Microenvironment control of prostate cancer            by an unconventional protein           ...
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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. ECM. ECM. ECM. ECM.

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Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

-

Initial signal might be

Growth factors/receptors

(e.g. ERK1,2)

MAP kinases (e.g. ERK1,2)

*H-RAS


Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

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)


Eva turley london regional cancer program london health sciences center

  • 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


Eva turley london regional cancer program london health sciences center

  • 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


Eva turley london regional cancer program london health sciences center

Hyaluronan is a simple polysaccharide

(Hascall and Laurent,Hyaluronan Today,

Seikagaku Glycoforum Website)


Eva turley london regional cancer program london health sciences center

Hyaluronan synthesis is de-regulated with tumor

progression

(Hyaluronan Today,

Seikagaku Glycoforum Website)


Eva turley london regional cancer program london health sciences center

For Hyaluronan, size is everything

structural function

Hyaluronidases,

ROS, and

Different HA synthases

signalling function

(Hyaluronan Today, Seikagaku Glycoform

Website)


Eva turley london regional cancer program london health sciences center

Hyaluronan fragments promote cell division and cell motility

HA fragments


Eva turley london regional cancer program london health sciences center

Hyaluronan fragments require RHAMM for binding

to cells

(Hascall and Laurent,Hyaluronan Today,

Seikagaku Glycoforum Website)


Eva turley london regional cancer program london health sciences center

  • 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


Eva turley london regional cancer program london health sciences center

The “Hyaluronome” in prostate cancer

CD44

HA

RHAMM

Hyase1


Eva turley london regional cancer program london health sciences center

RHAMM and CD44 are expressed in human prostate cancer cell lines

PC3M-LN4

PC3M-LN4

CD44 protein expression

LNCAP

RHAMM protein expression


Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

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

Hyaluronidase 1


Eva turley london regional cancer program london health sciences center

RHAMM and CD44 co-localize through an HA bridge


Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

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)


Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

However, RHAMM resembles a cytoplasmic protein

? No signal peptide ?


Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

Clotting

Angiogenesis

Inflammation

Skin Excisional Wound Repair

Re-epitheliallization

Fibroplasia, Matrix

Production


Eva turley london regional cancer program london health sciences center

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)


Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

Wounded cells produce factors and remodel ECM

e.g. Activated fibroblasts

Extracellular matrix

Production/remodelling

wound repair

Cytokines and

growth factors


Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

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


Eva turley london regional cancer program london health sciences center

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)


Eva turley london regional cancer program london health sciences center

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)


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