**3. Centrosome abnormalities in the development of drug resistance**

As signaling center, centrosome plays important roles in the development of drug resistance. Many centrosome-associated proteins are involved in chemo-resistance process, such as Her-2/neu, bcl-2, c-myc, ras, c-jun, MDM2, p210 BCR-abl, or mutant p53. In fact, abnormal centrosome itself may lead to formation of poly- or monopolarity spindle resulting in chromatin mis-segregation, which further result in or accelerate inactivation of tumor suppressor genes and/or activation of tumor genes, thereby leading to the development of chemoresistance. Support of this idea comes from the recent finding that p53 status determines tumor response to anti-angiogenic therapy and heat shock proteins (HSPs) varies with tumor progressions (Chen & Kong, 2009; Ciocca & Calderwood, 2005).

#### **3.1 Centrosome clustering pathway as a target in cancer therapy**

Centrosome clustering pathway is indispensable in cells with supernumerary centrosomes ensuring the success of cell division. Interference in this process could be lethal to tumor cells containing extra centrosomes (Kwon et al., 2008). Therefore, proper interference centrosome clustering pathway may raise the possibility of developing a new therapeutic strategy. HSET, the human homologue of the KAR3 family of minus end-directed kinesinlike motors, may be one of the most appropriate such candidates, as HSET depletion destroys centrosome clustering pathway and induces multipolar divisions and hence abnormal chromosome segregation or aneuploidy. Besides, HEST is essential only for clustering extra centrosomes in cancer cell but not in normal cells, by bundling the minus end of MT in acentrosomal spindles (Mountain et al., 1999). These results indicate that inhibition of HSET can selectively kill cells with extra centrosomes without affecting the viability of cells that contain normal centrosome numbers. In addition, HEST has been found to be involved in cell-cell adhesion by influencing the cells shape, then inducing low integrin -1 expression, and eventually resulting in tumor environment changes (Amendola et al., 2001). Taken together, HSET inhibitor may have a relative low toxicity compared with other mitosis-blocking agents involving centrosme, including checkpoint with forkhead and ring finger domains (CHFR), Aurora A, B, and C, Polo-like kinases (Plk1-4), and Nek kinases (NIMA1-11).

#### **3.2 Targeting the centrosome as a whole in HCC therapy**

As stated above, most of the key proteins are associated with cancer development. Selective inhibitors of these proteins such as p53, kinase C (PKC), proteasome, Aurora, NEDD1, and centrosome-associated regulators, therefore, have recently been tried in drug development (Graff et al., 2005; Montagut et al., 2005; Godl et al., 2005; Warner et al., 2006; Wang et la., 2009; Tillement et al., 2009). Since most of the key cellular proteins are localized to the centrosome, and centrosome abnormalities has long been found to be one of the most common features in a variety of human cancers and to be one of the earliest events in cancer development, as compared to p53 mutation and telomerase up-regulation that have been long regarded as the major factors contributing to the development of carcinogenesis. Centrosome is naturally becoming a candidate target in cancer therapy. In addition, chromosome instability (CIN) may be the fundamental cause in the development of drug resistance, and centrosome together with centrioles abnormalities are closely associated CIN, the whole complex consisting of the centrosome and centrioles may be a most promising candidate in cancer therapy.

Since increasing key proteins are found to be localized on centrosome and/or centrioles. And each protein exerts its yet unknown functions alone or through centrosome and/or centrioles. Selective targeting centrosome as a whole like mentioned previously (Kong, 2003a, 2003b, 2003c) or through combination of chemotherapeutic drugs that work through different mechanisms is expected to be reasonable and promising. Kong proposed that centrosome can be crystallized with tetrazolium salts (Kong et al., 2002). Although there is no further evidence to affirm whether it works or not in clinic, it seems to be reasonable that the crystallized centrosome may not function as the centre of the cell to mediate important cellular events. In other words, all key enzymes located in the centrosome will not function normally, and the cellular structures that are rich in the enzymes will be functionally and structurally frozen or restrained (Kong et al., 2002; Chen & Kong, 2006). Therefore, selective targeting centrosome as a whole unlike traditional approaches aiming at single protein or pathway is worthy of trying.

### **4. Conclusion**

226 Hepatocellular Carcinoma – Basic Research

(Nelson & Nusse, 2004). No matter which way β-catenin involves, the intracellular β-catenin level is critical to its functions, therefore, HBx can regulate β-catenin, which plays an important role in various aspects of liver biology including cancer development, either by GSK-3, which directly suppress its activation via Src, or indirectly inhibit its activation by ERK signaling, or by p53, in which process HBx stabilize p53 expression leading to β-catenin degeneration (Hsieh et al., 2011; Wu et al., 2008; Jung et al., 2007). Importantly, β-catenin, a component of centrosome, interacts with centrosomal proteins to regulate mitotic centrosome separation (Bahmanyar, 2010) by forming a complex with the centrosomal proteins Nek2, C-Nap1 and Rootletin (Bahmanyar et al., 2008; Hadjihannas et al., 2010). Depletion of β-catenin in asynchronous cells results in monopolar spindles with unseparated centrosomes (Bahmanyar et al., 2008), whereas expression of mutation βcatenin causes increased centriole splitting in G1-S (Bahmanyar et al., 2008; Hadjihannas et al., 2010). These findings suggest that cell adhesion is a major target for HBx both on cell

**3. Centrosome abnormalities in the development of drug resistance** 

**3.1 Centrosome clustering pathway as a target in cancer therapy** 

As signaling center, centrosome plays important roles in the development of drug resistance. Many centrosome-associated proteins are involved in chemo-resistance process, such as Her-2/neu, bcl-2, c-myc, ras, c-jun, MDM2, p210 BCR-abl, or mutant p53. In fact, abnormal centrosome itself may lead to formation of poly- or monopolarity spindle resulting in chromatin mis-segregation, which further result in or accelerate inactivation of tumor suppressor genes and/or activation of tumor genes, thereby leading to the development of chemoresistance. Support of this idea comes from the recent finding that p53 status determines tumor response to anti-angiogenic therapy and heat shock proteins (HSPs) varies with tumor progressions (Chen & Kong, 2009; Ciocca &

Centrosome clustering pathway is indispensable in cells with supernumerary centrosomes ensuring the success of cell division. Interference in this process could be lethal to tumor cells containing extra centrosomes (Kwon et al., 2008). Therefore, proper interference centrosome clustering pathway may raise the possibility of developing a new therapeutic strategy. HSET, the human homologue of the KAR3 family of minus end-directed kinesinlike motors, may be one of the most appropriate such candidates, as HSET depletion destroys centrosome clustering pathway and induces multipolar divisions and hence abnormal chromosome segregation or aneuploidy. Besides, HEST is essential only for clustering extra centrosomes in cancer cell but not in normal cells, by bundling the minus end of MT in acentrosomal spindles (Mountain et al., 1999). These results indicate that inhibition of HSET can selectively kill cells with extra centrosomes without affecting the viability of cells that contain normal centrosome numbers. In addition, HEST has been found to be involved in cell-cell adhesion by influencing the cells shape, then inducing low integrin -1 expression, and eventually resulting in tumor environment changes (Amendola et al., 2001). Taken together, HSET inhibitor may have a relative low toxicity compared with other mitosis-blocking agents involving centrosme, including checkpoint

migration and on signaling transduction.

Calderwood, 2005).

Centrosome works as an integrated complex in regulating important cellular events. Disrupting centrosome structurally and functionally may trigger malignant transformation. Although the roles of centrosome in carcinogenesis have been elucidated in some types of cancer, the roles of the centrosome in HCC development, particularly in cancer therapy, are largely uncovered. As discussed above, centrosome serves as a platform for HBV virus infection through centrosome-associated proteins, then transforming cell to immortalization. It is reasonable to believe that the drugs targeting centrosome-associated proteins should be developed to stop cancer cells proliferation and exert their efficacy when combined with conventional therapeutic agents. However, centrosome is an open prison, where proteins can bind and release in a precisely time-dependent manner in different cell cycle. Selective

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**11** 

*1,2Japan 3China* 

*Graduate School of Medicine* 

**Cytoplasmic Connexin32 and Self-Renewal of** 

**Cancer Stem Cells: Implication in Metastasis** 

*2Department of Otorhinolaryngology, Akita University Graduate School of Medicine 3Department of Pathology, College of Basic Medical Sciences, China Medical University* 

Gap junction is a unique intercellular channel which connects directly the cytoplasm of two neighbouring cells, and allows small (*Mr* < 1000) water-soluble molecules to travel between the cells throughout a tissue, thus serving as a tool of cell-cell communication (Goodenough et al., 1996). In general, gap junction plays crucial roles in tissue and cellular homeostasis and has long been known to suppress carcinogenesis in many tissues (Crespin et al., 2009; Leithe et al., 2006). In the liver, one of the organs where gap junctions are well developed, down-regulation of gap junction between hepatocytes is one of hallmarks for hepatocarcinogenesis (V.A. Krutovskikh et al., 1991). Gap junction is completely disrupted in not only hepatocellular carcinoma but also even precancerous lesions such as GST-P foci (Fitzgerald et al., 1989). A gap junction channel is composed of two hemichannels, which dock with each other to make a complete channel. Hemichannels are provided by each of two neighbouring cells and are called "connexons." The connexon is a hexamer of connexin protein, which forms connexin family consisting of more than 20 members in mammals (Beyer & Berthoud, 2009; Sohl & Willecke, 2003). Among them, connexin26 and connexin32 proteins are co-expressed in the hepatocyte (Nicholson et al., 1987; Vinken et al., 2008). During hepatocarcinogenesis, expression of connexin26 protein is abolished. On the other hand, connexin32 protein is reduced in expression but remains expressed not in plasma membrane but in cytoplasm, resulting in total loss of functional gap junction from both hepatocellular carcinoma and its precancerous lesions. More interestingly, the amount of connexin32 protein in cytoplasm often increases in the correspondence with tumour progression and/or the grade of malignancy (Fig. 1) (V. Krutovskikh et al., 1994). Therefore, although connexin32 protein localised in cytoplasm is non-functional as a gap junction component, it may contribute to tumour progression such as invasion and metastasis.

It has long been believed that the tumour is composed of monoclonal cells and thus is a homogenous cell population. According to this idea, every tumour cell should have the ability to develop a new tumour elsewhere and possible heterogeneity should be made only

**1. Introduction** 

Toshiaki Yoshioka1, Yohei Yamamoto1 and Katsuhiko Enomoto1

Yasufumi Omori1, Yohei Kawasaki1,2, Qingchang Li1,3,

*1Department of Molecular and Tumour Pathology, Akita University* 

