Recoding RNA editing of AZIN1 predisposes to

hepatocellular carcinoma

Leilei Chen, Yan Li, Chi Ho Lin, Tim Hon Man Chan, Daniel G Tenen,
Xin-Yuan Guan

Francisco Huizar
March 21, 2018
BIOS60532: RNA Editing
 Background
• RNA Editing: Any process, other than splicing, that results in a
change in the sequence of an RNA transcript and still produces a
functional transcript[1]

• Two types of deaminase RNA editing:

• C-to-U editing mediated by APOBEC enzymes
• A-to-I editing mediated by ADAR proteins

• A-to-I editing is the most frequent type of RNA editing in humans that
converts adenosine to inosine[1]
• End result: A-to-G point mutation

[1] Prosperi, J. (2019, March). RNA Editing. Lecture presented at the BIOS60532 Lecture, University of Notre Dame.
2
 Background
• Hepatocellular carcinoma (HCC) is the most common type of
primary liver cancer that is one of the fastest growing causes of death
in the US and makes up 65% of all liver cancers[2]

• Liver cancer has ~42,000 new cases per year with death rates
increasing by 3% each year since 2000 to an estimated ~30,000 per
year[3]

• Early detection has a low 5-year survival rate of 31%, dropping to

11% for mid-stage detection, and 3% after metastasis[4]

[2] Ghouri, Y. A., Mian, I., & Rowe, J. H. (2017). Review of hepatocellular carcinoma: Epidemiology, etiology, and carcinogenesis. Journal of Carcinogenesis, 16.
[3] Key Statistics About Liver Cancer. (n.d.). Retrieved March 16, 2019, from https://www.cancer.org/cancer/liver-cancer/about/what-is-key-statistics.html
[4] Liver Cancer - Statistics. (2012, June 25). Retrieved March 16, 2019, from https://www.cancer.net/cancer-types/liver-cancer/statistics 3
 Background
• Hepatocellular carcinoma (HCC) is the most common type of
primary liver cancer that is one of the fastest growing causes of death
in the US and makes up 65% of all liver cancers[1]
“It is often hard to find liver cancer early
• Liver cancersigns
because has ~42,000
and symptomsnew cases
oftenper
do year
not with death rates
appear by
increasing until
3%it each
is in its later
year stages.”
since 2000 to an estimated ~30,000 per
year[2]
“The American Cancer Society does not have
recommendations
• Early for liver
detection has a low cancer
5-year screening.”
survival rate of 31%, dropping to
11% for mid-stage detection, and 3% after metastasis[3]

[1] Ghouri, Y. A., Mian, I., & Rowe, J. H. (2017). Review of hepatocellular carcinoma: Epidemiology, etiology, and carcinogenesis. Journal of Carcinogenesis, 16.
[2] Key Statistics About Liver Cancer. (n.d.). Retrieved March 16, 2019, from https://www.cancer.org/cancer/liver-cancer/about/what-is-key-statistics.html
[3] Liver Cancer - Statistics. (2012, June 25). Retrieved March 16, 2019, from https://www.cancer.net/cancer-types/liver-cancer/statistics 4
Author’s Goals
 Author’s Goals
• Gap in knowledge: HCC development is thought to progress through
a multistep process of genetic and epigenetic alterations, BUT
pathogenesis at the molecular level is not well defined

• Specific Aim: In efforts to provide a better understanding of

tumor-initiating events in HCC at the molecular level, Chen et al.
utilized RNA-sequencing analysis to compare HCC tissue to normal
tissue

6
Methods
 Methods
• Two separate cohorts:
• Guangzhou (GZ), China: 135 paired human HCC and adjacent
nontumor tissues
• Shanghai (SH), China: 46 paired human HCC and matched
nontumor specimens
• None of the patients received chemotherapy or radiotherapy
prior to the study

• Liver cancer is much more common in sub-Saharan Africa and

Southeast Asia[3]
8
 Methods
• Four HCC cell lines:
• Hepatitis B Virus Positive Cell lines:
• QGY-7703
• PLC8024
• Hepatitis B Virus Negative Cell lines:
• HepG2
• H2M

• The most common risk factor for liver cancer is chronic infection
with HBV or HCV[5]

[5] Liver Cancer Risk Factors. (n.d.). Retrieved March 17, 2019, from https://www.cancer.org/cancer/liver-cancer/causes-risks-prevention/risk-factors.html
9
 Methods
• Real-time quantitative PCR: Extract RNA using Trizol for reverse transcription
to make cDNA for experiments
• Lentiviral transduction: for overexpression assays in cell lines
• RNA interference: Protein knockdown and rescue experiments using
short-hairpin RNA
• Western blot analysis: Observe relative protein abundance in cell lysates
• Immunohistochemistry: Observe distribution and localization of biomarkers in
paired tumor and non-tumor tissues
• In vivo tumorgenicity assay: Inject tumor cells into SCID mice and use
xenografts
• Fluorescence-activated cell sorting: Determine state of cell cycle progression
• Coimmunoprecipitation: Study protein-protein interactions
10
Results
 AZIN1 overediting is strongly associated with HCC
pathogenesis
• A) 63/135 patients in the GZ
cohort and 23/46 patients in the
SH cohort of the primary HCC
specimens had AZIN1
Rationale: “Through
overediting
genotyping validation of the
DNA samples, we found that AZIN1 had a high
• frequency
B) Frequency of nonsynonymous
of AZIN1 editing A→I transcript
in different liver specimens
editing, leading to a Ser→Gly amino acid
• C-E) substitution”
AZIN1 overediting in
tumors was significantly
correlated with liver cirrhosis,
tumor recurrence (and worse
prognosis 12
 AZIN1 overediting is strongly associated with HCC
pathogenesis
• A) 63/135 patients in the GZ
cohort and 23/46 patients in the
SH cohort of the primary HCC
specimens had AZIN1
overediting

• B) Frequency of AZIN1 editing

in different liver specimens

• C-E) AZIN1 overediting in

tumors was significantly
correlated with liver cirrhosis,
tumor recurrence (and worse
prognosis 13
 AZIN1 overediting is strongly associated with HCC
pathogenesis
• A) 63/135 patients in the GZ
cohort and 23/46 patients in the
of the primaryAZIN1
Takeaway:
SH cohort HCC editing frequency increases
specimens had AZIN1
during progression from cirrhosis and primary
overediting
liver cancer to advanced HCC with recurrence and
• metastasis;
B) Frequency of AZIN1 thus,
editingoverediting is strongly associated
in different
withliverHCC
specimens
pathogenesis
• C-E) AZIN1 overediting in
tumors was significantly
correlated with liver cirrhosis,
tumor recurrence ( and worse
prognosis 14
 ADAR1 is responsible for AZIN1 RNA editing in
human cancers
• A) p110 and p150 isoforms of
ADAR1 were upregulated in
Rationale: “On the
83% of the patients with HCC
basis of our RNA-Seq
analysis, the two ADAR1 transcript variants,
• encoding
B,C) AZIN1 110-kDa (p110) and 150-kDa (p150)
editing frequency
was positively correlated with the
isoforms, respectively, had relatively high
expression of ADAR1 but not
ADAR2 abundances in liver tissue.”

15
 ADAR1 is responsible for AZIN1 RNA editing in
human cancers
• A) p110 and p150 isoforms of
ADAR1 were upregulated in
83% of the patients with HCC

• B,C) AZIN1 editing frequency

was positively correlated with the
expression of ADAR1 but not
ADAR2

16
 ADAR1 is responsible for AZIN1 RNA editing in
human cancers
• D) AZIN1 editing in esophageal
(ESCC) Rationale: “To
tissues was higher investigate the role of ADAR1
than in
adjacent the
nontumor
AZIN1 (NT)editing
tissues regulation during cancer

• E) qPCRdevelopment, we also
ΔCT values for ADAR1 in determined the editing and
the HCC,expression of ADAR1
ESCC, and NPC tissues in two other types of
show higher expression
human in tumor
cancers, esophageal squamous cell
samples (lower ΔCT value)
carcinoma (ESCC) and nasopharyngeal carcinoma
(NPC).”

17
 ADAR1 is responsible for AZIN1 RNA editing in
human cancers
• D) AZIN1 editing in esophageal
(ESCC) tissues was higher than
adjacent nontumor (NT) tissues

• E) qPCR ΔCT values for ADAR1 in

the HCC, ESCC, and NPC tissues
show higher expression in tumor
samples (lower ΔCT value)

18
 ADAR1 is responsible for AZIN1 RNA editing in
human cancers
• D) AZIN1 editing in esophageal (ESCC) and
nasopharyngeal carcinoma (NPC) tissues were
Takeaway:
higher than These(NT)
adjacent nontumor data suggest
tissuesthat ADAR1 has a
major role in A→I (G) AZIN1 editing during
• E) qPCR ΔCT values for ADAR1 in the HCC,
cancer development for multiple cancers
ESCC, and NPC tissues show higher expression in
tumor samples (lower ΔCT value)

19
 ADAR1 is responsible for AZIN1 RNA editing in
human cancers
• F-H) Overexpression of
ADAR1 p110 but not ADAR2
led to a fivefold increase in
Rationale: “The nuclear
AZIN1 editing ADAR1 p110 isoform
and ADAR2 are expressed constitutively.
• I,J) Silencing ADAR1we
Therefore, in the
investigated whether the ADAR1
H2M cell line decreased
p110 isoform or ADAR2 is responsible for A→I
AZIN1 editing and was
rescuedediting of the AZIN1
by overexpressing an pre-mRNA.”
ADAR1 p110 mutant

20
 ADAR1 is responsible for AZIN1 RNA editing in
human cancers
• F-H) Overexpression of
ADAR1 p110 but not ADAR2
led to a fivefold increase in
AZIN1 editing

• I,J) Silencing ADAR1 in the

H2M cell line decreased
AZIN1 editing and was
rescued by overexpressing an
ADAR1 p110 mutant

21
 ADAR1 is responsible for AZIN1 RNA editing in
human cancers
• F-H) Overexpression of
ADAR1 p110 but not ADAR2
fivefold increaseStudies
led to aTakeaway: in of the expression of ADAR
AZIN1 family
editing members and functional studies support the
notion that the ADAR1 p110 isoform induces
• I,J) Silencing ADAR1 in the
H2M cellAZIN1 overediting
line decreased
AZIN1 editing and was
rescued by overexpressing an
ADAR1 p110 mutant

22
 AZIN1 RNA editing confers enhanced
tumorigenicity

Rationale: “To ascertain whether there is a

causative relationship between RNA editing and
gain-of-function phenotypes in cancer
development, we introduced V5-tagged wild-type
• AZIN1
A-C) Cells (wt/AZI)
transduced oredt/AZI
with the AZIN1
edited(edited) (edt/AZI)
lentivirus had accelerated growth
rates and higher frequencies
expression of focusinto
constructs and colony
two HCC formation
cellinlines
soft agar than cells
transduced with the wt/AZI (wildtype)
(PLC8024 and QGY-7703).”
• D) 8024-edt/AZI and 7703-edt/AZI cells had increased invasive capability,
consistent with AZIN1 overediting and tumor recurrence 23
 AZIN1 RNA editing confers enhanced
tumorigenicity

• A-C) Cells transduced with the edt/AZI (edited) lentivirus had accelerated growth
rates and higher frequencies of focus and colony formation in soft agar than cells
transduced with the wt/AZI (wildtype)

• D) 8024-edt/AZI and 7703-edt/AZI cells had increased invasive capability,

consistent with AZIN1 overediting and tumor recurrence 24
 AZIN1 RNA editing confers enhanced
tumorigenicity
• E-H) 8024-Edt3 cells,
harboring 85.3% edited
Rationale: “We compared the tumorigenic
AZIN1 transcripts, showed
properties
the most aggressiveof the wt/AZI transfectant (8024-Wt1)
with three edt/AZI transfectants (8024-Edt1,
phenotype.
8024-Edt2
• As little and 8024-Edt3) with different AZIN1
as an 18% increase
editing
in AZIN1 frequencies (24.8%, 42.9% and 85.3%,
editing
(8024-Edt1 compared to
respectively). ”
8024-Edt2 cells) was
sufficient to promote
tumorigenic potential
25
 AZIN1 RNA editing confers enhanced
tumorigenicity
• E-H) 8024-Edt3 cells,
harboring 85.3% edited
AZIN1 transcripts, showed
the most aggressive
phenotype.
• As little as an 18% increase
in AZIN1 editing
(8024-Edt1 compared to
8024-Edt2 cells) was
sufficient to promote
tumorigenic potential
26
 AZIN1 RNA editing confers enhanced
tumorigenicity
• E-H) 8024-Edt3 cells,
harboring 85.3% edited
Takeaway: These data demonstrate that
AZIN1 transcripts, showed
aggressive properties correlate with AZIN1
tumorigenic
the most
editing.
phenotype.
• As little as an 18% increase
in AZIN1 editing
(8024-Edt1 compared to
8024-Edt2 cells) was
sufficient to promote
tumorigenic potential
27
 AZIN1 editing contributes to augmented
tumor-initiating potential
• B) Mice xenograft showed
tumor incidence by
8024-edt/AZI cells was higher
than those induced by
8024-wt/AZI and 8024-LacZ
cells.

• C) Tumors induced by
8024-edt/AZI cells grew
significantly faster than those
induced by 8024-LacZ or
8024-wt/AZI cells
28
 AZIN1 editing contributes to augmented
tumor-initiating potential
• D-F) 4 weeks after
intrahepatic inoculation,
mice injected with
8024-edt/AZI cells formed
more liver nodules

29
 AZIN1 editing contributes to augmented
tumor-initiating potential
• D-F) 4 weeks after
intrahepatic inoculation,
Takeaway: All these data demonstrate that this
mice injected with
AZIN1 cells
8024-edt/AZI editing has an important role in tumor
formed
initiation.
more liver nodules

30
 RNA editing of AZIN1 changes subcellular
localization
• A) GFP-tagged wild-type
AZIN1 protein (GFP-wt/AZI)
was expressed in the cytoplasm,
and the majority of the edited
protein (GFP-edt/AZI) was
expressed in the nucleus

• B) Western blot of GFP-tagged

wild-type and edited AZIN1
protein in total lysate (TL) and
cytoplasmic (Cyto) and nuclear
(Nuc) fractions
31
 RNA editing of AZIN1 changes subcellular
localization
• A) GFP-tagged wild-type
AZIN1 protein (GFP-wt/AZI)
Takeaway:
was expressed All these data indicate that the AZIN1
in the cytoplasm,
and the majority
editingofmaythe edited
result in a cytoplasmic-to-nuclear
protein (GFP-edt/AZI) was
expressedtranslocation
in the nucleus

• B) Western blot of GFP-tagged

wild-type and edited AZIN1
protein in total lysate (TL) and
cytoplasmic (Cyto) and nuclear
(Nuc) fractions
32
 RNA editing of AZIN1 changes subcellular
localization
• C) Immunohistochemistry showed
cytoplasmic-to-nuclear translocation of AZIN1 in
5/14 patients with HCC with AZIN1 overediting

• D) Predicted conformational switch regions of

the AZIN1 protein as a result of editing

33
 Edited AZIN1 neutralizes antizyme-mediated
degradation of target oncoproteins
• A) PLC8024 cells transfected
with GFP-edt/AZI could pull
Rationale: “Antizyme-1 inhibits cell growth by
down more antizyme-1 protein
binding to and inducing degradation of proteins
than GFP-wt/AZI
such as ODC. AZIN1 is an ODC homolog and
shares its capacity to bind to antizyme. We then
investigated whether AZIN1 editing could affect
the binding affinity of AZIN1 protein to
antizyme.”

34
 Edited AZIN1 neutralizes antizyme-mediated
degradation of target oncoproteins
• A) PLC8024 cells transfected
with GFP-edt/AZI could pull
down more antizyme-1 protein
than GFP-wt/AZI

35
Edited AZIN1 neutralizes antizyme-mediated
degradation of target oncoproteins

Takeaway: This suggests that edited AZIN1 had

a higher binding affinity to antizyme-1 than did
wild-type AZIN1. Less antizyme, means more cell
growth.

36
 Edited AZIN1 neutralizes antizyme-mediated
degradation of target oncoproteins
• C-D) Increased binding
affinity to antizyme allowed
edited AZIN1 to be more
stable than wild-type

• Degradation of ODC and

CCND1 was reduced in cells
expressing GFP- or V5-tagged
edited AZIN1 compared to
wild-type

37
 Edited AZIN1 neutralizes antizyme-mediated
degradation of target oncoproteins
• C-D) Increased binding
affinityTakeaway:
to antizyme allowed
AZIN1 editing confers greater
edited AZIN1 to be more
stability to the AZIN1 protein as a result of
stable than wild-type
the
higher affinity of the edited form toward antizyme
• Degradation of ODCto
compared andthe wild-type form
CCND1 was reduced in cells
expressing GFP- or V5-tagged
edited AZIN1 compared to
wild-type

38
 Edited AZIN1 neutralizes antizyme-mediated
degradation of target oncoproteins
• E) The percentage of
xeno-edt/AZI cells in S
phase was consistently
higher than that of
xeno-wt/AZI cells at 6 h
and 9 h after release

39
 Edited AZIN1 neutralizes antizyme-mediated
degradation of target oncoproteins
• At the G1/S boundary,
phosphorylation of Rb activates
downstream targets CCNA,
Cdc2, and c-Myc23

• F-G) xeno-edt/AZI cells had

elevated expression of ODC
and CCND1, leading to
increased Rb phosphorylation
and CCNA upregulation

40
 Edited AZIN1 neutralizes antizyme-mediated
degradation of target oncoproteins
• F-G) xeno-edt/AZI cells had
elevated Takeaway:
expression of ODCThese data suggest that there is a tight
and CCND1, leading to
connection between the edt/AZI-ODC or -CCND1
increased Rb phosphorylation
and CCNA cellupregulation
proliferation axis and tumor initiation and
•
progression.
At the G1/S boundary,
phosphorylation of Rb activates
downstream targets CCNA,
Cdc2, and c-Myc23

41
Conclusions
 Conclusions
• AZIN1 overediting is strongly associated with HCC pathogenesis

• ADAR1 directs A→I AZIN1 RNA editing

• AZIN1 RNA editing confers enhanced tumorigenicity

• AZIN1 editing contributes to augmented tumor-initiating potential

and enhanced in vivo tumorigenic ability

43
 Conclusions
• RNA editing of AZIN1 changes subcellular localization and protein
conformation

• Edited AZIN1 neutralizes antizyme-mediated degradation of target

oncoproteins in vitro and in vivo

• Collectively, A→I RNA editing of AZIN1 may be a potential driver in the

pathogenesis of HCC

• How would you use this as a screening tool or create a novel therapeutic?
44
Discussion Questions
 Discussion Questions
• Were the authors successful in completing their specific aim? Successful
in advancing the field?

• Are there any limitations you can see in the study, if so, how would you
improve them? Were there any gaps in their methodology?

• The paper was published in 2013, are there any new techniques you would
use to re-do or confirm the experiments done here?

46
Thank You!