**4. Cancer stem cells**

The similarities between adult stem cells and CSC have led to confusion regarding their identity and it has often not been clear in the literature whether CSCs represent transformed progenitor or stem cells or whether both cell types are distinct cell populations that only share the expression of certain cell markers and display a similar biology. Adult tissue stem cells and CSCs are both defined by (i) highly efficient self-renewing ability through asymmetrical cell division and (ii) differentiation capacity along at least two if not more cell lineages. CSCs manifest the additional property of tumour initiation and/or maintenance. Nowadays the consensus is that the term CSC simply describes a cell's potential for selfrenewal and ability to give rise to the hierarchic organisation of the heterogeneous lineages of cancer cells that constitute the tumour and does not consider the cell's origin. CSCs may arise from the differentiation arrest and transformation of a normal adult stem cell through oncogenic and/or epigenetic aberrations or the dedifferentiation of a mature cell that subsequently acquires self-renewing capacity. The CSC concept has been debated for many decades and compelling evidence of their existence has only emerged in the past decade.

#### **4.1 Haematopoietic cancer stem cells**

The existence of CSCs was first demonstrated in the haematological malignancy acute myeloid leukaemia (AML). Dick and colleagues isolated human AML cells from peripheral blood and transplanted them into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The vast majority of cells was unable to induce leukaemia, even when transplanted in larger numbers, despite the fact that they displayed a leukaemic blast phenotype such as the CD34+/CD38+ subpopulation. Only 0.01-1% of all AML cells, the CD34+CD38- fraction, initiated AML and gave rise to a heterogeneous leukaemia tumour cell mass, classifying them as CSCs. The CD34+CD38- cells could be serially transplanted and reliably developed AML with the same morphology and cell surface marker expression as the original tumour (Bonnet & Dick, 1997). Additional tumour–initiating AML cell populations were later identified and described to be of a CD34+CD19- or CD34+CD10- and CD34+CD4- or CD34+CD7- phenotype (Cox *et al*., 2004; Cox *et al*., 2007).

#### **4.2 Solid tumour cancer stem cells**

Using similar approaches involving cell transplantation into immunodeficient mice, CSCs have subsequently been identified in a variety of solid tumours, including breast, brain and liver cancer.

#### **4.2.1 Breast cancer stem cells**

26 Liver Regeneration

strongly suggests they originate from LPCs (Roskams *et al.*, 2003a; Roskams *et al.*, 2003b, Falkowski *et al.*, 2003). LPCs always emerge in pathologies with a predisposition to cancer and their proliferation in an environment rich in inflammatory mediators, growth factors or reactive oxygen species renders them likely targets for transformation. Furthermore, inhibition of the LPC response has been demonstrated to reduce the formation of cancerous lesions, strongly supporting a role for LPCs in hepatocarcinogenesis (Davies *et al*., 2006; Knight *et al*., 2005; Knight *et al*., 2008). Very recently LPCs have not only been discussed as cellular precursors for liver cancer but also as potential liver cancer stem cells, which could be responsible for tumour maintenance and recurrence (Marquardt *et al.*, 2011; Rountree *et*

The similarities between adult stem cells and CSC have led to confusion regarding their identity and it has often not been clear in the literature whether CSCs represent transformed progenitor or stem cells or whether both cell types are distinct cell populations that only share the expression of certain cell markers and display a similar biology. Adult tissue stem cells and CSCs are both defined by (i) highly efficient self-renewing ability through asymmetrical cell division and (ii) differentiation capacity along at least two if not more cell lineages. CSCs manifest the additional property of tumour initiation and/or maintenance. Nowadays the consensus is that the term CSC simply describes a cell's potential for selfrenewal and ability to give rise to the hierarchic organisation of the heterogeneous lineages of cancer cells that constitute the tumour and does not consider the cell's origin. CSCs may arise from the differentiation arrest and transformation of a normal adult stem cell through oncogenic and/or epigenetic aberrations or the dedifferentiation of a mature cell that subsequently acquires self-renewing capacity. The CSC concept has been debated for many decades and compelling evidence of their existence has only emerged in the past decade.

The existence of CSCs was first demonstrated in the haematological malignancy acute myeloid leukaemia (AML). Dick and colleagues isolated human AML cells from peripheral blood and transplanted them into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The vast majority of cells was unable to induce leukaemia, even when transplanted in larger numbers, despite the fact that they displayed a leukaemic blast phenotype such as the CD34+/CD38+ subpopulation. Only 0.01-1% of all AML cells, the CD34+CD38- fraction, initiated AML and gave rise to a heterogeneous leukaemia tumour cell mass, classifying them as CSCs. The CD34+CD38- cells could be serially transplanted and reliably developed AML with the same morphology and cell surface marker expression as the original tumour (Bonnet & Dick, 1997). Additional tumour–initiating AML cell populations were later identified and described to be of a CD34+CD19- or CD34+CD10- and

Using similar approaches involving cell transplantation into immunodeficient mice, CSCs have subsequently been identified in a variety of solid tumours, including breast, brain and

CD34+CD4- or CD34+CD7- phenotype (Cox *et al*., 2004; Cox *et al*., 2007).

*al.*, 2012).

**4.

Metastatic breast cancer was the first solid tumour in which CSCs were identified and prospectively isolated. The CD44+CD24-/lowLineage- cell population initiated tumours upon transplantation into mice with as few as 100 cells per injection. Importantly, they could be serially passaged and reliably reproduced the heterogeneous phenotype of the original breast cancer. In contrast, unsorted cells from the primary tumour or injection of a large number of alternate phenotypes, such as CD44+CD24+ cells, failed to form tumours (Al-Hajj *et al*., 2003). Furthermore, it was established that increased expression of the detoxifying enzyme aldehyde dehydrogenase (ALDH) identifies the tumorigenic breast stem cell fraction and high ALDH1 activity correlates with poorer prognosis (Ginestier *et al*., 2007).

#### **4.2.2 Central nervous system cancer stem cells**

The discovery of breast CSC was reported in the same year as the identification of tumourinitiating stem cells in the brain. Singh and colleagues identified and prospectively isolated a CD133+ population of cells from a range of human brain tumours including medulloblastomas, pilocytic astrocytoma, glioblastoma and anaplastic ependymoma that *in vitro* exhibited stem cell properties and gave rise to heterogeneous cell populations with the same phenotype as the original tumour cells. Upon transplantation of as few as 100 CD133+ glioma cells into the frontal lobes of NOD/SCID mice, serially transplantable tumours were initiated that mirrored the original tumour phenotype, whereas no tumours developed after injection of a much larger number of CD133 cells from the same tumour (Singh *et al*., 2003; Singh *et al*., 2004).

#### **4.2.3 Liver cancer stem cells**

Only very recently have liver CSCs been described. However the mounting evidence is compelling and ever more markers are suggested to describe the population of cells that may be responsible for liver cancer initiation, maintenance and potentially tumour recurrence after HCC resection, as described below.

#### **<IP_ADDRESS> Side population (Hoechst 33342 dye efflux)**

The first evidence for the existence of liver CSCs came from Haraguchi and colleagues who performed Hoechst 33342 side population (SP) analyses of various human gastrointestinal cell lines and identified a subpopulation of cells with CSC properties. The SP approach is based on the finding that cells without stem cell characteristics accumulate the fluorescent nucleic acid-binding dye Hoechst 33342, whereas stem cells and CSCs do not as they are capable of effectively effluxing the dye through high activity of adenosine triphosphate (ATP)-binding cassette (ABC) transporters such as the multidrug resistance transporter 1 (MDR1) or breast cancer resistance protein (BCRP, also known as ABCG2). These ABC transporters employ ATP hydrolysis to facilitate substrate export across membranes against steep concentration gradients and thereby protect cells from cytotoxic agents and importantly from chemotherapeutic drugs such as cisplatin and doxorubicin. The authors report that the HCC lines HuH7 and Hep3B contained 0.9% to 1.8% SP cells with CSC properties, respectively, whereas no SP cells could be purified from the less aggressive hepatoma cell line HepG2 (Haraguchi *et al*., 2006). These results were confirmed shortly after by Chiba and colleagues who identified SP cells in some human liver cell lines, which

Liver Progenitor Cells, Cancer Stem Cells and Hepatocellular Carcinoma 29

End-stage complications of chronic liver disease (cirrhosis and HCC) are the 9th commonest global cause of death and will remain so for at least the next 20 years. Of great concern is the prediction by the World Health Organisation that by 2030, deaths from HCC will for the first time exceed those from non-malignant complications of cirrhosis, such as liver failure

Most cases of HCC in the western world arise in the setting of established cirrhosis (Bruix & Sherman, 2005; Olsen *et al.*, 2010; Sherman, 2011). The median survival of untreated HCC is in the order of 6-16 months. In view of the poor survival in the absence of therapy, strategies have been implemented to reduce the incidence of HCC through immunisation to prevent chronic HBV infection and screening of high-risk groups (i.e. those with cirrhosis). Despite these approaches, we are still faced with an escalation in the number of cases and requirement for treatment (El-Serag & Mason, 1999; Bruix & Sherman, 2005; Llovet *et al.*,

Presently the treatment of choice for HCC is liver resection or orthotopic liver transplantation (OLT), either with or without adjunctive chemotherapy or non-surgical ablative therapy. Liver resection is the treatment of choice for HCC in non-cirrhotic livers, and accounts for 5% of HCC cases in western countries and 40% of cases in non-western countries. Patients with well compensated cirrhosis and who do not have portal hypertension may also be considered for resection, provided that lesions are confined to the liver and enough "functional reserve" of liver is retained to ensure survival of the patient (Bruix & Sherman, 2005; Llovet *et al.*, 2005). Currently, tumour size, number and vascular invasion are still the strongest predictors of survival with up to 70% of subjects surviving five years. Tumour recurrence complicates 70% of cases at five years, reflecting either intrahepatic metastases (true recurrences) or the development of *de novo* tumours (Llovet *et al.*, 2005). Based on comparative genomic hybridisation, DNA fingerprinting using loss of heterozygosity assays, or DNA microarray studies, it is estimated that just over half of recurrences correspond to intrahepatic metastases undetected by the time of resection, whereas less than half are *de novo* HCCs (Chen *et al.*, 2000; Finkelstein *et al.*, 2003; Ng *et al.*, 2003). OLT is indicated in individuals who fulfil the "Milan criteria": patients with a single HCC of up to five centimetres in size or up to three nodules not larger than three centimetres each. Strict adherence to these criteria results in 5-year survival of up to 70% with recurrence rates usually less than 15%(Bismuth *et al.*, 1993; Mazzaferro *et al.*, 1996;

The activation and proliferation of LPCs during chronic liver injury is associated with an inflammatory response that involves activation of resident and recruited inflammatory cells (Fig. 4). These inflammatory cells initiate tissue regeneration by promoting the removal of cellular debris and by stimulating LPCs to proliferate through release of mitogenic growth factors and cytokines (Lowes et al., 2003; Knight et al., 2005). Whilst LPCs play an important role in normal liver repair processes, dysregulation of their proliferation and differentiation has been linked to fibrogenesis and carcinogenesis (Lowes *et al.*, 1999; Clouston *et al.*, 2005; Knight *et al.*, 2008; Ruddell *et al.*, 2009; Tirnitz-Parker *et al.*, 2010). Clear demonstration of a role for LPCs, and possibly CSCs, in HCC development was reported by Shachaf and

**5.1 HCC: A clinically important end-stage complication of chronic liver disease**

and portal hypertension (Mathers *et al*., 2006).

2005; Mathers & Loncar, 2006; Llovet *et al.*, 2008).

Llovet *et al.*, 2005; Mazzaferro *et al.*, 2009).

**5.2 Pathogenesis of HCC: The emerging role of LPCs and CSCs**

successfully induced xenograft tumours in NOD/SCID mice upon transplantation of as few as 1000 SP cells, while attempts to produce tumours with 1 x 106 non-SP cells failed consistently (Chiba *et al*., 2006).

#### **<IP_ADDRESS> CD133 (Prominin 1)**

Several recent studies have used this glycoprotein initially identified as a marker for CD34+ haematopoietic stem cells and later as a marker of LPCs for the isolation of liver CSCs. Suetsugu *et al*. reported that both the hepatoblastoma cell line HepG2 as well as the human fetal hepatoblast cell line Hc lacked CD133 expression, and that CD133+ cells could only be demonstrated in the human HCC line Huh7. CD133+ cells showed a higher proliferative potential in culture but also a greater ability to initiate tumour growth *in vivo* compared to the CD133- population (Suetsugu *et al*., 2006). Hepatic cells with a CD133 phenotype have been shown to be more resistant to chemotherapeutic drugs such as doxorubicin and 5 fluorouracil than their CD133 counterparts through preferential activation of the Akt/protein kinase B and Bcl-2 cell survival pathways. Furthermore, resistance of normal stem cells to cyclophosphamide is facilitated by the differentially expressed marker ALDH. Studies on ALDH and CD133+ cells found ALDH expression only in the CD133+ subpopulation and suggested a hierarchical cell organisation with regard to tumorigenicity in the order CD133+ALDH+ > CD133+ALDH- > CD133- ALDH- , which suggests ALDH as an additional marker useful for liver CSC identification (Ma *et al*., 2007, Ma *et al*., 2008). In addition, it has been demonstrated that TGFβ signalling can induce CD133 expression in the HCC cell line Huh7 through epigenetic regulation, which results in a significant increase in tumour initiation capacity in these cells compared to CD133- Huh7 cells (You *et al.*, 2010).

#### **<IP_ADDRESS> Epithelial cell adhesion molecule (EpCAM)**

Myajima and colleagues identified EpCAM as a biliary and LPC marker, which is expressed in biliary epithelial cells and becomes upregulated in liver upon 2-AAF/PHx and DDC treatment (Okabe *et al*., 2009). Since EpCAM expression has been reported in many normal epithelial as well as in tumour cells, it is not surprising that it has been suggested as a useful CSC marker. EpCAM+ cells isolated from human HCC tissues were shown to be more tumorigenic and invasive than EpCAM cells and consistently formed invasive tumours in NOD/SCID mice, even after serial transplantation, whereas the EpCAM population did not (Yamashita *et al*., 2009). EpCAM is a direct transcriptional target of Wnt/β-catenin signalling, which has been implicated as a CSC self-renewal pathway (Yamashita *et al*., 2007). Activation of the Wnt/β-catenin pathway increased the EpCAM+ cell population, whereas knockdown of EpCAM resulted in decreased proliferation, colony formation, migration and drug resistance (Yamashita *et al*., 2009).

#### **5. Hepatocellular carcinoma**

Mortality from chronic liver disease is the most rapidly increasing cause of death in many western nations. The commonest aetiologies contributing to this escalation are chronic viral hepatitis C or B infection, alcoholic and non-alcoholic fatty liver disease. All these conditions can cause fibrosis and, subsequently, cirrhosis and HCC. Much evidence has been gathered demonstrating that HCC can arise from deregulated LPC proliferation and maturation during chronic liver injury in humans and in animal models of liver disease and carcinogenesis.

successfully induced xenograft tumours in NOD/SCID mice upon transplantation of as few as 1000 SP cells, while attempts to produce tumours with 1 x 106 non-SP cells failed

Several recent studies have used this glycoprotein initially identified as a marker for CD34+ haematopoietic stem cells and later as a marker of LPCs for the isolation of liver CSCs. Suetsugu *et al*. reported that both the hepatoblastoma cell line HepG2 as well as the human fetal hepatoblast cell line Hc lacked CD133 expression, and that CD133+ cells could only be demonstrated in the human HCC line Huh7. CD133+ cells showed a higher proliferative potential in culture but also a greater ability to initiate tumour growth *in vivo* compared to the CD133- population (Suetsugu *et al*., 2006). Hepatic cells with a CD133 phenotype have been shown to be more resistant to chemotherapeutic drugs such as doxorubicin and 5-

kinase B and Bcl-2 cell survival pathways. Furthermore, resistance of normal stem cells to cyclophosphamide is facilitated by the differentially expressed marker ALDH. Studies on ALDH and CD133+ cells found ALDH expression only in the CD133+ subpopulation and suggested a hierarchical cell organisation with regard to tumorigenicity in the order

ALDH-

marker useful for liver CSC identification (Ma *et al*., 2007, Ma *et al*., 2008). In addition, it has been demonstrated that TGFβ signalling can induce CD133 expression in the HCC cell line Huh7 through epigenetic regulation, which results in a significant increase in tumour initiation

Myajima and colleagues identified EpCAM as a biliary and LPC marker, which is expressed in biliary epithelial cells and becomes upregulated in liver upon 2-AAF/PHx and DDC treatment (Okabe *et al*., 2009). Since EpCAM expression has been reported in many normal epithelial as well as in tumour cells, it is not surprising that it has been suggested as a useful CSC marker. EpCAM+ cells isolated from human HCC tissues were shown to be more tumorigenic and invasive than EpCAM- cells and consistently formed invasive tumours in

(Yamashita *et al*., 2009). EpCAM is a direct transcriptional target of Wnt/β-catenin signalling, which has been implicated as a CSC self-renewal pathway (Yamashita *et al*., 2007). Activation of the Wnt/β-catenin pathway increased the EpCAM+ cell population, whereas knockdown of EpCAM resulted in decreased proliferation, colony formation,

Mortality from chronic liver disease is the most rapidly increasing cause of death in many western nations. The commonest aetiologies contributing to this escalation are chronic viral hepatitis C or B infection, alcoholic and non-alcoholic fatty liver disease. All these conditions can cause fibrosis and, subsequently, cirrhosis and HCC. Much evidence has been gathered demonstrating that HCC can arise from deregulated LPC proliferation and maturation during chronic liver injury in humans and in animal models of liver disease and

NOD/SCID mice, even after serial transplantation, whereas the EpCAM-

Huh7 cells (You *et al.*, 2010).

counterparts through preferential activation of the Akt/protein

, which suggests ALDH as an additional

population did not

consistently (Chiba *et al*., 2006).

**<IP_ADDRESS> CD133 (Prominin 1)**

fluorouracil than their CD133-

CD133+ALDH+ > CD133+ALDH- > CD133-

capacity in these cells compared to CD133-

**<IP_ADDRESS> Epithelial cell adhesion molecule (EpCAM)**

migration and drug resistance (Yamashita *et al*., 2009).

**5. Hepatocellular carcinoma**

carcinogenesis.

#### **5.1 HCC: A clinically important end-stage complication of chronic liver disease**

End-stage complications of chronic liver disease (cirrhosis and HCC) are the 9th commonest global cause of death and will remain so for at least the next 20 years. Of great concern is the prediction by the World Health Organisation that by 2030, deaths from HCC will for the first time exceed those from non-malignant complications of cirrhosis, such as liver failure and portal hypertension (Mathers *et al*., 2006).

Most cases of HCC in the western world arise in the setting of established cirrhosis (Bruix & Sherman, 2005; Olsen *et al.*, 2010; Sherman, 2011). The median survival of untreated HCC is in the order of 6-16 months. In view of the poor survival in the absence of therapy, strategies have been implemented to reduce the incidence of HCC through immunisation to prevent chronic HBV infection and screening of high-risk groups (i.e. those with cirrhosis). Despite these approaches, we are still faced with an escalation in the number of cases and requirement for treatment (El-Serag & Mason, 1999; Bruix & Sherman, 2005; Llovet *et al.*, 2005; Mathers & Loncar, 2006; Llovet *et al.*, 2008).

Presently the treatment of choice for HCC is liver resection or orthotopic liver transplantation (OLT), either with or without adjunctive chemotherapy or non-surgical ablative therapy. Liver resection is the treatment of choice for HCC in non-cirrhotic livers, and accounts for 5% of HCC cases in western countries and 40% of cases in non-western countries. Patients with well compensated cirrhosis and who do not have portal hypertension may also be considered for resection, provided that lesions are confined to the liver and enough "functional reserve" of liver is retained to ensure survival of the patient (Bruix & Sherman, 2005; Llovet *et al.*, 2005). Currently, tumour size, number and vascular invasion are still the strongest predictors of survival with up to 70% of subjects surviving five years. Tumour recurrence complicates 70% of cases at five years, reflecting either intrahepatic metastases (true recurrences) or the development of *de novo* tumours (Llovet *et al.*, 2005). Based on comparative genomic hybridisation, DNA fingerprinting using loss of heterozygosity assays, or DNA microarray studies, it is estimated that just over half of recurrences correspond to intrahepatic metastases undetected by the time of resection, whereas less than half are *de novo* HCCs (Chen *et al.*, 2000; Finkelstein *et al.*, 2003; Ng *et al.*, 2003). OLT is indicated in individuals who fulfil the "Milan criteria": patients with a single HCC of up to five centimetres in size or up to three nodules not larger than three centimetres each. Strict adherence to these criteria results in 5-year survival of up to 70% with recurrence rates usually less than 15%(Bismuth *et al.*, 1993; Mazzaferro *et al.*, 1996; Llovet *et al.*, 2005; Mazzaferro *et al.*, 2009).

#### **5.2 Pathogenesis of HCC: The emerging role of LPCs and CSCs**

The activation and proliferation of LPCs during chronic liver injury is associated with an inflammatory response that involves activation of resident and recruited inflammatory cells (Fig. 4). These inflammatory cells initiate tissue regeneration by promoting the removal of cellular debris and by stimulating LPCs to proliferate through release of mitogenic growth factors and cytokines (Lowes et al., 2003; Knight et al., 2005). Whilst LPCs play an important role in normal liver repair processes, dysregulation of their proliferation and differentiation has been linked to fibrogenesis and carcinogenesis (Lowes *et al.*, 1999; Clouston *et al.*, 2005; Knight *et al.*, 2008; Ruddell *et al.*, 2009; Tirnitz-Parker *et al.*, 2010). Clear demonstration of a role for LPCs, and possibly CSCs, in HCC development was reported by Shachaf and

Liver Progenitor Cells, Cancer Stem Cells and Hepatocellular Carcinoma 31

The cellular target of transformation leading to HCC is currently undefined. Potential candidates include the hepatocyte and the LPC and they need not be mutually exclusive. However, there is substantial circumstantial as well as some direct evidence implicating LPCs. This view would also be compatible with the increasingly popular theory of the stem cell basis of cancer. In the context of HCC, a variety of animal models, which induce chronic liver injury ultimately produce liver cancers and most of these pathologies display increased proliferation of LPCs. To conform to current views on carcinogenesis i.e. it is a rare event that affects a few cells and there are multiple stages in the process, it is necessary to hypothesise that a minority of LPCs are tumorigenic and that these have incurred the early genetic alterations that have initiated their progression to cancer. The challenge for future strategies to treat liver cancer is to identify these initiated LPCs and to show their direct link to HCC. This should be followed up with studies to elucidate progressive changes at the molecular level, which govern their behaviour and to exploit their vulnerability. Such knowledge will facilitate better diagnosis as well as treatment and prevention of HCC.

The authors would like to thank Ian Dickson for technical assistance with preparation of the

Akhurst, B., Croager, E. J., Farley-Roche, C. A., Ong, J. K., Dumble, M. L., Knight, B. & Yeoh,

regeneration induced by chronic and acute injury. *Hepatology,* 41, 327-335. Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J. & Clarke, M. F. (2003).

Alison, M., Golding, M., Lalani, E. N., Nagy, P., Thorgeirsson, S. & Sarraf, C. (1997).

Arber, N., Zajicek, G. & Ariel, I. (1988). The streaming liver. II. Hepatocyte life history. *Liver,*

Bisgaard, H. C., Parmelee, D. C., Dunsford, H. A., Sechi, S. & Thorgeirsson, S. S. (1993).

Bonnet, D. & Dick, J. E. (1997). Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. *Nature Medicine,* 3, 730-737.

mouse monoclonal antibody OV-6. *Molecular Carcinogenesis,* 7, 60-66. Bismuth, H., Chiche, L., Adam, R., Castaing, D., Diamond, T. & Dennison, A. (1993). Liver

progeny of biliary stem cells. *Journal of Hepatology,* 26, 343-352.

effectively induces oval cells in mouse liver. *Hepatology,* 34, 519-522. Akhurst, B., Matthews, V., Husk, K., Smyth, M. J., Abraham, L. J. & Yeoh, G. C. (2005).

*National Academy of Sciences U S A,* 100, 3983-3988.

G. C. (2001). A modified choline-deficient, ethionine-supplemented diet protocol

Differential lymphotoxin-beta and interferon gamma signaling during mouse liver

Prospective identification of tumorigenic breast cancer cells. *Proceedings of the*

Wholesale hepatocytic differentiation in the rat from ductular oval cells, the

Keratin 14 protein in cultured nonparenchymal rat hepatic epithelial cells: characterization of keratin 14 and keratin 19 as antigens for the commonly used

resection versus transplantation for hepatocellular carcinoma in cirrhotic patients.

**6. Conclusion**

**7. Acknowledgement**

8, 80-87.

*Annals of Surgery,* 218, 145-151.

figures.

**8. References**

colleagues (Shachaf *et al.*, 2004). Inactivation of the Myc oncogene was sufficient to induce sustained regression of invasive HCC in a murine model. Tumour cells differentiated into hepatocytes and biliary epithelial cells. This process was associated with rapid loss of expression of the tumour marker α-fetoprotein, increase in expression of liver cell markers CK8 and carcinoembryonic antigen, and in some cells the biliary LPC marker CK19. Many of the "reverted" tumour cells remained dormant as long as Myc remained inactivated; however, Myc reactivation immediately restored their neoplastic features. Using array comparative genomic hybridisation, Shachaf and coworkers confirmed that the dormant liver cells and the restored tumour retained the identical molecular signature and hence were clonally derived from the tumour cells. Thus, tumours have pluripotent capacity to differentiate into normal cellular lineages and tissue structures, while retaining their latent potential to become cancerous

Several other studies have confirmed a LPC phenotype in a substantial number of HCCs. Detailed immunophenotyping revealed that 28–50% of HCCs express markers of LPCs, such as CK7 and CK19. Histologically, these tumours consist of cells that have an intermediate phenotype between LPCs and mature hepatocytes. Furthermore, HCCs that express both hepatocyte and biliary cell markers such as albumin, CK7 and CK19, carry a significantly poorer prognosis and higher recurrence after surgical resection and liver transplantation (Roskams, 2006; Yao & Mishra, 2009). The "precursor-product" relationship between LPCs, CSCs and HCC is further strengthened by the observation that 55% of small dysplastic foci, which represent the earliest premalignant lesions, are comprised of LPCs and intermediate hepatocytes (Weinstein *et al.*, 2001). Finally, inhibition of the LPC response to liver injury using a broad range of pharmacological therapies such as interferon alpha 2b (Lim *et al.*, 2006), COX-II inhibitors (Davies *et al.*, 2006), or tyrosine kinase inhibitors (Knight *et al.*, 2008) is associated with a reduction in the severity of hepatic fibrosis and incidence of HCC. These observations provide more evidence in support of a critical role for LPCs and CSCs in the carcinogenic process. Collectively these studies suggest that anti-inflammatory agents may be useful therapeutically in reducing the incidence of liver cancer among patients with chronic liver pathologies.

Fig. 4. Co-regulation of inflammatory response and LPC proliferation in hepatitis C patients. Haematoxylin and eosin (H&E) staining of a liver section from a hepatitis C virus-infected patient demonstrates disrupted liver architecture through infiltration and proliferation of small basophilic cells as well as steatotic changes in hepatocytes (A). Staining for the common leukocyte marker CD45 (B) and the biliary LPC marker CKpan (C) suggests co-regulation of the inflammatory response with the Ductular Reaction.

#### **6. Conclusion**

30 Liver Regeneration

colleagues (Shachaf *et al.*, 2004). Inactivation of the Myc oncogene was sufficient to induce sustained regression of invasive HCC in a murine model. Tumour cells differentiated into hepatocytes and biliary epithelial cells. This process was associated with rapid loss of expression of the tumour marker α-fetoprotein, increase in expression of liver cell markers CK8 and carcinoembryonic antigen, and in some cells the biliary LPC marker CK19. Many of the "reverted" tumour cells remained dormant as long as Myc remained inactivated; however, Myc reactivation immediately restored their neoplastic features. Using array comparative genomic hybridisation, Shachaf and coworkers confirmed that the dormant liver cells and the restored tumour retained the identical molecular signature and hence were clonally derived from the tumour cells. Thus, tumours have pluripotent capacity to differentiate into normal cellular lineages and tissue structures, while retaining their latent

Several other studies have confirmed a LPC phenotype in a substantial number of HCCs. Detailed immunophenotyping revealed that 28–50% of HCCs express markers of LPCs, such as CK7 and CK19. Histologically, these tumours consist of cells that have an intermediate phenotype between LPCs and mature hepatocytes. Furthermore, HCCs that express both hepatocyte and biliary cell markers such as albumin, CK7 and CK19, carry a significantly poorer prognosis and higher recurrence after surgical resection and liver transplantation (Roskams, 2006; Yao & Mishra, 2009). The "precursor-product" relationship between LPCs, CSCs and HCC is further strengthened by the observation that 55% of small dysplastic foci, which represent the earliest premalignant lesions, are comprised of LPCs and intermediate hepatocytes (Weinstein *et al.*, 2001). Finally, inhibition of the LPC response to liver injury using a broad range of pharmacological therapies such as interferon alpha 2b (Lim *et al.*, 2006), COX-II inhibitors (Davies *et al.*, 2006), or tyrosine kinase inhibitors (Knight *et al.*, 2008) is associated with a reduction in the severity of hepatic fibrosis and incidence of HCC. These observations provide more evidence in support of a critical role for LPCs and CSCs in the carcinogenic process. Collectively these studies suggest that anti-inflammatory agents may be useful therapeutically in reducing the incidence of liver cancer among patients with

Fig. 4. Co-regulation of inflammatory response and LPC proliferation in hepatitis C patients. Haematoxylin and eosin (H&E) staining of a liver section from a hepatitis C virus-infected patient demonstrates disrupted liver architecture through infiltration and proliferation of small basophilic cells as well as steatotic changes in hepatocytes (A). Staining for the common leukocyte marker CD45 (B) and the biliary LPC marker CKpan (C) suggests

co-regulation of the inflammatory response with the Ductular Reaction.

potential to become cancerous

chronic liver pathologies.

The cellular target of transformation leading to HCC is currently undefined. Potential candidates include the hepatocyte and the LPC and they need not be mutually exclusive. However, there is substantial circumstantial as well as some direct evidence implicating LPCs. This view would also be compatible with the increasingly popular theory of the stem cell basis of cancer. In the context of HCC, a variety of animal models, which induce chronic liver injury ultimately produce liver cancers and most of these pathologies display increased proliferation of LPCs. To conform to current views on carcinogenesis i.e. it is a rare event that affects a few cells and there are multiple stages in the process, it is necessary to hypothesise that a minority of LPCs are tumorigenic and that these have incurred the early genetic alterations that have initiated their progression to cancer. The challenge for future strategies to treat liver cancer is to identify these initiated LPCs and to show their direct link to HCC. This should be followed up with studies to elucidate progressive changes at the molecular level, which govern their behaviour and to exploit their vulnerability. Such knowledge will facilitate better diagnosis as well as treatment and prevention of HCC.

#### **7. Acknowledgement**

The authors would like to thank Ian Dickson for technical assistance with preparation of the figures.

#### **8. References**

Liver Progenitor Cells, Cancer Stem Cells and Hepatocellular Carcinoma 33

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Farber, E. (1956b). Similarities in the sequence of early histological changes induced in the

Fausto, N. & Campbell, J. S. (2003). The role of hepatocytes and oval cells in liver regeneration and repopulation. *Mechanisms of Development,* 120, 117-130. Fausto, N., Campbell, J. S. & Riehle, K. J. (2006). Liver regeneration. *Hepatology,* 43, S45-53. Finkelstein, S. D., Marsh, W., Demetris, A. J., Swalsky, P. A., Sasatomi, E., Bonham, A.,

Forbes, S. J., Russo, F. P., Rey, V., Burra, P., Rugge, M., Wright, N. A. & Alison, M. R. (2004).

Fujio, K., Evarts, R. P., Hu, Z., Marsden, E. R. & Thorgeirsson, S. S. (1994). Expression of

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**3**

*USA*

Eva Schmelzer

**Hepatic Progenitors of the Liver**

*McGowan Institute for Regenerative Medicine, Department of Surgery,*

The liver has a tremendous capacity to regenerate at all developmental stages (for reviews, see (1-3)). Liver cell mass can be restored even after repeated partial hepatectomies as well as after toxic injury. The contribution of stem cells to these processes is still under debate. Adult liver cells have been shown to regenerate liver tissue repeatedly when transplanted serially (4). However, hepatocytes cannot be considered stem cells because they are unipotent (for a glossary of terms, see **Table 1**). This chapter describes various liver progenitors that have been found by different researchers in humans and other mammalian species. Intra and extra-hepatic progenitors are discussed that can give rise to liver lineages. Intra-hepatic progenitors of non-hepatic lineages, such as endothelial or hematopoietic restricted progenitors, are not discussed. Although the focus of this chapter is on progenitors that have been characterized in normal, non-pathological conditions of the liver,

Totipotent Capable to give rise to cells of all three embryonic germ layers (i.e.

Pluripotent Capable to give rise to cells of all three embryonic germ layers (i.e.

Multipotent Capable to give rise to multiple but not all lineages. For example,

Bipotent Able to give rise to two fates. In liver, hepatoblasts are considered

Unipotent Able to give rise to only one cell type. Hepatocytes are considered

Progenitor Broad term to describe various types of precursors with different

derive from the inner cell mass of the blastocyst.

tissue of the placenta.

multipotent.

unipotential.

potential.

endoderm, mesoderm and ectoderm) as well as extra-embryonic

endoderm, mesoderm and ectoderm) but not to extra-embryonic tissue. Most commonly used e.g. for embryonic stem cells, which

bone marrow mesenchymal stem cells are considered

bipotential as they can develop into biliary and hepatic lineages.

**1. Introduction**

oval cells will be described briefly.

**Term Description**

**and Extra-Hepatic Tissues**

*University of Pittsburgh, Pennsylvania,*

hormone-producing cells. *Proceedings of the National Academy of Sciences U S A,* 99, 8078-8083.


**5. Conclusion**

Combination of inorganic and organic material is a potential approach to generate more stable NLO crystals. The chemistry correlated with this group of materials is such that the designing of new systems is extremely versatile and potentially contributing to improved optical response. Various semi-organic crystal satisfies the basic criteria's of NLO applications. The remarkable properties of these crystals suggest that this crystal can be used in various optical device applications.


**Author details**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

effective, less toxic, and better tolerated therapies.

Cáncer, CIBERONC, Instituto de Salud Carlos III (ISCIII).

**6. Conclusions**

**Acknowledgements**

**Conflict of interest**

There are not conflict of interest.

**Notes/thanks/other declarations**

**Acronyms and abbreviations**

DNMT DNA methyltransferase

HDR homology-dependent DNA repair LRES Long-range epigenetic silencing PARP poly [ADP-ribose] polymerase

PRC2 polycomb repressive complex 2 SDH succinate dehydrogenase TCA tricarboxylic acid cycle TCGA The cancer genome atlas

PPGL paraganglioma and pheochromocytoma

supplementation, has been shown to result in inhibited growth of hypersuccinylated tumors [59], thus shedding lights on alternative approaches for *SDHx*-mutated-PPGLs.

Metastasis is the most letal attribute of PPGLs, especially in patients with compromised SDH activity. Since the initial discovery of succinate as an oncometabolite that induces DNA hypermethylation, the knowledges that illustrate its role on epigenetic reprogramming and metastasis development continues to expand. The best characterized changes, DNA and histone methylation, could be efficiently and globally neutralized by DNA or histone hypomethylating agents, well-known epi-drugs that could be tested as single- or multi-drug therapy in metastatic SDHdeficient PPGLs. The activity of these epigenetic therapies, however, is not limited to cancer cells but have broad cellular effects leading to global loss of DNA methylation and off-target effects. Emerging scientific knowledges on the impacts that succinate-induced modification of the epigenetic code has on cancer development and progression is certainly empowering the research community to develop more

MDC is funded by Fondo de Investigación Sanitaria (grant numbers FIS PI17/01901 and PI20/01754) and the Red Temática de Investigación Cooperativa en

L.C. thanks the Spanish Ministerio de Ciencia, Innovación y Universidades for the FPU predoctoral contract. T.C. thanks to the Red Temática de Investigación Cooperativa en Cáncer, CIBERONC, Instituto de Salud Carlos III (ISCIII) for her contract. A.S.J.M. thanks to Principado de Asturias and Fondo Europeo de Desarrollo Regional (GRUPIN) for his contract. **Figures 1**–**3** were created with BioRender.com.

**38**

María-Dolores Chiara1,2,3\*, Lucía Celada1,3, Andrés San José Martinez1 , Tamara Cubiella1,3, Enol Álvarez-González1,2,4 and Nuria Valdés1,5

1 Institute of Sanitary Research of Principality of Asturias, Oviedo, Spain

2 Institute of Oncology of the Principality of Asturias, University of Oviedo, Oviedo, Spain

3 CIBERONC, Madrid, Spain

4 Department of Functional Biology, University of Oviedo, Oviedo, Spain

5 Section of Endocrinology and Nutrition, Hospital Universitario de Cabueñes, Gijón, Spain

\*Address all correspondence to<EMAIL_ADDRESS>
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