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

ECM Chambers A.F. et al. Nat Rev Cancer. 2002; 2(8): The extracellular matrix (ECM) is critical to cancer initiation and 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 A “remodelling” or “Inside-Outside” paradigm of tumor progression inside Outside

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

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

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 Foci formation is blocked by loss of HA/RHAMM interactions Tumor formation is blocked by loss of HA/RHAMM interactions + HA/RHAMM -HA/RHAMM Hall et al., 1995 Cell

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

 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

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: ) ]  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

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

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

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

Hyaluronan fragments promote cell division and cell motility HA fragments

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

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

CD44 RHAMM HA Hyase1 The “Hyaluronome” in prostate cancer

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

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

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

RHAMM and CD44 co-localize through an HA bridge

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

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

Anti-Rh Ab1Anti-Rh Ab2 Non-immune IgG RHAMM occurs in multiple compartments and has at least dual functions e.g. Samuel SK et al., J Cell Biol 1993 Harrison R, and Turley E Hyaluronan Today Seikagaku glycoforum website

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

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

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

Re-epitheliallization Clotting InflammationAngiogenesis Fibroplasia, Matrix Production Skin Excisional Wound Repair

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)

[HA] (ug/gm total protein+SE) unwounded skin h after wounding Hyaluronan synthesis is consistently and transiently increased immediately after tissue injury Injury hyaluronan Collagen 1 and fibronectin Day 3

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

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

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

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

We also isolated hyaluronan binding peptides using Recombinant phage display Wound hyaluronan isolated, purified and biotinylated Clones sequenced HA binding (Isothermal calorimetry) R. Savani (U. Pennsylvania) and Francoise Winnik (U. Montreal)

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)