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Co-expression of monoamine oxidase A and prostate cancer stem cell markers in Pten knockout mice

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Co-expression of monoamine oxidase A and prostate cancer stem cell markers in Pten knockout mice

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Co-expression of monoamine oxidase A and prostate
cancer stem cell markers in Pten knockout mice

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 in
Pharmaceutical Sciences

Peirou Chu
May 2019

2
ACKNOWLEDGEMENTS

First and foremost, I would like to thank my thesis advisor, Dr. Jean C. Shih of the
School of Pharmacy at University of Southern California for her continuous support
and guidance. During my research and writing process, the door to Dr. Shih’s office
was always open whenever I ran into a trouble or had questions. She consistently
encouraged me to challenge myself, and steered me in the right direction at all times.

I would like to thank the experts who helped me along the way for their involvement
and assistance in this research project and the insight and expertise I received from
them: Drs. Ronald Irwin, Pei-Chuan Li, Chun-Peng Liao and Mr. Bin Qian. Their
passionate participation and input have greatly improved the quality of this thesis.

I would also like to acknowledge Dr. Enrique Cadenas and Dr. Yong Zhang for
serving on my thesis committee and providing me with valuable insights and
comments.

Finally, I must express my most sincere gratitude to my parents, who have
provided me with unfailing support and nonstop encouragement throughout my
years of study and through my process of researching and writing this thesis. My
accomplishment would not have been possible without them. Thank you.

3
TABLE OF CONTENTS
Acknowledgements 2
List of Figures 4
Abbreviations 5
Abstract 6
Chapter 1: Introduction 7
1.1) Prostate Cancer 7
1.2) Monoamine Oxidase A 8
1.3) Prostate Cancer Stem Cells 10
1.4) Prostate Stem Cell Markers 17
1.5) Prostate Cancer Cell Spheroids 20
1.6) Specific Aims 21
Chapter 2: Results 23
2.1) Nanog and Oct4 Expression in Pten KO Mice 23
2.2) Co-expression of MAOA and Nanog/Oct4 25
2.3) Spheroid Formation of LNCaP Cells 29

Chapter 3: Discussion and Conclusion 32
Chapter 4: Materials and Methods 36
Bibliography 43

4
LIST OF FIGURES
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Figure 6

Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Differentiation of prostate epithelium.
Effects of MAOA overexpression on prostate CSCs.
PCa development and relapse depending on PCa cells.
Expression of Nanog and Oct4 in mouse prostate tissue.
Co-expression of MAOA and Nanog in Pten KO mouse
prostate tissue detected by IHC.
Co-expression of MAOA and Nanog or Oct4 in Pten KO
mouse prostate tissue detected by fluorescent IHC.
Spheroid formation from LNCaP cells.
Proposed mechanism of MAOA regulating CSCs.
Biotin-detection method of IHC.
Simplified procedures of IHC.
Simplified procedure of spheroid formation.
14
15
16
24
26

28

31
35
39
40
42

5
ABBREVIATIONS
ABCG2
ADT
AR
ATCC
CAF
ChrA
CRPC
CSC
DAPI
DNA
EMT
FACS
FBS
HIF1α
IF
IHC
KO
MACS
MAOA
MAOI
MCS
miRNA
MMAC1
NCI
NE
P-S
PCa
PIN
PSA
PSC
Pten
ROS
RT-PCR
SPOP
TBS
ATP binding cassette sub-family G member 2
Androgen-deprivation therapy
Androgen receptor
American Type Culture Collection
Cancer-associated fibroblast
Chromogranin A
Castration-resistant prostate cancer
Cancer stem cell
4’,6-diamidino-2-phenylindole
Deoxyribonucleic acid
Epithelial-mesenchymal transition
Fluorescence-activated cell sorting
Fetal bovine serum
Hypoxia-inducible factor 1α
Immunofluorescence
Immunohistochemistry
Knockout
Magnetic-activated cell sorting
Monoamine oxidase A
Monoamine oxidase inhibitor
Multicellular spheroid
microRNA
Mutated in multiple advanced cancers
National Cancer Institute
Neuroendocrine
Penicillin-streptomycin
Prostate cancer
Prostatic intraepithelial neoplasia
Prostate-specific antigen
Prostate stem cell
Phosphatase and tensin homolog
Reactive oxygen species
Reverse-transcriptase polymerase chain reaction
Speckle-type POZ protein
Tris-buffered saline

6
ABSTRACT
Monoamine oxidase A (MAOA) is a mitochondrial enzyme that degrades
monoamines and produces hydrogen peroxide. Recent studies have shown an
elevated expression of MAOA in prostate cancer (PCa). Inhibition of MAOA
reduces the tumorigenicity and stemness of prostate cancer stem cells (CSCs), which
confer the tumorigenesis, proliferation and metastasis of PCa. Nanog and Oct4
maintain the pluripotent status and activate stemness pathways in prostate CSCs. In
this study, we found elevated expression of Nanog and Oct4 in malignant mouse
prostate tissues compared to wild type. We also found the co-expression of MAOA
and Nanog/Oct4 in the prostate epithelia cells of Pten knockout (KO) mouse model.
Further, we performed spheroid formation from LNCaP PCa cell line and found a
cell concentration-dependent manner of the spheroid size and number. Taken
together, we propose that MAOA promotes PCa proliferation by upregulating CSC
stemness pathways in which Nanog and Oct4 are involved, and point to the potential
use of MAO inhibitors for therapy against prostate CSCs.

7
Chapter 1: Introduction
1.1) Prostate Cancer
Prostate cancer (PCa), the development of malignant cells in the gland of the male
reproductive system, is one of the most common cancers in males. The most
common ways to diagnose PCa are by transrectal biopsies, medical imaging, and
prostate-specific antigen (PSA) test. Tests are conducted to determine the spread of
PCa following the diagnosis. If the patient has signs that PCa has spread, such as
high PSA levels and high Gleason scores, the PCa may be grouped into Stage I, II,
III or IV according to the Gleason score (2019a).

Common risk factors for PCa include obesity, age, and genetic background.
Infection can also increase the chance of PCa development through constant
exposure of the tissue to an inflammatory environment (Hsing and Chokkalingam,
2006). Biopsies of the prostate tissue under precancerous conditions can reveal
prostatic intraepithelial neoplasia (PIN). Low-grade PIN can be an indication of
benign prostatic hyperplasia (BPH), which is not cancerous or fatal. On the other
hand, high-grade PIN are more likely to indicate the development of PCa (2019a).

Early-stage PCa that is localized only in partial prostate can be well-treated by a
combination of good prognosis and radical prostatectomy to remove the tumor
8
(2019b). Since the growth of prostate glands require androgen hormones, late-stage
PCa is treated with hormone therapies such as androgen-deprivation therapy (ADT)
(Seruga et al., 2011). As PCa progresses more aggressively, it can become metastatic
and spread to surrounding tissues, the bones and lymph nodes. Over time, PCa can
develop resistance to hormone therapy and become castration-resistant prostate
cancer (CRPC). Treatment of metastatic PCa can be challenging with limited options.
Currently, there is no treatment that can cure progressive hormone-refractory
metastatic PCa. Therefore, understanding the stages of cell differentiation in prostate
epithelium is crucial for the identification of target cells that are important in PCa
tumorigenesis, proliferation and metastasis.

1.2) Monoamine Oxidase A
Monoamine oxidase A (MAOA) is a flavoenzyme that is anchored to the
mitochondrial outer membrane. The MAOA gene that encodes the enzyme is located
on chromosome Xp11.3 (Lan et al., 1989). MAOA degrades monoamine
neurotransmitters including serotonin, norepinephrine and dopamine, as well as
dietary amines in the brain and peripheral tissues, and produces the reactive oxygen
species (ROS) byproduct hydrogen peroxide (Shih, 2018; Shih et al., 1999). ROS
can damage the DNA and can reduce the expression of tumor suppressor genes such
as p53 and Pten, and predispose cells to cancer by promotion of tumor initiation and
9
proliferation (Gupta et al., 2012). ROS also stabilizes hypoxia-inducible factor 1 α,
which plays a role in tumorigenesis, invasion and metastasis of cancers by activation
of hypoxia-inducible genes (Kaelin and Ratcliffe, 2008).

The roles of MAOA in behavioral and neurological disorders such as depression,
aggression and autism have been extensively studied over the past few decades.
Additionally, recent studies have showed that MAOA plays a role in several cancers
including PCa, glioma and Hodgkin lymphoma (Li et al., 2017; Shih, 2018).

Studies have identified MAOA as one of the most highly expressed genes in
epithelial cells in aggressive high-grade PCa, indicating that MAOA may contribute
to the growth of high-grade PCa (Peehl et al., 2008). In addition, evidence has proved
the importance of MAOA in the proliferation, invasion and metastasis of PCa (Liao
et al., 2018; Wu et al., 2014). Overexpression of MAOA in primary PCa cells results
in epithelial-mesenchymal transition (EMT), which is characterized by loss of cell
polarity and cell-cell adhesion, and occurs during the initiation of PCa metastasis
(Wu et al., 2015). The inhibition of MAOA by MAO inhibitors (MAOIs) or by gene
knockdown has shown decreased or even eliminated PCa tumorigenesis and
metastasis (Wu et al., 2014). Furthermore, MAOA plays a regulatory role in subset
of PCa cells known as cancer stem cells (CSCs) (Liao et al., 2018).
10
1.3) Prostate Cancer Stem Cells
In normal human prostate, the basement membrane of the prostatic epithelium is
lined by of 3 types of cells: basal, neuroendocrine (NE) and luminal cells (Seruga et
al., 2011). Each type of cells is distinguished based on their localization, morphology
and phenotypic characteristics, serves different functions and expresses different
surface markers. Luminal cells are androgen-dependent cells that release PSA into
the lumen. They are characterized by the expression of androgen receptor (AR),
CD24, CD26, CK8 and CK18. On the other hand, basal cells are incapable of
releasing AR and they express CD44, p63, CK5, CK14, and low levels of AR.
Neuroendocrine (NE) cells are secretory cells localized in both the basal and
neuroendocrine cell layers. They express NE markers such as synaptophysin and
chromogranin A (ChrA) (Sherwood et al., 1990).

Prostate stem cells (PSCs) are androgen-independent unipotent or multipotent cells
capable of differentiating into the epithelial cell types. They are characterized by the
expression of stem cell markers such as Nanog and Oct4. PSCs have a constant self-
renewal capability; they proliferate to produce progeny including both non-
differentiated and distinctly differentiated cells. In human prostate epithelium, the
PSCs are shown to belong to the basal cell. It was found that MAOA is highly
expressed in basal cells of the prostate epithelium, but not in NE or luminal cells.
11
Overexpression of MAOA induces the formation of the basal cell phenotype, and
suppress the differentiation of PSCs into NE and luminal secretory cells (Zhao et al.,
2008).

During PCa progression, malignant cells grow uncontrollably around the basement
membrane and eventually metastasize to other tissues (Rybak et al., 2015). The
cancer stem cell (CSC) theory hypothesizes that a subset of tumor cells plays a
crucial role in the tumorigenesis, progression and metastasis of cancer. These cells
are in a stage that has PSC-like characteristics and establishment of the
heterogeneous structure of cells within the tumor (Visvader and Lindeman, 2008).
CSCs have an unlimited self-renewal ability and can also generate non-tumorigenic
progeny. In addition, CSCs are resistant to common PCa therapies including chemo-
or radiotherapy (Marhold et al., 2015). Similar to PSCs, the CSCs can be identified
by the expression of stem cell markers including CD44, Nanog and Oct4. It was
shown that MAOA is highly expressed in prostate CSCs (Liao et al., 2018).

As the cancer becomes advanced, the prostate epithelium becomes less and less
distinctly differentiated with large amounts of CSCs aggregated. Eventually, in
CRPC and PCa metastasis, the prostate epithelium loses its heterogeneous structure
and becomes consisted of undifferentiated cells with uncontrolled growth. Some
12
evidence suggests the CSCs that pre-exist in tumors may have castration-resistant
properties. Since the CSCs are highly tumorigenic, they are capable of initiating
regeneration of a new tumor that recapitulates the original tumor histology and
heterogeneity even after the ADT (Chen et al., 2013; Tang et al., 2007). In addition,
the cancer cells that are preserved after the initial therapy can form genetically
modified progeny to drive tumorigenesis and promote PCa relapse (Adamowicz et
al., 2017).

Due to the highly proliferative and therapy-resistant characteristics of prostate CSCs,
various therapies targeting CSCs are under extensive research effort. Over the past
decade, investigators have identified numerous therapeutic targets that play roles in
maintaining prostate CSCs. Studies have proposed potential treatment regimen to
target CSCs in advanced PCa, especially for CRPC (Yun et al., 2016; Zhang et al.,
2016b) by targeting cells, protein complexes, as well as microRNAs. Cancer-
associated fibroblasts (CAFs) isolated from mouse PCa tumors were shown to
enhance the stemness and growth of prostate CSCs (Liao et al., 2010). Adipocytes
were also found to promote prostate CSC self-renewal and growth by induction of
an autocrine amplification loop (Tang et al., 2016).

13
Numerous microRNAs (miRNAs) have also been identified as a critical regulatory
factor in the stemness of prostate CSCs by regulate the stemness pathways of CSCs
(Fang et al., 2015; Song et al., 2018). For instance, inhibition of miRNA-128 was
found to reduce the spheroid formation and self-renewal of prostate CSCs, and
suppress the expression of pluripotency-maintaining factors including Nanog and
Oct4 (Nanta et al., 2013). On the other hand, miRNA-34a inhibits the self-renewal
of prostate CSCs by targeting genes involved in maintenance of CSC stemness (Liu
et al., 2011).

In addition, hopoxic pathway by HIF1α can regulate CSC properties and PCa
metastasis. It was shown that an elevated HIF1α level in prostate CSCs under
hypoxic condition can lead to a decrease in mTOR (mammalian target of rapamysin)
level, which then inhibits the viability of prostate CSCs (Marhold et al., 2015). Also,
the androgen-efflux transporter ABCG2 (ATP binding cassette sub-family G
member 2) enriched in ADT-resistant CSCs was shown to maintain the
undifferentiated state and the growth of prostate CSCs (Sabnis et al., 2017).
Inhibition of ABCG2 can disrupt the androgen efflux of the CSCs and force them to
undergo differentiation into ADT-sensitive luminal cells. Also, the phosphorylation-
mediated Nanog stability was proved to regulate the stemness of prostate CSCs, and
destruction of Nanog mediated by the SPOP (Speckle-type POZ protein) suppresses
14
the prostate CSC stemness and PCa progression (Wang et al., 2019; Zhang et al.,
2019). These studies suggest that Nanog can be used as a drug target in PCa therapy.
In addition, it is known that the deletion of MAOA suppresses invasive PCa and
leads to a significantly reduced CSC population (Liao et al., 2018). However, the
mechanism of this action and the stemness pathways of prostate CSCs regulated by
MAOA remain unknown. Therefore, in order for MAOIs to become therapeutic
agents targeting CSCs, the correlation between MAOA and proteins uniquely
expressed in CSCs needs to be further studied.

Figure 1. Differentiation pattern of prostate epithelium. Various subtypes of
prostatic cells are shown. The basal cells express MAOA. The stem cells and CSCs
are located in the basal layer and express MAOA, Nanog and Oct4. The luminal cells
express AR, CD24 and CK18. The NE cells are located in both the luminal and basal
layers and express synaptophysin and ChrA. (Figure adapted from Toivanen and
Shen (2017) and prepared by P. Chu on https://app.biorender.com/).

15

Figure 2. The effects of MAOA overexpression in differentiation, division, mutation
and dedifferentiation of CSCs during PCa. PSCs can self-renew into new PSCs or
differentiate into mature epithelial cell subtypes. MAOA overexpression promotes
basal, luminal, NE and PSCs to transform into malignant CSCs through mutagenesis
and/or dedifferentiation. MAOA overexpression inhibits differentiation of PSCs into
luminal or NE cells. CSCs interact with the tumor environment to develop
heterogeneity and promote proliferation of the tumor. (Figure prepared by P. Chu on
https://app.biorender.com/).

16
Figure 3. PCa development depending on PCa cell and CSCs. Both cell types
can survive the initial therapy. A potential PCa relapse can be driven by: (2)
tumorigenesis promoted by CSCs to recapitulate the original tumor; (3) non-stem
PCa cells with tumorigenic potential; (1) genetically modified CSC progeny; and (4)
genetically modified non-stem PCa cell progeny. Therapy against CSCs are often
needed for PCa relapse patients. (Figure adapted from Adamowicz et al. (2017) and
prepared by P. Chu on https://app.biorender.com/).

17
1.4) Prostate Stem Cells Markers
In the last several decades, numerous methods have been discovered to identify the
population of normal PSCs and prostate CSCs. Biomarkers are often used alone or
in combination to identify the population of CSCs. The CSCs are frequently purified
using either fluorescence-activated cell sorting (FACS) or magnetic-activated cell
sorting (MACS). The expression of the CSC biomarkers can also be analyzed by
immunohistochemistry (IHC), immunofluorescence (IF) and reverse-transcriptase
polymerase chain reaction (RT-PCR).

Cell surface markers are often used to isolate CSCs from primary tumors of PCa or
PCa cell lines. CD44 is a single-pass type I transmembrane protein that is a cellular
adhesion molecule important in extracellular signal transduction by binding to
hyaluronan and other extracellular molecules. It is extensively located on the
membrane of CSCs (Zhang et al., 2016a). CD44 is considered as a common marker
of prostate CSCs. Most studies have demonstrated that CD44
+
cells extracted from
PCa tumors possess stem cell-like characteristics. Recent studies have reported a
correlation between MAOA and CD44 expression in high-grade metastatic PCa
biopsy samples (Peehl et al., 2008). The CD44
+
phenotype is also considered as a
feature of tumors with more aggressive characteristics. It was discovered that in PCa
cell lines, expression of CD44 is positively correlated with expression of genes
18
involved in tumorigenesis and metastasis. CD44
+
CSCs also have a much greater
proliferative potential compared to CD44
-
cells (Kasper, 2009).

CD133 is a 5-domain transmembrane glycoprotein that is expressed on the surface
of stem cells (Zhang et al., 2016a). In normal prostate epithelial cells, CD133 has
been observed in both basal and luminal cells with low expression levels (Missol-
Kolka et al., 2011). Studies have reported a subpopulation of CD133
+
cells isolated
from PCa cell lines being highly clonogenic, malignant and resistant to radiation and
chemotherapeutic agent, docetaxel (Kanwal et al., 2018). However, another study
demonstrated that CD133
+
-enriched stem cells have less tumorigenic potential and
are less susceptible to tumorigenesis than CD133
-
basal cells. These results indicate
that further studies are needed to understand the complicated role of CD133 in PCa
tumorigenesis. Nonetheless, CD133 is widely used as a PSC and CSC marker in
combination with CD44.

Integrins are a family of transmembrane receptors important in cell adhesion and
cell-extracellular matrix (ECM) signaling (Zhang et al., 2016a). Through interacting
with specific ligands to stimulate cell-cell and cell-ECM signaling, integrins can
activate the expression of downstream proteins. Integrins are overexpressed in PCa
cells. More specifically, the α2β1 integrin plays an important role in the interaction
19
between prostatic epithelial and stromal cells, which contributes to bone metastasis
(Van Slambrouck et al., 2014). It was discovered that PSCs localized at the basal
layer have positive expression of α2β1 integrin. The expression of α2β1 integrin was
observed in a small fraction of PCa cells, with approximately 1% cells being α2β1
integrin
+
and displaying the basal cell phenotype (Van Slambrouck et al., 2014).
Over the past decade, α2β1 integrin has become a new biomarker for the
identification of PSCs. It has been widely used in combination with CD44 and
CD133 to isolate CSCs from PCa primary tumors and metastatic tissues. The
CD44
+
/CD133
+
/α2β1 integrin
+
biomarker combination has become an emerging
detection method for PCa evaluation and prostatic CSC identification.

Nanog is a homeodomain transcription factor that plays an important role in
maintaining the self-renewal, undifferentiating, proliferation and the pluripotency
status of normal and cancer stem cells (Gong et al., 2012). It is not expressed in
differentiated adult cells. Nanog functions in concert with other proteins including
Oct4 to establish the stemness of the cells (Gong et al., 2015). High expression of
Nanog can prevent differentiation of the stem cells. Nanog is negatively regulated
by the tumor suppressor P53 and has a role in tumorigenesis and metastasis in
cancers (Lin et al., 2005). Research has revealed that Nanog is highly expressed in
CD44
+
/CD133
+
/α2β1 integrin
+
prostate CSCs (Gong et al., 2012). Therefore, Nanog
20
has become a new marker and target of prostate CSCs.

Oct4 (also known as OCT3/4) is another transcriptional factor that regulates genes
involved in maintaining the pluripotent status of cells (Lee et al., 2006). It is present
in two isoforms: nucleus-localized Oct4 A and cytoplasm-localized Oct4 B. In
human PCa, Oct4 and Nanog can bind together to form a functional complex to
activate downstream stemness pathways in CSCs. In PCa, CD44
+
CSCs have high
expression of Oct4 (Zhang et al., 2016a). Therefore, Oct4 can be used in combination
with Nanog as CSC markers.

1.5) Prostate Cancer Stem Cell Spheroids
In recent cancer research, multicellular spheroid culture models have been used
extensively in studies of tumorigenesis and metastasis. Conventionally, two-
dimensional cancer cell culture models are frequently used as in vitro models in the
studies of cancer biology. However, the two-dimensional cultures lack features
within the tumor microenvironment including the typical structural architecture,
which are crucial conditions for growth of CSCs. More importantly, two-
dimensional cultures of cancer cells are often incapable of producing an abundant
quantity of CSCs. Compare to the two-dimensional cultures, the spheroid cultures
provide many experimental advantages. They are easier to manipulate and analyze
21
compared to in vivo cultures that are usually preferred, yet the properties of tumors
grown in vivo are still highly represented in spheroids (Kurioka et al., 2011). Above
all, the spheroid cultures can enrich the population of CSC phenotype by activation
of ΔNp63α-mediated developmental pathways involved in maintenance of the
stemness of PCa cell lines (Portillo-Lara and Alvarez, 2015). Studies on spheroids
grown from the PC3, LNCaP, 22Rv1 and DU145 PCa cell lines showed the
formation of PCa spheroids can lead to a dramatic enrichment of CD133
+
CSCs with
a highly proliferative, invasive and resistant profile (Ballangrud et al., 1999; Liao et
al., 2018; Portillo-Lara and Alvarez, 2015). Previously, it was shown that MAOA
can promote the stemness of prostate CSCs, and the inhibition of MAOA by both
genetic silencing and MAOIs significantly reduces the spheroid-forming ability of
LNCaP cells (Liao et al., 2018). Assays testing the spheroid-forming ability have
become widely used to characterize the phenotype of prostate CSCs. Therefore, the
spheroid culture system can be used in investigation of the molecular mechanism of
CSC growth inhibition caused by MAOA.

1.6) Specific Aims
Previous work has demonstrated the deletion of MAOA can reduce the stem cell
population and inhibit tumor formation in the Pten KO mouse model (Liao et al.,
2018). Similarly, the knockdown of MAOA in human PCa cell lines resulted in
22
reduced Oct4 and Nanog levels. In this study, we hypothesized that MAOA is
colocalized with the CSC markers Nanog and Oct4 in the Pten knockout (KO)
murine PCa model. These results will indicate a potential role of MAOIs in
disrupting the pluripotent status of CSCs maintained by Nanog and Oct4, and
provide preliminary evidence for establishing the role of MAOA in regulation of
CSCs, thus better understand the mechanism of MAOIs for PCa treatment.

23
Chapter 2: Results
2.1) Nanog and Oct4 expression in Pten KO mice
The phosphatase and tensin homolog deleted on chromosome ten (Pten), also known
as mutated in multiple advanced cancers (MMAC1), is a phosphatase that acts as a
tumor suppressor gene located at chromosome 10q23 (Chu and Tarnawski, 2004). It
is the most commonly mutated target in human PCa. Loss of heterozygosity (LOH)
at chromosome 10q23 are detected in half of all PCa patients, whereas homozygous
deletion of Pten is present in approximately 1/10 of these patients (Chu and
Tarnawski, 2004). In murine models, homozygous deletion of Pten results in
embryonic death, whereas LOH of Pten leads to spontaneous tumorigenesis in the
prostate and accelerated PCa progression.

A Pten KO mouse model mimicking the development of high-grade PIN and tumor
progression of human PCa has been established in Shih Lab (Liao et al., 2018). Here,
we compared the expression of Nanog and Oct4 CSC markers in wild type versus
Pten KO mouse prostate tissue phenotypes by chromogenic IHC. The results (Fig.
4) showed that the Pten KO model with spontaneous tumor formation had increased
expression of both Nanog and Oct4 compared to the wild type.

24

Figure 4. The expression of (A) Nanog and (B) Oct4 in 6-month old wild type
and Pten KO mouse dorsal-lateral prostate tissue sections detected by IHC. PIN
areas are visible. (A) Positive Nanog expression shown as dark nuclear staining
pointed by orange arrows. (B) Positive Oct4 expression shown as brown staining in
cytoplasm. Both Nanog and Oct4 have elevated expression in Pten KO phenotype
than wild type.

25
2.2) Co-expression of MAOA and Nanog/Oct4
In addition, we investigated the co-expression of MAOA and Nanog or Oct4 in Pten
KO mouse prostate tissue phenotypes. The chromogenic detection method for IHC
can be difficult in distinguishing co-localized targets since the chromogen forms
only a single color. In these experiments, we used the fluorescent IHC detection
method, which allows the separate identification of co-localized targets. The earliest
attempts of the experiments were conducted on 4-month Pten KO mouse prostate
tissues using various modified conditions such as differences in prostate lobes,
secondary antibody selections, and antibody dilutions. The results indicated positive
expression of MAOA in malignant PIN cells and sporadic expression of Nanog
among these cells (Fig. 5). However, the experimental conditions needed to be
improved to confirm the co-localization of both proteins.

26

Figure 5. Detection of MAOA and Nanog expression in 4-month Pten KO
mouse (A) apical and (B) dorsal-lateral prostate tissue sections by fluorescent IHC.
Blue: DAPI nucleus stain. Red: MAOA. Green: Nanog. MAOA is universally
expressed in the cytoplasm of cells of the PINs. The cells in PINs with concurrent
expression of MAO and Nanog are indicated by arrows.

PIN
DAPI

Nanog

MAOA

Overlay

A

PIN
Overlay

MAOA

Nanog

DAPI

B

27
Later attempts of the fluorescent IHC experiments were performed with optimized
conditions, such as changing antibodies and antibody dilution concentration. We
also used 6-month Pten KO mouse prostate tissues, which have generated more
malignant tissues compared to 4-month tissues. In CSCs, Nanog and Oct4 function
together by forming a complex. Therefore, to confirm the co-expression of Nanog
and MAOA, we also attempted to detect the co-expression of Oct4 and MAOA.
Higher magnification was used to image the cells on a confocal microscope to
observe the localization of the proteins.

The results showed a correlation in the expression of MAO and Nanog or Oct4 (Fig.
6). Previous work has shown decreased Nanog and Oct4 expression in MAOA-
deleted prostate tissues (Liao et al., 2018). Hence the results here are consistent with
the previous study. Similar to the earlier attempts (Fig. 5), MAOA is universally
localized in the cytoplasm (Fig. 6). Nanog and Oct4 are only expressed in a small
fraction of the cells (Fig. 6), indicating a low population of CSCs in these tissues.
Nanog is localized in the nucleus (Fig. 6A) of a MAOA
+
cell, and Oct4 is seen in the
nucleus and cytoplasm of MAOA
+
cells (Fig. 6B), indicating the co-expression of
MAOA and Nanog or Oct4.

28

Figure 6. Fluorescent IHC detection of co-expression of MAOA and (A)
Nanog or (B) Oct4 in 6-month Pten KO mouse dorsal-lateral prostate tissue PINs.
MAOA is localized in the cytoplasm. Cells with concurrent expression of MAOA
and Nanog or Oct4 are indicated by arrows. (A) MAOA and Nanog co-expression.
Nanog is expressed in the nucleus. Blue: DAPI. Red: Nanog. Green: MAOA. (B)
MAOA and Oct4 co-expression. Oct4 is expressed in the nucleus and cytoplasm.
Blue: DAPI. Red: Oct4. Green: MAOA.

DAPI

MAOA

Nanog

Overlay

A

DAPI

MAOA

Oct4

Overlay

B

29
2.3) LNCaP Spheroid Formation
It has been shown that that the multicellular spheroid cultures of cancer cell lines
can expand the population of prostate CSCs. Previous studies on spheroids grown
from the PC3, LNCaP, 22Rv1 and DU145 PCa cell lines have demonstrated the
capability of spheroids to result a dramatic enrichment of CSCs (Ballangrud et al.,
1999; Liao et al., 2018; Portillo-Lara and Alvarez, 2015).

The LNCaP cell model is a commonly used human cell line for in vitro study of PCa.
These AR and PSA-expressing androgen-dependent cells were derived from cells of
prostate adenocarcinoma lymph node metastasis (Horoszewicz et al., 1983). Unlike
other “classic” PCa cell lines such as PC-3 and DU145, which do not express AR or
PSA (van Bokhoven et al., 2003), LNCaP closely mimics the biological behaviors
of the vast majority of human PCa tissues encountered clinically.

Previous study has shown the inhibition of MAOA decreases the spheroid-forming
ability in LNCaP PCa cells, resulting in a decrease in the size and number of
spheroids (Liao et al., 2018). In this project, 500, 1,000 and 2,000 LNCaP cells per
well were seeded and cultured as three-dimensional spheroids. After 17 days
culturing in Matrigel, there was successful spheroid formation from the single cells
(Fig. 7A). The result from the 2,000 cell/well group showed that nearby spheroid
30
colonies tended to fuse together (Fig. 7A), indicating the interaction between
spheroid microenvironments during tumorigenesis. The number of spheroids formed
from LNCaP cells was also shown to be dependent on the number of cells planted
in the well. After 17 days of growth, the 2,000 cells/well group had a significantly
larger number of spheroids than the groups with less cells planted (Fig. 7B). In
addition, the sizes of the spheroids in each group were determined every 2-3 days of
growth. The results showed a cell concentration-dependent manner of spheroid size
(Fig. 7C). In general, the 1,000 cells/well group had larger spheroids sizes than the
500 cells/well group, and the spheroids of 2,000 cells/well group had overall larger
sizes than the former two.

31

Figure 7. Spheroid formation from different concentration of LNCaP cells
after 17 days of culture in Matrigel. Images of spheroids were captured every 2-3
days. (A) 500 LNCaP cells per well were seeded. (B) 1,000 LNCaP cells per well
were seeded. The number and size of the spheroids are visibly bigger than the 500
cells per well group. (C) 2,000 LNCaP cells per well were seeded. The number and
size of the spheroids are significantly larger than the 500 and 1,000 cells per well
groups. Day 14 and 17 show the interaction of two nearby spheroid colonies fusing
into one spheroid.

B

C

A

500 cells/well

1,000 cells/well

2,000 cells/well

32
Chapter 3: Discussion and Conclusion
PCa is one of the most common cancers in males. Early-stage PCa can be well-
treated, but clinical treatment options for aggressive and metastatic ADR-resistant
PCa are still very limited (2019b). It is known that CSCs contribute to the metastatic
and invasive potential of high-grade PCa. In the past decade, many studies have
aimed to target prostate CSCs by disrupting their stemness pathways (Yun et al.,
2016).

It was identified that the flavooxidase MAOA is one of the most highly expressed
proteins in high-grade PCa, it plays a critical role in the proliferation, invasion and
PCa metastasis (Liao et al., 2018; Peehl et al., 2008; Wu et al., 2014). The inhibition
or deletion of MAOA reduce PCa tumorigenesis and metastasis, and suppress the
population and stemness of prostate CSCs (Liao et al., 2018). However, the
mechanism of reduction of stemness caused by MAOA inhibition is unknown.

In this study, we have demonstrated the co-expression of MAOA and two CSC
markers, Nanog and Oct4, in Pten KO mouse prostate tissues. The expression of
MAOA was shown to be distributed among the cytoplasm of cells of malignant PINs.
On the other hand, only a small subset of cells of the expressed Nanog (Fig. 5A) and
Oct4 (Fig. 5B). The results indicate that these cells with co-expression of
33
Nanog/Oct4 and MAOA are highly tumorigenic, metastatic CSCs. Previous studies
have shown treatments targeting Nanog in CSCs are effective against PCa, and
deletion of MAOA results in decreased levels of Nanog and Oct4 (Liao et al., 2018).
Thus, the co-localization of the stem cell markers and MAOA shown in this project
provides a new piece of evidence to support potential role of MAOA in the
regulation of the CSC stemness pathways that Nanog and Oct4 are involved with.

It is known that CSCs build a network by sending signals back and forth within the
tumor environment and initiate the tumor growth and survival (Ciardiello et al.,
2018). Maintenance of the stemness of CSC is required to promote the
differentiation and proliferation of CSCs. The formation and expansion of tumor are
dependent on the cell-cell attachment and cell-ECM signaling of CSCs. The spheroid
model system is known to represent in vivo tumors and promote the population of
CSCs. In this study, we chose LNCaP PCa cells to perform spheroid formation for
its exceptional characteristics that highly represent in vivo PCa. The spheroid
formation method used in this study produced successful spheroid growth of LNCaP
spheroids. The results indicated cell-cell and spheroid-spheroid interactions within
the microenvironment of the cultures (Fig. 7A). The number and size of the
spheroids were shown to be cell concentration-dependent. Since spheroid growth
can drastically expand the population of CSCs (Kerr and Hussain, 2014), future
34
analysis of the mechanism of MAOA inhibition in prostate CSCs can be done by
IHC imaging or flow cytometry of these spheroid cultures with MAOI treatments.

Overall, it is known that the loss of MAOA in PCa decreases the CSC population
and inhibits the growth of tumor (Liao et al., 2018). However, the mechanism of
MAOA in CSCs remains unclear. This study showed the co-expression of MAOA
and Nanog or Oct4 in Pten KO mouse prostate tissues, suggesting that MAOA may
promote PCa proliferation by upregulating the prostate CSC stemness pathways in
which Nanog and Oct4 are involved. Here, we propose a mechanism for how MAOA
engages in regulation of prostate CSC stemness (Fig. 8).

35
Figure 8. A proposed working model for how MAOA regulates the stemness
of prostate CSCs by engaging ROS, hypoxia, PI3K/AKT/mTOR pathway and
Nanog/Oct4 expression. ROS produced from MAOA overexpression or during
hypoxia induces activity of HIF1α. Hypoxic pathways induced by HIF1α promote
CSC formation. The PI3K/AKT/mTOR pathway regulates self-renew of prostate
CSCs. The expression of Oct4 and Nanog is involved in EMT and maintenance of
stemness of prostate CSCs. These CSCs promote tumorigenesis and metastasis.

Future studies are required to determine the stemness pathways disrupted by MAOA
inhibition and the outcomes of the disruption, as well as the specific mechanism of
MAOA molecular interaction with CSC markers including Oct4 and Nanog. These
pieces of information will provide a novel direction in targeting CSC in PCa therapy,
and give an insight into potential therapeutic strategies of MAOIs in treating
aggressive, metastatic PCa or CRPC.
36
Chapter 4: Materials and Methods
Mouse generation and genotyping
The generation of the Pten KO and wild type mice is described by Liao et al., 2018.
Mice carrying ARR2PB-Cre and floxed Pten (Pten
f/f
) alleles on C54BL/6xDBA2/
129 background were used. The animals were housed and maintained under identical
conditions. Animal experimentation was conducted in accordance with the ethical
federal guidelines mandated by the University of Southern California Institutional
Aminal Care and Use Committee. The method for genotyping of the mice was also
described in Liao et al. (2018). In short, the genomic DNA was collected from the
tail and prostate. The samples were purified using DNA Mini Kit (Qiagen), and
amplified by PCR. This project used the tissue blocks from the wild type and Pten
KO mice that were previous generated to produce results in Liao et al. (2018).

Mouse prostate tissue histopathology sections
The generation of the mouse prostate tissue sections was described by Liao et al.,
2018. The Pten KO and wild type mice were sacrificed at the ages of 4 and 6 months.
The prostate tissues of Pten KO and wild type mice were collected and categorized
into apical, ventral and dorsolateral prostate. and incubated in unbuffered zinc
formalin solution (ThermoFisher) at 4 °C overnight. After the incubation, the tissues
were washed 3 times in PBS and 1 time in ethanol for 10 minutes each. The tissues
37
were fixed in 70% ethanol, then embedded in paraffin, before cut to 5-µm sections
onto glass microscope slides and covered with wax at 25 °C. This project used the
tissue sections previous generated in Liao et al. (2018).

IHC analysis
The paraffin-embedded mouse prostate tissue sections were deparaffinized in xylene,
a mixture of ethanol and wax, and rehydrated in the following decreasing
concentrations of ethanol: 100%, 90%, 80%, 70%, 50%, 30%, 10%, and eventually
in H
2
O for 5 minutes each. Antigen retrieval was done in 10mM sodium citrate
buffer at 95 °C for 15 minutes to break the protein crosslinks and unmask the
epitopes to enhance the staining intensity of antibodies and reduce false positive
results. The sections were then incubated in 3% H
2
O
2
in methanol for 20 minutes to
block the endogenous peroxidase activity, before blocking in 5% non-fat dry milk
or 5% normal goat serum to prevent nonspecific binding in Tris-buffered saline
(TBS) with 0.3% Triton X-100 for 2 hours at 4 °C. Triton X-100 was added as a
detergent for permeabilization and access to the antigen inside the cell membrane.
The slides were washed in TBS for 5 minutes 3 times at room temperature before
incubation in primary antibody working solutions at 4 °C overnight. Antibodies and
concentrations used in working solutions: Oct4, 1:400 (Abcam); Nanog, 1:400
(Abcam); MAOA, 1:200 (Santa Cruz). The sections were washed in TBS for 3 times,
38
5 minutes each at room temperature. To detect specific staining, the tissues were
incubated in biotinylated secondary antibody solutions (Vector Laboratories)
prepared in VECTASTAIN ABC Elite kit (Vector Laboratories) at 1:1000 fold
dilution, or in fluorescence labeled secondary antibody solutions prepared in TBS at
1:200 fold dilution, for 2 hours at 4 °C.

*For tissue sections incubated in biotinylated secondary antibodies (Fig. 9):
After washing with TBS for 3 times, 5 minutes each, liquid DAB+ substrate
chromogen system (Dako) was applied to the sections with biotinylated secondary
antibodies for development of specific staining for 5 minutes, and the sections were
washed in TBS for 5 minutes. The DAB substrate binds to peroxidase and become
oxidized, producing a brown precipitate. The slides were transferred into
hematoxylin nuclear counterstain for 1 minute and washed in TBS until no pigment
was present. Oxidized hematoxylin combines with aluminum ions to form an active
metal-dye complex that stains the nuclei of mammalian cells blue color by binding
to lysine residues on nuclear histones. The slides were dehydrated in increasing
concentrations of ethanol and in xylene. 1-2 drops of Permount toluene solution
(Fisher Scientific) was used to mount the sections with cover slides to preserve the
sections for storage and enhance the imaging quality. Digital images were captured
39
under 40X objective using a brightfield microscope (Leica) on randomly chosen
fields of each section.

Figure 9. Biotin detection method in IHC. The primary antibody binds to the
epitopes of tissue antigen, it is recognized by the secondary antibody conjugated
with biotin. Avidin that is labeled with peroxidase forms a complex with biotin. The
DAB substrate can be oxidized by the peroxidase to produce a brown color
precipitate as a label to indicate specific staining of the tissue antigen.

*For tissue sections incubated in fluorescence labeled secondary antibodies:
After washing with TBS for 3 times, 5 minutes each, 1-2 drops of VECTASHIELD
Antifade Mounting Medium with DAPI (Vector Laboratories) was applied to each
slide with cover slides on top. The DAPI stains the nuclei of the cells and develop a
fluorescent blue color. The slides were stored at 4 °C. Digital images were captured
40
under 40X objective using Leica SP-8 confocal microscope on randomly chosen
fields of each section.

Figure 10. Simplified procedures of IHC. Each step is necessary for
successful specific detection of antigen.

41
Cell culture
Human PCa LNCaP cells were obtained from American Type Culture Collection
(ATCC). The cells were cultured in RPMI 1640 medium (Genesee Scientific)
supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin
(P-S) on 100-mm dishes. The cells were grown in a 5% CO
2
and 95% air incubator
at 37 °C. The medium was changed three times per week.

Spheroid formation
Spheroids were initiated using the liquid overlay technique, which cultures the
multicellular spheroid (MCS) model as spontaneous aggregation formed from
clusters on the surface of an agarose gel matrix that blocks adherence of the cells
(Kurioka et al., 2011). In specific, LNCaP cells were obtained by trypsinization from
growing monolayer cultures. The cells were grown and sub-cultured for at least three
generations before used in spheroid formation. The concentration of cells was
determined using a hemocytometer (INCYTO). 2,000 LNCaP cells resuspended in
1:1 Matrigel:RPMI 1640. The cells were seeded to wells of a 24-well plate coated
with a layer of 100% Matrigel. After the Matrigel was solidified, RPMI 1640
medium supplemented with 1% FBS was added into each well. The plate was
incubated at 37 °C in a 5% CO
2
and 95% air incubator for 17 days. The liquid RPMI
42
1640 medium was changed every 2-3 days. Images of spheroids were captured using
a VWR VistaVision compound microscope under 40X objective.

Figure 11. Simplified procedure of spheroid formation. LNCaP cells grown
from a monolayer culture were transferred to a 24-well plate coated with Matrigel.
The cells are incubated in RPMI 1640 medium and form spheroids on the Matrigel
layer.

43
Bibliography
1. (2019a). Prostate Cancer (National Cancer Institute).
2. (2019b). Prostate Cancer Treatment (National Cancer Institute).
3. Adamowicz, J., Pakravan, K., Bakhshinejad, B., Drewa, T., and Babashah, S.
(2017). Prostate cancer stem cells: from theory to practice. Scand J Urol 51, 95-
106.
4. Ballangrud, A.M., Yang, W.H., Dnistrian, A., Lampen, N.M., and Sgouros, G.
(1999). Growth and characterization of LNCaP prostate cancer cell spheroids.
Clin Cancer Res 5, 3171s-3176s.
5. Chen, X., Rycaj, K., Liu, X., and Tang, D.G. (2013). New insights into prostate
cancer stem cells. 12, 579-586.
6. Chu, E.C., and Tarnawski, A.S. (2004). PTEN regulatory functions in tumor
suppression and cell biology. Med Sci Monit 10, RA235-241.
7. Ciardiello, C., Leone, A., and Budillon, A. (2018). The Crosstalk between
Cancer Stem Cells and Microenvironment Is Critical for Solid Tumor
Progression: The Significant Contribution of Extracellular Vesicles. Stem Cells
International 2018, 11.
8. Fang, Y.X., Chang, Y.L., and Gao, W.Q. (2015). MicroRNAs targeting prostate
cancer stem cells. Exp Biol Med (Maywood) 240, 1071-1078.
44
9. Gong, C., Liao, H., Guo, F., Qin, L., and Qi, J. (2012). Implication of
expression of Nanog in prostate cancer cells and their stem cells. 32, 242-246.
10. Gong, S., Li, Q., Jeter, C.R., Fan, Q., Tang, D.G., and Liu, B. (2015).
Regulation of NANOG in cancer cells. Mol Carcinog 54, 679-687.
11. Gupta, S.C., Hevia, D., Patchva, S., Park, B., Koh, W., and Aggarwal, B.B.
(2012). Upsides and downsides of reactive oxygen species for cancer: the roles
of reactive oxygen species in tumorigenesis, prevention, and therapy. Antioxid
Redox Signal 16, 1295-1322.
12. Horoszewicz, J.S., Leong, S.S., Kawinski, E., Karr, J.P., Rosenthal, H., Chu,
T.M., Mirand, E.A., and Murphy, G.P. (1983). LNCaP model of human
prostatic carcinoma. Cancer research 43, 1809-1818.
13. Hsing, A.W., and Chokkalingam, A.P. (2006). Prostate cancer epidemiology.
Front Biosci 11, 1388-1413.
14. Kaelin, W.G., Jr., and Ratcliffe, P.J. (2008). Oxygen sensing by metazoans: the
central role of the HIF hydroxylase pathway. Mol Cell 30, 393-402.
15. Kanwal, R., Shukla, S., Walker, E., and Gupta, S. (2018). Acquisition of
tumorigenic potential and therapeutic resistance in CD133+ subpopulation of
prostate cancer cells exhibiting stem-cell like characteristics. Cancer Lett 430,
25-33.
45
16. Kasper, S. (2009). Identification, characterization, and biological relevance of
prostate cancer stem cells from clinical specimens. 27, 301-303.
17. Kerr, C.L., and Hussain, A. (2014). Regulators of prostate cancer stem cells.
Curr Opin Oncol 26, 328-333.
18. Kurioka, J., Takagi, A., Yoneda, M., Y., H., Shiraishi, T., and Watanabe, M.
(2011). Multicellular Spheroid Culture Models: Applications in Prostate Cancer
Research and Therapeutics. Journal of Cancer Science & Therapy 3, 60-65.
19. Lan, N.C., Chen, C.H., and Shih, J.C. (1989). Expression of functional human
monoamine oxidase A and B cDNAs in mammalian cells. J Neurochem 52,
1652-1654.
20. Lee, J., Kim, H.K., Rho, J.Y., Han, Y.M., and Kim, J. (2006). The Human
OCT-4 Isoforms Differ in Their Ability to Confer Self-renewal. 281, 33554-
33565.
21. Li, P.C., Siddiqi, I.N., Mottok, A., Loo, E.Y., Wu, C.H., Cozen, W., Steidl, C.,
and Shih, J.C. (2017). Monoamine oxidase A is highly expressed in classical
Hodgkin lymphoma. The Journal of Pathology 243, 220-229.
22. Liao, C.P., Adisetiyo, H., Liang, M., and Roy-Burman, P. (2010). Cancer-
Associated Fibroblasts Enhance the Gland-Forming Capability of Prostate
Cancer Stem Cells. Cancer research 70, 7294-7303.
46
23. Liao, C.P., Lin, T.P., Li, P.C., Geary, L.A., Chen, K., Vaikari, V.P., Wu, J.B.,
Lin, C.H., Gross, M.E., and Shih, J.C. (2018). Loss of MAOA in epithelia
inhibits adenocarcinoma development, cell proliferation and cancer stem cells
in prostate. Oncogene 37, 5175-5190.
24. Lin, T., Chao, C., Saito, S., Mazur, S.J., Murphy, M.E., Appella, E., and Xu, Y.
(2005). p53 induces differentiation of mouse embryonic stem cells by
suppressing Nanog expression. Nat Cell Biol 7, 165-171.
25. Liu, C., Kelnar, K., Liu, B., Chen, X., Calhoun-Davis, T., Li, H., Patrawala, L.,
Yan, H., Jeter, C., Honorio, S., et al. (2011). The microRNA miR-34a inhibits
prostate cancer stem cells and metastasis by directly repressing CD44. Nat Med
17, 211-215.
26. Marhold, M., Tomasich, E., El-Gazzar, A., Heller, G., Spittler, A., Horvat, R.,
Krainer, M., and Horak, P. (2015). HIF1alpha Regulates mTOR Signaling and
Viability of Prostate Cancer Stem Cells. Mol Cancer Res 13, 556-564.
27. Missol-Kolka, E., Karbanová, J., Janich, P., Haase, M., Fargeas, C.A., Huttner,
W.B., and Corbeil, D. (2011). Prominin-1 (CD133) is not restricted to stem
cells located in the basal compartment of murine and human prostate. 71, 254-
267.
28. Nanta, R., Kumar, D., Meeker, D., Rodova, M., Van Veldhuizen, P.J., Shankar,
S., and Srivastava, R.K. (2013). NVP-LDE-225 (Erismodegib) inhibits
47
epithelial–mesenchymal transition and human prostate cancer stem cell growth
in NOD/SCID IL2Rγ null mice by regulating Bmi-1 and microRNA-128. 2,
e42.
29. Peehl, D.M., Coram, M., Khine, H., Reese, S., Nolley, R., and Zhao, H. (2008).
The Significance of Monoamine Oxidase-A Expression in High Grade Prostate
Cancer. 180, 2206-2211.
30. Portillo-Lara, R., and Alvarez, M.M. (2015). Enrichment of the Cancer Stem
Phenotype in Sphere Cultures of Prostate Cancer Cell Lines Occurs through
Activation of Developmental Pathways Mediated by the Transcriptional
Regulator DeltaNp63alpha. PLoS One 10, e0130118.
31. Rybak, A.P., Bristow, R.G., and Kapoor, A. (2015). Prostate cancer stem cells:
deciphering the origins and pathways involved in prostate tumorigenesis and
aggression. 6, 1900-1919.
32. Sabnis, N.G., Miller, A., Titus, M.A., and Huss, W.J. (2017). The Efflux
Transporter ABCG2 Maintains Prostate Stem Cells. Molecular Cancer Research
15, 128-140.
33. Seruga, B., Ocana, A., and Tannock, I.F. (2011). Drug resistance in metastatic
castration-resistant prostate cancer. 8, 12-23.
48
34. Sherwood, E.R., Berg, L.A., Mitchell, N.J., McNeal, J.E., Kozlowski, J.M., and
Lee, C. (1990). Differential cytokeratin expression in normal, hyperplastic and
malignant epithelial cells from human prostate. J Urol 143, 167-171.
35. Shih, J.C. (2018). Monoamine oxidase isoenzymes: genes, functions and targets
for behavior and cancer therapy. J Neural Transm (Vienna) 125, 1553-1566.
36. Shih, J.C., Chen, K., and Ridd, M.J. (1999). MONOAMINE OXIDASE: From
Genes to Behavior. 22, 197-217.
37. Song, X.-L., Huang, B., Zhou, B.-W., Wang, C., Liao, Z.-W., Yu, Y., and Zhao,
S.-C. (2018). miR-1301-3p promotes prostate cancer stem cell expansion by
targeting SFRP1 and GSK3β. Biomedicine & Pharmacotherapy 99, 369-374.
38. Tang, D.G., Patrawala, L., Calhoun, T., Bhatia, B., Choy, G., Schneider-
Broussard, R., and Jeter, C. (2007). Prostate cancer stem/progenitor cells:
identification, characterization, and implications. Mol Carcinog 46, 1-14.
39. Tang, K., Liu, J., Jovanovic, L., An, J., Hill, M.M., Vela, I., Lee, T.K., Ma, S.,
Nelson, C., Russell, P.J., et al. (2016). Adipocytes promote prostate cancer stem
cell self-renewal through amplification of the cholecystokinin autocrine loop.
Oncotarget 7, 4939-4948.
40. Toivanen, R., and Shen, M.M. (2017). Prostate organogenesis: tissue induction,
hormonal regulation and cell type specification. Development 144, 1382-1398.
49
41. van Bokhoven, A., Varella-Garcia, M., Korch, C., Johannes, W.U., Smith, E.E.,
Miller, H.L., Nordeen, S.K., Miller, G.J., and Lucia, M.S. (2003). Molecular
characterization of human prostate carcinoma cell lines. Prostate 57, 205-225.
42. Van Slambrouck, S., Groux-Degroote, S., Krzewinski-Recchi, M.A., Cazet, A.,
Delannoy, P., and Wim (2014). Carbohydrate-to-carbohydrate interactions
between α2,3-linked sialic acids on α2 integrin subunits and asialo-GM1
underlie the bone metastatic behaviour of LNCAP-derivative C4-2B prostate
cancer cells. Bioscience Reports 34, 546-557.
43. Visvader, J.E., and Lindeman, G.J. (2008). Cancer stem cells in solid tumours:
accumulating evidence and unresolved questions. Nat Rev Cancer 8, 755-768.
44. Wang, X., Jin, J., Wan, F., Zhao, L., Chu, H., Chen, C., Liao, G., Liu, J., Yu,
Y., Teng, H., et al. (2019). AMPK Promotes SPOP-Mediated NANOG
Degradation to Regulate Prostate Cancer Cell Stemness. Dev Cell 48, 345-360
e347.
45. Wu, J.B., Lin, T.-P., Gallagher, J.D., Kushal, S., Chung, L.W.K., Zhau, H.E.,
Olenyuk, B.Z., and Shih, J.C. (2015). Monoamine Oxidase A Inhibitor–Near-
Infrared Dye Conjugate Reduces Prostate Tumor Growth. 137, 2366-2374.
46. Wu, J.B., Shao, C., Li, X., Li, Q., Hu, P., Shi, C., Li, Y., Chen, Y.T., Yin, F.,
Liao, C.P., et al. (2014). Monoamine oxidase A mediates prostate
tumorigenesis and cancer metastasis. J Clin Invest 124, 2891-2908.
50
47. Yun, E.J., Zhou, J., Lin, C.J., Hernandez, E., Fazli, L., Gleave, M., and Hsieh,
J.T. (2016). Targeting Cancer Stem Cells in Castration-Resistant Prostate
Cancer. Clin Cancer Res 22, 670-679.
48. Zhang, J., Chen, M., Zhu, Y., Dai, X., Dang, F., Ren, J., Ren, S., Shulga, Y.V.,
Beca, F., Gan, W., et al. (2019). SPOP Promotes Nanog Destruction to
Suppress Stem Cell Traits and Prostate Cancer Progression. Dev Cell 48, 329-
344 e325.
49. Zhang, K., Zhou, S., Wang, L., Wang, J., Zou, Q., Zhao, W., Fu, Q., and Fang,
X. (2016a). Current Stem Cell Biomarkers and Their Functional Mechanisms in
Prostate Cancer. 17, 1163.
50. Zhang, Y., Jin, Z., Zhou, H., Ou, X., Xu, Y., Li, H., Liu, C., and Li, B. (2016b).
Suppression of prostate cancer progression by cancer cell stemness inhibitor
napabucasin. Cancer Med 5, 1251-1258.
51. Zhao, H., Nolley, R., Chen, Z., Reese, S.W., and Peehl, D.M. (2008). Inhibition
of monoamine oxidase A promotes secretory differentiation in basal prostatic
epithelial cells. Differentiation 76, 820-830.

Abstract (if available)

Abstract Monoamine oxidase A (MAOA) is a mitochondrial enzyme that degrades monoamines and produces hydrogen peroxide. Recent studies have shown an elevated expression of MAOA in prostate cancer (PCa). Inhibition of MAOA reduces the tumorigenicity and stemness of prostate cancer stem cells (CSCs), which confer the tumorigenesis, proliferation and metastasis of PCa. Nanog and Oct4 maintain the pluripotent status and activate stemness pathways in prostate CSCs. In this study, we found elevated expression of Nanog and Oct4 in malignant mouse prostate tissues compared to wild type. We also found the co-expression of MAOA and Nanog/Oct4 in the prostate epithelia cells of Pten knockout (KO) mouse model. Further, we performed spheroid formation from LNCaP PCa cell line and found a cell concentration-dependent manner of the spheroid size and number. Taken together, we propose that MAOA promotes PCa proliferation by upregulating CSC stemness pathways in which Nanog and Oct4 are involved, and point to the potential use of MAO inhibitors for therapy against prostate CSCs.

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Creator Chu, Peirou (author)

Core Title Co-expression of monoamine oxidase A and prostate cancer stem cell markers in Pten knockout mice

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 05/06/2019

Defense Date 05/10/2019

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

Tag cancer stem cell,MAOA,NANOG,OAI-PMH Harvest,Oct4,prostate cancer

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Advisor Shih, Jean (committee chair), Cadenas, Enrique (committee member), Zhang, Yong (committee member)

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