Epigenetic Regulation of HOXB13 in ì Castration Resistant Prostate Cancer
Synergistic Targeting of Androgen Receptor Circuits in Metastatic Prostate Cancer
Kiran Mahajan Ph.D. TB and GU Departments
HOXB13 Learner’s Objectives
1. The Androgen Receptor (AR) is expressed and func onal in a majority of lethal castra on resistant prostate cancers (CRPCs). 2. An important AR co-regulator in CRPCs is the homeodomain containing sequence specific transcrip on factor, HOXB13. 3. The expression of HOXB13 is epigene cally regulated by the BET bromodomain protein, BRD4. Introduction
• HOXB13 is cri cal for the differen a on of the ventral lobe of the prostate gland in mice. • In human cancers, HOXB13 expression correlates with advanced pT stage, high Gleason grade, posi ve lymph node status, high pre-opera ve PSA levels, TMPRSS2:ERG fusion, PTEN dele ons, AR expression, cell prolifera on and early PSA recurrence. • Co-expression analysis iden fied a subset of tumors with high HOXB13 and AR but low PSA expression that had a par cularly poor prognosis. • The epigene c mechanism by which HOXB13 is regulated in AR posi ve prostate cancers is not known. Mechanism of Action of HOXB13
Norris et al, Molecular Cell, 2009 HOXB13-A critical regulator of CRPC growth
n 2.0 Parental HOXB13pKO io C4-2B
ress C4-2B HOXB13 pKO 1
p 1.5 C4-2B ex HOXB13 C4-2B HOXB13 pKO 2 1.0 RNA m in t 0.5 c Actin A / e n
e 0.0 g HOXB13 C4-2B
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2000 **** C4-2B Intact
1000 HOXB13 pKO Intact
0 Tumor weights (mgs) weights Tumor ** C4-2B Castrated
C4-2B C4-2B HOXB13p KO Castrated HOXB13 pKO HOXB13 pKO Castrated Intact HOXB13 direct targets in PC HOTPAM9-Stratification of prostate adenocarcinomas from metastatic prostate cancers Validation of HOTPAM9 in Metastatic Prostate Cancer
C4-2B Control siRNA 8 7 6 C4-2B HOXB13 siRNA 5 4 CIT 1.0 0.8 0.6
0.4 BUB1
GENE/ACTIN mRNA 0.2 0.0 CIT NUF2 NEK2 BUB1 c-Myc MKI67 HSPB8 TRPM8 AURKA NCAPG HOXB13 TRPM8
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NCAPG BRD4 epigenetically regulates HOXB13 expression in PC
HOXB13 gene BRD4 ChIP t 0.3 IgG ChIP ChIP npu
anti-BRD4 I f
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DMSO % 0.1 JQ1 ChIP 0.0 anti-IgG 1 4 A R JQ MA AB Exon2 Exon1 DMSO ENZ C4-2B 6
A EGF IGF HRG 1.5 - - + - + - + BET inhibitor 4 Control siRNA A IP: HOXB13 HOXB13 siRNA 1.0 IB: AR * BRD4 siRNA 2 IB: HOXB13
0.5 IB: AR HOXB13/ACTIN mRN ** ** **
0 HOXB13/ACTIN mRN DMSO 0.1 0.5 1 2.5 uM 0.0 IB: Actin HOXB13 JQ1 Detection of HOXB13 in CTCs
A. VCAP cells No Ab control B. VCAP cells plus 5ug/ml HOXB13 Alexa Fluor 488 Ab DAPI/CK-PE CK-PE DAPI CD45-APC HOXB13 FITC DAPI/CK-PE CK-PE DAPI CD45-APC HOXB13 AF488
C. VCAP cells plus 5ug/ml HOXB13 FITC Ab D. VCAP cells plus 5ug/ml Alexa Fluor 488 IgG1 Ab
DAPI/CK-PE CK-PE DAPI CD45-APC HOXB13 FITC DAPI/CK-PE CK-PE DAPI CD45-APC IgG1 AF488 Non-invasive Detection of HOXB13 CTCs
HOXB13+ HOXB13-
40 DMSO CTCs % of Condi o Total Unassigne JQ1 s
total total n % n % t 30
n Events d events n (n) events ve E
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% 10
0 DMSO 1987 1310 677 34% 184 27 493 73 DMSO JQ1 DMSO JQ1 %CTCs %HOXB13+ve
JQ1 1386 1323 63 5% 9 14 54 86 ì Summary
1. BRD4, is an epigene c regulator of HOXB13 expression. 2. HOXB13 expression can be targeted in prostate cancers with BET bromodomain inhibitors such as JQ1. 3. We iden fied a subgroup of mito c kinases as key transcrip onal targets of HOXB13-a target gene signature termed as HOTPAM9 (HOXB13 Target genes separa ng Prostate Adenocarcinomas from Metastasis) in CRPCs. .
Acknowledgements
K. Mahajan lab Biostats Brent Kuenzi Dr. Sellers Neha Agarwal Yunyun Chen Ernst Schonbrunn Dr. Cleveland Duy Nguyen William Ma Nick Lawrence Dr. Chellappan Past members Youngchul Kim Harshani Lawrence Dr. Julio Pow-Sang Niveditha N. Jiqiang Yao Mohammed Ayaz Dr. Conor Lynch Ami Patel Jamie Teer Steven Gunawan John Vicente (SPARK) Comparative Medicine Uwe Rix Funding Samina Ismail (SPARK) Devon DeLoach Nupam Mahajan PC141124, PC141298 CDMRP, Department of Drug Discovery Department of Anatomic GU Physicians Defense Brent Kuenzi Pathology Jingsong Zhang Moffitt Support Account Ernst Schonbrunn Jasreman Dhillon Michael Poch Nick Lawrence Domenico Coppola Administration Steven Gunawan Anthony Magliocco Rose Reyes Uwe Rix Sanford Burnham Kimberly Heffner Translational Core Prebys Institute Chemical Biology Core John Puskas Ranjan Perera Harshani Lawrence Subbu S Mohammed Ayaz Alexey Eroshkin Drug Discovery Bioinformatics and Special Thanks References
1. Pomerantz, M. M. et al. The androgen receptor cistrome is extensively reprogrammed in human prostate tumorigenesis. Nature gene cs 47, 1346-1351, doi: 10.1038/ng.3419 (2015). 2. Frieling, J. S., Basanta, D. & Lynch, C. C. Current and emerging therapies for bone metasta c castra on-resistant prostate cancer. Cancer control : journal of the Moffi Cancer Center 22, 109-120 (2015). 3. Grasso, C. S. et al. The muta onal landscape of lethal castra on-resistant prostate cancer. Nature 487, 239-243, doi:10.1038/nature11125 (2012). 4. Mallo, M. & Alonso, C. R. The regula on of Hox gene expression during animal development. Development 140, 3951-3963, doi:10.1242/dev.068346 (2013). 5. Shah, N. et al. HOXB13 mediates tamoxifen resistance and invasiveness in human breast cancer by suppressing ERalpha and inducing IL-6 expression. Cancer research 73, 5449-5458, doi:10.1158/0008-5472.CAN-13-1178 (2013). 6. 12 Ylitalo, E. B. et al. Subgroups of Castra on-resistant Prostate Cancer Bone Metastases Defined Through an Inverse Rela onship Between Androgen Receptor Ac vity and Immune Response. European urology, doi:10.1016/j.eururo.2016.07.033 (2016). ì Thank you for your a en on! Post-Test/Questions
Which is a key epigene c regulator of HOXB13 expression in prostate cancer cells that can be targeted with the prototype inhibitor, JQ1?
1. AKT 2. BRD4 3. c-MYC 4. FOXA1