Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality worldwide, develops almost exclusively in patients with chronic liver disease and advanced fibrosis
. Here we interrogated ...functions of hepatic stellate cells (HSCs), the main source of liver fibroblasts
, 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.
Cancer-associated fibroblasts (CAF) are a poorly characterized cell population in the context of liver cancer. Our study investigates CAF functions in intrahepatic cholangiocarcinoma (ICC), a highly ...desmoplastic liver tumor. Genetic tracing, single-cell RNA sequencing, and ligand-receptor analyses uncovered hepatic stellate cells (HSC) as the main source of CAF and HSC-derived CAF as the dominant population interacting with tumor cells. In mice, CAF promotes ICC progression, as revealed by HSC-selective CAF depletion. In patients, a high panCAF signature is associated with decreased survival and increased recurrence. Single-cell RNA sequencing segregates CAF into inflammatory and growth factor-enriched (iCAF) and myofibroblastic (myCAF) subpopulations, displaying distinct ligand-receptor interactions. myCAF-expressed hyaluronan synthase 2, but not type I collagen, promotes ICC. iCAF-expressed hepatocyte growth factor enhances ICC growth via tumor-expressed MET, thus directly linking CAF to tumor cells. In summary, our data demonstrate promotion of desmoplastic ICC growth by therapeutically targetable CAF subtype-specific mediators, but not by type I collagen.
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•The majority of CAF in ICC are derived from hepatic stellate cells•Inflammatory CAF promote ICC through HGF and its receptor MET•myCAF promote ICC through Has2/hyaluronic acid•CAF-derived type I collagen contributes to stiffness but does not promote ICC growth
Intrahepatic cholangiocarcinoma (ICC) is an extraordinarily stiff liver tumor due to abundant scar-forming cancer-associated fibroblasts (CAF). Here, Affo et al. determine the origin and functions of CAF, and uncover distinct CAF subsets, promoting ICC growth via different therapeutically targetable mediators. Thus, CAF and their mediators may serve as therapeutic targets for ICC.
The novel coronavirus SARS-CoV-2 (coronavirus disease 19, or COVID-19) primarily causes pulmonary injury, but has been implicated to cause hepatic injury, both by serum markers and histologic ...evaluation. The histologic pattern of injury has not been completely described. Studies quantifying viral load in the liver are lacking. Here we report the clinical and histologic findings related to the liver in 40 patients who died of complications of COVID-19. A subset of liver tissue blocks were subjected to polymerase chain reaction (PCR) for viral ribonucleic acid (RNA). Peak levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were elevated; median ALT peak 68 U/l (normal up to 46 U/l) and median AST peak 102 U/l (normal up to 37 U/l). Macrovesicular steatosis was the most common finding, involving 30 patients (75%). Mild lobular necroinflammation and portal inflammation were present in 20 cases each (50%). Vascular pathology, including sinusoidal microthrombi, was infrequent, seen in six cases (15%). PCR of liver tissue was positive in 11 of 20 patients tested (55%). In conclusion, we found patients dying of COVID-19 had biochemical evidence of hepatitis (of variable severity) and demonstrated histologic findings of macrovesicular steatosis and mild acute hepatitis (lobular necroinflammation) and mild portal inflammation. We also identified viral RNA in a sizeable subset of liver tissue samples.
Immune checkpoint inhibitors have limited efficacy in many tumors. We investigated mechanisms of tumor resistance to inhibitors of programmed cell death–1 (PDCD1, also called PD-1) in mice with ...gastric cancer, and the role of its ligand, PD-L1.
Gastrin-deficient mice were given N-methyl-N-nitrosourea (MNU) in drinking water along with Helicobacter felis to induce gastric tumor formation; we also performed studies with H/K-ATPase-hIL1B mice, which develop spontaneous gastric tumors at the antral–corpus junction and have parietal cells that constitutively secrete interleukin 1B. Mice were given injections of an antibody against PD-1 or an isotype control before tumors developed, or anti–PD-1 and 5-fluorouracil and oxaliplatin, or an antibody against lymphocyte antigen 6 complex locus G (also called Gr-1), which depletes myeloid-derived suppressor cells MDSCs), after tumors developed. We generated knock-in mice that express PD-L1 specifically in the gastric epithelium or myeloid lineage.
When given to gastrin-deficient mice before tumors grew, anti–PD-1 significantly reduced tumor size and increased tumor infiltration by T cells. However, anti–PD-1 alone did not have significant effects on established tumors in these mice. Neither early nor late anti–PD-1 administration reduced tumor growth in the presence of MDSCs in H/K-ATPase-hIL-1β mice. The combination of 5-fluorouracil and oxaliplatin reduced MDSCs, increased numbers of intra-tumor CD8+ T cells, and increased the response of tumors to anti–PD-1; however, this resulted in increased tumor expression of PD-L1. Expression of PD-L1 by tumor or immune cells increased gastric tumorigenesis in mice given MNU. Mice with gastric epithelial cells that expressed PD-L1 did not develop spontaneous tumors, but they developed more and larger tumors after administration of MNU and H felis, with accumulation of MDSCs.
In mouse models of gastric cancer, 5-fluorouracil and oxaliplatin reduce numbers of MDSCs to increase the effects of anti–PD-1, which promotes tumor infiltration by CD8+ T cells. However, these chemotherapeutic agents also induce expression of PD-L1 by tumor cells. Expression of PD-L1 by gastric epithelial cells increases tumorigenesis in response to MNU and H felis, and accumulation of MDSCs, which promote tumor progression. The timing and site of PD-L1 expression is therefore important in gastric tumorigenesis and should be considered in design of therapeutic regimens.
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Anorectal melanoma is a rare disease that carries a poor prognosis. To date, limited genetic analyses confirmed KIT mutations as a recurrent genetic event similar to other mucosal melanomas, ...occurring in up to 30% of anorectal melanomas. Importantly, a subset of tumors harboring activating KIT mutations have been found to respond to c-Kit inhibitor-based therapy, with improved patient survival at advanced tumor stages. We performed comprehensive targeted exon sequencing analysis of 467 cancer-related genes in a larger series of 15 anorectal melanomas, focusing on potentially actionable variants based on gain- and loss-of-function mutations. We report the identification of oncogenic driver events in the majority (93%) of anorectal melanomas. These included variants in canonical MAPK pathway effectors rarely observed in cutaneous melanomas (including an HRAS mutation, as well as a BRAF mutation resulting in duplication of threonine 599), and recurrent mutations in the tumor suppressor NF1 in 20% of cases, which represented the second-most frequently mutated gene after KIT in our series. Furthermore, we identify SF3B1 mutations as a recurrent genetic event in mucosal melanomas. Our findings provide an insight into the genetic diversity of anorectal melanomas, and suggest significant potential for alternative targeted therapeutics in addition to c-Kit inhibitors for this melanoma subtype.
In many solid tumors, parasympathetic input is provided by the vagus nerve, which has been shown to modulate tumor growth. However, whether cholinergic signaling directly regulates progression of ...pancreatic ductal adenocarcinoma (PDAC) has not been defined. Here, we found that subdiaphragmatic vagotomy in LSL-
;
-Cre (KC) mice accelerated PDAC development, whereas treatment with the systemic muscarinic agonist bethanechol restored the normal KC phenotype, thereby suppressing the accelerated tumorigenesis caused by vagotomy. In LSL-
;LSL-
;
-Cre mice with established PDAC, bethanechol significantly extended survival. These effects were mediated in part through CHRM1, which inhibited downstream MAPK/EGFR and PI3K/AKT pathways in PDAC cells. Enhanced cholinergic signaling led to a suppression of the cancer stem cell (CSC) compartment, CD11b
myeloid cells, TNFα levels, and metastatic growth in the liver. Therefore, these data suggest that cholinergic signaling directly and indirectly suppresses growth of PDAC cells, and therapies that stimulate muscarinic receptors may be useful in the treatment of PDAC.
Subdiaphragmatic vagotomy or
knockout accelerates pancreatic tumorigenesis, in part via expansion of the CSC compartment. Systemic administration of a muscarinic agonist suppresses tumorigenesis through MAPK and PI3K/AKT signaling, in early stages of tumor growth and in more advanced, metastatic disease. Therefore, CHRM1 may represent a potentially attractive therapeutic target.
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Non-alcoholic steatohepatitis (NASH) is a leading cause of hepatocellular carcinoma (HCC), but mechanisms linking NASH to eventual tumor formation remain poorly understood. Herein, we investigate the ...role of TAZ/WWTR1, which is induced in hepatocytes in NASH, in the progression of NASH to HCC.
The roles of hepatocyte TAZ and its downstream targets were investigated in diet-induced and genetic models of NASH-HCC using gene-targeting, adeno-associated virus 8 (AAV8)-H1-mediated gene silencing, or AAV8-TBG-mediated gene expression. The biochemical signature of the newly elucidated pathway was probed in liver specimens from humans with NASH-HCC.
When hepatocyte-TAZ was silenced in mice with pre-tumor NASH using AAV8-H1-shTaz (short-hairpin Taz), subsequent HCC tumor development was suppressed. In this setting, the tumor-suppressing effect of shTaz was not dependent of TAZ silencing in the tumors themselves and could be dissociated from the NASH-suppressing effects of shTaz. The mechanism linking pre-tumor hepatocyte-TAZ to eventual tumor formation involved TAZ-mediated induction of the NOX2-encoding gene Cybb, which led to NADPH-mediated oxidative DNA damage. As evidence, DNA damage and tumor formation could be suppressed by treatment of pre-tumor NASH mice with AAV8-H1-shCybb; AAV8-TBG-OGG1, encoding the oxidative DNA-repair enzyme 8-oxoguanine glycosylase; or AAV8-TBG-NHEJ1, encoding the dsDNA repair enzyme non-homologous end-joining factor 1. In surrounding non-tumor tissue from human NASH-HCC livers, there were strong correlations between TAZ, NOX2, and oxidative DNA damage.
TAZ in pre-tumor NASH-hepatocytes, via induction of Cybb and NOX2-mediated DNA damage, contributes to subsequent HCC tumor development. These findings illustrate how NASH provides a unique window into the early molecular events that can lead to tumor formation and suggest that NASH therapies targeting TAZ might also prevent NASH-HCC.
Non-alcoholic steatohepatitis (NASH) is emerging as the leading cause of a type of liver cancer called hepatocellular carcinoma (HCC), but molecular events in pre-tumor NASH hepatocytes leading to HCC remain largely unknown. Our study shows that a protein called TAZ in pre-tumor NASH-hepatocytes promotes damage to the DNA of hepatocytes and thereby contributes to eventual HCC. This study reveals a very early event in HCC that is induced in pre-tumor NASH, and the findings suggest that NASH therapies targeting TAZ might also prevent NASH-HCC.
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•Silencing hepatocyte TAZ in pre-tumor NASH suppresses subsequent HCC.•Cybb is the key TAZ-induced gene in NASH hepatocytes that triggers tumor formation.•Cybb encodes NOX2, which promotes HCC by inducing oxidative DNA damage.•Silencing hepatocyte Cybb in pre-tumor NASH, or blocking DNA damage, suppresses HCC.•TAZ, NOX2, oxidative DNA damage are strongly correlated in human NASH-HCC liver.
Gynecologic cancers are routinely screened for DNA mismatch repair (MMR) gene mutations using immunohistochemistry (IHC) and/or polymerase chain reaction (PCR) for microsatellite instability (MSI) to ...enable selection of immune checkpoint inhibitor therapy and screen for Lynch syndrome. The limited data that compare IHC and MSI in endometrial tumors have shown discordance rates of 5–10%. We reviewed MMR/MSI results in gynecologic cancers and used next-generation sequencing (NGS) to interrogate discrepancies. Of the 328 cases with both IHC and MSI results, 256 (78.0%) were microsatellite stable (MSS) with preserved MMR (pMMR), 64 (19.5%) cases were MSI-High (MSI-H) with MMR deficient (dMMR), 2 cases showed subclonal loss of MLH1 and PMS2 with MSI-H, and 6 cases were discordant. Overall, there was a 98.2% (322/328) IHC/MSI concordance. Discordant cases were retested and/or subject to NGS. Of the six discrepant cases, five showed dMMR with MSS and one showed pMMR with MSI-H. One dMMR/MSI-L case showed loss of PMS2 with a germline pathogenic mutation. The pMMR/MSI-H case was found to harbor pathogenic variants in MLH1 and MSH6. One of the two cases with subclonal populations demonstrated MSI-H in the dMMR area and MSS in the pMMR area. These results emphasize the importance of selecting the appropriate tumor tissue for both IHC and molecular testing and demonstrate that NGS can help resolve discrepant MMR and MSI results.
•Gynecologic cancers show high concordance rate between MMR IHC and MSI PCR overall.•Subclonal loss of MMR proteins can lead to IHC/MSI discrepancies.•Selection of appropriate tumor and normal tissue for IHC and MSI testing reduces discrepancies.•NGS can help resolve MMR and MSI discordance.
MicroRNAs (miRNA) are abundant in the circulation and play a central role in diverse biologic processes; they may be useful for early diagnosis of hepatocellular carcinoma.
We conducted a two-phase, ...case-control study (20 pairs for the discovery set and 49 pairs for the validation set) to test the hypothesis that genome-wide dysregulation of circulating miRNAs differentiates hepatocellular carcinoma cases from controls. Taqman low-density arrays were used to examine genome-wide miRNA expression for the discovery set, and quantitative real-time PCR was used to validate candidate miRNAs for both discovery and validation sets.
Sixty-six miRNAs were found to be significantly overexpressed in plasma of hepatocellular carcinoma cases compared with controls after adjusting for false discovery rate (P < 0.05). A volcano plot indicated that seven miRNAs had greater than 2-fold case-control differences with P < 0.01. Four significant miRNAs (miR-150, miR-30c, miR-483-5p, and miR-520b) detectable in all samples with varied expression levels were further validated in a validation set. MiR-483-5p was statistically significantly overexpressed in hepatocellular carcinoma cases compared with controls (3.20 vs. 0.82, P < 0.0001). Hepatocellular carcinoma risk factors and clinic-pathological characteristics did not influence miR-483-5p expression. The combination of plasma miR-483-5p level and hepatitis C virus status can significantly differentiate hepatocellular carcinoma cases from controls with an area under the curve of 0.908 (P < 0.0001). The sensitivity and specificity were, respectively, 75.5% and 89.8%.
These preliminary results suggest the importance of dysregulated circulating miR-483-5p as a potential hepatocellular carcinoma biomarker.
Confirmation of aberrant expression of miR-483-5p in a large prospective hepatocellular carcinoma study will provide support for its application to hepatocellular carcinoma detection.
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