Consequences of ARID1A inactivation in bladder cancer · Consequences of ARID1A inactivation in...

Ryoichi Saito, MD, PhD

Kim Lab

Lineberger Comprehensive Cancer Center

University of North Carolina at Chapel Hill

Chapel Hill, NC

Consequences of ARID1A

inactivation in bladder cancer

8/11-13/2016 (Denver)

Gui, Nat Gen, 2011TCGA, Nature 2014

TCGA, Nature, 2014Gui, Nat Gen, 2011

ARID1A is frequently inactivated

in bladder cancer

SWI/SNF complex

Xiaofeng Wang, et al. Clin Cancer Res, 2013

SWI/SNF complex regulates a variety of signaling pathways

SWI/SNF complex activates DNA damage signaling

Cancer Discovery (2015)

Gabriele Sulli, et al.

Nature Reviews

Cancer (2012)

SWI/SNF complex has important roles in the

maintenance of genomic integrity

Brownlee, et al. DNA Repair (2015)

What is unknown?

• Does inactivation of ARID1a promote bladder carcinogenesis

in vivo?

– If so, how?

• At the transcription level?

• Impaired DNA damage response ?

• Increased chromosomal instability?

Mouse bladder

cancer model

Bladder

cancer

cell line

Normal

urothelial

cell line

Functional analysis

Human UC cell linesMouse UC cell lines

UC: Urothelial cancer = bladder cancer

Arid1a +/+ vs -/-

RNA-seq

Arid1a +/+ vs -/-

RNA-seq

Functional analysis

Normal urothelial cell lines

BBN in

drinking

water

Normal Bladder Cancer

Arid1a +/+

vs -/-BCPN

Liver

KAT or KT mice

Krt5-CreERT2;Arid1A (f/f or +/+); TdTomato mice (KAT or KT)

Umbrella cellIntermediate cell

Basal cell Krt5+Tamoxifen

Arid1a +/+ (KT)

Arid1a -/- (KAT)

Nat Cell Biol 2014

Nat Cell Biol 2014

Krt5+ cells contain the cellular origin of BBN-induced bladder cancer.

KAT or KT mice

Tamoxifen

5mg

week-1 0 8 10 12 14 16

Krt5-CreERT2;Arid1A (f/f or +/+); TdTomato mice (KAT or KT)

4

Carcinoma in situ

20

Invasive cancer

Umbrella cellIntermediate cell

Basal cell Krt5+Tamoxifen

Arid1a +/+ (KT)

Arid1a -/- (KAT)

Krt5+ cells contain the cellular origin of BBN-induced bladder cancer.

1227 KAT (= Arid1a -/-)12w10w

Bladder tumor formation was regularly monitored by ultrasound

1231 KT (= Arid1a +/+)

16w10w

Krt5-CreERT2;Arid1A (f/f or +/+); TdTomato (KAT or KT) mice

KAT and KT mice

Treated with BBN

continuously

Loss of Arid1a promoted carcinogenesis in a mouse bladder.

KT

KAT


KAT and KT mice

Loss of Arid1a promoted carcinogenesis in a mouse bladder.

At the step of initiation or progression?

Treated with BBN

continuously

KT

KAT


Tamoxifen BBN

Tamoxifen 5mg before BBN treatment

KT

KAT


Tamoxifen 5mg after BBN

treatment for 4week

Tamoxifen BBN BBN Tamoxifen

Loss of Arid1a promoted mouse bladder carcinogenesis when tamoxifen

was given before, but not after, BBN treatment.

Arid1a would have important roles in preventing initiation by BBN in

normal urothelial cells.

Tamoxifen 5mg before BBN treatment

KT

KAT

KT

KAT

Mouse bladder

cancer model

Bladder

cancer

cell line

Normal

urothelial

cell line

Functional analysis

Human UC cell linesMouse UC cell lines

UC: Urothelial cancer = bladder cancer

Arid1a +/+ vs -/-

RNA-seq

Arid1a +/+ vs -/-

RNA-seq

Functional analysis

Normal urothelial cell lines

Normal Bladder CancerBBN in

drinking

water

BCPN

Liver

Normal urothelial KT and KAT cell lines (= Tomato+ cells)KT1970 (+/+)

KAT1791 (-/-)

KT1970

KT1970

-Arid1aKO

KT1970

-Cas9

KT-1970

Ca

s9

Arid1a

Vinculin

Ari

d1

a-K

O

KT KAT

Isogenic Arid1a knockout cell line from KT1970

Epithelial island

Feeder cells

Epithelial island

Feeder cells

Transcriptome changes in normal cell lines Arid1a WT vs KO

KT-1970 (Arid1a WT)

KT-1970-KO (Arid1a KO)

N=3

N=3

SAM (FDR 0.00)

IPA

KT-1970 (Arid1a WT)

KT-1970-KO (Arid1a KO)

N=3

N=3

SAM (FDR 0.00)

IPA

Transcriptome changes in normal cell lines Arid1a WT vs KO

6 KT- and 4 KAT-

BBN cell lines

established

KT- and KAT-BBN cell lines (Bladder cancer cell lines)

IB:Arid1A

IB: Actin

11

50

12

31

12

91

12

96

13

53

13

54

12

27

12

87

12

92

12

94

+/+ f/f

KATBBN1227

Bright field TdTomato

KT-BBN KAT-BBN

Epithelial island

Feeder cells

Krt5+ cells are the cellular origin of BBN-induced bladder cancer

Transcriptome changes in cell lines from Arid1a WT and KO tumors

KT-BBN (Arid1a WT)

KAT-BBN (Arid1a KO)

N=6

N=4

SAM (FDR 0.00)

100 genes up

351 genes down

Pathway analysis suggests that Arid1a regulates genes involved in DNA damage repair

SAM (FDR 0.00)

100 genes up

351 genes down

BARDLTIPINFANCAFANCBPCNARAD51L1UBE2TXRCC2

WT KO

FANCONI pathwayKO/WTGSEATCGA_DDR

NER pathway MMR pathwayTCGA_DDR TCGA_DDR

Enrichment Score (ES) -0.6911

Normalized Enrichment

Score (NES)-1.6862

Nominal p-value 0.0

FDR q-value 0.0263

FWER p-Value 0.008



Score (NES)-1.617

Nominal p-value 0.02531

FDR q-value 0.03389

FWER p-Value 0.022



Score (NES)-1.5653137


FDR q-value 0.05021834

FWER p-Value 0.019



Score (NES)-1.5998


FDR q-value 0.0248

FWER p-Value 0.006



Score (NES)-1.65


FDR q-value 0.0208

FWER p-Value 0.001

Normal cell lineKT-1970

Ca

s9

Arid1a

VinculinA

rid

1a

-KO

#1

2

Loss of Arid1a did not significantly influence the sensitivity of normal cell lines to DNA damaging drugs.

Cancer cell line: KT1354 (KB6) isogenic cell lines

IB:Arid1A

IB: Vinculin

Cas9

#4 #11 #12 #13

Arid1a-CRISPR

Loss of Arid1a did not significantly influence the sensitivity of bladder cancer cell lines to DNA damaging drugs.

Pathway analysis suggests that Arid1a regulates genes involved in centromere maintenance

CENPICENPACENPQCENPL

WT KO

Front. Genet., 11 July 2013

Normal

Abnormal

Micronucleus = Chromosomal instability

BBN0.05% 16w BBN0.05% 16w

High grade, pTa Dysplasia

+ / + + / +KT-BBN1150 KT-BBN1231

Micronuclei50/365 (13.69%)

Micronuclei23/289 (7.9%)

KT- and KAT-BBN cell lines

Chromosomal instability is increased in Arid1a-dificient

mouse bladder cancer cell lines

KT- and KAT-BBN cell lines

Chromosomal instability is increased in Arid1a-dificient

mouse bladder cancer cell lines

Is it possible to reproduce this result in another settings?

Normal urothelial cells have enhanced chromosomal

instability when exposed to BBN in vivo

In vivo

Initiated urothelial cells

BBN4W

WT KO

Normal urothelial cells have enhanced chromosomal

instability when exposed to BBN in vivo or BCPN in vitro

In vitro In vivo

Normal cell line

BCPN for 28 days

BBN4W

WT KO

WT KOWT KO

Initiated cells

BCPN is the major metabolite of BBN

Conclusion

1. Loss of Arid1a promoted BBN-induced mouse bladder

tumorigenesis possibly at the step of tumor initiation.

2. We established normal mouse urothelial cell lines and BBN-

induced mouse bladder cancer cell lines by feeder cell culture

technique.

3. Loss of Arid1a increased chromosomal instability possibly

through incomplete DNA-damage response or inappropriate

chromosome segregation.

Future Direction

Transcriptome

?

ChIP-seq (SNF5)

Chromatin landscape

FAIRE-seq

Input samples

Acknowledgement

Kim Lab

Current members

– Jordan Kardos

– Janet Leung

– Xingnan Zheng

– Bhavani Krishnan

– Aleisha Smith

– Takanobu Utsumi

– Tracy Rose

– Jim Manocha

– William Y Kim

Former members

Harper Wilson

Jeffrey Damrauer

Sean Bailey

Bing Zhou

Hyo Jin Lee

UNC Flow cytometry core facility

– Evan Trudeau

Terry Magnuson (UNC)- Ron Chandler - Jesse Raab

Jen Jen Yeh(UNC)- Richard Moffitt- Kristin Voltzke

Young investigator award 2014

Consequences of ARID1A inactivation in bladder cancer · Consequences of ARID1A inactivation in...

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