**5.2.1 Gankyrin**

In human HCC, a small proteasome regulatory subunit called gankyrin is frequently overexpressed at both mRNA and protein levels. A study showed gankyrin expression levels were highly upregulated in hepatoma cell lines, and its level was higher in HCC samples compared to normal livers, and premalignant or cirrhotic livers (Higashitsuji et al., 2000). Gankyrin is found to interact with retinoblastoma, increasing its phosphorylation level so as

Novel Therapeutic Targets for Hepatocellular Carcinoma Treatment 43

(Deveraux et al., 1997). All in all, inhibition of XIAP or blockage of interaction between XIAP

It is gradually accepted that the progression and aggressiveness of cancer cells are defined by the tumor-stromal interaction. In HCC, the tumor microenvironment plays a pivotal role in affecting cancer development. Through paracrine and autocrine mechanisms, the stromal components communicate with the tumor, promoting the HCC cell proliferation, survival, and allowing them to invade and metastasize. In the past, majority of targeting therapies are derived from the research focusing on intracellular events of cancer cells. However, limited studies are able to be translated into effective therapies, because they ignored the influence from the surrounding components. Although the field is still in its embryonic stage waiting to be explored, targeting the interaction between tumor-stromal may be a more logical

Stromal remodeling occurs routinely during the development of hepatic fibrosis, cirrhosis and HCC, featured with the infiltration of activated hepatic stellate cells (HSC). Upon hepatic injury, HSCs is stimulated and transformed to acquire an activated myofibroblastlike phenotype that is responsible for the excessive hepatic matrix deposition in chronically damaged livers. They are densely located in tumor sinusoids, fibrous septa and HCCgenerated capsule. Activation of HSC is recognized as a key event during

Activated HSCs considerably increase the activity of NF-κB and ERK in HCC. It is known that both NF-κB and MAP kinase/ERK pathways are involved in the progression of human HCC, and they induce the proliferation of HCC cells, and protect HCC cells from apoptosis (Amann et al., 2009). The paracrinal communication between HSC and HCC forms the major linkage for the induction of HCC development. Several soluble factors secreted by activated HSC are identified to be responsible for the tumorigenic effects. HSC released a substantial amount of protumorigenic factors, including the hepatic growth factor (HGF), which enhances the invasiveness of HCC cells. The growth and the migration capability of HCC were impaired once the binding of HGF to HCC cells was disrupted (Barnaeva et al., 2007). Other studies demonstrated that TGF-β secreted by HSC accelerated tumor progression in neoplastic hepatocyte (Sano et al., 2005). TGF-β was able to induce epithelial to mesenchymal transition and augment PDGF signaling in oncogenic Ras-transformed hepatocyte. It is believed that a combination of HSC-released growth factors consisting of FGF-1 and -2, PDGF and IGF are responsible for promoting HCC tumorigenesis (Bataller & Brenner, 2005). The emerging evidences support that the activated HSC/myofibroblasts in tumor microenvironment have huge impact on HCC development and progression, and this stromal components should be regarded as one of the primary targets in HCC therapy.

Heparan sulfate proteoglycans (HSPG) play important biological roles in both cellular and extracellular context, contributing to the proper communication between cells and their

and TAK1 may be one of the best HCC management strategies.

**6. HCC tumor microenvironment** 

approach against HCC.

**6.1 Hepatic stellate cells** 

hepatotumorigenesis (Zhao et al., 2011).

**6.2 Heparan sulfate proteoglycan modulating enzymes** 

to reduce cellular retinoblastoma stability (Li et al., 2005). In addition, gankyrin increases both the association and activity of MDM2 for p53. This inactivates p53 by increasing the ubiquitylation level of p53 and in turn driving proteasomal degradation of p53 (Higashitsuji et al., 2005). Gankyrin also promotes HCC growth through the activation of oncoprotein D cyclin-dependent kinase 4 (CDK4). The activity of the kinase is negatively regulated by p16 during stress condition, but this inhibitory effect is removed when gankyrin competes with p16 for the binding of CDK4 and thus allows the activation of CDK4 (Dawson et al., 2002). Other than tumor promoting effect, gankyrin contributes to cancer drug resistance. It desensitizes cancer cells to the effect of DNA-damaging chemical agents by preventing p53 dependent apoptosis (Higashitsuji et al., 2005).

The use of RNAi to knock down gankyrin in HCC resulted in a decrease of cell growth, as well as reduction in observed levels of hyperphosphorylated retinoblastoma (Li et al., 2005) and restoration of caspase 8/9-dependent apoptosis (Higashitsuji et al., 2005). Silencing of gankyrin expression also reportedly attenuated epithelial to mesenchymal transition together with cell migration and invasion. The inhibition of gankyrin also reduced the level of nuclear β-catenin (Dong et al., 2011), c-myc, cyclin D1 (Fu et al., 2011) and insulin-like growth factor binding protein 5 (Umemura et al., 2008). The close relationship with several pro-tumorgenic events makes gankyrin a rationale target during HCC treatment.

#### **5.2.2 X-linked inhibitor of apoptosis**

X-linked inhibitor of apoptosis (XIAP) belongs to the inhibitor of apoptosis (IAP) domaincontaining family, and is famous of its anti-apoptotic ability. They are induced by NF-κB signaling to circumvent the pro-apoptotic effect induced by JNK pathway (Kaur et al., 2005), by inhibiting caspase-mediated apoptosis. Apart from this, XIAP participates in the regulation of transforming growth factor β (TGF-β) -induced apoptosis through an ubiqutinproteasomal regulating machine. XIAP is able to complex with TGF-β activated kinase 1 (TAK1), as such this negatively regulates the TGF-β signaling (Chen, 2005). The RING domain of XIAP is responsible for the poly-ubiquitylation of TAK1, resulting the proteasome-mediated degradation of TAK1. Subsequently, it disrupts the activation of JNK signaling and halts apoptosis. It is speculated that the involvement of XIAP in HCC is common given that most HCCs acquire resistance to TGF-β-mediated cell killing (Chen, 2005). Moreover, TAK1 is important for the phosphorylation and activation of the IKK complex. Increased activity of IKK leads to the degradation of the IκB-α inhibitor of NF-κB and subsequent activation of classical NF-κB signaling. Activation of IKK also causes the degradation of MKK7, the upstream kinase essential for activation of JNK signaling. Reduction of MKK7 level could ablate the JNK signaling and inhibit apoptosis (Kaur et al., 2005).

Inhibition of XIAP sensitizes HCC cells to apoptotic signal owing to retaining of TAK1. In doing so, persistent activation of JNK signaling is resulted whenever the TGF-β-mediated apoptotic signal is induced. Stabilized TAK1 also potentially attenuate the influence of NFκB signals (Chen, 2005). Besides, it is reported XIAP inhibition in HCC enhanced TRAILmediated cell killing. The combination of XIAP silencing shRNA and tumor-necrosis factorrelated apoptosis TRAIL is reported to generate potent antitumor effect in HCC cells and tumors in animal models (Pan et al., 2008). Targeting XIAP further renders HCC cells vulnerable to other therapeutic effect by releasing the break for caspase-mediated apoptosis (Deveraux et al., 1997). All in all, inhibition of XIAP or blockage of interaction between XIAP and TAK1 may be one of the best HCC management strategies.
