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
The extracellular matrix (ECM) is critical to cancer initiation and progression
ECM
ECM
ECM
ECM
ECM
ECM
Chambers A.F. et al. Nat Rev Cancer. 2002; 2(8):563-572
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
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
Initial signal might be
Growth factors/receptors
(e.g. ERK1,2)
MAP kinases (e.g. ERK1,2)
*H-RAS
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
We thought this happened because……..
Hyaluronan
e.g. PDGF
RHAMM
c-Src
PKC
FAK
Ras
MEK1,2
Erk1,2
RSK1,2
AP-1
(c-Jun/c-fos)
But more on this later……
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 111:1357-65) ]
 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
Hyaluronan is a simple polysaccharide
Chemical structure
of hyaluronan
Computer model of
hyaluronan
(Hascall and Laurent,Hyaluronan Today,
Seikagaku Glycoforum Website)
Hyaluronan synthesis is de-regulated with tumor
progression
(Hyaluronan Today,
Seikagaku Glycoforum Website)
For Hyaluronan, size is everything
structural function
signalling function
(Hyaluronan Today, Seikagaku Glycoform
Website)
Hyaluronidases,
ROS, and
Different HA synthases
Hyaluronan fragments promote cell division and cell motility
HA fragments
Hyaluronan fragments require RHAMM for binding
to cells
Chemical structure
of hyaluronan
Computer model of
hyaluronan
(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
The “Hyaluronome” in prostate cancer
CD44
RHAMM
HA
Hyase1
RHAMM and CD44 are expressed in human prostate cancer cell lines
PC3M-LN4
PC3M-LN4
CD44 protein expression
LNCAP
RHAMM protein expression
Both Hyaluronan receptors are required for tumor cell growth in 3D
Furthermore, hyaluronidase stimulates PC3M-LN4 growth………..
Hyaluronidase 1
RHAMM and CD44 co-localize through an HA bridge
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
Prostate Cancer
HA fragments
RHAMM
MONOMERS
AND DIMERS
ERK ERK
ERK ERK
Strong signaling
We thought this happened because……..
Hyaluronan
e.g. PDGF
RHAMM
c-Src
PKC
FAK
Ras
MEK1,2
Erk1,2
RSK1,2
AP-1
(c-Jun/c-fos)
But more on this later……
RHAMM occurs in multiple compartments and has at least
dual functions
Anti-Rh Ab1
Anti-Rh Ab2
Non-immune IgG
Non-immune IgG
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?
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
Known mechanisms of unconventional protein export
Protein-release complex
Transporter channels
Extracellular
Extracellular
intracellular
Flippase activity
Extracellular
intracellular
Transporter protein
Phosphotidylserine
Cytoplasmic protein
intracellular
intracellular
Skin Excisional Wound Repair
Re-epitheliallization
Clotting
Fibroplasia, Matrix
Production
Angiogenesis
Inflammation
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)
Hyaluronan synthesis is consistently and transiently
increased immediately after tissue injury
3000
2000
1000
0
0
2
8
24
48
96 168
h after wounding
hyaluronan
Injury
Day 3
Collagen 1 and fibronectin
Wounded cells produce factors and remodel ECM
e.g. Activated fibroblasts
Extracellular matrix
Production/remodelling
wound repair
Cytokines and
growth factors
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
We isolated rapidly moving fibroblasts in culture
Explanted tissue
fragment
Initial rapid migration out, and
high hyaluronan production
dividing
24-36 hr
tissue explant
72 hr
Rapidly migrating
fibroblasts
ANALYSIS of supernatant media
slower
moving
fibroblasts
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
We also isolated hyaluronan binding peptides using
Recombinant phage display
Wound hyaluronan isolated,
purified and biotinylated
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
releasedw
ith
medical
grade HA
HA binding (Isothermal calorimetry)
Clones sequenced
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)