**6.2 The Ras/raf/MEK/ ERK/MAP kinase pathway**

212 Hepatocellular Carcinoma – Basic Research

Fig. 6. C. Consecutive hepatic section after Digoxigenin-labeled sense IGF II mRNA

Fig. 6. D. IGF II-expressed one of the early preneoplastic lesions in an animal of initiation

group. (Mukherjee et al.2005)

treatment during *in situ* hybridization method. (Mukherjee et al. 2007)

The MAP kinase pathway has probably undergone the most extensive characterization in the process of development of hepatocellular carcinoma. Binding of a growth factor to a tyrosine kinase receptor causes receptor phosphorylation, leading to the formation of a molecular complex with an adaptor protein growth factor receptor bound-2 (Grb2), Grb-2 associated binder 1 and signal relay protein SH-2 domain-containing tyrosine phosphatase-2. This is then localized in the plasma membrane. Other protein such as an exchange factor, Son-of-sevenless (SOS) joins. This complex activates ras while exchange GDP to GTP in the ras/raf/MEK/ERK/MAP kinase pathway. Ras/raf/MEK/ ERK/MAP kinase pathway is known to involve in cell proliferation, dedifferentiation, angiogenesis and cell survival process (Rapp, 1991). Activation of the components of this pathway has been reported to contribute to tumorigenesis, including liver cancer. The GTPase (Guanine neucleotides triphosphate)-Ras and the serine/threonine kinase raf (signaling regulators) regulate the signaling process immediately by activating raf which then phosphorylates the

Chemically Induced Hepatocellular Carcinoma and Stages of Development with Biochemical and Genetic Modulation: A Special Reference to Insulin-Like-Growth Factor II and Raf Gene Signaling 215

activation of Raf and Ras (Zang et al., 2002).

**7. Conclusion** 

**8. Acknowledgement** 

**9. References** 

(ICMR), grant no. 58/7/2009-BMS

wanted to investigate the stage(s) at which c-raf.1 overexpression occurs. Our findings (as studied by in-situ hybridization) suggest that overexpression of the gene (Figure 8) occurs at the late stage basophilic focal lesions and in hepatocellular carcinoma. Further, c-raf.1 mediated activation of ras/raf/MEK/ERK/MAP kinase pathway may be a late stage phenomenon during the development of hepatocellular carcinoma and the activation may be either through c-raf.1 oncogenic mutation or by constitutive c-raf.1 activation by other deregulated proteins such as growth factors during hepatocarcinogenesis. Thus, constitutive activation of this pathway at one or more steps, particularly at ras or raf, can lead to a malignant state. However, no predominant difference is noticed between constitutive

Unlike IGF II, c-raf.1 overexpression was observed in the late basophilic lesions associated with hepatocellular carcinoma. Thus, IGF II may have a role in activation of c-raf.1 signaling in the late stage of development of cancer. The role of raf.1 protein in IGF-induced signaling has been reported (Evert et al., 2004). But, when does it happen during the process of development of hepatocellular carcinoma? The c-raf.1 gene overexpression was predominantly found in hepatocellular carcinoma and late basophilic foci. Thus, the overexpression of c-raf.1 has been considered as a late-stage phenomenon during hepatocarcinogenesis (Bannasch, 2010). However, overexpression of c-raf.1 in very early lesion was also reported (He & Gascon- Barre, 1997). In our study, the dissection of animals after 7-8 weeks of carcinogenic insult did not show any raf.1-expressed lesions. Further, it was reported that c-raf.1 expressed in the basophilic tumors (Hwang et al., 2004). IGF II gene overexpression was noticed in the preneoplastic lesions and in hepatocellular carcinoma. On the other hand, overexpression of c-raf.1 gene was seen in the basophilic lesions associated with hepatocellular carcinoma as well as in tumor. While correlating the expression patterns of IGF II and c-raf.1, it suggests that IGF II-induced cellular signaling may be mediated through and/or affected by c-raf.1 in hepatocellular carcinoma and in the late stage of development of cancer. Thus, IGF II mediated c-raf.1

The work has been carried out, using the fund from Indian Council of Medical Research

Bannasch, P. (1995). Sequential cellular and molecular changes during hepatocarcinogenesis.

Bannasch, P. (2010). Hepatocellular glycogenosis and hepatic neoplasms.*Toxicol Pathol*. 38, 6,

Bannasch,P., D'Intron,A., Leonetti, P., Metzgen, C., Klimek,F., Mayer,D. (1998). Early

aberrations of energy metaboloisim in carcinogenesis. In: *Cell growth and* 

activation may drive late preneoplastic lesions towards neoplasia.

*Verh Dtsch Ges Patho,*79, pp.(72-83), ISSN: 0070-4113.

pp. (1000-1002), ISSN: 1533-1601.

mitogen/extracellular protein kinase kinases, MEK-1 and MEK-2. MEK proteins then phosphorylate the downstream extracellular signal-regulated kinase (ERK) signaling molecules, ERK-1 and ERK-2. GTPase-Ras is a switch protein which alternates between an active on state with a bound GTP and an inactive off state with a bound GDP (Polakis & McCormick, 1992). Activation of ERK-1 and ERK-2 regulates many target proteins and gene regulation proteins in cytoplasm and nucleus. Ras protein of this pathway is found to involve other signaling pathways such as phosphoinositol-3-kinase/Akt pathway, Phospholipase C/protein kinase C pathway and Ral guanine nucleotide dissociation stimulator pathway.

The c-raf.1 is a direct downstream effector of ras. The signaling molecule c-raf.1 is one of the three highly conserved members (raf A, raf B and c-raf.1) of the raf gene family, which code for serine threonine-specific protein kinases in ras-mediated signal transduction pathway (Daum et al., 1994; Rapp, 1991; Sebolt-Leopold, 2000). The c-raf.1 has a crucial role in diverse signal transduction pathways (Rapp, 1991). The c-raf.1 protein kinase has oncogenic potential and is found to be up-regulated in tumors (Störm SM et al. 1993; Stanton VP Jr et al. 1987) and highly expressed in hepatocellular carcinoma too (Rapp,1991; Störm et al., 1993). An excessive activation of the MAPK pathway was observed in hepatocellular carcinoma (Rapp,1991). This findings and another findings that shows overexpression of c-raf in hepatocellular carcinoma in all the 30 different tissue specimens as tested by Hwang et al. (Hwang et al., 2004) thus suggest the predominant role of the raf protein as well as ras/raf/MEK/ERK/MAP kinase pathway in hepatocellular carcinoma. In a phase-wise study (initiation/ promotion/ hepatocellular carcinoma) in rat hepatocarcinogenesis model (Mukherjee et al., 2007), we

Fig. 8. A predominant raf-expressed late basophilic lesion (shown by arrows) in animal of cancer control group. (Das et al. 2009)

wanted to investigate the stage(s) at which c-raf.1 overexpression occurs. Our findings (as studied by in-situ hybridization) suggest that overexpression of the gene (Figure 8) occurs at the late stage basophilic focal lesions and in hepatocellular carcinoma. Further, c-raf.1 mediated activation of ras/raf/MEK/ERK/MAP kinase pathway may be a late stage phenomenon during the development of hepatocellular carcinoma and the activation may be either through c-raf.1 oncogenic mutation or by constitutive c-raf.1 activation by other deregulated proteins such as growth factors during hepatocarcinogenesis. Thus, constitutive activation of this pathway at one or more steps, particularly at ras or raf, can lead to a malignant state. However, no predominant difference is noticed between constitutive activation of Raf and Ras (Zang et al., 2002).
