**2.5 PI3k/AKT/mTOR pathway**

Phosphoinositide 3 kinase (PI3K) is an intracellular signal transducer enzyme that can phosphorylate the hydroxyl group of phosphatidylinositol (Vanhaesebroeck and Waterfield, 1999). It belongs to a large family of PI3K-related kinases (Kuruvilla and Schreiber, 1999). PI3K is comprised of a catalytic subunit and a regulatory subunit. The regulatory subunit p85 can interact with phosphotyrosines on activated RTKs that recruit the enzyme to the plasma membrane and activate the enzymatic activities (Paez and Sellers, 2003). PI3K produces the lipid second messenger phosphatidylinosoltriphosphate, which is absent in resting cells but can be acutely produced in response to activated PI3K (Vanhaesebroeck and Waterfield, 1999).

Akt is a serine-threonine kinase downstream of PI3K. It contains a pleckstrin homology (PH) domain in the N-terminus, a central catalytic kinase domain and a C-terminus regulatory domain (Paez and Sellers, 2003). The PH domain will bind to phosphatidylinosoltriphosphate with high affinity. Upon PI3K activation and phosphatidylinosoltriphosphate production, Akt is recruited to the plasma membrane through its PH domain together with another PH domain containing protein, phosphoinositide dependent kinase 1 (PDK1). PDK1 then phosphorylate key residues in the kinase domain activation loop of Akt to activate Akt kinase activity (Paez and Sellers, 2003).

Activated Akt phosphorylates multiple protein substrates and regulates a variety of critical cellular activities (Paez and Sellers, 2003; Whittaker et al., 2010). Mammalian target of rapamycin (mTOR) is one of the most important downstream effectors of Akt. Akt phosphorylates the tuberous sclerosis complex (TSC1/TSC2) which activates the small G protein, Ras homolog enriched in brain (Rheb). Rheb, in its GTP-bound state, can activate mTOR. mTOR is a serine-threonine protein kinase that also belongs to PI3K-related kinase family. It is a large protein consists of tandem HEAT repeats, FAT (FRAP-ATM-TRRAP) and FATC (FAT C-terminus) domains, FKBP12-rapamycin binding domain (FRB), and Cterminus catalytic kinase domain that resembles the catalytic domain of PI3K (Wullschleger et al., 2006). For mTOR to activate its signaling cascade, it must form the ternary complex mTORC1 (mTOR Complex-1) or mTORC2 (mTOR Complex-2), but mTOR is the catalytic subunit of both of these two complexes. mTORC1 complex contains mTOR, RAPTOR (Regulatory Associated Protein of mTOR), mammalian LST8/G-protein β-subunit like protein (mLST8/GβL), PRAS40 and DEPTOR. mTORC2 complex consists of mTOR, rapamycin-insensitive companion of mTOR (Rictor), GβL, and mammalian stressactivated protein kinase interacting protein 1 (mSIN1) (Wullschleger et al., 2006; Paez and Sellers, 2003).

Another very important component of PI3K/Akt/mTOR pathway is PTEN (phosphatase and tensin homolog). It consists of a phosphatase domain which carries out the enzymatic function

with control mice. Moreover, inactivation of JNK pathway with -JNKI1 suppressed the

In summary, numerous data have clearly demonstrated the deep involvement of MAPK signaling pathways during liver carcinogenesis. Identifying pharmacological intervention points along these pathways could be considered as a very promising strategy for

Phosphoinositide 3 kinase (PI3K) is an intracellular signal transducer enzyme that can phosphorylate the hydroxyl group of phosphatidylinositol (Vanhaesebroeck and Waterfield, 1999). It belongs to a large family of PI3K-related kinases (Kuruvilla and Schreiber, 1999). PI3K is comprised of a catalytic subunit and a regulatory subunit. The regulatory subunit p85 can interact with phosphotyrosines on activated RTKs that recruit the enzyme to the plasma membrane and activate the enzymatic activities (Paez and Sellers, 2003). PI3K produces the lipid second messenger phosphatidylinosoltriphosphate, which is absent in resting cells but can be acutely produced in response to activated PI3K (Vanhaesebroeck and Waterfield, 1999). Akt is a serine-threonine kinase downstream of PI3K. It contains a pleckstrin homology (PH) domain in the N-terminus, a central catalytic kinase domain and a C-terminus regulatory domain (Paez and Sellers, 2003). The PH domain will bind to phosphatidylinosoltriphosphate with high affinity. Upon PI3K activation and phosphatidylinosoltriphosphate production, Akt is recruited to the plasma membrane through its PH domain together with another PH domain containing protein, phosphoinositide dependent kinase 1 (PDK1). PDK1 then phosphorylate key residues in the kinase domain activation loop of Akt to activate Akt kinase activity (Paez

Activated Akt phosphorylates multiple protein substrates and regulates a variety of critical cellular activities (Paez and Sellers, 2003; Whittaker et al., 2010). Mammalian target of rapamycin (mTOR) is one of the most important downstream effectors of Akt. Akt phosphorylates the tuberous sclerosis complex (TSC1/TSC2) which activates the small G protein, Ras homolog enriched in brain (Rheb). Rheb, in its GTP-bound state, can activate mTOR. mTOR is a serine-threonine protein kinase that also belongs to PI3K-related kinase family. It is a large protein consists of tandem HEAT repeats, FAT (FRAP-ATM-TRRAP) and FATC (FAT C-terminus) domains, FKBP12-rapamycin binding domain (FRB), and Cterminus catalytic kinase domain that resembles the catalytic domain of PI3K (Wullschleger et al., 2006). For mTOR to activate its signaling cascade, it must form the ternary complex mTORC1 (mTOR Complex-1) or mTORC2 (mTOR Complex-2), but mTOR is the catalytic subunit of both of these two complexes. mTORC1 complex contains mTOR, RAPTOR (Regulatory Associated Protein of mTOR), mammalian LST8/G-protein β-subunit like protein (mLST8/GβL), PRAS40 and DEPTOR. mTORC2 complex consists of mTOR, rapamycin-insensitive companion of mTOR (Rictor), GβL, and mammalian stressactivated protein kinase interacting protein 1 (mSIN1) (Wullschleger et al., 2006; Paez and

Another very important component of PI3K/Akt/mTOR pathway is PTEN (phosphatase and tensin homolog). It consists of a phosphatase domain which carries out the enzymatic function

hyperproliferation in p38-deficient animals (Hui et al., 2007).

combating HCC.

and Sellers, 2003).

Sellers, 2003).

**2.5 PI3k/AKT/mTOR pathway** 

and a C2 domain which binds the phospholipid membrane. PTEN dephosphorylates phosphatidylinosoltriphosphate and serves as a negative regulator of PI3K/AKT/mTOR pathway (Paez and Sellers, 2003).

mTOR controls several important cellular processes including regulation of protein translation. Abberent protein translation often leads to abnormal cell growth and tumorigenesis (Petroulakis et al., 2006; Wullschleger et al., 2006). mTOR enhances translation initiation via two major targets, the eIF4E binding proteins (4E-BPs) and the ribosomal protein S6 kinases (S6K1 and S6K2). Eukaryotic mRNAs contain a 'cap' structure, m7GpppN at the 5' end and can be specifically recognized by the initiation factor eIF4E, which associates with eIF4G and eIF4A to form eIF4F complex and initiate cap-dependent translation. 4E-BP binds to eIF4E and inhibits eIF4F complex formation. Upon phosphorylation of 4E-BP by mTOR, eIF4E is released to stimulate translation initiation. S6 kinase is activated by mTOR and phosphorylates 40S ribosomal protein S6, which leads to increased translation of a subset of mRNAs containing 5' tract of oligopyrimidine (TOP). 5' TOP mRNAs encode ribosomal proteins, elongation factors, the poly-A binding protein and other components of the translational machinery. Therefore, stimulation of the 5' TOP mRNA translation by S6 results in up-regulation of the overall cellular translation capacity (Petroulakis et al., 2006).

The PI3K/Akt/mTOR signaling pathway is known to be up-regulated in various carcinoma cell lines, as well as in human ovarian and breast carcinomas (Altomare et al., 2004) (McAuliffe et al., 2010). For HCC, one study has shown overexpression of phospho-mTOR in 15% of liver tumors. Phospho-mTOR also positivity correlated with increased expression of total S6 kinase, which was found in 45% of the cases (Sahin et al., 2004). Elevated Akt phosphorylation was also found in 23% of HCC and implicated early HCC recurrence and poor prognosis (Boyault et al., 2007). There is also a high frequency (35.6%) of somatic PI3K mutations in HCC specimens (Lee et al., 2005). PTEN, the negative component of the PI3K/Akt/mTOR, is mutated in 5% of HCC and its expression is reduced in half of all HCC tumors, leading to the over activation of the pathway (Whittaker et al., 2010). In HCC patients, reduced PTEN expression has been associated with advanced tumor stage, high recurrences rate and poor survival outcome, suggesting inactivation of PTEN is involved in the pathogenesis of HCC (Hu et al., 2003). A study using hepatocyte-specific PTEN deficient mice further supported such a connection, by demonstrating that at 80 weeks of age, 66% of PTEN-deficient mice developed HCC (Horie et al., 2004).

Given the strong association between aberrant PI3K/Akt/mTOR signaling and HCC, pharmacological inhibition of this pathway could be a viable HCC treatment strategy. The mTOR inhibitor everolimus has been shown to decrease the growth of HCC cell line (Villanueva et al., 2008). Everolimus also induced a significant delay of tumor growth in a HCC xenograft mice model (Villanueva et al., 2008). In a separate study, the mTOR inhibitor sirolimus was tested in a rat HCC model and the treatment resulted in significantly longer survival time, smaller tumor size, and fewer extrahepatic metastases in those animals (Semela et al., 2007). Sirolimus has also been tested in human HCC patients, and it induced a partial response in 5% of the patients and tumor stabilization for at least 3 months in 24% of the patients. Another study has shown that 33% of patients partially response to sirolimus treatment (Rizell et al., 2008; Semela et al., 2007). Several new clinical trials are currently testing mTOR inhibitors and its combination with other therapies among HCC patients.

### **2.6 Other miscellaneous pathways**

The EGFR (epidermal growth factor receptor) is a receptor tyrosine kinase which is activated by ligands including epidermal growth factor (EGF) and transforming growth factor α (TGF-α) (Herbst, 2004). Upon activation, EGFR forms a dimer and autophosphorylates the tyrosine residues in its intracellular cytoplasmic domain, which in turn leads to initiation of many downstream signal transduction cascades (Herbst, 2004). The EGFR signaling pathway is one of the most important pathways that regulate growth, survival, proliferation, and differentiation in mammalian cells. Numerous studies have shown that aberrant EGFR signaling plays a vital role in tumor angiogenesis and proliferation and agents that specifically block this pathway showed efficacy in several types of solid tumors (Zhang et al., 2007). For HCC, EGFR overexpression was detected among 40–70% of the tumors and TGF-α level was also elevated in pre-neoplastic HCC (Feitelson et al., 2004). Thus, targeting EGFR may also show beneficial effect for HCC patients. EGFR blocking agents include both small molecule tyrosine kinase inhibitors and monoclonal antibodies targeting the receptor. Erlotinib is a low molecular weight inhibitor of EGFR kinase (Thomas et al., 2007). It is able to suppress the growth of HCC cell lines *in vitro*. Furthermore, during a Phase 2 clinical trial, 17 out of 40 patients with unresectable HCC achieved stable disease at 16 weeks of erlotinib treatment. Progression-free survival at 16 weeks was 43% and median overall survival was 43 weeks, longer than the historical controls. Monoclonal antibody cetuximab which targets the extracelluar domain of EGFR has also been tested in HCC (Zhu et al., 2007). Although cetuximab was able to inhibit cell growth and induce apoptosis in some HCC cell lines, the results from clinical trials testing its efficacy among HCC patients have been inconsistent. Currently, several more anti-EGFR pathway compounds are being tested in clinic, in some cases in combination with other therapeutic methods. The results from those trials might further clarify the benefit of the EGFR blocking agents for HCC.

Deregulation of the insulin-like growth factor (IGF) pathway has also been implicated in the development of HCC (Scharf et al., 2001). IGF-1 and 2 bind to IGF receptor IGF-1R and activate downstream signaling (Alexia et al., 2004). IGF signaling pathway regulates cell proliferation, motility and apoptosis. Pronounced alterations in the expression of components of the IGF pathway have been reported during hepatocarcinogenesis (Whittaker et al., 2010). IGF-2 is overexpressed in 16-40% of HCC and around 30% of HCCs overexpress IFG-1R (Cariani et al., 1988). Neutralizing IGF-2 has been shown to reduce cell proliferation and increase apoptosis in HCC cell lines (Lund et al., 2004). Furthermore, a monoclonal antibody that selectively inhibits IGF-1R was not only able to decrease viability and proliferation of liver cancer cells *in vitro* but also delay tumor growth and prolong survival in HCC xenograft mice model (Tovar et al., 2010). Several small molecules and monoclonal antibodies targeting IGF-1R are now under early clinical development.

In HCC, the transforming growth factor-beta (TGF-β) pathway regulates several steps in tumor progression, including angiogenesis, production of the extracellular matrix and immune suppression (Giannelli et al., 2011). It is also involved in initiating signaling cascade which promotes liver fibrosis, cirrhosis and subsequent progression to HCC (Giannelli et al., 2011). Increased levels of TGF-β in HCC patients' sera and urine are associated with disease progression (Yasmin Anum et al., 2009; Tsai et al., 1997). Specific small molecule inhibitors targeting TGF-β type I receptor (TGF-βRI) kinase LY2109761 reduce migration of HCC cells

The EGFR (epidermal growth factor receptor) is a receptor tyrosine kinase which is activated by ligands including epidermal growth factor (EGF) and transforming growth factor α (TGF-α) (Herbst, 2004). Upon activation, EGFR forms a dimer and autophosphorylates the tyrosine residues in its intracellular cytoplasmic domain, which in turn leads to initiation of many downstream signal transduction cascades (Herbst, 2004). The EGFR signaling pathway is one of the most important pathways that regulate growth, survival, proliferation, and differentiation in mammalian cells. Numerous studies have shown that aberrant EGFR signaling plays a vital role in tumor angiogenesis and proliferation and agents that specifically block this pathway showed efficacy in several types of solid tumors (Zhang et al., 2007). For HCC, EGFR overexpression was detected among 40–70% of the tumors and TGF-α level was also elevated in pre-neoplastic HCC (Feitelson et al., 2004). Thus, targeting EGFR may also show beneficial effect for HCC patients. EGFR blocking agents include both small molecule tyrosine kinase inhibitors and monoclonal antibodies targeting the receptor. Erlotinib is a low molecular weight inhibitor of EGFR kinase (Thomas et al., 2007). It is able to suppress the growth of HCC cell lines *in vitro*. Furthermore, during a Phase 2 clinical trial, 17 out of 40 patients with unresectable HCC achieved stable disease at 16 weeks of erlotinib treatment. Progression-free survival at 16 weeks was 43% and median overall survival was 43 weeks, longer than the historical controls. Monoclonal antibody cetuximab which targets the extracelluar domain of EGFR has also been tested in HCC (Zhu et al., 2007). Although cetuximab was able to inhibit cell growth and induce apoptosis in some HCC cell lines, the results from clinical trials testing its efficacy among HCC patients have been inconsistent. Currently, several more anti-EGFR pathway compounds are being tested in clinic, in some cases in combination with other therapeutic methods. The results from those trials might further clarify the benefit of the

Deregulation of the insulin-like growth factor (IGF) pathway has also been implicated in the development of HCC (Scharf et al., 2001). IGF-1 and 2 bind to IGF receptor IGF-1R and activate downstream signaling (Alexia et al., 2004). IGF signaling pathway regulates cell proliferation, motility and apoptosis. Pronounced alterations in the expression of components of the IGF pathway have been reported during hepatocarcinogenesis (Whittaker et al., 2010). IGF-2 is overexpressed in 16-40% of HCC and around 30% of HCCs overexpress IFG-1R (Cariani et al., 1988). Neutralizing IGF-2 has been shown to reduce cell proliferation and increase apoptosis in HCC cell lines (Lund et al., 2004). Furthermore, a monoclonal antibody that selectively inhibits IGF-1R was not only able to decrease viability and proliferation of liver cancer cells *in vitro* but also delay tumor growth and prolong survival in HCC xenograft mice model (Tovar et al., 2010). Several small molecules and

monoclonal antibodies targeting IGF-1R are now under early clinical development.

In HCC, the transforming growth factor-beta (TGF-β) pathway regulates several steps in tumor progression, including angiogenesis, production of the extracellular matrix and immune suppression (Giannelli et al., 2011). It is also involved in initiating signaling cascade which promotes liver fibrosis, cirrhosis and subsequent progression to HCC (Giannelli et al., 2011). Increased levels of TGF-β in HCC patients' sera and urine are associated with disease progression (Yasmin Anum et al., 2009; Tsai et al., 1997). Specific small molecule inhibitors targeting TGF-β type I receptor (TGF-βRI) kinase LY2109761 reduce migration of HCC cells

**2.6 Other miscellaneous pathways** 

EGFR blocking agents for HCC.

and blocks invasion of HCC cells into the tissue microenvironment and blood vessels. LY2109761 also is effective in blocking tumor growth in a HCC xenograft chick embryo model and this antitumor activity was associated with anti-angiogenic effect (Mazzocca et al., 2009).

Hepatocyte growth factor (HGF) is a cytokine secreted by mesenchymal cells. It can stimulate mitogenesis, cell motility and has been implicated in tumor invasion. HGF is secreted as a single inactive polypeptide and is cleaved by serine proteases into a 69-kDa alpha-chain and 34-kDa beta-chain (Matsumoto and Nakamura, 1996). Active HGF is a herterodimer between alpha-chain and beta-chain linked by disulfide bond. HGF is homologous to the plasminogen subfamily of S1 peptidases but has no detectable protease activity. The proto oncogene c-Met is a receptor for HGF that is a heterodimer composed of a 50-kDa alpha-chain and a membrane spanning 145-kDa beta-chain with tyrosine kinase activity (Matsumoto and Nakamura, 1996). Activation of c-Met by HGF has been shown to activate MAPK, PI3K and Wnt signaling (Whittaker et al., 2010; Apte et al., 2006). Overexpression of c-Met was noted in 20-48% HCC tissues compared to surrounding noncancerous liver tissues, and the overexpression levels correlated with worsening behavior of HCC and decreased 5-year survival in HCC patients (Whittaker et al., 2010; Ueki et al., 1997). A preclinical study reported that inhibition of c-Met by small molecule inhibitor SU11274 decreased HCC cell growth (Inagaki et al., 2011).
