**4. CD44 as a multifunctional marker of HCC late stages and metastatic disease, also useful for targeted therapy and drug delivery**

The high mortality rate in patients with HCC is mainly due the lack of effective treatment options, especially for those with advanced or unresectable disease. These patients generally have a very poor prognosis and treatments, such as transarterial chemo-embolization, intraarterial or systemic chemotherapy, radiotherapy, immunotherapy or hormonal therapy, are mainly used as palliative. Thus, the development of more effective therapeutic tools and strategies is much needed. The conventional chemotherapy, implying the use of systemic administration or non-targeted distribution of the drug, has numerous drawback such as the limited accessibility of drug to the tumor tissue, that reduces its therapeutic efficacy, the requirement of high doses, and undesirable side effects, primarily the high mielotoxicity and the development of multidrug resistance. To overcome these problems, in the last decades numerous researches focused on developing cancer-specific drugs or systems of antitumor drug delivery (Allen, 2002; Gabizon, 2002; Gabizon et al., 2003; Mohanty et al., 2011; Sapra & Allen, 2003). This therapeutic strategy may allow a controlled release of the drug and a high targeting selectivity on tumor cells, increasing drug cytotoxicity and decreasing its undesirable side effects. In this context, targeted drug delivery involving the use of drugs covalently conjugated to macromolecular carriers, that are able to specifically link to overexpressed molecules on tumor cells, is one the most promising approach in developing innovative therapies against cancer.

CD44, the receptor for hyaluronic acid-mediated cell motility, is a highly glycosylated transmembrane protein involved in cell–cell and cell–matrix interactions. The standard isoform (CD44s), participates to several functions including lymphocyte homing, tissue regeneration, signal transmission involved in cell proliferation, migration and apoptosis (Goodison et al., 1999; Ponta et al., 2003). Besides its involvement in physiological activities of normal cells, CD44 is associated with pathologic functions of tumor cells. Increased expression of CD44 (the standard isoform CD44s and the splice variant CD44v) has been associated to advanced stages not only of hepatocellular carcinoma (Endo & Terada, 2000) but also of breast cancer, colorectal cancer, thyroid carcinoma, lung cancer, renal cell carcinoma, gallbladder carcinoma, ovarian carcinoma, endometrial cancer and melanoma (Akisik et al., 2002; Bendardaf et al., 2006; Jothy , 2003; Naor et al., 2002; Seiter et al., 1996). For this reason, CD44 is emerging as a valuable metastatic tumor marker, also associated with an unfavorable prognosis for a variety of cancers, including HCC (Beckebaum et al., 2008). Therefore, agents specifically targeting CD44 should be promising drug for inhibiting tumor spread and for treatment of metastatic disease. It has been demonstrated that targeting CD44 with specific anti-CD44 monoclonal antibodies is able to inhibit proliferation and to induce terminal differentiation or apoptosis in leukemic cell lines (Charrad et al., 2002; Jin et al., 2006). Furthermore, inhibition of CD44 expression by CD44 antisense oligonucleotide significantly induced apoptosis, decreased tumorigenesis and invasion, and increased chemosensitivity in a CD44 over-expressing human HCC cell line (Xie et al., 2008).

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

The high mortality rate in patients with HCC is mainly due the lack of effective treatment options, especially for those with advanced or unresectable disease. These patients generally have a very poor prognosis and treatments, such as transarterial chemo-embolization, intraarterial or systemic chemotherapy, radiotherapy, immunotherapy or hormonal therapy, are mainly used as palliative. Thus, the development of more effective therapeutic tools and strategies is much needed. The conventional chemotherapy, implying the use of systemic administration or non-targeted distribution of the drug, has numerous drawback such as the limited accessibility of drug to the tumor tissue, that reduces its therapeutic efficacy, the requirement of high doses, and undesirable side effects, primarily the high mielotoxicity and the development of multidrug resistance. To overcome these problems, in the last decades numerous researches focused on developing cancer-specific drugs or systems of antitumor drug delivery (Allen, 2002; Gabizon, 2002; Gabizon et al., 2003; Mohanty et al., 2011; Sapra & Allen, 2003). This therapeutic strategy may allow a controlled release of the drug and a high targeting selectivity on tumor cells, increasing drug cytotoxicity and decreasing its undesirable side effects. In this context, targeted drug delivery involving the use of drugs covalently conjugated to macromolecular carriers, that are able to specifically link to overexpressed molecules on tumor cells, is one the most promising approach in developing

CD44, the receptor for hyaluronic acid-mediated cell motility, is a highly glycosylated transmembrane protein involved in cell–cell and cell–matrix interactions. The standard isoform (CD44s), participates to several functions including lymphocyte homing, tissue regeneration, signal transmission involved in cell proliferation, migration and apoptosis (Goodison et al., 1999; Ponta et al., 2003). Besides its involvement in physiological activities of normal cells, CD44 is associated with pathologic functions of tumor cells. Increased expression of CD44 (the standard isoform CD44s and the splice variant CD44v) has been associated to advanced stages not only of hepatocellular carcinoma (Endo & Terada, 2000) but also of breast cancer, colorectal cancer, thyroid carcinoma, lung cancer, renal cell carcinoma, gallbladder carcinoma, ovarian carcinoma, endometrial cancer and melanoma (Akisik et al., 2002; Bendardaf et al., 2006; Jothy , 2003; Naor et al., 2002; Seiter et al., 1996). For this reason, CD44 is emerging as a valuable metastatic tumor marker, also associated with an unfavorable prognosis for a variety of cancers, including HCC (Beckebaum et al., 2008). Therefore, agents specifically targeting CD44 should be promising drug for inhibiting tumor spread and for treatment of metastatic disease. It has been demonstrated that targeting CD44 with specific anti-CD44 monoclonal antibodies is able to inhibit proliferation and to induce terminal differentiation or apoptosis in leukemic cell lines (Charrad et al., 2002; Jin et al., 2006). Furthermore, inhibition of CD44 expression by CD44 antisense oligonucleotide significantly induced apoptosis, decreased tumorigenesis and invasion, and increased chemosensitivity in a CD44 over-expressing human HCC cell line (Xie et al., 2008).

attractive molecular target to eradicate liver cancer stem cells (Ji et al., 2011)

**disease, also useful for targeted therapy and drug delivery** 

innovative therapies against cancer.

**4. CD44 as a multifunctional marker of HCC late stages and metastatic** 

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

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

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

New Molecular Biomarkers Candidates for the Development of Multiparametric

grateful to Dr. Giuseppe Nicotera for his scientific secretariat support.

this goal.

**6. Acknowledgment** 

**7. References** 

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Platforms for Hepatocellular Carcinoma Diagnosis, Prognosis and Personalised Therapy 91

biomarkers useful for early diagnosis and prognosis, such as the NGF, and for target therapy and drug delivery, such as the CD44, as well as the deepening of knowledge on pathways actively involved in hepatocarcinogenesis, helpful for HCC staging and targetspecific cancer therapy, such as the WNT/β-catenin signaling, are essential steps to achieve

We would like to thank Federica Andreola, Noemi Moroni and Manuela Zonfrillo, for their valuable contribution to the researches carried out in our laboratory on NGF and on CD44 and to the ongoing study on the role of WNT/β-catenin signaling in cancer. We are also

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cytoplasmic vesicles-like and lysosome-like structures, suggesting that processing of the HA-drug molecules might be coupled with a recycling of the CD44 receptor (**Fig. 7B**). We have also demonstrated that, using this drug delivery strategy, the delivered drug exerts a strong and irreversible *in vitro* inhibitory effect on growth of CD44 over-expressing cancer cells, higher than that exerted by free drug.

As mentioned above, the effectiveness of the drug delivery strategy using HA as a carrier targeting its CD44 receptor has been demonstrated in several kinds of CD44 over-expressing tumors, including colorectal, ovarian, bladder, gastric, breast, oesophageal and lung cancers, not only *in vitro* but also *in vivo* (Banzato et al., 2008; Luo & Prestwich, 1999; Luo et al., 2000; Luo et al., 2002; Rosato et al., 2006; Serafino et al., 2011). Due the association of increased expression of CD44 to advanced stages of hepatocellular carcinoma, this therapeutic approach might be also applicable for treatment of metastatic HCC. In addition, the growing evidences concerning the CD44 expression on liver cancer stem cells (Liu et al., 2011) not only improve the prognostic significance of CD44 but also make the drug delivery strategy through CD44/HA binding interaction useful for eradicating hepatic cancer stem cells.
