The inhibitory effect of rapamycin on the oval cell response and development of preneoplastic foci in the rat

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Abstract

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.

Introduction

The adult mammalian liver displays an immense capacity for regeneration in response to injury caused by chemicals or toxins, and to surgical reduction in mass. The regenerative response is a complex, coordinated process whereby pre-existing, mature hepatocytes, biliary cells, and stromal cells re-enter the cell cycle to replace functional liver mass while maintaining their differentiated phenotype (Taub, 2004). In cases where adult liver cell proliferation is impaired, such as in chronic liver injury or acute liver failure, a progenitor cell compartment is activated and gives rise to transit amplifying cells (Mangnall et al., 2003). These cells, termed oval cells by Farber because of their ovoid nucleus and large nuclear to cytoplasmic ratio, are now known to be bipotential progenitor cells capable of differentiating into hepatocytes and ductal cells (Dabeva and Shafritz, 1993, Farber, 1956, Hixson, 2003, Hixson et al., 2000).

Oval cells can be induced in the rat by feeding a choline-deficient diet in combination with ethionine or by treating with 2-acetylaminofluorene (2-AAF) followed by 2/3 partial hepatectomy (PHx) (Hixson, 2003). Hepatic oval cells, referred to as progenitor cells in humans, have been identified in a number of human liver diseases, including hepatitis, alcoholic and non-alcoholic steatohepatitis, and extra-hepatic biliary atresia. Their abundance is correlated with disease severity (Lowes et al., 1999, Roskams et al., 2003). Many human liver diseases in which progenitor cell expansion occurs are associated with the development of liver cancer. In patients with viral hepatitis, proliferating oval cells are found in close proximity to hepatocellular carcinoma (HCC). Furthermore, a portion of the HCCs from these patients express oval cell markers, suggesting that persistent oval cells may acquire mutations that contribute to the development of HCC (Hixson, 2003, Lowes et al., 1999). This notion is supported by recent studies that have shown that a subset of HCC expresses progenitor cell markers, suggesting that these tumors arise from oval cells. This has special significance in light of the data supporting the existence of cancer stem cells and their role in tumor recurrence, metastasis, and resistance to chemotherapeutics (Alison et al., 2011, Bomken et al., 2010). In agreement with the cancer stem cell hypothesis, hepatic tumors expressing progenitor cell antigens have been correlated with a high rate of recurrence and poor patient survival (Yamashita et al., 2008, Yang et al., 2010).

Although the circumstances that result in oval cell activation have been well studied, the signaling pathways regulating their activation, proliferation, and differentiation remain unclear. To date, a TNF family member, TWEAK, has been the only mitogen identified to be selective for oval cells. However, inhibition of TWEAK by blockade or deletion of its receptor, Fn14, does not prevent oval cell activation or proliferation (Jakubowski et al., 2005). The Wnt/β-catenin pathway has also been shown to be activated in proliferating oval cells but, as with TWEAK, deletion of β-catenin only partially inhibits the oval cell response in mice placed on the 2-AAF/partial hepatectomy protocol (Apte et al., 2008).

Previous studies in our laboratory have focused on the signaling mechanisms that regulate hepatocyte growth and proliferation during development. We have found that the pathways regulating adult hepatocyte proliferation after 2/3 PHx are not active in late gestation fetal liver. In addition, fetal hepatocyte proliferation is insensitive to rapamycin, a specific inhibitor of the nutrient- and energy-sensing kinase mTOR (Boylan et al., 2001). Rapamycin is a macrolide antibiotic with immunosuppressive and antitumor activities (Eng et al., 1984, Thomson et al., 2009, Vezina et al., 1975). In mammalian cells, mTOR exists in two complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (Abraham, 2002). mTORC1 is rapamycin sensitive while mTORC2 is rapamycin resistant and is primarily involved in the control of the actin cytoskeleton (Guertin and Sabatini, 2009).

Rapamycin inhibits mTOR's serine/threonine kinase activity, resulting in changes in the phosphorylation state and activity of downstream effectors of mTOR, including S6 kinase (S6K) and eukaryotic translation initiation factor 4E binding protein 1 (Fingar and Blenis, 2004, Hay and Sonenberg, 2004). Rapamycin's anti-cancer properties reflect the drug's ability to inhibit the normal biological functions of mTOR, including ribosome biogenesis and protein translation, promotion of cellular proliferation, and angiogenesis (Baldo et al., 2008, Gingras et al., 2001, Wiederrecht et al., 1995). Our previous work on the mTORC1/S6K pathway has shown that signaling via the pathway is sensitive to rapamycin during late gestation development in a number of tissues including liver, intestine, and kidney. However, cellular proliferation and normal tissue remodeling that occur as part of normal growth in the late gestation and early postnatal periods in the rat are rapamycin resistant (Sanders et al., 2008). In addition, we found that sensitivity to the anti-proliferative effects of rapamycin varied across a panel of hepatic cell lines and that sensitivity did not correlate with the degree of transformation (Jimenez et al., 2009). On the basis of these observations on the differential role of mTOR in fetal and adult hepatocyte proliferation, we examined the role of mTOR signaling in oval cell activation and proliferation.

Section snippets

Animals

Timed pregnant female and 6–7 week old male Fisher F344 rats were obtained from Charles River Laboratories (Wilmington, MA). Animals were housed under standard conditions with access to food and water ad libitum. All animal experiments were performed in accordance with the guidelines of the National Institutes of Health and the Rhode Island Hospital Institutional Animal Care and Use Committee. Cesarean sections were performed on timed-pregnant rats under isoflurane anesthesia on embryonic day 19

A modified CDE protocol for oval cell activation

To induce oval cells, rats were placed on a modified CDE protocol. For this protocol, rats were fed a choline deficient diet and received daily intraperitoneal injections of ethionine (12 mg), instead of the traditional method where ethionine is administered orally. Liver was harvested on day 11 or day 15 of the protocol. Oval cell expansion was assessed by indirect immunofluorescence for the oval cell marker OC.10 and a marker of mitosis, phospho-histone H3. After 11 days on the CDE protocol,

Discussion

The signaling mechanisms that mediate the hepatic oval cell response are poorly understood. Given that previous studies have shown that oval cell differentiation recapitulates hepatoblast differentiation during development, and that oval cells express cell-surface markers that are also found on fetal hepatocytes (Farber, 1956), we set out to test the hypothesis that the signaling phenotype of oval cells would resemble that of late gestation fetal hepatocytes in vivo.

One of the most striking

Grant support

These studies were supported by National Institutes of Health grants P20 RR017695 (J.A.S.), R01 HD24455 (P.A.G.) and R01 CA93840 (D.C.H.) and by the Department of Pediatrics.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

We thank Virginia Hovanesian for assistance with image acquisition and Joan Boylan for critical reading of this manuscript.

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    1

    Present address: Department of Pathology, MacNeal Hospital, Berwyn, IL 60402, USA.

    2

    Present address: Feculdade de Ciências Médicas da Santa Casa de São Paulo, São Paulo 01221-020 Brazil.

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