Activated hepatic stellate cells induce tumor progression of neoplastic hepatocytes in a TGF‐β dependent fashion

The interaction between tumor cells and their microenvironment fundamentally affects cancer development by triggering cell proliferation and survival as well as the capability to invade the surrounding tissue for subsequent spreading and colonization (Bissell and Radisky, 2001; Bhowmick et al., 2004). In the majority of tumors, the aberrant networks of cytokines and chemokines including their cognate receptors are critically involved in the progression of primary cancer to fatal metastatic disease (Balkwill, 2004). The key cascades are regulated by paracrine and autocrine mechanisms which are central to the communication between cancer and neighboring cells, the latter collectively known as tumor-stroma (Coussens and Werb, 2002; Bhowmick and Moses, 2005). Depending on the complexity of the tissue context, the stroma includes activated fibroblasts, blood vessel cells and immune cells such as tumor-associated macrophages (TAMs) or leukocytes (Pollard, 2004). In the liver, hepatocytes represent the major cell type of the parenchyme, whereas the non-parenchymal compartment is composed of various cell types including hepatic stellate cells (HSCs; also referred to as Ito cells) and liver-specific macrophages, termed Kupffer cells. During liver injury due to viral infection or long-term insult of hepatotoxins, HSCs get activated to myofibroblasts (MFBs) in response to platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-β (Friedman, 1999; Ramadori and Armbrust, 2001). These changes in cell fate of HSCs towards MFBs provide the cellular basis for the establishment of hepatic fibrosis and cirrhosis (Friedman et al., 2000; Pinzani et al., 2005), which are characterized by the vast remodeling of the extracellular matrix (ECM) and altered expression of growth factors (Friedman, 2003). Upon progression of hepatocellular carcinomas (HCCs), which develop from the aberrant growth of hepatocytes (Fausto, 1999; Thorgeirsson and Grisham, 2002), MFBs abundantly localize in fibrotic deposits and surround the parenchyme intra- and peritumorally. At this stage, malignant hepatocytes of human HCCs frequently show autocrine secretion of TGF-β1 (Shirai et al., 1994; Bedossa et al., 1995; Factor et al., 1997), loss of tumor suppressors such as E-cadherin (Osada et al., 1996) and stabilization of nuclear β-catenin (Buendia, 2000). We previously established a cellular model of HCC progression which reflects changes in epithelial plasticity leading to a metastatic phenotype of either cytoplasmic Met transgenic (Amicone et al., 1997) or p19ARF null (MIM) hepatocytes (Mikula et al., 2004). The progression to higher malignancy of hepatocytes occurs through the collaboration of hyperactive Ras and TGF-β signaling. While MIM-R cells expressing oncogenic Ha-Ras display epithelial organization and show moderate tumorigenicity, MIM-R hepatocytes treated with TGF-β exhibit a fibroblastoid phenotype accompanied by a dramatic increase in malignancy towards metastastic properties (Gotzmann et al., 2002, 2006; Fischer et al., 2005). This model of hepatocellular cancer progression is particularly associated with downregulation of E-cadherin, nuclear accumulation of β-catenin as well as autocrine secretion of TGF-β, and is thus considered as a significant cellular correlate to human HCC progression (Gotzmann et al., 2004; Lee et al., 2004; Eger and Mikulits, 2005). In addition, we recently established p19ARF deficient immortalized HSCs, referred to as M1-4HSCs, which show expression of characteristic marker proteins of activated HSCs such as alpha-smooth muscle actin (α-SMA), glial fibrillary acidic protein (GFAP), pro-collagen I and desmin (Proell et al., 2005). These non-tumorigenic M1-4HSCs undergo a further transition to MFBs in vitro upon treatment with TGF-β (termed M-HTs), and thus provide a highly suitable cellular tool to analyze the molecular and cellular mechanisms involved in liver fibrogenesis. Non-parenchymal liver cells have been described as the major source of TGF-β, while hepatocytes of the healthy adult liver fail to express detectable levels of this cytokine (Rossmanith and Schulte-Hermann, 2001). The cell cycle inhibitory and proapoptotic function of TGF-β on healthy hepatocytes might, therefore, depend on paracrine regulation. In contrast, HCCs most frequently show high levels of TGF-β along with malignant progression (Bedossa et al., 1995), indicating a tumor-promoting role of TGF-β in liver tumorigenesis. Moreover, elevated TGF-β concentrations have been observed in sera of HCC patients (Shirai et al., 1994) and previous findings indicate that TGF-β1 constitutively activates Smad2 in an autocrine fashion (Matsuzaki et al., 2000a). Yet, the ambiguous role of TGF-β in hepatocarcinogenesis as well as the specific contributions of the various non-parenchymal cell types in the paracrine to autocrine TGF-β regulation of hepatocytes in vivo still remains a matter of debate. Here we employed a cellular transplantation model to analyze the epithelial-mesenchymal interaction by the crosstalk of malignant hepatocytes with the fibrotic stroma. This model of microenvironmental interaction with cancerous cells is based on (i) the TGF-β dependent tumor progression of MIM-R hepatocytes (Gotzmann et al., 2002; Mikula et al., 2003, 2004; Fischer et al., 2005), (ii) activated M1-4HSCs or myofibroblastoid M-HTs which mimic the major fibrotic cell types (Proell et al., 2005), and (iii) the subcutaneous cotransplantation of these hepatic cell populations which allows to study a defined intercellular communication in vivo. We show that M1-4HSCs, and even more pronounced M-HT cells, strongly enhance tumor progression of epithelial MIM-R hepatocytes as observed by nuclear accumulation of Smad2/3 as well as β-catenin. These data implicate that the pathophysiological relationship between hepatic fibrosis and cancer progression critically relies on TGF-β which might be of particular relevance for the therapy of liver carcinoma.