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Hepatic progenitor cells, senescence and IL-6 as the main players in combined hepatocellular-cholangiocarcinoma development

  • María Arechederra
    Correspondence
    Corresponding authors. Address: Program of Hepatology, Centre of Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain.
    Affiliations
    Program of Hepatology, Centre of Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain

    National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Carlos III Health Institute), 28029 Madrid, Spain

    IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain
    Search for articles by this author
  • Maite G. Fernández-Barrena
    Correspondence
    Corresponding authors. Address: Program of Hepatology, Centre of Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain.
    Affiliations
    Program of Hepatology, Centre of Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain

    National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Carlos III Health Institute), 28029 Madrid, Spain

    IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain
    Search for articles by this author
Published:September 20, 2022DOI:https://doi.org/10.1016/j.jhep.2022.09.008

      Linked Article

      See Article, pages 1631–1641
      Primary liver cancer (PLC) is the fourth leading cause of cancer-related deaths and is having an increasing impact on society (https://gco.iarc.fr/). When thinking of liver malignancies, hepatocellular carcinoma (HCC), the pediatric tumor hepatoblastoma, and cholangiocarcinoma (CCA) first come to mind. However, a neoplasm exhibiting elements of both HCC and CCA within the same nodule had already been reported in 1903, later being called “mixed” or “combined” hepatocellular-cholangiocarcinoma (cHCC-CCA).
      • Brunt E.
      • Aishima S.
      • Clavien P.
      • Fowler K.
      • Goodman Z.
      • Gores G.
      • et al.
      cHCC-CCA: consensus terminology for primary liver carcinomas with both hepatocytic and cholangiocytic differentation.
      Although less frequent, the incidence of this malignant entity has been underestimated over the years, mainly due to its difficult histological diagnosis and discrimination from HCC and intrahepatic CCA (iCCA). As such, a significant number of cases have been misclassified as HCC or CCA,
      • Wege H.
      • Schulze K.
      • Felden von J.
      • Calderaro J.
      • Reig M.
      RARE-LIVER) for the rare liver tumors working group of the ERN on HD (ERN. Rare variants of primary liver cancer: fibrolamellar, combined, and sarcomatoid hepatocellular carcinomas.
      ,
      • Calderaro J.
      • Tommaso L.D.
      • Maillé P.
      • Beaufrère A.
      • Nguyen C.T.
      • Heij L.
      • et al.
      Nestin as a diagnostic and prognostic marker for combined hepatocellular-cholangiocarcinoma.
      therefore hindering its molecular characterization and the design of specific cHCC-CCA clinical trials. However, relevant studies are emerging.
      • Calderaro J.
      • Tommaso L.D.
      • Maillé P.
      • Beaufrère A.
      • Nguyen C.T.
      • Heij L.
      • et al.
      Nestin as a diagnostic and prognostic marker for combined hepatocellular-cholangiocarcinoma.
      • Nguyen C.T.
      • Caruso S.
      • Maillé P.
      • Beaufrère A.
      • Augustin J.
      • Favre L.
      • et al.
      Immune profiling of combined hepatocellular-cholangiocarcinoma reveals distinct subtypes and activation of gene signatures predictive of response to immunotherapy.
      • Xue R.
      • Chen L.
      • Zhang C.
      • Fujita M.
      • Li R.
      • Yan S.-M.
      • et al.
      Genomic and transcriptomic profiling of combined hepatocellular and intrahepatic cholangiocarcinoma reveals distinct molecular subtypes.
      A genomic landscape and a comprehensive comparison of cHCC-CCA with HCC and iCCA
      • Xue R.
      • Chen L.
      • Zhang C.
      • Fujita M.
      • Li R.
      • Yan S.-M.
      • et al.
      Genomic and transcriptomic profiling of combined hepatocellular and intrahepatic cholangiocarcinoma reveals distinct molecular subtypes.
      revealed that this rare variant constitutes a distinct subtype with different molecular and clinical characteristics. Like the other PLCs, the asymptomatic nature of incipient cHCC-CCA often results in its detection at an advanced stage; its prognosis is worse than that of HCC, but similar to that of iCCA.
      • Beaufrère A.
      • Calderaro J.
      • Paradis V.
      Combined hepatocellular-cholangiocarcinoma: an update.
      Still much remains to be known about cHCC-CCA tumors.
      Whereas it is mostly accepted that mature hepatocytes and cholangiocytes are the cells of origin of HCC and CCA, respectively,
      • Sia D.
      • Villanueva A.
      • Friedman S.L.
      • Llovet J.M.
      Liver cancer cell of origin, molecular class, and effects on patient prognosis.
      the cell of origin of cHCC-CCA remains a matter of debate. This is an essential issue when it comes to our understanding of the biology and pathogenesis of this rare tumor. Although cHCC-CCA tumors comprise a heterogenous group, their “general” molecular signature of stemness, together with observations in different mouse models, have indeed supported the concept of a stem cell origin.
      • Brunt E.
      • Aishima S.
      • Clavien P.
      • Fowler K.
      • Goodman Z.
      • Gores G.
      • et al.
      cHCC-CCA: consensus terminology for primary liver carcinomas with both hepatocytic and cholangiocytic differentation.
      However, other studies suggested the dedifferentiation and subsequent transformation of mature hepatocytes.
      • Beaufrère A.
      • Calderaro J.
      • Paradis V.
      Combined hepatocellular-cholangiocarcinoma: an update.
      Moreover, it has recently been agreed that the presence of typical stem-cell features is no longer sufficient to classify a tumor as cHCC-CCA, as this phenotype is also observed in certain subtypes of HCCs and CCAs.
      • Brunt E.
      • Aishima S.
      • Clavien P.
      • Fowler K.
      • Goodman Z.
      • Gores G.
      • et al.
      cHCC-CCA: consensus terminology for primary liver carcinomas with both hepatocytic and cholangiocytic differentation.
      ,
      • Beaufrère A.
      • Calderaro J.
      • Paradis V.
      Combined hepatocellular-cholangiocarcinoma: an update.
      Therefore, the origin and characteristics of this type of tumor are still poorly understood.
      In this issue of Journal of Hepatology, Rosenberg et al.
      • Beaufrère A.
      • Calderaro J.
      • Paradis V.
      Combined hepatocellular-cholangiocarcinoma: an update.
      examine in detail the contribution of hepatic progenitor cells (HPCs) to the development of PLC in the context of chronic inflammation. The study points to this progenitor compartment as the cell of origin of cHCC-CCA, and provides relevant insights into the underlying mechanism.
      • Rosenberg N.
      • Haele M.V.
      • Lanton T.
      • Brashi N.
      • Bromberg Z.
      • Adler H.
      • et al.
      Combined hepatocellular-cholangiocarcinoma derives from liver progenitor cells and depends on senescence and IL6 trans-signaling.
      The authors employed the multidrug resistance 2 knockout (Mdr2-KO) mouse model that develops progressive liver disease with portal inflammation, sclerosing cholestatic hepatitis and ductular proliferation.
      • Mauad T.H.
      • Nieuwkerk van C.M.J.
      • Dingemans K.P.
      • Smit J.J.M.
      • Schinkel A.H.
      • Notenboom R.G.E.
      • et al.
      Mice with homozygous disruption of the mdr2 P-glycoprotein gene. A novel animal model for studies of nonsuppurative inflammatory cholangitis and hepatocarcinogenesis.
      It is generally accepted that Mdr2-KO mice spontaneously develop HCC by 12-16 months of age.
      • Rosenberg N.
      • Haele M.V.
      • Lanton T.
      • Brashi N.
      • Bromberg Z.
      • Adler H.
      • et al.
      Combined hepatocellular-cholangiocarcinoma derives from liver progenitor cells and depends on senescence and IL6 trans-signaling.
      • Mauad T.H.
      • Nieuwkerk van C.M.J.
      • Dingemans K.P.
      • Smit J.J.M.
      • Schinkel A.H.
      • Notenboom R.G.E.
      • et al.
      Mice with homozygous disruption of the mdr2 P-glycoprotein gene. A novel animal model for studies of nonsuppurative inflammatory cholangitis and hepatocarcinogenesis.
      • Potikha T.
      • Stoyanov E.
      • Pappo O.
      • Frolov A.
      • Mizrahi L.
      • Olam D.
      • et al.
      Interstrain differences in chronic hepatitis and tumor development in a murine model of inflammation-mediated hepatocarcinogenesis.
      However, the authors explored whether at later stages, when the ductular reaction is prolonged, other tumors, in particular cHCC-CCA, could emerge from HPCs in this chronic inflammatory background.
      To trace HPCs and their progeny the authors generated a Mdr2-KO mouse line in which HPCs were labelled with YFP using the Foxl1 reporter system. When these mice were followed-up for 18 months, half of them developed cHCC-CCA tumors, as indicated by concomitant detection of hepatocytic and cholangiocytic markers. Importantly, these cHCC-CCA tumors arose from HPCs, as shown by the expression of YFP and the progenitor markers CD24 and CD44. These findings were elegantly corroborated in an engineered version of Mdr2-KO mice in which HPCs can be specifically ablated in a controlled manner, as these mice showed a significant reduction in the number of cHCC-CCA tumors. At this point, it was important to establish the relevance of the Mdr2-KO mouse model to the human disease, and this was achieved by transcriptomic analyses comparing murine and human tumors. These assays not only confirmed the similarity between human and murine cHCC-CCAs, they also clearly differentiated these lesions from HCCs, confirmed the enrichment in stemness-related genes and also established a significant overlap between inflammatory and progenitor pathways in cHCC-CCAs across species.
      Most interestingly, these transcriptomic analyses together with immunohistological studies identified the IL-6-gp130-STAT3 pathway, which is known to sustain the stemness niche, as a potentially key molecular player in cHCC-CCA tumors. Consistently, functional studies demonstrated that IL-6-neutralizing antibodies indeed reduced cHCC-CCA tumors in Mrd2-KO mice, albeit treated mice developed more HCCs. These observations confirm the complex role of IL-6 in hepatocarcinogenesis and prompted the authors to further explore the underlying mechanisms of its involvement.
      Single-cell transcriptomic analyses on liver tissues from 18-month-old Mdr2-KO mice revealed a higher expression of IL-6 and IL-6R in immune and parenchymal cells, whereas gp130, and the downstream effectors STAT3 and ERK, were found in the HPC population, suggesting a predominant contribution of the IL-6 trans-signaling pathway
      • Garbers C.
      • Rose-John S.
      Dissecting interleukin-6 classic- and trans-signaling in inflammation and cancer.
      in HPC proliferation and cHCC-CCA development. Indeed, in vitro activation of IL-6 trans-signaling significantly increased HPC proliferation, while its specific blockade reduced the number of cHCC-CCA tumors in mice. This protumorigenic response of HPCs to IL-6 trans-signaling was already known, but IL-6 was specifically produced by tumor-associated macrophages, and as far as it was characterized, only HCC tumors developed.
      • Wan S.
      • Zhao E.
      • Kryczek I.
      • Vatan L.
      • Sadovskaya A.
      • Ludema G.
      • et al.
      Tumor-associated macrophages produce interleukin 6 and signal via STAT3 to promote expansion of human hepatocellular carcinoma stem cells.
      In the case of cHCC-CCA, the authors observed a positive correlation between age, senescence and IL-6 production in the chronically inflamed Mdr2-KO livers, thus pointing to senescent cells as the source of IL-6, as part of the senescence-associated secretory phenotype (SASP).
      Consistently, in vivo ablation of senescent cells with a senolytic agent strongly reduced IL-6 levels and the number of cHCC-CCA tumors.
      This and previous studies highlight the complex, and a priori contradictory, context-dependent role of IL-6 in liver tumorigenesis. Thus, when acute liver injury occurs, involving hepatocellular loss-of-function and compensatory liver regeneration, a protumorigenic role for IL-6 has been described.
      • Naugler W.E.
      • Sakurai T.
      • Kim S.
      • Maeda S.
      • Kim K.
      • Elsharkawy A.M.
      • et al.
      Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production.
      ,
      • Lanton T.
      • Shriki A.
      • Nechemia-Arbely Y.
      • Abramovitch R.
      • Levkovitch O.
      • Adar R.
      • et al.
      Interleukin 6–dependent genomic instability heralds accelerated carcinogenesis following liver regeneration on a background of chronic hepatitis.
      IL-6 promotes hepatocyte proliferation through the enhancement of DNA synthesis (in cooperation with growth factors)
      • Nechemia-Arbely Y.
      • Shriki A.
      • Denz U.
      • Drucker C.
      • Scheller J.
      • Raub J.
      • et al.
      Early hepatocyte DNA synthetic response posthepatectomy is modulated by IL-6 trans-signaling and PI3K/AKT activation.
      and an increase in genomic instability, defining its protumorigenic character. However, in chronic liver injury associated with high levels of fibrosis and senescence, IL-6, as part of the SASP, has been found to protect against HCC development. In fact, it was demonstrated that the absence of IL-6 signaling leads to a collapse of senescence and the SASP, followed by increased tumorigenesis (HCC) in Mdr2-KO mice.
      • Shriki A.
      • Lanton T.
      • Sonnenblick A.
      • Levkovitch-Siany O.
      • Eidelshtein D.
      • Abramovitch R.
      • et al.
      Multiple roles of IL-6 in hepatic injury, steatosis, and senescence aggregate to suppress tumorigenesis.
      Thus, in chronic injury, IL-6 acts to suppress hepatocyte cell division and hepatocarcinogenesis. Strikingly, this same SASP-related IL-6, through the trans-signaling pathway in HPCs, drives cHCC-CCA development.
      • Rosenberg N.
      • Haele M.V.
      • Lanton T.
      • Brashi N.
      • Bromberg Z.
      • Adler H.
      • et al.
      Combined hepatocellular-cholangiocarcinoma derives from liver progenitor cells and depends on senescence and IL6 trans-signaling.
      Previous reports showed how IL-6 derived from this inflammatory environment can also promote the retro-differentiation of tumor-derived hepatocytes or cholangiocytes into stem/progenitor cells,
      • Dubois-Pot-Schneider H.
      • Fekir K.
      • Coulouarn C.
      • Glaise D.
      • Aninat C.
      • Jarnouen K.
      • et al.
      Inflammatory cytokines promote the retrodifferentiation of tumor-derived hepatocyte-like cells to progenitor cells.
      which may also further undergo full malignant transformation into cHCC-CCA. Actually, this event represents another hypothesis regarding the identity of the cell of origin of this rare variant of PLC, and constitutes an area of active research.
      The study of Rosenberg et al. is nevertheless a significant contribution to unravelling the natural history of these combined tumors. It reinforces the hypothesis of the HPC compartment as their cellular origin, and identifies a new role for the complex IL-6 pathway as part of the SASP in the outcome of chronic liver disease (Fig. 1). Moreover, the authors confirm Mdr2-KO mice as an excellent model to study HCC development,
      • Rosenberg N.
      • Haele M.V.
      • Lanton T.
      • Brashi N.
      • Bromberg Z.
      • Adler H.
      • et al.
      Combined hepatocellular-cholangiocarcinoma derives from liver progenitor cells and depends on senescence and IL6 trans-signaling.
      • Mauad T.H.
      • Nieuwkerk van C.M.J.
      • Dingemans K.P.
      • Smit J.J.M.
      • Schinkel A.H.
      • Notenboom R.G.E.
      • et al.
      Mice with homozygous disruption of the mdr2 P-glycoprotein gene. A novel animal model for studies of nonsuppurative inflammatory cholangitis and hepatocarcinogenesis.
      • Potikha T.
      • Stoyanov E.
      • Pappo O.
      • Frolov A.
      • Mizrahi L.
      • Olam D.
      • et al.
      Interstrain differences in chronic hepatitis and tumor development in a murine model of inflammation-mediated hepatocarcinogenesis.
      and, most interestingly, identify these mice as a faithful experimental tool to mimic human cHCC-CCA. However, open questions still remain, mainly concerning the human relevance of these findings and their translation to the clinic. What is the specific context in chronic liver disease that defines the role of IL-6 acting in one way or another? Do cHCC-CCA tumors also develop in a senescent microenvironment in humans? If so, are these senescent cells the unique source of IL-6 with protumorigenic trans-signalling effects on HPCs? Would treatment with a senolytic agent be an alternative for patients with cHCC-CCA? A complex role of senescence in liver homeostasis and disease has also been shown to be context-dependent, since targeting senescent cholangiocytes and stellate cells ameliorates fibrosis at early stages,
      • Moncsek A.
      • Al-Suraih M.S.
      • Trussoni C.E.
      • O’Hara S.P.
      • Splinter P.L.
      • Zuber C.
      • et al.
      Targeting senescent cholangiocytes and activated fibroblasts with B-cell lymphoma-extra large inhibitors ameliorates fibrosis in multidrug resistance 2 gene knockout (Mdr2−/−) mice.
      while hepatocyte senescence likely impedes protumorigenic steatohepatitis-associated phenotypes
      • Shriki A.
      • Lanton T.
      • Sonnenblick A.
      • Levkovitch-Siany O.
      • Eidelshtein D.
      • Abramovitch R.
      • et al.
      Multiple roles of IL-6 in hepatic injury, steatosis, and senescence aggregate to suppress tumorigenesis.
      at more advanced phases of the disease. Additional factors and their environmental interconnections should be identified in order to properly understand the dichotomous activity of IL-6 and senescence in liver carcinogenesis. All these considerations make senescence and IL-6 challenging therapeutic targets.
      Figure thumbnail gr1
      Fig. 1The Mdr2-KO mouse is proposed as an excellent model to study cHCC-CCA tumors.
      On the background of chronic liver inflammation, senescence contributes to IL-6 secretion as part of the SASP. IL-6 trans-signaling on HPCs promotes their proliferation and cHCC-CCA development. Together with HCC, iCCA and stemness markers, pSTAT3 and pERK activity might contribute to the accurate cHCC-CCA diagnosis. The similarities between the mouse and human cHCC-CCA tumors are corroborated at the level of gene and protein expression, thus postulating the aged Mdr2-KO mouse as a faithful experimental tool to mimic human cHCC-CCA. cHCC-CCA, combined hepatocellular-cholangiocarcinoma; HCC, hepatocellular carcinoma; HPCs, hepatic progenitor cells; iCCA, intrahepatic cholangiocarcinoma; SASP, senescence-associated secretory phenotype.
      Finally, as mentioned at the beginning, one of the major challenges in cHCC-CCA today is discriminating it from HCC and CCA. In this regard, a very recent work also published in the Journal of Hepatology evaluated the largest cohort of patients with cHCC-CCA ever recruited and provided valuable information. In agreement with previous studies,
      • Xue R.
      • Chen L.
      • Zhang C.
      • Fujita M.
      • Li R.
      • Yan S.-M.
      • et al.
      Genomic and transcriptomic profiling of combined hepatocellular and intrahepatic cholangiocarcinoma reveals distinct molecular subtypes.
      ,
      • Malvi D.
      • Biase D.
      • Fittipaldi S.
      • Grillini M.
      • Visani M.
      • Pession A.
      • et al.
      Immunomorphology and molecular biology of mixed primary liver cancers: is Nestin a marker of intermediate-cell carcinoma?.
      this study strengthened data on the stem cell of origin of these combined tumors and validated the discriminatory potential of the progenitor cell marker nestin to tell cHCC-CCA apart from HCC, although not from iCCA[3]. Taking into account Rosenberg et al.’s results, we can speculate that pSTAT3 and pERK activity, together with HCC, iCCA and stemness markers, would help to accurately discriminate cHCC-CCA from HCC and iCCA (Fig. 1). Therefore, Rosenberg et al.’s study provides new and relevant data on the cell of origin and the mechanisms implicated in the development of cHCC-CCA tumors as well as a potential signature for their correct clinical identification.

      Financial support

      This work was funded by CIBERehd; Instituto de Salud Carlos III (ISCIII) co-financed by “Fondo Europeo de Desarrollo Regional” (FEDER) “Una manera de hacer Europa” (PI19/00613, PID2020-117116RB-I00); AECC LAB Grant 2020; Gobierno de Navarra (2018-055); AECC post-doctoral fellowship POSTD18014AREC to MA and Ramón y Cajal Program contract RYC2018-024475-1 to MGF-B.

      Authors’ contributions

      MA and MGF-B: wrote the manuscript.

      Conflicts of interest

      The authors declare no conflict of interest.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following are the supplementary data to this article:

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