cholesterol synthesis. Consistent with our findings in the mouse model, the P53-USP19-SOAT1 signaling axis is also dysregulated in human HCCs. CONCLUSIONS: Collectively, our findings demonstrate that SOAT1 participates in HCG by increasing cholesterol esterification, thus indicating that SOAT1 is a potential biomarker and therapeutic target in P53-deficient HCC.

Collagenolysis-dependent DDR1 signalling dictates pancreatic cancer outcome.

Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic, aggressive cancer that frequently progresses and spreads by metastasis to the liver1. Cancer-associated fibroblasts, the extracellular matrix and type I collagen (Col I) support2,3 or restrain the progression of PDAC and may impede blood supply and nutrient availability4. The dichotomous role of the stroma in PDAC, and the mechanisms through which it influences patient survival and enables desmoplastic cancers to escape nutrient limitation, remain poorly understood. Here we show that matrix-metalloprotease-cleaved Col I (cCol I) and intact Col I (iCol I) exert opposing effects on PDAC bioenergetics, macropinocytosis, tumour growth and metastasis. Whereas cCol I activates discoidin domain receptor 1 (DDR1)-NF-κB-p62-NRF2 signalling to promote the growth of PDAC, iCol I triggers the degradation of DDR1 and restrains the growth of PDAC. Patients whose tumours are enriched for iCol I and express low levels of DDR1 and NRF2 have improved median survival compared to those whose tumours have high levels of cCol I, DDR1 and NRF2. Inhibition of the DDR1-stimulated expression of NF-κB or mitochondrial biogenesis blocks tumorigenesis in wild-type mice, but not in mice that express MMP-resistant Col I. The diverse effects of the tumour stroma on the growth and metastasis of PDAC and on the survival of patients are mediated through the Col I-DDR1-NF-κB-NRF2 mitochondrial biogenesis pathway, and targeting components of this pathway could provide therapeutic opportunities.

Opposing roles of hepatic stellate cell subpopulations in hepatocarcinogenesis.

Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality worldwide, develops almost exclusively in patients with chronic liver disease and advanced fibrosis1,2. Here we interrogated functions of hepatic stellate cells (HSCs), the main source of liver fibroblasts3, during hepatocarcinogenesis. Genetic depletion, activation or inhibition of HSCs in mouse models of HCC revealed their overall tumour-promoting role. HSCs were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analyses of mouse and human HSC subpopulations by single-cell RNA sequencing together with genetic ablation of subpopulation-enriched mediators revealed dual functions of HSCs in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSCs, protected against hepatocyte death and HCC development. By contrast, type I collagen, enriched in activated myofibroblastic HSCs, promoted proliferation and tumour development through increased stiffness and TAZ activation in pretumoural hepatocytes and through activation of discoidin domain receptor 1 in established tumours. An increased HSC imbalance between cytokine-producing HSCs and myofibroblastic HSCs during liver disease progression was associated with increased HCC risk in patients. In summary, the dynamic shift in HSC subpopulations and their mediators during chronic liver disease is associated with a switch from HCC protection to HCC promotion.