Targeting Cancer Stem Cells and Peritumoral Stromal Response in Liver Cancer with Nanoparticles and Hyaluronidase

THE UAEU INTERDISCIPLINARY CENTER-BASED RESEARCH GRANT
COMPETITION
2014
Proposal Submitted for: CO2 Restoration Agency
Principal Investigator: Dr. X
Signature:
College: Science
Department: Chemistry
Telephone: xxxxxxxxxxx
E-mail address: xxxxxxxxxxxxxx
Names and Signatures of the College Administration
Dean/Assistant Dean for Research: Professor Peter Werner
Proposal Title:
“Liver cancer”
Key words: Cancer, Drug, Diagnosis,
Co-Investigators:
Name Department/Institution Contribution
Dr. X Molecular Genetics 95%
Department / UAEU
Prof. Y Biology / UAE University 2%
Prof. Z Anthropology Department /
UAE University 3%
Total Budget (AED)

ABSTRACT:
Pancreatic and hepatocellular carcinoma are two of the worst malignancies with
expectation of life measures as a few months from diagnosis. Since current therapies have made
little impact on survival, alternative strategies are desperately needed. Two major problems with
current cancer therapy have been identified. Firstly, chemotherapeutic agents do not eradicate the
cancer stem cells, which are the only cells able to recapitulate the tumor and are also responsible
for forming distant metastases. Secondly, cancer cells recruit peritumoral stromal cells which
induce interactions that benefit the cancer and reduce penetration of therapeutic drugs.
The current proposal will address these problems by targeting the cancer stem cells and
the stroma. Chitosan nanoparticles carrying a payload of metformin and the smoothened inhibitor
vismodegic, both of which have been shown to eradicate cancer stem cells. These nanoparticles
will target the cancer stem cells by incorporation of monoclonal antibodies to the cancer stem cell
marker EpCAM and will also carry rhodamine 123 dye for tracking the nanoparticles after injection.
In addition, pegylated hyaluronidase will be employed to target and disrupt the peritumoral storma
and prevent its reformation.
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INTRODUCTION:
Pancreatic ductal adenocarcinoma (PDAC) and hepatocellular carcinoma (HCC) are the
most deadly forms of cancer with the shortest expectations of life after diagnosis [1]. PDAC is the
fourth leading cause of death in the western world and incidence of this cancer is increasing,
probably due to the epidemic of obesity and diabetes which are predisposing factors [1]. HCC has
the fifth highest incidence and third highest mortality rate of all malignancies worldwide [2]. Most
cases of both cancers are inoperable and the only treatment that can be offered is chemotherapy
or palliative procedures [1-3]. Targeted therapy has been of limited success. The epidermal growth
factor (EGF) receptor kinase inhibitor, erlotininib in combination with gemcitabine modestly
improves life expectancy in a sub-group of patients [4]. At the present time, only one drug,
sorafenib, is FDA approved drug for treating advanced HCC. It blocks different receptors,
particularly VEGFR and c-KIT [5].
While late diagnosis undoubtedly contributes to the lack of response of these cancers to
therapy, it has become apparent that the presence of cancer stem cells (CSC), which is not easily
eradicated by standard chemotherapeutics are ultimately responsible for the failure of existing
strategies [6-8]. Recent understanding of the heterogeneous makeup of the cancer cells in a tumor
has revealed the presence of CSCs [9-10]. CSCs can be characterized their self-renewing ability
via asymmetric division, ability to differentiate into diverse phenotypes, ability to initiate tumors
from minute numbers, and chemoresistance [9-10]. As expected, the discovery of CSCs in cancer
development has reshaped our understanding of cancer biology and is revolutionizing the current
efforts of developing new therapeutics. Recent studies have demonstrated the unique properties of
CSCs in many tumor types, including breast, colon, melanoma, brain, bone, ovary, prostate as well
as in PDAC [6,7], and HCC [8] By FACS analysis, Li et al isolated CD44+/CD24+/EpCAM+
(CD326) pancreatic CSCs which counted for 0.2–0.8% of total cancer cells, showed stem-cell like
properties and had the capability of form tumors in animal models when as few as 100 cells [6].
Hermann et al characterized another subpopulation of pancreatic CSCs expressing the surface
marker CD133 and described it to be exclusively tumorigenic and highly resistant to standard
chemotherapy [Hermann et al., 2007]. Similarly, hepatic CSCs have been shown to be enriched
via different cell surface markers, eg, CD13, CD24, CD44, CD90, CD133, and EpCAM (CD326).
Functional assays such as screening cells with a high activity of aldehyde dehydrogenase could
also be used to identify CSCs [8]. Being a viable cause of metastasis along with their high
resistance to conventional chemotherapy has led to the growing belief that CSCs can be the
ultimate foe in the combat against cancer. It is, therefore, becoming the common wisdom that
therapies that would specifically eradicate those CSCs are being actively investigated. CSCs are
known to contribute to tumor initiation, self-renewal, chemoresistance, and metastasis [10].
Indeed, they are the only cells from a tumor that are able to recapitulate the disease, indicating
that their eradication is essential [10].
Epithelial cell adhesion molecule (EpCAM) proves to be a very useful marker and target for
CSCs, EpCAM is overexpressed in epithelial cell cancer stem cells, particularly in PDAC and HCC.
Targeting EpCAM+ HCC cells has been shown to be possible using the antibodies, which inhibit
the growth of pancreatic carcinomas.
Metformin is an AMP kinase inhibitor which has become the mainstay of therapy for type 2
diabetes, belongs to a class of compounds called biguanine that was first isolated from a medicinal
plant known as Galega officinalis. The anticancer effects of metformin were first documented by
one of the investigators on the present project, when this drug completely prevented the formation
of pancreatic tumors in a hamster model of PDAC [24]. A recent epidemiological study revealed
that diabetics taking metformin had a 46% and 78% decreased the risk of PDAC and HCC,
respectively [Zhang et al., 2013]. Two theories have been proposed to explain metastasis:
epithelial–mesenchymal transition (EMT) and CSC. Regardless which mechanism prevails, reports
showing metformin-mediated transcriptional repression of EMT, a cellular phenotype associated
with CSC, support the potential use of metformin in preventing metastasis [15]. Metformin has also
been shown to inhibit colony and sphere formation in cancer stem cells from breast, ovary,
prostate lung as well as pancreas and liver carcinomas [14-19, 26-27]. These findings have
already been translated into clinical trials and there are currently at least 26 ongoing clinical trials
with metformin in various cancers [15].
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Other approaches to the eradication of cancer stem cells include targeting the hedgehog
(Hh), Wnt-β-catenin, and notch pathways [12, 28-30].Reactivation of these developmental
pathways is common in both PDAC and HCC [12, 28-30]. Recent studies have shown that both
temporal and spatial control of Hh and Wnt activity is involved in specifying the lineage that can
progress to cancer [31]. Activation of the Hh in PDAC and HCC has also been shown to regulate
HCC invasiveness and the response to chemotherapy [28, 30-31]. Suppression of the Hh pathway
by its inhibitor, vismodegib (GDC-0499), a smoothened (SMO) antagonist, not only decreased the
number of progenitors but also caused regression of these tumors [32-33]. This agent recently
received FDA approval having shown remarkable activity in clinical trials [30]. Vismodegib is
currently undergoing clinical trials for the treatment of a variety of cancers including PDAC and
HCC [34].
Another major hurdle in the treatment of solid malignancies; in particular PDAC and to a
lesser extent in HCC is the stromal response to the tumor [35]. One of the hallmarks of PDAC is
the presence of extensive desmoplasia consisting of stellate cells, fibroblasts, immune cells
vasculature and extensive extracellular matrix [35]. The cancer cells exploit this microenvironment
for support of growth, invasion and metastatic spread [35]. More importantly from the therapeutic
point of view are that the stoma provides an effective barrier to prevent therapeutic agents
penetrating the cancer cells [35-37]. Studies have shown that the stroma impairs vasculature and
perfusion of the tumors in a mouse model of PDAC and that inhibition of hedgehog signaling that
depleted the tumor stromal response markedly improved the tumor response to gemcitabine [37].
This was accompanied by a marked increase in intratumoral gemcitabine concentrations,
indicating that the stroma was contributing to chemoresistance [37].
Several approaches have been used to disrupt the peri-tumoral stroma in order to enhance
the effect of conventional therapeutics. These include inhibitors of the hedgehog pathway, the
TGFB pathway, targeting of specific matrix metalloproteinases, blocking angiogenesis, targeting
the notch pathway, or enzymatically destroying the hyaluronan component of the extracellular
matrix [38-41]. Of these approaches, the use of hyaluronidase appears to be the most effective
method of destroying the extracellular matrix and enhancing the effects of conventional
chemotherapy [39]. Since hyaluronidase has a short half-life in the circulation, a pegylated human
recombinant form of the enzyme, PEGPH20 has been developed [40-41]. Administration of
PEGPH20 rapidly normalizes the elevated interstitial fluid pressure surrounding tumors and
markedly enhances the therapeutic effects of the standard chemotherapeutic agent, gemcitabine
in a pancreatic cancer model [40]. In a separate study, PEGPH20 was shown to rapidly and
sustainably deplete HA in the extratumoral matirix, induce the expansion of blood vessels and
Increase the intratumoral concentrations of gemcitabine [41]. The combination of PEGPH20 with
gemcitabine inhibited cancer growth and markedly prolonged survival in the animals [41]. Phase 2
clinical trials with PEGPH20 and gemcitabine are currently underway in pancreatic cancer [42].
It is clear from the failure of conventional chemotherapy to control PDAC and HCC, that
novel approaches in the therapy and involvement of agents targeting several different aspects of
the disease, particularly, the cancer stem cells and the peritumoral stroma are needed if we are
going to make any impact on these devastating diseases. Here we plan to address this problem by
targeting the CSCs using a novel nano-technological approach as well as targeting the peritumoral
stroma.
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