Pancreatic cancer: a review on biology, genetics and therapeutics

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PANCREATIC CANCER: A REVIEW ON BIOLOGY, GENETICS AND THERAPEUTICS

By
Yasaman Aletomeh

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

December 2014
II

Table of Contents
List of Tables IV
List of Figures V
Abstract VI
Chapter 1: The Pancreas 1
1.1 Gross anatomy of the pancreas 2
1.2 Blood supply of the pancreas 3
1.3 Pancreatic ducts 4
1.4 Endocrine and exocrine parts of the pancreas 5
1.5 Developmental biology of the pancreas 7
Chapter 2: Pancreatic cancer 11
2.1 Hallmarks of cancer 11
2.2 Pancreatic Cancer 14
2.3 Endocrine and Exocrine tumors 15
Chapter 3: Biology of Pancreatic Cancer 19
3.1 Stromal Biology of Pancreatic Cancer 19
Chapter 4: Genetics and pathology of pancreatic cancer 24
4.1 Pancreatic cancer precursor lesions 24
4.2 Molecular genetics of pancreatic cancer 28
4.2.1 Genetic mutations in pancreatic cancer 29
4.2.2 Signaling Pathways in Pancreatic Cancer 37
4.2.3 Genetic Mutations in Pancreatic Precursor Lesions 44
Chapter 5: Genetically engineered mouse (GEM) models of PDAC: Literature Review 46
III

Chapter 6: An Overview on Current drugs and trials 50
6.1 Current Drugs 50
6.2 Treatment Strategies and Clinical Trials 52
References 59
IV

List of Tables

Table 1 The role of pancreatic cells and transcription factors involved in differentiation 10
Table 2 Endocrine Tumors 17
Table 3 Exocrine Tumors 18
Table 4 Exocrine Tumors Cont’d 18
V

List of Figures

Figure 1 Location of the pancreas 2
Figure2 Gross anatomy and blood supply of the pancreas 3
Figure3 Pancreatic ducts 4
Figure4 Endocrine and Exocrine pancreas 6
Figure5 Transcription factors involved in pancreas development 9
Figure6 Normal pancreatic duct, PanIN1-A ,PanIN1-B ,PanIN-2, p53 26
immunohistochemistry for PanIN-3

Figure7 K-Ras pathway in pancreatic cancer 30
Figure8 Pancreatic Cancer Signaling Pathways 43
VI

Abstract
Pancreatic cancer is the fourth leading cause of cancer death in the United States and
remains a major unsolved health problem in the 21st century. Since it lacks specific symptoms
in the beginning of its development and diagnostic methods are limited, the disease is often
detected during its formative stages. As a result, the disease prognosis is extremely poor.
Consequently not many patients have resectable disease and survival rate is low. Even though
combination therapies may have short-term effects, conventional treatments such as surgery,
radiation, chemotherapy, or combinations of these, has little long-term impact on pancreatic
cancer due to its intense resistance to all extant treatments. Almost all patients who have
pancreatic cancer develop metastases and lose their battle to cancer. Researchers have been
studying the challenges in the field addressing many hurdles and opportunities. They have
realized that developing a detailed understanding of the molecular biology and genetics of
pancreatic cancer, generating refined animal models and investigating signaling pathways
involved in cellular growth, survival and differentiation during the disease course will be
effective in analysis of this complex disease. Such strategies seem to hold a great promise in
diagnosis prevention and treatment of this disease.

1

Chapter 1: The Pancreas
The word pancreas comes from two Greek words pan (all) and kreas (flesh).This organ
was first recognized by Herophilus (335-280 BC), a Greek anatomist and surgeon and a few
hundred years later, Ruphos, another Greek anatomist, gave the pancreas its name. The
pancreas is 12-15–cm in length and 75-100 grams in weight in human adults.it is a J-shaped
glandular, lobulated, soft, retroperitoneal organ which is part of both endocrine and digestive
system in all vertebrates and therefore has dual function.
83
As shown in the following figure
the pancreas is located partially behind the stomach on the left side of the body, 3-6 inches
above the “belly button” on the back wall of the abdominal cavity. The other part of the
pancreas is located in the C-loop of the duodenum and is connected to the first section of the
small intestine.

Figure1. Location of the pancreas

2

There are two ways by which the pancreas is divided into parts: by parts of the overall shape
and by the function of its cells. In the context of overall shape knowledge of the structure of the
pancreas from gross anatomy down to the level of molecular aspect s is essential to understand
how the human body functions and how the structure and function is modified by disease.
Therefore this section is dedicated to a brief study of the pancreatic parts, vasculature, ducts
and cell types
1.1 Gross anatomy of the pancreas
Grossly the pancreas is divided to 5 parts which are uncinated process, head, neck, body
and tail. portal vein, mesenteric vessels, aorta, and vena cava which are the most important
vessels of the abdomen pass through or lie next to the parts of the pancreas .
16
Uncinate
process is located beneath the body of the pancreas. Two of the very important blood vessels,
the superior mesenteric artery and vein cross this section. It lies anterior to the aorta and
inferior to vena cava and is covered superiorly by the superior mesenteric vessels that emerge
below the neck of the pancreas. Neck of the pancreas is the thin section between the head and
the body of the gland which is related to the origin of the portal vein. Pancreatic branches of
splenic artery supply the neck which drains into splenic vein. Head of the pancreas is the widest
section of the organ which is nestled in the curve of the duodenum. The superior pancreatic-
duodenal artery from gastro-duodenal artery and the inferior pancreatic-duodenal artery from
superior mesenteric artery run between the pancreas and the duodenum and supply the head
of pancreas. The head drains into the superior mesenteric and portal veins. Body of the
pancreas is located in the middle part of gland between the neck and the tail and lies behind
the stomach. Pancreatic branches of splenic artery supplies this part. The body of the pancreas
3

drains into splenic vein. The tail of the organ is the thin tip of gland which is in close proximity
with the spleen. Pancreatic branches of splenic artery supply the tail.
1.2 Blood Supply of Pancreas
The location of the pancreas deep within the abdomen places it close to numerous large
blood vessels that are necessary for life. Therefore involvement of arteries and veins is
important in the treatment of pancreatic cancer. These cancers do not need to grow large in
order to invade. There are two main arteries close to the pancreas which are called the celiac
artery and the superior mesenteric artery. The celiac artery gives rise to the splenic artery and
the hepatic artery and supplies blood to the liver, pancreas, spleen and stomach. The superior
mesenteric artery gives rise to numerous branches that supply the small bowel, part of the
colon and the pancreas. The venous system in this area is the portal vein and its tributaries. It
drains blood from the gastrointestinal track back to the liver and to the heart through the
hepatic veins. Figure below shows pancreatic blood supply.

Figure2. Gross anatomy and blood supply of the pancreas

4

1.3 Pancreatic Ducts
There are several ducts associated with the pancreas. The most important of them is the
major pancreatic duct of Wirsung which runs the length of the pancreas and is about one-
sixteenth of an inch in diameter and has many small side branches. The main pancreatic duct is
the functional duct of the pancreas that carries pancreatic juices provided by the "exocrine
pancreas", a part of the gland which will further be discussed later. This duct joins the common
bile duct and together they form ampulla of Vater. This ampulla empties into the duodenum at
the major duodenal papilla. Most people only have the major pancreatic duct. However, some
have an additional accessory pancreatic duct, called the Duct of Santorini above the major
papilla, which is directly connected to the duodenum at the minor duodenal papilla. Figure
below illustrates these ducts.

Figure3. Pancreatic ducts

5

1.4 Endocrine and Exocrine Pancreas

Endocrine Pancreas
The pancreas is a dual function organ with both endocrine and exocrine features.
Approximately two percent of the mass of the whole organ is an endocrine gland. It comprises
of millions of cell clusters called islets of Langerhans which are intertwined by a network of
capillaries.
95
The islets are connected to blood vessels and made up of four main cell types, each
of which responsible for secretion of specific hormones directly to the blood stream and the
capillaries around them. The secreted hormones can be further transported to their site of
action from the systemic circulation. One group of the islet cells is called α cells. These cells
secrete glucagon and as a result increase blood glucose levels. β cells of the islets secrete
insulin to decrease levels of glucose in blood. They also produce amylin to slow down gastric
emptying and promote satiety which further results in regulation of carbohydrate absorption.
Another group of cells are called delta cells. These cells secrete somatostatin which is
responsible for regulation of endocrine system. Also PP cells or gamma cells secrete pancreatic
polypeptide whose secretion varies in different conditions. For example after protein intake,
fasting, physical activity, and acute hypoglycemia such secretion is increased. PP secretion
regulates both endocrine and exocrine pancreatic secretion activities. More recently, ghrelin-
secreting epsilon cells which are less than 1% of total islet cells have been discovered.
39
It has
been reported that ghrelin levels depend on nutritional state and are the highest right before
6

food intake and the lowest right after a meal as a result it has an appetite inducing function
among others.
33

Exocrine pancreas
The human pancreas is predominantly consists of exocrine section. The exocrine
pancreas consists of acinar and duct cells. It is considered as a part of the digestive system and
is responsible for assisting nutrients for better absorption and breakdown in the small intestine.
Acinar cells of the pancreas accomplish this duty by secreting pancreatic juice containing
digestive enzymes. These enzymes help breakdown the carbohydrates proteins and lipids in the
chime. The acinar cells secrete three different types of enzymes: Proteolytic enzymes for
protein digestion, pancreatic amylase for carbohydrate digestion and pancreatic lipase for fat
digestion. The duct cells on the other hand secrete an aqueous alkaline solution rich in sodium
bicarbonate (NaHCO3). Sodium bicarbonate neutralizes the acidity of the chyme so that it won't
burn the intestine.
99
The following figure illustrates parts of functional parts of the pancreas.

Figure4. Endocrine and Exocrine pancreas
7

1.5 Developmental Biology of the Pancreas
Understanding the development of the pancreas has a vital effect on gaining insight into
diseases associated with the organ, and developing innovative therapeutic agents. Therefore
many studies have been conducted to examine developmental biology of the pancreas in
sheep, mice, pigs and humans. It is considered that the pancreas is a single endodermal organ
that is embryologically derived from one dorsal and two ventral anlagen.
44
26 days after
conception in humans and approximately 9.5 days after gestation in mice the endoderm
evaginates into the mesenchyme on top of it. At this time the first projection on the dorsal part
of the foregut is evident and continues to elongate. Six days later in humans and 12 hours later
in mice another outgrowth which is called ventral bud starts to grow on the opposite side of the
foregut. In this phase as a result of buds elongation and gut rotation the ventral and dorsal buds
start to fuse with each other to form the pancreas. The dorsal bud becomes the tail and body
and a portion of the head while the ventral bud gives rise to uncinate process and the other
portion of the pancreatic head. The main ducts of the ventral and dorsal buds also merge upon
fusion, establishing the main pancreatic duct. The dorsal pancreatic bud gives rise to the
accessory pancreatic duct, while the ventral pancreatic bud gives rise to the major pancreatic
duct.
101

As mentioned in the previous section the pancreas is a dual function organ with both endocrine
and exocrine sections. These sections have specific fully differentiated cells that arise from the
pancreatic progenitor cells. Differentiation of the progenitors that are in the “center” between
8

stem cells and fully differentiated cells is regulated by various compartments and transcription
factors. Once the ventral and dorsal pre-pancreatic domains are specified, several transcription
factors become expressed. Some of these transcription factors such as Pdx1, Ptf1a, and Sox9
are specific to the pancreas and promote differentiation of pancreatic cells. These transcription
factors are expressed early in the region.
3, 55, 90
There are also other transcription factors that
their expression is not limited to the pancreatic domains. Gata4/6, Foxa1/2, Tcf2, Onecut-1/2,
Hes1, Prox1, and Mnx1 are said to be among these transcription factors as reviewed in the
literature.
41
As reported in these studies all early pancreatic transcription factors are necessary
for further development of pancreatic buds. Also lineage decision during pancreas development
is mediated by various transcription factors during the primary and secondary transitions.
During the primary transition, the transcription factors Pdx1, Prox1, Onecut-1(Oc1), Ptf1a,
Foxa1/2, Tcf2, Sox9, Gata4/6, and Hes1 play essential role as mediators that help expansion of
pancreatic progenitor cells. They also uphold pancreatic identity. On the other hand the
pancreatic epithelium undergoes dynamic structural changes during the secondary transition
stage. At this stage Notch activity promotes the trunk which is marked by of Ptf1a, c-Myc, and
Cpa and suppresses tip identity which is marked by expression of Nkx6.1/6.2,Sox9, Tcf2,
Onecut-1, Prox1, and Hes1. Transcriptional Cross-repression between Ptf1a and Nkx6.1 further
facilitates separation of tip and trunk upon positive-regulatory loop between PTF1-L and Nr5a2.
Tip cells give rise to acinar cells and trunk cells on the other hand can give rise to ductal or
endocrine cell fate.
97
Notch activity can control whether the ductal or endocrine cells are going
to develop. High Notch activity further activates both Hes1, a repressor of Ngn3, and Sox9, an
Ngn3 activator leading to ductal cell development. Whereas low Notch activity, activates only
9

Sox9 leading to endocrine differentiation.
18 ,108
In the later secondary transition state Ngn3+
cells endocrine precursors differentiate into five different cell types including α- and β-cells.
Cross-repression between Arx and Pax4, Nkx6.1, and Pdx1 separates these two cell lineages.
While the transcription factors Pax4, Pdx1, and Nkx6.1 are β-cell determinants, Arx determines
α-cell identity.
30, 43, 60, 98
Progenitors will further differentiate into δ-cells, and finally PP-cells.
Figure below shows these transcription factors.

Figure5. Transcription factors involved in pancreas development

As mentioned in the previous section even though there are many studies conducted in the
literature on the role of various transcription factors on pancreatic development, not much is
10

known about the signals that specifically control their regulation. It has been reported that
Notch signaling which is essential in regulation of factors such as Hes1 and Sox9, is an important
pathway for early pancreas growth.
2
Table 1 shows the role of pancreatic cells and related
transcription factors.
Table 1 The role of pancreatic cells and transcription factors involved in differentiation

11

Chapter 2: Pancreatic Cancer

2.1 Hallmarks of Cancer
Cancer is not just one disease but it is a broad range of many diseases. Even though the
cause and outcomes of various cancers are different they all share the same features, which are
recognized as the hallmarks of cancer. Human body has millions of cells found in various
tissues. These cells can make exact replicas and that is how life goes. These cells divide during
embryonic and fetal development, and periods of growth .Many of them in the body eventually
enter a senescent phase, the point where there is no more cell division or growth. Regulation of
the activity of each cell within the human body is important for survival. On the other hand,
cells in the body can become malignant when mechanisms regulating cell division, proliferation
and differentiation are lost due to life style changes, genetic disposition, prolonged exposure to
chemical and pollutant exposure as well as past exposures to viral carcinogens. Also activation
of oncogenes or suppression of tumor suppressor genes and exposure to ionizing radiation may
cause damage to DNA and cancer development. One of the major cell groups that accounts for
many carcinomas is epithelial cell. Therefore understanding the cellular processes and
mechanisms causing malignancies will help scientists and physicians to come up with novel
treatments. Even though the underlying reason that causes malignant changes to a normal cell
is different among different cancers, in all cancers the cells have the same characteristics in
transition from normal to malignant. This was discussed in detail in a paper and was defined as
the hallmark of cancer. Understanding the hallmark of cancer is a good help to comprehend
cancer biology.
58
the hallmarks of cancer include proliferation, overcoming growth suppression,
12

resisting cell death, immortality, angiogenesis, invasion and metastasis. Below is the brief
description of each of these hallmarks.
Proliferation: Different tissues have different structure and function. In each tissue there is a
balance between division and cell death. Cell proliferation and division which is regulated by
specific growth factors in normal cells is necessary to give each tissue its specific function and
structure. Malignant cells on the other hand can produce growth factors of their own which
triggers autocrine proliferative signals. Cancer cells may also send signals to stimulate normal
cells within tumor stroma which can further provide the malignant cells with growth
factors.
28,19
Additionally expression of growth factor receptors on the surface of cancer cells
leads to responsiveness of such cells to growth factors.
Overcoming growth suppression: Loss of tumor suppressor genes could be a significant step in
the development of cancer. There are proteins encoded by tumor suppressor genes that act as
gatekeepers for cell proliferation, trigger senescence or cell death and monitor stress and
abnormal function within the cell. Upon receiving inputs such as genetic damages, nutrients or
decreased oxygen levels these proteins can activate apoptosis. Cancer cells on the other hand
avoid programs that negatively control cell proliferation. This results in inappropriate
replication of cells due to loss of function of the proliferation suppressors
Resisting cell death: It has been shown in studies that the programmed cell death also known
as apoptosis acts as a natural barrier to cancer development .Oncogenic signaling and DNA
damage lead to activation of apoptotic proteins while suppressing anti-apoptotic proteins in
normal cells. Tumor cells on the other hand develop a variety of approaches to limit or avoid
Apoptosis. Among these approaches is loss of function of TP53 tumor suppressor, increased
13

expression of anti-apoptotic regulators such as Bcl-2, Bcl-xL, up-regulation of survival signals
(Igf1/2) or down-regulation of pro-apoptotic factors such as Bax, Bim. Additionally in the case of
unordered cell death or necrosis, cells release inflammatory mediators into the local tissue
microenvironment. Such process has definitely some tumor promoting potential.
Immortality: Normal cells replicate a limited number of times until they enter senescence. This
is regulated by telomerase shortening with each division. In contrast cancer cells replicate
unlimitedly to form malignant tumors.
Angiogenesis: Like normal cells, highly metabolic tumor cells need nutrient and oxygen. They
also need to release waste and carbon dioxide. Angiogenesis is therefore vital in malignancies in
order for cells to maintain nutrient. Oncogenes and inflammatory mediators trigger formation
of the new leaky blood vessels from the quiescent vasculature through the process which is
known as angiogenesis.
Invasion and metastasis: In normal cells molecules such as E - cadherin are essential for cell
adhesion or cell-extra cellular matrix interaction to maintain the structure of epithelia cells
sheets. These molecules are therefore considered to be antagonists of invasion and metastasis.
Down-regulation of these molecules triggers invasion and metastasis. There are other
molecules such as N-cadherin which are overly expressed in cancer cells. These molecules are
found in migrating neurons and mesenchymal cells. Also malignant cells may invade to local
tissues and further systemic circulation or lymphatic capillaries, escape from the leaky vessels
and adapt to foreign tissue microenvironments resulting in successful colonization and survival
in other tissues, through the process known as metastasis.
14

In addition to the six common hallmarks of cancer studies have proposed two new hallmarks
which are genetic alteration in cells that leads to tumor progression and inflammatory
responses which are evident in neoplastic progression. These two consequences of neoplasms
suggest that there may be two new hallmarks that are probably involved in all cancers. Since
these new hallmarks have not been completely confirmed, they are considered as emerging
hallmarks. The first new hallmark that supports neoplastic proliferation implicates the capability
to change or reprogram cellular metabolism. The second suggests cancer cells to avoid
immunological destruction by T and B lymphocytes, macrophages, and natural killer cells.
70

2.2 Pancreatic cancer
Now that we have gained some information about the structure and function of
pancreas and cancer biology we should move forward combining such information to study
biology and genetics of pancreatic cancer, its treatment strategies and potential therapeutic
agents.
Pancreatic adenocarcinoma is a disease that is associated with advancing age.
6
it is the fourth
cause of cancer death in the United States. Approximately 28,000 people are diagnosed and die
of pancreatic cancer each year.
49
Like other malignancies, pancreatic cancer is a result of
interaction between genetic and environmental risk factors. Cigarette smoking, chronic and
hereditary pancreatitis, late-onset diabetes mellitus, and familial cancer syndromes are among
the most important risk factors of the disease. Generally pancreatic cancer is developed by
activation of oncogenes, inactivation of tumor suppressor genes, and deregulation of many
signaling pathways. Consequently in order to design prevention or treatment strategies,
15

scientists have proposed to target these molecules or their downstream signaling pathways.
Although such studies show promising results in animal models or in vitro, they are not yet very
successful in clinical trials. This is due to lack of sufficient knowledge of pancreatic cancer
biology and targeted agents. In 1935 the first PANCREATICODUODENECTOMY was reported by
Whipple and colleagues. Ever since, surgery was suggested as the most potential curative
strategy that buys patients more time than other strategies. However, even in successful
resection surgeries followed by adjuvant chemotherapy in 15–20% of patients the 5-year
survival is only 20%.
4

Gemcitabine is used as standard adjuvant chemotherapy for patients with advanced pancreatic
cancer .Combination of this drug with 5-FU, irinotecan, and oxaliplatin (FOLFIRINOX) was shown
to improve survival rates with higher but still manageable toxicity. Since the overall survival has
been reported less than 12 months even in the case of combination therapy, there is still need
to design novel therapeutic agents to overcome such problems and improve patients' survival.
For this purpose, the knowledge on the molecular aspects of pancreatic cancer is very
important and is likely to be helpful in the design of new drugs and the molecular selection of
existing drugs for targeted therapy.

2.3 Pancreatic Endocrine and Exocrine tumors:
It is known that pancreatic cancer is not one disease. It arises as number of different
tumors. Each of these tumors which can arise in either endocrine or exocrine part of the
pancreas has a different microscopic characteristic and prognosis. Since these parts have
16

different normal functions, arising tumors in different parts of the pancreas will result in
different symptoms. In order to come up with rational treatment, understanding of these
various tumors is required.
Exocrine pancreas tumors: Tumors of the pancreas majorly arise in the exocrine part.
Microscopically these tumors are in form of glands that can grow large enough to invade nerves
or metastasize to the liver or lymph nodes. This form of tumor is usually very dismal and has a
poor prognosis due to a lack of specific symptoms and limitations in diagnostic methods. There
are various forms of malignant tumors of the exocrine pancreas the most common form of
which is pancreatic ductal adenocarcinoma. Other rare types of exocrine tumors are acinar cell
carcinomas, adenosquamous carcinomas, colloid carcinomas, giant cell tumors, hepatoid
carcinomas, intraductal papillary-mucinous neoplasms, mucinous cystic neoplasms,
pancreatoblastoma, serous cyst adenomas, signet ring cell carcinomas, solid pseudopapillary
tumors and undifferentiated carcinomas.
7
Endocrine pancreas tumors: The endocrine tumors can originate within the pancreas from the
usual cells of the islet of Langerhans or from similar neuroendocrine cells outside of the
pancreas. Compared to non-endocrine tumors these tumors are less common. Most of these
tumors are benign. However, tumor behavior is not very predictable. Most of these tumors are
non-secretory or nonfunctional. This means that they don't secrete any products or even if they
do, the amount and type of products would not cause symptoms. On the other hand, some of
the endocrine tumors are functional and may produce active hormones arising severe
symptoms. The two common forms of functional endocrine tumors are insulinomas and
glucagonomas.
17, 48
17

As discussed in chapter one, the pancreas is located deep within the stomach close to large
blood vessels. There are two main arteries in the area of the pancreas; one is the celiac artery
that gives rise to the splenic artery and the hepatic artery that supplies blood to the liver,
pancreas, spleen and stomach. The other one is superior mesenteric artery that gives rise to
numerous branches that supply the small bowel, part of the colon and the pancreas. Even
though these vessels are vital for life, they make pancreatic cancer have an easily invasive
characteristic. Therefore cancers of the pancreas do not need to grow very large before
invading the lymphatic system and vessels. This gives the pancreatic cancer its lethal
characteristic and its resistance to therapeutic agents and hence leading to poor prognosis and
highly incurable characteristic of the disease.
62
Tables 2, 3, 4 provide information on different
pancreatic tumors and brief description of their characteristics.
Table2. Endocrine Tumors
18

Table3. Exocrine Tumors
Table4. Exocrine Tumors Cont’d
19

Chapter 3: Biology of pancreatic cancer

In spite of the fact that PDAC Pancreatic cancer remains lethal, Researchers in the last
few years have brought significant advances at the molecular and biological levels to better
understand pancreatic cancer. Molecular pathology and cancer genetics give researchers
insight on how cellular perturbations could be linked to pancreatic adenocarcinoma. In order to
better understand the biology of pancreatic cancer, study of pancreatic cancer stem cells as
well as study of the tumor microenvironment is of great importance. This leads to finding of
potential new therapeutic targets as well as improving treatment strategies. Additionally, due
to predominant needs to in depth analysis of pancreatic cancer biology, researchers have also
utilized high-resolution genomic profiles and developed genetically engineered animal models
of pancreatic adenocarcinoma and patient derived xenografts to further study genetic
mutations that lead to emergence of specific biological features of the disease.
63, 82
The
following chapter will be focusing on details about how each of these studies involved in
pancreatic cancer studies and development of novel treatment strategies.

3.1 Stromal Biology of pancreatic cancer and related therapeutic targets
Tumor microenvironment
An important characteristic of pancreatic cancer is formation of desmoplastic stroma by
pancreatic stellate cells. This stroma not only acts as a barrier but it is also involved in tumor
20

formation, progression and metastasis.

Stellate cells get activated by growth factors such as
TGFβ-1, PDGF (platelet derived growth factor), and FGF (fibroblast growth factor) which results
in secretion of collagen and other extra cellular matrix components. They express proteins such
as Cox-2, PDGF receptor, vascular endothelial growth factors (VEGF), stromal derived factor
(SDF), chemokines, integrins, SPARC (secreted protein-acid rich in cysteine), and hedgehog
pathway elements which are associated with drug resistant, poor vascularization and poor
prognosis in patients.
40
Pancreatic cancer has dense stroma with cellular components such as
cells of mesenchymal and immune origin and acellular components of extracellular matrix
proteins (ECM), growth factors and cytokines. The stromal cells that contribute to pancreatic
cancer growth could be targeted by potential therapeutic agents. For example disruption of the
stroma with inhibitors of the hedgehog may result in increased vascular supply and
improvement in drug delivery. Unfortunately this method is not yet successful in human clinical
trials. SPARC also known as osteonectin is a stroma target in pancreatic cancer that is able to
bind to albumin coated nanoparticle of paclitaxel and gets accumulated in tumor tissue.
preclinical studies show that this method eliminates the pancreas cancer stroma leading to
improved delivery of chemotherapeutic agents.
107
In genetically engineered mouse models of
pancreatic cancer this complex blocks breaking down gemcitabine, leading to accumulation of
the drug within the tumor. This has further taken to phase I, II and III in preclinical trials. Other
studies have focused on immunosuppressive characteristic of the tumor microenvironments
and attempted to reverse it by therapeutic agents. For example since CD40 activation can
reverse immunosuppression and drive antitumor T-cell responses, scientists have used agonist
CD40 antibody combined with gemcitabine chemotherapy in patients with surgically incurable
21

PDAC. It has been reported that such method work by infiltration of macrophages that use up
the stroma.
14

Cancer-Associated Fibroblasts
The dense stroma of pancreatic tumor is formed by cancer-associated fibroblasts (CAFs),
which are reported to be derived from various cell types and represent a heterogeneous
population of cells. These Cancer-Associated Fibroblasts play role in tumorgenesis, metastasis
and resistance to conventional therapies via various signaling pathways which involves CXCR4
(chemokine receptor). Additionally in cancer stem cells presence of CD133 and CXCR4 gives the
stem cells highly invasive characteristic.
61
which makes it a potential target for therapy.
.
Another component of tumor stroma which makes it resistant to drug delivery is the
extracellular matrix component, hyaluronic acid (hyaluronan). As a result using a PEGylated
form of the hyaluronic acid- degrading enzyme (PEGPH20) combined with gemcitabine helped
the researchers restore a normalized tumor vasculature and increase the efficacy of
chemotherapy.
72, 76
These findings highlight the importance of tumor stroma as a potential
target for therapies.
Immune Cells
Another component of tumor stroma is various types of immune cells which tumor
promoting properties such as mast cells, neutrophils, T and B lymphocytes

or antagonistic
properties whose balance is associated with tumor growth. These immune cells get infiltrated
in all stages from precancerous lesions to malignant invasive tumors. Secreting regulators of
tumor and stromal growth such as VEGF, FGF2, chemokines , cytokines, pro-angiogenic factors
22

such as MMP-9 and other matrix metalloproteases and heparinase.
159,55
The importance of
immune system leads researchers to target the immunosuppressive environment present in
pancreatic cancer which will be discussed in detail later.
Cancer Stem Cells
Another component of many solid tumors is a subset of cancer cells that can spread and
form new heterogenous tumors with the same features of original malignant tumor. These cells
are called cancer stem cells (CSCs).Since such cells are resistant to conventional therapies even
with responsiveness of the bulk tumor to such therapies, it is important to distinguish between
cancer stem cells and bulk of tumor for designing effective treatments. CSCs were identified in
various cancer types such as acute myeloid leukemia (AML) and pancreatic cancer among other
malignancies.
21
Just like other stem cells pancreatic cancer stem cells have the capacity of self-
renewal and asymmetric division. Presence of cancer stem cells in primary tumors is associated
with shorter overall survival, resistance to the standard chemotherapy and invasion and
metastasis.
80
Preclinical studies have reported developmental pathways such as Hedgehog, Wnt
and Notch, apoptotic pathway targets such as DR5 and novel pathways such as nodal-activin as
therapeutic targets of pancreatic cancer stem cells.
78
It has been hypothesized that while
standard chemotherapy with Gemtacibine causes tumor regression for a short period of time ,
targeting such pathways may cause prolonged treatment. However the idea has not been
successfully used in clinical trials. In the case of pancreatic cancer, markers such as
CD44/CD24/ESA , CD133 or ALDH have been also reported in a subset of pancreatic cancer
stem cells and c-Met+/CD44+ pancreatic cancer stem cells have been recently identified.
61
As
23

mentioned earlier cancer stem cells form heterogeneous tumors with the same pathologic
characteristics of the original tumor. This genetic heterogenecity of tumors could be
determined as a barrier to design of targeted therapies. On the other hand, in the context of
cancer stem cells, one could identify the main up-regulated signaling pathways involved in
progression or recurrence of the disease. Such pathways represent potential therapeutic
targets utilized as potential treatment when combined with chemotherapeutic agents such as
gemcitabine by reducing CSCs. For example in case of c-Met+ pancreatic cancer stem cells
inhibitors of Met has been proposed to reduce pancreatic cancer stem cells and
tumorgenicity.
61

Nutritional requirements of pancreatic cancer
Similar to other cancers, pancreatic cancer cells need nutritional sources such as glucose
and glutamine in order to proliferate. As a result interrupting such nutrients may act as novel
therapeutic approaches. For example LDHA which regenerate nicotinamide adenine
dinucleotide (NAD+) to convert glucose to pyruvate to generate ATP, could be used as a
potential target. Another target protein is glutaminase which could be inhibited by BPTES (bis-
2-[5-( phenylacetamido)-1,3,4-thiadiazol-2-ethyl sulfide) inhibitor
82
. Additionally they are able
to survive and grow under hypoxia. Consequently blocking proteins involved in the process may
provide therapeutic advantages.

24

Chapter 4: Genetics and Pathology of Pancreatic Cancer

Most pancreatic cancer cases are diagnosed at later stages when the disease is more
advanced. As a result even with some promising therapeutic regimens, pancreatic cancer
patients usually would not respond to most forms of treatments. Therefore unlike other
malignancies such as breast or colorectal cancer in which early detection and more effective
drugs decrease the rate of mortality, the prognosis for patients with pancreatic cancer is not
good and the mortality rates are high
93
. As a result in the past decade scientists have carefully
investigated pancreatic cancer precursor lesions and molecular and genetic characterization of
such lesions. These findings are definitely useful in diagnostic examination of patients with
these lesions and may lead to identifying new targets and designing new therapeutic agents for
pancreatic cancer. Therefore the goal of the following chapters would be to recognize
pancreatic noninvasive precursor lesions, analyze histopathological features of such lesions,
examine various gene alterations associated with the precursor lesions, identify genomic
landscape of pancreatic cancer and analyze phenotypic alteration of pancreatic cancer model
using genetically engineered mice.
4.1 Pancreatic Cancer Precursor Lesions
It is vital but still challenging to identify pancreatic cancer in an early stage before
development of invasive carcinoma. The invasive pancreatic cancer develops from different
precursor lesions. This underscores the importance of studying pancreatic precursor lesions.
Understanding the genetic alterations of such lesions and involved biomarkers allow for
25

detection of the patients who are at high risk of developing invasive pancreatic cancer.
Furthermore treatment of the lesions may reduce the mortality rates of the disease. The best
characterized histological precursor of pancreatic cancer is the microscopic Pancreatic
Intraepithelial Neoplasia. (PanIN) .
67
On the other hand, macroscopic cystic precursor lesions
intraductal papillary mucinous neoplasia (IPMN), intraductal tubulopapillary neoplasm (ITPN),
and mucinous cystic neoplasia/mucinous cystadenoma (MCN) are less common.
64, 68
The
following section briefly reviews the histopathology and clinical aspects of these precursor
lesions .
Pancreatic Intraepithelial Neoplasia
More than a century ago scientists identified microscopic Pancreatic Intraepithelial
Neoplasia. PanINs are mostly located in the head of the pancreas and in smaller pancreatic
ducts
.69
and are divided in three grades based on their architecture.

As opposed to cuboidal
characteristics of epithelium in normal pancreatic ducts, in low grade PanIN-1A, small ducts are
lined by flat epithelium consisting of columnar mucinous cells which have uniform round to oval
nuclei and supranuclear mucin. Pancreatic ducts in PanIN-1B are lined by epithelium consisting
of columnar mucinous cells and micropapillary architecture. In PanIN-2, pancreatic ducts are
lined by columnar cells with nuclear hyperchromasia, pseudostratification, and papillary
architecture. In higher grade PanIN-3, pancreatic ducts are lined by columnar cells with
cytonuclear pleiomorphism and loss of nuclear polarity. These lesions have complex
architecture with micropapillary epithelium and cribriform growth pattern as shown in the
figure.
69

26

Figure6. A)Normal pancreatic duct B) PanIN1-A C)PanIN1-B D)PanIN-2 E)PanIN-F)p53
immunohistochemistry for PanIN-3

Intraductal Papillary Mucinous Neoplasm
Unlike microscopic characteristics of PanINs, IPMNs which are more than 1 cm in
diameter are macroscopically visible. These lesions which mainly arise in the main pancreatic
duct (mostly in the pancreatic head) often produce copious thick mucin. As a result pancreatic
duct gets dilated following some symptoms such as abdominal or back pain, nausea, vomiting,
weight loss, or recurrent episodes of pancreatitis
37
Another form of these lesions appears as
multi-cystic structures in the branch-duct (mainly in the head and uncinate process).

While
main-duct IPMNs are often associated with high-grade dysplasia and invasive carcinoma, most
27

branch-duct IPMNs are low-grade lesions.
87
Since the high-grade dysplasia in main duct is more
associated with invasive carcinoma, detailed investigation is required to grossly and
histologically distinguish between main-duct and branch-duct IPMNs in order to better predict
patients’ prognosis.
Intraductal Tubulopapillary Neoplasm
ITPN lesions are solid nodular tumors that are macroscopically visible due to their larger
size. As opposed to mucin producing IPMNs with papillary characteristics, these tumors
produce much less mucin and mainly have tubular growth pattern
113
40 % of cases with such
lesions develop an invasive carcinoma. Also less than 1 % of all exocrine pancreatic neoplasms
and 3 % of pancreatic intraductal neoplasms show ITPN lesions.
113
Although these studies
show the association between such lesions and pancreatic cancer, there is still limited data
available about prognosis for patients with ITPNs.

Mucinous Cystic Neoplasm
Like IPMNs and ITPNS, single spherical cystic MCN lesions are macroscopically visible.
These lesions may produce thick mucin. In Low-grade MCNs a smooth and glistering internal
surface is visible, whereas higher grade neoplasms show papillary projections. Like other types,
in higher grade lesions more association with invasive carcinoma can be identified. These high
grade neoplasms are larger and multilocular and contain papillary projections.
115.
The body and
tail of the pancreas are where these lesions mostly develop with no direct contact to the
28

pancreatic ductal system. Depending on the size of MCNs, these lesions are detected either by
imaging for another reason in case of smaller MCNs or due to patients complaints about
abdominal discomfort or sensation of mass in case of larger MCNs .
115

4.2 Molecular genetics of pancreatic cancer
Several alterations in oncogenes, tumor-suppressor genes and genome-maintenance
genes are required to modulate gene transcription, cell proliferation and cell cycle and alter the
downstream physiological responses which further results in development of pancreatic tumor.
These alterations may be either gain or loss of function as a hallmark of cancer, which occurs
either later during adulthood or germ line mutations during embryonic development that
permit the development of familial pancreatic cancer over the lifetime of these patients.
While some of these mutations are known to occur at a higher frequency, there are some other
genetic alterations that occur less often. As a result each case of pancreatic cancer may be
characterized by a unique biology and genetics. These characteristics of pancreatic cancer
highlight the importance of detailed understanding of the molecular genetics underlying the
pathogenesis of this malignancy which enables scientists to improve prognosis and therapeutic
approaches.
There have been several studies that used the candidate gene approach to identify
genes based on their biological, physiological or functional relevance to pancreatic cancer.
Although the candidate gene approach has been useful to study the role of frequently mutated
genes and to identify important pathways known to be involved in development of other tumor
29

types, it would not enable researchers to discover new genetic modifications and underlying
pathways. Recently the advent of high-throughput technologies such as next-generation
sequencing (NGS) has enabled scientists and oncologists to improve their knowledge on genetic
landscape of cancers and to use personalized medicine for patients based on their unique
genetic modifications. The focus of the following section is to describe spectrum of genetic
mutations which are more or less common in pancreatic cancer and to further identify the
genetic alterations associated with different pancreatic precursor lesions which may develop
cancer.

4.2.1 Genetic Mutations in Pancreatic
Oncogenes: Approximately 30 % of human tumors contain RAS oncogene mutations
making it the most frequently mutated oncogene in human cancer.KRAS2 gene ( Kirsten rat
sarcoma viral oncogene homolog) which is one isoform of Ras oncogene family is located on
chromosome 12p and encodes a GTP-binding and hydrolyzing protein. In 90% of pancreatic
adeno carcinoma cases as well as PanIN lesions activating mutations in the KRAS2 gene is
detected.
96
These KRAS mutations are believed to originate from somatic mutations during
adulthood rather than germline mutations.
The Biology of K-Ras Signaling Pathways in Pancreatic Cancer
Under physiological conditions, K-ras is activated due to binding to GTP. This protein is
known as prototypical small GTPase and due to its intrinsic property of GTP hydrolyzation, it
rapidly gets inactivated. The GTP/GDP cycle is controlled by guanine nucleotide exchange
factors. GEFs promote the release of GDP and allow more abundant GTP to bind which results
30

in activation of K-Ras GTPase activation protein. It will then promote the intrinsic GTP
hydrolysis, resulting in K-Ras inactivation.

Due to genetic mutations in the k Ras protein, it gets
locked in the GTP-bound activated form which further results in a conformational change in the
Ras protein that affects the binding affinity to downstream effectors. As a result of such
mutations k-Ras remains in its activated form and interacts with effector molecules such as the
mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/Akt and Ral
signaling pathways.This interaction leads to activation of downstream pathways required for
oncogenesis including survival, proliferation, invasion and metabolism. Figure below shows a
detailed depiction of this pathway.

Figure7. K-Ras pathway in pancreatic cancer. (KRAS*) represents the mutational activation of
Tumor-Suppressor Genes

31

Additionally there are studies showing selective loss of the wild type allele of K-Ras in human
tumors. This imbalance, which can occur by either copy number gains or uniparental disomy,
was identified in various diseases including pancreatic cancers.
96
KRAS2 mutations in biological
samples such as pancreatic juice, stool, and blood can be used as biomarkers for the diagnosis
of pancreatic cancer. Although the roles of specific K-RAS effector pathways in pancreatic
cancer pathogenesis have not been resolved and their mutations are not that usual in
pancreatic cancer, it is noteworthy that alterations in downstream effectors generally may lead
to carcinogenesis.
Overexpression of EGF-family ligands such as transforming growth factor-α(TGF-α) and EGF
and their receptors EGFR,ERBB2 (also known as HER2/neu) and ERBB3 is evident in pancreatic
adenocarcinoma as well as low grade PanINs.
34
Gain-of-function mutaions in the catalytic
subunit of PI3K may also increases enzymatic function and stimulate downstream signaling
elements responsible for oncogenesis. It is reported that 9 % of patients with PDA have
mutations in the catalytic subunit of PI3K .
116

CDKN2A/p16 tumor suppressor gene is one of the most frequently inactivated tumor
suppressor genes located on chromosome 9p21 which can be associated with pancreatic
cancer.

Like K-Ras mutation, which was discussed earlier and with the help of ultrasensitive
detection technologies, the frequently mutant CDKN2A/p16 identified in the pancreatic juice of
patients can be used as a biomarker.
Another tumor suppressor gene, TP53 which is located on chromosome 17p plays a critical role.
It is responsible for controlling the G1/S cell cycle phase and G2/M checkpoints. These
32

Checkpoints prevent cell cycle progression at specific points which results in induction of cell
cycle arrest. When DNA is damaged the inability of repair leads to apoptosis in presence of p53.
It is reported that in 50–75 % of invasive pancreatic cancers TP53 gene is mutated due to
missense alterations of the DNA binding domain. As a result the protein translated from the
mutant gene can’t bind to DNA to activate the downstream transcriptional network. Several
TP53 mutations have been reported in cancers. Majority of TP53 mutations result in
accumulation of stabilized translated protein in the nucleus which can be detected with
immunohistochemistry.
9
Since the p53 mutations occur very frequently in pancreatic cancer, it
can be used as a potential biomarker in biological samples such as pancreatic juice samples.

In
recent years scientists have developed small molecules that selectively target p53 R175H
mutations in order to retrieve its function as a tumor suppressor.

Other researchers have
developed small molecule that acts as an antagonist to selectively acts against p53.
77

DPC4/SMAD4 is another tumor suppressor gene on chromosome 18q21.Intracellular protein
encoded by this gene gets activated upon binding of transforming growth factor β (TGFβ) to its
membrane receptors. This leads to activation of downstream growth inhibitory signal
transduction pathways by G1–S cell-cycle transition or by promoting apoptosis.
92
Mutation of
DPC4/SMAD4 is among the most important mutations in pancreatic cancer. It occurs due to
homozygous deletion or inactivating mutation in 30-50% of pancreatic adenocarcinomas.
92
the
mechanism by which this mutation leads to cancer involves its role in TFG-β in growth
regulation. Unfortunately the roles of TGF-β signaling in pancreatic adenocarcinoma
pathogenesis are not well defined yet and further investigation is needed to deeply understand
how it is related to pancreatic cancer. It is also reported that such mutation is related to
33

systemic metastases of the advanced pancreatic adenocarcinoma and can be used as a
biomarker for that matter.

Like previously mentioned cases, there are some studies focusing
on designing small molecules that target cells with DPC4/SMAD4 mutations providing targeted
potential targeted therapies.
Identification of the frequently mutated genes such as KRAS, CDKN2A/p16 , SMAD4/ DPC4 and
TP53 as discussed above has been done using the candidate gene approach. This method even
though useful, did not enable scientists to find less frequently altered genes in pancreatic
adenocarnoma. With advancing research in 2008, exomic sequencing was conducted. Data
obtained from sequencing of the sporadic pancreatic cancer genome may be useful in
advancing the knowledge of genomic alterations in precursor lesions,
111
discovery of genes in
familial pancreatic cancer

and clarifying the genes involved in metastases
112
which all affect the
drug discovery efforts in pancreatic cancer in the future.

Genome-Maintenance Genes
In addition to oncogenes and tumor-suppressor genes, alteration of a third class of
genes has been identified in cancers such as pancreatic cancer. These cancer susceptibility
genes are known as "caretakers" and are responsible for genomic and chromosomal stability
and therefore cellular health under normal conditions.

These genes stabilize the genome by
preventing the accumulation of these mutations. Upon DNA damage caretaker genes encode
maintenance operations such as nucleotide excision repair, base excision repair, non-
34

homologous end joining recombination pathways, mismatch repair pathways, and telomere
metabolism. As a result, when such genes are mutated, their altered products cause formation
of neoplasia.

Caretaker genes control cell division and apoptosis and thus indirectly regulate cell
proliferation. For example, a mutation in a caretaker gene coding for a DNA repair pathway
leads to the inability to properly repair such damage, which further results in excessive cell
growth. It has been proposed that the caretaker genes are responsible for many hereditary
predispositions to cancers. According to Johns Hopkins Hospital

BRCA2 gene located on
chromosome 13q is one of the “caretaker” genes inactivated in 5 % of sporadic pancreatic
cancers. Also germ line mutations of BRCA2 are observed in 5–10 % of people with hereditary
pancreatic cancer. Such mutations are more common among individuals with a family history of
pancreatic cancer and have been shown to be associated with an increased risk of breast,
ovarian, prostate cancer besides pancreatic cancer.
103

Also Mutations in the PALB2 gene, partner and localizer of BRCA2, have been associated with
breast and pancreatic cancer.
104
Binding of The PALB2 protein with the product of BRCA2 is
important in connecting BRCA1 and BRCA2 and formation of “BRCA complex” which is
stabilized in the nucleus. The integrity of the complex is essential for the maintenance of
genomic stability .This results in the avoidance of cancer. The above process will be reversed
due to mutations.
51
Other genes involved in DNA repair that have been identified in hereditary
pancreatic cancers are hMLH1 and hMSH2.
45
Mutations or silencing in hMLH1 and hMSH2
result in replication errors in microsatellites , leading to microsatellite instability which will be
discussed in more detail in the following section.

35

Chromosomal Aberrations
The previous section has focused on gain or loss of function genomic modifications as a
hallmark of pancreatic cancer. Even though genetic mutations at the nucleotide level are very
common in cancers, there are many instances in which genes or pathways are changed at the
chromosomal level. This highlights the importance of studying Genomic instability as another
hallmark of pancreatic cancer. The alterations could occur either as chromosomal instability
(CIN) or microsatellite instability (MIN). Such variations can be determined by histological
features of the alterations.

Chromosomal instability
Centrosome abnormalities, which results in defects in the mitotic-spindle apparatus, may
also lead to genomic instability at the level of chromosomes.

It has been identified in almost all
pancreatic cancer cases by cytogenetic analysis methods such as COMPARATIVE GENOMIC
HYBRIDIZATION (CGH). It is due to copy-number gains and losses translocations, inversions,
amplifications and homozygous deletions. At the level of chromosomes, this instability occurs
as losses on chromosomes 6p, 9p, 13q, 17p, and 18q, as well as gains on chromosomes 7q and
20 ,losses on chromosomes 17p, 9p and 18q, deletions of chromosomes 8p and 6q and 4q, and
amplifications of chromosomes 8q, 3q, 20q and 7p.
37
Several studies have investigated
chromosomal alterations. For example in one study scientists identified such alterations in
pancreatic cancer xenografts by using genome wide allelotyping. In that study they have
36

reported that the most common copy number alterations occur on chromosomes 9p, 18p and
17p with overlaaping regions of some major genes involved in pancreatic cancer such as
CDKN2A, SMAD4/DPC4 and TP53 , respectively also various patterns of CIN have been
identified in pancreatic cancer such as mixture of allelic loss and copy number changes or
regions of homozygous deletions.
25

Microsatellite instability (MIN)
The second form of genomic stability is known as microsatellite instability caused by
impaired DNA mismatch repair genes such as MLH1, MSH2 and MSH6. Phenotypically it shows
that the DNA mismatch repair is not functioning properly in some cells and therefore such cells
accumulate errors instead of correcting them. This leads to formation of microsatellites
fragments in which genetic sequences are not preserved properly and therefore can be
characterized by very few alterations in chromosome ploidy. Such instability exhibits a unique
histological fashion known as "medullary” with poorly differentiated histology, pushing borders,
and large numbers of tumor infiltrating lymphocytes.
37
This alteration is reported to put
patients at higher risk of getting less aggressive pancreatic cancer.
1

Telomere Alterations
Telomere is a region of repetitive noncoding nucleotide sequences TTAAGGG at each
end of a chromatid, which protects the end of the chromosome from breakage or fusion with
neighboring chromosomes.

The length of these telomeres becomes shorter after each cell cycle
37

which further results in apoptosis. In most human cancers on the other hand, activation of
telomerase enzyme prevents telomere shortening and progressively elongates the telomeres.
Telomere shortening is identified at the early stage of noninvasive lesions, while at later stages
of pancreatic cancer progression telomerase enzyme gets activated.
106

Attrition in telomere length is one of the earliest detectable molecular alterations in pancreatic
cancer, observed at even lower grade PanINs. Loss of fluorescence intensity by TEL-FISH
compared to stromal cells close to the adenocarcinoma with bright telomere signals show
telomere attrition.

4.2.2 Signaling pathways in Pancreatic Cancer

Through the progression from pancreatic precursor lesion to cancer, genetic mutations
such as KRAS mutations, p16/INK4A, p53 and DPC4/SMAD4 could occur. Besides such genetic
mutations, there are developmental signaling pathways which are altered in pancreatic cancer.
As a result, therapeutic agents could be developed to target pathways instead of individual
mutated genes. The most important pathways involved in pancreatic cancer are Hedgehog,
NOTCH, Wnt, MET, and TGF-β pathways which are depicted in the figure at the end of this
section. These pathways and tumor microenvironment cells not only cause tumor growth but
they also cause resistance to chemotherapy. Changes in pancreatic tumors are not due to single
mutation or pathway but combination of many mutations. As a result targeting a single
pathway or molecule is not successful. Other cells in the tumor microenvironment must also be
38

targeted. Understanding these genetic alterations and signaling pathways is important in
pancreatic cancer targeted drug development. Further improvement of treatment efficacy
involves normalizing stroma and reducing pancreatic cancer stem cells. Currently Gemcitabine
is used as a chemotherapeutic agent alone or in combination with other agents. FOLFIRINOX is
another chemotherapy regimen made up of folinic acid, fluorouracil, irinotecan and oxaliplatin
drugs. Since these therapies are useful for short term treatment, development of new therapies
that target the signaling pathways or desmoplastic stroma is of great importance in pancreatic
cancer treatment.

The Hedgehog Signaling Pathway
The Hedgehog signaling pathway is an important pathway during embryonic
development.

Binding of one of the three hedgehog (HH) ligands (Sonic, Indian, and Desert) to
a transmembrane protein Patched (PTCH) will activates the signaling pathway and inhibits
repression of transmembrane receptor-like protein called Smoothened (SMO).As a result SMO
leads to signal transduction via GLI transcription factors which are the effectors of this signaling
pathway.
54, 86
which in turn activates hedgehog transcriptional genes such PTCH , GLI and
Hedgehog Interaction protein HHIP. Deregulation of this signaling pathway is associated with
different cancers via different mechanisms. In case of high HH ligand expression will get
activated in a ligand dependent manner. This leads to formation of solid tumors in different
organs such as pancreas.

In these cases HH ligand expression increases as the early PanIN 1
lesions leading to pancreatic adenocarcinoma. For example Sonic HH (SHH) which is the
39

dominant HH ligand is overly expressed in 70 % of patients causes premalignant lesions.
102
It is
also responsible for desmoplastic features of pancreatic cancers.
12

On the other hand in the epithelial compartment, non-canonical overexpression of GLI1 leads
to activation of HH pathway. HH signaling is also responsible for formation of dense stroma in
primary pancreatic tumors. This characteristic is reported to make this cancer a very harmful
malignancy with metastatic progression. Another negative effect of the dense stroma is its
contribution to resistance to drug delivery in pancreatic cancer. As a result to improve drug
delivery and drug efficacy in this kind of malignancy, researchers have proposed ways to block
HH signaling pathways. One way to block the pathway is by using SMO antagonists to inhibit
SMO in mouse models of pancreatic cancer. According to clinicaltrial.gov studies have proposed
that targeting the Hedgehog signaling pathway with smoothened antagonist IPI 926 combined
with gemcitabine in pancreatic cancer mouse models may increase survival rates.

Additionally
according to clinicaltrials.gov website studies have used different HH pathway inhibitors such as
LDE225, LEQ506, and GDC-0449 in combination with chemotherapy in early phase trials of
pancreatic cancer.
The NOTCH Signaling Pathway
NOTCH signaling pathway is important in cell differentiation

as well as regulation of
adult stem cell homeostasis and maintenance. Four members of the NOTCH receptors
(NOTCH1-4) can be activated by NOTCH ligands such as Dll-1 (Delta-like1), Dll-3 (Delta-like3) Dll-
4 (Delta-like4), Jagged-1, and Jagged-2. Upon activation NOTCH receptors can be cleaved by the
metalloprotease tumor necrosis factor α-convertase enzyme (TACE) and γ-secretase. This
results in release of intracellular domain of NOTCH which further moves to the nucleus and
40

competes with inhibitory proteins to bind to CSL transcription factor. Further recruitment of co-
activators such as p300, mastermind-like 1–3 (MAML1-3), and histone acetyltransferases
converts CSL from a transcriptional repressor to transcriptional activator.
42, 116
Based on the
effect of NOTCH signaling up-regulation in pancreatic cancer, development of targeted
therapies to target this pathway is proposed. MRK-003 which is γ-secretase inhibitors is
suggested to have therapeutic potential when combined with gemcitabine chemotherapy in
mouse models of the disease.
31
Combination of MRK003 or MK-0752 which are both γ-
secretase inhibitors, with gemcitabine is also being tested in clinical trials in patients with
pancreatic cancer according to clinicaltrials.gov.

The Wnt Signaling Pathway
Wnt-β-catenin signaling is required for morphogenesis, proliferation and differentiation of
many organs. The product of Wnt gene is responsible for embryonic development and
homeostasis and self- renewal. To initiate signaling pathway, Wnt ligands bind to Frizzeled
receptors which in turn activates transmembrane co-receptors LRP5/6.This results in
Dishevelled (Dsh) protein recruitment. The downstream signaling pathway is then divided into
canonical and non-canonical pathways. Activated LRP5/6 then recruits the protein Dishevelled
(Dsh), at this point Wnt signaling can branch into two different path ways, a canonical and non-
canonical pathway. In the canonical pathway β-catenin gets degraded upon phosphorylation by
a protein complex of the adenomatou polyposis coli (APC) tumor suppressor protein, Axin, and
the glycogen synthase kinase, GSK3β. In the presence of Wnt, unphosphorylated β-catenin gets
41

accumulated in the nucleus. It then binds to TCF/LEF (T-cell factor/lymphoid enhancing factor)
to activate downstream target genes.
110
Also, its role in cancer in different tissues has been
proposed in various studies especially via inherited or sporadic mutations in APC tumor
suppressor gene. While APC mutations or mutations in the gene encoding β-catenin may lead
to colorectal adenomas, loss of function mutations in APC or gain of function mutations in β-
catenin are often rare in pancreatic cancer.
50
Based on the role that Wnt signaling pathway
plays in development of pancreatic cancer, researchers should try to target the pathway in
different levels such as by antibodies against Fzd or other antitumor agents. According to
clinicaltrials.gov in phase 1 clinical trials of patients with pancreatic solid tumors PRI-724
inhibitor is being used to block the interaction of β-catenin with CBP.

The MET Signaling Pathway
The MET Signaling Pathway plays a key role in modification of cellular proteins,
intercellular junctional molecules and the cell cytoskeleton that leads to epithelial
mesenchymal transition (EMT). Such transition integrates several pathways that control cell
proliferation essential for normal processes such as embryogenesis organ regeneration, wound
healing and cancer invasion. In this signaling pathway MET which is an integral plasma
membrane protein expressed by progenitors as well as epithelial and endothelial cells, is
activated when it is bound to HGF ligand through its extracellular domain. Binding of the
hepatocyte growth factor to MET and receptor dimerization and transphosphorylation of two
tyrosine residues will stimulate receptor tyrosine kinase activity of MET. Consequently further
phosphorylation of two additional docking tyrosines in the carboxyl terminal tail leads to
42

recruitment of signaling molecules. MET signaling activates other downstream pathways such
as MAP kinase PI3K-AKT, STAT, and NF-κB pathways resulting in genetic expression. In terms of
cancer, germline mutations and activating mutations of MET have been identified in hereditary
and sporadic cancers respectively.
88
On the other hand overexpression of MET has been
related to cancers in different organs such as pancreas.
38
Based on MET signaling role in
tumorgenesis and cancer progression, It is considered as one of the important targets in
therapy. Many preclinical and clinical studies have been focusing on such targeted drug
development. For example MET inhibition or ligand neutralization may decrease tumotgenic
and metastatic characteristics of pancreatic cancer.

The TGF-β Signaling Pathway
TGF-β cytokine controls cell differentiation, proliferation, and angiogenesis during
embryonic development and later in adult tissues. To initiate signaling, TGF-β ligands interact
with two receptors, TGFβRI and TGFβRII. Upon interaction of the ligand with TGFβRII, TGFβRI
gets phosphorylated and recruited, which further leads to activation and phosphorylation of
SMAD2 and SMAD3. Phosphorylated SMAD2 and SMAD3 then combine with SMAD4 to move to
the nucleus.

In the nucleus, this complex along with cofactors regulates genetic transcription. In
case of pancreatic cancer SMAD4 may be inactivated through homozygous deletion or
mutation.
73
It is suggested that such inactivation occurs more frequently in later stages of the
disease which is associated with worse prognosis.
70
Besides SMAD4 which is involved in TGF-β
signal transduction, inhibitory members such as SMAD 6, 7 have been upregulated in half of
43

pancreatic cancer cases.
8
The role of this signaling pathway is more complex than other
pathways since it has growth inhibitory function in early stages of tumor development which is
switched to promotion of invasion and metastasis in the later stages. Based on such
information it is of great importance to target this signaling pathway in pancreatic cancer. As a
result blocking production of TGF-β ligands and inhibition of kinase activity of the receptors
may be appropriate. Interestingly combined therapies of small-molecule inhibitors with
immune-stimulating vaccines or a phosphorothioate antisense mRNA targeting TGF-β2 ((AP
12009) are being utilized.
102
Further evaluation of these TGF-β inhibitory agents is necessary for
development of agents with better efficacy in clinical trials.
Figure8. Pancreatic Cancer Signaling Pathways
44

4.2.3 Genetic Mutations in Pancreatic Precursor Lesions
Over the past few years scientists' knowledge of the genomic alterations in sporadic
pancreatic cancer has been improved. It has been reported that pancreatic lesions such as
PanIN and IPMN as well as pancreatic ductal adenocarcinoma present altered genes. These
genes may show gain of function or loss of function mutations. Minimally dysplastic epithelium
PanIN1A and B may progress to higher grade PanIN- 3.These mutations in the lesions along with
mutations of oncogenes and tumor suppressor genes as discussed earlier may further lead to
development of malignant tumors in the pancreas.

Genetically engineered animal models with
such mutations will further support the idea that such genetic alterations may lead to cancer
development in animal models. Studies have also identified the relevance of such mutations in
different lesions and malignancies. For example it is reported that KRAS mutations is seen 36 %
in PanIN-1A, 44 % in PanIN-1B, 87 % in PanIN- 2/3, and >90 % in PDAC cases. This further
suggests that K-RAS mutation is more involved after PanIN initiation rather than initiation of
tumorigenesis.
66
Genetic mutations, promoter methylation and allelic deletion of p16 is also
observed in 30 % of PanIN-1, 55 % of PanIN-2 and 70 % of PanIN-3.
66
One mutation which is usually identified in the more advanced lesions with features of
dysplasia is loss of function of CDKN2.
13
Another abnormality which is detected at later stages
of PanINs is nuclear p53 accumulation. Retention of p53 tumor suppressor in lower grade
lesions and its occurrence at later stages would further support the idea that pancreatic cancers
are very uncommon even with common lower grade lesions especially in older population.
32
45

Besides all these genetic mutations in precursor lesions and malignant tumors, chromosomal
instability which was discussed earlier in this chapter is observed in the progression of lower
grade lesions to invasive adenocarcinoma of the pancreas. Moreover, telomer shortening in the
early stages of pancreatic carcinogenesis is evident in more than 90 % of low-grade PanIN
lesions leading to chromosomal instability and neoplasia.
106
Similarly the genomic profiling of
other lesions such as IPMNs and MCNs have been reported to have inactivation of RNF43 , a
gene encoding for RING domain containing ubiquitin ligase in almost 50% of cases. RNF43
protein functions as a Wnt pathway inhibitor as a result inactivation mutations of RNF43 along
with activation of CTNNB1 in a subset of IPMNs lead to Wnt activation through which
researchers became able to differentiate cystic neoplasms from usual ductal adenocarcinoma.
59

In conclusion, tremendous advances have been achieved over the last few years in the
knowledge of the genomic alterations in sporadic pancreatic cancer.

46

Chapter 5: Genetically engineered mouse models of PDAC (literature overview)

In the last few years, based on genetic studies of activating mutations in K-Ras or TGF-β
and/or inactivation of tumor suppressors such as p53, INK4A/ARF BRCA2 and Smad4,
genetically engineered mouse models of pancreatic neoplasia have been developed to mimic
the disease. Pathogenic changes from intraepithelial neoplasia to progressive lesions of invasive
and metastatic disease have been developed in the transgenic mice to characterize cellular and
molecular pathology of the neoplasia. This further helps the researchers to investigate new
therapeutic approaches and treatments. Among the important models for pancreatic cancer,
are genetically engineered mouse models of prenatal PDAC, genetically engineered mouse
models of postnatal PDAC and mouse models of hereditary pancreatic cancer. As mentioned
earlier the activating mutation of the K-Ras oncogene is the most common alteration in
pancreatic cancer. As a result genetically engineered mouse models studied are mostly based
on the K-Ras oncogene leading to development of human pancreatic cancers. There are several
genetically engineered mouse models in pancreatic cancer. This section is only briefly focusing
on prenatal, postnatal hereditary models of the disease in mice based on the literature.
Mouse models of PDAC: Prenatal
In the beginning, attempts for expression of K-Ras in ductal cells were not successful. The
first model to induce PDAC in mice which was similar to human PDAC was developed by
expressing knocked-in KRasG12D oncogene during the embryonic development of pancreatic
lineages.
63
The LSL-KrasG12D mouse has been developed for the K-Ras model, which has a
47

glycine to aspartic acid transition in codon 12. Upstream of this codon is a conditional STOP
cassette flanked by LoxP sites that prevents the mutant allele expression. Result of combination
with a tissue-specific Cre is excise of the STOP cassette leading to activation of K-Ras at
physiological levels which is the most frequent activating K-Ras mutation in human PDAC. It is
reported by crossing LSL-KrasG12D mice with transgenic mice expressing a bacterial Cre
recombinase and with either the Pdx1 or Ptf1a (P48) promoters in control, PanIN lesions were
developed. Such lesions showed similar markers to human PDAC such as the Notch signaling
target Hes1, COX2 and MMP7. In this model PanIN lesions progress with long latencies. As a
result addition of mutations in loci encoding tumor suppressors such INK4A, TP53, LKB1 or
SMAD4 accelerate the progression of these PanIN lesions to invasive or even metastatic lesions.
Notch or Hedgehog signaling pathways are important in progression of pancreatic cancer. The
role of Notch signaling which is upregulated in pancreatic cancer has been studied in genetically
engineered mouse models. It has been reported that Expression of an active form of Notch1
(NIC) in Kras mouse models leads to formation of PanINs.
36
besides the Notch pathway,
Hedgehog pathway is also important in development of PanINs due to elevated expression of
canonical Gli target genes, which may further result in cancer development.
102

Mouse models of PDAC: Postnatal
The previously discussed model of PDAC is of great importance. However there are
limitations. As we know PDAC is not a pediatric disease and tumors arise mainly due to
mutations in adults. Also K-Ras mutations appear not in all but only in specific cells of pancreas.
Such limitations led to development of another mouse model. This model was generated by
48

crossing mice carrying a knocked-in KRasLSLG12Vgeo allele with double transgenic mice (Elas-
tTA; Tet-O-Cre) that express a Cre recombinase under the control of the Elastase promoter
following an inducible Tet-Off strategy.
53
In such model expression of K-Ras oncogene would be
in a controlled temporal manner. The mouse model develops the knocked-in KRasG12V
oncogene in some of the acinar cells during embryonic development
53
suggesting acinar and
not ductal development of PDAC at one year of age. Addition of mutations in INK4a and TP53
tumor suppressors further decreases tumor latency while increasing rate of death in mice
during early development.
52
However, expression of resident K-Ras oncogene in 60 day old did
not develop lesions even in presence of tumor suppressor genes.
52
Another activation mutation
is BRAF or PIK3CA mutation. Even though uncommon in PDAC,

such mutations would activate
K-Ras downstream effectors leading to PDAC development. It has been reported that
expression of the BRafV600E mutation will lead to embryonic death. It also leads to PanIN
development but not PDAC when expressed upon activation of the Pdx1-CreERT2 transgene by
exposure of P14 mice to tamoxifen.
29
In case of PIK3CA activation mutation (PIK3CAH1047R)
under the control of the same Pdx1-CreERT2 transgene failed to develop PanINs. This further
suggests that due to importance of RAF/MEK/ERK signaling pathway in tumor initiation,
therapeutic strategies should be investigated.

49

Mouse models of hereditary pancreatic cancer
Pancreatic cancer occurs with a higher risk in families with the disease especially when
families carry the germ line mutations in genes such as BRCA2, CDKN2A/P16INK4a, STK11/LKB1,
PRSS1 and PALB2.
65
This suggests the importance of mouse models of hereditary pancreatic
cancer. Such mouse models have been developed by crossing the LSL-KRasG12D with mice with
truncated Brca2 locus leading to tumors with shorter latency compared to the ones with wild
type Brca2 alleles. More importantly, these tumors retained the wild typeBrca2 allele,
indicating that LOH is not an essential requirement for tumor development.
94
In a similar study
however, homozygous inactivation of Brca2 in a KRasG12D background led to the generation of
acinar carcnomas, not PDAC.
84
Addition of a conditional Lkb1 floxed allele to the KC LSL-
KRasG12D mouse model also leads to increased PanIN numbers and decreased latency without
detectable LOH. Interestingly, homozygous loss of Brca2 or Lkb1 in early pancreatic precursors
in the absence of KRas oncogenes has distinct consequences. Whereas ablation of Brca2 does
not induce histological alterations.
84
A very important conclusion would be that there are
multiple important pathways to control transformation of pancreatic cells. Knowing that
pancreatic cancer is not one gene one disease, there may be various possible genetically
engineered mouse models that could be developed for the disease to further improve research
in the field of drug development. Also incorporation of human immune sys to genetically
engineered mice models may help us obtain a more biologically appropriate model to study the
disease and drug effects.

50

Chapter 6: An Overview on Current Drugs and Trials

Pancreatic cancer is a systemic disease which usually develops abdominal metastasis.
Even in the 20% of cases which has the solitary localized tumor, distant metastasis is frequent.
Unfortunately up to mid-1990s the available drugs for palliative chemotherapy such as 5-
fluorouracil (5-FU) solely or in combination with other drugs such as methotrexate, vincristine,
cyclophosphamide, cisplatin, mitomycin C, or doxorubicin for these patients, shown high
toxicity and poor long term survival.
11
Generally no successful treatment has been reported
with the use of 5-FU in a palliative situation of pancreatic cancer.
5
other drugs have been later
suggested a better result.
6.1 Current Drugs
First line chemotherapy
Pancreatic cancer is a very complicated cancer genetically, with several genetic
abnormalities. As mentioned before K-RAS is often considered the signature mutation in
pancreatic cancer and occurs in majority of cases, but there are also other abnormalities in
many other pathways such as Hedgehog, SMAD4 and p16.This heterogeneity suggests that in
order to come up with solutions to treat the disease in the future, combination therapy is what
should be the focus of researchers. Based on Xenografts each individual shows a different
profile of genetic changes including deletions, amplifications and mutations in the key pathways
which were discussed earlier. Consequently personalized therapy based on individual profiles
should be designed. As it is known pancreatic cancer defies most treatments and patients with
51

the metastatic disease usually lose the battle six months after diagnosis. Consequently any new
progress in metastatic pancreatic cancer is always welcome. First-line chemotherapy with
Gemcitabine has been used for patients with either locally advanced or metastatic pancreatic
cancer following the drug’s approval in 1997. It is suggested to be standard of care for the
disease. Gemcitabine was studied in a relatively small study with only 126 patients. Despite
that, the results were clear, with a one-year survival of 18% with gemcitabine versus 2% with 5-
FU.
56

Second-Line Chemotherapy
A number of relatively large phase III studies of gemcitabine with a second cytotoxic agent
have been conducted in the subsequent years which mostly had little success in improvement
in survival outcomes. Up until now the only combination approved by FDA is the
Gemcitabine/Erlotinib (Tarceva). The drug ABRAXANE which is an albumin-bound form of
paclitaxel is manufactured using patented technology and is formulated with albumin, a human
protein to enhance delivery of the drug to tumors and reduce side effects. It is reported to
prolong the lives of patients with advanced pancreatic cancer by almost two months in one
clinical trial called Celgene’s trial. In this trial, patients were given Abraxane in combination with
gemcitabine. Survival has been reported with median of 8.5 months, compared with 6.7
months for those who received gemcitabine alone. Further survival in the first and second year
was 35 and 9 percent compared with 22 and 4 percent for those under gemcitabine treatment.
The results of the study which involved 861 patients were released by Celgene and the
American Society of Clinical Oncology. In September 2013, the FDA approved ABRAXANE in
52

combination with gemcitabine as first–line treatment of patients with metastatic
adenocarcinoma of the pancreas. This was a better success compared to the 2011 study
published in the New England Journal of Medicine which reported FOLFIRINOX with a four
months longer survival compared to gemcitabine alone. Also in some cases of un-resectable
pancreatic cancer chemo-radiation enhances overall survival in comparison to chemotherapy
alone.
105

6.2 Treatment Strategies and Clinical Trials
Targeting VEGF receptor
As mentioned earlier, combination therapies have had little success as a course of
treatment for pancreatic cancer. As a result different strategies have been studied to enhance
the outcome of the treatment. One of the most important strategies being studied are anti-
angiogenic treatment strategies of the pancreas adenocarcinoma. So far, several agents
developed to inhibit tumor angiogenesis. The goal is to inhibit the extensive growth of growth
factors with high intrinsic tyrosine kinase activities that induce cell proliferation, dissemination,
and neo-angiogenesis. It has been reported that angiogenesis involves cytokines such as
vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and
angiopoietin 1.
10, 20
It has been reported that VEGF and its two principal receptors were
expressed in pancreatic cancer. These receptors are associated with disease progression. For
example there is an association between VEGF-R2 and poor prognosis. As a result patients with
this receptor mainly express the more aggressive form of the disease.
24
VEGF inhibition
53

strategy suggests decreased pancreatic cancer growth by inhibition of neovascularization by
targeting the nutritional support of tumor cells. It also leads to reduction of interstitial pressure
within the tumor and thereby increases the delivery of chemotherapeutic agents. Consequently
anti-angiogenic agents have additive effect on conventional therapies. Small molecule tyrosine
kinase inhibitors such as PD173074 block VEGF-R2 as well as bFGF R1 signaling and thus have
anti-angiogenic effect. This small molecule inhibits the cell progression at the G0/G1
transmission point leading to increased apoptosis.
100
However, data for patients is not
available for such study. Another potential strategy would be to create antitumor activity by
inhibition of several pathways such as epidermal growth factor receptor (EGFR) and the VEGF-
receptor pathways by means of different chemotherapeutic agents or small molecules. Pre-
clinical studies also show the effect of combination therapy with the mammalian target of
Rapamycin (mTOR) which is a serine-threonine kinase and an anti-VEGF antibody in growth
inhibition.
100
another approach would be designing small molecules with effect on multiple
pathways. In pre-clinical models of pancreatic cancer in human an MMP inhibitor called
Marimastat, has been demonstrated to inhibit tumor growth.
26
Besides pre-clinical studies,
there are several clinical studies using anti-angiogenic agents for inhibition of growth in
patients with adenocarcinoma of the pancreas. According to clinicaltrials.org in a phase ll study
using combination of Bevaczumab (10 mg/kg every second week) with gemcitabine as the
Partial response was seen in 21% and 46% of patients became stable. This trial showed a
median progression-free survival of 5.4 months and a median overall survival of 8.8 months
which was better than Gemcitabine alone. Even though the trial proceeded to phase lll, it
stopped since it did not support the superiority of Bevacizumab plus gemcitabine.
54

Targeting the Epidermal Growth Factor Receptor
As it is known EGFR is a cell surface receptor which belongs to the family of protein
tyrosine kinases that have a big influence on the cellular regulation of growth, differentiation
and apoptosis by regulating signaling pathways. In pancreatic cancer overexpression of this
receptor is associated with the aggressiveness of the disease.
114
As a result therapeutic targets
that inhibits such receptor alone or in combination with first- line chemotherapy or
radiotherapy or both should be suggested. EGFR also induces vascular endothelial growth
factor and therefore is important in tumor vascularizaton.
46
To prevent the effect of this
receptor small molecules such as Erlotinib which is a tyrosine kinase inhibitor have been
developed for pancreatic cancer and other malignancies. The other strategy to inhibit EGFR is
by prevention of ligand binding to the receptor by designing monoclonal antibodies such as
cetuximab which has been used in case of pancreatic cancer. There are several pre-clinical
studies in which animals were treated. Such as with a combination of Gemcitabine,
Wortmannin which is a kinase inhibitor and intravenous erlotinib.
74
in a phase lll clinical trial
patients were treated with gemcitabine plus Erlotinib from Roche Pharmaceuticals and
compared the results with gemcitabine plus placebo. Patients received either gemcitabine
1,000 mg/m2 weekly on days 1 to 43, followed by 1 week‘s rest and then, on days 1, 8, and 15
of a 4-week cycle with Erlotinib, given at a dose of 100 or 150 mg/day orally or plus placebo.
According to clinicaltrials.gov website, the trial resulted in progression-free survival with no
major toxicity issue in the next eight weeks in 50 of the patients.

This led to authorization of
this combination therapy in USA in 2006. Cetuximab which is an anti-EGFR antibody has also
been combined with standard gemcitabine chemotherapy in pancreatic cancer. A phase II trial
55

has been performed in 41 patients, receiving Cetuximab (initial dose 400 mg/m2, then 250
mg/m2 weekly) followed by gemcitabine 1,000 mg/m2 weekly for 7 weeks plus 1 week‘s rest.
Subsequent cycles were 4 weeks long with gemcitabine given on days 1, 8 and 15 and a rest on
day 22. Patients were treated until progression or intolerable toxicity. These data showed a 1-
year survival rate of 18% and a 1-year progression-free-survival of 9%, which further proved the
superiority of combination therapy compared to Gemcitabine monotherapy.
22

Targeting NF-κB
With all the research on pancreatic cancer treatment strategies are still poorly
effective. During the past few years scientists have paid much attention to the transcription
factor nuclear factor kappa B (NF-κB) which determines chemoresistance of pancreatic cancer
to therapeutic agents. In normal conditions it resides in the cytoplasm but upon activation by
cytokines, growth factors or viral proteins, the IκB kinase (IKK) complex becomes activated and
phosphorylates IκB proteins. Upon polyubiquitination, followed by degradation by the 26S-
proteasome, NF-κB becomes released from IκB and translocates into the nucleus where it acts
as a transcription factor which is important in variety of cellular processes such as proliferation,
apoptosis, cell growth and differentiation. It also leads to expression of pro-inflammatory and
pro-oncogenic proteins such as inducible nitric oxide synthetase (iNOS), IL-1β, IL-8 or cyclin
D1.
91
Expression of k-Ras oncogene and epidermal growth factor (EGF) receptor might
contribute to tumor progression and aggressiveness due to activation of NFκB.
71, 91
Generally
inhibition of activation of NF-κB act can occur with three different approaches that are
Inhibition of NF-κB protein expression, interference with DNA binding of NF-κB and inhibition of
56

its activation. One of the most promising approaches is to inhibit the activation of protein by a
proteasome inhibitor, Bortezomib. Bortezomib sensitizes tumor cells to apoptosis in
combination with other therapeutic agents such as Gemcitabine and Docetaxel for which there
was no good result reported.
35
Another potential agent is Anticancer agent CHS 828 which
suppresses nuclear factor-kappa B activity in cancer cells through down-regulation of IKK
activity. Until now, two phase I studies have been conducted evaluating the maximum tolerated
dose and toxicity of CHS-828 in solid tumors but neither of those focused on pancreatic
cancer.
81
Consequently it will be worthwhile to use combination of CHS-828 or improved forms
of it with less toxicity and Gemcitabine as the first-line treatment in patients with pancreatic
cancer.
Immunotherapeutic Strategies
In pancreatic cancer patients, tumor-specific T4 and T8 cells as well as antibodies
against tumor-associated antigens such as MUC-1 have been identified which suggests the
immunogenicity of pancreatic cancer.
89
However, since pancreatic cancer is generally
diagnosed in a late stage, the tumor must have found ways to overcome the hosts immune
response through escape mechanisms such as Secretion of transforming growth factor (TGF)-
beta, interleukin (IL)-6 and IL-10.
15
As a result immunotherapy has been suggested to be a
potential to enhance immune response in pancreatic cancer patients. One of the most effective
strategies in cancer immunotherapy is use of monoclonal antibodies, which are directed against
growth factors or their receptors. In case of pancreatic cancer, antibodies are being tested in
clinical trials. For example EGFR antibody, Matuzumab, is under evaluation.
47
According to
57

clinicaltrials.gov , Bevacizumab antibody that binds to free VEGF and may therefore reduce
VEGF receptor (VEGFR) activation and neovascularization was used in a phase ll trial in
combination with gemcitabine for which superior efficacy was reported.

Peptides Vaccine
Peptide vaccines derived from tumor-associated antigens have been reported
somehow effective in various studies. For example a peptide vaccine derived from gastrin
which is a growth factor in pancreatic cancer cells, has been reported to enhance immunity and
recruitment of dendritic cells in a phase II study of the vaccine.
23

As mentioned earlier, about 90% of pancreatic adenocarcinomas show ras mutations.
Therefore researchers have also focused on development of mutated Ras-peptide vaccine.
Immune response was produced and measured by mutated Ras-specific IgG levels but it is still
unclear whether it can be transferred to clinical responses in patients.
27
MUC-1
mucopolysaccharide which is usually expressed in the apical area of ductal pancreatic cells have
been used to form vaccines in the form of antibody by Freund‘s adjuvants which passed phase I
trials.
79

Besides peptide vaccines, viral vector and immunocytokines may be used to develop antibodies
against a tumor associated antigen in pancreatic cancer. Although several novel targets have
been developed in the field of antitumor immunity which has immunological response and
survival effects, Immunotherapy has not yet replaced chemotherapy and can be only used in
combination with chemotherapy, radiotherapy or both. Even though there have been many
studies and trials, the field of research in pancreas cancer remains wide open with the need of
58

better understanding of mutations in each individual, and better study designs that may
proceed to phase lll trials. Based on multiple genetic alterations associated with the disease,
the development of multiple targeted therapies that could be delivered with the knowledge of
the tumor’s molecular abnormalities would be the goal. Unfortunately there will always be a
challenge in development of combination therapies. Pharmacokinetic, pharmacodynamics,
potential drug interactions that may increase toxicity as well as economic factor are among the
challenges that should be taken in to consideration.

59

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Creator Aletomeh, Yasaman (author)

Core Title Pancreatic cancer: a review on biology, genetics and therapeutics

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School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 11/17/2014

Defense Date 12/17/2014

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cancer biology,genetics,OAI-PMH Harvest,pancreatic cancer,therapeutics

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Language English

Advisor Haworth, Ian S. (committee chair), Neamati, Nouri (committee member), Okamoto, Curtis Toshio (committee member)

Creator Email yaletome@usc.edu

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Abstract (if available)

Abstract Pancreatic cancer is the fourth leading cause of cancer death in the United States and remains a major unsolved health problem in the 21st century. Since it lacks specific symptoms in the beginning of its development and diagnostic methods are limited, the disease is often detected during its formative stages. As a result, the disease prognosis is extremely poor. Consequently not many patients have resectable disease and survival rate is low. Even though combination therapies may have short-term effects, conventional treatments such as surgery, radiation, chemotherapy, or combinations of these, has little long-term impact on pancreatic cancer due to its intense resistance to all extant treatments. Almost all patients who have pancreatic cancer develop metastases and lose their battle to cancer. Researchers have been studying the challenges in the field addressing many hurdles and opportunities. They have realized that developing a detailed understanding of the molecular biology and genetics of pancreatic cancer, generating refined animal models and investigating signaling pathways involved in cellular growth, survival and differentiation during the disease course will be effective in analysis of this complex disease. Such strategies seem to hold a great promise in diagnosis prevention and treatment of this disease.