Cytokeratin-19 positivity is acquired along cancer progression and does not predict cell origin in rat hepatocarcinogenesis.
ABSTRACT: Although the expression of the stem/progenitor cell marker cytokeratin-19 (CK-19) has been associated with the worst clinical prognosis among all HCC subclasses, it is yet unknown whether its presence in HCC is the result of clonal expansion of hepatic progenitor cells (HPCs) or of de-differentiation of mature hepatocytes towards a progenitor-like cell phenotype. We addressed this question by using two rat models of hepatocarcinogenesis: the Resistant-Hepatocyte (R-H) and the Choline-methionine deficient (CMD) models. Our data indicate that the expression of CK-19 is not the result of a clonal expansion of HPCs (oval cells in rodents), but rather of a further step of preneoplastic hepatocytes towards a less differentiated phenotype and a more aggressive behavior. Indeed, although HCCs were positive for CK-19, very early preneoplastic foci (EPFs) were completely negative for this marker. While a few weeks later the vast majority of preneoplastic nodules remained CK-19 negative, a minority became positive, suggesting that CK-19 expression is the result of de-differentiation of a subset of EPFs, rather than a marker of stem/progenitor cells. Moreover, the gene expression profile of CK-19-negative EPFs clustered together with CK-19-positive nodules, but was clearly distinct from CK-19 negative nodules and oval cells.i) CK-19-positive cells are not involved in the early clonal expansion observed in rat hepatocarcinogenesis; ii) CK-19 expression arises in preneoplastic hepatocyte lesions undergoing malignant transformation; iii) CK-19 positivity in HCCs does not necessarily reflect the cell of origin of the tumor, but rather the plasticity of preneoplastic cells during the tumorigenic process.
Project description:Analysis of early changes in the R-H model of carcinogenesis in order to investigate the relationship between oval cell proliferation and preneoplastic foci 5 controls, 5 CK-19-negative preneoplastic nodules, 5 CK-19-positive preneoplastic nodule, 5 Preneoplastic foci
Project description:T3 induces a global shift of the expression profile of KRT-19+ lesions towards that of fully differentiated hepatocytes Overall design: 8 controls, 10 CK-19-negative preneoplastic nodules, 10 CK-19-positive preneoplastic nodule, 10 CK-19-positive preneoplastic nodule +T3 2 d,10 CK-19-positive preneoplastic nodule +T3 4 d.
Project description:UNLABELLED:Human hepatocellular carcinoma (HCC) is a heterogeneous disease of distinct clinical subgroups. A principal source of tumor heterogeneity may be cell type of origin, which in liver includes hepatocyte or adult stem/progenitor cells. To address this issue, we investigated the molecular mechanisms underlying the fate of the enzyme-altered preneoplastic lesions in the resistant hepatocyte (RH) model. Sixty samples classified as focal lesions, adenoma, and early and advanced HCCs were microdissected after morphological and immunohistochemical evaluation and subjected to global gene expression profiling. The analysis of progression of the persistent glutathione S-transferase (GSTP)(+) focal lesions to fully developed HCC showed that approximately 50% of persistent nodules and all HCCs expressed cytokeratin 19 (CK19), whereas 14% of remodeling nodules were CK19(+). Unsupervised hierarchical clustering of the expression profiles also grouped the samples according to CK19 expression. Furthermore, supervised analysis using the differentially expressed genes in each cluster combined with gene connectivity tools identified 1308 unique genes and a predominance of the AP-1/JUN network in the CK19(+) lesions. In contrast, the CK19-negative cluster exhibited only limited molecular changes (156 differentially expressed genes versus normal liver) consistent with remodeling toward differentiated phenotype. Finally, comparative functional genomics showed a stringent clustering of CK19(+) early lesions and advanced HCCs with human HCCs characterized by poor prognosis. Furthermore, the CK19-associated gene expression signature accurately predicted patient survival (P < 0.009) and tumor recurrence (P < 0.006). CONCLUSION:Our data establish CK19 as a prognostic marker of early neoplastic lesions and strongly suggest the progenitor derivation of HCC in the rat RH model. The capacity of CK19-associated gene signatures to stratify HCC patients according to clinical prognosis indicates the usefulness of the RH model for studies of stem/progenitor-derived HCC.
Project description:Combined hepatocellular and cholangiocarcinoma (CHC) is a unique subtype of liver cancer comprising both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC); however, its cellular origin remains unclear. The purpose of this study was to investigate the clinicopathologic features and the clonal relationship between HCC and ICC in 34 patients with CHC.The clinicopathologic features and prognosis of the 34 CHC patients were compared with those of 29 patients with separated HCC and ICC (SHC). Loss of heterozygosity (LOH) at 10 highly polymorphic microsatellite markers was detected in 16 CHC and 10 SHC tissues for determination of the clonal origin of CHC. Expression of hepatocyte markers [hepatocyte paraffin 1 (Hep Par 1) and glypican 3 (GPC3)] and cholangiocyte markers [cytokeratin (CK)7 and 19] in tumor tissues was examined by immuno histochemical analysis.In the 16 CHC specimens, the difference in LOH patterns between HCC and ICC was less than 30%, suggesting the same clonal origin of HCC and ICC. Consistent with this finding, immunohistochemical analysis revealed that hepatocyte markers (Hep Par 1 and GPC3) and cholangiocyte markers (CK7 and CK19) were simultaneously expressed in both the HCC and ICC components in 52.9% of CHC specimens, suggesting that the two components shared a similar phenotype with hepatic progenitor cells (HPCs). On the contrary, in all 10 SHC cases, the difference in LOH patterns between the HCC and ICC components was greater than 30%, suggesting different clonal origins of HCC and ICC. Overall survival and disease-free survival were shorter for patients with CHC than for patients with SHC (P < 0.05).Our results suggest that the HCC and ICC components of CHC may originate from the same clone, having the potential for dual-directional differentiation similar to HPCs. CHC tended to exhibit the biological behaviors of both HCC and ICC, which may enhance the infiltrative capacity of tumor cells, leading to poor clinical outcomes for patients with CHC.
Project description:Most hepatocellular carcinomas (HCCs) develop in patients with chronic hepatitis, which creates a microenvironment for the growth of hepatic progenitor cells (HPCs) at the periportal area and subsequent development of HCCs. We investigated the signal from the inflammatory liver for this pathogenic process in the hepatic conditional ?-catenin knockout mouse model. Senescent ?-catenin-depleted hepatocytes in aged mice create an inflammatory microenvironment that stimulates periportal HPC expansion but arrests differentiation, which predisposes mice to the development of liver tumors. The release of complement C1q from macrophages in the inflammatory niche was identified as the unorthodox signal that activated the ?-catenin pathway in periportal HPCs and was responsible for their expansion and de-differentiation. C1q inhibitors blocked the ?-catenin pathway in both the expanding HPCs and the liver tumors but spared its orthodox pathway in pericentral normal hepatocytes. This mechanism has been validated in human liver specimens from patients with chronic hepatitis. Taken together, these results demonstrate that C1q- mediated activation of ?-catenin pathway in periportal HPCs is a previously unrecognized mechanism for replenishing hepatocytes in the inflammatory liver and, if unchecked, for promoting hepatocarcinogenesis. C1q may become a new target for blocking carcinogenesis in patients with chronic hepatitis.
Project description:Although inhibition of autophagy has been implicated in the onset and progression of cancer cells, it is still unclear whether its dysregulation at early stages of tumorigenesis plays an oncogenic or a tumor suppressor role. To address this question, we employed the Resistant-Hepatocyte rat model to study the very early stages of hepatocellular carcinoma (HCC) development. We detected a different autophagy-related gene expression and changes in the ultrastructural profile comparing the most aggressive preneoplastic lesions, namely those positive for the putative progenitor cell marker cytokeratin-19 (KRT-19) with the negative ones. The ultrastructural and immunohistochemical analyses of KRT-19-positive preneoplastic hepatocytes showed the presence of autophagic vacuoles which was associated with p62, Ambra1 and Beclin1 protein accumulation suggesting that a differential modulation of autophagy occurs at early stages of the oncogenesis in KRT-19-positive vs negative lesions. We observed an overall decrease of the autophagy-related genes transcripts and a strong up-regulation of miR-224 in the KRT-19-positive nodules. Interestingly, the treatment with the autophagy inducer, Amiodarone, caused a marked increase in the proliferation of KRT-19 positive preneoplastic lesions associated with a strong increase of their size; by contrast, Chloroquine, an inhibitor of the autophagic process, led to their reduction. These results show that autophagy modulation is a very early event in hepatocarcinogenesis and is restricted to a hepatocytes subset in the most aggressive preneoplastic lesions. Our findings highlight the induction of autophagy as a permissive condition favouring cancer progression indicating in its inhibition a therapeutic goal to interfere with the development of HCC.
Project description:<h4>Purpose</h4>Recently, it has been emphasized that hepatic progenitor/oval cells (HPCs) are significantly involved in liver fibrogenesis. We evaluated the multipotential population of HPCs by transmission electron microscope (TEM), including relations with adherent hepatic nonparenchymal cells (NPCs) in rats with biliary fibrosis induced by bile duct ligation (BDL).<h4>Methods</h4>The study used 6-week-old Wistar Crl: WI(Han) rats after BDL for 1, 6, and 8 weeks.<h4>Results</h4>Current ultrastructural analysis showed considerable proliferation of HPCs in experimental intensive biliary fibrosis. HPCs formed proliferating bile ductules and were scattered in periportal connective tissue. We distinguished 4 main types of HPCs: 0, I, II (bile duct-like cells; most common), and III (hepatocyte-like cells). We observed, very seldom presented in literature, cellular interactions between HPCs and adjacent NPCs, especially commonly found transitional hepatic stellate cells (T-HSCs) and Kupffer cells/macrophages. We showed the phenomenon of penetration of the basement membrane of proliferating bile ductules by cytoplasmic processes sent by T-HSCs and the formation of direct cell-cell contact with ductular epithelial cells related to HPCs.<h4>Conclusions</h4>HPC proliferation induced by BDL evidently promotes portal fibrogenesis. Better understanding of the complex cellular interactions between HPCs and adjacent NPCs, especially T-HSCs, may help develop antifibrotic therapies in the future.
Project description:Proliferating ducts, termed "oval cells," have long been thought to be bipotential, that is, produce both biliary ducts and hepatocytes during chronic liver injury. The precursor to oval cells is considered to be a facultative liver stem cell (LSC). Recent lineage tracing experiments indicated that the LSC is SRY-related HMG box transcription factor 9 positive (Sox9(+) ) and can replace the bulk of hepatocyte mass in several settings. However, no clonal relationship between Sox9(+) cells and the two epithelial liver lineages was established. We labeled Sox9(+) mouse liver cells at low density with a multicolor fluorescent confetti reporter. Organoid formation validated the progenitor activity of the labeled population. Sox9(+) cells were traced in multiple oval cell injury models using both histology and fluorescence-activated cell sorting. Surprisingly, only rare clones containing both hepatocytes and oval cells were found in any experiment. Quantitative analysis showed that Sox9(+) cells contributed only minimally (<1%) to the hepatocyte pool, even in classic oval cell injury models. In contrast, clonally marked mature hepatocytes demonstrated the ability to self-renew in all classic mouse oval cell activation injuries. A hepatocyte chimera model to trace hepatocytes and nonparenchymal cells also demonstrated the prevalence of hepatocyte-driven regeneration in mouse oval cell injury models.Sox9(+) ductal progenitor cells give rise to clonal oval cell proliferation and bipotential organoids, but rarely produce hepatocytes in vivo. Hepatocytes themselves are the predominant source of new parenchyma cells in prototypical mouse models of oval cell activation.
Project description:Hepatic progenitor cells (HPCs) are small cells with a relative large oval nucleus and a scanty cytoplasm situated in the canals of Hering that express markers of (immature) hepatocytes and cholangiocytes. HPCs are present in large numbers in alcoholic steatohepatitis (ASH), one of the leading causes of chronic liver disease. To date, the mechanisms responsible for proliferation and differentiation of human HPCs are still poorly understood and the role of HPCs in ASH development is unknown. In this study, we aimed to characterise human HPCs and their interactions with other cells through comparison, on both protein and RNA level, of HPC-enriched cell populations from adult human liver tissue using different isolation methods. Fresh human liver tissue was collected from ASH explant livers and HPC-enriched cell populations were obtained via four different isolation methods: side population (SP), epithelial cell adhesion molecule (EpCAM) and trophoblast antigen 2 (TROP-2) membrane marker isolation and laser capture microdissection. Gene expression profiles of fluorescent-activated cell-sorted HPCs, whole liver extracts and laser microdissected HPC niches were determined by RNA-sequencing. Immunohistochemical evaluation of the isolated populations indicated the enrichment of HPCs in the SP, EpCAM+ and TROP-2+ cell populations. Pathway analysis of the transcription profiles of human HPCs showed an enrichment and activation of known HPC pathways like Wnt/?-catenin, TWEAK and HGF. Integration of the HPC niche profile suggests autocrine signalling by HPCs (TNF?, PDGFB and VEGFA) as well as paracrine signalling from the surrounding niche cells including MIF and IGF-1. In addition, we identified IL-17?A signalling as a potentially novel pathway in HPC biology. In conclusion, we provide the first RNA-seq-based, comparative transcriptome analysis of isolated human HPCs from ASH patients and revealed active signalling between HPCs and their surrounding niche cells in ASH livers and suggest that HPCs can actively contribute to liver inflammation.
Project description:Oval cell activation occurs under conditions of severe liver injury when normal hepatocyte proliferation is blocked. Recent studies have shown that a subset of hepatocellular carcinomas expresses oval cell markers, suggesting that these cells are targets of hepatocarcinogens. However, the signaling pathways that control oval cell activation and proliferation are not well characterized. Based on the role of the nutrient signaling kinase complex, mTORC1, in liver development, we investigated the role of this pathway in oval cell activation. Oval cell proliferation was induced in male Fisher rats by a modification of the traditional choline deficient plus ethionine model (CDE) or by 2-acetylaminoflourene treatment followed by 2/3 partial hepatectomy with or without initiation by diethylnitrosamine. To assess the role of mTOR in the oval cell response and development of preneoplastic foci, the effect of the mTORC1 inhibitor, rapamycin, was studied in all models. Rapamycin induced a significant suppression of the oval cell response in both models, an effect that coincided with a decrease in oval cell proliferation. Rapamycin administration did not affect the abundance of neutrophils or natural killer cells in CDE-treated liver or the expression of key cytokines. Gene expression studies revealed the fetal hepatocyte marker MKP-4 to be expressed in oval cells. In an experimental model of hepatic carcinogenesis, rapamycin decreased the size of preneoplastic foci and the rate of cell proliferation within the foci. mTORC1 signaling plays a key role in the oval cell response and in the development of preneoplastic foci. This pathway may be a target for the chemoprevention of hepatocellular carcinoma.