Developmental Pathways and Transcriptional Networks in Prostate Cancer Progression

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1 Developmental Pathways and Transcriptional Networks in Prostate Cancer Progression ASIP Cotran Established Investigator Award Lecture April 21, 2009 Carlos S. Moreno, Ph.D. Department of Pathology and Laboratory Medicine Emory University

2 Prostate Cancer Over 218,000 new cases and 27,000 deaths in US (2007) Second leading cause of cancer deaths in American men Gold Standard Pathology Gleason Score Gleason 6 Gleason 7 Gleason 8+ Excellent prognosis Prognosis uncertain Poor prognosis

3 Prostate Cancer Expression Profiling Identify Genes Correlated with Gleason Score Identify Genes Predictive of Outcome Identify Novel Therapeutic Targets

4 Expression Pattern of 143 Probe Sets Significantly Altered in Tumor vs. Normal Liu et al, Cancer Res 46:

5 Upstream Signals CONFAC Analysis Software Tx Factors Up/Down Gene Clusters Downstream Events Cellular Consequences Genetic Pathways GO Ontologies

6 CONFAC Analysis of Genes Increased in Prostate Cancer Identified Enrichment of Homeobox Sites Karanam and Moreno, NAR, 32:W475 W475 84, 2004.

7 HOXC6 gene was the most highly correlated with Gleason Score Spearman's Rho P Value Corrected p value E Ramachandran et al, Oncogene, 24(1):188, 2005.

8 HOXC6 sirna Transfection Decreases Viability of LNCaP and C4 2 2 Cells Ramachandran et al, Oncogene, 24(1):188, 2005.

9 HOXC6 Represses IGFBP3 Expression Ramachandran et al, Oncogene, 24(1):188, 2005.

10 Are these HOXC6 targets Direct or Indirect?

11 ChIP chip assay Global Analysis of TF Binding Requires a Highly Specific Antibody Buck and Leib, Genomics. 83(3):

12 Stable HOXC6 HA HA LNCaP Cell Lines McCabe et al, Cancer Research, 68(6): , 96, 2008.

13 NimbleGen 25K Promoter Array Set Queries 25,000 Promoters 4kb upstream 1kb downstream of TSS 50mer probes tiled every 150 bp

14 HOXC6 HA HA ChIP chip 468 Promoters Reproducibly Enriched in HOXC6 HA HA LNCaP cells over input chromatin (p( < 1.0E 3), But NOT in YFP LNCaP cells HOXC6 direct targets: BMP7 PDGFRA CARD8 PSEN1

15 ChIP chip Validation McCabe et al, Cancer Research, 68(6): , 96, 2008.

16 ChIP QPCR Validation McCabe et al, Cancer Research, 68(6): , 96, 2008.

17 HOXC6 Activates Expression of Luciferase Reporters McCabe et al, Cancer Research, 68(6): , 96, 2008.

18 What about HOXC6 Function in vivo? Hoxc6 / mice Demetri Spyropoulos, MUSC Whole Genome Expression Profiling Hoxc6 / prostates vs wild type prostates

19 HOXC6 Activates Expression of PDGFRα, FGFR2, DKK3, and WIF1 in mouse prostates McCabe et al, Cancer Research, 68(6): , 96, 2008.

20 WIF 1, DKK, and SFRP block Wnt Signaling Journal of Cell Science 116, , 2003.

21 HOXC6 Represses Expression of BMP7 in mouse prostates McCabe et al, Cancer Research, 68(6): , 96, 2008.

22 BMP7 Blocks Notch Signals and Prostate Cancer Metastasis Buijs et al, AJP 171(3):1047, 2007

23 Model of HOXC6 Network in Prostate Cancer McCabe et al, Cancer Research, 68(6): , 96, 2008.

24 Can Recombinant BMP7 Block the effects of HOXC6 overexpression? BMP7 inhibits pro proliferative proliferative and anti apoptotic apoptotic effects of HOXC6 overexpression McCabe et al, Cancer Research, 68(6): , 96, 2008.

25 Does inhibition of PDGFRα block pro proliferative proliferative effects of HOXC6 overexpression? McCabe et al, Cancer Research, 68(6): , 96, 2008.

26 HOXC6 activates Wnt Repressor Genes How Are WIF1, SFRP1, SFRP2, DKK3 Downregulated in Aggressive Cancers when HOXC6 is Overexpressed?

27 WIF1, DKK3, SFRP1, and SFRP2 Promoters are Hypermethylated in Prostate Cancers and Lung Metastases

28 Model of HOXC6 and Wnt Pathway

29 How can we better model HOX6 overexpression in vivo? Doxycycline inducible HOXC6 transgenic expression system Cross Probasin2 rtta x HOXC6 GFP 8 week old Pb2 rtta GFP male (4 weeks of Dox; 1 mg/ml) B, bladder; U, urethra; SV, seminal vessicle; lobes of the prostate: anterior (AL), dorsal (DL), lateral (LL), ventral (VL).

30 SOX4 SOX4 mrna is Strongly Correlated with Gleason Score Liu et al, Cancer Res 46:

31 SOX4 Protein is Strongly Correlated with Gleason Score Liu et al, Cancer Res 46:

32 SOX4 sirna Decreases Viability and Induces Apoptosis in LNCaP Cells Liu et al, Cancer Res 46:

33 What is SOX4? Highly conserved, single exon transcription factor Homology to TCF/LEF Family High Mobility Group (HMG) Binds AT rich sequences Induces extreme DNA bending Required for development of Heart, Pancreas, B cells, CNS, Bone DNA binding domain Prostate??? Overexpressed in many cancers (Prostate, Breast, Colon, Lung, Leukemia, Lymphoma, Medulloblastoma, Glioblastoma). Little functional molecular information exists

34 Effects of SOX4 Perturbation on Global Gene Expression Liu et al, Cancer Res 46:

35 The Wnt Pathway TLE1

36 SOX4 stably binds to β catenin in a Wnt3A dependent manner

37 SOX4 cooperates with β catenin to activate TOP FLASH Luciferase Reporters Scharer et al, Cancer Research, 69(2): , 2009.

38 SOX4 Direct Target Genes Play Roles in Survival, Apoptosis, and Wnt Signaling Scharer et al, Cancer Research, 69(2): , 2009.

39 SOX4 ChIP chip Analysis Create an HA tagged SOX4 cell lines LNCaP RWPE 1 Confirm SOX4 expression and presence at known target Scharer et al, Cancer Research, 69(2): , 2009.

40 SOX4 Transcriptional Targets 282 High Confidence Direct Targets Scharer et al, Cancer Research, 69(2): , 2009.

41 SOX4 ChIP chip Assay Validation 24/28 (86%) of candidate SOX4 targets were confirmed Scharer et al, Cancer Research, 69(2): , 2009.

42 SOX4 ChIP chip Target gene expression levels Are changed by QRT PCR Scharer et al, Cancer Research, 69(2): , 2009.

43 SOX4 ChIP chip Target gene expression levels Are changed by QRT PCR Scharer et al, Cancer Research, 69(2): , 2009.

44 The MicroRNA Pathway Jinek and Doudna, Nature, 457(7228):405, 2009 Robb and Rana, Molecular Cell, 26(4):523, 2007.

45 SOX4 Regulates Dicer expression Scharer et al, Cancer Research, 69(2): , 2009.

46 SOX4 Transcriptional Network Scharer et al, Cancer Research, 69(2): , 2009.

47 The Notch Pathway SOX4 and HOXC6 both Feed into the Notch Pathway Radtke, F., and K. Raj Nat Rev Cancer 3:

48 SOX4 and HOXC6 both Induce Notch 1 Cleavage

49 Dominant Negative SOX4 inhibits Wnt3a Induction of DLL1

50 SOX4 and HOXC6 Feed Into The PI3K AKT Pathway

51

52 SOX4 is important for metastasis SOX4 partial shrna knockdown reduces transwell invasion and lung metastases in MDA MB 231 Breast Cancer Cells

53 Prostate Cancer Biomarkers of Recurrence Long term Follow up data required Largest and best annotated samples are from Formalin Fixed Fixed Paraffin Embedded (FFPE) Tissues Standard Microarrays Cannot Use RNA Prepared from FFPE tissues Illumina DASL Assay Enables RNA profiling of FFPE samples

54 DASL assay (cdna NA mediated Annealing, Selection, extension and Ligation) Total RNA isolated from FFPE tissue b cdna synthesis P1 P2 Address b P3 Query oligo annealing, extension, and ligation P1 P2 P3 PCR with common primers Address /WV /WV Product capture by hybridization to array Yeakley et al. Nature Biotechnology. (2002) Fan et al. Genome Research. (2004)

55 Illumina DASL Assay Enables RNA profiling of FFPE samples Designed our own Custom DASL Prostate Cancer Gene Panel 1536 probes querying 522 genes Illumina human mirna v2 DASL panel Queries 1,146 human small RNAs (> 97% coverage of mirbase release 12) Profiled 85 radical prostatectomies 71 with long term (4 10 year) follow up 46 with Gleason Score 7

56 Clustering of Samples using Predictive Biomarkers

57 Combined Biomarker Panels (14 genes, all cases)

58 What regulates the changes in mirna levels?

59 Does SOX4 Directly Regulate mirnas associated with PCa recurrence?

60 SAM mirna Survival Analysis Top 6 mirnas from Cox Regression Analysis Recurrence vs. Non Recurrence Symbol Predicted Target Genes Score mir 103 BTG2, 3.51 mir 339 BCL mir 183 BTG mir 182 ITPR mir 221 FOS 3.02 mir 136 HOXC

61 BTG 2 Staining is Reduced in PCa

62 SOX4 is required for p53 activation in response to DNA damage SOX4 regulates p53 protein stability and activity SOX4 stabilizes p53 by blocking MDM2 ubiquitination of p53 SOX4 regulates cell cycle arrest, apoptosis, and tumorigenesis in a p53 dependent manner

63 SOX4 Directly Regulates PUMA Liu et al, Cancer Res 46:

64 Model of SOX4 mirna BTG Pathway

65 Summary SOX4 and HOXC6 Regulate Growth Factor Receptors SOX4 and HOXC6 Regulate Components of the Wnt Pathway SOX4 and HOXC6 Regulate Components of the Notch Pathway SOX4 and HOXC6 Regulate Components of the mirna Pathway

66 Acknowledgements Moreno Lab Dr. Colleen McCabe Noelani Anderson Christopher Scharer Yu Heng Lai Dr. Mohamed Ali Seyed Suresh Karanam Dr. Pengbo Liu Dr. Sumathi Ramachandran Holly Williams MUSC Demetri Spyropoulos University of Toronto Dr. Arun Seth Dr. Robert Nam Funding: NIH R01 CA Georgia Research Alliance Winship Cancer Institute Dept. Pathology & Lab Medicine Emory University / Winship Cancer Institute Dr. Brian Leyland Jones Dr. Wei Zhou Dr. John Petros Dr. Adeboye O Osunkoya Dr. Brent Johnson Daron Williams Dr. W. David Martin Dr. Andrew Young Dr. Fray Marshall Dr. David Jaye Dr. Milton Datta Emory Biomarker Service Center Dr. Mark Bouzyk Dr. Maja Ordanic Kodani Benjamin Barwick Nicholas Wagar Mingjing Xia