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Effects of moderate alcohol intake and role of stearoyl-CoA desaturase on the development of pancreatic tumors

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Effects of moderate alcohol intake and role of stearoyl-CoA desaturase on the development of pancreatic tumors

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Effects of moderate alcohol intake and role of stearoyl-CoA desaturase on the development of
pancreatic tumors

By

Kaitlin Skrypek

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(EXPERIMENTAL AND MOLECULAR PATHOLOGY)

December 2020

Copyright 2020 Kaitlin Skrypek
ii
Table of Contents
Abstract .....................................................................................................................................iv
Part I ..........................................................................................................................................v
Moderate alcohol intake promotes pancreatic ductal adenocarcinoma development in mice
expressing oncogenic Kras ................................................................................................................ v
Abstract .....................................................................................................................................vi
Introduction............................................................................................................................... 1
Materials and Methods .............................................................................................................. 5
PDAC mouse models ......................................................................................................................... 5
Histological analysis .......................................................................................................................... 5
Immunofluorescence staining ........................................................................................................... 6
RNA-seq analysis .............................................................................................................................. 6
Statistical Analysis ............................................................................................................................ 7
Results ....................................................................................................................................... 8
Oncogenic mutated Kras expression induces PanIN formation and PDAC development .............. 8
Moderate alcohol consumption induces tumor formation in Pdx1
Cre
;LSL-Kras
G12D
mice .............. 9
Alcohol up-regulates mRNA expression of genes involved in tumor progression......................... 10
Figures .................................................................................................................................... 12
Figure 1 ........................................................................................................................................... 12
Figure 2 ........................................................................................................................................... 13
Figure 3 ........................................................................................................................................... 14
Figure 4 ........................................................................................................................................... 15
Figure 5 ........................................................................................................................................... 16
Discussion ............................................................................................................................... 17
Part II ...................................................................................................................................... 21
Inhibition of stearoyl-CoA desaturase suppresses growth of pancreatic tumors in vitro and in vivo
......................................................................................................................................................... 21
Abstract ................................................................................................................................... 22
Introduction............................................................................................................................. 23
Mice ................................................................................................................................................. 27
Immunofluorescence staining ......................................................................................................... 27
Tumor organoid culture ................................................................................................................. 27
Treatment of tumor organoids ....................................................................................................... 29
iii
Quantitative polymerase chain reaction (qPCR) ........................................................................... 29
Detection of apoptosis in pancreatic tumors .................................................................................. 30
Statistical Analysis .......................................................................................................................... 30
Results ..................................................................................................................................... 31
Isolated EPCAM+ pancreatic tumor cells form organoids in matrigel culture ............................ 31
The inhibition of SCD induces degeneration of tumor organoids in vitro ..................................... 32
Inhibition of SCD in tumor organoids induces the unfolded protein response (UPR) ................. 33
Inhibition of SCD induces pancreatic tumor cell death via apoptosis in vivo ............................... 34
Tables ...................................................................................................................................... 35
Table 1 ............................................................................................................................................. 35
Figures .................................................................................................................................... 36
Figure 6 ........................................................................................................................................... 36
Figure 7 ........................................................................................................................................... 37
Figure 8 ........................................................................................................................................... 38
Figure 9 ........................................................................................................................................... 39
Discussion ............................................................................................................................... 40
Conclusion .............................................................................................................................. 47
References ............................................................................................................................... 48

iv
Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a relatively rare cancer type, but is the fourth-
leading cause of cancer death in the U.S. Although chemotherapy and immunotherapy have been
used for patients with PDAC, the overall five-year survival rate is 8% and there is an urgent need
for effective therapies. Genetic risk factors, such as Kras, have been identified as key mutations
that induce PDAC development. Environmental risk factors including obesity, pancreatitis, and
tobacco smoking have also been discovered to promote PDAC. Heavy alcohol drinking has been
suspected to be a risk factor due to its role in causing pancreatitis, but it has not been known if
moderate alcohol drinking could promote PDAC. In our study, we examined the effects of a
moderate alcohol diet on the development of pancreatic neoplasia and invasive PDAC in mice
expressing oncogenic Kras. We found that moderate alcohol drinking induces the formation of
advanced neoplasia as well as PDAC compared to a regular chow diet in Kras-expressing mice.
Our data indicate that moderate alcohol intake is a risk factor for pancreatic cancer. RNA-seq
analysis suggests up-regulation of stearoyl-CoA desaturase 1 (SCD1) in pancreatic tumors
developed in mice fed alcohol. SCD is an enzyme localized to the endoplasmic reticulum that
functions by converting saturated fatty acid to monounsaturated fatty acid. We found that the
inhibition of SCD disrupts lipid metabolism in pancreatic tumor cells, leading to their death in
vitro and in vivo. Our data shows the selectivity of tumor cell death by SCD inhibition, making it
a novel target for PDAC suppression.

v

Part I

Moderate alcohol intake promotes pancreatic ductal adenocarcinoma
development in mice expressing oncogenic Kras

vi
Abstract

Mutations in genes, such as Kras, account for the genetic influences in pancreatic ductal
adenocarcinoma (PDAC) development. Environmental factors including tobacco smoking,
pancreatitis, diabetes, and obesity are known risk factors contributing to PDAC pathogenesis, but
the impact of moderate alcohol consumption on PDAC still remains elusive. In the present study,
we investigated the effects of moderate alcohol feeding on the promotion of pancreatic tumors
using Pdx1
Cre
;LSL-Kras
G12D
mice that spontaneously develop pancreatic intraepithelial neoplasia
(PanIN). Following feeding with a liquid diet containing 3.5% alcohol for 5 months,
Pdx1
Cre
;LSL-Kras
G12D
mice had developed advanced PanIN and PDAC compared to those fed
regular chow. We tested the effects of a Western diet on Pdx1
Cre
;LSL-Kras
G12D
mice as well,
exhibiting similar effects on pancreatic tumor development comparative to alcohol feeding.
RNA-Seq analysis revealed that alcohol intake increases the expression of tumor markers
(Epcam, Krt19, Prom1) and cytokines (Tgfb1, Tnf) in the pancreatic tumor. Alcohol decreased
the expression of genes Fgf21 and Il6 in pancreatic tumor tissue. Our data indicates that
moderate alcohol intake is a risk factor for PDAC development.

1

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the 4
th
leading cause of cancer death in the United
States (Chiaravalli et al., 2017; Siegel et al., 2018) and is predicted to become the 2
nd
leading
cause of cancer death by 2030 (Rahib et al., 2014). PDAC is a relatively rare cancer type, but
threatening due to its lack of sensitive diagnostic markers. This leaves a large majority of
patients, about 80%, not eligible for surgical resection (Kleef et al., 2016). Chemotherapy and
immunotherapy have been used to treat patients with PDAC yet the overall five-year survival
rate remains at 8%. The combination of these elements indicates the necessity for alternative,
more effective therapies.
The pancreas functions in both the digestive and endocrine system. The digestive
function is facilitated by the exocrine portion of the pancreas. Acinar cells secrete digestive
enzymes and bicarbonate through pancreatic ducts to the duodenum. The endocrine portion of
the pancreas consists of the islets of Langerhan’s. The islets function is to secrete hormones and
regulate blood glucose levels. Pancreatic ductal cells were believed to be the origin of PDAC due
to its ductal phenotype, but this was disproven when cell lineage tracing studies were conducted
and instead indicated that the major source of PDAC in mice were acinar cells (Kopp et al.,
2012). For acinar cells to differentiate into ductal-like cells, they need to undergo acinar-ductal
metaplasia (ADM). This process is an early stage in PDAC development that gives rise to the
early pancreatic lesions, PanINs (Liou et al., 2013). PanINs gradually lose their polarity and their
nuclei become pleomorphic and eventually invasive PDAC develops, which has a high
metastasizing capacity (Morris et al., 2010).
Over 90% of PDAC patients show oncogenic mutations of KRAS GTPase (di Magliano
2
and Logsdon, 2013). Studies have shown that by inducing the overexpression of oncogenic
mutated KRAS in the progenitor cells of a mouse pancreas, this will lead to the development of
early preinvasive lesions and eventually invasive PDAC (Hingorani et al., 2013). Pancreatic and
duodenal homeobox 1 (Pdx1) is expressed in progenitor cells that give rise to all of the mature
cells of the pancreas. The Cre-recombinase system is used to insert a Lox-STOP-Lox (LSL)
construct, a LoxP site, into the KRAS locus that has been modified to contain an amino acid
switch from Glycine to Aspartic acid in codon 12 (Kras
G12D
). When mice expressing this
mutation are interbred with mice that express the enzyme Cre recombinase from the specific
progenitor promoter Pdx1, the Pdx1
Cre
; LSL-KRAS
G12D
mouse is generated. These mice
endogenously express the KRAS mutation only in epithelial cells of the pancreas (Hingorani et
al., 2013).
Throughout tumor formation, important phenotypical markers that are specific to
pancreatic cancer can be detected by immunohistochemical staining. These include keratin 19
(KRT19), epithelial cell adhesion molecule (EPCAM), prominin-1 (PROM1/CD133), and
doublecortin-like kinase 1 (DCLK1). KRT19 is an intermediate filament protein that plays an
important role in maintaining the structural integrity of epithelial cells. It is expressed in normal
tissue, but is found to be overexpressed in cancer tissue including breast, colon, liver, and
intestinal cancers (Saha et al., 2018). KRT19 is postulated to play a vital role in malignant
transformation and the mechanism by which this is achieved has been widely studied. It is
suggested that KRT19 expression and differentiation may be important in the reprogramming of
cancer stem cells (CSCs) (Brembeck and Rustgi, 2000). CSCs are cells within tumors that have
traits in self-renewal, differentiation, tumorigenicity, and play a role in metastasis (Yu et al.,
2012). EPCAM is a transmembrane glycoprotein found in epithelial cells that facilitates in cell
3
adhesion and also contributes to signaling, migration, and proliferation of tumors. EPCAM is
expressed in epithelial cells in normal tissues and is a known marker of progenitor cells and stem
cells. EPCAM is overexpressed in proliferative cells compared to differentiated cells and has
been postulated to be a marker for CSCs in human cancers such as pancreatic, colorectal, and
hepatocellular carcinoma (Huang et al., 2018). PROM1 is another transmembrane glycoprotein
and the most frequently used cell surface protein to detect CSCs from multiple solid tumors in
cancers including brain, colon, lung, liver, and pancreas. It’s expression is localized to the
surface of adult stem cells in normal tissues. It is similar to EPCAM in its role in CSC
proliferation and regulation in that they both induce the Wnt signaling pathway (Glumac and
LeBeau, 2018). DCLK1 is a kinase protein that is normally expressed in tuft cells of the
epithelium and a normal stem cell marker of the intestines (Nakanishi et al., 2013). DCLK1 has
been observed to be overexpressed in pancreatic cancer and indicates that these DCLK1+ cells
are capable of initiating tumorigenesis through Ras activation (Qu et al., 2019). DCLK1 is found
to be co-expressed with other known CSC markers further proving its potential role in
tumorigenesis and invasion in the presence of mutation (Qu et al., 2019). The observation of the
expression or overexpression of these proteins is vital in proving our mouse model is sufficient
in developing invasive PDAC.
In addition to genetic risk factors, such as mutations in Kras, Trp53, Cdkn2a, Smad4,
and BRCA2 (Hezel et al., 2006), environmental risk factors exist for PDAC as well. Obesity,
type II diabetes, chronic pancreatitis, and tobacco smoking are all established risk factors for
promoting pancreatic tumor development in mice that express mutant Kras (Guerra et al, 2007).
Alcohol drinking has been suspected to be a potential risk factor for PDAC since heavy alcohol
drinking can lead to chronic pancreatitis (Apte et al., 2005). Consumption of large quantities of
4
alcohol can cause fibrosis of the pancreas by increasing the production of transforming growth
factor beta (TGFb) in response to lipopolysaccharide. This sequence of events negatively affects
the gut wall and enhances pancreatic injury (Gu et al., 2013).
In the present study, we explore the potential role of moderate alcohol intake on
pancreatic tumor development and the promotion of PDAC in Pdx1
Cre
;LSL-Kras
G12D
mice. We
observe that alcohol fed mice develop larger tumors of higher grades in the pancreas as well as
PDAC compared to mice fed regular chow. Alcohol up-regulates the expression of important
tumor markers as well as pro-tumorigenic cytokines, indicating moderate alcohol intake to be a
risk factor for PDAC. This experimental data is the first proof of this postulation and is insightful
for future studies involving moderate alcohol drinking.

5
Materials and Methods

PDAC mouse models

Pdx1
Cre
;LSL-Kras
G12D
mice were purchased from Jackson laboratory (Bar Harbor, ME).
Pdx1
Cre
;LSL-Kras
G12D
or Pdx1
Cre
control mice (2-3 months of age) were used for testing
promotion of PDAC in pancreas. Mice were fed regular chow diet (710027; Dyets, Bethlehem.
PA), a Lieber-DeCarli diet containing 3.5% ethanol (710260; Dyets), or a Western diet
containing high-cholesterol and high-saturated fat (710142; Dyets) for 5 months. After this time
period, the pancreas and blood were collected. Plasma samples prepared from the blood were
used for measurement of blood alcohol levels using an AMI Alcohol Analyzer (Analox
Instruments, Stourbridge, UK). The plasma ALT values were measured using an ALT reagent
(Cliniqa, San Marcos, CA) and a PowerWave 200 spectrophotometer (BioTech, Winooski, VT).
All animal experiments were performed in accordance with the National Institutes of Health
(NIH) guidelines under the protocol approved by the Institutional Animal Care and Use
Committee at the University of Southern California.

Histological analysis

Pancreas tissues were fixed with 4% paraformaldehyde at 4
o
C overnight. Fixed tissues were
incubated with 30% sucrose in PBS overnight and were embedded in freezing medium. We
made cryosections (7 µm) with a Cryostat (CM1900; Leica, Buffalo Grove, IL), separated at
least 10 sections from each sample, and stained with hematoxylin and eosin (H&E). Images were
captured with a Nikon 90i microscope and DS-Fi1 digital camera (Nikon, Melville, NY). To
quantify the area of tumors or stroma in the pancreas, 10 images were randomly captured from 4
sections stained with H&E using a 10X objective. The tumor and pancreas areas were measured
6
using NIS-Element software. Two observers blindly examined tumor grades in H&E-stained
sections based on morphology of the tumors.

Immunofluorescence staining

Cryosections were blocked with 5% donkey serum and 0.2% bovine serum albumin for 30 min
and incubated with primary antibodies at 4
o
C overnight. The primary antibodies used included:
CDH1 conjugated with AlexaFluor 488 (100-fold dilution, 560061, BD Biosciences, San Jose,
CA), DCLK1 (100-fold dilution, PA5-20908, ThermoFisher Scientific), PROM1 (50-fold
dilution, 14-1331), EPCAM (100-fold dilution, G88, Developmental Studies Hybridoma Bank,
Iowa City, IA), and KRT19 (50-fold dilution, TROMA-III). The unconjugated primary
antibodies were detected with secondary antibodies conjugated with AlexaFluor 488 and 568
dyes (Thermo Fisher Scientific). The sections were counterstained with DAPI.

RNA-seq analysis

Total RNAs were isolated from the pancreas tissues from the Pdx1
Cre
and Pdx1
Cre
;LSL-Kras
G12D
mice fed with or without alcohol for 5 months using RNeasy kit (Qiagen, Germantown, MD).
Samples were submitted to Genewiz (South Plainfield, NJ) for RNA-Seq. The data were
normalized with the reads per kilobase per million method and analyzed with Flow software
(Partek, Chesterfield, MO). The data were deposited in the Gene Expression Omnibus database
(GSE139357).

7
Statistical Analysis

Statistical tests for the significance of differences were assessed by one-way ANOVA followed
by a Tukey HSD post-hoc test. A p-value of less than 0.05 was considered statistically
significant.

8
Results

Oncogenic mutated Kras expression induces PanIN formation and PDAC
development

We used Pdx1
Cre
; LSL-Kras
G12D
mice to investigate the progression from early benign lesions
into invasive PDAC (Fig. 1A) (Hingorani et al. 2003). From both the control Pdx1
Cre
versus
Pdx1
Cre
; LSL-Kras
G12D
mice, we resected the pancreas along with the spleen and part of the
duodenum to visualize if tumors evolved. The pancreas was enlarged in the Pdx1
Cre
; LSL-
Kras
G12D
mouse compared to the control Pdx1
Cre
mouse indicating presence of a tumor (Fig. 1B).
H&E staining allowed us to characterize the progression of pancreatic tumors within the tissue.
Tissue from the Pdx1
Cre
control mouse pancreas illustrates acinar cells and islets of Langerhan’s
that have not gone under any morphological changes (Fig. 1C). In the Pdx1
Cre
; LSL-Kras
G12D

mouse, the acinar cells of the pancreas begin to undergo ADM, the preliminary stage of
pancreatic tumor development. Acinar cells gradually lose their original morphology and form a
ductal-cell phenotype (Fig. 1C). Following this process of metaplasia, preinvasive lesions,
PanIN, begin to develop as polarity is lost and their nuclei become pleiomorphic. PanIN stages
ranging from 1 to 2 begin to show cellular atypia and stromal reactivity (Fig. 1C).
Through fluorescence immunostaining, we characterized pancreatic tumors using
antibodies against tissue-specific proteins and antigens including KRT19, PROM1/CD133,
DCLK1, CDH1, and EPCAM. It is confirmed that tumors are highly positive for E-cadherin
(CDH1) (Fig.1D). Expression of KRT19 in the normal pancreas is limited to ductal epithelial
cells and PDAC is known to retain this expression (Cao et al., 2005). Immunofluorescence
staining of KRT19 shows overexpression due to the highly stained cytoplasmic and membranous
areas of epithelial cells of the lesions (Fig. 1D). PROM1/CD133 is known to be expressed in
9
duct cells and PDAC tumor cells (Glumac et al., 2018) and its expression is localized to the
apical membrane of these preinvasive lesions (Fig. 1D). EPCAM shows an overexpression in the
membranous and cytoplasmic regions of the epithelial cells in pancreas cancer tissue (Fig. 1D).
DCLK1 is suggested to be a marker for pancreatic cancer stem cells (Qu et al. 2019) and we
found its expression in several EPCAM+ tumors (Fig. 1D). The expression of these established
PDAC markers in neoplasms of the mouse pancreas indicates that our Pdx1
Cre
;LSL-Kras
G12D
model is effective in developing pancreatic cancer.

Moderate alcohol consumption induces tumor formation in Pdx1
Cre
;LSL-Kras
G12D

mice

To test if moderate alcohol intake could be a potential risk factor for PDAC, Pdx1
Cre
;LSL-
Kras
G12D
mice were fed a Lieber-DeCarli diet containing 3.5% alcohol for a period of 5 months
(n=14). It was concluded that our alcohol feeding model achieves a moderate alcohol diet by
measuring blood alcohol levels after the feeding period. Using H&E staining, we observe mostly
early stages of tumor development in Pdx1
Cre
;LSL-Kras
G12D
mice fed regular chow, such as
ADM (Fig. 2A). In a representative picture of Pdx1
Cre
;LSL-Kras
G12D
mouse pancreas fed
alcohol, we observe the development of PanIN-2 (Fig. 2B). We also observed further
development to PanIN-3 (Fig. 3C) and invasive PDAC in pancreas tissue of mice expressing
Kras fed alcohol (Fig. 2D).
By quantifying the data from H&E staining, we analyzed tumor size and grade.
Pdx1
Cre
;LSL-Kras
G12D
mice fed alcohol resulted in the formation of tumors that occupied a larger
area of the pancreas compared to Pdx1
Cre
;LSL-Kras
G12D
mice fed regular chow (n=28) (Fig. 3A).
Western diet feeding was observed to have a more potent effect on tumor growth, leading to the
development of a tumor occupying over 80% of the total pancreas. The combination of alcohol
10
and western diet feeding showed little to no change in tumor size compared to alcohol feeding
alone. Not only were tumors larger in Kras mice fed alcohol, these mice also developed tumors
containing lesions of higher grades. About 40% of these tumors were PanIN-2, but mice fed
alcohol were able to develop PanIN-3 and PDAC (Fig. 3B). In Kras mice fed regular chow, only
20% of tumors were PanIN-2 and PanIN-1B, the remainder of the tumor PanIN-1A only (Fig.
3B). Western diet feeding induced PDAC as well as advanced neoplasia and combination with
alcohol feeding had the greatest observation of PDAC, occupying around 20% of the tumor. Our
experimental data proves that moderate alcohol consumption can be risk factor for PDAC
development.

Alcohol up-regulates mRNA expression of genes involved in tumor progression

To examine how alcohol intake promotes pancreatic tumor development, we analyzed RNA
expression in pancreas tissues of Pdx1
Cre
;LSL-Kras
G12D
mice with (Group 2) or without alcohol
feeding (Group 1) by RNA-Seq. Using principal component analysis to visualize variability
amongst the RNAs of these two groups, we observed a 71% variance between RNA expression
isolated from the pancreas tissues of Group 2 (Fig. 4A). This PC1 variance declares that there is
significant difference in gene expression in pancreas tissue treated with alcohol versus without
alcohol. With this information, we analyzed differential gene expression via a volcano plot (Fig.
4B). This scatter plot measures the ratio of genes in alcohol-treated pancreas tissue compared to
control pancreas highlighting that there are many genes expressed in similar quantities, but some
are up-regulated (red) and few are down-regulated (blue). The differences between these genes
could be important in the role they play regarding the genetic influence of moderate alcohol
consumption on PDAC progression.
11
Cytokines, such as Tgfb1 and Tnf, were found to be heavily up-regulated in the pancreas
of our mice fed alcohol compared to regular chow (Fig.5). Both of these cytokines can play vital
roles in tumor promotion and development by inducing tumor cell migration. Epcam, Krt19, and
Prom1 were also found to be up-regulated in the alcohol model, and these genes are known to
have positive impacts on pancreatic tumor development (Fig.5). The high counts of RNA
detected in these genes indicate their alcohol-induced expression and that alcohol has a positive
impact on the promotion and development on pancreatic tumors and, therefore, PDAC in Kras
mice. In contrast to these genes, we also found two genes heavily down-regulated in pancreas
tissues treated with alcohol: Fgf21 and Il6 (Fig.5).

Alcohol up-regulates the expression of stearoyl-CoA desaturase 1

Along with tumor markers and pro-tumor cytokines, we observed the up-regulation of Scd1, but
not Scd2, in pancreas tumor tissues of Kras-expressing mice fed alcohol compared to regular
chow. The induction of SCD1 is relevant since we believe this enzyme to play an important role
in lipid metabolism and could have a protective role in pancreatic tumor cell survival. We
analyzed the role of SCD in pancreatic tumor in Part II.

12
Figures

Figure 1

Fig 1. Histological analysis of tumor and PDAC development in Pdx1
Cre
; LSL-Kras
G12D
mice. We wanted to verify the efficiency of our Pdx1
Cre
; LSL-Kras
G12D
in forming preinvasive
lesions and invasive PDAC. (A) The mouse model utilizing the Cre-Lox recombinase system in
order to establish the expression of the mutation, Kras
G12D
. By using Pdx1 as a promoter for Cre,
we limit this expression to epithelial cells of the mouse pancreas. (B) Resection of the pancreas,
spleen, and part of the duodenum from both the control Pdx1
Cre
and mutant Pdx1
Cre
; LSL-
Kras
G12D
mice. The pancreas is enlarged in our model expressing the Kras mutation. (C) H&E
was performed on the pancreas tissues in (B) to confirm that tumor developed in the mutant
Pdx1
Cre
; LSL-Kras
G12D
model and not in the control Pdx1
Cre
model. The control model shows
healthy exocrine and endocrine pancreas morphology. The Pdx1
Cre
; LSL-Kras
G12D
model
displays the early preinvasive stages of PDAC development including ADM and the formation
of stages of PanIN’s, ranging from 1 to 2. (D) Immunohistochemistry of pancreatic tissues
bearing tumors were stained with common markers for pancreatic cancer. KRT19 is
overexpressed in the cytoplasmic and membranous regions of the lesion. CD133 is localized to
the apical membrane of tumor lesions expressing CDH1. DCLK1 and EPCAM are co-expressed
with one another inside the lesion. EPCAM staining is seen in the cytoplasmic and membranous
regions, similar to KRT19. Nuclei were counterstained with DAPI.

13
Figure 2

Fig. 2. H&E staining displaying tumor development in control and alcohol-fed mice.
Pdx1
Cre
;LSL-Kras
G12D
mice fed with or without a Lieber-DeCarli diet containing 3.5% alcohol.
(A) Kras
G12D
mouse fed a regular chow diet showing acinar cells undergoing ADM (arrows), an
early stage of PanIN development. (B-D) Kras mutant mouse fed 3.5% alcohol display later
tumor development in the pancreas, including PanIN-2 lesions (B, arrows), PanIN-3 lesion (C,
arrow), and invasive PDAC (D, arrow).

14

Figure 3

Fig. 3. Effects of alcohol feeding on tumor size and grade in Pdx1
Cre
;LSL-Kras
G12D
mice.
Pdx1
Cre
;LSL-Kras
G12D
mice were fed a Lieber-DeCarli diet containing 3.5% alcohol, a Western
diet, and a combination of the two. From H&E sections, (A) percentages of the tumor area
occupying the pancreas and (B) tumor grades were determined against control. *P < 0.05 and
**P < 0.001 against None.

15
Figure 4

Fig. 4. RNA-Seq analysis of pancreas tissue from control and alcohol-fed mice. Total RNAs
were collected from Pdx1
Cre
; LSL-Kras
G12D
mice pancreas without alcohol feeding (Group 1)
and with alcohol feeding (Group 2). (A) Principal component analysis shows that there are slight
differences amongst gene expression between both groups looking at PC1 versus PC2. (B) The
volcano plot measures differential gene expression between Group 1 and Group 2 showing genes
up-regulated (red) and down-regulated (blue).

16
Figure 5

Fig. 5. RNA-Seq analysis from Pdx1
Cre
; LSL-Kras
G12D
mice fed regular chow (Ctr) and
alcohol (Alc). RNAs from Pdx1
Cre
; LSL-Kras
G12D
mice fed a regular chow (Ctr, n=3) and
alcohol (Alc, n=3) were subjected to RNA-seq analysis. Counts were normalized and the mean
normalized counts were compared amongst the 2 groups.

17
Discussion

Heavy alcohol consumption has been known to be a risk factor for PDAC, but this study
provides the first elements of experimental data that highlights moderate alcohol drinking as a
risk factor in promoting PDAC development in Pdx1
Cre
;LSL-Kras
G12D
mice. By lowering the
standard Lieber-DeCarli diet containing 4-5% alcohol (vol/vol) to 3.5%, the alcohol intake is
reduced, leading to only a moderate level of intake since blood alcohol levels in mice are
relatively low. After 5 months of alcohol feeding, blood alcohol levels increased 76.6 + 33.3
mg/dL in mice, indicating that our alcohol model achieves a moderate alcohol diet. Even with
lowering the percentage of alcohol, PanINs still developed as well as PDAC; proving moderate
alcohol intake to be a risk factor.
Alcohol is an already known risk factor for pancreatitis (Habtezion, 2015). When alcohol
is metabolized, reactive oxygen species are generated in the pancreas, which can lead to a
determinantal cascade of events causing mitochondrial damage and failure (Shalbueva et al,
2013). Not only that, but metabolism of alcohol can generated fatty acid ethyl esters that can also
damage mitochondria and alter its function (Huang et al, 2014). Even though chronic pancreatitis
is a known risk factor for PDAC (Bansal and Sonnenberg, 1995), alcohol feeding alone is not
efficient enough for inducing pancreatitis in rodents (Tsukamoto et al., 1988). With this
knowledge, we assume that alcohol feeding does not cause pancreatitis and PDAC develops from
PanIN lesions independently in Pdx1
Cre
;LSL-Kras
G12D
mice. It is then our goal to investigate the
potential mechanism behind alcohol-induced pancreatic tumor progression.
To analyze if there are differences amongst pancreas tissues of Kras mice fed with or
without alcohol, we analyzed changes amongst gene expression patterns by RNA-seq. RNA-Seq
is important for quantifying gene expression between a normal and ‘disease-state’ in tissues, our
18
normal being a regular chow diet and the ‘disease-state’ is alcohol-feeding . This analysis allows
us to visualize differential gene expression and further elucidate the genetic mechanism behind
alcohol-promoted PDAC (Kukurba and Montgomery, 2015). The data showed significant
differences amongst gene expression patterns when analyzed via principal component analysis
and displayed on a volcano plot. In our RNA-seq analysis, we found that alcohol heavily down-
regulates the expression of Fgf21. It is a metabolic hormone that has complicated and complex
functions due to its expression in various tissues (Tezze et al., 2019). It’s found to be expressed
in liver, adipose tissue, and the pancreas and its functions in each tissue appear to be different
(Singhal et al., 2018). It was recently discovered that FGF21 has a protective role in the
promotion of PDAC in mice (Luo et al., 2019). Alcohol metabolism could be potentially
interfering with Fgf21 expression, leading to its dysfunction, thereby causing inflammation of
the pancreas. The protective mechanism behind Fgf21 is still unknown so more studies are still
needed to further elucidate the interactions between Fgf21 and alcohol.
Interestingly, another gene we found to be down-regulated was Il6. Il6 is pro-
inflammatory cytokine known to drive PDAC progression via the JAK/STAT3 pathway (Lesina
et al., 2011). Some studies demonstrate the anti-inflammatory role of Il6 in response to
inflammation by controlling the levels of pro-inflammatory cytokines. It could be possible that
when alcohol heavily up-regulates Tnf, the down-regulation of Il6 is a response to the strong
inflammatory response of the pro-inflammatory cytokine Tnf during PDAC initiation (Xing et
al., 1998). The down-regulation of these genes is insightful and requires more studies to
elucidate their mechanisms and interactions with alcohol.
Important genes involved with promoting tumor growth were observed to be induced via
our RNA-Seq analysis. Epcam, Krt19, and Prom1 are all genes known to be involved and
19
expressed in PDAC (Huang et al., 2018; Saha et al., 2018; Glumac and LeBeau 2018). RNA-Seq
analysis affirmed that alcohol up-regulates these genes in Pdx1
Cre
;LSL-Kras
G12D
mice fed
alcohol compared to regular chow. Alcohol also induces the expression and up-regulation of pro-
inflammatory and tumorigenic cytokines Tnf and Tgfb1, further proving the positive effect
alcohol has on PDAC development. Tnf has been known to be highly expressed at the earlier
stages of PDAC induction and plays a role in initiating desmoplasia in vivo (Zhao et al., 2016).
Chronic inflammation induces pancreatic injury, which in turn, can lead to the development of
pancreatitis (Habtezion, 2015). TNF is a known regulator and inducer of inflammation. It is a
member of a large superfamily of cytokines that function by activating inflammatory signaling
pathways including NF-kB, JNK, and p38-MAPK via their respective receptors (Shadhu and Xi,
2018). Studies have shown that tumor cells can produce TNF and enhance their growth,
metastases, and immune evasion in the tumor microenvironment (Shadhu and Xi, 2018).
Therapies have been developed against TNF to inhibit the growth and metastasis of pancreatic
cancer (Egberts et al., 2008). Tgfb1 is the member of a family of complex cytokines. TGFb
cytokines can play two roles: tumor promoting and tumor suppressive. The tumor stage and its
microenvironment are what indicate what function Tgfb will perform (Shen et al., 2017). Three
isoforms of Tgfb exist in mammals: Tgfb1, Tgfb2, and Tgfb3. Tgfb1 is the most abundant and its
expression indicates promotion of cell growth and proliferation (Shen et al., 2017). Since we
observe heavily up-regulated Tgfb1 by alcohol, we believe it’s functioning as tumor promoting in
early stages of PDAC initiation.
SCD is an enzyme localized to the ER that functions in fatty acid metabolism by
converting saturated fatty acid to monounsaturated fatty acid via the insertion of a single double
bond (Paton and Ntambi, 2008). Four isoforms exist in mice: Scd1, Scd2, Scd3, and Scd4, but the
20
shared isoform amongst mice and humans is Scd1. With moderate alcohol intake, we can see that
Scd1 expression is up-regulated, indicating that alcohol and the metabolic enzyme have a
positive relationship in promoting PDAC. We analyzed expression of Scd2 as well, but see little
to no change amongst mice fed with or without alcohol and this is because Scd2 is predominantly
expressed in the brain (Paton and Ntambi, 2008). In addition to the increase of Scd1 expression
in alcohol-fed mice, we also discovered the up-regulation of precursor genes including Acox1.
Acyl-CoA oxidase is involved in the first step of fatty acid beta-oxidation pathway and in the
breakdown of long-chain fatty acids. This finding indicates that alcohol plays an important role
in overall dysregulation of fatty acid metabolism, including at early stages. We further analyzed
the role of SCD in Part II of the thesis.
Our experimental data is the first to demonstrate that moderate alcohol drinking is an
independent risk factor promoting PDAC. This knowledge can provide opportunity to further
examine and elucidate the complex molecular mechanisms behind alcohol-induced PDAC. The
addition of this risk factor to the several other environmental risk factors already known can add
insight to certain populations of people who are predisposed to pancreatic cancer.

21

Part II

Inhibition of stearoyl-CoA desaturase suppresses growth of pancreatic
tumors in vitro and in vivo

22
Abstract

Although chemotherapy and immunotherapy have been used for patients with PDAC, the overall
five-year survival rate is only 8%, indicating an urgent need for effective therapies. Stearoyl-
CoA desaturase (SCD) is an enzyme localized in the endoplasmic reticulum and generates
monounsaturated fatty acid from saturated fatty acid by introducing a single double bond. In this
study, we examined the role of lipid metabolism in PDAC growth and survival using Pdx1
Cre
;
LSL-Kras
G12D
mice that spontaneously generated pancreatic tumors. We found that a specific
inhibitor for SCD (A939572, 4-(2-chlorophenoxy)-N-[3-[(methylamino)carbonyl]phenyl]-1-
piperidinecarboxamide) induces degeneration of pancreatic tumor organoids formed in matrigel
culture. Treatment with the SCD inhibitor increased mRNA expression of unfolded protein
response (UPR) markers, such as Atf4, Atf6, Ddit3, spliced Xbp1, Gadd34 and Grp78 in
organoids. The increased expression of these genes was reversed by the addition of oleic acid,
but not stearic acid. After oral administration of the SCD inhibitor to Pdx1
Cre
; LSL-Kras
G12D

mice bearing pancreatic tumors, we observed TUNEL+ cells in pancreatic tumors, but not in
islets or acinar cells. Our data suggest that the inhibition of SCD induces the UPR and cell death
in pancreatic tumors due to the accumulation of saturated fatty acid.

23
Introduction

Pancreatic ductal adenocarcinoma has high metastasizing capabilities and is expected to become
the second-leading cause of cancer death by 2030 (Rahib et al., 2014). Due to the aggressive
nature of PDAC and lack of effective treatment, the overall survival rate is only 8% and
alternative, novel therapies are urgently needed. Over 90% of patients with PDAC exhibit
oncogenic mutations of Kras GTPase (di Magliano and Logsdon, 2013). By overexpressing
mutant Kras in the acinar cells of the pancreas, we replicated the Pdx1
Cre
;LSL-Kras
G12D
mouse
model used in Part I. These mice will spontaneously develop PanINs and eventually invasive
PDAC allowing us to study the effects of our specific inhibitor on pancreatic tumor development
and survival.
Lipid metabolism has been linked to cancer growth and metastasis (Beloribi-Djefaflia et
al., 2016; Manccini et al., 2018). Cancer cells need to proliferate in order to disseminate and this
is achieved by increasing their uptake of exogenous lipids or stimulating their endogenous
synthesis. If this metabolism is disrupted or halted, it could lead to negative effects on their
growth and survival. Stearoyl-CoA desaturase (SCD) is an enzyme localized in the endoplasmic
reticulum (ER) and functions in generating monounsaturated fatty acid from saturated fatty acid
by introducing a single double bond (Paton and Ntambi, 2009). Palmitoleic acid (16:1) and oleic
acid (18:1) are monounsaturated fatty acids generated from palmitic acid (16:0) and stearic acid
(18:0), respectively. As described in Part I, we found that Scd1 is up-regulated in pancreatic
tumors developed in Pdx1
Cre
;LSL-Kras
G12D
mice fed alcohol. Although SCD enzymes have been
shown to play important roles in various cancers, little is known about its role in pancreatic
cancer. Tumor tissue resected from breast and hepatocellular carcinoma indicated high
expression levels of Scd1 as well as an association with a poor survival rate (Tracz-Gaszewska
24
and Dobrzyn, 2019). SCD has been known to have effects on inflammation and be a main player
in the production of active palmitoleated Wnt proteins. In hepatocellular carcinoma, it has been
observed that SCD expression in hepatic stellate cells promotes liver fibrosis and tumor
progression via Wnt signaling (Lai et al., 2017). In colorectal cancer (CRC), Scd1 expression is
up-regulated in colorectal tissues and promoted CRC via regulation of epithelial-mesenchymal
transition, thereby, stimulating metastasis (Ran et al., 2018). SCD1 was observed to have a role
in cancer stem cell survival and propagation in human lung adenocarcinoma (Noto et al., 2017).
Interestingly, the higher levels of expression of Scd in cancers previously mentioned all
correlated with poor survival rates, indicating the important of lipid metabolism in tumor
survival.
ER stress is a physiological and pathological response to stimuli including hypoxia,
shortage of glucose metabolism, and genome instability. These stimuli are often associated with
tumor progression, which can lead to the induction of the unfolded protein response (UPR)
(Corazzari et al., 2017). The UPR is a vital cell survival process induced to maintain cellular
homeostasis. There are three major signaling pathways and sensors of the UPR: inositol
requiring enzyme 1 (IRE1), double-stranded RNA-activated protein kinase-like ER kinase
(PERK), and activating transcription factor 6 (ATF6). Under normal conditions, Grp78, also
known as the ‘binding immunoglobulin protein’, retains the stress sensors in their inactive state.
During periods of ER stress, Grp78 dissociates itself from each sensor allowing for their
activation in their relative signaling pathways to collectively act together in returning the cell
back to homeostasis (Madden et al., 2019). Each individual pathway acts differently in relieving
ER stress by inducing the transcription of specific genes, pro-apoptotic factors, and by reducing
overall protein translation. IRE1a initiates the splicing of Xbp1 mRNA to generate active sXbp1,
25
which induces the expression of genes that function in protein folding and lipid synthesis. PERK
activates the phosphorylation of eIF2a, which suppresses global protein translation. The
phosphorylation of eIF2a also induces the translation of ATF4, and this cascade of events leads
to the expression of Ddit/Chop, a proapoptotic transcription factor. Chop induction is known to
be involved in the onset of cell death as well as re-establishing protein translation via Gadd34,
which would aggravate ER stress and trigger apoptosis (Madden et al., 2019). Lastly, ATF6
activation induces the transcription of multiple genes involved in protein folding and ER-
associated degradation. If ER stress persists and homeostasis cannot be attained from UPR
induction, the cell will switch to a death-inducing response and execute apoptosis (Madden et al.,
2019). The up-regulation of these markers indicates UPR activation, which has been represented
as a hallmark of several cancers including melanoma, squamous cell carcinoma, colorectal
carcinoma, and prostate cancer (Corazzari et al., 2017).
Matrigel culture has been developed for culturing epithelial cells from the intestine, liver
and pancreas (Huch et al., 2013). For the purpose of our study, we isolated EPCAM+ pancreatic
tumor cells by magnetic-activated cell sorting (MACS) from Kras-expressing mouse pancreas.
These cells were cultured in matrigel in the presence of R-Spondin 1, which is a protein
important in the activation and promotion of Wnt signaling. EPCAM+ pancreatic tumor cells
formed tumor organoids in matrigel. Using the tumor organoids that develop in the matrigel
culture, we examined the role of lipid metabolism in PDAC growth and survival.
In the present study, we examined the role of SCD in pancreatic tumor growth using
matrigel culture. We found that an inhibitor specific to SCD strongly induces the degeneration of
pancreatic tumor organoids in matrigel culture via induction of the UPR. An important finding
was that the addition of oleic acid (18:1), but not stearic acid (18:0), rescued the inhibitor-
26
induced degeneration. Our data indicate that SCD is required for early development of pancreatic
cancer growth and is a novel therapeutic target for the suppression of pancreatic cancer.

27
Materials and Methods
Mice
Pdx1
Cre
;LSL-Kras
G12D
and Rosa26-tdTomato
flox
mice were purchased from Jackson Laboratory
(Bar Harbor, ME)

(di Magliano and Logsdon, 2013). From these mice, we generated Pdx1
Cre
;
LSL-Kras
G12D
and Pdx1
Cre
; LSL-Kras
G12D
; Rosa26-tdTomato
flox
mice. All animal experiments
were performed in accordance with the NIH guidelines under the protocol approved by the
IACUC at the University of Southern California.

Immunofluorescence staining

Cryosections were blocked with 5% donkey serum and 0.2% bovine serum albumin for 30 min
and incubated with primary antibodies at 4
o
C overnight. The primary antibodies used included:
amylase (AMY, 200-fold dilution, PA5-50358, ThermoFisher Scientific, Waltham, MA) as well
as all of the antibodies previously listed in Part I.

Tumor organoid culture

Pancreas tissues were from Pdx1
Cre
; LSL-Kras
G12D
mice (5 months of age) were digested with
125 µg/ml collagenase type XI (Sigma, St. Louis, MO) and 125 µg/ml Dispase II (ThermoFisher
Scientific, Waltham, MA) at 37
o
C for 1 hour

(Boj et al., 2015). Digested cells were collected by
centrifugation and incubated with rat monoclonal EPCAM antibody (G8.8, DSHB, Iowa City,
IA) at 4
o
C for 30 min. After washing, cells were incubated with anti-rat IgG microbeads
(Miltenyi Biotech, Auburn, CA) and EPCAM+ tumor cells were separated by MACS Pro
separator (Miltenyi Biotech). EPCAM+ cells (2x10
4
cells) were suspended in 20 µl of matrigel
28
(Corning, Tewksbury, MA), plated on a round bottom 96-well plate, and incubated in a CO2
incubator at 37
o
C for 30 min. After polymerization of the matrigel, cells were cultured in 100 µl
of advanced DMEM/F-12 glutamax (ThermoFisher Scientific) supplemented with 1X
penicillin/streptomycin, 1X B27 supplement, 10 mM HEPES (Sigma), 1.25 mM N-acetylcystein,
10 mM nicotinamide, 10 nM Gastrin I, 0.5 µM A83-01 (R&D Systems), 0.1 ng/ml FGF-10, 100
ng/ml Noggin, 50 ng/ml EGF, 1 µg/ml R-Spondin-1 (PeproTech, Rocky Hill, NJ) and 10 nM Y-
27632 (Tocris, Minneapolis, MN)

(Huch et al., 2013). Tumor organoids formed in matrigel were
disrupted by pipetting and were passaged for experiments from 1 well to 3 wells.
To examine the structure of tumor organoids, we isolated EPCAM+ tumors from Pdx1
Cre
;
LSL-Kras
G12D
; Rosa26-tdTomato
flox
mice (5-months old) and formed organoids. The
morphology of tdTomato+ organoids were analyzed by confocal microscope. Confocal
microscopy provides us with live 3D imaging of our organoids to view their composition, shape,
and cell-cell interactions (Dekkers et al., 2019). Live and untreated organoids were brought in
their original 96-well plate to the Cell Imaging Core to be analyzed by the Leica SP8 confocal
microscope. Z-stacks between 100-150 µm for 10 steps were made of each organoid to measure
their thickness and overall volume. These images were then analyzed and exported off the Leica
SP8 confocal microscope imaging software.
The organoids established from Pdx1
Cre
; LSL-Kras
G12D
mice (5-months old) were fixed
with 4% paraformaldehyde and embedded in freezing medium. Cryosections made from the
organoid were analyzed by fluorescence immunostaining as described above.

29
Treatment of tumor organoids

An SCD1 inhibitor (A939572), 4-(2-chlorophenoxy)-N-[3-[(methylamino)carbonyl]phenyl]-1-
piperidinecarboxamide (Tocris) was dissolved in dimethylsulfoxide (DMSO) and added to the
culture medium (20 µM at a final concentration). Oleic acid (18:1, O1008), stearic acid (18:0,
S4751), and fatty acid free-BSA (A8806) were purchased from Sigma. BSA-conjugated fatty
acids were prepared by diluting 50 mM stocky fatty acids in DMSO with 100 mg/ml BSA in 1X
PBS to produce a 3 mM working solution (Lai et al., 2017). Cells were then treated with a final
concentration of 30 µM BSA-conjugated fatty acid (a final concentration of 0.1% BSA). Cells
were treated with or without A939572 or 30 µM of fatty acid for 16 hours.

Quantitative polymerase chain reaction (qPCR)

RNAs were isolated using RNA miniprep kit (Zymo Research, Irvine, CA). cDNA was
synthesized using Maxima first strand cDNA synthesis kit (ThermoFisher Scientific). qPCR was
performed with SYBR FAST qPCR kit (KAPA Biosystems, Wilmington, MA) in ViiA7
RealTime PCR System (Applied Biosystems, Carlsbad, CA). Primer sequences for mouse genes
are listed in Table 1 (Atf4, Atf6, Chop, Grp78, Gadd34, sXbp1, Gapdh). The samples were run in
triplicate. The relative mRNA levels per sample were calculated by subtracting the detection
limit (40 Ct) from the cycle threshold value (Ct) of each gene in the same sample to obtain the
DCt value. Taking the log2 of -DCt resulted in the relative expression value of each gene for each
sample expressed in arbitrary units. Each value was normalized to that of Gapdh.

30
Detection of apoptosis in pancreatic tumors

Pdx1
Cre
;LSL-Kras
G12D
mice were given the SCD inhibitor orally (5 mg/kg body weight) 2 days
before collecting the pancreas (n=4). Pancreas tissues were fixed with 4% paraformaldehyde in
PBS overnight at 4
o
C. Cryosections (7 µM) were used for detection of apoptosis using In Situ
Apoptosis Detection Kit (Trevigen, Caithersburg, MD). DNA fragments were visualized with
antibodies conjugated with horseradish peroxidase and diaminobenzidine according to a
manufacturer’s instruction. Imagers were captured with a Nikon 90i microscope and DS-Fi1
digital camera (Nikon, Melville, NY). To quantify the tumor cells showing apoptosis, 5 images
were randomly captured from 3 independent samples using a 10X objective and used ImageJ to
count positively stained cells.

Statistical Analysis

Statistical tests for the significance of differences were assessed by one-way ANOVA followed
by a Tukey HSD post-hoc test. A p-value of less than 0.05 was considered statistically
significant.

31
Results

Isolated EPCAM+ pancreatic tumor cells form organoids in matrigel culture

Matrigel organoid culture has been developed for culturing epithelial stem cells from the
intestine, liver, and pancreas

(Huch et al., 2013). From Pdx1
Cre
;LSL-Kras
G12D
mouse pancreas,
we separate pancreatic tumor cells expressing EPCAM by magnetic-activated cell sorting
(MACS) and culture them in matrigel in the presence of R-Spondin 1, which activates Wnt
signaling. EPCAM+ pancreatic tumor cells formed tumor organoids in matrigel (Fig. 6A). To
visualize pancreatic tumors, we generated Pdx1
Cre
;LSL-Kras
G12D
; Rosa26-tdTomato
flox
mice in
which pancreatic tumor cells express tdTomato. Using a confocal microscope, we found that
tdTomato+ pancreatic tumor cells form organoids consisting of a few epithelial cell layers
without branching like those from intestine (Fig. 6B).
Next, we embedded tumor organoids in freezing medium, prepared cryosections, and
further characterized them by immunofluorescence staining. In the organoid model, all tumor
epithelial cells are positive for E-cadherin (CDH1) and EPCAM (Fig. 6C). KRT19 is stained and
localized to the apical membrane of epithelial cells (Fig. 6C) similar to mouse pancreas seen in
Part I (Fig. 1D). CD133 staining is highly expressed in tumor cells located at the lumen side,
which is similar to in vivo PDAC, suggesting the formation of apical-basal polarity. The
epithelial cells on the lumen are evenly positive for DCLK1 (Fig. 6C). Staining of Amylase
(AMY), a marker for acinar cells, is limited in a few cells. This staining pattern is comparable to
in vivo (Fig. 1D), but distinct in that it suggests culture condition drives tumor cells adjacent to
the lumen near DCLK1+ cells.

32
The inhibition of SCD induces degeneration of tumor organoids in vitro

Using these organoids in matrigel culture, we examined the role of lipid metabolism in PDAC
growth and survival. SCD is an enzyme that functions in the ER by converting monounsaturated
fatty acid from saturated fatty acid by introducing a single double bond (Paton and Ntambi,
2009). Interestingly, we found that a specific inhibitor for SCD (A939572) induces degeneration
of pancreatic tumors in matrigel culture. After organoids assembled, we treated them with the
SCD inhibitor dissolved in dimethyl sulfoxide (DMSO). For our control, organoids were treated
with DMSO of equal concentration (Fig. 7A). After 16 hours of treatment with the specific SCD
inhibitor (A939572, 20 µM), the tumor spheroids decrease in size and morphology becomes
abnormal, indicating degeneration (Fig. 7D).
Since SCD converts saturated fatty acids to monounsaturated fatty acids, we wanted to
verify whether the SCD inhibitor induces degeneration of the organoids by disrupting fatty acid
metabolism. We treated organoids with the SCD inhibitor in the presence or absence of
monounsaturated fatty acids (oleic acid, 18:1) or saturated fatty acids (stearic acid, 18:0). We
expected if lack of monounsaturated fatty acids is responsible for degeneration of the organoids,
addition of oleic acid can rescue the negative effect of the inhibitor on the organoids. Addition of
oleic acid or stearic acid alone did not induce degeneration on the organoids (Fig. 7B,C),
suggesting no induction of lipotoxicity in the tumor organoids by stearic acid. Interestingly, we
found that the addition of oleic acid reverses the negative effect of the SCD inhibitor (Fig. 7E).
However, stearic acid did not show such effect (Fig. 7F). This experiment demonstrates that
reduction of de novo cellular biogenesis of monounsaturated fatty acids inhibits the growth of
pancreatic tumor organoids.

33

Inhibition of SCD in tumor organoids induces the unfolded protein response (UPR)

Next, we tested whether degeneration observed in pancreatic tumor cells after treatment with the
SCD inhibitor is due to cellular stress. We hypothesized that dysregulated lipogenesis due to the
lack of enzymatic activity from SCD would cause ER stress, leading to the induction of the UPR
and resulting in apoptotic cell death. The UPR is a connection of multiple signaling pathways
that monitor and respond to conditions in the ER. When the ER undergoes certain stressors, such
as misfolding of proteins and protein buildup, these signaling pathways function to express genes
that will establish and maintain homeostasis. If the UPR is overactivated and therefore, cannot
achieve cellular homeostasis, the cell will undergo apoptosis. The three main UPR signal
transducers include: IRE1, PERK, and ATF6. To observe if the UPR is induced in organoids
undergoing degeneration, we treated organoids with 20 µM of SCD inhibitor (A939572)
dissolved in dimethyl sulfoxide (DMSO) in the presence or absence of 30 µM of oleic acid
(18:0) or stearic acid (18:1) for 16 hours. Following treatment, we isolated total RNAs from
organoids and measured important UPR marker genes, including, sXbp1, Chop/Dddit3, Atf4,
Atf6, Gadd34, and Grp78, by qPCR. The relative mRNA of each gene was measured in
organoids with different treatment conditions and each measurement was normalized to Gapdh.
Downstream effectors of Perk; Atf4, Chop, and Gadd34 mRNAs, were induced following SCD
inhibition. sXbp1, which is the spliced and active form of Xbp1, is mediated by IRE1 and was
up-regulated similarly. Atf6 and Grp78 function as chaperones for misfolded proteins and their
expression was also increased via SCD inhibition. Organoids treated with oleic acid (18:1) and
stearic acid (18:0) alone showed no induction of these markers in organoids, indicating that these
fatty acid additions do not induce the UPR (Fig. 8). In organoids treated with the SCD inhibitor,
34
co-treatment with stearic acid did not further increase the expression of UPR markers. In
contrast, co-treatment with oleic acid did have an effect and reduced the expression of these
markers induced by SCD inhibition suggesting that the reduction of de novo lipogenesis of
monounsaturated fatty acid and the accumulation of saturated fatty acid suppress the growth and
survival of tumor organoids through UPR induction. This data compares to our previous results
suggesting that oleic acid may play a cytoprotective role.

Inhibition of SCD induces pancreatic tumor cell death via apoptosis in vivo

To test whether the SCD inhibitor induces cell death in pancreatic tumors in vivo, Pdx1
Cre
; LSL-
Kras
G12D
mice bearing PanIN were given the SCD inhibitor orally (A939572, 5 mg/kg body
weight). Two days following treatment, we assessed cell death in the pancreas by TUNEL assay.
When cells undergo apoptosis, DNA is fragmented due to cleavage by endonuclease leading to
the exposure of free hydroxyl residues at the 3’ end. The enzyme, terminal deoxynucleotidyl
transferase, adds biotinylated nucleotides to this free end allowing visualization of DNA
fragmentation. There is no positive staining seen in the control pancreas without treatment (Fig.
9A), but the inhibitor-treated pancreas showed TUNEL+ apoptotic cells in PanIN (Fig. 9B). The
SCD inhibitor did not induce apoptosis in islet or acinar cells. This experiment indicates that the
accumulation of saturated fatty acid selectively induces apoptosis in pancreatic cancer in vivo
and SCD would be a novel therapeutic target for suppression pancreatic cancer.

35
Tables

Table 1

Gene Forward Reverse
Atf4 5’-GAAACCTCATGGGTTCTCCA-3’ 5’-GCCAATTGGGTTCACTGTCT-3’

Atf6 5’-TTTCGAAGGGATCATCTGCT-3’

5’-GGGTCGTCTCTGTGGTTGTT-3’

Chop 5’-TCCTGTCCTCAGATGAAATTGG-
3’

5’-CCTAGTTCTTCCTTGCTCTTCC-3’
Gadd34 5’-GAGGGACGCCCACAACTTC-3’

5’-TTACCAGAGACAGGGGTAGGT -3’

Gapdh 5’-CGTCCCGTAGACAAAATGGT-3’ 5’-GAATTTGCCGTGAGTGGAGT-3’
Grp78 5’-TACTCGGGCCAAATTTGAAG-3’ 5’-GGGGACAAACATCAAGCAGT-3’

sXbp1 5’-GAGTCCGCAGCAGGTG-3’

5’-GTGTCAGAGTCCATGGGA -3’

Table 1. Primer sequences for genes analyzed by qPCR in Figure 8.

36

Figures

Figure 6

Fig. 6. Formation of pancreatic tumor organoids from Pdx1
Cre
; LSL-Kras
G12D
mice. (A)
EPCAM+ pancreatic tumor cells sorted by MACS formed organoids in matrigel culture. (B)
Confocal microscopy of organoid expressing tdTomato. (C) Immunofluorescent staining of
organoids using specific proteins known to be expressed in PDAC. Negative (Neg) was stained
with DAPI only. Nuclei were co-stained with DAPI.

37

Figure 7

Fig. 7. SCD inhibition induces degeneration in pancreatic tumor in vitro. Degeneration of
pancreatic tumor spheroids by SCD inhibitor after 16 hours of treatment. Addition of oleic acid
(18:1), but not stearic acid (18:0), rescued the inhibitory effect of the SCD inhibitor. Control
organoids treated with DMSO (A). We demonstrated that the addition of fatty acids alone does
not induce degeneration of organoids (B, C). Organoids treated with the SCD inhibitor (20 µM)
alone, exhibited degeneration (D). Then, organoids were treated with SCD inhibitor (20 µM) and
oleic acid (30 µM) and the inhibitory effect was recovered due to the addition of oleic acid (E).
Organoids were treated with the SCD inhibitor (20 µM) and stearic acid (30 µM) and
degeneration of organoids was observed (F).
38

Figure 8

Fig. 8. SCD inhibition induces the UPR in pancreatic tumor organoids. Pancreatic organoids
were treated with or without the SCD inhibitor for 16 hours in the presence or absence of oleic
acid (OA, 18:1) or stearic acid (SA, 18:0). Expression values were normalized against Gapdh.

39
Figure 9

Fig. 9. SCD inhibition induces apoptotic cell death in pancreatic tumor in vivo. TUNEL
assay performed on cryosections of pancreas tissue taken from Pdx1
Cre
; LSL-Kras
G12D
mice
bearing PanIN, observed at 20X. (A) Pancreas tissue not treated with SCD-inhibitor showing no
positive staining in lesions. (B) SCD-treated pancreas displaying dark, brown staining indicating
the presence of apoptotic cells only in pancreatic lesions.

40

Discussion

Even with years of studies conducted on elucidating PDAC in efforts to develop more effective
and efficient therapies, it has remained one of the most aggressive, fatal malignancies and its
incidence continues to rise. The only curative treatment is by surgical resection and
unfortunately, only 20% of patients are eligible. This leaves the remaining 80% of patients left to
undergo adjuvant chemotherapy (Adamska et al., 2017). The top chemotherapeutic drug used to
treat PDAC presently is Gemcitabine, occasionally used in combination with Cisplatin
(Heinemann, 2001). Even with chemotherapy treatment, most PDAC patients have a dismal
prognosis indicating the urgent need for more efficacious treatment. Due to the fact that most
patients cannot undergo surgical resection, a more useful route may be to look for small
molecules that can act as suppressors of PDAC in combination with present chemotherapeutic
drugs.
In testing novel therapeutic drugs, it’s critical to investigate their toxicity, molecular
mechanisms involved, and appropriate dosage in in vitro and in vivo models. Cell culture of
cancer cell lines has previously been used for these analyses of the in vitro data, but over time a
more relevant and accomplished culture and model system has arose to more adequately portray
the tumor microenvironment: organoids. Organoids in matrigel have been used to culture
epithelial cells of the intestine, liver and pancreas (Huch et al., 2013). The nature of the 2D
monoculture of cell lines has its advantages, but compared to organoids, do not encompass the
same features that allow organoids to be more comparable to more realistic models. For instance,
organoid models are used for imitating cell-cell, cell-matrix interactions, and highlighting the
signaling pathways that are used to initiate tumorigenesis in tissue (Yin et al., 2017). The
41
structure and formation of organoids varies amongst the organs they are recapitulating. For
example, matrigel culture of epithelial cells from the intestine form ‘enteroids’, another term for
intestinal organoids. Enteroids form a defined ‘crypt-vili’ structure to imitate the normal
intestinal architecture and function (Wallach and Bayrer, 2017). Organoids derived from
epithelial cells of the pancreas differ because they form a much simpler, spherical structure made
of few tumor cells. By inducing the Wnt signaling pathway via addition of R-Spondin 1, our
organoids derived from tumor cells of mouse pancreas, grew in matrigel culture. We wanted to
prove that PDAC was capable of being promoted and fully developed in this in vitro model, so
we utilized the same immunofluorescent markers previously used on pancreas tissue sections
(Part I) on organoid sections. The expression patterns of KRT19, PROM1/CD133, EPCAM,
CDH1, and DCLK1 were similar and comparable amongst our organoid model and mouse
pancreas tissue, proving both models to be sufficient in examining PDAC pathogenesis and
eventually the effects of our specific inhibitor.
Lipid metabolism has been indicated to play a role in cancer metastasis. Cancer cells
proliferate at high rates and require increasing their uptake of exogenous lipids or
overstimulating their endogenous synthesis (Beloribi-Djefaflia et al., 2016). Our focus is on the
conversion step between saturated fatty acids and monounsaturated fatty acids, which is
catalyzed by the enzyme SCD. By inhibiting SCD, saturated fatty acids will accumulate and we
observed this effect in our in vitro and in vivo models to see if this is toxic to pancreatic tumor
cells. After treatment, obvious organoid degeneration was observed, indicating that disrupted
lipid metabolism has a negative effect on the survival of pancreatic tumors. In literature, it has
been proven that long-chain saturated fatty acid accumulation can induce lipotoxicity in cells.
Not only that, but monounsaturated fatty acids have been hypothesized and proven to have a
42
cytoprotective role (Nolan and Larter, 2009). With this in mind, we believed that the SCD
inhibition lead to the accumulation of saturated fatty acids and influenced organoid degeneration.
Oleic acid (18:1) and stearic acid (18:0) are a monounsaturated fatty acid and saturated fatty
acid, respectively. The addition of these fatty acids alone to organoid culture had no effect,
meaning that saturated fatty acids do not induce lipotoxicity in our model. The lack of
lipotoxicity could be due to the endogenously synthesized monounsaturated fatty acids from
pancreatic cancer cells that are proliferating in the organoid. The addition of the SCD inhibitor in
combination with stearic acid (18:0) is even more detrimental compared to SCD-treatment alone.
Interestingly, the combination of the SCD inhibitor with oleic acid (18:1) rescues the inhibitor-
induced degeneration, proving that de novo biosynthesis of monounsaturated acid is vital for
tumor cell survival. A potential underlying mechanism behind the cytoprotective role of
monounsaturated acids is that of the activation of peroxisome proliferator-activator receptors
(PPARs) (Nolan and Larter, 2009). This family of nuclear receptors is involved in sensing the
lipid or nutritional state of a cell and are transcriptional regulators of lipid metabolism. These
receptors can be activated by either fatty acid, but monounsaturated acids are believed to be more
potent PPAR ligands, which is a valid reason as to why PPAR activation could explain the
differences that each fatty acid has on cell survival. If PPAR is activated by oleic acid, this
interaction can promote the oxidation or sequestration of saturated fatty acids, which would
prevent cytotoxicity. Not only does PPAR activation by oleic acid detoxify saturated fatty acids,
it also suppresses the inflammatory response by inhibiting the nuclear factor kappa-light-chain-
enhancer of activated B cells (NF-kB) signaling pathway. NF-kB signaling pathway has been
linked to promoting lipotoxicity, so the combination of these events following PPAR activation
by oleic acid could explain the reason behind the positive role in cell survival that we observe via
43
monounsaturated fatty acids (Nolan and Larter, 2009). In future studies, it would be important to
conduct western blotting data on the possible activation of certain PPAR receptors, like
PPARa, from organoids treated with oleic acid versus stearic acid. Investigating the effects of
treating with other long-chain fatty acids, such as palmitic acid (16:0) and palmitoleic acid
(16:1), on the growth and viability of organoids in the presence and absence of our SCD inhibitor
would also be useful in expanding the evidence of our experimental data.
A theory we investigated is that the dysregulated lipid metabolism via SCD inhibition
induces ER lipotoxic stress and cell death in pancreatic tumors. In our data, we found that
organoids treated with our SCD inhibitor as well as in combination with stearic acid (18:0), show
moderate levels of mRNA expression of a variety of important markers involved in the UPR, a
pro-survival mechanism induced when ER stress is present. Interestingly, in combination with
oleic acid (18:1), there is a reduction in the expression of each UPR gene. These include Atf4,
Atf6, Chop, Grp78, sXbp1, and Gadd34 (a proapoptotic factor). When cellular homeostasis and
normal physiological status cannot be sustained, the UPR is prolonged until the cell is instructed
to undergo apoptosis. This suggests that the accumulation of saturated fatty acids, like stearic
acid, causes ER lipotoxic stress and the eventual cell death in pancreatic tumors via induction of
UPR. Monounsaturated fatty acid production may help alleviate cellular stress overall and the
induction of the UPR, reiterating that oleic acid may have a cytoprotective role. It is possible that
the accumulation of saturated fatty acids may not only cause lipotoxicity via ER stress, but could
have effects on other pathways by altering the fluidity and composition of the plasma membrane.
To verify if UPR induction is playing the dominant role in cell-mediated death, a future study
would be to use specific inhibitors of molecules involved in each pathway to observe the effects
on organoids treated with SCD inhibitor. Since IRE1 has been known to activate the c-Jun N-
44
terminal kinase signaling pathway by its interaction with adaptor protein TNF receptor-
associated factor 2, it would be insightful to use a JNK inhibitor to observe if apoptosis is
suppressed in organoids treated with SCD inhibitor (Madden et al., 2019).
Following the data exhibiting UPR induction in SCD-treated organoids, we wanted to
investigate if the degeneration we were observing was due to tumor cells undergoing apoptotic
cell death. The TUNEL assay is an efficient tool in detecting apoptosis in cells using a
fluorescent-labeling enzyme terminal deoxynucleotidyl transferase that catalyzes the addition of
nucleotides to the fragmented DNA ends that are generated during apoptosis (Kyrylkova et al.,
2012). Both our untreated and SCD-treated mice bore PanIN’s and TUNEL analysis was
conducted. TUNEL+ cells were only observed in the SCD-treated pancreatic lesions and no
positive staining was seen in acinar cells or islets, indicating the selectivity of cell death in tumor
cells. We believe cell death is limited to tumor cells due to the studies that show there is not as
high of a demand for uptake of endogenous monounsaturated fatty acids in non-cancer cells
compared to cancer cells (Beloribi-Djefaflia et al., 2016). Cancer cells are highly proliferative
and require increase metabolic activity for their growth and survival (Baenke et al., 2013). It has
been observed that cancer cells are able to manufacture their own lipids by de novo lipogenesis,
making their levels of lipid biosynthesis of fatty acids comparable to that of liver cells. This
feature in tumor cells is not seen in non-tumor cells due to restrictions established by the tumor
microenvironment. From lack of oxygen and nutrient supply, tumor cells overcome these losses
by enhancing their lipid metabolism machinery, which involves increasing the activity of key
metabolic enzymes, including SCD (Tracz-Gaszewska and Dobrzyn, 2019). Further studies have
confirmed that neoplastic lesions show the aberrant induction of de novo lipogenesis, which
could explain what is happening in the PanINs of our Pdx1
Cre
;LSL-Kras
G12D
mice. The inhibition
45
of SCD, and potentially other enzymes involved in fatty acid synthesis, could lead to the
suppression of tumor cell growth (Baenke et al., 2013). The combination of literature and our
experimental data shows the selectivity of SCD-inhibition on pancreatic tumor cells, making
SCD a targetable candidate for therapy.
Cancer cells have been known to alter their metabolic activity via reprogramming in
order to increase glucose uptake, biosynthesis of lipids, and other metabolic processes (Baenke et
al., 2013). We believe that if SCD-inhibition has a positive role in the suppression of pancreatic
cancer in human patients, then it could eventually be a potential therapy in various cancers as
well. The deregulation of lipid metabolism has been linked to cancer metastasis and the
inhibition of enzymes involved in the synthesis of individual lipids could open the doors to new
treatment options that could be used in combination with chemotherapy, radiotherapy, and/or
immunotherapy. For example, SCD inhibitor, A939572, has been used in combination with a
‘mammalian target of rapamycin’ inhibitor for renal cell carcinoma and resulted in the hindrance
of cell proliferation colony formation and abated tumor volume (Von Roemeling et al., 2013).
The inhibition of SCD also proved to overcome chemoresistance of glioblastoma cells when
used in combination with the drug Temozolomide (Dai et al., 2018). Proteasome inhibitors have
been used as treatment for anaplastic thyroid carcinoma since this can lead to the accumulation
of misfolded proteins, inducing ER stress and eventually the UPR. It has been suggested that
using protease inhibitors in combination with SCD inhibitor may enhance this ER stress
response, thus leading to the death of these cancer cells (Altmann et al., 2012; Mitsiades et al.,
2006). This combination would need to be further studied to observe the presence or absence of
synergistic effects. Not only could SCD inhibition be utilized in cancer treatment, but it has been
studied in treating diseases like Parkinson’s (Vincent et al., 2018). The inhibition of SCD is not
46
only linked to induce lipotoxicity in cancer cells, but can be a protective role in human neurons
from a-synuclein (Vincent et al., 2018). SCD inhibition has been used to treat multiple metabolic
syndromes, such as diet-induced obesity, hepatic steatosis, and insulin resistance (Brown and
Rudel, 2010).
Unfortunately with most novel therapies, there are adverse effects that need to be
addressed and overcome. With SCD inhibition; inflammation, atherosclerosis, steatohepatitis,
and pancreatic beta cell dysfunction have been observed in preclinical animal models due to the
accumulation of saturated fatty acids (Brown and Rudel, 2010). Though in our data with the
Kras mouse, we did not see any effect in pancreatic islet cells, so this would need to be further
examined in various models including human-derived cell lines and tissue. It was previously
mentioned that oleic acid could activate PPAR and lead to the suppression of inflammation by
shutting off the NF-kB signaling pathway, therefore, SCD would play a role in suppressing
inflammatory diseases. The role of SCD in highly proliferating cells, such as gut epithelial cells,
is to be addressed. Fatty acid production is necessary to regulate cell proliferation so inhibition of
SCD has been observed to upregulate proinflammatory markers due to saturated fatty acid
accumulation. With the addition of dietary supplements with oleic acid, this can counteract and
reduce intestinal inflammation in mice (Ducheix et al., 2018). To further lower the risk of
inflammation, SCD inhibitors given as treatment should be given in conjunction with anti-
inflammatory agents to potentially prevent these pernicious adverse effects.

47

Conclusion

Our experimental data provide insightful information on new risk factors and a potential
suppressive therapy. Multiple environmental risk factor including obesity, pancreatitis, and
tobacco smoking have already been identified as coaxers of PDAC. Heavy alcohol drinking has
been suspected to play a role as well since it can cause pancreatitis, but our study is the first to
examine moderate alcohol drinking as a risk factor. Alcohol feeding induced the up-regulation of
multiple genes involved in PDAC pathogenesis and that are known to be expressed in PanIN
lesions. Up-regulated genes also included pro-tumor cytokines that induce PDAC tumor
formation via their respective signaling pathways. Interestingly, two genes (Fgf21 and Il6) were
heavily down-regulated, and their interactions with alcohol remain unknown. This data is the
first to exploit moderate alcohol drinking as another risk factor for PDAC. The up-regulation of
Scd1 by alcohol in Kras-expressing mice lead to our focus on SCD as a potential target for
PDAC tumor suppression. The addition of the SCD inhibitor to pancreatic tumor organoids leads
to their degeneration, indicating the importance of SCD in pancreatic tumor cell survival. Our
data demonstrates that the accumulation of saturated fatty acid and lack of monounsaturated fatty
acid causes lipotoxicty leading to the induction of UPR and eventual apoptosis of tumor cells.
The observed selectivity of SCD inhibition on tumor cells makes it a targetable candidate for
PDAC therapy.

48

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Abstract (if available)

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a relatively rare cancer type, but is the fourth-leading cause of cancer death in the U.S. Although chemotherapy and immunotherapy have been used for patients with PDAC, the overall five-year survival rate is 8% and there is an urgent need for effective therapies. Genetic risk factors, such as Kras, have been identified as key mutations that induce PDAC development. Environmental risk factors including obesity, pancreatitis, and tobacco smoking have also been discovered to promote PDAC. Heavy alcohol drinking has been suspected to be a risk factor due to its role in causing pancreatitis, but it has not been known if moderate alcohol drinking could promote PDAC. In our study, we examined the effects of a moderate alcohol diet on the development of pancreatic neoplasia and invasive PDAC in mice expressing oncogenic Kras. We found that moderate alcohol drinking induces the formation of advanced neoplasia as well as PDAC compared to a regular chow diet in Kras-expressing mice. Our data indicate that moderate alcohol intake is a risk factor for pancreatic cancer. RNA-seq analysis suggests up-regulation of stearoyl-CoA desaturase 1 (SCD1) in pancreatic tumors developed in mice fed alcohol. SCD is an enzyme localized to the endoplasmic reticulum that functions by converting saturated fatty acid to monounsaturated fatty acid. We found that the inhibition of SCD disrupts lipid metabolism in pancreatic tumor cells, leading to their death in vitro and in vivo. Our data shows the selectivity of tumor cell death by SCD inhibition, making it a novel target for PDAC suppression.

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Asset Metadata

Creator Skrypek, Kaitlin (author)

Core Title Effects of moderate alcohol intake and role of stearoyl-CoA desaturase on the development of pancreatic tumors

School Keck School of Medicine

Degree Master of Science

Degree Program Experimental and Molecular Pathology

Publication Date 10/23/2020

Defense Date 06/29/2020

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

Tag Alcohol,lipid metabolism,monounsaturated fatty acid,OAI-PMH Harvest,pancreatic cancer,PDAC,saturated fatty acid,SCD,spheroids,stearoyl-CoA desaturase

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Asahina, Kinji (committee chair), Ouellette, Andre (committee member), Tsukamoto, Hidekazu (committee member)

Creator Email katieskrypek@gmail.com,skrypek@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-383285

Unique identifier UC11666271

Identifier etd-SkrypekKai-9054.pdf (filename),usctheses-c89-383285 (legacy record id)

Legacy Identifier etd-SkrypekKai-9054.pdf

Dmrecord 383285

Document Type Thesis

Rights Skrypek, Kaitlin

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA