MSO Young Investigator Award

Salvatore Venuta Young Scientist Prize

Rome, January 28, 2011

BAG3 protein as novel marker and therapy target in Glioblastoma

Alessandra Rosati, PhDDepartment of Pharmaceutical

SciencesDivision of Biomedicine “Arturo

Leone”University of Salerno

The BAG Family

BAG NLS WW UBL PXXP repeats

BAG1L

BAG1M

BAG1S

BAG2

BAG3

BAG4

BAG5

BAG6

345

274

230

211

575

457

447

1132

BAG3 protein and bag3 gene

BAG3 protein

bag3 gene

WW domain

Prolin-rich domain

BAG domain

Exons

Introns

575

25 52 304 417 421 4981

87 101 200 213

IPV motif

BAG3 expression in human adult tissues

Lee J., et al. 1999, Oncogene

BAG3 KO mice (1)

Homma S. et al. 2006, AJP

Experimental model References

Human leukaemia cell line (Molt-4) Tabucchi Y. et al. 2007

Retinas from Balb/c mice Chen L. et al. 2004

Human normal and neoplastic leukocytes Bonelli P. et al. 2004

Human epithelial cells from cervical carcinoma (HeLa) Pagliuca M.G. et al. 2003

Bortezomib-treated proximal tubular epithelial cells (PTC) Sarkozi R. et al. 2008

Neuroblastoma cells (SK-N-MC) Gentilella A. et al. 2008

Thyroid cancer cells Du Z.X. et al. 2008

Human epithelial cells from cervical carcinoma (HeLa) Franceschelli S. et al. 2008

Human colon cancer cells (HCT116), fibrosarcoma cells (HT1080), epithelial cells from cervical carcinoma (HeLa), breast cancer cells (MCF7), ovarian cancer cells (OVCAR8)

Wang H.Q. et al. 2008

Prostate epithelial cells (LNCaP) Ben Aicha S. et al. 2007

Pulmonary epithelium Wang X.Y. et al. 2007

Bu-resistant leukaemia cell lines (CML B5-AML KBM3) Valdez B.C. et al. 2008

Human colon cancer cells (HCT116) Zhou J. et al. 2008

Cardiac hypertrophy, cardiomyocytes Sakamoto M. et al. 2006

Chondrons and chondrocytes Zang Z. et al. 2006

HIV-infected microglia and lymphocytes Rosati et al., 2006

Regulation of bag3 expression by stressful

stimuli or drugs

Rosati A. et al. 2007, IJBCB

bag3 gene expression is regulated by Heat Shock Factor 1

GTCCCCTCCTTACAAGGAAAGTAATTTTGAAACAACCAATCCAGTCTTGGTTCTTTGTTTCTGCTTTCTTCAGCCCTTCTCTGTCTATAAAACCAACCTCTTCTGCTCAGCTCATGAGAGCACTTATTCTAATTTAATGGAGATGTTGCCCAATTTCTAGAATCACAAATAAGATCTTTAACAAAAGTTAAAAAATGAAAATCAACAACAACAACAACAAAAAAGATCTTTAAATGTGTTGTAATTTTCTCCTTTGACAGGTTACAAGTGCAATTGAATAAACCAAACAAAAAATGTCATGGTTATTCGGTCTTTACTTCCCAAATCAGGGTCAAGATATAAGATAGCAAGAACCAATCTCTACCATAAAAAAGATCCAGACCCAAGAAAATGTGATTTATCTTTAAAGCTGCAACGAAACTTTCTTTTTAGTCTAGGAGACTAAAAGCCATCAGTTGATACAATCTCTTTTTTTCTTGAAGTTTCCAAAGGAAGGCAGGTGAGTTTGCACTGACTTCCATTTTCTCCCTGGCTTCCTTCCGGTGTCAGATGAGAGTCCCCGCGCTCCTCCCGGACAGCCCTCAGGGTCCTCGGCTATCATATATGGGGGAGGGGGTGGAGGTGGGCGAGATGGTTTTGTTTGTACAGGAAGGACGGGCGGCAGGCACGGGGAGCACGGGCTCCGGCCCCGGGAACACTCACTCGGCGCAAAGGAGAGGGAGAGAGCGACTATGAAAGCCGAAACCCGCCCGGGGCGTACGGAGGACGGCCCCAGTCCAGCTCGTCCGGCTTCCCACCCGCAGGCGGACCCCGAAGCCGCGCCCGCGCCCGCCCGCGGCGACTCCGGCCCGGGCACGCGCACGAGTCGGAAGGGGGAGGGGCGGGAGGAGGTGGGGGAGGGGCGAGCGGGCCGAGGCCCCGCCCCCGCTCCTCTGCCCCGCCCGCCGCGATTATAGCCGATGACTCAGGGCGGAGCTCCGCATCCAACCCCGGGCCGCGGCCAACTTCTCTGGACTGGACCAGAAGTTTCTAGCCGGCCAGTTGCTACCTCCCTTTATCTCCTCCTTCCCCTCTGGCAGCGAGGAGGCTATTTCCAGACACTTCCACCCCTCTCTGGCCACGTCACCCCCGCCTTTAATTCATAAAGGTGCCCGGCGCCGGCTTCCCGGACACGTCGGCGGCGGAGAGGGGCCCACGGCGGCGGCCCGGCCAGAGACTCGGCGCCCGGAGCCAGCGCCCCGCACCCGCGCCCCAGCGGGCAGACCCCAACCCAGC

HSE

Sp1

Ap1

Franceschelli S. et al. 2007, JCP

BAG3 association with human primary tumour types

Experimental models References

Thyroid carcinomas Chiappetta G. et al. 2007

Acute lymphoblastic leukaemias

Romano M.F. et al. 2003b

B-chronic lymphocytic leukaemias

Romano M.F. et al. 2003a

Pancreatic carcinomas Liao Q. et al. 2001

Glioblastoma Festa M et al., submitted pending revision

Melanomas Franco R. et al. submitted paper

Experimental models References

Human primary lymphoid and myeloblastic leukaemias

Normal and neoplastic leukocytes

Acute lymphoblastic leukaemia cells

B-chronic lympocytic leukaemia cells

Human epithelial cells from cervical carcinoma (HeLa)

Rosati A. et al. 2007

Bonelli P. et al. 2004

Romano M.F. et al. 2003b

Romano M.F. et al. 2003a

Lee J.C. et al. 1999

Human melanoma cells (M14) Ammirante M. et al. 2010

Human and rat glioblastoma cells Festa M. et al., submitted pending revision

BAG3 is an anti-apoptotic protein

BAG3 regulates NF-kB activity

Ammirante M. et al. 2010, PNAS

IKK- is a target of BAG3 (1)

Ammirante M. et al. 2010, PNAS

Ammirante A. et al. 2010, PNAS

IKK- is a target of BAG3 (2)

Hsp70IKK-

Hsp70Proteasome

Hsp70

BAG3

IKK-IKK-

Rosati A. et al. 2007, IJBCB

IKK- is a target of BAG3 (3)

Human Glioblastoma

•Glioblastoma multiforme is the most aggressive and most frequent tumor of the brain

•Despite significant advances in neurosurgical techniques and aggressive multimodal treatments, the median survival for this tumor is approximately 56 weeks

•Therapies targeting a single pathway seem to lack clinical benefits

Normal brain Low Grade Astrocytoma Glioblastoma Multiforme

BA

G3

Anaplastic Astrocytoma

BAG3 is overexpressed in human Glioblastoma (1)

Pathology diagnosisN° of

samplesBAG3 negative

n (%) BAG3 low

positive n (%)BAG3 high

positive n (%)

Astrocytoma grade I 13 3 (23) 6 (46) 4 (31)

Astrocytoma grade II 85 6 (7) 43 (50.6) 36 (42.4)

Anaplastic Astrocytoma (grade III)

17 1 (5.9) 6 (35.3) 10 (58.8)

Glioblastoma Multiforme 36 4 (11.1) 9 (25) 23 (63.9)

BAG3 is overexpressed in human Glioblastoma (1)

% o

f B

AG

3 p

osit

ive c

ells P** VS grade I

P* VS grade II

BAG3 regulates apoptosis in rat glioblastoma cells

*

N°

of

via

ble

cells

X 1

00

cleaved caspase-3

a-tubulin

BAG3

a-tubulin

untr

ansf

ecte

dNTs

iRNA

BAG3s

iRNA

*

% o

f su

b G

1 c

ells

untr

ansf

ecte

dNTs

iRNA

BAG3s

iRNA

BAG3

a-tubulin

BAG3 regulates apoptosis in human glioblastoma cells

% o

f su

b G

1 c

ells

*

**

T98G

control CDDP

DBTRG-05MG

control CDDP

% o

f su

b G

1 c

ells

*

FBS

10%

FBS

1%BAX

BAD

-tubulin

BAX

BAD

HSP60

GAPDH

membrane

cytosol

BAG3s

iRNA

NTs

iRNA

BAG3s

iRNA

NTs

iRNA

BAG3 affects BAX subcellular localization and mitochondrial

membrane polarization

BAG3 effects on BAX are mediated by HSP70

BAG3

HSP70

BAX

Tota

l pro

tein

sPr

ot.A

Cont

rol I

gGs

IP:B

AX

GAPDH

HSP70

Tota

l pro

tein

s

Cont

rol I

gGs

BAG3s

iRNA

NTs

iRN

A

IP : BAX

An orthotopic glioblastoma model to study BAG3 knockdown effect in vivo

BAG3 knockdown affects glioblastoma tumor growth

NTsiRNA

Tu

mor

are

a (m

m2)

**

BAG3siRNA

BAG3 knockdown induce apoptosis in glioblastoma tumors

NTsiRNA

BAG3siRNA

Hem

ato

xyli

n &

Eo

sin

T

un

el A

ssay

Mechanisms for BAG3 anti-apoptotic activity

cell compartment

BAG3 HSP70

clientprotein

clientprotein

clientprotein

clientproteinclient

proteinenzyme

Proteasome

Acknoweldgements

Prof. Maria Caterina Turco

Prof. Maria PascaleProf. Liberato Marzullo

Dr. Michela FestaDr. Anna BasileDr. Morena d’AveniaDr. Antonia FalcoDr. Margot De Marco

Prof. Roberto ParenteProf. Raffaele D’AlessioDr. Michele Petrone

Dr. Claudio ArraAnimal Facility Istituto Pascale, NapoliDr. Antonio BarbieriDr, Marianna GalaDr. Giuseppe PalmaDr. Domenica ReaAntonio Luciano

Prof. Vincenzo De LaurenziUniversity of Chieti-Pescara

Prof. Kamel Khalili Dr. Luis Del ValleTemple University, Phila, PA

In fond memory of Prof. Arturo Leone