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