**7. Epigenetic modulator**

Abnormal epigenetic events are frequently observed in HCC, which can alter gene expression through modification of histone tails or DNA. The major players contributing to these aberrations such as DNA methyltransferase and histone deacetylase are under intensive investigations. In fact, there are many other players involved during the establishment of aberrant epigenetic status. Among them, polycomb repressive complexes (PRC) are catching more attention recently due to their significant roles during cancer development via suppression of various tumor suppressor genes (Steele et al., 2006). In human, there are two polycomb repressive complexes namely PRC1 and PRC2. Despite their unique gene repression mechanism, both of them are frequently involved in the oncogenesis of HCC. Targeting of epigenetic modulators in theory generates persistent effects on tumors as heritable changes are induced. Such an approach is superior to targeting other molecular players that only bring out transient effects.

#### **7.1 BMI1**

44 Hepatocellular Carcinoma – Basic Research

surrounding components. While extracellular HSPGs function to maintain extracellular matrix (ECM) self assembly and integrity with other ECM molecules, cell surface HSPGs are responsible for the binding of growth factors, chemokines, cytokines and enzymes. In addition to normal biological process, HSPGs also influence a number of pathological events

Evidences suggested that the expression of heparanase, an enzyme that degrade the side chain heparin sulfate, is closely related to tumor invasion, angiogenesis and metastasis in HCC (El-Assal et al., 2001). Heparanase level is high both in HCC patient serum and tumor tissues. Heparanase level in serum is linked with the aggressiveness of HCC (Wang et al., 2010), and that in tumors is positively correlated with tumor size, staging and portal vein invasiveness (El-Assal et al., 2001). It is speculated that the major pro-tumorigenic effect of heparanase is derived from the ability to cleave HSPG, resulting in the release of HS-bound molecules such as ECM digesting enzymes and angiogenic factors. Consequent ECM degradation and angiogenic factor released combine to construct a microenvironment

Extensive cleavage of heparin sulfate might release other cell surface bound factors such as growth factors and chemokines that potentially generate diverse biological effects in both autocrine and paracine manners. Upregulation of heparanase is associated with increased releasing of basic fibroblast growth factor (bFGF). bFGF released in this way contributes to tumor progression through the activation of oncogenic signaling and construction of a

Targeting heparanase provides a novel perspective in managing HCC by modulating the tumor-stromal communication. Knocking down of heparanase can significantly inhibit the invasiveness, metastasis, and angiogenesis of HCC cell both in vitro and in vivo (Zhang et al., 2007). Several molecule inhibitors of heparanase can also attenuate the progression of hepatoma cells. The antitumor effect is possibly generated by preventing the degradation of ECM and basal membrane. Another study showed inhibiting heparanase could effectively stop the release of bFGF so as to inactivate the bFGF signaling effect and suppress subsequent angiogenesis (Zhao et al., 2006). These findings have gradually switched the attention in cancer therapy research, from focusing solely in intracellular targets to the

Another important feature of heparin sulfate chains is related to its substrate binding capacity. 6-O-sulfation, a type of heparin sulfate modification, is known to play a specific role in modulating ligand binding. The enzyme SULF2 is a member of the sulfatase family that modulates critical cellular signaling pathways by the removal of 6-O-sulfation (Morimoto-Tomita et al., 2002). In contrast to another sulfatase member tumor suppressor SULF1, SULF2 has an oncogenic role in cancer, and its expression is elevated in HCC. Upregulation of SULF2 is observed in 57% HCC tissues and 73% HCC cell lines. Level of SULF2 is positively correlated with a more aggressive tumor phenotype and poorer patient survival (Lai et al., 2008). Ectopic expression of SULF2 promoted cell proliferation and

including inflammation, tumor growth, metastasis and angiogenesis.

favorable for HCC cell migration and invasion (Zhang et al., 2007).

interplay between cancer cells and the surrounding microenvironment.

favourable tumor niche (Zhao et al., 2006).

**6.2.1 Heparanase** 

**6.2.2 Sulfatase 2** 

PRC aroused increasing attention recently as they are shown to contribute heavily in the maintenance of stem cell and the determination of cell fate. BMI1 is a critical component of PRC1 in mediating the ubiquitination of histone in order to regulate local gene expression. BMI1 is not detected in normal hepatocyte but is overexpressed in HCC. Dysregulation of BMI1 is speculated to promote activation of cancer stem cell in HCC. BMI1 has a higher basal level in the side-population (SP) cell where such a subgroup of cancer cells is characterized by the ability to exclude Hoechst 33342 dye via the ABC cassette transporter. This subpopulation is believed to harbor stem cell properties, and BMI1 is shown to play a crucial role in their self-renewal process (Chiba et al., 2008).

Novel Therapeutic Targets for Hepatocellular Carcinoma Treatment 47

of EZH2 role in HCC suggested that strategies built around EZH2 is definitely

EZH2 is theoretically suitable for pharmalogical targeting as it contains a SET domain responsible for the histone methyltransferase activity. Targeting of the enzymatic domain is proved to reduce histone methylation and de-repress expression of tumor suppressor genes. In addition to the catalytic domain, there are two N-terminal domains mediating proteinprotein interactions and promoting nuclear localization within EZH protein, which are druggable targets in ablating EZH2 activity. Disruption of the PRC2 is also effective to attenuate the tumorigenic effect of EZH2. The formation of functional PRC2 requires other protein subunits such as EED and SUZ12. Report revealed that the use of agent disrupting PRC2 subunits is a relevant way to affect PRC2 function (Tan et al., 2007). Although currently there is no EZH2 specific inhibitor, but agent such as DZnep is also able to deplete the cellular EZH2, inhibit EZH2 functions and lower the H3K27 trimethylation level (Chiba et al., 2011). This fundamental knowledge surely enables researchers to design potent agents

MicroRNAs are small non-coding RNAs that regulate the translation of many genes. They not only regulate normal cell development but also play important roles in cancer development and progression by affecting cell survival, angiogenesis and metastasis. Many studies illustrated the potential of manipulating microRNA expression in cancer therapy. It is believed that microRNA-based remedy can have a huge impact on cancer cells, as they regulate whole programs of gene expression via suppressing hundreds of genes simultaneously (Farazi et al., 2011). In human HCC, numerous microRNAs are identified to give major contributions, either having oncogenic or tumor suppressing ability. Here, those

OncomiR is a novel term coined for microRNAs possessing proto-oncogenic effects in cancers. In HCC, a number of oncomiRs are identified and their roles are characterized. Among them, the roles of microRNA-21 (miR-21) in HCC development were welldocumented (Liu et al., 2010). miR-21 is universally overexpressed in majority of cancers and is phenomenally involved in approximately all tumorigenic processes. miR-21 is able to induce cell transformation, mediate cancer cell growth, cell cycle and self-renewal, prevent apoptosis, promote metastasis and generate drug-resistance (Liu et al., 2010). High expression of miR-21 is correlated with advanced tumor stage, frequent metastasis and poor patient prognosis. In HCC, miR-21 is overexpressed, and has been proved to promote malignant cell growth and spreading by targeting tumor suppressor PTEN and inducing FAK phosphorylation (Meng et al., 2007). Furthermore, miR-21 induces resistance to anticancer effect of interferon-α and 5-fluorouracil in HCC (Tomimaru et al., 2010). Importantly, inhibition of miR-21 is able to reduce the aggressiveness in HCC, and relieves the suppressive effect to several tumor suppressor genes targeted. Besides miR-21, targeting other oncomiRs such as miR-29 (Xiong et al., 2010) and miR-221 (Pineau et al., 2010) is also

microRNAs having great potential as HCC therapeutic targets will be discussed.

robust in reversing the malignant phenotypes in HCC.

advantageous.

to target EZH2 in HCC.

**8. Non-coding RNA** 

**8.1 MicroRNA** 

BMI1 mediates stemness features in HCC cells. In HCC, SP cells expressing BMI1 showed enhanced tumorigenic potential compared to the corresponding non-SP cells. Knockdown of BMI1 markedly abolished the tumor-initiating ability of SP cells in non-obese diabetic/severe combined immunodeficiency mice, leading to a 100 fold decrease of tumorigenic activity (Chiba et al., 2008). Such decrease in tumorigenic activity was accompanied with a reduction of SP cell number in different HCC cell lines. It is shown that BMI1 mediates the suppression of INK4A/ARF and drives self-renewal. Inhibition of BMI1 resulted in the derepression of INK4A/ARF, and in turn disrupted self-renewal in SP cells, hence suppressed SP cells survival upon long time culture. BMI1 additionally regulates diverse cellular processes including cell cycle, apoptosis and senescence by the repression of the INK4A/ARF expression (Xu et al., 2009).

Since there are numerous targets of BMI1 in human genome, it is predictable that the oncogenic effect of BMI1 should not simply depend on INK4A and ARF repression. BMI1 was able to cooperate with activated RAS to transform hepatocytes into malignant cells (Xu et al., 2009). Furthermore, BMI1 expression in HCC is significantly associated with the expression of ABC transporter B1 (ABCB1) which was consistently reported to generate the multiple drug-resistant phenotype (Effendi et al., 2010). It is possible that one of the downstream targets of BMI1 is ABCB1. The ability to eliminate cancer progenitor cell in HCC by BMI1 inhibition is a potent anticancer mediation, and potentially provides a cure for HCC patients.
