The winner of the Anthony Dipple Carcinogenesis Award for 2016 is Dr. Michael Karin. View his presentation now.

1. NEW INSIGHTS to LIVER CANCER
DEVELOPMENT and TREATMENT
MICHAEL KARIN
UCSD SCHOOL of MEDICINE
La Jolla CA
Anthony Dipple Carcinogenesis Award

2. Obesity increases cancer risk,
especially HCC

3. Mortality trends of patients with different malignancies in the USA (1990–2009)
Clin. Cancer Res. 19, S4–S98 (2013).

4. Types of Liver Cancer

5. DEN Induced HCC:
8 Months
Dose= 25mg/kg i.p.
Vehicle or DEN
PBSDEN
14d old

6. TUMOR
INITIATION
Dead
cells
TUMOR
PROMOTION
Dead
cells
+ DEN
hepatocytes
KC
Mutation
Dead
cells
IL-6

7. Isolation of HCC Progenitor Cells (HcPC):
Karin M & Dhar D. Carcinogenesis 2016 Jun;37(6):541-6

8. CD44 is Upregulated in HcPC and HCCWT+PBSWT+DEN5mTak1Δhep
5m
IHC: Pan-CD44
Human HCCMouse
0
5
10
15
20
25
N
orm
alH
ep
N
on-H
cPC
H
cPC
H
C
C
CD44pan
mRNAFold

9. CD44 (+)
CD44 (-)
HCC
No HCC
Transplant to
Mup-uPA
Disperse
Only CD44(+) HcPCs Progress to HCC
He G*, Dhar D* et. al. Cell. 2013 Oct 10;155(2):384-
96.

10. Malignancy Markers Segregate with the CD44+ HcPC Population
CD44: More than just a stem cell MARKER in HCC

11. CD44: A Multi-Functional Cell-Surface Glycoprotein
 Adhesion Molecule
 Acts as a ligand binding receptor.
Ligands: HA, osteopontin, collagens
 Function as Co-receptor for EGFR, Met
 Function as a specialized “platform” for
assembling enzymes and
substrates….recruitment of MMP9,7
 C-Terminus associates with various
signaling molecules e.g., Src, Rho
GTPase, Rho Kinase, ERM (ezrin, radixin
and meosin) proteins.
! " ##$%&' ( ) %*( ++%) , &' ( -%. ( / ( &%, / 0%(
Embryonic stem (ES) cells are charac
expression of aset of transcription facto
! "#$%&'( ')!" # $$!%&' &!( ' ) !* +, -&.' !/-+01-0+&2!( !)!! " ##'*+, -"-.-'+/'-&0&%
+/'67"*7'1%&'*+, -.1, .'%&#"+, '&3+, -'.71.'1%&'$-&8'", '&0&%9'! " ##'5 : ; <'1,
?1%-@'1, 8'+.7&%-'1%&'01%"1, .'&3+, -'>%&8'?1%-@'.71.'1%&'$-&8'", '.7&'ABCC'01%"
1%&'-&2&*.&8'?9'12.&%, 1."0&'-=2"*", #D!3')'E315 =2&-'+/'12.&%, 1."0&29'-=2"*&8'A
'1')'F7&'ABCC'=%+.&", '"-'*+5 =+-&8'+/'1, '&3.%1*&22$21%'2", G'8+'5 1", 4'1'-.12GH2
&3.%1*&22$21%'8+5 1", '*2+-&'.+'.7&'.%1, -5 &5 ?%1, &'%&#"+, 4'67&%&'.7&'01%"1,
>%&8@'1%&'", -&%.&84'.7&'.%1, -5 &5 ?%1, &'%&#"+, '>FI @'1, 8'.7&'*9.+=21-5 "*'.1"
5 $2."=2&'-".&-'/+%'$H#29*+-921."+, '>?%+6, '*"%*2&-@'1, 8'%H#29*+-921."+, '>+%1
.6+'1*."0&'#29'*+-15 ", +#29*1, '>K<K@H?", 8", #'-".&-'>9&22+6'*"%*2&-@4'+, &'2+*
&3+, '=%+8$*.D'F7&'2", G'5 +8$2&'*+, '.1", -'.7&'?", 8", #'-".&'/+%'7912$%+, 1, '>M
*9.+=21-5 "*'.1"2'71-'?", 8", #'5 +."/-'/+%'*9.+-G&2&.12'2", G&%'=%+'.&", -4'1-'6&22
G", 1-&-D'E: I 4'&O%", 4'%18"3", '1, 8'5 +&-", P'NQN4'8"-$2=7"8&'?+, 8-D
Zoller M; Nature Reviews Cancer 11, 254-267 (April 2011)

12. CD44-/- Mice are Resistant to DEN Induced HCC
WTB6CD44-/-
DEN 9m

13. X CD44ΔHep
CD44f/f Alb-Cre
Hepatocyte Specific Deletion of CD44
DEN
CD44F/F mice from Dr. Jan Tuckermann, Germany
0
20
40
60
Tumors/Liver
**
n=10
n=16
C
d44F/F
C
d44ΔH
ep
Tumor Number
0
5
10
15
Diameter(mm)
C
d44F/F
C
d44ΔH
ep
Maximal Size

14. Central Vein
Hepatic
Vein
Portal
Vein
Hepatic
Artery
Zone I Zone II Zone III
DEN
Cell Death MutationIL-6
TNFa
HGF
TUMOR
DEN Induced Liver Cancer

15. P-p53
(Ser15)
Tubulin
50
50
DEN 24h
WT Cd44-/-
Tubulin
P-p53
(Ser15)
WT
0h 48h 0h 48h
Cd44-/-
50
50
P-p53 (Ser15)
Tubulin
50
50
0h 6h
WT Cd44-/-
WT Cd44-/-
DEN24hDEN0h
40μm
40μm
40μm
IHC: p53
W
T
C
d44
-/-
0
20
40
60
80
100
Positivecells/Field
ns
DEN 24h
40μm
WT Cd44-/-
DEN 48h
40μm 40μm
DEN48h
0
5
10
15
20
25
W
TC
d44-/-
Positivecells/Field
***

16. P-p53
(Ser15)
Tubulin
50
50
DEN 24h
WT Cd44-/-
Tubulin
P-p53
(Ser15)
WT
0h 48h 0h 48h
Cd44-/-
50
50
P-p53 (Ser15)
Tubulin
50
50
0h 6h
WT Cd44-/-
WT Cd44-/-
DEN24hDEN0h
40μm
40μm
40μm
IHC: p53
W
T
C
d44
-/-
0
20
40
60
80
100
Positivecells/Field
ns
DEN 24h
40μm
WT Cd44-/-
DEN 48h
40μm 40μm
DEN48h
0
5
10
15
20
25
W
TC
d44-/-
Positivecells/Field
***
0h
6h
24h
48h
0
200
400
600
800
1000
WT
CD44-/-
Serum ALT
Time post DEN Inj.
ALT(U/L)

17. Zone 1
Zone 3
Zone 3
Zone 1
CD44 InSITU Hybridization
24h post DENNo DEN (0h)
CD44 expression begins within 24h post DEN injection and is
localized to zone III (pericentral)

18. p53 Target Genes are More Potently Induced
in the Absence of CD44
WT Cd44-/-
p21
Tubulin
DEN 24h
1 2 3 1 2 3
25
50

19. Increased Damage in the CD44-/- Liver is
Prevented by either p21 or p53 Deletion
0h
48h
0
200
400
600
800
1000
p53ΔHep
Cd44-/-
p53F/F
Cre(+)
Cd44-/-
p53F/F
Cre(-)
ALT(U/L)
Time post DEN
***
***
0h
48h
0
200
400
600
800
1000
p21-/-
Cd44-/-p21-/-
Cd44-/-
Time post DEN
***
***

20. Deletion of Either p53 or p21 Rescues the CD44 Phenotype
0
10
20
30
Tumors/Liver
C
d44-/-p53ΔH
ep
p53ΔH
ep
/C
d44-/-
n=28
n=13
n=16
*** ns
***
0
10
20
30
Tumors/Liver
Cd44
-/-
p21
-/-
p21
-/- /Cd44
-/-
n=28
n=22
n=10
*
ns
***
Tumor Numbers Tumor Numbers

21. CD44 mediated p53 antagonism is critical for HCC
development.
In Summary…

22. Non-alcoholic steatohepatitis; NASH
HCC!!
Simple steatosis NASH
2nd hit?
NAFLD (non-alcoholic fatty liver disease)

23. MUP-uPA transgenic mouse;
urokinase-type plasminogen activator (uPA)
controlled by major urinary protein (MUP) promoter

24. Ballooning hepatocytes and bridging fibrosis in HFD
fed MUP-uPA mice
MUP MUP+HFDWT
Ballooning hepatocytes Oil Red O

25. Significantly accelerated tumorigenesis in HFD-fed MUP-uPA mice

26. HFD
MUP-uPA mice
TNFa↑
TNFR1
NFkB↑
TNFa
IL-6
Summary
TUNEL Ki67 Sirius RedHE
ERK↑
STAT3↑
S6↑
CCL2
CCL7
CXCL13
LTa LTb
HGF
JNK↑

27. WT
MUP MUP+HFD
WT+HFD
ROS accumulation in HFD fed MUP-uPA mice
DHE
staining
16 w

28. MUP-uPA
+ HFD
ER stress↑
Cell death↑
TNF↑
Kupffer cell
activation
Fibrosis↑
TGFb↑
PDGFb↑
Steatosis↑
Tumorigenesis ↑
Summary
NASH
or
Viral hepatitis + obesity
like environment
ROS
SREBP1↑

29. Hypernutrition
AMPK
TORC1
ATG1 ATG1- P
Autophagy
Lipid droplets p62
Hepatosteatosis Cancer ER stress

30. p62,the key component of Mallory Denk Bodies(MDB),
suppresses inflammation but promotes tumor development
InflammationCancer
PB1
Z
Z
TB LIR KIR UBAp62
Mitophagy
IL-1βNLRP3 inflammasome
Keap1
NRF2
Survival of oxidatively
stressed cancer-initiating
cells

31. HFD induces p62 accumulation only in NASH-prone mice

32. TSC1 Δhep mice
Sci. Signal., 27 March 2012
Vol. 5, Issue 217, p. ra24

33. p62 is needed for HCC development in Tsc1Δhep mice

34. p62 is required for NASH to HCC progression,
but not for hepatic steatosis

35. p62 activates the NRF2-dependent anti-oxidant response in
mouse liver and human HCC
Mouse liver Human HCC

36. By activating NRF2 p62 promotes survival of ROS-
accumulating HCC progenitor cells

37. A model
p62
Rescue of dying HCC
initiating cells from
oxidative stress
allowing mutations to
accumulate
KEAP1
NRF2
37

38. Kras
6 1 2
Kras
6 6
p53
2
Kras
6 6
p53
6
p16
To win the cancer
jackpot the same
tumor progenitor
needs to accumulate
multiple oncogenic
mutations, up to 11
in HCC!
How can this happen
,especially when
numerous tumor
suppressive
mechanisms are at
play?

39. p62 in non-tumor liver predicts HCC recurrence after
ablation therapy