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An essential role of argininosuccinate synthase 1 in Kaposi’s sarcoma-associated herpesvirus-induced cellular transformation

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An essential role of argininosuccinate synthase 1 in Kaposi’s sarcoma-associated herpesvirus-induced cellular transformation

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Content An Essential Role of Argininosuccinate Synthase 1 in Kaposi’s Sarcoma-Associated
Herpesvirus-Induced Cellular Transformation
By
Tingting Li

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In partial Fulfilment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
May 2016
Copyright 2016 Tingting Li
ACKNOWLEDGEMENTS

I would like to express my special appreciation and thanks to my adviser Dr.
Shou-Jiang Gao for his constant encouragement and guidance towards research and
writing this thesis. Your word of advice on both research and my career has been very
important for me.
I would also like to thank other thesis committee members Dr. Zoltan Tokes and
Dr. Michael R. Stallcup for their guiding comments and suggestions. I would like to
express my regards to all member of Dr. Gao’s lab for being a wonderful supportive
team.

Thank you everyone for your help and support.

Table of Contents
Abstract .............................................................................................................................. 1
Chapter 1: Introduction ................................................................................................... 2
Cancer metabolism ................................................................................................................... 2
Virus infection and cell metabolism ........................................................................................ 4
Viral oncogenesis and cell metabolism .................................................................................... 5
Kaposi’s sarcoma-associated herpesvirus (KSHV) ................................................................ 7
KSHV and cell metabolism .................................................................................................... 11
Model of KSHV-induced cellular transformation of primary cells .................................... 16
KSHV-infected human cell models ........................................................................................ 18
Chapter 2: Objectives of the project ............................................................................. 20
Chapter 3: Results........................................................................................................... 21
ASS1 is upregulated in KSHV-infected/transformed cells .................................................. 21
ASS1 knockdown inhibits cell proliferation, formation of colonies in soft agar and
induces apoptosis ..................................................................................................................... 23
Arginine depletion inhibits cell proliferation and colony formation in softagar of KMM
cells ........................................................................................................................................... 26
The synergistic effect of ASS1 knockdown and arginine deprivation on cell proliferation
................................................................................................................................................... 28
KSHV miRNAs mediate the upregulation of ASS1 ............................................................. 30
Chapter 4: Discussion ..................................................................................................... 32
Chapter 5: Methods and materials ................................................................................ 35
Cell culture and reagents ........................................................................................................ 35
Cell proliferation and softagar colony assays ....................................................................... 35
Lentiviral vectors and lentiviral infections ........................................................................... 36
Reverse transcription quantitative real-time polymerase-chain reaction (RT-qPCR) ..... 36
Western blot analysis .............................................................................................................. 37
Apoptosis assay and BrdU incorporation ............................................................................. 37
References ........................................................................................................................ 39

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Abstract

Cancer cells undergo metabolic reprogramming to sustain cell proliferation and
survival. Argininosuccinate synthase 1 (ASS1), catalyzes the synthesis of
argininosuccinate from L-citrulline and aspartate, which is a critical step in the urea
cycle. In this pathway, argininosuccinate is further converted to L-arginine and fumarate,
both of which participate in cellular metabolism [1]. Thus, ASS1 is the rate-limiting
enzyme in te de novo synthesis of L-arginine [2]. Several studies have shown an essential
role of ASS1 in cancer but no study has examined its role in cancers associated with
Kaposi’s sarcoma-associated herpesvirus (KSHV) [3-5]. Here, we show that ASS1 is
upregulated in KSHV-transformed cells, and is required for the cell survival and cellular
transformation of these cells. Knockdown ASS1 induces apoptosis, inhibits cell growth
and reduces the efficiency of colony formation in soft agar. Furthermore, arginine
deprivation induces similar effects. Finally, results of reverse genetics show that ASS1
upregulation is mediated by a cluster of KSHV-encoded microRNAs. These results
indicate that both ASS1 and arginine are required for the survival and proliferation of
KSHV-transformed cells, which might serve as effective therapeutic targets for
KSHV-associated cancers.

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Chapter 1: Introduction

Cancer metabolism
The description of aerobic glycolysis by Sir Otto Warburg, which is recognized as
the famed Warburg effect [6], points to the existence of fundamental differences in the
central metabolic pathways between malignant and normal tissues. Indeed, cancer cells
rely on aerobic glycolysis to maintain cell growth and proliferation [7-8]. However, the
Warburg effect alone is far away from perfect for explaining anabolic growth of all
cancer cells. Recent studies have demonstrated mutations in a number of signaling and
metabolic pathways, which drive the anabolic growth of different types of cancers [9].
These discoveries have driven the recent excitements in the cancer and metabolism fields.
Metabolic adaptations in tumors extend far beyond the Warburg effect. Cancer
cells, particularly proliferating cells, adapt to microenvironment in order to efficiently
uptake and incorporate nutrients to support their anabolic growth, which requires rapid de
novo synthesis of macromolecules including nucleotides, amino acids, and lipids [10].
Particularly, the rapid growth of cancer cells requires high levels of exogenous essential
and non-essential amino acids [11-14]. The highest concentration of amino acid in the
plasma is L-glutamine, which is required for protein synthesis, generation of the
non-essential amino acids (through transamination), replenishing the citric acid cycle
(TCA cycle) metabolite levels (through anapleurosis), maintaining redox homeostasis and
nucleotide biosynthesis [11, 15]. Moreover, glutamine reinforces cellular oncogenic
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signaling pathways. In some cancers, glutamine regulates the mammalian target of
rapamycin complex 1 (mTORC1) for initiating protein translation and cell growth [11,
15]. Further, glutamine-derived nitrogen is a component of amino sugars, known as
hexosamines, which are used for the glycosylation of receptors of growth factors and thus
localization to the cell surface [16]. As a result, glutamine is regarded as an essential
amino acid in cancer cells rather than a nonessential as in the normal cells.
Proline can be synthesized from glutamine as well as derived from collagen
degradation [17]. It can also feed the TCA cycle through the urea cycle, and its oxidation
by proline dehydrogenase (PRODH) leads to the formation of reactive oxygen species
(ROS) [17]. For cancers, these functions play a role in apoptosis, autophagy and in
response to nutrient and oxygen deprivation. Importantly, proline-derived ROS served as a
driving signal for reprogramming [18]. The proto-oncogene c-Myc, which is expressed at
high levels in many human cancers, activates proline synthesis pathway using glutamine
as a substrate by suppressing proline dehydrogenase (POX/PRODH) expression [18].
Another important amino acid derived from glutamine and proline is arginine,
which is often dysregulated in cancer cells due to the loss of function of
argininosuccinate synthase 1 (ASS1) [3, 5]. These cancers, called arginine auxotrophic
tumors, include malignant melanoma, hepatocellular carcinoma, prostate cancer, breast
cancer and acute lymphoblastic leukemia, etc. [3, 5, 18]. In fact, this is the basis for
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arginine depletion therapy, which is to convert arginine to citrulline using ADI-PEG20
[19]. Arginine metabolism intersects with many other metabolic pathways, including
nitric oxide, creatine, urea, and polyamine metabolism [20].
Despite the intensive investigation of cancer metabolic pathways in the last
decade, how cancer cells regulate metabolic pathways to adapt to different stress
conditions remains not entirely clear.

Virus infection and cell metabolism
Viral infections often reprogram metabolic pathways of the host cells to support
the demand of their rapid replication [21]. This is reminiscent of the metabolic switch of
cells shifting from a quiescent phase to a proliferative state. For examples, several studies
have documented metabolic reprogramming by human cytomegalovirus (HCMV) and
herpes simplex virus-1 (HSV-1), which is required for their replication. As infection by
HCMV progresses, several metabolic pathways, including glycolysis, the TCA cycle,
mTOR-dependent lipogenesis and pyrimidine biosynthesis, are markedly activated,
indicating that HCMV disrupts metabolic homeostasis [22-25]. HSV-1 also alters host
metabolism to provide substrates required for viral replication. For example, HSV-1
upregulates the de novo pyrimidine nucleotide biosynthesis from glucose via the pyruvate
carboxylase-catalyzed anaplerotic and the aspartate transaminase 2 catalyzed-cataplerotic
reactions of the TCA cycle [25, 26]. Thus, exploring the underlying molecular
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mechanisms of metabolic reprogramming induced by viruses can not only provide
insights into the mechanism of viral replication but also discover novel potential
noninvasive biomarkers for detecting viral infections, particularly at the early stage of
infection.
Viral oncogenesis and cell metabolism
Infections by a number of viruses can cause cancer. There are seven human tumor
viruses: Epstein-Bar Virus (EBV), Hepatitis B Virus (HBV), Human T-Lymphotropic
Virus 1 (HTLV1), Human Papillomaviruses (HPV), Hepatitis C Virus (HCV), Kaposi’s
Sarcoma-Associated Herpesvirus (KSHV or HHV8), Merkel cell polyomavirus (MCV).
Oncogenic viruses trigger tumorigenesis by targeting the cellular signaling pathways that
are responsible for controlling cell cycle progression, cell invasion, migration, apoptosis,
etc. For example, HCV, a single, positive-stranded RNA virus of the Flaviviridae family,
targets the liver, replicates in hepatocytes and represents the main etiologies of
hepatocellular carcinoma (HCC) development [27]. Infection by HCV results in oxidative
stress and inflammation, and triggers rearrangements of the liver tissue architecture [28,
29]. Furthermore, HCV can activate growth and survival pathways including the
PI3K/AKT/mTOR and the EGFR/RAS/MAPK pathways, and inhibit p53 and pRb tumor
suppressive pathways [28, 29]. Deregulation of these signaling pathways also regulates
metabolic reprogramming. Patients with chronic HCV infection often develop secondary
metabolic disorders such as insulin resistance and steatosis [30]. It has been shown that
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HCV infection induces early perturbations in glycolysis, the pentose phosphate pathway,
and the TCA, which favored host biosynthetic activities to support viral replication and
propagation [31, 32]. This is followed by a compensatory shift in metabolism aimed at
maintaining energy homeostasis and cell viability during elevated viral replication and
increased cellular stress [31, 32]. A number of lipid species (e.g. phospholipids and
sphingomyelins) are temporally perturbed and predicted to play important roles in viral
replication, downstream assembly and secretion events [33, 34]. The elevation of
lipotoxic ceramide species suggests a potential link between HCV-associated
biochemical alterations and the direct cytopathic effect observed in this in vitro system
[33-35].
EBV, the first-characterized human tumor virus, is a herpesvirus with preferential
tropism for B cells. EBV has been associated with several lympho-proliferative disorders:
Hodgkin’s lymphoma, Burkitt’s lymphoma, gastric cancer and nasopharyngeal carcinoma
[36]. EBV-infected lymphocytic cell lines exhibit Warburg effect by increasing the
expression of glucose transporter GLUT1 and activating several glycolytic enzymes [37].
EBV latent membrane protein 1 (LMP1) mediates a PI3K-dependent induction of Myc,
leading to the upregulation of HK2 and an increase in aerobic glycolysis and resistance to
apoptosis [38].
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Thus, metabolic reprogramming appears to be a hallmark of viral infection.
Understanding the virus-induced metabolic reprogramming is essential for delineating the
mechanism of viral life cycle as well as the resulting cellular consequences. For
oncogenic viruses, dissecting virus-induced metabolic reprogramming can provide
insights into the mechanism of viral oncogenesis. In this project, we examine the
molecular basis of KSHV-induced metabolic reprograming with focus on the arginine
pathway.

Kaposi’s sarcoma-associated herpesvirus (KSHV)
Kaposi’s sarcoma-associated herpesvirus (KSHV), a double-stranded DNA virus,
also known as human herpesvirus 8 (HHV8), belongs to the -herpesvirinae subfamily
[39]. KSHV infection is associated with three malignancies: Kaposi’s sarcoma (KS), and
multicentric Castleman’s disease (MCD), primary effusion lymphoma (PEL). The size of
the KSHV genome ranges from 165 to 170 kb [40]. The long unique region (LUR) of
KSHV, which is 140.5 kb in length, contains over 90 ORFs, flanked by terminal repeat
(TR) sequences at both ends of the viral genome [41].
KSHV contains two different phases of its viral lifecycle: latent phase and lytic
phase [42, 43]. The lytic phase is characterized by the replication of linear viral genomes,
and the expression of most of the viral transcripts in a highly orchestrated temporal order
of immediate-early, early, and late categories [42]. Latent KSHV infection is
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characterized by a circularized, extra-chromosomal viral genome (episome) and the
expression of a very small subset of latent transcripts in the infected cells without the
production of any infectious viral particles [43]. In latently infected cells in all three
KSHV-associated malignancies, the expression of K13/ORF71/vFLIP (viral FLICE
inhibitory protein), ORF72/vCyclin (viral cyclin), ORF73/LANA (latency-associated
nuclear antigen) and a cluster of 12 precursor microRNAs (miRNAs) has been detected
[43]. These viral latent products play a critical role in KSHV-induced cellular
transformation and tumorigenesis (Figure 1). Indeed, KSHV miRNAs are essential for
KSHV-induced cellular transformation [44]. Both KSHV miRNAs and vFLIP promote
cell survival and proliferation by activating the NF-κB pathway [45, 46]. Moreover, the
KSHV miRNAs inhibit the anti-angiogenic molecule thrombospondin 1, possibly
contributing to KS-related angiogenesis [47]. vFLIP is responsible for spindle cell
morphology of KSHV-transformed endothelial cells in vitro [48]. KSHV LANA
promotes cell proliferation and survival by inhibiting the p53 and pRb tumor suppressor
pathways [49, 50]. LANA also enhances the transcription of HIF-1α and activates several
oncogenic signaling pathways [51]. LANA deregulates Wnt signaling by nuclear trapping
of glycogen synthase kinase 3β (GSK3β), which results in the stabilization of β-catenin.
Additionally, LANA also inhibits anti-proliferative transforming growth factor-β
(TGF-β) signaling by inactivating TGF-β receptors [52]. KSHV vCyclin promotes cell
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proliferation by activating cyclin-dependent kinase 6 (CDK6) and inactivation of p27.
Together, KSHV latent products contribute to the malignant transformation and growth
of KSHV-induced tumors (Figure 1) [52].

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Figure 1: Regulation of cell proliferation and survival in latently KSHV-infected
cells. Some soluble factors such as VEGF can bind and activate host cell receptors, which
can stimulate KSHV latent genes to be functional, including direct pro-oncogenic and
anti-apoptotic activities of KSHV latent genes. For example, vFLIP can activate NF-κB
pathway to express FLIP and IAPs to inhibit cell apoptosis. LANA is capable of
regulating Wnt pathway by trapping GSK3β to upregulate Myc to stimulate cell
proliferation. β-cat, β-catenin; CDK, cyclin-dependent kinase; GSK3β, glycogen synthase
kinase 3β; HIF, hypoxia-inducible factor; IAPs, inhibitor of apoptosis proteins; NF-κB,
nuclear factor-κB; PKC, protein kinase C; PLC, phospholipase C; ROS, reactive oxygen
species [52].

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KSHV and cell metabolism
Recent studies have shown that KSHV infection induces Warburg effect in
endothelial cells [53, 54]. Expression of a cluster of KSHV miRNAs decreases the
number of mitochondria and induces HIF1α, a key metabolic modulator, in lymphatic
endothelial cells [55]. Further, KSHV infection elevates the levels of metabolites
involved in the synthesis of fatty acids leading to increased lipid droplet organelle
formation in the infected cells [53]. The synthesis of fatty acids is necessary for the
survival of latently infected endothelial cells [53]. PI3K-dependent increased aerobic
glycolysis and synthesis of fatty acids are also present in PEL cell lines [56]. Elevated
glutamate secretion and mGluR1 expression are present in KSHV-infected endothelial
cells, which is correlated to increased proliferation of KSHV-infected cells, and is
mediated by LANA and kaposin A [57].
Our lab has recently found that KSHV-transformed cells are addicted to
glutamine but not glucose for cell survival and transformation. Examination of gene
expression in the metabolic pathways by RNA-seq and microarray profiling reveals
upregulation of arginine de novo synthesis pathway in KSHV-transformed cells
compared to the uninfected cells. Among these genes, ASS1 is highly upregulated.
Because ASS1 is the rate-limiting enzyme in this pathway, we have decided to further
investigate its role in KSHV-induced metabolic reprograming.

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Argininosuccinate synthase 1 (ASS1)
ASS1 was first found in liver. As a rate-limiting enzyme in urea cycle, ASS1 is
now known as a ubiquitous enzyme in mammalian cells [1]. Numerous studies have
confirmed that ASS1, considered as a house-keeping gene in normal cells and a
rate-limiting biosynthetic enzyme in hepatocytes and endothelial cells, is absent in many
cancers [3, 5]. ASS1 has numerous functions including urea synthesis, de novo synthesis
of arginine and nitric oxide (NO) metabolism, etc. [1, 58].
The urea cycle, also known as the Krebs urea cycle or ornithine cycle, was first
discovered in 1932 by Hans Krebs [59]. The cycle is comprised of five enzymes that are
localized to the liver in mammals albeit kidney also has low level of urea metabolism.
Hence, ASS1 has the highest expression levels in kidney and liver cells [60]. The first
two enzymes in the urea cycle, carbamoyl phosphate synthetase I and ornithine
transcarbamoylase, are located within the mitochondrial matrix of periportal hepatocytes,
while the remaining three enzymes are located in the cytosol [61]. ASS1 utilizes the
aspartate and citrulline to synthesize argininosuccinate that always stay transient
intracellularly. Argininosuccinate lyase (ASL) catalyzes the cleavage of
argininosuccinate to produce arginine and fumarate (Figure 2) [62]. Subsequently,
arginase splits the arginine to ornithine and urea, which ensures that the arginine
generated by the actions of ASS1 and ASL is, for the most part, directed to urea
production (Figure 2) [62]. Urea is excreted and the ornithine is transported back into the
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mitochondria to complete the cycle. Loss of function of ASS1 will cause genetic diseases
including type I citrullinemia (CTLN1) and type II citrullinemia (CTLN2) [63, 64].
In addition, ASS1 is a critical enzyme for endogenous synthesis of arginine [2].
Its expression, localization, and regulation might differ significantly according to the
needs for arginine in the tissue. ASS1 plays a fundamental role in providing arginine for
both catabolic and anabolic metabolisms [65]. For example, ASS1 provides the essential
arginine for creatine and polyamine biosynthesis. The supply of arginine provided
through ASS1 can enhance the incorporation of glutamine into nucleotides in colorectal
cancers [66, 67]. Moreover, ASS1 is involved in nitric oxide (NO) metabolism [68].
ASS1 is expressed in an entire range of cell types, often related to their involvements in
providing the essential arginine for NO production [68]. In fact, the capacity to recycle
citrulline to produce arginine appears to be a prerequisite for all NO producing cells [69].
NO, as the two-edge sword, can affect cell functions in various aspects. At a low
concentration, it can promote cell survival and anti-apoptotic effect whereas at a high
concentration, it does the opposite.
As ASS1 plays many roles in cells, it is not a surprising that it is related to
tumorigenesis. Normally, ASS1 is downregulated in many different cancers, including
melanoma, gastric cancer, lung cancer, etc, for which the reasons are unclear [3, 5]. This
leads to a critical dependence on exogenous arginine, suggesting that ASS1 deficiency
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may be both a prognostic biomarker and predictor of sensitivity to arginine deprivation
therapy [70]. Several phase I/II clinical trials with the arginine-lowering drug, pegylated
arginine deiminase (PEG-ADI20) in ASS1-negative tumors have shown encouraging
results with low toxicity in patients [71]. ASS1 can be inactivated by
methylation-dependent transcriptional silencing or reversible nitrosylation at Cys-132 in
response to lipopolysaccharide-treatment in both cultured smooth muscle cells and in
mice [5,72]. Moreover, it is shown in vascular endothelial cells, TNF-alpha induced a
dramatic decrease in ASS1, which was mediated by NF-κB [73]. One possible
explanation for ASS1 downregulation in cancer cells is to facilitate pyrimidine synthesis
via CAD (carbamoyl-phosphate synthase 2, aspartate transcarbamylase, and
dihydroorotase complex) activation since ASS1 mutation will increases cytosolic
aspartate levels, which leads to the increase of CAD activation as a result of substrate
availability and phosphorylation by S6K1 through the mTOR pathway [74]. Additionally,
ASS1 upregulation in some cancer cells has also been reported. Epithelial ovarian tumor
tissue was highly dependent on elevated ASS1 expression for carcinogenesis and tumor
progression [4].
In this project, we also have shown that ASS1 is significantly upregulated and is
essential for the cell survival of KSHV-transformed cells.

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Figure 2: ASS1 catalyzes the urea cycle and is involved in TCA cycle through
shunting aspartate and fumarate. ASS1 participates the critical step in the urea cycle.
ASS1 deficiency will block the urea cycle and accumulate ammonia in the body and thus
cause citrullinaemia. Further, the intermediates of urea cycle-aspartate and fumarate can
be concerted into the intermediate products in the TCA cycle, which connects the urea
cycle with the TCA cycle. Additionally, ASS1 is essential to provide arginine for NO
production. More importantly, aspartate, as the substrate for synthesizing pyrimidine,
relates the urea cycle to the one-carbon metabolism, which gives ASS1 more significance
in cancer metabolism [62, 65, 68, 74].

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Model of KSHV-induced cellular transformation of primary cells
The exact mechanisms of KSHV-induced tumorigenesis are unclear, partly due to
the lack of a suitable model for studying cellular transformation of primary cells. Our lab
has found that KSHV can efficiently infect and transform primary rat embryonic
metanephric mesenchymal precursor cells (MM) [75]. Cellular transformation occurred at
as early as day 4 after infection and almost in all infected cells. KSHV-transformed cells
(KMM) express markers of lymphatic endothelial, vascular endothelial and mesenchymal
cells [75]. KMM cells are latently infected by KSHV. KMM cells proliferate at a faster
rate than MM cells. KMM cells are immortalized and transformed as shown by their
ability to grow in softagar and serum-free medium. Subcutaneous inoculation of KMM
cells into nude mice efficiently induced tumors [75]. KMM tumors have slit-like spaces;
and tumor cells manifest spindle morphology and express lymphatic endothelial, vascular
endothelial, and mesenchymal markers, which are reminiscent of KS tumors. This model
of KSHV-induced cellular transformation of primary cells is extremely useful for
delineating the mechanism of KSHV-induced oncogenesis [75].
Using the MM and KMM model, our lab found that KMM cells have altered
metabolisms and activation of many pro-oncogenic pathways, e.g., KMM cells hijack
BMP-Smad 1-Id pathway for tumorigenesis [76]; and KSHV microRNA clusters in
KMM cells target cellular apoptotic and survival pathways [77]. However, the link
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between the metabolic changes and tumorigenic pathways in this model still need be
further studied.
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KSHV-infected human cell models
Primary effusion lymphomas (PELs) are specifically associated with KSHV
infection and most frequently occur in human immunodeficiency virus (HIV)-positive
individuals as lymphomatous effusions in the serous cavities without a detectable solid
tumor mass [78]. Most PELs have concomitant EBV infection. Several KSHV-infected
PEL cell lines have been established and commonly used for studying KSHV. These
include BC3, BCBL1 and BCP1 cell lines [79-81]. A Burkitt’s lymphoma cell line BJAB
has been experimentally infected with KSHV, and the resulting BJAB-KSHV has also
been used for KSHV studies [82]. Nevertheless, infection by KSHV is inefficient in B
cells in vitro, and those infected cells are not transformed by KSHV [79-81]. As PEL
lines are immortalized cancer lines, there are usually no appropriate controls for their
characterizations limiting their uses for KSHV studies. While PEL-derived cell lines
maintain KSHV indefinitely, all KS tumor-derived cells to date have lost viral genomes
upon ex vivo cultivation.
SLK cells are uninfected endothelial cells derived from a gingival KS lesion of an
HIV-negative renal transplant recipient [82]. iSLK, a derivative of SLK, constitutively
expresses puromycin N-acetyl-transferase for drug selection, and GFP and RFP for
monitoring viral replication, and a doxycycline-inducible transgene RTA, whose
expression is sufficient to induce KSHV lytic replication [82].
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To study KSHV latency and tumorigenesis in endothelial cells,
telomerase-immortalized human umbilical vein endothelial (TIVE) cells and
telomerase-immortalized microvascular endothelial cells (TIME) have been generated
[83]. However, KSHV infection of these cells do not lead to cellular transformation
except a cell line derived from long-term KSHV-infected TIVE cells (LTC-TIVE), which
contains genetic alterations.

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Chapter 2: Objectives of the project
Based on our previous microarray data and RNA-seq data, ASS1 was highly
upregulated in KSHV-transformed rat primary mesenchymal stem cells. In this project,
our main goal is to investigate the role of ASS1 in cell proliferation and cellular
transformation in KSHV-transformed cells, and the underlying mechanisms. We propose
that ASS1, unregulated by KSHV, promotes KSHV-transformed cell proliferation.

The objectives for this project are:
1. To determine the role of ASS1 in the proliferation and survival of
KSHV-transformed cells;
2. To determine the mechanism by which ASS1 mediates the proliferation and survival
of KSHV-transformed cells;
3. To identify the KSHV genes/products that induce ASS1 expression.

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Chapter 3: Results

ASS1 is upregulated in KSHV-infected/transformed cells
Our RNA-seq results show that ASS1 is upregulated 43 fold in KMM cells
compared to MM cells. In order to confirm these results, we used RT-qPCR and Western
blotting methods to examine the expression of ASS1 (Figure 3A). Indeed, ASS1 was
upregulated at both transcript and protein levels in KMM cells compared to MM cells
(Figure 3A). Examination of KSHV-infected human cells showed that ASS1 was strongly
upregulated in KSHV-infected BJAB cells. In KSHV-infected iSLK cells, there is a slight
upregulation compared to their uninfected controls. Furthermore, compared to BJAB, the
expression of ASS1 was also moderately upregulated in three KSHV-infected PEL cell
lines (BC3, BCBL1 and BCP1) (Figure 3B). In conclusion, the expression of ASS1 is
significantly upregulated in KSHV-infected/transformed cells.

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Figure 3: ASS1 is upregulated in KSHV-infected/transformed cells. A. RT-qPCR and
Western blot results showing that the expression of ASS1 was induced in both mRNA
and protein levels in KSHV-transformed cells, compared to untransformed MM cells. B.
Upregulation of ASS1 in KSHV-infected iSLK cells and in PEL cells. Western-blot
detection of ASS1 protein levels in PEL cell lines BCBL1, BC3 and BCP1, in uninfected
and KSHV-infected BJAB cells, and in uninfected and KSHV-infected iSLK cells.
Statistical symbols “***” represents p-values < 0.001.

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ASS1 knockdown inhibits cell proliferation, formation of colonies in soft agar and
induces apoptosis
To investigate the role of ASS1 in KSHV-transformed cells, we applied shRNA
technique to knockdown ASS1. RT-qPCR and Western-blot results showed that ASS1
expression level was reduced by three different shRNAs compared to non-targeting
control (NT) (Figure 4A-B). While knockdown of ASS1 resulted in the inhibition of cell
proliferation in both MM and KMM cells (Figure 4C). On average, ASS1 knockdown
decreased KMM cell proliferation to almost 61.6% whereas 45.3% in MM cells, ranging
from 50%-85%. Thus, there is a significantly differential effect between MM and KMM
cells with more dramatically inhibitory effect shown in KMM cells with p value=0.40.
Furthermore, pharmacological inhibition with an ASS1 inhibitor MDLA significantly
inhibited cell proliferation in KMM cells but had no effect on MM cells. Base on our
student t test, MDLA has almost three times suppressive effect on KMM cells as on MM
cells (the significance of the differential effect indicated by p value= 0.20) (Figure 4D).
To determine the role of ASS1 in KSHV-induced cellular transformation, softagar assay
was carried out. As reported in previous studies, KMM but not MM cells lost
contact-inhibition and formed colonies in softagar. ASS1 knockdown efficiently inhibited
the efficiency of colony formation of KMM cells (Figure 4E). Consistent with the above
results, ASS1 knockdown reduced BrdU incorporation (Figure 4F) and induced apoptotic
cells in both MM and KMM cells (Figure 4G). Collectively, these results indicate that
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ASS1 is required for the proliferation, survival and cellular transformation of
KSHV-transformed cells.

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Figure 4: ASS1 knockdown inhibits cell proliferation and formation of colonies in
softagar, and induces apoptosis. A-B, Analysis of ASS1 expression in MM and KMM
cells upon ASS1 knockdown by RT-qPCR (A) and Western blot (B). C. ASS1
knockdown inhibits cell proliferation in MM and KMM cells. Cells were seeded in 6-well
plates in complete medium. In the following day cells were infected with three ASS1
shRNAs (sh1, sh2 and sh3). 24h after transduction, cells were split and re-seeded at
1.5x10
4
cells/well in 24 well-plates and counted daily. D. ASS1 inhibitor (MDLA)
suppresses cell proliferation in KMM but not MM cells. Cells were seeded at 3x10
4
/well
in 24 well-plates and in the following day were treated with MDLA at 0, 10 and 25 mM
and counted daily. E. ASS1 knockdown inhibited colony formation of KMM cells in
softagar. Representative pictures at 40x magnification were shown. Colonies with
diameter >50 were quantified in each field and the results were graphed in the right panel.
F-G. ASS1 knockdown reduced BrdU incorporation (F) and induced apoptosis (G) in
both MM and KMM cells. BrdU incorporation was analyzed by FACS 48 h after ASS1
shRNA transduction. Apoptotic cells were detected by Annexin V staining 72 h after
ASS1 shRNA transduction. Statistical symbols “*”, “**” and “***” represent p-values <
0.05, < 0.01 and < 0.001, respectively, while “NS” indicates “not significant”.

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Arginine depletion inhibits cell proliferation and colony formation in softagar of
KMM cells
Since ASS1 is the critical enzyme required for de novo synthesis of endogenous
arginine and its knockdown can inhibit cell proliferation and survival, we postulated that
arginine deprivation might have similar effect. Indeed, in the absence of arginine, the
proliferation of both MM and KMM cells were strongly inhibited (Figure 5A), indicating
that extracellular source of arginine is required for the proliferation of both MM and
KMM cells. Arginine deprivation also inhibited colony formation of KMM cells in
softagar (Figure 5B). Consistent with these results, FACS analyses showed that arginine
deprivation reduced BrdU incorporation (Figure 5C) and increased apoptotic cells (Figure
5D) in MM and KMM cells. Together, these results indicated that the arginine metabolic
pathway is essential for the proliferation and cellular transformation of KMM cells.

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Figure 5: MM and KMM cells require arginine for proliferation, survival and
formation of colonies in softagar. A. MM and KMM cells require arginine for
proliferation. Cells were seeded at 3x10
4
per well in 24 well-plates in complete medium
(+Arg) which were replaced with arginine-free (-Arg) or complete medium the following
day, and cell numbers were counted for 4 consecutive days. B. KMM cells cannot form
colonies in the absence of arginine. MM and KMM cells were plated in softagar in the
absence or presence of arginine. Representative pictures at 40x magnification were
shown. C. Arginine deprivation significantly reduce BrdU incorporation in MM and
KMM cells. BrdU incorporation was analyzed by flow cytometry 48 h following arginine
deprivation. D. Arginine deprivation dose not induce apoptosis in MM and KMM cells.
Apoptotic cells were detected by Annexin V staining 72 h following of arginine
deprivation. Statistical symbols “**” and “***” represent p-values < 0.01 and < 0.001,
respectively, while “NS” indicates “not significant”.

28 | P a g e

The synergistic effect of ASS1 knockdown and arginine deprivation on cell
proliferation
Intracellular arginine can come from the uptake of extracellular medium and de
novo synthesis. Our results so far indicate that both sources are important for the
proliferation and survival of KSHV-transformed cells. Thus, we examined the combined
effect of ASS1 knockdown and arginine deprivation. As expected, ASS1 knockdown and
arginine deprivation synergized with each other, causing profound effects on cell
proliferation (Figure 6). Consistent with previous data, once knockdown ASS1, the cell
proliferation of both MM and KMM cells were highly suppressed and more effective in
KMM cells (the differential effect indicated by p value=0.048). Further, after knockdown
ASS1, the arginine depletion could induce an even more dramatically inhibitory effect on
cell proliferation in both MM and KMM cells (p value<0.001). That combined inhibition
was much stronger observed in KMM cells than MM cells (indicated by p value<0.02)
(Fig 6, table 1). These results indicate that both exogenous source of arginine and de
novo synthesis through the ASS1 pathway are essential for the proliferation of
KSHV-transformed cells. Whether addition of a higher concentration of arginine can
rescue inhibition of cell proliferation by ASS1 knockdown remains to be determined.

29 | P a g e

Table 1: Statistical analysis of the synergistic effect of ASS1 knockdown and arginine
deprivation.

Figure 6: ASS1 knockdown and arginine deprivation have additive effect on cell
proliferation. MM and KMM cells were seeded in 6-well plates in complete medium.
Cells were transduced with ASS1 shRNA the following day. At 24 h post-transduction,
MM and KMM cells were split and re-seeded at 1.5x10
4
cells/well in the 24 well-plates
in complete medium. In the following day, the medium was replaced with fresh complete
medium or arginine-free medium, and cell numbers were counted for 4 consecutive days.
The statistical t test indicated the differential effect between ASS1 knockdown alone and
ASS1 knockdown combined with arginine depletion in MM and KMM cells with three
different shRNA treatments (p<0.001). Also, the differential effect of the combined
treatments of ASS1 knockdown and arginine deprivation between MM cells and KMM
cells was tested by t test (p<0.02). Statistical symbols “***” represent p-values < 0.001,
while “NS” indicates “not significant”.

30 | P a g e

KSHV miRNAs mediate the upregulation of ASS1
KMM cells are latently infected by KSHV and express only viral latent
genes/products including vFLIP, vCyclin, LANA and a cluster of 12 pre-miRNAs. To
identify which KSHV latent genes might be responsible for ASS1 upregulation in the
KSHV-transformed cells, we generated MM cells latently infected by KSHV mutants
containing individual deletion of either vFLIP, vCyclin or a cluster of 10 of the 12
pre-miRNAs (miR-K1-9 and 11) [84, 85]. We were not able to obtain cells that were
stably infected by a KSHV mutant containing a deletion of LANA because of its essential
role in KSHV persistent infection [86, 87]. Deletion of the miRNA cluster but not vFLIP
and vCyclin abolished the expression of ASS1 (Figure 7A) indicating that the miRNA
cluster was required for KSHV induction of ASS1. Indeed, deletion of vFLIP also has
moderate effect on decreasing ASS1 expression whereas vCyclin does not. As LANA
remained expressed in the miRNA cluster mutant, these results also indicated that LANA
alone was insufficient to induce ASS1 expression. To determine if expression of the
miRNAs cluster was sufficient to induce ASS1, we generated MM cells stably expressing
the miRNA cluster [77]. Expression of the miRNA cluster indeed is sufficient to induce
the expression of ASS1 in MM cells (Figure 7B). Together, these data suggest that
miRNA cluster mediates the upregulation of ASS1 in KSHV-transformed cells.

31 | P a g e

Figure 7: KSHV microRNA clusters mediate ASS1 upregulation. A. Western blotting
of ASS1 expression in MM cells and those infected by different recombinant KSHV
including wide-type (KMM), and mutants with a deletion of a cluster of 10 precursor
miRNA (dmiRs), vFLIP (dvFLIP) or vCyclin (dvCyclin). B. Western-blotting to detect
ASS1 expression in MM cells overexpressed with KSHV miRNA cluster or MM cells
transduced with the empty vector pITA (MM+pITA) as the control for MM-miR.
β-tubulin was used as internal controls for Western-blot. Mock: MM, WT: KMM.

32 | P a g e

Chapter 4: Discussion
In this study, we have shown that ASS1, upregulated in KSHV-transformed cells
by the miRNA cluster, is required for their proliferation and cell survival. KMM cells
have much higher expression level of of ASS1 than that of MM cells. Knockdown of
ASS1 inhibited the cell proliferation and cell survival of both MM and KMM cells. These
results are supported by reduced BrdU incorporation and increased apoptotic cells
following ASS1 knockdown. These results indicate that both MM and KMM cells
depend on ASS1, which is most likely due to their dependence on the de novo arginine
synthesis pathways. Interestingly, we observed an inhibitory effect on KMM but not MM
cells using MDLA, an ASS1 inhibitor. This discrepancy is likely due to the limited
specificity of the inhibitor. In fact, we had to use 25 mM of MDLA in order to observe its
effect on KMM cells. It is possible that KMM cells are more susceptible than MM cells
to the inhibition of the ASS1 pathway because of the higher expression level of ASS1.
We have also observed a differential effect on cell proliferation following ASS1
knockdown. Moreover, our softagar results have shown that ASS1 is required for
maintaining the transformation phenotype of KMM cells. Interestingly, arginine
deprivation induces auxotroph albeit the expression level of ASS1 indicating that
endogenous synthesis of arginine is insufficient to support the anabolic growth of KMM
cells, and extracellular uptake of arginine is required to support their optimal growth. As
a results, ASS1 knockdown and arginine deprivation has synergistic effect on the
33 | P a g e

proliferation and survival of KSHV-transformed cells. Furthermore, by using reverse
genetics and overexpression approaches, we have shown that the KSHV miRNA cluster
is required for KSHV upregulation of ASS1, and its expression is sufficient for inducing
ASS1 expression. The specific miRNA(s) and the mechanism by which it induces ASS1
upregulation remains to be determined.
Our results indicate that ASS1 might promote the proliferation and survival of
KSHV-transformed cells. Nevertheless, these results contradict with some of those
reported in other cancers. For example, ASS1 was identified as the tumor suppressor
gene in myxofibrosarcomas [88]. Loss of ASS1 expression via epigenetic DNA
methylation in the promoter region confers cells aggressive phenotypes [88]. Further
investigations are required to determine the roles of ASS1 in KSHV-induced
tumorigenesis in vivo and in other cancers.
ASS1, as the critical metabolic enzyme, can be connected to TCA cycle and
glutaminolysis, both of which are core metabolic pathways in cancer. Upregulation of
ASS1 enhances the flux of arginine into glutamine and proline, which favors cell
proliferation [15-17]. Upregulation of ASS1 can result in higher level of de novo
synthesis of arginine and subsequent conversion NO and fumarate. NO itself can act as a
signaling molecule to directly promote cell proliferation [55], while fumarate can regulate
the redox homeostasis to benefit cancer cell survival [56]. Thus, ASS1 knockdown can
34 | P a g e

have an impact on the NO production contributing to cell apoptosis [89]. We have shown
that the arginine starvation also induces cell arrest indicating that endogenous de novo
synthesis is insufficient to meet the requirement of anabolic growth of the cells despite
high level of ASS1 expression. Together, these results demonstrate the importance of
arginine pathway in KSHV-transformed cells.
ASS1 also plays a key role in the urea cycle resulting in the secretion of ammonia.
Results from our lab have shown that KMM cells prefer glutamine over glucose,
implicating higher levels of metabolism of amino acids, hence ammonia production. The
detoxification of ammonia requires functional ASS1 in the urea cycle as ASS1
knockdown can lead to accumulation of ammonia. As a result, high level of ammonia can
lead cell arrest in the S phase [90].
In a conclusion, we have identified two essential metabolic components for the
proliferation and survival of KSHV-transformed cells: arginine and ASS1. It can be
speculated that an approach based on arginine starvation such as using ADI-PEG20 and
ASS1 inhibition might be an effective therapy for KSHV-induced malignancies.

35 | P a g e

Chapter 5: Methods and materials

Cell culture and reagents
Rat primary embryonic metanephric mesenchymal stem cells (MM),
KSHV-transformed MM cells (KMM) [75], MM cells infected by KMM deleted with
KSHV latent genes [84, 85]: vFLIP deleted (dvFLIP), vCyclin deleted (dvCyclin), MM
cells overexpressing KSHV latent genes: MM-pITA, miRNAs, and 293T cells were
maintained in Dulbecco’s modified Eagle’s medium [77] (DMEM; with 25 mM glucose,
4 mM L-glutamine and 2 Mm sodium pyruvate) supplemented with 10% fetal bovine
serum (FBS; Sigma-Aldrich, St. Louis, Mo) and antibiotics (100 μg/mL penicillin and
100 μg/mL streptomycin). For arginine deprivation, cells were cultured in DMEM
without arginine, glutamine, lysine and sodium pyruvate (supplemented with 4 mM
glutamine and 2 mM sodium pyruvate), supplemented with 10% FBS (Sigma-Aldrich).
PEL cells (BJAB, BCBL1, BC3, BCP1), BJAB and BJAB-KSHV are kept in RPMI
medium with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, Mo) and
antibiotics (100 μg/mL penicillin and 100 μg/mL streptomycin) [79-82]. MDLA(Sigma)
are dissolved in water in 1M.

Cell proliferation and softagar colony assays
Soft agar assay was performed as previously described. Briefly, a total of 2x10
4

36 | P a g e

cells suspended in 1 ml of 0.3% top agar (Cat. A5431, Sigma-Aldrich) were plated onto
one well of 0.5% base agar in 6 well-plates and maintained for 2-3 weeks. Colonies with
diameter >50 μm were counted and photographed at 40Å~ magnification using a
microscope.
Lentiviral vectors and lentiviral infections
ASS1 shRNA plasmids were co-tranfected with pMDLg/pRRE, pRSVRev and
pMD2.G packaging plasmids into actively growing HEK293T cells by using
Lipofectamine 2000 transfection reagent. Virus-containing supernatants were collected
72 hr after transfection and filtered to remove cells, and target cells were infected in the
presence of 8 μg/mL polybrene. MM-Vector, KMM-Vector were selected with 10 μg/mL
Blasticidin after transduction.

Reverse transcription quantitative real-time polymerase-chain reaction (RT-qPCR)
Total RNA was isolated with TRI Reagent (Cat. T9424, Sigma) according to the
instructions of the manufacturer. Reverse transcription was performed with total RNA
using Maxima H Minus First Strand cDNA Synthesis Kit (Cat. K1652, Thermo Fisher
Scientific, Waltham, MA). qPCR analysis was performed on Eppendorf Real Plex using
KAPA SYBR FAST qPCR Kits (Cat. KK4602, Kapa Biosystems, Wilmington, MA).
The relative expression levels of target genes were normalized to the expression of
internal control genes, which yielded a 2
-ΔΔCt
value. All reactions were run in triplicates.
37 | P a g e

The cycle threshold (Ct) values should not differ more than 0.5 among triplicates. Rat
β-actin was used as an internal control.

Western blot analysis
Total cell lysates were separated in SDS-polyacrylamide gels, electrophoretically
transferred to nitrocellulose membranes (GE Healthcare, Piscataway, NJ). The
membranes were incubated sequentially with primary and secondary antibodies. The
signal was developed using Luminiata Crescendo Western HRP substrate (cat.
WBLUR0500, EMD Millipore, Billerica, MA). The antibody used for Western blot is
mouse monoclonal antibodies (mAbs) for ASS1 (cat. ab124465, Abcam, Cambridge, MA
and β-tubulin (7B9, Sigma).

Apoptosis assay and BrdU incorporation
BrdU incorporation were performed at the indicated time points as previously
described. Cell cycle was analyzed by propidium iodide (PI) staining. BrdU incorporation
was performed by pulsing cells with 10 μM BrdU for 2h and then stained with a Pacific
Blue monoclonal antibody to BrdU (Cat. B35129, Life technologies, Grand Island, NY).
Apoptotic cells were detected by Fixable Viability Dye eFluor 660 staining (Cat. 650864,
eBioscience, San Diego, CA) and with a PECy7 Annexin V. PECy7 Annexin V single
positive staining indicates the early apoptotic cells. PECy7 Annexin V and Fixable
38 | P a g e

Viability Dye eFluor 660 doule positive staining refers to late apoptosis. Fixable Viability
Dye eFluor 660 single positive staining means dead cells. Apoptosis Detection Set (Cat.
88810374, eBioscience) following the instructions of the manufacturer. Flow cytometry
was performed in a FACS Canto System (BD Biosciences, San Jose, CA) and analysis
was performed with FlowJo (FlowJo, LLC, Ashland, OR).

Statistical analysis
Data were expressed as the mean ± standard error of the mean (SEM) from at
least three independent experiments. Unless otherwise noted, the differences between
groups were analyzed using Student’s t-test when two groups were compared and using
one-way ANOVA when more than two groups were compared. Correlation was
determined using Spearman’s correlation coefficient. All statistical tests were two-sided.
A p < 0.05 was considered statistically significant. Statistical symbols “*”, “**” and
“***” represent p-values < 0.05, < 0.01 and < 0.001, respectively, while “NS” indicates
“not significant”. All analyses were performed using the Microsoft excel (GraphPad
Software Inc., San Diego, CA). All of experiments are repeated at least three times.

39 | P a g e

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Abstract (if available)

Abstract Cancer cells undergo metabolic reprogramming to sustain cell proliferation and survival. Argininosuccinate synthase 1 (ASS1), catalyzes the synthesis of argininosuccinate from L-citrulline and aspartate, which is a critical step in the urea cycle. In this pathway, argininosuccinate is further converted to L-arginine and fumarate, both of which participate in cellular metabolism [1]. Thus, ASS1 is the rate-limiting enzyme in te de novo synthesis of L-arginine [2]. Several studies have shown an essential role of ASS1 in cancer but no study has examined its role in cancers associated with Kaposi’s sarcoma-associated herpesvirus (KSHV) [3-5]. Here, we show that ASS1 is upregulated in KSHV-transformed cells, and is required for the cell survival and cellular transformation of these cells. Knockdown ASS1 induces apoptosis, inhibits cell growth and reduces the efficiency of colony formation in soft agar. Furthermore, arginine deprivation induces similar effects. Finally, results of reverse genetics show that ASS1 upregulation is mediated by a cluster of KSHV-encoded microRNAs. These results indicate that both ASS1 and arginine are required for the survival and proliferation of KSHV-transformed cells, which might serve as effective therapeutic targets for KSHV-associated cancers.

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Asset Metadata

Creator Li, Tingting (author)

Core Title An essential role of argininosuccinate synthase 1 in Kaposi’s sarcoma-associated herpesvirus-induced cellular transformation

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 04/20/2016

Defense Date 03/22/2016

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

Tag argininosuccinate synthase 1,ASS1,Kaposi’s sarcoma-associated herpesvirus,KSHV,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Gao, Shou-Jiang (committee chair), Stallcup, Michael R. (committee member), Tokes, Zoltan (committee member)

Creator Email tli339@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-234341

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