Eva Turley London Regional Cancer Program London Health Sciences Center

1. Microenvironment control of prostate cancer by an unconventional protein RHAMM/HMMR Eva Turley London Regional Cancer Program London Health Sciences Center The University of Western Ontario

2. ECM ECM ECM ECM ECM ECM The extracellular matrix (ECM) is critical to cancer initiation and progression Chambers A.F. et al. Nat Rev Cancer. 2002; 2(8):563-572

3. A “remodelling” or “Inside-Outside” paradigm of tumor progression Oncogenic mutations and tumor microenvironment “collaborate” to coordinate Tumor progression Tumor microenvironment , particularly one that is remodelling (e.g. wounds) is likely dominant over mutations Outside inside

4. The transformed phenotype is dependent on signaling from the ECM EGFR, B1 integrin, cadherin 11 B4 integrin Tumorigenic and invasive MDA-MB-231 breast tumor cells [multiple oncogenic mutations (including H-Ras, p53 loss) and genomic instability] EGFR, B1 integrin, cadherin 11 B4 integrin Non-tumorigenic in culture and in vivo

5. - Initial signal might be Growth factors/receptors (e.g. ERK1,2) MAP kinases (e.g. ERK1,2) *H-RAS

6. One of the first demonstrations for a role of microenvironment in tumor progression was the demonstration that Hyaluronan:RHAMM interactions are necessary for Ras-transformation + HA/RHAMM -HA/RHAMM Foci formation is blocked by loss of HA/RHAMM interactions Tumor formation is blocked by loss of HA/RHAMM interactions Hall et al., 1995 Cell

7. We thought this happened because…….. Hyaluronan e.g. PDGF RHAMM But more on this later…… c-Src PKC FAK Ras MEK1,2 Erk1,2 RSK1,2 AP-1 (c-Jun/c-fos)

8. One of the functions of RHAMM is as a hyaluronan receptor and if this function • is ablated RHAMM shuts down the Ras transformation pathway • Wildtype RHAMM is oncogenic when hyperexpressed

9. RHAMM (also known as HMMR and CD168) IS: • a tumor antigen [use of RHAMM peptides in phase I clinical trials have been completed (Schmitt et al., Blood 111:1357-65)] • a novel breast cancer susceptability gene • associated with poor prognosis and enhanced peripheral metastasis in breast and other cancers • highly expressed in response-to-injury • not highly expressed during normal tissue homeostasis

10. Hyaluronan is a simple polysaccharide (Hascall and Laurent,Hyaluronan Today, Seikagaku Glycoforum Website)

11. Hyaluronan synthesis is de-regulated with tumor progression (Hyaluronan Today, Seikagaku Glycoforum Website)

12. For Hyaluronan, size is everything structural function Hyaluronidases, ROS, and Different HA synthases signalling function (Hyaluronan Today, Seikagaku Glycoform Website)

13. Hyaluronan fragments promote cell division and cell motility HA fragments

14. Hyaluronan fragments require RHAMM for binding to cells (Hascall and Laurent,Hyaluronan Today, Seikagaku Glycoforum Website)

15. Prostate cancer progression is driven, in part, by hyaluronan metabolism • Experimentally: • Hyaluronan synthases and RHAMM mRNA are increased at the G2M boundary • blocking hyaluronan fragment:prostate cancer cell interactions or inhibiting HAS/RHAMM expression arrests prostate cancer cell mitosis and inhibits invasion • Clinically: • Elevated levels of hyaluronan within primary prostate tumors is an independent negative prognostic indicator • high hyaluronan levels are associated with perineural infiltration, seminal vesicle invasion by tumors and PSA recurrence • An increased ratio of hyaluronidase 1:hyaluronan is an independent indicator of poor prognosis

16. The “Hyaluronome” in prostate cancer CD44 HA RHAMM Hyase1

17. RHAMM and CD44 are expressed in human prostate cancer cell lines PC3M-LN4 PC3M-LN4 CD44 protein expression LNCAP RHAMM protein expression

18. Both Hyaluronan receptors are required for tumor cell growth in 3D

19. Furthermore, hyaluronidase stimulates PC3M-LN4 growth……….. Hyaluronidase 1

20. RHAMM and CD44 co-localize through an HA bridge

21. ERK ERK ERK ERK Extracellular RHAMM and CD44 together promote prostate cancer progression HA CD44 CD44 endocytosis and HA metabolism Normal Prostate ERK Weak signaling RHAMM Lysosomal degradation RHAMM DIMERS HA fragments Prostate Cancer RHAMM MONOMERS AND DIMERS Strong signaling

22. We thought this happened because…….. Hyaluronan e.g. PDGF RHAMM But more on this later…… c-Src PKC FAK Ras MEK1,2 Erk1,2 RSK1,2 AP-1 (c-Jun/c-fos)

23. Anti-Rh Ab1 Anti-Rh Ab2 Non-immune IgG Non-immune IgG Harrison R, and Turley E Hyaluronan Today Seikagaku glycoforum website RHAMM occurs in multiple compartments and has at least dual functions e.g. Samuel SK et al., J Cell Biol 1993

24. However, RHAMM resembles a cytoplasmic protein ? No signal peptide ?

25. A conundrum?Or a novel form of inside-outside signaling? cell surface functions = invasion/motility e.g. Tolg et al 2006 J Cell Biol 175:1017-28 Mitotic spindle/centrosome functions = genomic instability e.g. Joukov et al., 2006 Cell 127:453-5

26. Transporter channels Protein-release complex Extracellular Extracellular Extracellular intracellular intracellular intracellular intracellular Flippase activity Transporter protein Phosphotidylserine Cytoplasmic protein Known mechanisms of unconventional protein export

27. Clotting Angiogenesis Inflammation Skin Excisional Wound Repair Re-epitheliallization Fibroplasia, Matrix Production

28. The gene signature of serum activated (e.g. wounded) fibroblasts predicts progression of some human cancers (e.g. Chang et al, 2005 Proceed. Natl. Acad Sci. USA)

29. 3000 2000 [HA] (ug/gm total protein+SE) 1000 0 0 2 8 24 48 96 168 unwounded skin h after wounding hyaluronan Injury Day 3 Collagen 1 and fibronectin Hyaluronan synthesis is consistently and transiently increased immediately after tissue injury

30. Wounded cells produce factors and remodel ECM e.g. Activated fibroblasts Extracellular matrix Production/remodelling wound repair Cytokines and growth factors

31. Strategy to identify wound and tumor specific genes • focus upon ECM remodeling events that are tightly • temporally regulated • select those that are de-regulated in tumorigenesis • simulate remodeling event in vitro • link analysis to this remodeling event

32. We isolated rapidly moving fibroblasts in culture Explanted tissue fragment Initial rapid migration out, and high hyaluronan production dividing 72 hr 24-36 hr Rapidly migrating fibroblasts slower moving fibroblasts tissue explant ANALYSIS of supernatant media

33. We isolated fibroblast hyaluronan binding proteins • hyaluronan sepharose affinity chromatography • monoclonal and polyclonal antibody preparation to isolated proteins • monoclonal and polyclonal antibodies screened for migration blocking • functions

34. Phage and Biotinylated HA-Streptavidin-Sepharose Growth of clones Phage and Biotinylated HA-Streptavidin-Sepharose Growth of clones Phage and Biotinylated HA-Streptavidin-Sepharose Growth of clones Clones releasedwithmedical grade HA We also isolated hyaluronan binding peptides using Recombinant phage display Wound hyaluronan isolated, purified and biotinylated HA binding (Isothermal calorimetry) Clones sequenced R. Savani (U. Pennsylvania) and Francoise Winnik (U. Montreal)

35. Acknowledgements Conny Toelg Fu-Sheng Wang Sara Hamilton Jenny Ma Sara Crump Qi Yang Collaborators Dr. Mina Bissell (Lawrence Berkeley National Laboratories) Dr. J. Koropatnick (London Regional Cancer Program) Dr. J. McCarthy (University of Minnesota) Dr. Len Luyt and Dr. T. Lee (Regional Cancer Program/Robarts)