**3.1 Main signaling pathways implicated in HCC**

During hepatocarcinogenesis, two main pathogenic mechanisms predominate: 1) cirrhosis associated with hepatic regeneration after tissue damage caused by hepatitis infection, toxins such as alcohol or aflatoxin, or metabolic syndromes such as insulin resistance, obesity, type 2 diabetes or dyslipidemia in non-alcoholic steatohepatitis (Bugianesi, 2005); 2) mutations occurring in single or multiple oncogenes or tumor suppressor genes (Thorgeirsson & Grisham, 2002; Villanueva et al., 2007; Wang et al., 2002). These two mechanisms have been related to aberrations in various critical molecular signaling pathways that participate to the carcinogenic process. The most important of these pathways include the growth factor-mediated angiogenic signaling (mainly the VEGF receptor signaling), the epidermal growth factor receptor (EGFR), the insulin growth factor receptor (IGFR), the hepatocyte growth factor receptor HGF/c-MET signaling, and the platelet-derived growth factor receptor (PDGFR) signaling (**Fig. 5**) (Whittaker et al., 2010).

Since liver is a highly vascular organ, HCC growth and invasion is highly dependent on dysregualtion of angiogenensis (Semela & Dufour, 2004), and targeting molecular components of pathway signaling involved in the angiogenetic process are currently the main therapeutic strategy exploited for HCC treatment. Actually, targeted drug selectively hitting the VEGF/VEGFR and PDGFR signaling (Sunitinib, Bevacizumab, Cediranib and Vatalanib) or the EGF/EGFR and IGFR signaling (Lapatinib, Cetuximab, Erlotinib Gefitinib, Everolimus, Sirolimus) (Fig. 5) are under evaluation in phase I-III clinical trials as monotherapy or in combination with other chemotherapeutics (see for review, Whittaker et al., 2010). Sorafenib (Nexavar; Bayer HealthCare Pharmaceuticals Inc., Wayne, NJ, USA), a potent inhibitor of VEGFR and PDGFR, has been approved for treatment of HCC and is the only option of effective systemic treatment currently available for management of the advanced malignancy (Llovet et al., 2008).

In the last years, great attention has been given to some signaling pathways which deregulation or constitutive activation have been demonstrated to have a role in cancer insurgence and progression. These pathways could be of interest for therapeutic perspectives, because targeting them may contribute to prevent tumorigenesis or achieve tumor reversion. Drugs directly acting on components of the signaling pathways implicated in tumorigenesis have exhibited clinical benefit in patients with various tumor types, including colorectal, renal, breast and lung cancers, and more recently, HCC (Whittaker et al., 2010). Thus, deepening of knowledge on the molecular pathways actively involved in HCC insurgence and progression could potentially provide new targets for drug delivery and therapy, allowing to overcome the poor response to the current therapeutic strategies. Moreover, owing the role of these pathways in the carcinogenetic process, crucial molecules of this signaling should be validate as new HCC-related biomarkers for the improvement of

During hepatocarcinogenesis, two main pathogenic mechanisms predominate: 1) cirrhosis associated with hepatic regeneration after tissue damage caused by hepatitis infection, toxins such as alcohol or aflatoxin, or metabolic syndromes such as insulin resistance, obesity, type 2 diabetes or dyslipidemia in non-alcoholic steatohepatitis (Bugianesi, 2005); 2) mutations occurring in single or multiple oncogenes or tumor suppressor genes (Thorgeirsson & Grisham, 2002; Villanueva et al., 2007; Wang et al., 2002). These two mechanisms have been related to aberrations in various critical molecular signaling pathways that participate to the carcinogenic process. The most important of these pathways include the growth factor-mediated angiogenic signaling (mainly the VEGF receptor signaling), the epidermal growth factor receptor (EGFR), the insulin growth factor receptor (IGFR), the hepatocyte growth factor receptor HGF/c-MET signaling, and the platelet-derived growth factor receptor (PDGFR) signaling (**Fig.** 

Since liver is a highly vascular organ, HCC growth and invasion is highly dependent on dysregualtion of angiogenensis (Semela & Dufour, 2004), and targeting molecular components of pathway signaling involved in the angiogenetic process are currently the main therapeutic strategy exploited for HCC treatment. Actually, targeted drug selectively hitting the VEGF/VEGFR and PDGFR signaling (Sunitinib, Bevacizumab, Cediranib and Vatalanib) or the EGF/EGFR and IGFR signaling (Lapatinib, Cetuximab, Erlotinib Gefitinib, Everolimus, Sirolimus) (Fig. 5) are under evaluation in phase I-III clinical trials as monotherapy or in combination with other chemotherapeutics (see for review, Whittaker et al., 2010). Sorafenib (Nexavar; Bayer HealthCare Pharmaceuticals Inc., Wayne, NJ, USA), a potent inhibitor of VEGFR and PDGFR, has been approved for treatment of HCC and is the only option of effective systemic treatment currently available for management of the

**3. Components of signaling pathways involved in HCC insurgence and progression as innovative biomarkers for diagnosis, prognosis and drug** 

**targeting** 

the current diagnostic/prognostic tools.

**5**) (Whittaker et al., 2010).

advanced malignancy (Llovet et al., 2008).

**3.1 Main signaling pathways implicated in HCC** 

Fig. 5. Cellular signaling pathways implicated in the pathogenesis of HCC and therapeutics targeting molecular components of these pathway, useful for HCC treatment. EGF, epidermal growth factor; EGFR, EGF receptor; IGFR, insulin-like growth factor receptor; PDGFR, platelet-derived growth factor receptor; WNT, family of secreted glycoproteins that act as ligands of the Frizzled receptor; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor. Details and main function/s of AKT, BAD, c-JUN, c-MYC, DSH, ERK 1/2, FOXO, GSK-3β, GBP, MEK 1/2, mTOR, PI3K, PTEN, p53, RAF, RAS, β-catenin, are reported in **Table 3**. Adapted from Whittaker et al., 2010

Besides the mentioned pathways, directly or indirectly involved in the angiogenic signaling, in the last years numerous studies demonstrated that the WNT/β-catenin pathway is actively involved in initiation and progression of several kinds of human cancers, including HCC (De La et al., 1998; Polakis, 1999; Waltzer & Bienz, 1999) and growing attention has been given to new anti-tumor therapeutic approaches targeting components of this signaling pathway (Gonsalves et al., 2011; Luu et al., 2004; Moon et al., 2004).

New Molecular Biomarkers Candidates for the Development of Multiparametric

**biomarkers and therapeutic targets** 

functions of the adult liver (Sekine et al., 2006).

modulating cell proliferation and invasion.

Platforms for Hepatocellular Carcinoma Diagnosis, Prognosis and Personalised Therapy 85

Wnts are secreted glycoproteins that act as ligands to stimulate receptor-mediated signal transduction pathways in both vertebrates and invertebrates. Activation of Wnt pathways can modulate cell proliferation, survival, cell behavior, and cell fate in both embryos and adults. Wnt signaling pathway, and its signaling cascade is one of the core signal transduction pathway driving tissue morphogenesis during both development and progression of human cancers (see for reviews on Wnt: Moon et al., 2004; Nelson et al., 2004). Wnt signaling also plays a critical role in regulating liver cell proliferation during development (Monga et al., 2003; Suksaweang et al., 2004) and in controlling crucial


Many of the molecular component of the WNT/-catenin signaling have been reported to be modified in HCC, and are proposed as HCC diagnostic/prognostic markers or as

Mutations of Axin or stabilizing mutations of -catenin genes, leading to constitutive activation of the Wnt/-catenin pathway, have been recovered in various cancers, including

Conversely, inactivating mutations of the APC gene, frequently implicated in other tumor and particularly in colorectal cancer, have not been described in HCC. However, loss of APC function activating the WNT/β-catenin signaling seems to be implicated in liver carcinogenesis (Colnot et al., 2004). Moreover, aberrant reactivation of Wnt signaling due to accumulation of β-catenin is evident in many different tumors of the liver (Colnot et al., 2004). Frequent overexpression of the Wnt receptor Frizzled-7 has been detected in HCC and mainly in hepatitis B virus–related HCCs, and this overexpression seems to be an early event in hepatocarcinogenesis (Merle et al., 2004). It has been recently reported that serum βcatenin levels were significantly elevated in patients with HCC compared to those with chronic hepatitis or healthy controls, and it has been proposed as a potential marker for early diagnosis of HCC in HCV infected patients (Zekri et al., 2011). Moreover, in human

therapeutic target for treatment of the primary or metastatic malignancy (**Table 4**).

hepatoblastoma and HCC (Buendia, 2000; De La et al., 1998; Whittaker et al., 2010).

**3.2 Molecular component of the WNT/β-catenin signaling as innovative diagnostic** 


Table 3. List of the main molecular component of cellular signaling pathways implicated in the pathogenesis of HCC.

regulating cell survival

regulating apoptosis

AKT EGF/EGFR, IGFR serine/threonine protein kinase involved in

BAD EGF/EGFR, IGFR BCL-2-associated death promoter, involved in

apoptosis.

WNT downstream effector of WNT sinaling

GSK-3β WNT glycogen synthase kinase-3β, component of β-

PTEN EGF/EGFR , IGFR phosphatase and tensin homolog that regulates

RAS prototypical member of the RAS superfamily of

signaling Table 3. List of the main molecular component of cellular signaling pathways implicated in

β-catenin WNT integral component of the WNT/β-catenin

Main role and function

in combination with c-FOS, forms the activator protein-1 (AP-1) early-response transcription factor; involved in cell proliferation and

Encodes for a transcription factor that regulates the expression of many genes involved in cell proliferation; overexpression of c-MYC is

genes involved in cell survival and proliferation

kinases that phosphorylate mitogen-activated

serine/threonine protein kinase that regulates cell growth, proliferation, motility, and survival

tumor suppressor protein, regulates the cell cycle

MAP kinase kinase kinase (MAP3K); functions in the MAPK/ERK signal transduction pathway

proteins; RAS signaling causes cell growth,

associated with carcinogenesis.

EGF/EGFR , IGFR transcription factor regulating the expression of

catenin destruction complex

protein (MAP) kinase (MAPK)

cell-survival pathway

differentiation and survival

extracellular signal-regulated kinases

Main Cellular signaling

Molecular components

DSH

(Dishevelled)

FOXO (Forkhead box subclass O)

c-JUN VEGF/VEGFR,

c-MYC EGF/EGFR , IGFR, WNT

ERK 1/2 VEGF/VEGFR,

MEK 1/2 VEGF/VEGFR,

p53 VEGF/VEGFR,

RAF VEGF/VEGFR,

the pathogenesis of HCC.

PDGFR

PDGFR

PDGFR

PDGFR,

PDGFR

EGF/EGFR, IGFR

GBP WNT GSK3-binding protein

mTOR EGF/EGFR , IGFR mammalian target of rapamycin, a

PI3K EGF/EGFR , IGFR phosphatidylinositol-3-kinase

#### **3.2 Molecular component of the WNT/β-catenin signaling as innovative diagnostic biomarkers and therapeutic targets**

Wnts are secreted glycoproteins that act as ligands to stimulate receptor-mediated signal transduction pathways in both vertebrates and invertebrates. Activation of Wnt pathways can modulate cell proliferation, survival, cell behavior, and cell fate in both embryos and adults. Wnt signaling pathway, and its signaling cascade is one of the core signal transduction pathway driving tissue morphogenesis during both development and progression of human cancers (see for reviews on Wnt: Moon et al., 2004; Nelson et al., 2004). Wnt signaling also plays a critical role in regulating liver cell proliferation during development (Monga et al., 2003; Suksaweang et al., 2004) and in controlling crucial functions of the adult liver (Sekine et al., 2006).


Many of the molecular component of the WNT/-catenin signaling have been reported to be modified in HCC, and are proposed as HCC diagnostic/prognostic markers or as therapeutic target for treatment of the primary or metastatic malignancy (**Table 4**).

Mutations of Axin or stabilizing mutations of -catenin genes, leading to constitutive activation of the Wnt/-catenin pathway, have been recovered in various cancers, including hepatoblastoma and HCC (Buendia, 2000; De La et al., 1998; Whittaker et al., 2010).

Conversely, inactivating mutations of the APC gene, frequently implicated in other tumor and particularly in colorectal cancer, have not been described in HCC. However, loss of APC function activating the WNT/β-catenin signaling seems to be implicated in liver carcinogenesis (Colnot et al., 2004). Moreover, aberrant reactivation of Wnt signaling due to accumulation of β-catenin is evident in many different tumors of the liver (Colnot et al., 2004). Frequent overexpression of the Wnt receptor Frizzled-7 has been detected in HCC and mainly in hepatitis B virus–related HCCs, and this overexpression seems to be an early event in hepatocarcinogenesis (Merle et al., 2004). It has been recently reported that serum βcatenin levels were significantly elevated in patients with HCC compared to those with chronic hepatitis or healthy controls, and it has been proposed as a potential marker for early diagnosis of HCC in HCV infected patients (Zekri et al., 2011). Moreover, in human

New Molecular Biomarkers Candidates for the Development of Multiparametric

currently explored include:

**Molecular component of the WNT signaling**

diagnosis/therapy

Platforms for Hepatocellular Carcinoma Diagnosis, Prognosis and Personalised Therapy 87

specifically inhibit Wnt signaling cascade are not currently available. However, owing the crucial role in cancer ascribed to this pathway, in the last years the researches on the molecular mechanisms driving this signaling are in increasing and conspicuous funds are invested by several pharmaceutical and biotech companies for the development of innovative drugs targeting its molecular components. The main therapeutic strategies

1. The use of small-molecules able to regulate the catenin responsive transcription (Chen et al., 2009a; Lepourcelet et al., 2004; Thorne et al., 2010; Vo & Goodman, 2001). 2. Compounds that inhibit Wnt signaling by influencing the stability and expression levels

3. Molecules that inhibit Wnt signaling by acting on events upstream of the axin/APC/GSK-3β complex, such as the secretion or reception of Wnt ligands at the plasma membrane (Chen et al., 2009a; Chen et al., 2009b) or transduction of the Wnt

> pre-cancerous lesion and increased in tumor; increased mRNA expression compared to

translocation in the early stages; increased protein expression in

compared to those with chronic hepatitis (CH) and healthy

evidenced; loss of function

compared to normal liver, already at early stages

differentiated tumors compared to poorly differentiated cancers; Expression levels inversely correlated with histological grade and prognosis

implicated in liver carcinogenesis;

normal liver; nuclear

the late stage

controls

Table 4. List of the main molecular component of the WNT signaling useful for HCC

**Trends found in HCC Main possible use/s**

Indicative of WNT signaling activation (early diagnosis); diagnostic marker of

Early diagnosis of HCV-associated

Early diagnosis; therapeutic target

Early diagnosis; therapeutic target

Predictive of early recurrence after hepatic resection and metastatization of HCC cells; marker

of tumor differentiation

metastasis ; therapeutic target

HCC

of β-catenin (Chen et al., 2009a; Huang et al., 2009; Thorne et al., 2010)

signal by Dishevelled (Dvl) (Chen et al., 2009b, Shan et al., 2005)

β-catenin Tissue Gene mutation recovered in

β-catenin Serum Elevated in patients with HCC

APC Tissue Gene mutation not frequently

E-cadherin Tissue Expression increased in well-

Frizzled receptor Tissue Overexpressed in HCC

**Biological material analyzed**

HCC tissues, higher levels of β-catenin expression was found in the tumor area compared to the non-tumor area and the level of expression and nuclear translocation of β-catenin was increased in HCC late-stage. Thus, β-catenin have been proposed as a suitable diagnostic marker of metastasis in human HCC (Lai et al., 2011).

Fig. 6. Schematic representation of the Wnt/β-catenin signaling activation. APC, adenomatous polyposis coli; β-cat, β-catenin; CBP, CREB-binding protein; CK, casein kinase; DKK, Dickkopf; DSH, Dishevelled; GBP, GSK3-binding protein; GSK-3β, glycogen synthase kinase 3β; LRP, LDL receptor-related protein; P, phosphorylation; sFRP, secreted Frizzledrelated protein; TCF, T-cell factor.

Finally, due to the tight interaction of β-catenin with E-cad at the cell-cell junction, activation of WNT signaling has also been related to dysregulation of cadherin expression, which is often associated with dysplasia, tumor formation, and metastasis. This causal relationship between E-cad and Wnt signaling makes E-cad an additional molecular marker that should be taken into account in the setting up a multiparametric diagnostic/prognostic platform for HCC. The E-cad expression levels have been reported to inversely correlate with histological grade and prognosis, and might be a prognostic marker of early recurrence of HCC after hepatic resection (Huang et al. 1999; Matsumura et al. 2001). Since E-cad expression is higher in well-differentiated tumors compared to poorly differentiated cancers, that exhibit lost of the intercellular junction integrity and development of metastasis (Shiozaki et al., 1996; Wijnhoven et al., 2000), it may also be predictive of invasion and metastatization of HCC cells.

While several drugs targeting the VEGF/VEGFR, PDGFR , EGF/EGFR and IGFR signaling have been approved or are in late-stage clinical trials (**Fig. 5**), clinically useful agents that

HCC tissues, higher levels of β-catenin expression was found in the tumor area compared to the non-tumor area and the level of expression and nuclear translocation of β-catenin was increased in HCC late-stage. Thus, β-catenin have been proposed as a suitable diagnostic

Fig. 6. Schematic representation of the Wnt/β-catenin signaling activation. APC,

related protein; TCF, T-cell factor.

HCC cells.

adenomatous polyposis coli; β-cat, β-catenin; CBP, CREB-binding protein; CK, casein kinase; DKK, Dickkopf; DSH, Dishevelled; GBP, GSK3-binding protein; GSK-3β, glycogen synthase kinase 3β; LRP, LDL receptor-related protein; P, phosphorylation; sFRP, secreted Frizzled-

Finally, due to the tight interaction of β-catenin with E-cad at the cell-cell junction, activation of WNT signaling has also been related to dysregulation of cadherin expression, which is often associated with dysplasia, tumor formation, and metastasis. This causal relationship between E-cad and Wnt signaling makes E-cad an additional molecular marker that should be taken into account in the setting up a multiparametric diagnostic/prognostic platform for HCC. The E-cad expression levels have been reported to inversely correlate with histological grade and prognosis, and might be a prognostic marker of early recurrence of HCC after hepatic resection (Huang et al. 1999; Matsumura et al. 2001). Since E-cad expression is higher in well-differentiated tumors compared to poorly differentiated cancers, that exhibit lost of the intercellular junction integrity and development of metastasis (Shiozaki et al., 1996; Wijnhoven et al., 2000), it may also be predictive of invasion and metastatization of

While several drugs targeting the VEGF/VEGFR, PDGFR , EGF/EGFR and IGFR signaling have been approved or are in late-stage clinical trials (**Fig. 5**), clinically useful agents that

marker of metastasis in human HCC (Lai et al., 2011).

specifically inhibit Wnt signaling cascade are not currently available. However, owing the crucial role in cancer ascribed to this pathway, in the last years the researches on the molecular mechanisms driving this signaling are in increasing and conspicuous funds are invested by several pharmaceutical and biotech companies for the development of innovative drugs targeting its molecular components. The main therapeutic strategies currently explored include:



Table 4. List of the main molecular component of the WNT signaling useful for HCC diagnosis/therapy

New Molecular Biomarkers Candidates for the Development of Multiparametric

Rosato et al., 2006; Serafino et al., 2011).

internalization by cancer cells.

Platforms for Hepatocellular Carcinoma Diagnosis, Prognosis and Personalised Therapy 89

The described overexpression of CD44 in advanced stages of several kinds of cancer including HCC, makes hyaluronic acid (HA), the well-known component of the extracellular matrix to which CD44 binds for driving the cell motility, an excellent macromolecular carrier for anticancer drug delivery. HA is a natural and biodegradable polysaccharide formed by D-glucuronic acid and *N*-acetyl-D-glucosamine repetitive units (**Fig. 7A**), used for the development of pharmaceutical carriers and biomedical systems. HA plays crucial roles in cell adhesion, growth, and migration, by interacting with specific cellular receptors (CD44, RHAMM, ICAM), and acts as a signaling molecule in cell motility, inflammation, wound healing, and cancer metastasis (Marhaba & Zoller, 2004; Nedvetzkiet al., 2004; Toole, 2004; Weigel et al., 2003). In this context, HA-drug bioconjugates inherently show a marked selectivity for cancer cells, also providing advantages in drug solubilization, stabilization, localization, and controlled release. Bioconjugates of hyaluronic acid with different antineoplastic drugs, such as paclitaxel, doxorubicin and SN-38 (the active metabolite of Irinotecan) have been reported to posses promising anti-tumor effects both *in vitro* and *in vivo* (Banzato et al., 2008; Luo & Prestwich, 1999; Luo et al., 2000; Luo et al., 2002;

Fig. 7. **A**: Molecular structure of hyaluronic acid. **B**: mechanism of HA-drug biocojugate

In our recent paper (Serafino et al., 2011) we showed that the HA-drug bioconjugates, after interaction of the HA backbone with CD44, enter the tumor cells through a receptormediated endocytosis, followed by release of the active drug in the cytosol, where it directly reach its site of action. The internalized bioconjugate/CD44 complex has been recovered on

Moreover, it has been recently reported that microRNA-181s (miR-181s) are transcriptionally activated by the Wnt/β-catenin signaling in HCC and these miRs have been proposed as attractive molecular target to eradicate liver cancer stem cells (Ji et al., 2011)
