II. YB-1 knockdown induces apoptosis or G0/G1 cell cyclearrest, cisplatin sensitisation and impedes the migrationof MPM cells.Transfection of YB-1 siRNA induced (A) apoptosis or (B) an increasein G0/G1 population in MPM cells. (C) Cells undergoing apoptosisafter YB-1 downregulation were sensitised to cisplatin, one of the mostcommonly prescribed drugs to patients suffering this disease, while aslight increase in resistance was seen in cells undergoing cell cyclearrest. (D) YB-1 silencing significantly inhibited MPM cell migration.

A

C

BackgroundMalignant pleural mesothelioma (MPM) is an aggressive asbestos-related malignancy with extremely poor prognosis, with a 5 year survival of approximately 5%. MPM remains difficult to treat - the current standard of chemotherapy care (a combination of cisplatin and pemetrexed) has been largely unchanged over the last decade and in most cases, is essentially palliative. Novel therapeutic targets are therefore urgently needed in this disease. Y-box binding protein-1 (YB-1) is a multifunctional oncoprotein associated with all of the hallmarks of cancer. Here, we evaluate YB-1 as a therapeutic target in this disease by investigating its role in driving malignant behaviour and chemoresistance of MPM cells.

AcknowledgementsThis work was supported by a scholarship from the Asbestos Diseases Foundation of Australia, a Top-Up Scholarship from Sydney Catalyst and a Cancer Institute of NSW grant.

Contact detailsThomas Johnson – e: tjoh9110@uni.sydney.edu.auKarin Schelch – e: karin.schelch@meduniwien.ac.at

Figure II: (A) Cells were transfected with 5 nM YB-1, RRM1 (+Co) or control (-Co) siRNA andincubated for 96 h. Live cells were then stained with Annexin V-488 conjugate and propidium iodideand a TALI-image based cytometer was used to measure apoptosis, or (B) fixed and stained withpropidium iodide and cell flow cytometry was conducted. (C) 2000 cells were transfected with 5 nMof YB-1 or control (Co) siRNA. The next day, cells were treated with cisplatin and a SYBR greenbased proliferation assay was employed after 96 h, normalising to the cisplatin negative cells ofeach different siRNA group. (D) Cells were transfected with 5 nM of YB-1 siRNA, 25,000 cells wereplated and videomicroscopy performed. Single cell migration was manually tracked via Image Jover 25 h (n=3). A students t-test was used to determine statistical significance (***p>0.001).

III. YB-1 is important in MPM tumour formation in vivo.MSTO cells stably expressing luciferase were transfected with 5 nM ofeither YB-1 or control (Co) siRNA and the next day intraperitoneally(IP) injected injection into SCID mice (n=5 per group). (A) Tumourgrowth was monitored in situ using the IVIS imaging system afterluciferin substrate injection. (B) Detection of bioluminescence wasalso used to visualise tumour nodules when harvested. (C) Totaltumour weight was significantly lower in mice injected with YB-1 vs CosiRNA transfected cells.

Co si-YB-1

4 weeks after injectionB

Co si-YB-1

3 weeks after injectionA

IV. YB-1 is overexpressed in MPM cells with acquiredchemoresistance, and these cells have heightenedmigratory capacity compared to their parental lines.MPM cells were grown in medium supplemented with IC50concentrations of either cisplatin, gemcitabine or vinorelbine – allcommonly prescribed drugs in the management of MPM – until theydisplayed >2 fold increase in IC50 to that drug. (A) YB-1 wasupregulated in cell lines with acquired drug resistance (drugR cells)compared to respective parental cells. (B) There was no significantdifference in growth rate, but (C) drug resistant cells displayedsignificantly increased migratory capacity compared to parental cells.

Figure IV: (A) Protein was harvested from untreated cells and Western Blot analysis of YB-1 wasconducted using β-actin as loading control. Densitometry analysis on Image J allowed aquantitative comparison between parental and drug resistant cell lines, after normalising to β-actin. (B) 2000 untreated cells per well were grown for 96 h and a SYBR green basedproliferation assay was conducted. (C) 25000 cells were plated and single cell migration wasmanually tracked via videomicroscopy on Image J over 25 h. A students t-test was used todetermine statistical significance (*** p>0.001)

ResultsI. YB-1 is overexpressed in MPM and silencing inhibits MPM,but not non-malignant mesothelial proliferation.(A) YB-1 protein was overexpressed in most MPM cell lines compared tothe immortalised mesothelial cell line MeT-5A. (B) YB-1 knockdown viasiRNA transfection did not affect MeT-5A proliferation, but did significantlyinhibit MPM cell growth (4/6 cell lines) and (C) colony formation.

Figure I: (A) Protein was harvested from untreated MPM or MeT-5A cells and Western Blot analysis ofYB-1 was conducted using β-actin as loading control. Overexpression compared to MeT-5A wasconfirmed in at least 2 repeats per cell line (representative blot shown here)

YB-1

b-actin

A

Figure I:(C) Cells were transfectedwith 5 nM of YB-1 or control(-Co) siRNA, seeded (2,000cells per well) and grownuntil colonies formed (n=2,representative photos shownhere). RRM1 siRNA wasused as positive control(+Co)

C

Figure I:(B) 2000 cells (triplicates) per96-well were transfected with5 nM of YB-1, or control (Co)siRNA and incubated for 96 h.Proliferation was determinedusing a SYBR green basedgrowth assay (n=3). A studentst-test was used to determinestatistical significance(**p>0.01, ***p>0.001).

B

B

Figure III: Mice were injected with MPM cells transfected with 5 nM YB-1 (si-YB-1) or control (Co)siRNA, monitored regularly and humanely euthanised 4 weeks after injection (A) Mice wereanesthetised via IP injection of 100 mg/kg ketamine and 10 mg/kg xylazine. 200 µL VivoGlo™luciferin was injected IP, with IVIS images being taken 10 minutes later. Representative photographswere taken at 3 weeks post injection. (B) Mice were anesthetised and humanely euthanised viacervical dislocation. Tumour tissue was visualised on a ChemiDoc™ Gel imaging system by pipettingVivoGlo™ luciferin into the peritoneal cavity and resected. (C) Total tumour weight was measured(n=5 mice per group). A students t-test was used to determine statistical significance (*p>0.05)

YB-1:animportantdriverofmesotheliomadrugresistanceandapotentialnoveltherapeutictarget

ThomasJohnson1,2,3,KarinSchelch4,KadirSarun1,MarissaWilliams1,2,YuenYeeCheng1,AnnetteLasham5,GlenReid21TheAsbestosDiseasesResearchInstitute,Sydney,Australia;2SchoolofMedicine,TheUniversityofSydney,Australia;3SydneyCatalystTranslationalCancerResearchCentre,Australia;4InstitueofCancerResearch,MedicalUniversityofVienna,Austria;

5DepartmentofMolecularMedicineandPathology,SchoolofMedicalSciences,UniversityofAuckland,Auckland,NewZealand

YB-1 siRNA transfection

MeT-5A H28 Ren

MSTOMMO5

SPC212

VMC230

20

40

60

80

100

** ** ******

Cel

l gro

wth

(% C

o)

C

MSTO par

MSTO cisR

MSTO gemR

MSTO vinR

MMO5 par

MMO5 gemR0

100

200

% of pa

rental

cell line

YB-1

b-actin

1.0 1.34 2.41 1.87 1.0 1.47

A

p a r

g emR

v inR

0

5 0

1 0 0

1 5 0

2 0 0

mig

rate

d d

ista

nce

(µ

m)

M S T O

* * * * * *

p a rc is

R

g emR

v inR

0

2 5

5 0

7 5

1 0 0

1 2 5

ce

ll g

row

th (

% p

are

nta

l)

M S T O

B C

1 10 1000

2 5

5 0

7 5

1 0 0

C is p la tin (µM )

cell

gro

wth

(% v

eh

icle

tre

ate

d)

1 10 1000

2 5

5 0

7 5

1 0 0

C is p la tin (µM )

% c

ell

gro

wth

(ve

hic

le t

rea

ted

)

-C o

1 n M5 n M

D

-Co

s i-YB -1

0

2 0

4 0

6 0

8 0

1 0 0

mig

rate

d d

ista

nce

(µ

m)

* * *

-Co

s i-YB -1

0

5 0

1 0 0

1 5 0

mig

rate

d d

ista

nce

(µ

m)

* * *

ConclusionsYB-1 is a potential novel therapeutic target in MPM. Itplays an important role in the growth and migration ofMPM cells, can sensitise cells to cisplatin in vitro andis important in the growth of tumours in vivo. Ourfindings also indicate that this protein is involved in theacquired chemoresistance of MPM. Taken together,our data make a strong case to further investigate thisoncogene’s potential as a therapeutic target in MPM.

Co siYB-1

Apoptotic DeadLate apoptotic Alive

MSTO

REN

CoYB1

0.0

0.2

0.4

0.6

Tota

l tum

our w

eigh

t (g)

*

MSTO REN

MSTO REN

MSTO REN

Sub G-1 G0/G1S G2/M

Co

si-YB1

0

50

100

% o

f pop

ulat

ion

Co

si-YB1

0

50

100

% o

f pop

ulat

ion