Consequences of ARID1A inactivation in bladder cancer
Consequences of ARID1A inactivation in bladder cancer Ryoichi Saito, MD, PhD Kim Lab Lineberger Comprehensive Cancer Center University of North Caroli...
Consequences of ARID1A inactivation in bladder cancer Ryoichi Saito, MD, PhD Kim Lab Lineberger Comprehensive Cancer Center University of North Carolina at Chapel Hill Chapel Hill, NC
8/11-13/2016 (Denver)
ARID1A is frequently inactivated in bladder cancer TCGA, Nature 2014
Gui, Nat Gen, 2011
SWI/SNF complex
TCGA, Nature, 2014 Gui, Nat Gen, 2011
SWI/SNF complex regulates a variety of signaling pathways
Xiaofeng Wang, et al. Clin Cancer Res, 2013
SWI/SNF complex activates DNA damage signaling
Gabriele Sulli, et al. Nature Reviews Cancer (2012)
Cancer Discovery (2015)
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?
Normal
Bladder Cancer BBN in drinking water
Mouse bladder cancer model
Liver Arid1a +/+ vs -/-
BCPN
Normal urothelial cell line
Bladder cancer cell line
RNA-seq
RNA-seq
Arid1a +/+ vs -/-
Arid1a +/+ vs -/-
Functional analysis Normal urothelial cell lines
Functional analysis Human UC cell lines Mouse UC cell lines UC: Urothelial cancer = bladder cancer
Krt5-CreERT2;Arid1A (f/f or +/+); TdTomato mice (KAT or KT)
Nat Cell Biol 2014
KAT or KT mice Nat Cell Biol 2014
Umbrella cell
Basal cell
Intermediate cell
Krt5+
Arid1a +/+ (KT) Tamoxifen
Arid1a -/- (KAT)
Krt5+ cells contain the cellular origin of BBN-induced bladder cancer.
Krt5-CreERT2;Arid1A (f/f or +/+); TdTomato mice (KAT or KT)
Tamoxifen 5mg
Invasive cancer Carcinoma in situ
-1
0
4
8
10
12
14
16
KAT or KT mice
Umbrella cell
Basal cell
20 week
Intermediate cell
Krt5+
Arid1a +/+ (KT) Tamoxifen
Arid1a -/- (KAT)
Krt5+ cells contain the cellular origin of BBN-induced bladder cancer.
Bladder tumor formation was regularly monitored by ultrasound 1227 KAT (= Arid1a -/-) 10w
12w
1231 KT (= Arid1a +/+) 10w
16w
Krt5-CreERT2;Arid1A (f/f or +/+); TdTomato (KAT or KT) mice
KT
KAT and KT mice
KAT
Treated with BBN continuously
Loss of Arid1a promoted carcinogenesis in a mouse bladder.
Krt5-CreERT2;Arid1A (f/f or +/+); TdTomato (KAT or KT) mice
KT
KAT and KT mice
KAT
Treated with BBN continuously
Loss of Arid1a promoted carcinogenesis in a mouse bladder. At the step of initiation or progression?
Krt5-CreERT2;Arid1A (f/f or +/+); TdTomato (KAT or KT) mice Tamoxifen
BBN
KT KAT
Tamoxifen 5mg before BBN treatment
Krt5-CreERT2;Arid1A (f/f or +/+); TdTomato (KAT or KT) mice Tamoxifen
BBN
BBN
Tamoxifen KT
KT
KAT KAT
Tamoxifen 5mg before BBN treatment
Tamoxifen 5mg after BBN treatment for 4week
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.
Normal
Bladder Cancer BBN in drinking water
Mouse bladder cancer model
Liver BCPN Normal urothelial cell line
Bladder cancer cell line
RNA-seq
RNA-seq
Arid1a +/+ vs -/-
Arid1a +/+ vs -/-
Functional analysis Normal urothelial cell lines
Functional analysis Human UC cell lines Mouse UC cell lines UC: Urothelial cancer = bladder cancer
Normal urothelial KT and KAT cell lines (= Tomato+ cells) KT1970 (+/+)
Epithelial island KAT1791 (-/-)
Feeder cells Epithelial island
KT
KAT Feeder cells
Isogenic Arid1a knockout cell line from KT1970
Arid1a Vinculin
Arid1a-KO
Cas9
KT-1970 KT1970 -Cas9 KT1970
KT1970 -Arid1a KO
Transcriptome changes in normal cell lines Arid1a WT vs KO KT-1970 (Arid1a WT) N=3
KT-1970-KO (Arid1a KO) N=3
SAM (FDR 0.00)
IPA
Transcriptome changes in normal cell lines Arid1a WT vs KO KT-1970 (Arid1a WT) N=3
KT-1970-KO (Arid1a KO) N=3
SAM (FDR 0.00)
IPA
KT- and KAT-BBN cell lines (Bladder cancer cell lines) KATBBN1227
Epithelial island Feeder cells
TdTomato
Bright field
Krt5+ cells are the cellular origin of BBN-induced bladder cancer
IB:Arid1A
IB: Actin
KT-BBN
KAT-BBN
1294
1292
1287
1227
1354
1353
f/f 1296
1291
1231
+/+
1150
6 KT- and 4 KATBBN cell lines established
Transcriptome changes in cell lines from Arid1a WT and KO tumors KT-BBN (Arid1a WT) N=6
KAT-BBN (Arid1a KO) N=4
SAM (FDR 0.00) 100 genes up 351 genes down
Pathway analysis suggests that Arid1a regulates genes involved in DNA damage repair WT SAM (FDR 0.00) 100 genes up 351 genes down
KO BARDL TIPIN FANCA FANCB PCNA RAD51L1 UBE2T XRCC2
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 Cas9
Arid1a-CRISPR #4 #11 #12 #13
IB:Arid1A IB: Vinculin
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 WT
KO CENPI CENPA CENPQ CENPL
Normal
Abnormal
Micronucleus = Chromosomal instability Front. Genet., 11 July 2013
KT-BBN1150 + / + BBN0.05% 16w
KT-BBN1231 + / + BBN0.05% 16w
High grade, pTa
Micronuclei 50/365 (13.69%)
Dysplasia
Micronuclei 23/289 (7.9%)
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 KT- and KAT-BBN 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 WT
BBN4W
KO
Initiated urothelial cells
Normal urothelial cells have enhanced chromosomal instability when exposed to BBN in vivo or BCPN in vitro In vivo
In vitro
WT
WT
KO
WT
KO
KO
Normal cell line
BCPN for 28 days
BCPN is the major metabolite of BBN
BBN4W
Initiated cells
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 BBNinduced 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 Chromatin landscape
ChIP-seq (SNF5)
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
Jen Jen Yeh(UNC) - Richard Moffitt - Kristin Voltzke Terry Magnuson (UNC) - Ron Chandler Young investigator award 2014 - Jesse Raab UNC Flow cytometry core facility – Evan Trudeau
