IGF-1R: A key linker between chemoresistance and cancer stem cells in epithelial ovarian cancer

cellsRam Kumar Singh, Ankit Jinager, Ajit Dhadwe, Abhijit De and Pritha Ray*

Advanced Centre for Treatment Research and Education in Cancer,

Tata Memorial Centre, Navi Mumbai, India

5th Asia Pacific Summit on Cancer Therapy, 20th – 22nd July 2015

Ovarian Cancer Ovarian cancer (OC) is the 4th leading cause of gynecological deaths around the world. (Siegel et al., Cancer J Clin, 2012)

As per the Tata Memorial Center, Mumbai’s registry India, it is the third most lethal cancer amongst women.

Types Germ cell OC (3-5%) Epithelial OC (85-90%) Sex-cord stromal cell OC (5-7%)

Subtypes: Epithelial Ovarian Carcinoma

Chemotherapy Platinum (cisplatin and carboplatin) Taxane (paclitaxel)

Though the patients show response to 1st line therapy, 50-60 % ultimately exhibit recurrence and finally succumb to the disease.

Key players behind drug resistance

There are subset of cells within the heterogeneous tumor designated as cancer stem cells.These CSCs have high DNA repair efficiency and chemo resistance property.

Ovarian cancer-stem-like side population cells are tumorigenic and chemoresistant. (Hu et. al., British Journal of cancer, 2010) In vivo and in vitro studies have showed the role of Insulin Growth Factor -1Receptor (IGF-1R) cross-talk in

chemoresistance and have proven that IGF-1R inhibitors can overcome cytotoxic resistance. (Wong et. al., Gynecologic Oncology, 2012).

Ovarian cancer and chemoresistance

Neoadjuvent therapy

Debulking Surgery

Adjuvant Chemotherapy

Tumor Relapse

Chemo resistance

Tumor Relapse

Chemo resistance

Early detection ofChemo resistance

Targeting Right population

Cancer Stem Cells

IGF-1R Axis

Schematic representation of development of cellular resistant

models in A2780 cells

Understanding the role of the biological and molecular players during induction of

drug resistance

CSC characterization• SP assay• Biomarker assay• Spheroid assay• Tumor xenograft assay

Cell line used• A2780/APFT• OAW42• SKOV3• Hey

Side population assayCisplatin Model Paclitaxel Model Combination Model

Per

cent

Sid

e P

opul

atio

n

0

5

10

15

20

25

p<0.0001

p=0.0002

p=0.0004

p<0.0001

p=0.0002

p=0.0054

Self Renewal and Chemoresistance Property

Num

ber

of sp

her

oid

s

0

10

20

30

40

50

60

70

80

90

100

110

P=0.0093

P=0.0014

P=0.0082

P=0.0008

P=0.0004

P=0.0005

Num

ber

of s

pher

oids

0

10

20

30

40

50

60

70

80

90

100

110

***

******

*** ***

*** ***= P<0.001***

A B

Per

cent

Via

bilit

y

0

20

40

60

80

100MP

SP

NSP

**

* *

C

Stemness gene expression across the resistant models

Pluripotent Genes

Rela

tive

gene

expr

essi

on (2

- ct

)

0.000

0.005

0.010

0.015

0.020 APFTCIS-ERCIS-LRPAC-ERPAC-LRCOMBI-ERCOMBI-LR

* * ns**

ns

***

***

ns**

**** *

***

**

Biomarker analysis across the resistant models

+1

I Sense Loop antisense Terminator TAACATGTGTAAGCTGCGGCCCTTCAAGAGAGGGCCGCAGCTTACACATGTTTTTTTTGC

ATTGTACACATTCGACGCCGGGAAGTTCTCTCCCGGCGTCGAATGTGTACAAAAAAAACGCCGG

Oct4 target Sequence adapted from Zaheres et al, Stem Cells, 2005

Knock down of Oct4 gene using pll3.7 lentilox vector

Num

ber o

f ser

ial p

assa

ge

0

2

4

6

8 ***

**

****

**

= P<0.01***** = P<0.001

Effect of oct4 KD on its self renewal and drug resistanceA Spheroid formation assay B Spheroid formation at multiple passage

Num

ber

of sp

her

oid

s

0

20

40

60

80

**

***

****

**** P<0.05** P<0.005*** P<0.0005

Per

ce

nt

Via

bili

ty

APFT

APFT OCT4

KD

CISLR

CISLR O

CT4 K

D

PAC LR

PACLR OCT4

KD0

20

40

60

80

100

D MTT assay to monitor drug resistance E Oct4 KD and P-AKTC Oct4 KD and SP phenotype

p/s

/cm

²/s

r

104

106

108

1010

1012SPNSP

Tu

mo

r V

olu

me

(cm

3 )

0.0

0.5

1.0

1.5

2.0

2.5

3.0

In vivo imaging of PACER SP/NSP cells for tumor formation

Day 0 Day 25

Day 40

Day 60

Day 80

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

Mouse 1 Mouse 2 Mouse 3

Day 0 Day 25 Day 40 Day 60 Day 801.00E+03

1.00E+04

1.00E+05

Mouse1 Mouse2 Mouse3

In vivo imaging of PACLR SP/NSP cells for tumor formation

SP Tumor Xenograft

p/s/

cm²/s

r

p/s/

cm²/s

rNSP Tumor Xenograft

In vivo imaging of CISLR SP/NSP cells for tumor formationp/

s/cm

²/sr

102

104

106

108

1010

1012

1014 SPNSP

Tum

or V

olum

e (c

m3 )

0.0

0.5

1.0

1.5

2.0

2.5

3.0 SP

IGF-1 -/- : Small OvariesIGF1-R -/- : Infertile

IGF Signaling in ovarian follicular development

The insulin-like growth factor (IGF) family is an essential growth factor system in the development of tissues or organs and postnatal growth, and maintenance of normal function of many cell types of the body (Li and Geng, 2010).

IGF may sustain the stem cell's capacity of self-renewal and differentiation, promote their survival and migration, and prevent senescence acting downstream to PI3K pathway (Li and Geng, 2010).

Joanne s. Richards et al., Recent Prog Horm Res. 2002

Is IGF-1R crucial for initiation and maintenance of resistance

and stemness phenotype in ovarian cancer?

Elevated expression of IGF-1R at early stages of drug resistance

A B C

D

Singh et. al., Cancer Letters, 2014

Inhibition of IGF-1R signaling with PPP

PPP is a small molecule that selectively inhibits IGF-1R kinase activity without showing any effect against Insulin receptor and other RTKs.

C

Singh et. al., Cancer Letters, 2014

Potentiation of cytotoxicity using picropodophylin (PPP)

Cisplatin Model Paclitaxel Model Combination Model

Singh et. al., Cancer Letters, 2014

A B

Effect of IGF-1R inhibition on long term survival of cells

through clonogenic assay

Singh et. al., Cancer Letters, 2014

Correlation between IGF-1R signaling and stemness phenotype

Num

ber

of s

pher

oids

0

10

20

30

40

50

60

70

80

90

100

110

**

***

*****

***

***

***= P<0.001

= P<0.01*****

Fo

ld c

ha

ng

e

(2-

ct

)

OCT4

SOX2

NANOG0.0

0.5

1.0

1.5PAC-LR PAC-LR + PPP

** *** *

Fo

ld c

ha

ng

e (

2-

ct)

OCT4

SOX2

NANOG0.0

0.5

1.0

1.5PAC-ER PAC-ER PPP

*** *** ***

How do late resistant cells maintain their resistant and stemness phenotype?

Effect of AKT inhibition on stemness property

Stemness/Chemoresistance

IGF-1R

AKTP

Stemness/Chemoresistance

IGF-1R

Oct4 mRNA

Early resistant stage

Late resistant stage

Regulation of stemness and chemoresistance at early and late

resistant stage

Active IGF-1R signalling Suppressed IGF-1R signalling

AKT

25

Acknowledgement ACTREC and UGC for Funding

UGC Fellowship