**3. Conclusive remarks**

In this chapter, we reviewed the literatures describing cytoplasmic connexin proteins in different cancers including HCC and presented our previous studies demonstrating that accumulation of cytoplasmic conneixin32 protein enhanced self-renewal of CSCs in HCCderived cells and resulted in induction of metastasis. What is the impact of the number of CSCs upon metastasis?

In our studies, we hypothesised that expansion of the CSC population should mediate the pro-metastatic function of cytoplasmic connexin32 protein. It is incontestable that cell

Cytoplasmic Connexin32 and Self-Renewal of Cancer Stem Cells: Implication in Metastasis 247

this new era. In other words, we will acquire, in near future, novel important knowledge

The authors are very grateful to Ms. Reiko Ito and Ms. Yuko Doi for their technical assistance, and to Ms. Eriko Kumagai for her secretarial work. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y. Omori, Grant No. 21590427). Q. Li was

Aden, D.P., Fogel, A., Plotkin, S., Damjanov, I., & Knowles, B.B. (1979). Controlled synthesis

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that is presently masked by connexin.

**4. Acknowledgment** 

**5. References** 

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motility and invasiveness are essential factors for metastasis. What these two factors modulate most directly is the length of the latent period between development of a primary tumour and that of its first metastatic focus. On the other hand, the number of CSCs in a tumour should be related closely to the number of metastatic lesions that develop because CSCs are so-called functional seeds that are tumorigenic at a destination site of cell migration while non-CSCs are sterile (Fig. 9) (Jordan et al., 2006). Each metastatic lesion arises from a single CSC but not from non-CSCs. A recent report clearly demonstrated that tumour cells began to circulate in peripheral blood even at the early phase of cancer development, when no risk for metastasis was clinically estimated (Hüsemann et al., 2008; Riethdorf et al., 2008). Taken together, the proportion of CSCs to the whole population in a tumour should be more relevant to the extent of metastasis than the bulk of tumour cells in migration. So it is quite reasonable that cytoplasmic connexin32 protein should enhance the metastatic potential of HCC by expanding its CSC population.

Fig. 9. Each metastatic tumour should be originated from a single cancer stem cell.

For the past five decades, connexin-mediated gap junctional communication has been believed to be one of mechanisms for tumour suppression. This was actually the case. Gap junctional communication efficiently suppressed tumour development in many organs as revealed by both *in vitro* and *in vivo* experiments. Today, connexin proteins are, however, beyond gap junction (Goodenough & Paul, 2003)! A great variety of structures, functions, and behaviours of connexin proteins are known, *i.e.*, hemichannel (Jiang & Gu, 2005), mitochondrial connexin (Boengler et al., 2005), oncogenic connexin (Banerjee et al., 2010; Boengler et al., 2005; Ito et al., 2000), cytoplasmic connexin (Omori et al., 2007), fragmented connexin, and then, gap junction. Therefore, the functions of connexin proteins in cancers are not always suppressive to either cell proliferation or tumour progression (Naus & Laird, 2010). Complex and diverse functions of connexin proteins still remain to be elucidated in this new era. In other words, we will acquire, in near future, novel important knowledge that is presently masked by connexin.

#### **4. Acknowledgment**

246 Hepatocellular Carcinoma – Basic Research

motility and invasiveness are essential factors for metastasis. What these two factors modulate most directly is the length of the latent period between development of a primary tumour and that of its first metastatic focus. On the other hand, the number of CSCs in a tumour should be related closely to the number of metastatic lesions that develop because CSCs are so-called functional seeds that are tumorigenic at a destination site of cell migration while non-CSCs are sterile (Fig. 9) (Jordan et al., 2006). Each metastatic lesion arises from a single CSC but not from non-CSCs. A recent report clearly demonstrated that tumour cells began to circulate in peripheral blood even at the early phase of cancer development, when no risk for metastasis was clinically estimated (Hüsemann et al., 2008; Riethdorf et al., 2008). Taken together, the proportion of CSCs to the whole population in a tumour should be more relevant to the extent of metastasis than the bulk of tumour cells in migration. So it is quite reasonable that cytoplasmic connexin32 protein should enhance the

metastatic potential of HCC by expanding its CSC population.

Fig. 9. Each metastatic tumour should be originated from a single cancer stem cell.

For the past five decades, connexin-mediated gap junctional communication has been believed to be one of mechanisms for tumour suppression. This was actually the case. Gap junctional communication efficiently suppressed tumour development in many organs as revealed by both *in vitro* and *in vivo* experiments. Today, connexin proteins are, however, beyond gap junction (Goodenough & Paul, 2003)! A great variety of structures, functions, and behaviours of connexin proteins are known, *i.e.*, hemichannel (Jiang & Gu, 2005), mitochondrial connexin (Boengler et al., 2005), oncogenic connexin (Banerjee et al., 2010; Boengler et al., 2005; Ito et al., 2000), cytoplasmic connexin (Omori et al., 2007), fragmented connexin, and then, gap junction. Therefore, the functions of connexin proteins in cancers are not always suppressive to either cell proliferation or tumour progression (Naus & Laird, 2010). Complex and diverse functions of connexin proteins still remain to be elucidated in The authors are very grateful to Ms. Reiko Ito and Ms. Yuko Doi for their technical assistance, and to Ms. Eriko Kumagai for her secretarial work. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y. Omori, Grant No. 21590427). Q. Li was recipient of Japanese Government (MEXT) Scholarship (#022101).

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**12** 

*Greece* 

**The Involvement of the ERK-Hypoxia-**

**Angiogenesis Signaling Axis and HIF-1** 

*Laboratory of Biochemistry, School of Medicine, University of Thessaly &* 

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is associated with high resistance to drugs and high mortality. There are multiple factors that influence its molecular pathogenesis but two well established characteristics of malignant transformation in HCC are its hypervascular nature and the upregulation of the Raf/MEK/ERK signaling cascade. MAPK pathway activation can be triggered by important risk factors of hepatocarcinogenesis such as HBV or HCV infection. Furtermore, its deregulation is well documented in human HCC patients and is associated with poor prognosis (Bruix & Llovet, 2009). Constitutive stimulation of both ERK isoforms has been frequently observed in both HCC samples and hepatocarcinoma-derived cell lines and plays a prominent role in the proliferation, invasion and metastasis of HCC cells (Min et al., 2010). These processes are also associated with neoangiogenesis and aberrant vessel formation, which in turn depend on the development of hypoxic regions and VEGF overexpession frequently observed in tumor samples (Rosmorduc & Housset, 2010). The master regulators of the cellular response to oxygen deprivation are the hypoxia inducible transcription factors (HIFs). Their activation results in expression of many genes that contribute to survival and proliferation of malignant cells and, more importantly, resistance to conventional treatments and poor patient outcome (Poon, E. et al., 2009). Interestingly, hypoxia has been observed to lead to ERK activation, which can further stimulate HIF transcriptional activity. This can result in increased expression of HIF target genes that include pro-angiogenic factors and other proteins that facilitate adaptation of tumor cells to their environment (Dimova et al., 2009). In this chapter, we will discuss the cross-talk between these pathways, their contribution to HCC development and progression and their potential as targets of combined therapeutic

Hepatocellular Carcinoma (HCC) is the fifth most common and third in lethality cancer. It is characterized by intrinsic drug-metabolizing activity that confers resistance to

**1. Introduction** 

approaches.

**2. Hepatocellular carcinoma** 

 **in Hepatocellular Carcinoma** 

*Institute of Biomedical Research and Technology (BIOMED), Centre for Research and Technology – Thessaly (CE.RE.TE.TH)* 

Ilias Mylonis and George Simos

