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A truncating mutation in the autophagy gene UVRAG drives inflammation and tumorigenesis in mice

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A truncating mutation in the autophagy gene UVRAG drives inflammation and tumorigenesis in mice

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Content A truncating mutation in the autophagy gene
UVRAG drives inflammation and
tumorigenesis in mice
By
Christine Quach

A Thesis Presented to the Faculty of

THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA

In Partial Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE
MOLECULAR MICROBIOLOGY AND IMMUNOLOGY

August 2019

1
Table of Contents
ABBREVIATIONS ........................................................................................................................... 2
LIST OF FIGURES AND TABLES .................................................................................................. 3
ABSTRACT ..................................................................................................................................... 4
CHAPTER 1: INTRODUCTION ........................................................................................................ 5
AUTOPHAGY .................................................................................................................................. 5
UVRAG ....................................................................................................................................... 8
INFLAMMATORY BOWEL DISEASES AND COLORECTAL CANCER ....................................................... 10
CHAPTER 2: MATERIALS AND METHODS ................................................................................. 11
MOUSE MODELS .......................................................................................................................... 11
Generation of iUVRAG
FS
transgenic mice. ........................................................................... 11
In vivo bioluminescence imaging. ......................................................................................... 12
Generation of bone marrow chimeric mice. .......................................................................... 12
LPS sepsis model. ................................................................................................................ 13
Induction of DSS-induced colitis and treatment studies. ....................................................... 13
Induction of colitis-associated tumorigenesis. ....................................................................... 14
CELL CULTURE AND TRANSFECTION .............................................................................................. 14
Bone marrow-derived macrophages (BMDMs) isolation and culture. ................................... 15
PLASMID CONSTRUCTS ................................................................................................................ 16
ANTIBODIES, FLUORESCENT DYES, AND OTHER REAGENTS ............................................................. 16
AUTOPHAGY ANALYSES ................................................................................................................ 18
IMMUNOFLUORESCENCE AND CONFOCAL LASER SCANNING MICROSCOPY ........................................ 19
IMMUNOHISTOCHEMISTRY ............................................................................................................ 20
Oil red O staining. ................................................................................................................. 21
HISTOPATHOLOGY ANALYSIS OF COLON ........................................................................................ 21
CONVENTIONAL ELECTRON MICROSCOPY ..................................................................................... 22
IMMUNOBLOTTING AND IMMUNOPRECIPITATION .............................................................................. 22
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) ........................................................................ 23
BACTERIA COLONY FORMING UNITS (C.F.U.) .................................................................................. 24
CELL VIABILITY ............................................................................................................................ 24
MITOCHONDRIAL ROS DETECTION ............................................................................................... 24
MEASUREMENT OF CYTOSOLIC MITOCHONDRIAL DNA (MTDNA) ..................................................... 25
GENE KNOCKDOWN BY SHRNA AND LENTIVIRAL GENE DELIVERY .................................................... 25
RNA EXTRACTION, CDNA SYNTHESIS, AND REAL-TIME PCR ANALYSIS .......................................... 26
CNV ANALYSIS ............................................................................................................................ 26
STATISTICAL ANALYSIS ................................................................................................................. 27
CHAPTER 3: RESULTS ................................................................................................................. 29
UVRAG
FS
INHIBITS STARVATION-INDUCED AUTOPHAGY IN VIVO. ..................................................... 29
UVRAG
FS
PREVENTS TLR4 INDUCTION OF AUTOPHAGY IN VIVO. .................................................... 36
UVRAG
FS
BLOCKADE OF AUTOPHAGY ENHANCES INFLAMMATORY RESPONSES. .............................. 38
UVRAG
FS
EXACERBATES INFLAMMATORY RESPONSES IN EXPERIMENTAL COLITIS. .......................... 44
PROMOTION OF SPONTANEOUS TUMORIGENESIS BY UVRAG
FS
...................................................... 52
UVRAG
FS
PROMOTES PROLIFERATION AND b-CATENIN ACTIVATION ................................................ 56
UVRAG
FS
ACTIVATES b-CATENIN THROUGH AGE-RELATED AUTOPHAGY SUPPRESSION. ................... 60
CHAPTER 4: DISCUSSION ........................................................................................................... 64
ACKNOWLEDGMENTS ............................................................................................................... 70
REFERENCES .............................................................................................................................. 71

2
Abbreviations

AOM Azoxymethane
BMDM Bone marrow derived macrophages
Dox Doxycycline
DSS Dextran sodium sulfate
iUVRAG
FS
Inducible UVRAG frameshift
LC3 Microtubule-associated protein 1A/1B-light chain 3
LPS Lipopolysaccharides
MitoQ Mitoquinone
MSI Microsatellite instability
NLR NOD-like receptor
NLRP3 NOD-like receptor protein 3
p62/SQSTM1 Sequestosome1
PI3KC3 Class III phosphatidylinositol 3-kinase complex
PINK1 PTEN-induced putative kinase 1
PTEN Phosphatase and tensin homolog
UVRAG Ultraviolet Irradiation Associate Gene
UVRAG
FS
UVRAG frameshift
Vps34 Vesicular protein sorting 34

3
List of Figures and Tables

Figure 1: Five Key Steps of Autophagy
Figure 2: Lipophagy
Figure 3: Mitophagy
Figure 4: Dox-inducible expression of UVRAG
FS
in mice
Figure 5: UVRAG
FS
inhibits starvation-induced autophagy in mice
Figure 6: UVRAG
FS
prevents TLR4 induction of autophagy in mice
Figure 7: Effects of UVRAG
FS
on LPS-induced autophagy activation
Figure 8: UVRAG
FS
enhances LPS-induced lethal shock by promotion of
inflammatory responses
Figure 9: UVRAG
FS
blockade of LPS-induced autophagy enhances inflammatory
response
Figure 10: UVRAG
FS
exacerbates intestinal colitis in vivo.
Figure 11: UVRAG
FS
enhances inflammatory response in DSS-induced colitis
Figure 12: UVRAG
FS
expression inhibits cell differentiation in AOM-DSS model
Figure 13: UVRAG
FS
predisposes mice to colitis-associated colon cancer.
Figure 14: UVRAG
FS
promotes spontaneous tumorigenesis in mice.
Figure 15: UVRAG
FS
expression promotes spontaneous tumorigenesis
Figure 16: UVRAG
FS
activates b-catenin by promoting age-related autophagy
suppression.
Figure 17: UVRAG
FS
promotes cell proliferation by b-catenin activation
4
Abstract

Aberrant autophagy is a major risk factor for inflammatory diseases and cancer.
However, the genetic basis and underlying mechanisms are less well-established.
UVRAG is a tumor suppressor candidate involved in autophagy, which is truncated
in cancers by a frameshift (FS) mutation and expressed as a shortened UVRAG
FS
.
To investigate the role of UVRAG
FS
in vivo, we generated mutant mice that
inducibly express UVRAG
FS
(iUVRAG
FS
). These mice are normal in basal
autophagy but deficient in starvation- and LPS-induced autophagy by disruption of
UVRAG-autophagy complex. iUVRAG
FS
mice display increased inflammatory
response in sepsis and intestinal colitis, and colitis-associated cancer
development through NLRP3-inflammasome hyperactivation. Moreover,
iUVRAG
FS
mice show enhanced spontaneous tumorigenesis related to age-
related autophagy suppression and resultant b-catenin stabilization. Thus, UVRAG
is a crucial autophagy regulator in vivo, and autophagy promotion may help
prevent/treat inflammatory disease and cancer in susceptible individuals.

5
Chapter 1

Introduction

Autophagy
Autophagy is the major catabolic process that is responsible for regulated
degradation of damaged organelles and intracellular proteins in order to maintain
cellular homeostasis and adaptation to environmental stress
1,2
. Autophagy
involves the delivery of cytoplasmic cargo to the lysosome for degradation leading
to the release of essential metabolites for use as an energy source
2
. This process
is mediated by evolutionary conserved genes, ATG genes, the majority of which
are required for the formation of the autophagosome containing the cytoplasmic
cargo to be delivered to the lysosome
3
. Autophagy occurs at basal levels in nearly
all cell types and increases in response to a diverse set of cues such as nutrient
deprivation, organelle damage and exposure to pathogens.
Figure 1: Five Key Steps of Autophagy (Levine et al., 2019)
6
The autophagy pathway involves five key steps (Figure 1).
The pathway begins with the initiation and formation (nucleation) of the
phagophore. The phagophore forms a double membrane around the cytoplasmic
cargo to be degraded. This step notably involves Vps34, a PI3KC3, and its binding
partner Beclin-1. The third step is the elongation of the phagophore membrane
and LC3 processing. During autophagy, cytosolic LC3 (LC3-I) is conjugated to
phosphatidylethanolamine to form LC3-II which is integrated into the elongating
autophagosome membrane
4
. This processing step is mediated by the Atg5-Atg12
complex. The phagophore membrane ends fuses to complete the double
membrane autophagosome. The autophagosome fuses with the lysosome to
create the autolysosome in order for lysosomal acid proteases to be delivered into
the autophagosome for degradation of its cargo. Each step is mediated by specific
complexes and proteins including the ATG proteins. A defect in any protein may
compromise the entire pathway leading to
disease.
Autophagy has been linked to many
diverse processes such as intracellular lipid
metabolism (lipophagy) and selective
clearance of damaged mitochondria
(mitophagy)
5,6
. Lipophagy is an alternative
pathway of lipid metabolism that requires
the lysosome for degradation of lipids.
During starvation, lipid droplets are taken up
Figure 2: Lipophagy (Liu, K. and Czaja, MJ., 2012)
7
by autophagosomes and hydrolysed to free fatty acids and glycerol for energy
(Figure 2). Impaired lipophagy can lead to the accumulation of lipid droplets in
tissue especially the liver
7
.
Mitophagy is the clearance of damaged mitochondria to reduce generation of
mitochondrial ROS (mtROS) that triggers inflammation and prevent the release of
mitochondrial apoptotic signals
8-10
. Mitophagy can be induced by different stimuli
and progress through several
mechanisms. The PINK1-Parkin
mediated autophagy pathway is
ubiquitin dependent (Figure 3). In
healthy mitochondria, PINK1 is
transported into the inner
mitochondrial membrane where it
is cleaved. The truncated PINK1
is degraded by the ubiquitin-
proteasome system
11
. In
damaged mitochondria, usually following membrane potential disruption, PINK1 is
stabilized on the outer mitochondrial membrane and subsequently phosphorylated
and translocated to the mitochondrial surface. Parkin, an E3 ubiquitin ligase,
associates with PINK1 on the mitochondrial surface generating K63-linked
polyubiquitin chains to recruit p62/SQSTM1 to the damaged mitochondria
11
. The
autophagic substrate, p62, is responsible for interacting with LC3 on the
Figure 3: Mitophagy (Liu et al., 2017)
8
autophagosome membrane to promote mitophagy. In the process of autophagy,
p62 is degraded making it a useful marker for autophagic flux.
A lack of sufficient autophagy has been implicated in inflammatory pathologies
and immune-dysfunction such as sepsis, Crohn’s disease, diabetes, and
inflammation-associated cystic fibrosis lung disease
12
. Under unstressed
conditions, chronic autophagy suppression is associated with decreased lifespan
and healthspan in animal models, including age-related renal and cardiac
deterioration and spontaneous tumorigenesis
13
.

UVRAG
In the past decade, there has been a considerably increased interest in
autophagy. The 2016 Nobel Peace Prize was awarded to Dr. Yoshinori Ohsumi
for his discovery of the molecular mechanisms of autophagy in yeast. Many articles
have been published in that time leading to the discovery of new autophagy-related
genes. One such gene is the ultraviolent (UV) radiation resistance-associated
gene (UVRAG). It is an autophagy-related protein that forms a complex with
Beclin1 and the lipid kinase PI3KC3/Vps34 which are essential for phagophore
nucleation
14
.
UVRAG was first identified to partially complement UV sensitivity of xeroderma
pigmentosum (XP) cells
14
. UVRAG contains four major domains: a proline-rich
(PR) domain, a lipid-binding C2 domain, a Beclin-1 binding coiled-coil domain
(CCD), and a C-terminal domain. These four domains allow UVRAG to act as a
multifunctional protein and as a tumor suppressor
14
.
9
Different UVRAG complexes exist and modulate the tightly regulated
autophagosome progression to lysosomal degradation, as well as other
membrane trafficking events that either intersect or converge with the autophagy
pathway
15-18
. Overexpression of UVRAG activates autophagy and suppresses
tumor cell growth, whereas silencing of UVRAG causes failure of autophagy and
uncontrolled cell proliferation
14,19
. UVRAG also has autophagy-independent
functions in DNA repair, organelle integrity, and chromosomal stability
20-22
.
The human UVRAG gene contains a tract of 10 adenosine nucleotide repeats
in the CCD domain. This tract of repetitive DNA, called a microsatellite, are prone
to impaired DNA mismatch repair. A deletion of one adenosine results in a
frameshift (FS) mutation in UVRAG leading to a truncated form of UVRAG
(referred to as UVRAG
FS
). UVRAG
FS
is linked to susceptibility to different cancer
types
23
. The truncated UVRAG
FS
acts as a dominant negative mutant by
antagonizing WT UVRAG activity and autophagy suppressor in cell-based
assays
23
.
However, some studies and models have shown that deletion of yeast Vps38
and Arabidopsis Vps38p, which are considered as orthologs of mammalian
UVRAG and are a part of the PI3KC3 complex, impairs vacuolar protein sorting
but has minimal effect on autophagy
24.25
. This raises the concern whether UVRAG
regulates autophagy in vivo. Unfortunately, there is no genetic evidence in mice
showing a role of UVRAG in autophagy largely because genetic inactivation of
UVRAG resulted in early embryonic lethality
26
. Despite accumulated evidence of
mammalian UVRAG in autophagy regulation in vitro, definitive evidence that the
10
autophagic response succumbs to impaired UVRAG function and its impact on
tissue homeostasis and diseases propensity is lacking.

Inflammatory Bowel Diseases and Colorectal Cancer
The inflammatory bowel diseases, ulcerative colitis and Crohn’s disease, are
characterized by chronic inflammation of all or part of the digestive tract. While the
exact etiology is unknown, current research strongly supports a model of
inflammation driven by dysregulation of the mucosal immune system in genetically
susceptible individuals
27
. Specifically, the NLRP3 inflammasome has been found
to play an important role in the development of chronic colitis
27
. The NLRP3
inflammasome is responsible for detecting pathogens and endogenous danger
signals such as ATP in order to trigger downstream caspase-1 dependent
processing of IL-1b and IL-18. Individuals with inflammatory bowel diseases have
significantly increased risk of developing colorectal cancer.
Approximately 15% of sporadic colorectal cancer (CRC) and 90% of Lynch
syndrome patients exhibit microsatellite instability (MSI) in which there is a high
rate of insertions/deletions in the repetitive DNA tracts due to impaired DNA
mismatch repair
23
. MSI mutations in certain genes can be positively selected for
leading to tumorigenesis. Notably, 33% of CRC, 8% of endometrial and 7.8% of
gastric cancers with MSI show high frequencies of the UVRAG
FS
mutation
23
.

UVRAG
FS
in CRC was found to promote tumorigenesis and metastatic spread in
vitro independently of autophagy
23
.
11
Chapter 2

Materials and Methods

Mouse models
Generation of iUVRAGFS transgenic mice. A Flag-tagged human UVRAG
FS

mutant described in our previous work
23
was PCR amplified and subcloned into
Mlu I and Not I restriction sites of pTRE-Tight (631059, Clontech), which contained
a Tet-responsive Ptight promoter and a SV40 poly(A). The IRES-luciferase (Luc)
cDNA fragment was cloned into Not I site of pTRE-Tight. The resulting plasmid,
pTREtight-Flag-UVRAG
FS
-Luc, was digested with Xho I to release the transgenic
cassette. The gel-purified cassette was injected into the pronucleus of fertilized 1-
cell stage embryos (B6D2F1 background) with standard procedure. Injected
embryos were cultured in M16 medium (M6111, Cytospreen) at 37°C under 5%
CO2 overnight. All the two-cell stage embryos were then transferred into oviducts
of the pseudopregnant CD-1 female mice at 0.5 dpc by Norris Comprehensive
Cancer Center Transgenic Mice Core Facility (USC). Integration of the construct
was confirmed by PCR (Supplementary Table 1). Two independent founder lines
were identified and back-crossed for more than 20 generations to C57BL/6 mice
(Jackson Laboratories). Flag-UVRAG
FS
-Luc transgenic mice were crossed with
Rosa26-rtTA*M2 mice (Jackson Laboratories) in a pure C57BL/6 background to
generate the double-transgenic mice (Rosa26-rtTA*M2;Flag-UVRAG
FS
-Luc),
12
denoted as iUVRAG
FS
. Animals were maintained on the C57BL/6 background. To
turn on the expression of UVRAG
FS
, iUVRAG
FS
mice were administered a
doxycycline (Dox) diet (TD.01306, Envigo) beginning at 22 days of age. iUVRAG
FS

mice and its wild-type littermate control mice were further crossed with GFP-LC3
transgenic mice
28
on the C57BL/6 background and tissues of offspring were used
for autophagy analyses in vivo. Both male and female control and iUVRAG
FS
mice
were used in the studies.
Atg5
flox/flox
mice has been previously described
28,29
and were a gift from Dr. Noboru
Mizushima (Tokyo Medical and Dental University, Tokyo, Japan). Atg5
flox/flox
mice
were bred to Rosa26CreER
T2
mice (004847; Jackson laboratories) to generate
Atg5
-/-
mice upon administration of tamoxifen.
In vivo bioluminescence imaging. In vivo bioluminescence imaging was
performed using the IVIS Lumina LT Series III in vivo imaging system
(CLS136334, PerkinElmer), as previously described
30
. Images were captured
and analyzed with Living Image® software. Signal intensity y was measured over
the region of interest and quantified as flux (photons per s per cm
2
per sr).
Generation of bone marrow chimeric mice. Bone marrow in recipient mice was
ablated by lethal irradiation with 550 cGy (twice, 24 h apart) as described
31
before
transplantation. Bone marrow were flushed from the femurs and tibias from wild-
type control and iUVRAG
FS
donor mice on Dox and washed twice with warm PBS.
1 x 10
7
BM cells per mouse were infused intravenously into the tail veins of
recipient mice. Mice were housed in microisolator cages for 6 weeks for full
reconstitution and recovery before induction of DSS-colitis. Mice were provided
13
with water containing gentamycin sulfate (0.2 mg/ml) for the first two weeks after
transplantation. To assess BM reconstitution, peripheral blood was collected from
chimeric mice and RNA was isolated using the Mouse RiboPure™ Blood RNA
Isolation Kit according to the manufacturer’s protocol (AM1951, Invitrogen) for RT-
PCR with primers for mouse luciferase (Supplementary Table 1).
LPS sepsis model. 8-10-week old and sex-matched iUVRAG
FS
mice were injected
intraperitoneally with 20 mg kg
-1
body weight of lipopolysaccharides (LPS) from E.
Coli (L8274, Sigma). Tissues and blood were collected 3 h post injection. Survival
after LPS challenge was assessed every 12 h for 3 days. All survived mice were
euthanized at the end of the third day.
Induction of DSS-induced colitis and treatment studies. Experimental colitis was
induced by adding DSS (5% wt/vol) to the drinking water for 6 days, followed by a
4-day recovery period with water. Mice were weighed daily and monitored for
clinical signs of colitis (e.g., weight loss, stool consistency, and rectal bleeding).
To inhibit the NLRP3 inflammasome activation in vivo, control and iUVRAG
FS
mice
on Dox were treated i.p. with 20 mg kg
-1
MCC950
32
or saline vehicle daily on days
0-5 of DSS administration. Mice were euthanized 24 h after the last treatment on
day 6. A colitis disease activity index (DAI) was calculated for each mouse daily
based on the following criteria as described previously
33
: weigh loss from baseline
(0, no weight loss; 1, 1-3% weight loss; 2, 3-6% weight loss; 3, 6-9% weight loss;
4, >9% weight loss); stool consistency (0, normal; 2, loose stool; 4, diarrhea), and
fecal blood (0, none; 2, blood visible in stool; 4, gross bleeding). Gross bleeding
14
was defined as fresh perianal blood with obvious hematochezia. Upon necropsy,
colon length was measured.
Induction of colitis-associated tumorigenesis. Colitis-associated colon
tumorigenesis was induced according to the literature
34
. Briefly, Dox-
treated/untreated control and iUVRAG
FS
mice were administered AOM (10 mg kg
-
1
body weight) intraperitoneally on day 0, followed by three cycles of DSS (2.5%,
wt/vol) in the drinking water for five days with a 14-day water interval between each
DSS cycle. The animals were weighed daily and sacrificed on day 60.
All mice were maintained in a pathogen-free facility with ad libitum access to food
and water. All animal experiments were approved by the Institutional Animal Care
and Use Committee (IACUC) of the University of Southern California and
performed in accordance with IACUC guidelines for animal care and use.

Cell culture and transfection

293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (5796,
Sigma). Primary fetal human colon epithelial cells (FHC) (CRL-1831, ATCC) were
cultured in DMEM:F-12 medium, supplemented with 25 mM HEPES,
10 ng ml
−1
cholera toxin, 5 μg ml
−1
insulin, 5 μg ml
−1
transferrin,
100 ng ml
−1
hydrocortisone, and 20 ng ml
-1
human recombinant EGF. SW480 was
cultured in Leibovitz's L-15 in the absence of CO2. All media above were
supplemented with 10% fetal bovine serum (FBS) (Seradigm), 2 mM L-glutamine,
and 1% penicillin-streptomycin (Gibco-BRL). Murine fibroblast L-929 cells were
purchased from ATCC (CRL 6364) and cultured in DMEM containing 10% Tet
15
system approved FBS (631106, Clontech), 2 mM L-glutamine, and 1% penicillin-
streptomycin (Gibco-BRL). Transfections were performed using Calcium
Phosphate Transfection Kits (631312, Clontech) or PolyFect Reagent (301107,
Qiagen), following the manufacturer’s instructions. None of the cell lines used in
this study was found in the database of commonly misidentified cell lines that is
maintained by ICLAC and NCBI Biosample. All cell lines were tested and
confirmed to be free of mycoplasma.
Bone marrow-derived macrophages (BMDMs) isolation and culture. BMDMs were
prepared as previously described (ref). Bone marrow collected from mouse tibias
and femurs was plated on sterile petri dishes and was incubated for 7 days in
DMEM containing 10% Tet system approved FBS, 1% penicillin-streptomycin, 2
mM L-glutamine, and 20% (vol/vol) conditioned medium from L929 mouse
fibroblasts. To induce UVRAG
FS
expression, cells were treated with 1μg/mL of Dox
on the 3
rd
day after plating. For inflammation studies, BMDMs were incubated with
LPS (100 ng/ml) for 6 h and then were treated with ATP (1mM) for 1 h, as
previously described
8
. For inflammation inhibition, BMDMs were primed with 100
ng/ml of LPS for 6 h followed by treatment with 10 µM Z-YVAD-FMK (ALX-260-
154-R020, Enzo Life Sciences) or 1 µM of MitoQ (10-1363-0005, Focus
Biomolecules) for 1 h and followed by stimulation with 1 mM ATP treatment for 1
h.

16
Plasmid constructs
The mRFP-EGFP-LC3 plasmid and p40(phox)-PX-EGFP plasmid were kindly
provided by Drs. Jae U Jung (University of Southern California) and S. Field
(University of California, San Diego), respectively. All constructs were confirmed
by sequencing using an ABI PRISM 377 automatic DNA sequencer (Applied
Biosystems).

Antibodies, fluorescent dyes, and other reagents
The following antibodies were used in this study: polyclonal rabbit anti-UVRAG (C-
term) (AP1850b, Abgent, 1:1000 for WB, 1:200 for IP), polyclonal rabbit anti-LC3B
(2775S, Cell Signaling Technology, 1:1000 for WB), polyclonal rabbit anti-p62
(5114S, Cell Signaling Technology, 1:1000 for WB), polyclonal rabbit anti-p62
(18420-1-AP, Proteintech, 1:500 for IHC), monoclonal rabbit anti-Atg16 (8089T,
Cell Signaling Technology, 1:1000 for WB), monoclonal rabbit anti-Atg5
(GTX62601, GeneTex, 1:1000 for WB), Beclin-1 (11306-1-AP, Proteintech, 1:1000
for WB, 1:200 for IP), polyclonal rabbit anti-PI3KC3 (AP8014a, Abgent, 1: 1000 for
WB), polyclonal rabbit anti-Rubicon (GTX129096, GeneTex, 1:1000 for WB),
polyclonal rabbit anti-p-UVRAG (Ser498) (ABS1600, EMD-Millipore, 1:1000 for
WB), monoclonal rabbit anti-MyD88 (4283, Cell Signaling Technology, 1:1000 for
WB), polyclonal rabbit anti-TRAF6 (PA5-29622, Invitrogen, 1:1000 for WB),
polyclonal rabbit anti-TRIF (GTX13810, GeneTex, 1:1000 for WB), monoclonal
rabbit anti-Ubiquitin (linkage-specific K63) (ab179434, Abcam, 1:1000 for WB),
polyclonal rabbit anti-Bcl-2 (2876S, Cell Signaling Technology, 1:1000 for WB),
17
monoclonal rabbit anti-Caspase-1 (4199T, Cell Signaling Technology, 1:1000 for
WB, 1:200 for IP), polyclonal goat anti-IL-1β (AF-401-SP, R&D Systems, 1:1000
for WB), monoclonal rabbit anti-NLRP3 (15101S, Cell Signaling Technology,
1:1000 for WB), monoclonal rabbit anti-ASC (67824T, Cell Signaling Technology,
1:1000 for WB), monoclonal mouse anti-Parkin (PRK8) (sc-32282, Santa Cruz
Biotechnology, 1:1000 for WB, 1:200 for IF), monoclonal rabbit anti-Tom20
(42406S, Cell Signaling Technology, 1:1000 for WB, 1:200 for IP, 1:200 for IF),
monoclonal rabbit anti-p-IκBα (2859T, Cell Signaling Technology, 1:1000 for WB),
monoclonal mouse anti-IκBα (4814T, Cell Signaling Technology, 1:1000 for WB),
monoclonal rabbit anti-NFκB (8242T, Cell Signaling Technology, 1:400 for IF),
polyclonal rabbit anti-Ki67 (NB110-89719SS, Novus Biologicals, 1:500 for IHC),
polyclonal rabbit anti-cleaved caspase-3 (9661T, Cell Signaling Technology, 1:200
for IHC), polyclonal rabbit anti-γ-H2AX (NB100-384, Novus Biologicals, 1:3000 for
IHC), monoclonal rabbit anti-Keratin 20 (13063T, Cell Signaling Technology, 1:800
for IHC), polyclonal rabbit anti-E-cadherin (20874-1-AP, Proteintech, 1:200 for IHC
and 1:1000 for WB), polyclonal rabbit anti-N-cadherin (PA5-29570, Thermo Fisher,
1:1000 for IHC and 1:1000 for WB), monoclonal rabbit anti-Vimentin (5741T, Cell
Signaling Technology, 1:1000 for IHC & WB), monoclonal rabbit anti-GSK3β
(12456T, Cell Signaling Technology, 1:1000 for WB), monoclonal rabbit anti-p-
GSK3β (Ser9) (5558T, Cell Signaling Technology, 1:1000 for WB), monoclonal
mouse anti-β-catenin (MA1-300, Thermo Fisher, 1:1000 for WB), monoclonal
rabbit anti-phosphor-β-catenin (Ser33/37/Thr41) (8814S, Cell Signaling
Technology, 1:500 for IHC, 1:1000 for WB), monoclonal rabbit anti-Cyclin
18
D1(2978T, Cell Signaling Technology, 1:1000 for WB), monoclonal mouse anti-c-
Myc (626802, Biolegend, 1:1000 for WB), polyclonal rabbit anti-c-Myc (10828-1-
AP, Proteintech, 1:400 for IHC), polyclonal rabbit anti-CD20 (PA5-16701, Thermo
Fisher, 1:50 for IHC), polyclonal rabbit anti-Bcl-6 (21187-1-AP, Proteintech, 1:500
for IHC), polyclonal rabbit anti-Kappa (14678-1-AP, Proteintech, 1:400 for IHC),
polyclonal rabbit anti-Cytokeratin 7 (NBP1-88080, Novus Biologicals, 1:500 for
IHC), monoclonal rabbit anti-TTF-1 (ab76013, Abcam, 1:300 for IHC), monoclonal
mouse anti-TCF4 (GTX52873, GeneTex, 1: 1000 for WB), and monoclonal mouse
anti-Flag (F7425, Sigma; 1:1000 for IHC & WB). HRP-labelled or fluorescently
labelled secondary antibody conjugates, purchased from Molecular Probes
(Invitrogen). Purified rabbit IgG was purchased from Pierce. Z-YVAD-fmk was from
Enzo Life Sciences (ALX-260-154-R020); Mitoquinone (MitoQ) was from Focus
Biomolecules (10-1363-0005); Torin1 was from Selleckchem; 3-MA was from
Santa Cruz Biotechnology; MCC950 was from Adipogen; Dextran Sulfate Sodium
(DSS) was purchased from Affymetrix; Doxycycline, azoxymethane (AOM), ATP,
Oil Red O, and LPS (Escherichia coli), chloroquine (CQ) were from Sigma. Unless
otherwise stated, all chemicals were purchased from Sigma.

Autophagy analyses
For assessment of autophagy in vivo, 6-8-week-old Dox-treated control;GFP-LC3
or iUVRAG
FS
;GFP-LC3 mice were either subjected to starvation for 48 h or
challenged with 20 mg kg
-1
LPS from Escherichia coli intraperitoneally. Mice were
then perfused with 4% paraformaldehyde (PFA) in PBS and tissues were collected
19
and processed for frozen sectioning as described
27
. The total number of GFP-LC3
puncta was counted per 2,620 µm
2
area (20 randomly chosen fields were used per
mouse) or per colon crypt structure and the average value for each tissue for each
mouse was determined by two independent researcher blinded to genotype. The
mouse skeletal muscle, heart, liver, and colon tissue sections were imaged using
a 60x Nikon objective (PL APO, 1.4 NA).
For western blot analysis, frozen tissues were lysed in ice-cold RIPA lysis buffer
(50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5%
sodium deoxycholate, 0.1% SDS) containing protease inhibitor cocktail (A32953,
Pierce) for 30 min at 4 °C. Lysates were centrifuged at 16,000 x g for 10 min at 4
°C. Cleared lysates were diluted in 2X SDS-PAGE loading buffer and analyses
using antibodies against LC3 and p62 as previously described
10,79
.

Immunofluorescence and confocal laser scanning microscopy
Immunofluorescence microscopy was carried out as previously described
29
.
Briefly, cells plated on coverslips were fixed with 4% paraformaldehyde (30 min at
RT). After fixation, cells were permeabilized with 0.2% Triton X-100 for 5 min and
blocked with 10% goat serum (G9032, Sigma) for 2 h at RT. Primary antibody
staining was carried out using antiserum or purified antibody in 1% goat serum for
1-2 h at RT or overnight at 4°C. Cells were then extensively washed with PBS and
incubated with diluted Alexa 488-, Alexa 594-, and/or Alexa 633-conjugated
secondary antibodies in 1% goat serum for 1 h, followed by DAPI (4’, 6’-diamidino-
2-phenylindole) staining. Cells were mounted using Vectashield (Vector
20
Laboratories, Inc.). Confocal images were acquired using a Nikon Eclipse C1
laser-scanning microscope (Nikon, PA), fitted with a 60´ Nikon objective (PL APO,
1.4NA), and Nikon imaging software. Images were collected at 512 ´ 512 pixel
resolution. The stained cells were optically sectioned in the z-axis. For
multichannel imaging, fluorescent staining was imaged sequentially in line-
interlace modes to eliminate crosstalk between the channels. The step size in the
z-axis varied from 0.2 to 0.5 mm to obtain 16 slices/imaged file.
For image quantification, approximately 200 cells, randomly chosen from 10 high
power fields and pooled from three independent experiments, were evaluated for
the distribution pattern of the indicated molecules. The Pearson correlation
coefficient was calculated using the built-in colocalization analysis module of the
NIS-Elements AR software. All experiments were independently repeated several
times. The investigators conducted blind counting for each quantification-related
study.

Immunohistochemistry
Tissue sections were fixed in 10% neutral buffered formalin and embedded in
paraffin. Tissue sections were routinely stained with hematoxylin and eosin. For
immunohistochemistry staining, tissue slides were deparaffinized in xylene and
rehydrated in alcohol. Endogenous peroxidase was blocked with 3% hydrogen
peroxide. Antigen retrieval was performed by incubating the sections in 10 mM
sodium albumin (2960, Calbiochem) in PBS for 1 h and incubated with the
indicated primary antibody overnight at 4°C. Antibody binding was detected with
21
EnVision
TM
Dual Link System-HRP DAB kit (K4010, Dako). Sections were then
counterstained with hematoxylin. For negative controls, the primary antibody was
excluded. For evaluation and scoring of immunohistochemical data, we randomly
selected 10 fields within the tumor area under high power magnification (40x) for
evaluation. The investigators conducted blind counting for all quantification.
Oil red O staining. Oil red O staining was performed, as described previously
35
.
Slides were imaged on a Keyence bz-9000 microscope.

Histopathology analysis of colon
Histopathology assessment was performed by an anatomical pathologist (A. H)
from de-identified section slides, as previously described
36
. The features evaluated
covered: acute and/or chronic inflammation, hyperplastic changes of the colon
epithelium, and crypt distortion or damage, fibrosis, and neoplasia. Three
independent parameters were measured: inflammatory cell infiltrate, extent of
hyperplasia, and crypt damage. The total histological score was calculated by
summing of the three independent scores with a maximum score of 12.
Inflammation was assessed using a scoring system from the literature
37,38
: 0 = no
inflammation; 1 = mild chronic mucosal inflammation; 2 = mild acute or moderate
chronic mucosal and submucosal inflammation; 3 = severe acute or chronic
mucosal, submucosal and transmural inflammation. Hyperplastic changes was
scored as the increase in epithelial cell numbers in crypts relative to baseline
epithelial numbers per crypt as follows: 0 = none or minimal (< 20%); 1 = mild (21-
35%); 2 = moderate (36-50%); 3 = marked (> 50%). Crypt damage was scored as
22
0 = none; 1 = only surface epithelium damaged; 2 = surface crypt and epithelium
damaged; 3 = entire crypt lost and surface epithelium damaged; 4 = entire crypt
and epithelium lost.

Conventional Electron Microscopy
Electron microscopy was performed as previously described
39
. Briefly, mice were
euthanized, and liver was rapidly fixed overnight at 4˚C in 1/2 strength Karnovsky’s
(2% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer,
pH 7.4). Tissues were post-fixed in 2% osmium tetroxide in 200 mM sodium
cacodylate for 2 h at 4˚C and rinsed in 0.1 M cacodylate buffer. Samples were then
blocked and stained with 1% uranyl acetate overnight at 4˚C. Pellet was then
rinsed with 0.1 M sodium acetate. Samples were dehydrated through a graded
series of ethanol, and then infiltrated with Epon resin overnight at room
temperature. They were then embedded in resin overnight at 60°C. Thin sections
were cut on a Leica Ultracut R, and collected onto formvar-carbon coated slot grids.
Sections were examined on a JEOL 2100 transmission electron microscope.
Images were recorded on film at 5,000x magnification.

Immunoblotting and immunoprecipitation
For co-immunoprecipitation from mice tissues, frozen tissues were weighed and
homogenized in ice-cold lysis buffer (25 mM HEPES, 150 mM NaCl, 1 mM EDTA,
1% Triton X-100; 1 ml per 100 mg tissue) containing a complete protease inhibitor
cocktail (A32953, Pierce). Lysates were centrifuged (16,000 x g at 4 °C for 30 min)
23
and the supernatant were pre-cleared with protein A/G agarose beads for 2 h at
4 °C. Lysates were used for immunoprecipitation (IP) with the indicated antibodies.
Generally, 1-4 µg commercial antibody was added to 1 ml lysates and incubated
at 4°C for 8-12 h. After addition of protein A/G agarose beads, incubation was
continued for another 2 h. Immunoprecipitates were extensively washed with IP
wash buffer (10 mM Tris at pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.2% Triton X-100)
supplemented with protease inhibitor cocktail and then eluted with SDS–PAGE
loading buffer by boiling for 5 min.
For co-immunoprecipitation from cell culture, cells were washed with ice-cold PBS,
lysed in 2% Triton X-100 lysis buffer (20 mM Tris at pH 7.5, 150 mM NaCl, 1 mM
EDTA and 2% Triton X-100) supplemented with protease inhibitor cocktail before
co-immunoprecipitation using the same protocol described for mouse tissues.
For immunoblotting, eluates were resolved by SDS–PAGE and transferred to a
PVDF membrane (BioRad). Membranes were blocked with 5% non-fat milk or BSA,
and probed with the indicated antibodies. Horseradish peroxidase (HRP)-
conjugated goat secondary antibodies were used (1:3,000, Invitrogen).
Immunodetection was achieved with Hyglo chemiluminescence reagent (Denville
Scientific), and detected by ChemiDoc Imaging System (Bio-Rad).

Enzyme-linked immunosorbent assay (ELISA)
Mouse cytokines in serum or culture supernatants were measured with ELISA kits
(R&D Systems) for IL-1b (MLB00C), IL-6 (M6000B), TNF-a (MTA00B), and IFN-b
24
(MIFNB0), according to the manufacturer’s directions. Mouse IL-18 was measured
by ELISA (7625, MBL international).
Cytokine levels in the colon extracts were measured as described previously
40
.
Briefly, colon tissues were homogenized with RIPA buffer containing protease
inhibitor cocktail. The supernatant was collected and tested for IL-1b, IL-6, and
TNF-a according to the manufacturer’s directions.

Bacteria colony forming units (c.f.u.)
Levels of c.f.u. in freshly isolated mice feces were determined by homogenization
of feces in 0.01% Triton X-100 in PBS followed by serial dilution plating on non-
selective Luria-Bertani agar plates.

Cell viability
Cell viability was determined using the CellTiter 96
®
non-radioactive cell
proliferation assay (G4001, Promega) following the manufacturer’s instructions.

Mitochondrial ROS detection
MitoSOX (M36008, Invitrogen) was used to measure mitochondrial ROS
production. Briefly, Dox (1 µg/ml)-treated BMDMs were primed with LPS (100
ng/ml) for 2 h and stimulated with ATP (1 mM) for 30 mins. The cells were then
washed twice with PBS and incubated with 5 μM of MitoSOX for 10 min and
washed twice with PBS again. Fluorescence intensity was determined using a
25
Synergy 2 multimode plate reader (BioTek Instruments), and the data were
normalized to PBS controls.

Measurement of cytosolic mitochondrial DNA (mtDNA)
mtDNA release was measured as previously described
5,7
. Briefly, Dox (1 µg/ml)-
treated BMDM were primed with LPS (100 ng/ml) for 2 h and stimulated with ATP
(1 mM) for 30 mins. Cytosolic mtDNA was measured using a mitochondrial
isolation kit (89874, ThermoScientific) according to manufacturer’s instructions.
Mitochondrial DNA encoding cytochrome c oxidase 1 was measured by
quantitative qPCR (refer to Supplementary Table 1 for primers’ sequence) with
same volume of the DNA solution. Nuclear DNA encoding 18S ribosomal RNA was
used for normalization.

Gene knockdown by shRNA and lentiviral gene delivery
All shRNAs were purchased from Open Biosystem. Lentiviral-compatible shRNAs
against Beclin1 (sh1: V2LHS_241693, sense: TGTTGGTCATCTCCAGGCG; sh2:
V3LHS_332992, sense: TCGCTAGGCAGCTCCTGCT), UVRAG (sh1:
V2LHS_197759, sense: ATTGTAACTGGACTCCAGG; sh2: V3LHS_357540,
sense: ATGACATCATCAATCTCCT). For lentivirus production, HEK293T cells
were transfected with the transfer vector (e.g. pCDH-CMV-MCS-EF1-Puro or
pGIPZ), pCMV-dR8.91 packaging plasmid, and pCMV-VSV-G envelope plasmid
in a 5:1:4 ratio using the Calcium Phosphate Transfection Kit (Clontech). The
medium was replaced 12 h later. Viral particles were collected 48 h post-
26
transfection, filtered with 0.45 μm sterile filter, and concentrated overnight by Lenti-
X concentrator (631312, Takara) at a ratio of 3:1, followed by centrifugation at 4˚C
(28,800 x g, 2 h, ThermoFisher Sorval RC 6+). Viral particles were re-suspended
in fresh medium with 8 μM/mL polybrene, and were plated with target cells for 24
h. Lentiviral-transduced cells were selected in 2 μg/mL puromycin for 7 days with
the medium changed daily.

RNA extraction, cDNA synthesis, and Real-time PCR Analysis
Total RNA was extracted from mice tissue or cell culture using TRIzol (15596-026,
Invitrogen) and purified with RNeasy Plus Mini Kit (Qiagen 74104), following the
manufacturer’s instructions. 1 µg of total RNA was used for cDNA synthesis using
iScript™ cDNA Synthesis Kit (1708891, Bio-rad). Quantitative real-time PCRs
were carried out using the primers listed in Supplementary Table 1 and iQ SYBR
Green Master Mix (Bio-rad). Samples were obtained and analyzed on the CFX96
Touch Real-Time PCR Detection System (Bio-Rad). The gene expression levels
were normalized to actin.

CNV analysis
Genomic DNA was isolated from bulk sample with TRI Reagent BD from Sigma-
Aldrich (T3809). Concentration of DNA was quantified with Qubit Fluorometric
Quantification (Thermo Fisher). Amplified DNA was sheared using sonication
(Covaris S2/E210 focused-Ultrasonicater) with the microtube setup and the 200
bp target size protocol for DNA shearing. 30 ng of sonicated DNA from was used
27
for library construction using the NEBNext Ultra DNA Library Preparation Kit for
Illumina (New England Biolabs, Cat#. E7370L). The constructed library DNA
concentration was quantified with Qubit (Thermo Fisher) and the expected library
size distribution of 300–500bp was confirmed using the Agilent 2100 Bioanalyzer
(High-Sensitivity DNA Assay and Kit, Agilent Technologies, Cat#. 5067-4626). The
individual libraries from barcoded samples were pooled. The pooled libraries were
cleaned using AMPure XP Beads (Beckman Coulter Inc., Cat# A63882). Libraries
were sequenced using the Illumina NextSeq 500 or the HiSeq2500 SR50
generating fastq files. 30 bp was trimmed off the ’5 end of each read to remove the
WGA4 adapter sequence before alignment to the hg19 reference genome using
the Bowtie algorithm. The resulting BAM file was sorted and PCR duplicates were
removed using SAMtools. The number of reads falling into each of 5000 ‘bins’
comprising the entire UCSC reference genome, was calculated using a previously
published Python script
59
. Finally, an R script utilizing the Bioconductor package,
DNAcopy_1.26.0 (http://bioconductor.org/packages/DNAcopy/), was used to
normalize and segment the bin counts across each chromosome generating a
genome-wide CNV profile.

Statistical analysis
Statistical significance was performed using a Student’s t test for unpaired
samples or two-way analysis of variance for multiple comparisons and by log-
rank analysis for survival curve using GraphPad Prism 5.0 (GraphPad Software,
Inc.), unless otherwise stated. Data are presented as the mean ± SD. A p value
28
of £ 0.05 was considered statistically significant. All experiments were
independently repeated at least three times. To ensure adequate power and
decrease estimation error, we used large sample sizes and multiple independent
repeats by independent investigators. Multiple lines of experiments including
different quantification methods were used for consistent and mutually supportive
results. The sample size was chosen according to the well-established rule in
the literature as well as our ample experience in previous research.

29
Chapter 3

Results

UVRAG
FS
inhibits starvation-induced autophagy in vivo. To study the role of
UVRAG
FS
in a temporal-specific manner in vivo, we generated a conditional Flag-
tagged UVRAG
FS
-luciferase transgene under the control of a doxycycline (Dox)-
responsive element (designated TRE-UVRAG
FS
) (Figure 4a). These mice were
crossed to ROSA26-rtTA2-M2 mice
41-43
to enable doxycycline (Dox)-inducible
expression of human UVRAG
FS
in a tightly regulated fashion (Figure 4a). This
double transgenic strain is referred to hereafter as iUVRAG
FS
.

Dox treatment
effectively induced UVRAG
FS
expression at the mRNA and protein levels without
affecting endogenous UVRAG expression (Figure 4b,c). No UVRAG
FS
transgene
expression was detected in Dox-treated wild-type littermate control mice (Figure
4b,c). Luciferase expression was not detected in untreated samples but strongly
correlated with UVRAG
FS
expression, providing a visual biomarker for UVRAG
FS

expression (Figure 4d-f). The transgene expression was reversible, with loss of
luciferase and UVRAG
FS
expression 4 days following Dox withdrawal in different
organs (i.e., lung, liver, spleen, and colon) (Figure 4d-h). Dox-treated iUVRAG
FS

mice were of normal size and weight, and displayed normal histology in major
organs (Figure 4h,i).
30

31

To explore the impact of cancer-related UVRAG
FS
on autophagy in vivo, we
bred wild-type control or iUVRAG
FS
mice to GFP-tagged LC3 transgenic mice that
express a fluorescent marker of autophagosomes
28
, on C57BL/6J (B6)
background, and analyzed the tissues of resultant compound mice after starvation
(Figure 5a). In skeletal muscle, liver, heart, and colon, Dox-treated iUVRAG
FS
mice
had significantly decreased numbers of GFP-LC3 puncta compared to WT controls
and to Dox-untreated animals that showed a marked increase in GFP-LC3 puncta
in response to starvation (Figure 5a). We further confirmed mice with UVRAG
FS

expression having suppressed starvation-induced autophagy by western blot
analyses (Figure 5b). There were decreased levels of autophagosome-associated
lipidated LC3 (LC3-II)
44,45
and increased levels of the autophagy substrate p62
46
,
while Atg12 conjugation to Atg5 remained unaffected, in 48 h-starved iUVRAG
FS

mice on Dox (Figure 5b). The deficient starvation-induced autophagy in Dox-
treated iUVRAG
FS
mice was not associated with increased cell death (Figure 4j).
Using electron microscopy (EM), it was also observed that the numbers of
autophagic vacuole (autophagosome and autolysosome) were comparable at
Figure 4: Dox-induced expression of UVRAG
FS
in mice. (a) Schematic diagram of the system used for Dox-
inducible expression of UVRAG
FS
. Dox, doxycycline. Luc, luciferase. (b) Levels of UVRAG
FS
transcript were
evaluated by quantitative RT-PCR in the spleen and colon from mice of indicated genotype (n = 5 mice per
group). Data represents the mean ± SD. **, P < 0.01; ****, P < 0.0001 (Student’s t test).

(c) Western blot (WB)
analysis of transgenic Flag-UVRAG
FS
and endogenous UVRAG expression in spleens from mice of indicated
genotype with or without Dox treatment for 7 days. Data is the representative of three independent
experiments.

(d) in vivo bioluminescent imaging of the abdominal region of representative iUVRAG
FS
mice
treated with Dox as indicated. (e,f) Bioluminescent imaging of the spleen (e) and colon (f) from iUVRAG
FS

mice treated with Dox as indicated. (g) WB analysis of UVRAG
FS
expression in the spleens from iUVRAG
FS

mice treated with Dox as indicated. Actin serves as a loading control. (h) Representative H&E (left panel) and
Flag immunohistochemical (IHC) (right panel) sections of the colon, lung, liver, and spleen from iUVRAG
FS

mice treated with Dox as indicated. Scale bars, 100µm.

(i) Relative weight of control and iUVRAG
FS
mice with
and without Dox treatment over 14 days.

(j) Representative H&E-stained section (left panel),
immunohistochemical (IHC) staining of cleaved caspase 3 (middle panel), and Oil red O staining (right panel)
of the livers from fed and 48-h-fasted iUVRAG
FS
mice with or without Dox treatment. Arrows indicate apoptotic
cells. Scale bars, 100 µm.

32

33

baseline in liver of Dox-untreated and -treated mice but failed to increase in Dox-
treated iUVRAG
FS
mice following starvation (Figure 5c). In parallel with
compromised starvation-induced autophagy, Dox-treated iUVRAG
FS
mice showed
massive enlargement and accumulation of lipid droplets (LD) in liver when
compared to control mice (Figure 5c). Increased LDs were also observed in fasted
wild-type mice, but to a much lesser extent, and LDs were mostly surrounded by
autophagic vesicles that was not found in cells with UVRAG
FS
expression (Figure
5c). The marked increase of LDs was further confirmed by oil red O staining of
liver sections in starved iUVRAG
FS
mice on Dox (Figure 4j), supporting the
previous findings on the role of autophagy in the clearance of hepatic LDs
5,6
. These
results indicate that UVRAG
FS
prevents starvation-induced autophagy activation in
vivo, which may disturb metabolic adaptation of cells to nutrient deprivation and
thereof increase the risk of metabolic disorders.
To understand how UVRAG
FS
suppresses starvation-induced autophagy in
vivo, we examined WT UVRAG association with Beclin1 and Vps34, which are
Figure 5: UVRAG
FS
inhibits starvation-induced autophagy in mice. (a) Representative images (left panels)
and quantification (right panels) of GFP-LC3 puncta in indicated tissues from Dox-treated/untreated
iUVRAG
FS
mice and littermate control mice that had been crossed with GFP-LC3 transgenic mice, following
48 h of starvation. Data represent the mean ± SD of n = 20 tissue sections pooled from three independent
experiments. Scale bars, 10 µm. (b) Western blot analysis of LC3-I/II, p62 levels, Atg12-Atg5 conjugates, and
Atg16 levels in the liver from Dox-treated/untreated iUVRAG
FS
mice and littermate control mice following 48
h of starvation. The densitometric quantification of the LC3-II/LC3-I and the p62/actin ratios under the
indicated conditions are shown (right panels). Data represents the mean ± SD from n = 5 independent
experiments. (c) Representative electron microscopy (EM) images of the liver from Dox-treated/untreated
iUVRAG
FS
mice in fed or starving conditions for 48 h. Note the accumulation of lipid droplets (LDs) in starved
iUVRAG
FS
mice on Dox. Insets highlight LDs being surrounded by autophagic membrane structures.
Arrowheads denote autophagic vacuoles. The number and size of LD and the number of autophagic vacuoles
(AV) in liver from indicated mice were quantified (right panels). Data represents the mean ± SD from n = 20-
30 cells pooled from three independent experiments. Scale bar, 1.0 µm. (d) Co-immunoprecipitation (co-IP)
of autophagy-related proteins with UVRAG in 48 h-starved liver from control and Dox-treated/untreated
iUVRAG
FS
mice. Actin serves as a loading control. Data is the representative of three independent
experiments. IP, immunoprecipitated; WB, western blot; WCL, whole cell lysates. n.s., not significant; *, P <
0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s t-test).

34
parts of an autophagic-specific class III phosphatidylinositol-3-OH kinase
35

(PI3KC3) complex that has a key role in autophagosome formation
14,15-17
. There
was a marked reduction in UVRAG co-immunoprecipitation with Beclin1 and
Vps34, concomitant with an increase in its binding to Rubicon, a negative regulator
of the PI3KC3 complex
18,47
, in the 48 h-starved tissue of Dox-induced iUVRAG
FS

mice (Figure 5d). Notably, reduced assembly of the UVRAG-containing PI3KC3
complex was not due to alteration of UVRAG S497 phosphorylation
(corresponding to S498 in human UVRAG) in Dox-treated iUVRAG
FS
mice (Figure
5d), which, mediated by mTORC1, was previously shown to decrease the
autophagy activity of UVRAG in vitro
48
. Rather, it is associated with the dominant-
negative effect of UVRAG
FS
that was able to bind to and sequester UVRAG from
the PI3KC3 complex, leading to autophagy suppression (Figure 5d). These results
indicate that UVRAG
FS
inhibits starvation-induced autophagy by acting on the
UVRAG-autophagy complex, consistent with the previous findings in vitro
23
, and
that mammalian UVRAG, unlike its predicted orthologue of yeast Vps38
49
, is
required for starvation-induced autophagy activation in vivo.

Figure 6: UVRAG
FS
prevents TLR4 induction of autophagy in mice. (a) Representative images (left panels)
and quantification (right panels) of GFP-LC3 puncta in indicated tissues from Dox-treated/untreated iUVRAG
FS

mice and littermate control mice that had been crossed with GFP-LC3 transgenic mice, challenged with PBS
or LPS (20 mg per kg body weight; intraperitoneally). Scale bars, 10 µm. Data represents the mean ± SD of
n = 20 tissue sections pooled from three independent experiments. (b) UVRAG
FS
inhibits the Beclin1-
autophagy interactome and K63-linked ubiquitination in LPS-stimulated bone marrow-derived macrophage
(BMDM). BMDM derived from iUVRAG
FS
mice were incubated with PBS or LPS (100 ng/ml) for 4 h in the
presence/absence of Dox and subjected to IP with anti-Beclin1 followed by WB detection of indicated proteins.
Expression of indicated proteins in WCL were shown (right). Data is the representative of three independent
experiments.(c) UVRAG
FS
prevents LPS-induced PI3KC3 activation in BMDM. Cells cultured as in (b) were
transfected with p40(phox)-PX-GFP (to monitor PI3P formation) and subjected to confocal microscopy.
Representative images of p40(phox)-PX-GFP puncta in indicated BMDM are shown (top) and the number of
p40(phox)-PX-GFP puncta per cell was quantified (bottom). Scale bars, 10 µm. Data represents the mean ±
SD of n = 50 cells pooled from three independent experiments. n.s., not significant; ****, P < 0.0001 (Student’s
t-test).
36
UVRAG
FS
prevents TLR4 induction of autophagy in vivo. In addition to
starvation, autophagy is induced by Toll-like receptors (TLRs) signaling as part of
innate immunity in response to microbial components
9,50,51
. To evaluate the impact
of UVRAG
FS
on autophagy induced by lipopolysaccharide (LPS), a major
component of bacterial endotoxin that activates the TLR4 microbe sensor
52
, we
challenged control and UVRAG
FS
-expressing mice that had been crossed with
GFP-LC3 mice with LPS-induced septic shock. We observed that LPS stimulation
drastically increased GFP-LC3 puncta in different tissues (liver, colon, and lung)
of control mice, while Dox-treated iUVRAG
FS
mice displayed marked impairment
of LPS-induced upregulation of autophagy (Figure 6a). Analogous results were
also obtained in bone marrow-derived macrophages (BMDM) from iUVRAG
FS

mice, whereby LPS induced a bona fide increase in both numbers of
autophagosomes (indicated by GFP
+
RFP
+
puncta) and autophagic flux (indicated
by GFP
-
RFP
+
puncta) in wild-type mice but not in mice with UVRAG
FS
expression,
as indicated by the tandem fluorescently tagged mRFP-EGFP-LC3
53
(Figure 7a).
These results indicate that UVRAG
FS
prevents autophagy activation in response
to LPS.
LPS engagement of TLR4 and resultant autophagy induction requires MyD88-
and/or TRIF-dependent TRAF6 (E3 ubiquitin ligase) activation and TRAF6-
mediated K63-linked ubiquitination of Beclin1 in macrophage in vitro
54,55,56
. We
examined Beclin1 recruitment to the TLR4 receptor complex and consequent
modification by TRAF6 in LPS-treated iUVRAG
FS
BMDM. Co-immunoprecipitation
analysis showed that LPS treatment resulted in increased interaction of Beclin1
37
with MyD88, TRIF, and TRAF6, which was associated with increased K63-linked
Beclin1 ubiquitination in BMDM (Figure 6b). Dox treatment decreased LPS-

induced Beclin1 interaction with MyD88 and TRAF6, and less with TRIF, as well
as Beclin1 K63 ubiquitination (Figure 6b). By contrast, Beclin1 interaction with its
Figure 7: Effects of UVRAG
FS
on LPS-induced autophagy activation. (a) UVRAG
FS
expression inhibits LPS-
induced autophagic response. iUVRAG
FS
BMDM transfected with mRFP-EGFP-LC3 were treated with PBS
or LPS (100 ng/ml) for 4 h in the presence/absence of Dox. Representative confocal images of mRFP-EGFP-
LC3 puncta in cells were shown (left) and the numbers of GFP
+
RFP
+
puncta (yellow dots; neutral
autophagosome) and GFP
-
RFP
+
puncta (red dots; matured autophagosome) per cell were quantified (right).
Scale bars, 10 µm. Data represents the mean ± SD of 50-100 cells pooled from three independent
experiments. (b) Co-immunoprecipitation (IP) of indicated proteins with Beclin1 in the livers from control and
iUVRAG
FS
mice, treated with Dox and LPS as indicated. Expression of indicated proteins in WCLs were shown
(right panel).(c) Schematic diagram showing the mechanism by which UVRAG
FS
disrupts the LPS-induced
Beclin1 recruitment to TLR4 receptor complex and enhances its interaction with negative regulator Bcl-2 by
regulating TRAF6-mediated K63 ubiquitination of Beclin1.
38
negative regulator Bcl-2 was increased in LPS-stimulated iUVRAG
FS
BMDM
(Figure 6b). We also noted that UVRAG
FS
was able to dissociate the UVRAG-
Beclin1-PI3KC3 complex, but had minimal effect on the Atg14-Beclin1-PI3KC3
complex in vivo, in LPS-stimulated iUVRAG
FS
BMDM (Figure 6b). The suppression
of the Beclin1 interactome by UVRAG
FS
was associated with reduced PI3KC3
kinase activity in LPS-treated BMDM (Figure 6c), as illustrated by decreased
punctate staining of the Vps34 kinase product, phosphatidylinositol 3-phosphate
(PI3P)
57
. Similar results were obtained in livers of LPS-treated iUVRAG
FS
mice
(Figure 7b). Together, these results indicate that UVRAG
FS
plays an inhibitory role
in TLR4-induced autophagy by interfering with the dynamics of Beclin1-containing
TLR4 signaling complex formation and autophagy activity (Figure 7c).

UVRAG
FS
blockade of autophagy enhances inflammatory responses. In
parallel with impaired LPS-induced autophagy in Dox-treated iUVRAG
FS
mice,
there was increased mice vulnerability to intraperitoneally injected LPS (Figure 8a).
The survival of UVRAG
FS
-expressing mice in septic shock was significantly lower
than that of controls (no Dox) treated in the same way (Figure 8a). Notably, Dox-
treated iUVRAG
FS
mice had similar levels of serum TNF-a, IL-6, and IFN-b after
LPS stimulation as control mice (Figure 9a). Characterization of NF-kB activation
in BMDM from control and UVRAG
FS
-expressing mice revealed no discernable
difference in the phosphorylation and degradation of the NF-kB inhibitor IkBa
58

(Figure 8b) and in nuclear translocation of p65 (Figure 8c). Consistent with this,
the expression of Tnf-a, Il-6, and Ifn-b mRNA in spleens was comparable between
39

40
Figure 8: UVRAG
FS
enhances LPS-induced lethal shock by promotion of inflammatory responses. (a) Kaplan-
Meier curve showing survival of Dox-treated/untreated iUVRAG
FS
mice (n = 10 per group) challenged with
LPS (20 mg per kg body weight; intraperitoneally). **, P < 0.01 (Mantel-Cox test). (b) WB analysis (left) and
densitometric quantification (right) of phosphorylated (p-)IkBa and total IkBa in Dox-treated or untreated
iUVRAG
FS
BMDMs stimulated with LPS (100 ng/ml). (c) Immunofluorescence analysis (left) and quantification
(right) of relative distribution of endogenous p65 (green) in the nucleus in cells in (b). Scale bars, 50 µm. (d)
Quantitative RT-PCR analysis of cytokine genes as indicated in the spleen of Dox-treated/untreated
iUVRAG
FS
mice and littermate control challenged with LPS (20 mg per kg body weight; intraperitoneally). (e)
Cell viability of Dox-treated/untreated iUVRAG
FS
BMDMs stimulated with PBS or LPS (100 ng/ml) examined
by MTT assay. (f) Co-IP of ASC and NLRP3 with pro-caspase-1 and WB analysis of caspase-1 cleavage, IL-
1b production, LC3-I/II in WCLs of BMDM obtained from Atg5 wild-type and tamoxifen-induced knockout mice
(n = 10 per group), primed with PBS or LPS followed by 30 min of ATP (LPS+ATP) stimulation. Actin serves
as a loading control. Densitometric quantification (right) of relative production of cleaved form of caspase-1
and IL-1b to their precursors in cell culture. (g) Relative concentrations of mitochondrial DNA (mtDNA) in LPS-
primed ATP-stimulated iUVRAG
FS
BMDM in the presence/absence of Dox and MitoQ treatment (1 µM, 1 h).
Data represents the mean ± SD from three independent experiments (b-e; g). n.s., not significant; **, P < 0.01;
***, P < 0.001; ****, P < 0.0001 (Student’s t test).

LPS-stimulated UVRAG
FS
-expressing mice and LPS-stimulated control mice
(Figure 8d). Interestingly, unlike TNF-a, whose synthesis and secretion are
inflammasome-independent, production of IL-1b and IL-18, which requires
inflammasome-dependent caspase-1 activation
8,59
, was greater in abundance in
the serum of Dox-induced iUVRAG
FS
mice than littermate control mice in response
to LPS (Figure 9a), although their mRNA synthesis was not increased accordingly
(Figure 8d). To determine whether UVRAG
FS
plays a role in LPS-induced
inflammasome activation, we stimulated control and UVRAG
FS
-expressing BMDM
with LPS and the NLRP3 inflammasome activator ATP and observed more than
two folds increase in mature IL-1b production, but not pro-IL-1b levels, by Dox-
induced iUVRAG
FS
macrophages than control cells, suggesting that enhanced IL-
1b secretion is likely related to increased inflammasome activation (Figure 9b,c).
As expected, there was a notable increase in caspase-1 activation (indicated by
cleaved caspase-1) in LPS+ATP-treated iUVRAG
FS
BMDM on Dox than in control
cells (Figure 9b,c). Elevated caspase-1 activation and IL-1b production correlated
41
with increased rate of macrophage death (Figure 8e). Treatment of iUVRAG
FS

BMDM with z-YVAD-fmk, a caspase-1 inhibitor, ablated enhanced IL-1b secretion
by UVRAG
FS
(Figure 9d), confirming that UVRAG
FS
-associated increased levels of
mature IL-1b was the consequence of increased caspase-1 activation. Thus,
caspase-1 overactivation contributes to excessive inflammation in Dox-treated
iUVRAG
FS
mice after LPS stimulation.
Caspase-1 is activated when it is recruited and incorporated into
inflammasomes, which also contains a Nod-like sensor protein such as NLRP3
and the adaptor molecule ASC
60
. We observed that UVRAG
FS
-expressing BMDM
demonstrated a marked increase in NLRP3 and ASC co-immunoprecipitation
with the precursor pro-caspase-1 than in control BMDM, suggesting increased
NLRP3 inflammasome assembly upon UVRAG
FS
expression (Figure 9b). Despite
NF-kB-dependent upregulation of NLRP3 by LPS+ATP, no differences in NLRP3
inflammasome protein expression were observed in control versus UVRAG
FS
-
expressing cells (Figure 9b), suggesting that increased NLRP3 inflammasome
assembly by UVRAG
FS
is not due to altered protein expression. To examine
whether the observed increase in NLRP3 inflammasome assembly/activation is
due to specific effects of UVRAG
FS
, or due to defective autophagic response, we
conducted the same experiment in macrophages from mice that are deficient for
the autophagy gene Atg5 (Atg5
-/-
), an essential autophagy protein that associates
with Atg12 and Atg16 to conjugate LC3 to autophagosome membrane
61
.
42

43
Figure 9: UVRAG
FS
blockade of LPS-induced autophagy enhances inflammatory response. (a) ELISA of
inflammatory cytokines as indicated in serum from Dox-treated/untreated iUVRAG
FS
mice and littermate
control mice (n = 10 per group), assessed 3 h after intraperitoneal injection of LPS (20 mg per kg body weight).
(b) Co-IP of ASC and NLRP3 with pro-caspase-1 and WB analysis of caspase-1 activation and IL-1b
production in WCLs and supernatants (Sup.) of BMDM from Dox-treated/untreated iUVRAG
FS
mice and
control mice stimulated with PBS or LPS (100 ng/ml, 6 h) followed by 30 min of ATP (LPS+ATP). (c)
Densitometric quantification of relative production of cleaved caspase-1 and IL-1b to their precursors in BMDM
culture in (b). (d) ELISA of IL-1b secretion by LPS-primed control and UVRAG
FS
-expressing BMDM incubated
for 1 h with z-YVAD-fmk (10 µM) or vehicle (DMSO), followed by ATP stimulation for 1 h. (e) Relative
mitochondrial ROS (mtROS) amounts determined by MitoSOX staining of LPS-primed ATP-stimulated
iUVRAG
FS
BMDM in the presence/absence of Dox and MitoQ treatment (1 µM, 1 h).(f) Representative
confocal microscopy (left) and quantification (right) of intracellular distribution of Parkin (green) relative to
Tom20-labelled mitochondria (red) in LPS-primed (100 ng/ml, 6 h) ATP-stimulated (1 mM , 1 h) iUVRAG
FS

BMDM in the presence/absence of Dox (1 µg/ml). Scale bars, 10 µm. (g) ELISA of IL-1b secretion (left) and
WB analysis for caspase-1 in lysates (right) of iUVRAG
FS
LPS-primed BMDM incubated with MitoQ (1 µM, 1
h), followed by 1 h ATP treatment. Data represents the mean ± SD from three independent experiments (a,
c-g). n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s t-test).

As mice with constitutive Atg5 deletion die soon after birth
62
, we used BMDM from
an inducible Atg5 knockout mice, whereby addition of tamoxifen abrogated Atg5
expression (Figure 8f). As seen with UVRAG
FS
expression, Atg5-deficient BMDM
exhibited deficient autophagy activation, increased NLPR3:ASC:pro-caspase-1
complex formation, increased caspase-1 activation and IL-1b maturation than wild-
type controls after LPS+ATP treatment (Figure 8f). The similarity of the phenotypes
of cells from UVRAG
FS
mice and Atg5-deficient mice provides strong support for a
role of suppressed autophagy, rather than other UVRAG
FS
-regulated functions, in
NLRP3 inflammasome activation and increased inflammatory response in
iUVRAG
FS
mice.
Additionally, Dox-treated iUVRAG
FS
mice exhibited increased mtROS
production (Figure 9e) and cytosolic mtDNA release (Figure 8g) in LPS+ATP-
treated BMDM. This increase was associated with impaired mitochondrial
homeostasis, as indicated by increased recruitment and aggregation of Parkin,
which selectively labels impaired mitochondria
63
, to Tom20-labelled mitochondria
(Figure 9f). Treatment of cells with the mitochondrial-specific antioxidant MitoQ
64
,
44
which eliminated not only mtROS but also prevented mtDNA release (Figure 9e
and Figure 8g), effectively abrogated the apparent increase in both caspase-1
activation and IL-1b production in Dox-treated iUVRAG
FS
BMDM in response to
LPS+ATP (Figure 9g). These data are in agreement with the previous studies
8,10,65

demonstrating that autophagy, particularly mitophagy, prevents perpetuated
inflammasome activation by eliminating inflammasome activators such as mtROS
or mtDNA
60,65,66
. Thus, the impaired LPS-induced autophagy activation is
responsible for aberrant NLRP3-inflammasome activation and increased septic
shock in iUVRAG
FS
mice.

UVRAG
FS
exacerbates inflammatory responses in experimental colitis.
Abnormal inflammasome activation has been implicated in the development of
inflammatory bowel diseases (IBD)
67,68
. We next examined whether UVRAG
FS

suppression of autophagy exacerbates intestinal inflammation induced by oral
administration of dextran sodium sulfate (DSS), an epithelial irritant
69
. Dox-treated
cohorts of wild-type and iUVRAG
FS
mice were fed 5% DSS (w/v) in their drinking
water for 6 days, followed by 4 days of regular drinking water. Induced expression
of UVRAG
FS
made mice more susceptible to DSS-induced colitis (Figure 10a). In
addition, UVRAG
FS
-expressing mice exhibited markedly Increased disease
severity, as demonstrated by elevated disease activity indices (DAI) and severe
colonic shortening at necropsy on days 6 and 10 (Figure 10a-c). Enhanced colitis
corresponded with a marked increase in all indices of histopathological changes,
including crypt damage and inflammatory infiltrates (Figure 10d,e). This increased
45
susceptibility of UVRAG
FS
-expressing mice to DSS-induced colitis is not due to
inherent differences at baseline, because no difference in colon length and crypt
structure were observed between the two genotypes (Figure 11a,b). The number
of bacteria in the feces of both cohorts was also comparable (Figure 11c). To
assess whether UVRAG
FS
suppression of autophagy contributes to DSS-induced
intestinal inflammation, we analyzed autophagy activity in DSS-treated colons. As
expected, DSS treatment led to elevate levels of autophagy, as measured by
increased LC3-II conversion and p62 turnover, in control colon lysates, which was
suppressed in Dox-treated iUVRAG
FS
mice (Figure 10f,g). Deficient autophagy
was associated with increased production of active caspase-1 and IL-1b in DSS-
treated UVRAG
FS
mice colons (Figure 10f-h). In accord, amounts of
proinflammatory cytokines IL-1b, but not IL-6 and TNF-a, produced by colonic
tissue were significantly increased in DSS-fed UVRAG
FS
-expressing mice versus
DSS-fed control mice (Figure 10i). Supporting this, the UVRAG
FS
-expressing mice
had elevated induction of mRNA expression for IL-1b-inducible chemokines
CXCL1, CXCL2, and CCL2 in colon (Figure 11d) and enhanced neutrophils
infiltration (Figure 10d). As observed in sepsis, UVRAG
FS
did not alter the response
of mice to DSS with respect to NF-kB activation in colon (Figure 11e).

46

47
Figure 10: UVRAG
FS
exacerbates intestinal colitis in vivo. (a) Kaplan-Meier curve showing survival of Dox-
treated control and iUVRAG
FS
mice (n = 10 per group) fed with 5% DSS in drinking water for 6 days. **, P <
0.01 (Mantel-Cox test). (b) Disease activity index (DAI) of colitis in Dox-treated control and iUVRAG
FS
mice
treated in (a). The DAI value was calculated as described in Methods. (c) Colon lengths of DSS-treated mice
with indicated genotypes were assessed at the time of necropsy (day 6 and day 10) with representative
macroscopic images (left). Data represents the mean ± SD (n = 5-6 mice per genotype). (d) H&E sections of
colon from DSS-treated mice with indicated genotypes on day 10 after DSS. Inset magnification highlights
inflammation and crypt damage associated with UVRAG
FS
expression. Scale bars, 300 µm. (e) Histopathology
scores for tissue injury, inflammation, and overall pathology in the colon for control and UVRAG
FS
-expressing
mice after DSS-induced colitis. Data represents the mean ± SD (n = 5-6 mice per genotype). (f) WB analysis
of caspase-1, IL-1b, ASC, NLRP3, LC3-I/II, and p62 levels in colon tissues of Dox-treated control and
iUVRAG
FS
mice before or after DSS for 6 days. (g,h) Densitometric quantification of the ratios of LC3-II/LC3-
I and p62/actin (g) and the relative production of cleaved caspase-1 and IL-1b to their precursors (h) in the
colons in (f). Data represents the mean ± SD from three independent experiments. (i) ELISA of cytokines in
colon tissues of control and UVRAG
FS
-expressing mice after DSS treatment. Data represents the mean ± SD
(n = 5-6 mice per genotype). (j,k) Histopathology scores (j) and H&E sections (k) of colons from control and
UVRAG
FS
-expressing mice treated with MCC950 (20 mg kg
-1
body weight; intraperitoneally) during DSS-
induced colitis. (l) ELISA of cytokines in colon tissues of control and UVRAG
FS
-expressing mice in (j). Data
represents the mean ± SD (n = 3-5 mice per genotype) (j-l). n.s., not significant; *, P < 0.05; **, P < 0.01; ***,
P < 0.001(Student’s t-test).

To further determine the contribution of hematopoietic UVRAG
FS
versus non-
hematopoietic UVRAG
FS
in driving exacerbated colitis, we generated UVRAG
FS

bone marrow chimeras by reconstituting both control and UVRAG
FS
-expressing
recipients with bone marrow harvested from either control or UVRAG
FS
-expressing
donors, as previously describe
70
, and confirmed UVRAG
FS
expression in these BM
chimeras (Figure 11f). We observed that both sets of chimeric mice were
significantly more hypersensitive to DSS-induced colitis compared with the
control® control animals, as indicated by increased colon length reduction and
histological indices (Figure 11g-i). Histopathological analysis of colons isolated
from the chimeric mice revealed that all of the UVRAG
FS
-expressing animals (both
donor and recipient) exhibited significantly increased crypt damage, extensive
areas of ulceration, and enhanced inflammatory compared with the control®
control mice (Figure 11h,i). Thus, UVRAG
FS
functions through both hematopoietic
and the non-hematopoietic compartments in inflammasome activation.
48

49
Figure 11: UVRAG
FS
enhances inflammatory response in DSS-induced colitis. (a) Representative
macroscopic images (left) and length of colon (right) from mice of indicated genotypes at Day 0 in a colitis
model. (b) H&E sections of the colons from mice in (a). Scale bars, 300 µm (left) and 100 µm (right).(c) The
number of bacteria in feces of control and UVRAG
FS
-expressing mice. CFU, colony forming unit. (d) Relative
mRNA expression of the neutrophil chemokines CXCL1, CXCL2, and CCL2 as determined by quantitative
RT-PCR in the colons from mice of indicated genotypes. (e) WB analysis of phosphorylated (p-)IkBa and total
IkBa in the colon from Dox-treated mice of indicated genotype on day 0 and day 6 of DSS treatment. (f) RT-
PCR analysis for UVRAG
FS
reconstitution in peripheral leukocytes in 12-week-old UVRAG
FS
bone marrow-
chimeric mice. Control ® control, control mice reconstituted with control BM-derived cells; control ®
UVRAG
FS
, UVRAG
FS
-expressing mice reconstituted with control BM-derived cells; UVRAG
FS
® control,
control mice reconstituted with UVRAG
FS
-expressing BM-derived cells; UVRAG
FS
® UVRAG
FS
, UVRAG
FS
-
expressing mice reconstituted with UVRAG
FS
-expressing BM-derived cells.(g) Representative macroscopic
images of the colons from DSS-treated UVRAG
FS
BM chimera on day 10.(h,i) Histopathology scores (h) and
H&E sections (i) of the colons from UVRAG
FS
chimeric mice as indicated in DSS-induced colitis. (j) Schematic
representation of the experimental procedure for targeting NLRP3 inflammasome in DSS-induced colitis. Dox-
treated control and iUVRAG
FS
mice were treated with 5% DSS for six days, followed by 4 days of regular
water. These mice were injected daily from days 0 to 5 with MCC950 (20 mg per kg

body weight) or vehicle
control.(k,l) Relative weight change (k) and DAI (l) of Dox-treated control and iUVRAG
FS
mice in (j) in response
to MCC950 therapy in DSS-induced colitis. (m,n) Representative macroscopic images (m) and length of colon
(n) from MCC950-treated mice of indicated genotypes during DSS-induced colitis. Data represents the mean
± SD (n = 5-10 mice per genotype) (a, c, d, h, k, l, n). n.s, not significant; *, P < 0.05; **, P < 0.01; ***, P <
0.001; ****, P < 0.0001 (Student’s t test).

Administration of the NLRP3 inhibitor MCC950
55
significantly attenuated the
clinical and histological signs of colitis in Dox-treated iUVRAG
FS
mice, including
weight loss, colon length reduction, histological indices, tissue injury and
inflammatory infiltrates (Figure 11j-n; Fig. 10j-l). Although inflammatory cytokines
IL-6 and TNF-a were unaffected, colonic IL-1b was significantly lower after
MCC950 treatment (Fig. 10l). These results indicate that heighted NLRP3
activation with subsequent increased production of pro-inflammatory cytokines
account for increased colitis in iUVRAG
FS
mice.

UVRAG
FS
predisposes mice to colitis-associated colorectal cancer (CRC).
We next examined whether autophagy restriction of inflammasome activation
confers a protective role from colitis-associated neoplastic process in iUVRAG
FS

mice, using an established azoxymethane (AOM)-DSS model of CRC
34
, which

50

Figure 12: UVRAG
FS
expression inhibits cell differentiation in AOM-DSS model. (a) Schematic representation
of the experimental procedure for the AOM-DSS model. Mice were injected with AOM (10 mg per kg

body
weight) on day 0, followed by 3 cycles of 2% DSS given in the drinking water (black boxes) for 6 days
separated by 2 weeks of regular water. Mice were sacrificed on day 60.(b) Weight change during AOM-DSS
treatment in mice of indicated genotypes was expressed as the percentage of change from day 0. Data
represents the mean ± SD (n = 5-12 mice per genotype). (c) WB analyses (left) and densitometric quantitation
(right) of E-cadherin, N-cadherin, and Vimentin in the colons from Dox-treated mice of indicated genotypes
during AOM-DSS treatment (three randomly chosen samples per group; similar results observed in all 5-12
samples per group). Data represents the mean ± SD from three independent experiments (b,c). n.s, not
significant; **, P < 0.01; ****, P < 0.0001 (Student’s t test).

recapitulates the colitis and the crypt foci–adenoma–carcinoma sequence
occurring in CRC
71
. For this model, tumorigenesis is initiated with a single dose of
AOM and promoted by three cycles of oral 2% DSS administration that triggers
intestinal injury and colitis (Figure 12a). At this concentration of DSS, no difference
in body weight was observed between Dox-treated iUVRAG
FS
mice and wild-
typecontrol littermates (Figure 12b). However, we observed a more than two-fold
increase in the incidence of tumors in Dox-treated iUVRAG
FS
mice compared with
control mice, and the average tumor size and tumor load was significantly elevated
51
upon UVRAG
FS
expression (Figure 13a-d). Immunohistochemistry analyses
revealed increased cell proliferation, as measured by Ki67 staining of colonic
epithelium, and decreased apoptosis, as indicated by reduced active caspase-3
staining on day 60 after AOM-DSS treatment, in the colon of Dox-treated
iUVRAG
FS
versus control mice (Figure 13e). There were markedly decreased
levels of the epithelial cell markers, Keratin 20 (Krt20) and E-cadherin, but
increased levels of the mesenchymal markers, N-cadherin and vimentin, in colons
with UVRAG
FS
expression (Figure 13e and Figure 12c), suggesting an induction
of epithelial-to-mesenchymal transition (EMT) in the course of tumorigenesis. The
hyperproliferation and blocked differentiation by UVRAG
FS
is
consistent with early onset of AOM-DSS induced colitis-associated CRC in Dox-
treated iUVRAG
FS
mice.
In concordance with increased colonic tumorigenesis, Dox-treated
iUVRAG
FS
mice colon displayed more severe crypt disruption and inflammatory
infiltration after AOM-DSS treatment (Figure 13f,g). Supporting this, there were
increased caspase-1 activation and IL-1b production in the colon of Dox-treated
iUVRAG
FS
as compared to control mice, which correlated with suppressed
autophagy, as measured by LC3 conversion and p62 turnover (Figure 13e,h), in
support of the concept that disturbed autophagy by UVRAG
FS
has a mechanistic
role in unresolved inflammasome activation and inflammation-associated tumor
susceptibility.

52

53
Figure 13: UVRAG
FS
predisposes mice to colitis-associated colon cancer. (a) Representative macroscopic
images of the colon from AOM-DSS-treated control (Ctrl) and iUVRAG
FS
mice in the presence/absence of Dox
at the time of necropsy (day 60). Red circles highlight colonic tumors.(b-d) The number (b), size (c), and
overall load (d) of colonic tumors in AOM-DSS-treated mice in (a). Tumor load is evaluated by totaling the
diameters of all tumors in AOM-DSS-treated mice in (a).(e) IHC staining of Ki67, cleaved caspase 3, p62,
keratin 20, E-cadherin, N-cadherin, and vimentin in the colons from AOM-DSS-treated mice of indicated
genotypes. Data are from one animal that is representative of 5-12 animals in each group. The levels of Ki67
staining (top right) and tumor apoptosis (bottom right) in the indicated colon were quantified. Arrows denote
cells undergoing apoptosis. (f) H&E sections of the colon from AOM-DSS-treated mice in (a). Data are from
one animal that is representative of 5-12 animals in each group.(g) Histological scores for crypt damage (top)
and total histological score (bottom) of colons from mice in (a) were quantified at day 60 as described in the
Methods. Data represents the mean ± SD pooled from two independent experiments (n = 10 mice per
group).(h) WB analysis of caspase-1 cleavage, IL-1b production, LC3-I/II, and p62 levels in colon tissues of
Dox-treated control and iUVRAG
FS
mice after AOM-DSS treatment. Scale bars, 100 µm. n.s., not significant;
*, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (Student’s t test).

Promotion of spontaneous tumorigenesis by UVRAG
FS
. Despite the oncogenic
feature of UVRAG
FS
in vitro
23
, it remains untested whether UVRAG
FS
is sufficient
to initiate tumorigenesis in mammals. To this end, we aged cohorts of iUVRAG
FS

mice and wild-type littermates that were fed Dox starting from 2 month of age and
explore the potential impact of UVRAG
FS
on spontaneous tumorigenesis.
Dox-treated iUVRAG
FS
mice succumbed to the development of spontaneous
tumors starting at 30 weeks (Figure 14a). By 15 and 18 months of age,
approximately 90% of UVRAG
FS
-expressing mice as compared with 25% of wild-
type animals had malignancies (Figure 15a). The tumors arising in iUVRAG
FS
mice
on Dox comprised mainly lymphomas, squamous cell carcinoma, and
adenocarcinoma. We observed massive spleen and lymph node enlargement in
most (70% ~ 80%) of UVRAG
FS
-expressing mice autopsied at 6 to 18 months of
age, which was histologically confirmed to represent lymphomas (Figure 14b,c).
By contrast, few lymphoma was observed in Dox-treated wild-type mice (Figure
54
14c and Figure 15b). The lymphomas invaded a variety of tissues including the
liver and lungs (Figure 14b). The predominant type of lymphomas developed in
UVRAG
FS
-expressing mice was diffuse large B cell lymphomas (DLBCL), which
showed strong immunoreactivity for B cell marker CD20 but were largely negative
for the germinal center marker Bcl-6 (Figure 14d). Some Dox-induced iUVRAG
FS

mice also developed extranodal marginal zone B cell lymphoma with crystal storing
histiocytosis positive for monotypic kappa (Figure 14d).
Figure 14: UVRAG
FS
promotes spontaneous tumorigenesis in mice. (a) Kaplan-Meier plot of time to
development of any malignancy in Dox-treated iUVRAG
FS
mice versus Dox-treated control mice. Malignancy
is determined by complete histologic survey of all major internal organs. ***, P < 0.001(log-rank test). (b)
Representative images of DLBCL in different organs that developed in Dox-treated iUVRAG
FS
mice.(c)
Percentage of control and UVRAG
FS
-expressing mice with spontaneous tumors, including lymphoproliferative
disease (LPD), lymphomas, and non-lymphoid malignancies.(d) H&E and IHC analysis of the most frequently
observed neoplastic lesions from Dox-treated iUVRAG
FS
mice using the antibodies as indicated. Scale bars,
55
100 µm.

In addition to frank lymphomas, there was increased prevalence of splenic,
nodal, and extranodal lymphoproliferative disease (LPD) associated with
UVRAG
FS
expression (Figure 14c). Moreover, iUVRAG
FS
mice treated with Dox
had lung lesions in earlier stages of neoplasia, including poorly differentiated
adenocarcinomas with nuclear immunoreactivity for TTF-1, a tissue-specific
transcription factor in lung epithelial cells
72
, and squamous cell carcinomas with
strong cytokeratin 7 (CK7) and weak TTF-1 staining (Figure 14d). All malignancies
had detectable UVRAG
FS
expression in tumor cells (Figure 14d). Consistent with
human cancers, genome-wide copy number variation (CNV) analysis
73
of tumors
developed in Dox-treated iUVRAG
FS
mice revealed marked variability and
heterogeneity with more chromosomal amplifications and deletions than the
control (Figure 15e). Thus, expression of this cancer-derived frameshift mutant of
UVRAG results in increased susceptibility to spontaneous malignancies in vivo.

56

Figure 15: UVRAG
FS
expression promotes spontaneous tumorigenesis. (a) Prevalence of macroscopic
malignancies in 8-month-old and 18-month-old Dox-treated iUVRAG
FS
mice versus Dox-treated control mice.
P < 0.05 (Fisher’s exact test).(b) Kaplan-Meier plot of time to development of lymphoma in Dox-treated
iUVRAG
FS
mice versus Dox-treated control mice (n = 10 mice per group). (P < 0.0001; log-rank test).
Lymphoma is determined by complete histologic survey of all major internal organs.(c) Global CNA profile
shows heterogeneous amplifications (red) and deletions (blue) among different tumors (n = 13) from Dox-
treated iUVRAG
FS
mice. CNV profiles are created using the mouse genome (mm9) as reference. Copy number
is displayed as the ratio to the median.

UVRAG
FS
promotes proliferation and b-catenin activation. Although colon
tumors were not observed in our cohort of aged mice, histopathologic examination
of colons from Dox-treated iUVRAG
FS
mice revealed hyperplastic changes
compared with those from controls (Figure 16a). Crypt length was increased by
~1.3-fold along the entire length of the colon in Dox-treated iUVRAG
FS
mice along
with strong cytoplasmic expression of UVRAG
FS
at the colonic crypts (Figure
16a,b). No significant differences were detected in the number of cleaved caspase-
3-positive cells in the colon of control versus UVRAG
FS
-expressing mice (Figure
57
16c). However, there was increased Ki67 staining throughout the crypts in
UVRAG
FS
-expressing mice as compared to those in controls (Figure 16c,d),
suggesting that UVRAG
FS
expression results in abnormal cellular proliferation in
colon. We asked whether the pro-proliferative effect of UVRAG
FS
in the colon of
aged mice might also involve aberrant NLRP3-inflammasome activation, a risk
factor for intestinal tumorigenesis
74
. No notable caspase-1 activation and IL-1b
production was detected in both young (2-month-old) and older (18-month-old)
mice in either genotype (Figure 17a). Likewise, no changes were observed in the
expression of NLRP3-inflammasome proteins in these mice (Figure 17a). Similar
results were obtained in splenocytes that gave rise to increased spontaneous
malignancies (Figure 17b). These data indicate that UVRAG
FS
affects cell
proliferation and spontaneous tumorigenesis independently of NLRP3 activity.
The increased cell proliferation in iUVRAG
FS
mice suggested that an
UVRAG
FS
-dependent oncogenic signaling or mitogen might be involved in
spontaneous tumorigenesis. Because of the essential role of the Wnt/b-catenin
pathway in intestinal homeostasis and tumorigenesis
75,76
, we tested whether
UVRAG
FS
regulates b-catenin expression. UVRAG
FS
expression resulted in a
significant increase in overall b-catenin levels and in their nuclear accumulation in
colon (Figure 16e,f). Immunoblotting analysis of colonic b-catenin further
confirmed the increased expression of b-catenin and concomitant upregulation of
b-catenin transcriptional targets, c-Myc and Cyclin D1, in 18-month-old
58

59
Figure 16: UVRAG
FS
activates b-catenin by promoting age-related autophagy suppression. (a) H&E and IHC
analysis of colon from Dox-treated control and iUVRAG
FS
mice (12-month-old). (b) Crypt length of the colon
from the indicated group in (a).(c,d) Representative images of Ki67 (top) and cleaved caspase 3 (bottom)
staining of colons in 12-month-old control and iUVRAG
FS
mice on Dox and quantitation of percentage of Ki67-
positive cells per crypt in the colon of indicated genotype. (e,f) IHC staining of b-catenin and c-Myc (e) and
quantitation of the number of nuclear b-catenin per crypt (f) in the colon from Dox-treated control and
iUVRAG
FS
mice (12-month-old).(g) WB analysis of indicated protein expression in colons from mice of the
indicated genotype (two randomly chosen samples per group similar results observed in all ten samples per
genotype).(h) Quantitative RT-PCR analysis of b-catenin and b-catenin target gene expression in colons from
Dox-treated mice of indicated genotype.(i,j) WB analysis of b-catenin and autophagy marker proteins in
immortalized human colon epithelial cells (iFHC) treated with 3-MA (1 mM) (i) or with chloroquine (CQ, 20 µM)
for indicated time. Actin serves as a loading control. Data is the representative of three independent
experiments.(k) UVRAG
FS
inhibits b-catenin interaction with autophagy marker proteins. SW480 CRC cells
were transfected with increasing amounts of Flag- UVRAG
FS
. WCL were used for co-IP with anti-b-catenin,
followed by IB with the indicated antibodies. Data is the representative of three independent experiments.(l)
Quantitative RT-PCR analysis of indicated gene expression in cells in (k). Data represents the mean ± SD
from three independent experiments. (m) Co-IP of b-catenin with LC3 and TCF4 in the spleens from 18-month-
old mice of indicated genotype (two randomly chosen samples per group similar results observed in all ten
samples per genotype). Data represents the mean ± SD (n = 10 mice per group) (b, d, f, h). n.s., not significant;
*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s t test). Scale bars, 100 µm.

UVRAG
FS
-expressing mice but not in wild-type mice of the same age (Figure 16g).
Supporting this, Dox-treated iUVRAG
FS
mice (18-month-old) had elevated
induction of mRNA for b-catenin targets including c-Myc, Cyclind1, and Axin2,
while the mRNA abundance of b-catenin was not increased in colons (Figure 16h).
Analogous results were obtained in splenocytes, whereby increased levels of b-
catenin, and its target genes c-Myc and Cyclind1 were observed in the spleens of
18-month-old Dox-treated iUVRAG
FS
mice but not in the age-matched controls
(Figure 17c). Notably, in the colon of young mice, no genotype-specific differences
were observed in any of the b-catenin-related factors assessed (Figure 16g),
indicating that the enhanced b-catenin activation in older UVRAG
FS
-expressing
mice genuinely reflects an age-related changes.
The increase in b-catenin protein without a concomitant increase in mRNA level
in iUVRAG
FS
mice suggested that b-catenin is subjected to post-translational
regulation. Canonically, cytoplasmic b-catenin protein levels are controlled by the
60
Axin1-APC-GSK3 destruction complex (DC), and mutations in DC components
stabilizes and activates b-catenin, resulting in cancer, mostly notably of the colon
77-
79
. Exon sequencing of Axin1, APC, and GSK3 in colonic DNA from the 18-month-
old Dox-treated iUVRAG
FS
mice did not reveal any genetic changes (data not
shown). Both the young and older Dox-treated iUVRAG
FS
mice and wild-type
littermate controls expressed comparable levels of DC components and the b-
catenin DC complex assembly (Figure 17d). These results point to a role for
UVRAG
FS
in b-catenin activation in older mice through a mechanism
independently of the b-catenin DC properties, which induces transcription of its
target oncogenes and increases cancer susceptibility.

UVRAG
FS
activates b-catenin through age-related autophagy suppression.
Autophagy is recently implicated in the regulation of Wnt/b-catenin pathway
80-82
.
We postulated that UVRAG
FS
exerts its role in b-catenin activation by autophagy
suppression. Although UVRAG
FS
did not readily affect basal autophagy in young
mice, it exacerbates age-related decline in autophagic function in 18-month-old
mice, as indicated by LC3-II production and p62 turnover, in conjunction with
increased b-catenin activity in vivo (Figure 16g and Figute 17c). addtion, inhibition
of the autophagy pathway by 3-methyladenine (3-MA), chloroquine (CQ), or by
depletion of Beclin1 and UVRAG, markedly increased b-catenin protein levels in
immortalized fetal human colon epithelial cells (iFHC) and in SW480 colorectal
cancer cells (Figure 16i,j and Figure 17e,f), indicating a steady-state turnover of
b-catenin through the autophagy-lysosome pathway. This was consistent with the
61
previous observation demonstrating that the autophagy pathway is important in b-
catenin degradation
83
. We therefore tested whether UVRAG
FS
expression is
sufficient to stabilize b-catenin protein in colon epithelial cells. Concomitant with
the dose-dependent decrease of autophagy in UVRAG
FS
-expressing SW480 cells,
there were increase in b-catenin protein levels and in the transcriptional output of
the Wnt pathway, accordingly, while b-catenin mRNA levels remained unaffected
(Figure 16k,l). Indeed, elevated b-catenin protein levels correlated with a marked
decreased in b-catenin
62

63
Figure 17: UVRAG
FS
promotes cell proliferation by b-catenin activation. (a,b) WB analysis of caspase-1
cleavage, IL-1b production, ASC, NLRP3 in the spleen (a) and the colon (b) from 2-month-old and 18-
month-old Dox-treated control and iUVRAG
FS
mice (two randomly chosen samples per group; similar results
observed in all 10 samples per group).(c) WB analysis of indicated proteins in the spleen from 2-month-old
and 18-month-old Dox-treated control and iUVRAG
FS
mice (two randomly chosen samples per group; similar
results observed in all 10 samples per group).(d) Co-IP of GSK3b, APC, and b-catenin with Axin in the
colons from 2-month-old and 18-month-old Dox-treated control and iUVRAG
FS
mice. Data is representative
of three independent experiments. (e) WB analysis of b-catenin and autophagy markers in SW480 cells
treated with 3-MA (1 mM) or with CQ (20 µM) for the indicated time. Actin serves as a loading control. (f)
Knockdown of autophagy proteins stabilizes b-catenin. SW480 cells were transfected with control shRNA,
Beclin1- or UVRAG-specific shRNA, followed by WB analysis of indicated proteins. (g) Wild-type UVRAG
and autophagy induction antagonizes the effect of UVRAG
FS
on b-catenin stabilization. SW480 cells stably
expressing UVRAG
FS
were treated with Torin 1 (1 µM, 6 h) or transfected with wild-type UVRAG, followed
by IP with b-catenin and IB with indicated antibodies. (h) Co-IP of b-catenin with LC3 and TCF4 in the
colons from 18-month-old mice of indicated genotype (two randomly chosen samples per group; similar
results observed in all ten samples per genotype).
association with the autophagy marker proteins LC3-II (Figure 16k). Conversely,
overexpression of wild-type UVRAG or treating cells with the mTORC1 inhibitor
Torin 1, which reversed the decreased autophagy induced by UVRAG
FS
, promoted
b-catenin association with LC3-II and reduced b-catenin levels to those observed
in control cells (Figure 17g). These results support the concept that decreased
autophagy has a mechanistic role in UVRAG
FS
-associated b-catenin stabilization
and activation. We confirmed that these effects of autophagy suppression in vitro
on b-catenin regulation also occurred in vivo. In 18-month-old iUVRAG
FS
mice,
induced expression of UVRAG
FS
decreased autophagy and resulted in increased
levels of b-catenin (Figure 16m and Figure 17h). The reduced association of b-
catenin with autophagosomes in these mice was accompanied by increased
interaction with T-cell factor 4 (TCF4), leading to upregulation of the Wnt/b-catenin
signaling (Figure 16m and Figure 17h). Taken together, these results indicate that
the cancer-derived UVRAG
FS
mutant stabilizes b-catenin by inhibiting it
autophagic degradation, a mechanism that may contribute to spontaneous
tumorigenesis in UVRAG
FS
-expressing mice.

64
Chapter 4

Discussion
In this study, we demonstrate that a frameshift mutation in the autophagic tumor
suppressor UVRAG impairs starvation- and endotoxin-induced autophagy
resulting in increased inflammation and can promote age-related spontaneous
malignancies in mice. This study reveals that UVRAG has previously undescribed
roles in the in vivo regulation of starvation- and endotoxin-induced autophagy and
inflammation. Our discovery indicates that time-dependent autophagy suppression
by UVRAG
FS
and ensuant b-catenin stabilization/activation may serve as a tumor-
promoting mechanism in age-related cancer susceptibility.
The role for UVRAG in autophagy regulation, particularly the early stage of
autophagosome formation, has been debated
14,17,19,24,84
. Earlier studies
demonstrated that ectopic expression of UVRAG upregulates the levels of
autophagy by forming a complex with the core autophagy machinery Beclin1 and
PI3KC3
14,19,85
. Conversely, another study revealed that suppressing UVRAG in
cells had limited effects on autophagosome formation or autophagic flux
17
. Genetic
knockout of the mammalian UVRAG ortholog in yeast or Arabidopsis shows
controversial impact on starvation-induced autophagy
24,25,84
. Due to the embryonic
lethality of Uvrag knockout mouse, the exact in vivo role of mammalian UVRAG in
autophagy regulation remained largely unaddressed.
65
We generated an inducible UVRAG
FS
(iUVRAG
FS)
transgenic mouse model and
crossed it with GFP tagged LC3 mice to track autophagy. In this model, we
demonstrate that the dominant negative mutant, UVRAG
FS
, inhibits
starvation-induced autophagy in mice because Dox-induced iUVRAG
FS
mice
accumulate much fewer autophagosomes during starvation and display reduced
rates of autophagic flux as measured by the autophagy-mediated turnover of p62.
We also observed that lipophagy, lysosome-dependent degradation of
intracellular lipid droplets (LDs)
5
, is impaired in mice with UVRAG
FS
expression.
Electron microscopy image of the hepatocytes of starved control mice revealed
that LDs are primarily surrounded by autophagic vesicles. In contrast, enlarged
LDs can be found free in the cytoplasm in the hepatocytes of starved UVRAG
FS

mice, suggesting a failure of their targeting to lysosomal degradation. However,
we cannot definitely exclude other anti-autophagy effects of the UVRAG
FS
mutant
besides UVRAG inhibition.
Nonetheless, the decreased interaction of UVRAG with the Beclin1-Vps34
complex in vivo with increased interaction with Rubicon due to the truncating
mutation in UVRAG is consistent with the autophagy-promoting role of UVRAG
14,19
.
Our iUVRAG
FS
mice reveal that the effect of the UVRAG
FS
mutant on basal
autophagy is not as strong as that induced by starvation, implying that the UVRAG-
associated PI3KC3 activity is either not essential, or alternatively, other PI3KC3
complexes for instance that assembled with Atg14 is sufficient for the basal levels
of autophagy.
66
In addition to starvation, Dox-induced iUVRAG
FS
mice are impaired in
endotoxin-induced autophagy and more susceptible to death after LPS injection.
This increased susceptibility to endotoxin is associated with increased cell death,
caspase-1 activation, and caspase-1-dependent inflammatory cytokine production,
but not with elevated levels of NFkB activation. We isolated BMDMs from
iUVRAG
FS
mice, primed with LPS and treated with ATP, and found that UVRAG
FS
-
expressing cells have increased levels of mtROS production, mtDNA release, and
abnormal mitochondrial morphology decorated by Parkin. Given the emerging link
between mitophagy, mtROS, and NLRP3 inflammasome activation, we examined
whether the pathogenic alterations in mitochondrial homeostasis in UVRAG
FS
-
expressing cells contribute to aberrant NLRP3 inflammasome activation. The
NLRP3 inflammasome is responsible for caspase-1 dependent processing of
inflammatory cytokines, IL-1b and IL-18.
Primary Dox-induced iUVRAG
FS
BMDM primed with LPS and treated with ATP,
results in increased mtROS generation, NLRP3 inflammasome assembly and
caspase-1 cleavage which in turn is associated with hypersecretion of IL-1b. In
support, BMDM treated with MitoQ, a mtROS scavenger, significantly reduced
caspase-1 activation and IL-1b secretion. Mice expressing UVRAG
FS
show
consistent phenotypes with those observed in previous studies of mice with
deficiency of Atg16, LC3, or Beclin1 – all core components of the autophagy
pathway
8,9
. These results indicate that autophagy exerts a protective role during
inflammation by restricting caspase-1 overactivation and resultant cytotoxicity. It is
difficult to conclude whether UVRAG functions directly through mitophagy or
67
indirectly through other cellular factors in inflammatory response. However, the
diminished recruitment of Beclin1 to the TLR4-receptor complex and the
decreased UVRAG-Beclin1-Vps34 assembly/activity in Dox-induced iUVRAG
FS

mice during sepsis suggest that the autophagy function of UVRAG is intrinsically
required for mitochondrial homeostasis and adequate regulation of inflammasome
activation.
The role of UVRAG in inflammasome regulation may extend to other
inflammatory disorders. Dox-induced iUVRAG
FS
mice have enhanced
susceptibility to DSS-colitis displaying increased levels of IL-1b in colonic tissues,
correlating with disease severity. We show that selective blockade of NLRP3 with
MCC950 in iUVRAG
FS
mice confers a protective effect during acute colitis. This
is consistent with a model in which failure of UVRAG-mediated autophagy results
in hyperactivation of the inflammasome and enhanced secretion of IL-1b during
DSS-colitis. Intriguingly, we provide data that both hematopoietic and
nonhematopoietic UVRAG
FS
play a proinflammatory role in our colitis model. We
also observed that uncontrolled inflammation in iUVRAG
FS
mice is associated with
increased tumor growth and de-differentiation in the AOM-DSS-induced model of
colitis-associated colon cancer. These results suggest that UVRAG-mediated
autophagy plays an important role in inflammatory signaling and promotes anti-
inflammatory processes and tumor surveillance in the intestinal microenvironment.
In addition to tumor-promoting inflammation, cells deficient in autophagy-
related gene function show increased cancer incidence
86,87
. Loss-of-function
mutations on several essential autophagy genes result in spontaneous
68
tumorigenesis
87
. However, the precise molecular mechanisms underlying
autophagy-related tumor prevention remains elusive. Our iUVRAG
FS
mice allows
for whole body induction of UVRAG
FS
, resulting in the inactivation of UVRAG,
which leads to the premature onset of age-related neoplasia, particularly
spontaneous lymphoma independently of its role in inflammasome regulation.
Previous work has shown that human cancers especially those with
microsatellite instability show a global expression of the truncating UVRAG
FS

mutant compared with normal tissues
23
. Our work confirmed that this mutant can
promote tumorigenesis in vivo. We found that UVRAG
FS
expression enhances
age-related decline of autophagy, which stabilizes and thus increases the
abundance of b-catenin. These findings are also consistent with work showing the
autophagic clearance as a negative regulator of b-catenin levels in vitro
80
, while
extending its role in vivo and correlating age-related b-catenin accumulation with
increased tumorigenesis in iUVRAG
FS
mice. Of note, a previous study has shown
that increased Wnt impairs autophagic clearance
80
. This interrelationship may trap
cells in a deleterious cycle, whereby decreased autophagy promotes b-catenin
accumulation that then further suppresses autophagy, thereby additionally
increasing b-catenin retention. This sequence of events enhances cell proliferation
and may accelerate oncogenic transformation through the deregulation of b-
catenin target oncogenes (e.g. cyclind1, c-myc) in iUVRAG
FS
mice. More studies
are needed to determine whether this age-dependent regulation of b-catenin
observed in iUVRAG
FS
mice can be generalized to other autophagy-related tumor
models. Furthermore, considering the pleiotropic effects of UVRAG in cellular
69
homeostasis, we cannot rule out other pro-tumorigenic effects caused by UVRAG
inhibition other than the autophagy-Wnt/b-catenin pathway that may influence the
tumorigenic phenotype in iUVRAG
FS
mice. Nevertheless, the emerging facet of
oncogene-autophagy interactions highlight a functional role for autophagy, more
likely selective autophagy, in age-related diseases including cancer.

70
Acknowledgments

I would like to express my deepest gratitude to my mentor Dr. Chengyu Liang
for all of her support and providing me the opportunity to be a lab technician and
masters student in her lab. I greatly appreciate her teaching me how to conduct
scientific research as an independent scientist and always motivating me to be the
best I can. I would also like to thank my committee members Dr. Weiming Yuan
and Dr. Keigo Machida.
Most importantly I would like to thank Hongrui Guo and Ying Song for
conducting most of the biochemistry and molecular biology experiments. I would
like to thank Shun Li, Dr. Hadi Maazi, and Nathaniel Sands for helping me with the
bone marrow chimera experiments. I would like to thank to Douglas O’Connell,
Billy Chai, Marshall Fung and Dr. Nancy Wu for contributing to the creation of the
transgenic mice and their characterization. Thank you to Dr. Sara Restrepo-
Vassalli and Dr. James Hicks for the CNV analysis of the mice tumors. Thank you
to Dr. Sue Ellen Martin and Dr. Ashley Hagiya for help with IHC and pathological
analyses. Lastly, I would like to thank Dr. Vasu Punji, Dr. Omid Akbari, Dr. Gregory
E. Idos, Dr. Shannon Mumenthaler and Dr. Hengmin Cui for contributing their
expertise.

71
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Abstract (if available)

Abstract Aberrant autophagy is a major risk factor for inflammatory diseases and cancer. However, the genetic basis and underlying mechanisms are less well-established. UVRAG is a tumor suppressor candidate involved in autophagy, which is truncated in cancers by a frameshift (FS) mutation and expressed as a shortened UVRAG-FS. To investigate the role of UVRAG-FS in vivo, we generated mutant mice that inducibly express UVRAG-FS (iUVRAG-FS). These mice are normal in basal autophagy but deficient in starvation- and LPS-induced autophagy by disruption of UVRAG-autophagy complex. iUVRAG-FS mice display increased inflammatory response in sepsis and intestinal colitis, and colitis-associated cancer development through NLRP3-inflammasome hyperactivation. Moreover, iUVRAG-FS mice show enhanced spontaneous tumorigenesis related to age-related autophagy suppression and resultant β-catenin stabilization. Thus, UVRAG is a crucial autophagy regulator in vivo, and autophagy promotion may help prevent/treat inflammatory disease and cancer in susceptible individuals.

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Asset Metadata

Creator Quach, Christine (author)

Core Title A truncating mutation in the autophagy gene UVRAG drives inflammation and tumorigenesis in mice

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 07/31/2020

Defense Date 06/06/2019

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

Tag autophagy,frameshift,Inflammation,OAI-PMH Harvest,tumorigenesis,UVRAG

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Liang, Chengyu (committee chair), Machida, Keigo (committee member), Yuan, Weiming (committee member)

Creator Email cquach89@gmail.com,quachc@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-206151

Unique identifier UC11662762

Identifier etd-QuachChris-7716.pdf (filename),usctheses-c89-206151 (legacy record id)

Legacy Identifier etd-QuachChris-7716.pdf

Dmrecord 206151

Document Type Thesis

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Rights Quach, Christine

Type texts

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

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

Repository Name University of Southern California Digital Library

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