2 Biology of Hepatocellular Carcinoma 25 disease. The liver is different from the skin and gastrointestinal tract with regard to transit-amplifying cells, in that the most highly differentiated cells, the hepato- cytes, are not terminally differentiated and can respond to injury or loss by rapid, highly regulated proliferation. Thus, in the liver, differentiated hepatocytes per se can be viewed as the hepatic version of transit-amplifying cells. Given the postu- lated presence of a hepatic lineage of cells from periductal stem cells, to bipolar ductal progenitor cells, to hepatocytes, with each cell type proliferation competent, it would not be surprising to find that HCCs can arise from the stem cells, the bipo- lar ductal progenitor cells, or the hepatocytes [ 17]. The fact that stem cell activation precedes the development of HCC in almost all models of hepatocarcinogenesis and invariably accompanies chronic liver damage in humans makes it likely that the mature hepatocyte is not the cell of origin of all HCCs [18]. Detailed immunophe- notyping of HCCs indicated that 28–50% of HCCs express markers of progenitor cells such as CK7 and CK19 [19]. But what transforms a stem cell into a cancer stem cell (CSC)? There are at least two proposed mechanisms of CSC origin: onco- genic mutations may inactivate the constraints on normal stem cell expansion or, alternatively, oncogenic mutations in a more differentiated cell generate continual proliferation of cells that no longer enter a postmitotic differentiated state, thereby creating a pool of self-renewing cells in which further mutations can accumulate [20]. Several pathways have been proposed to be implicated in stem cell prolif- eration: TGFβ, Notch, Wnt and Hedgehog are some examples [19]. The detailed mechanisms directing transformation of stem cells to cancer stem cells and hepa- tocellular cancer, however, remain still to be elucidated and definitive markers for these putative cancer stem cells have not yet been established. Angiogenesis HCC is one of the most vascular solid cancers, associated with a high propen- sity for vascular invasion. In fact, its active neovascularization can be visualized in angiography and is used as a diagnostic criterion for HCC. The development of neovasculature in the tumour provides two essential functions for the growth and metastasis of a cancer. First, the vessels provide a route f or supply of nutrient and oxygen to sustain growth and excretion of metabolic waste. Second, the neovessels provide access for tumour cells to enter the circulation and spread as metastases. In fact, HCC is characterized by a high propensity for vascular invasion, and the angiogenic activity of HCC correlates with the risk of vascular invasion [21]. HCC typically develops from dysplastic nodules in a cirrhotic liver and the endothelial cells in these nodules undergo phenotypic changes during malignant transformation as demonstrated by changes in endothelial cell markers. The process of angiogene- sis is a complex multistep process initiated by the release of angiogenic factors from tumour cells. The angiogenic factors bind to specific receptors of endothelial cells of preexisting blood vessels and activate the endothelial cells, which then secrete enzymes to degrade the underlying basement membrane. The activated endothelial 26 M.L. Balmer and J F. Dufour cells then proliferate, migrate and assemble into new capillary tubes, followed by the synthesis of a new basement membrane. However, some recent studies suggested that some of the neovessels in tumours may be derived from circulating endothelial precursor cells that originate from the bone marrow [22, 23]. There is also evidence indicating that some tumours may be vascularized without significant angiogenesis, probably by using existing vessels through a process described as vascular co-option or even by forming vascular channels on their own through a nonendothelial cell process called ‘vascular mimicry’ [24]. Angiogenesis can be triggered by activation of oncogenes like ras or inactivation of tumour suppressor genes like p53 [25, 26]. In addition, a number of cellular stress factors such as hypoxia, nutrient deprivation or inducers of reactive oxygen species are important stimuli of angiogenic signalling [27]. Many angiogenic and antiangiogenic factors have been studied in recent years. Vascular endothelial growth factor (VEGF) is one of the first isolated angiogenic peptides and is the most well-studied angiogenic factor so far. It has a specific mito- genic effect on endothelial cells, and it also increases vascular permeability (hence also known as vascular permeability factor) and promotes extravasation of proteins from tumour vessels, leading to the formation of a fibrin matrix that supports the growth of endothelial cells and allows invasion of stromal cells into the developing tumour [28]. The expression of VEGF protein was found to correlate with clinico- pathological factors such as proliferation, vascular invasion and tumour multiplicity and was reported to associate with not only invasion and metastasis of HCC but also postoperative recurrence [29]. Expression of VEGF is regulated by microenviron- mental and genetic alterations in cancer cells. Hypoxia is a key microenvironmental factor of angiogenesis, and hypoxia-inducible factors (HIF) are known to stimulate VEGF expression [30, 31]. The upregulation of VEGF in HCC is controlled at tran- scriptional levels as well as by the mRNA stability of VEGF [32]. In addition, the p53 tumour suppressor and HBx genes might regulate VEGF expression in HCC [33, 34]. Fibroblast growth factors (FGFs) are a family of heparin-binding growth factors that includes at least 22 structurally related members, of which acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) are the best-known members. FGFs exert their pro-angiogenic activity by interacting with various endothelial cell surface receptors, including tyrosine kinase receptors FGFR1 and FGFR2, heparan sulphate proteoglycans and integrins [35]. bFGF appears to act synergistically with VEGF in the induction of angiogenesis [36]. Aside from its angiogenic effect, bFGF has also been shown to act as a mitogen for HCC cell proliferation via an autocrine mechanism [37]. Angiopoietins play an important role in angiogenesis. Angiopoietin-1 (Ang-1) is a survival signal for endothelial cells and it promotes recruitment of pericytes and smooth muscle cells to form mature blood vessels. In contrast to Ang-1, angiopoi- etin 2 (Ang-2) induces vascular regression in the absence of VEGF but increases vascular sprouting in its presence. It has been shown that the ectopic expression of Ang-2 in HCC cells promotes rapid development of tumour and aggravates its prognosis [38]. 2 Biology of Hepatocellular Carcinoma 27 Other angiogeneic factors have also been shown to be involved in tumour angiogenesis including platelet-derived endothelial cell growth factor [39], tissue factor [40], cyclooxygenase-2 [41] and angiogenin [42]. Telomere Shortening The progressive shortening of telomeres with each cell division serves in most somatic cells as a “mitotic clock,” indicating cell age and cellular senescence. After a certain number of cell doublings, when a threshold level of telomeric length is reached, a signal is initiated to cease cell division and further progression to S-phase is prevented. Loss of telomeres initiates or drives chromosomal instability, which in turn results in chromosomal abnormalities such as end-to-end fusion and rearrangement. In carcinogenesis, certain cells such as those that have undergone viral transformation, irradiation or mutagenesis will continue to divide, have their telomeres further shortened and eventually die. However, prior to death, the result- ing genomic instability causes a small number of these cells to undergo multiple mutations, including the regaining of telomerase activity which thereafter serves to maintain telomere length and genomic stability indefinitely [43]. Telomere shorten- ing is accelerated in chronic liver disease and critically short t elomeres characterize cirrhosis stage [44]. The cancer risk increases in response to telomere shorten- ing during aging and chronic liver disease. Furthermore, telomerase activity has been detected in human HCC while it is absent in adjacent non-tumour tissues [43]. Studies in telomerase knockout mice have provided experimental evidence that telomere shortening influences stem cell function, aging and carcinogene- sis. These mice exhibit an impaired maintenance and function of adult stem cells and reduced regenerative reserve in response to organ damage. Interestingly they show an increase in chromosomal instability and tumour initiation but impaired tumour progression [45]. Telomerase has been shown to be a critical component for in vivo progression of p53 mutant HCC with short telomeres in the chroni- cally damaged liver. In this molecular context, telomerase limits the accumulation of telomere dysfunction, the evolution of excessive aneuploidy and the activation of p53-independent checkpoints suppressing hepatocarcinogenesis [46]. This dual role of telomeres may point to new treatment options in patients with HCC. HCC in the Non-cirrhotic Liver The incidence of HCC arising in the non-cirrhotic liver varies greatly between different studies, ranging from 10 to 50% [47–50]. These tumours have been char- acterized as often uninodular, encapsulated and expansive growing and they seem to be bigger than normal HCC [50]. All factors that can induce a HCC with cirrho- sis can also lead to a non-cirrhotic HCC. Nevertheless, there are several conditions which are known to be associated predominantly with non-cirrhotic liver cancer: sexual steroids induce HCCs through the development of liver adenomas; patients 28 M.L. Balmer and J F. Dufour with Alagille syndrome (arteriohepatic dysplasia) [51], hypercitrullinemia [51], α1-antitrypsin deficiency [52] and glycogenosis type 1 [53] often develop HCC without cirrhosis. Iron overload seems to be a general risk factor for developing a non-cirrhotic HCC [54], as well as clinically unapparent mutations in the HFE gene [55]. It has been estimated that up to 40% of hepatitis B virus (HBV)-related HCC occur in persons who do not have cirrhosis, while almost all cases of hep- atitis C virus (HCV)-related HCC occur in the setting of cirrhosis [56, 57]. Direct oncogenic potential of HBV through chromosomal integration (cis-activation) or trans-activation of cellular genes seems to be an important feature in the pathogene- sis of these cancers [58]. Nevertheless, the two major risk factors for developing an HCC without underlying liver cirrhosis seem to be the metabolic syndrome (MS) with fatty liver disease and liver adenomas. The association between diabetes, obesity, steatosis, non-alcoholic fatty liver disease (NAFLD) and the develop- ment of HCC is not well elucidated so far. Changes in fat metabolism, including expression of adipocyte-like gene pathways, appear to play a role both in hepatic regeneration and sometimes in neoplastic transformation [59, 60]. This relation- ship to fat metabolism appears to be important both in NAFLD-related cancer and in HCV, where steatosis, steatohepatitis and associated oxidative stress are increasingly recognized as significant risk or cofactors in HCC development [61]. Furthermore steatosis is an independent predictor of postoperative HCC recurrence in HCV-associated HCC [62]. Additional epidemiological data indicate a signifi- cantly increased risk of hepatocellular carcinoma among diabetic patients [63]. The pathophysiological components of this disorder, especially when steatohepatitis is present, include lipid peroxidation, stem cell proliferation, and increased growth factors, such as insulin and TGF. Proliferation of cells in the setting of oxidative stress and increased trophic factors associated with the metabolic syndrome such as hyperinsulinemia seems to be the hallmark of non-cirrhotic HCC in context with the metabolic syndrome [61]. Recently it has been shown that most of the tumours in context with the metabolic syndrome develop in nonfibrotic livers [64]. Wnt signalling pathway deregulation did not represent the main carcinogenic process involved in this context [64]. A significant percentage of HCCs that develop in the context of MS without significant fibrosis arises from malignant transformation of liver adenoma, especially the TA (telangiectatic) subtype. It has been proposed that liver adenomas can transform into malignant liver tumours [53, 65–67]. However, the incidence and underlying molecular mechanisms still remain unclear. Moreover, the published data usually derive from patients with- out liver cirrhosis. This is rather due to the conceptual problem that adenomas are only diagnosed in non-cirrhotic livers and are labelled as macroregenerative nod- ules or adenomatous hyperplasias in patients with underlying liver cirrhosis, than due to a fundamentally different pathogenesis of these tumours. However, the con- ceptual adenoma-carcinoma sequence, as observed in other organs like the gut, seems to exist in a similar manner in HCC as well. Among the three genotypi- cally identified subtypes of liver adenoma HNF1α inactivated (35–50% of cases), β-catenin activated (15–18% of cases) and inflammatory (40–55% of cases), the β-catenin-activated adenomas seem to be at higher risk of HCC [68]. 2 Biology of Hepatocellular Carcinoma 29 Metastasis HCC is characterized by early development of intrahepatic metastasis, whereas distant organs are usually late involved in the disease. In the diagnosis of intra- hepatic HCC metastases, one problem is to separate metastatic dissemination from multifocal tumours in the liver. The possibility that more than one nodule may be detected in patients with HCC has been known for more than 50 years [69]. This is not only semantic but of biological importance. ‘Metastatic dissemination or mul- tifocal tumour?’ is not a Hamletic question, because the two possibilities are not mutually exclusive as cirrhotic liver can generate more than one cancer nodule with the same, still unknown mechanisms, during the history of the disease. However, the identification of these two distinct hypotheses seems to be an underestimated problem by clinicians, although a patient with a multifocal tumour has a better prog- nosis than a patient with a metastatic cancer [70, 71]. Multiple HCC nodules are an expression of metastasis rather than of multifocal cancer in more than 60% of cases [72], but more sensitive tests are needed to distinguish metastatic from multifocal HCC in the liver. The process of metastasis involves an intricate interplay between altered cell adhesion, survival, proteolysis, migration, lymph/angiogenesis, immune escape mechanisms and homing on target organs. Not surprisingly, the molecular mech- anisms that propel invasive growth and metastasis are also found in embryonic development and, however, to a less perpetual/chronic/aggressive/quantitatively dif- ferent extent, in adult tissue maintenance (e.g. involving stem cell differentiation) and repair processes [73]. Several molecular examples and pathways are involved and all act in concert to guide the tumour cell to its new home (Table 2.1). All these players are tightly orchestrated and interact through several molecular pathways: The Wnt/β-catenin pathway links cell–cell adhesion and downstream signalling and mutations in β-catenin genes can be detected in 12–26% of human HCC; p53 mutation is involved in determining dedifferentiation, proliferating activity and tumour pro- gression [74]; is strongly related to the invasiveness of HCC and also influences the postoperative course (particularly recurrence within 1 year) [75]. The mitogen- activated protein kinase pathway (MAPK) and the Raf kinase inhibitor protein Table 2.1 Molecular examples and pathways are involved and all act in concert to guide the tumour cell to its new home Biological capability Molecular examples/pathway entities Survival IGF survival factors Adhesion and deadhesion CAMs, cadherins, integrins Migration Met-SF/HGF signalling, FAK Proteolysis/ECM remodelling MMPs, uPA, ADAMs, heparanase Immune escape Downregulation of intrinsic immunogenicity, MHC loss Lymph/angiogenesis VEGF, PDGF, bFGF Homing on target organs Chemokines/chemokine receptors, CD44, osteopontin 30 M.L. Balmer and J F. Dufour (RKIP) as an inhibitor of this pathway revealed prominent roles during human HCC metastasis [76]. Signalling pathways in liver cancer metastasis are highly com- plex and little is known about the importance of every single cascade in metastasis development. A bioinformatics analysis of metastasis-related proteins in hepatocel- lular carcinoma resulted in a gigantic diversity of involved partners in metastasis development (506 proteins, 83 pathways) [77]. From a more macroscopic point of view, there are four proposed models of metastasis development: According to Chambers and coworkers, only a very small population of injected tumour cells in mice form micrometastases, although most of them are arrested in the liver. Furthermore, not all of the micrometastases persist, and the progressively growing metastases arise only from a small subset (0.02%) of cells [78]. Muschel and coworkers recently proposed a new model for pul- monary metastasis in which endothelium-attached tumour cells that survived the initial apoptotic stimuli proliferate intravascularly. Thus, a principal tenet of this new model is that the extravasation of tumour cells is not a prerequisite for metastatic colony formation and that the initial proliferation t akes place within the blood vessels [79]. The unique ability of aggressive tumour cells to generate patterned networks, similar to the patterned networks during embryonic vasculogenesis, and concomitantly to express vascular markers associated with endothelial cells, their precursors and other vascular cells has been termed ‘vasculogenic mimicry’ by Hendrix and coworkers [80]. It has been shown that tumour cells can migrate as tumour emboli that conserve a tissue architecture reminiscent of the primary tumour. Tumour cells are thus protected from anoikis and direct immunological engagement during dissemination [81]. All of these mechanisms are supposed to have an impact on the pathogenesis of HCC metastasis and should be taken into account when planning new treatment strategies. 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