**1. Introduction**

34 Hepatocellular Carcinoma – Basic Research

Wang XH., Meng XW., Sun X., Liu BR., Han MZ., DU YJ., Song YY. & Xu W. (2011). Wnt/β-

Whittaker S., Marais R. & Zhu AX., 2010, The role of signaling pathways in the development and treatment of hepatocellular carcinoma. *Oncogene*, Vol.29, pp.4989–5005 Yang, H., Xiong, FX., Lin, M., Qi, RZ., Liu, ZW., Rui, JA., Su, J. & Zhou, RL. (2010a).

hepatocellular carcinoma cells. *Neoplasma.* Vol.58, No.3, pp.239-44.

Hepatocellular Carcinoma. *J Surgical Oncology*, Vol.101, pp.363–369. Yang H, Xiong FX, Lin M, Yang Y, Nie X and Zhou RL. (2010b) LAPTM4B-35 over-

and in vivo. *Cancer Lett*,Vol.294, pp.236–244.

*Oncology Reports*, Vol.20, No.5, pp.1077-1083.

*Oncol*. Vol.104, No.1, pp.29-36.

*Cance,* Vol.129, No.3, pp.629-35

*Surg*,Vol.199, No.4, pp.515-21.

cells. *Surgery,* Vol. 150, No.1, pp.25-31

*Hepatogastroenterology*, Vol. 57, No.98, pp.207-11.

hepatocellular carcinoma, *J Cancer Res Clin Oncol.*,Vol.136, pp.275-281. Yang H., Xiong F., Wei X., Yang Y., McNutt MA. & Zhou RL. (2010c). Over-expression of

Yang Y., Yang H., Mcnutt MA., Xiong FX., Nie X., Li L. & Zhou RL. (2008). LAPTM4B

Yin M., Li C., Li X., Lou G., Miao B., Liu X., Meng F., Zhang H., Chen X., Sun M., Ling Q. &

Yin M., Xu Y., Lou G., Hou Y., Meng F., Zhang H., Li C. & Zhou RL. (2011b). LAPTM4B

Zhou L., He XD., Chen J., Cui QC., Qu Q., Rui JA. & Zhao YP. (2007) Overexpression of

Zhou L., He XD., Cui QC., Zhou WX., Qu Q., Zhou RL., Rui JA. & Yu JC. (2008), Expression

of extrahepatic cholangiocarcinoma. *Cancer Lett*. Vol.264, No.2, pp.209-217. Zhou L., He XD., Yu JC., Zhou RL., Yang H., Qu Q. &, Rui JA. (2010a). Over-expression of

Zhou L., He XD., Yu JC., Zhou RL., Xiong FX., Qu Q., Rui JA. (2010b). Expression of

Zhou L., He XD., Yu JC., Zhou RL., Shan Y. & Rui JA. (2011). Overexpression of LAPTM4B-

catenin signaling regulates MAPK and Akt1 expression and growth of

LAPTM4B-35 Is a Novel Diagnostic Marker and a Prognostic Factor of

expression is a risk factor for tumor recurrence and poor prognosis in

LAPTM4B-35 promotes growth and metastasis of hepatocellular carcinoma in vitro

over-expression is an independent prognostic marker in ovarian carcinoma,

Zhou RL. (2011a). Over-expression of LAPTM4B is associated with poor prognosis and chemotherapy resistance in stages III and IV epithelial ovarian cancer. *J Surg* 

over-expression is a novel predictor of epithelial ovarian carcinoma metastasis. *Int J* 

LAPTM4B-35 closely correlated with clinicopathological features and postresectional survival of gallbladder carcinoma. Eur J Cancer. Vol.43, No.4, pp.809-15.

of LAPTM4B-35: A novel marker of progression, invasiveness and poor prognosis

LAPTM4B promotes growth of gallbladder carcinoma cells in vitro. *Am J* 

LAPTM4B in gallbladder carcinoma cells: the role in invasive potential.

35 attenuates epirubucin-induced apoptosis of gallbladder carcinoma GBC-SD

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Surgery is a possible curative treatment, but most symptomatic HCC cases are in advanced stage where surgical resection is not possible. For this group of patients, the prognosis after any kind of therapy remains unsatisfactory due to high relapse rate (Llovet et al., 2003). Studies were rigorously conducted to tackle various obstacles in treating HCC, putting the focuses on targeting cancer cells that either disseminated from the tumor origin, or escaped from therapeutic effects. Recently, a multikinase inhibitor sorafenib was approved by FDA for the treatment of advanced HCC patients. It marks a major advance in the field as the first efficacious targeted therapy for HCC. The primary molecular targets of sorafenib include vascular endothelial factor receptor (VEGFR), platelet derived growth factor receptor (PDGFR) and Raf (Wilhelm et al., 2004). Although it significantly prolongs both patient survival and the time to progression, its overall survival benefit is modest (Llovet et al., 2008).

Other HCC associated targets, such as epidermal growth factor (EGF) signaling (Hampton, 2007), telomerase (Djojosubroto et al., 2005) and cyclooxygenase (Márquez-Rosado et al, 2005), were studied intensively with regard to their therapeutic effects. However, the benefits are far from satisfactory, so there is still a need to identify new therapeutic targets. The exploration of new targets against HCC involves multiple disciplines including hepatology, oncology, pathology and molecular studies. Increasing number of therapeutic targets which play crucial roles in HCC were identified. Identification of new targets not only improves the current HCC therapeutic modality, but also drives a deeper understanding of HCC that allows personalized treatment in the future. In this chapter, we will briefly review the novel molecular and cellular players that contribute to HCC tumorigenesis and progression, and evaluate their potential as additional therapeutic targets.

### **2. Growth receptor signaling**

The studies of sorafenib administration and other growth signaling inhibitors demonstrated the prowess of targeting growth signalings such as epidermal growth factor (EGF), VEGF and PDGF pathways. In HCC, many other growth signalings were identified that markedly contributes to tumorigenesis and pathogenesis. They include insulin-like growth factor

Novel Therapeutic Targets for Hepatocellular Carcinoma Treatment 37

pathogenesis of HCC, where aberration of mTOR pathway was seen in 15% to 41% of HCC cases ranged from 15% to 41% (Hu et al., 2003). In HCC, the commonly hyperactive EGF and IGF signaling is responsible for the induction of PI3K/AKT/mTOR pathway, promoting tumor progression. The mTOR signaling is mediated by mTOR complex 1 and 2 (mTORC1 and mTORC2). mTORC1 is comprised of mTOR, regulatory associated protein of mTOR (RAPTOR), and mammalian LST8/G-protein β-subunit–like protein. mTORC1 is a downstream signal of AKT, and has a pivotal role in regulating cell growth and proliferation. mTORC1 activates S6 kinase to regulate protein synthesis and induces cell

Besides, mTOR is also the subunit of mTORC2 which consists of a protein RAPTORindependent companion of mTOR (RICTOR), and proline-rich protein 5/G-protein βsubunit–like protein. Unlike mTORC1 which is inducible by AKT, mTORC2 plays a critical role in the phosphorylation and activation of AKT (Sarbassov et al., 2005). The serine/threonine kinase AKT acts as a cytoplasmic regulator of numerous signals. It is shown that AKT is frequently amplified and overexpressed in various cancers, and it demonstrates significant oncogenic properties in diverse cancer types. In homeostasis condition, AKT is negatively regulated by the tumor-suppressor PTEN. However, increased activation of AKT is often observed, because PTEN is frequently lost in cancers including HCC. Other than mTORC1, AKT regulates a wide-spectrum of targets such as cyclin D1 and MDM2/p53 (Vivanco & Sawyers, 2002). In HCC, aberration of mTORC2 enhances AKT activity, induces downstream AKT targets and promotes tumorigenesis. One can see that the AKT regulating effect of mTORC2 is as important as mTORC1 within the

Recently, it is suggested that the PI3K/AKT/mTOR pathway can be a major molecular target in cancer remedy. As a critical player in the mTOR signaling, the activity of mTOR often increases in HCC. Blockage of mTOR-mediated signaling showed antineoplastic activity in different experimental models of HCC. The use of mTOR inhibitors could reduce cell proliferation in vitro, and decrease tumor growth in xenografted mouse model (Villanueva et al., 2008). mTOR inhibitors such as sirolimus and everolimus demonstrated potent antitumor properties. Encouraging results were obtained when both mTOR inhibitors were studied in clinical trials, either as a single agent or as adjuvant. Furthermore, components in the mTOR complexes can also be the therapeutic targets. High level of RICTOR is correlated to early recurrence in HCC, and siRNA knockdown of RICTOR reduces HCC cells viability (Villanueva et al., 2008). Disruption of mTOR complexes might have additive benefit along

Glypican-3 (GPC3) is a protein anchored to the cell surface by a glycosylphosphatidylinositol link. Glypican-3 is highly expressed in HCC, and plays a role in stimulating various tumorigenic signaling pathways. GPC3 is specifically expressed in HCC, but not in cholangiocarcinoma or normal liver tissue. More than 70% of HCC tumors were observed with high GPC3 level compared to normal liver tissues (Hsu et al., 1997). Consistent with the high GPC3 protein expression found in clinical samples, numerous

cycle to proceed from G1 to S phase (Bjornsti and Houghton, 2004).

with mTOR inhibition to abrogate mTOR pathway in treating HCC.

PI3K/AKT/mTOR pathway.

**3. Cell-surface protein** 

**3.1 Glypican-3** 

signaling and mTOR pathway, and numerous studies suggested these pathways can be the targets against HCC.

#### **2.1 Insulin-like growth factor signaling**

The insulin-like growth factor (IGF) signaling pathway is frequently dysregulated in HCC. The activation of IGF signaling can be established in malignant cells through an autocrinal route when the activated signaling is induced by an overexpressed IGF ligand in HCC cells (Nussbaum et al., 2008). Insulin-like growth factor 2 (IGF-2) is increased after an inflammatory response to liver damage or viral transactivation (Feitelson et al., 2004), and it is the major ligand contributing to the increased IGF activity in HCC. IGF-2-mediated induction of IGF signaling is prevalent in human HCC, where IGF-2 is overexpressed in 16- 40%, whilst the level of competitive receptor for IGF-2 is decreased in around 80% (Whittaker et al., 2010). As such, IGF receptor-ligand binding is enhanced, and subsequent downstream signaling is activated in cancer cells. Activation of IGF signaling in HCC cells is associated with increase of cell proliferation rate (Schirmacher et al, 1992). While RNAimediated knockdown of IGF-2 could reduce the cell proliferation and induce apoptosis in HCC cells, small molecule inhibiting IGF-2-dependent IGF signaling was able to impair the growth of HCC cells and retard tumour growth in mice xenograft (Lund et al., 2004).

Altered IGF-2 bioavailability is another reason for the hyperactivation of IGF signaling in HCC. Normally, circulating IGF-2 is bound by IGF-binding protein (IGFBP) so that the efficiency of ligand-receptor binding is lowered. In HCC, members of IGFBPs are downregulated so that less IGF-2 is sequestrated which allow uncontrolled IGF-2-receptor interaction (Hanafusa et al, 2002). Hence, reducing the level of IGF-2 in circulation is another valid approach to abrogate the IGF signaling. Re-introduction of recombinant human IGFBP-3 was tested and showed potent effect in lowering the activity of IGF-2 (Aishima et al., 2006). IGFBP-3 was able to inhibit cancer cell growth and attenuate mitogenic activity of HCC cells. It is also reported that IGFBP-3 decreased the phosphorylation and activity of numerous pro-tumorigenic proteins such as IRS-1, MAPK, Elk-1, Akt-1 and phosphatidylinositol 3'-kinase (Huynh et al., 2002).

In addition, inhibition of IGF signaling can also be achieved by disrupting other players along the IGF signaling axis. IGF signal transduction is mediated by the Insulin receptor, IGF-IR and a hybrid of both receptors. In HCC, there is detectable level of IGF receptors ready for the signal generation stimulated by the overexpressed IGF-2. Studies showed that blocking of the receptors was able to give antitumoral effect in HCC cells (Nussbaum et al., 2008). Selective blockage of IGF-IR by monoclonal antibody effectively disrupted IGF signaling, reduced cell viability and proliferation. The inhibition of IGF-IR signal initiation was able to delay tumor growth and prolonged survival in vivo (Tovar et al., 2010). With understanding of IGF signaling mechanism in HCC, it is possible to employ various strategies to effectively inhibit IGF signaling, and in turn suppress cell proliferation and increase apoptosis in HCC.

#### **2.2 mTOR pathway**

mTOR pathway is a downstream growth signal induced by EGF and IGF signaling, and is coupled with PI3K/AKT pathway. mTOR pathway has an important role in the

signaling and mTOR pathway, and numerous studies suggested these pathways can be the

The insulin-like growth factor (IGF) signaling pathway is frequently dysregulated in HCC. The activation of IGF signaling can be established in malignant cells through an autocrinal route when the activated signaling is induced by an overexpressed IGF ligand in HCC cells (Nussbaum et al., 2008). Insulin-like growth factor 2 (IGF-2) is increased after an inflammatory response to liver damage or viral transactivation (Feitelson et al., 2004), and it is the major ligand contributing to the increased IGF activity in HCC. IGF-2-mediated induction of IGF signaling is prevalent in human HCC, where IGF-2 is overexpressed in 16- 40%, whilst the level of competitive receptor for IGF-2 is decreased in around 80% (Whittaker et al., 2010). As such, IGF receptor-ligand binding is enhanced, and subsequent downstream signaling is activated in cancer cells. Activation of IGF signaling in HCC cells is associated with increase of cell proliferation rate (Schirmacher et al, 1992). While RNAimediated knockdown of IGF-2 could reduce the cell proliferation and induce apoptosis in HCC cells, small molecule inhibiting IGF-2-dependent IGF signaling was able to impair the

growth of HCC cells and retard tumour growth in mice xenograft (Lund et al., 2004).

Altered IGF-2 bioavailability is another reason for the hyperactivation of IGF signaling in HCC. Normally, circulating IGF-2 is bound by IGF-binding protein (IGFBP) so that the efficiency of ligand-receptor binding is lowered. In HCC, members of IGFBPs are downregulated so that less IGF-2 is sequestrated which allow uncontrolled IGF-2-receptor interaction (Hanafusa et al, 2002). Hence, reducing the level of IGF-2 in circulation is another valid approach to abrogate the IGF signaling. Re-introduction of recombinant human IGFBP-3 was tested and showed potent effect in lowering the activity of IGF-2 (Aishima et al., 2006). IGFBP-3 was able to inhibit cancer cell growth and attenuate mitogenic activity of HCC cells. It is also reported that IGFBP-3 decreased the phosphorylation and activity of numerous pro-tumorigenic proteins such as IRS-1, MAPK, Elk-1, Akt-1 and

In addition, inhibition of IGF signaling can also be achieved by disrupting other players along the IGF signaling axis. IGF signal transduction is mediated by the Insulin receptor, IGF-IR and a hybrid of both receptors. In HCC, there is detectable level of IGF receptors ready for the signal generation stimulated by the overexpressed IGF-2. Studies showed that blocking of the receptors was able to give antitumoral effect in HCC cells (Nussbaum et al., 2008). Selective blockage of IGF-IR by monoclonal antibody effectively disrupted IGF signaling, reduced cell viability and proliferation. The inhibition of IGF-IR signal initiation was able to delay tumor growth and prolonged survival in vivo (Tovar et al., 2010). With understanding of IGF signaling mechanism in HCC, it is possible to employ various strategies to effectively inhibit IGF signaling, and in turn suppress cell proliferation and

mTOR pathway is a downstream growth signal induced by EGF and IGF signaling, and is coupled with PI3K/AKT pathway. mTOR pathway has an important role in the

targets against HCC.

**2.1 Insulin-like growth factor signaling** 

phosphatidylinositol 3'-kinase (Huynh et al., 2002).

increase apoptosis in HCC.

**2.2 mTOR pathway** 

pathogenesis of HCC, where aberration of mTOR pathway was seen in 15% to 41% of HCC cases ranged from 15% to 41% (Hu et al., 2003). In HCC, the commonly hyperactive EGF and IGF signaling is responsible for the induction of PI3K/AKT/mTOR pathway, promoting tumor progression. The mTOR signaling is mediated by mTOR complex 1 and 2 (mTORC1 and mTORC2). mTORC1 is comprised of mTOR, regulatory associated protein of mTOR (RAPTOR), and mammalian LST8/G-protein β-subunit–like protein. mTORC1 is a downstream signal of AKT, and has a pivotal role in regulating cell growth and proliferation. mTORC1 activates S6 kinase to regulate protein synthesis and induces cell cycle to proceed from G1 to S phase (Bjornsti and Houghton, 2004).

Besides, mTOR is also the subunit of mTORC2 which consists of a protein RAPTORindependent companion of mTOR (RICTOR), and proline-rich protein 5/G-protein βsubunit–like protein. Unlike mTORC1 which is inducible by AKT, mTORC2 plays a critical role in the phosphorylation and activation of AKT (Sarbassov et al., 2005). The serine/threonine kinase AKT acts as a cytoplasmic regulator of numerous signals. It is shown that AKT is frequently amplified and overexpressed in various cancers, and it demonstrates significant oncogenic properties in diverse cancer types. In homeostasis condition, AKT is negatively regulated by the tumor-suppressor PTEN. However, increased activation of AKT is often observed, because PTEN is frequently lost in cancers including HCC. Other than mTORC1, AKT regulates a wide-spectrum of targets such as cyclin D1 and MDM2/p53 (Vivanco & Sawyers, 2002). In HCC, aberration of mTORC2 enhances AKT activity, induces downstream AKT targets and promotes tumorigenesis. One can see that the AKT regulating effect of mTORC2 is as important as mTORC1 within the PI3K/AKT/mTOR pathway.

Recently, it is suggested that the PI3K/AKT/mTOR pathway can be a major molecular target in cancer remedy. As a critical player in the mTOR signaling, the activity of mTOR often increases in HCC. Blockage of mTOR-mediated signaling showed antineoplastic activity in different experimental models of HCC. The use of mTOR inhibitors could reduce cell proliferation in vitro, and decrease tumor growth in xenografted mouse model (Villanueva et al., 2008). mTOR inhibitors such as sirolimus and everolimus demonstrated potent antitumor properties. Encouraging results were obtained when both mTOR inhibitors were studied in clinical trials, either as a single agent or as adjuvant. Furthermore, components in the mTOR complexes can also be the therapeutic targets. High level of RICTOR is correlated to early recurrence in HCC, and siRNA knockdown of RICTOR reduces HCC cells viability (Villanueva et al., 2008). Disruption of mTOR complexes might have additive benefit along with mTOR inhibition to abrogate mTOR pathway in treating HCC.
