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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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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 an unconventional protein

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


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


- ECM

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…….. microenvironment in tumor progression

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)



  • RHAMM (also known as HMMR and CD168) and if this function 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 and if this function

(Hascall and Laurent,Hyaluronan Today,

Seikagaku Glycoforum Website)


Hyaluronan synthesis is de-regulated with tumor and if this function

progression

(Hyaluronan Today,

Seikagaku Glycoforum Website)


For Hyaluronan, size is everything and if this function

structural function

Hyaluronidases,

ROS, and

Different HA synthases

signalling function

(Hyaluronan Today, Seikagaku Glycoform

Website)



Hyaluronan fragments require RHAMM for binding and if this function

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


The “Hyaluronome” in prostate cancer hyaluronan metabolism

CD44

HA

RHAMM

Hyase1


RHAMM and CD44 are expressed in human prostate cancer cell lines

PC3M-LN4

PC3M-LN4

CD44 protein expression

LNCAP

RHAMM protein expression



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

Hyaluronidase 1



ERK growth………..

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


We thought this happened because…….. growth………..

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)


Anti-Rh Ab1 growth………..

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


However, RHAMM resembles a cytoplasmic protein growth………..

? No signal peptide ?


A conundrum? growth………..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


Transporter channels growth………..

Protein-release complex

Extracellular

Extracellular

Extracellular

intracellular

intracellular

intracellular

intracellular

Flippase activity

Transporter protein

Phosphotidylserine

Cytoplasmic protein

Known mechanisms of unconventional protein export


Clotting growth………..

Angiogenesis

Inflammation

Skin Excisional Wound Repair

Re-epitheliallization

Fibroplasia, Matrix

Production


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)


3000 fibroblasts predicts progression of some human cancers

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


Wounded cells produce factors and remodel ECM fibroblasts predicts progression of some human cancers

e.g. Activated fibroblasts

Extracellular matrix

Production/remodelling

wound repair

Cytokines and

growth factors


Strategy to identify wound and tumor specific genes fibroblasts predicts progression of some human cancers

  • 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 fibroblasts predicts progression of some human cancersin 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


We isolated fibroblast hyaluronan binding proteins fibroblasts predicts progression of some human cancers

  • hyaluronan sepharose affinity chromatography

  • monoclonal and polyclonal antibody preparation to isolated proteins

  • monoclonal and polyclonal antibodies screened for migration blocking

  • functions


Phage and fibroblasts predicts progression of some human cancers

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)


Acknowledgements fibroblasts predicts progression of some human cancers

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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