Duncan Simmons1, Leah O’Shea1, Ronald J Neufeld2, and Myron R Szewczuk1*.
Depts of 1Biomedical and Molecular Sciences, and 2Chemical Engineering, Queen’s University, Kingston, ON K7L 3N6, Canada.
*speaker
Cancer therapeutics in the clinical setting
Need for alternate therapeutics
Target multiple cancer pathways
Circumvent the genetic mutations
Identified a new anti-cancer therapeutic approach:
Oseltamivir phosphate (OP) monotherapy or in combination with chemotherapeutics against human ovarian cancer regulates tumour neovascularization, growth and invasiveness.
OP: Oseltamivirphosphate
EGFR Trk insulin
TLR-4, -7 & -9Published:
C o n tro l
E G F 3 0 n g /ml
O P 1 6 4 µg /m
L + E G F
A n ti-Ne u 1 1 0 0 µ
g /mL + E G F
M M P 9 i 50 µg /m
L + E G F
Me
an
flu
ore
sc
en
ce
C o n trol
E G F 33 n g /m
l
O P 33 3 µ
g /mL +
EG F
C o n trol
E G F 33 n g /m
l
O P 33 3 µ
g /mL +
EG F
Mea
n f
luo
resc
ence
Neu1 sialidase activity is associated with epidermal growth factor (EGF) stimulation of A2780 ovarian carcinoma cells
A2780
LD50 = 4 μM at 48 hrLD50 = 7 μM at 48 hr
A2780cisA2780Stable A2780 shRNA Slug KD
Stable A2780 shRNA Snail KD
LD50 > 488 μM at 48hr LD50 > 488 μM at 48 hr
Ce
ll V
iab
ilit
y(%
of
Co
ntr
ol)
+S
EM
Cel
l Via
bili
ty (
% o
f C
on
tro
l)+
SE
M
Cel
l Via
bili
ty(%
of
Co
ntr
ol)
±S
EM
Cel
l Via
bili
ty(%
of
Co
ntr
ol)
±S
.E.
OP LD50 of 7μM for A2780 OP LD50 of 4μM for cisplatin-resistant A2780cis OP LD50 of 488μM for A2780 shRNA Slug and Snail KD
Oseltamivir phosphate (OP) decreases the cell viability of A2780, A2780cis, A2780-shRNA Slug and A2780-shRNA
Snail cells
Cisplatin 5-FU Gemcitabine Paclitaxel
u n trea te
d co n tro
l
5 -FU 1
µM
OP 3
0 0 µg /m
L + 5-F
U 1 µM
OP 6
0 0 µg /m
L + 5
-FU 1 µ
M
OP 8
0 0 µg /m
L + 5
-FU 1 µ
M
Cel
l Via
bili
ty(%
of
Co
ntr
ol)
±S
.E.
u n trea te
d co n tro
l
P a c lita x e l 1
µM
OP 3
0 0 µg /m
L + P
a c 1µM
OP 6
0 0 µg /m
L + P
a c 1µM
OP 8
0 0 µg /m
L + P
a c 1µM
Ce
ll V
iab
ilit
y(%
of
Co
ntr
ol)
±S
.E.
u n trea te
d co n tro
l
Gem
1µM
OP 3
0 0 µg /m
L + G
em 1 µ
M
OP 6
0 0 µg /m
L + G
em 1 µ
M
OP 8
0 0 µg /m
L + G
em 1 µ
M
Ce
ll V
iab
ilit
y(%
of
Co
ntr
ol)
±S
.E.
u n trea te
d co n tro
l
C isp la
t in 1
µM
OP 3
0 0 µg /m
L + C
is 1µ
M
OP 6
0 0 µg /m
L + C
is 1µ
M
OP 8
0 0 µg /m
L + C
is 1µ
M
Ce
ll V
iab
ilit
y(%
of
Co
ntr
ol)
±S
.E.
Cell viability of A2780 cells treated with different dosages of OP in combination with 1μM of cisplatin, 5-FU, gemcitabine and paclitaxel
Beneficial effect using combination therapy of OP and standard clinical chemotherapeutics on amplifying chemo-drug sensitivity.
Hypersensitivity reactions to oxaliplatin, gemcitabine are noted complications in cancer patients.
A2780 Untreated Control A2780 30mg/kg OP A2780 50mg/kg OPA
B
Tu
mo
r vo
lum
e (m
m3)
C o n tr o l T V
Tum
or v
olum
e (m
m3 )
Tu
mo
r v
olu
me
(m
m3)
A2780 human ovarian cancer cell line implanted cutaneously in RAGxCγ double mutant mice
B
u n trea te
d
OP 3
0 mg /k
g
OP 5
0 mg /k
g
Nu
mb
er
me
tas
tati
cc
lus
ters
pe
r lu
ng
H&E staining of microtome 5µm sections of paraffin-embedded necropsy lungs taken from A2780 tumor-bearing RAGxCy double
mutant miceLungs
Untreated OP 50mg/kg OP 30mg/kg
Ovarian A2780
Ovarian A2780 SLUG KD
Ovarian A2780 SNAIL KD
A2780 human ovarian cancer cell line in RAGxCy double mutant mice
SNAIL: repressor of E-cadherin gene expression in epithelial tumour cells
SLUG: represses E-cadherin expression and induces epithelial to mesenchymal transitions
OVARIAN CANCER
B
A 2 7 8 0
A 2 7 8 0 Slu
g KD
A 2 7 8 0 SN A IL
KD
Nu
mb
er
me
tas
tati
cc
lus
ters
pe
r lu
ng
H&E staining of microtome 5µm sections of paraffin-embedded necropsy lungs taken from tumor-bearing RAGxCy double mutant mice
Necropsy Lungs
Untreated Slug shRNA Snail shRNA
A2780UntreatedA4 mouse
Tumor
TumorH&E
Bkg
VE-cad
A2780SLUG KD
B2 mouse
E-cad
N-cad
A2780SNAIL KDC2 mouse
A 2 7 8 0
A 2 7 8 0 SL U G
KD
A 2 7 8 0 SN A IL
KD
rela
tiv
e t
ota
ls
tain
ing
de
ns
ity
A
B
C
D
E
E-cadherin N-cadherin
Necropsy tumorsUntreated Slug Snail
Untreated
Signaling cascade
Tumor vasculature
Slug
Snail
MMP9
Snail-MMP-9 signaling axis potentiates Neu1 sialidase and MMP-9 cross-talk in regulating RTK receptors, tumor neovascularization,
growth and invasiveness
Abdulkhalek et al 2014 Clinical and Translational Medicine 3:28 (open access)
C4B4A4
Tu
mo
r v
olu
me
(m
m3)
A B
Confidential unpublished data
A2780 Sialidaseactivity
EGF 30ng/ml
MMP9i 50 μg/mL+ EGF
Anti-Neu1 100 μg/mL+ EGF
Phase
Control
Tamiflu400 μM+ EGF
Sialidaseactivity
A2780Snail shRNA
A2780 human ovarian cancer cell line was established from tumour tissue from an untreated patient
OVARIAN CANCER
Confidential unpublished data
Ce
ll V
iab
ilit
y(%
of
Co
ntr
ol)
+S
.E.
Ce
ll V
iab
ilit
y(%
of
Co
ntr
ol)
+S
.E. LD50 = 8 μMLD50 = 50 μM
A2780cisA2780
u n trea te
d co n tro
l
C isp la
t in 1µM
T amif l
u 0.7
3mM
+ C
isp la
t in 1µM
T amif l
u 1.4
6mM
+ C
isp la
t in 1µM
T amif l
u 1.9
5mM
+ C
isp la
t in 1µM
T amif l
u 0.7
3mM
T amif l
u 1.4
6mM
Ce
ll V
iab
ilit
y(%
of
Co
ntr
ol)
±S
EM
(n
=6
)
A2780A B C
Confidential unpublished data
Tu
mo
r v
olu
me
(m
m3)
A
BConfidential unpublished data
u n trea te
d
T ami f l
u 30 m
g /kg
T ami f l
u 50 m
g /kg
Nu
mb
er
me
tas
tati
cc
lus
ters
pe
r lu
ng
u n trea te
d
T amif l
u 30 m
g /kg
T ami f l
u 50 m
g /kg
Nu
mb
er
me
tas
tati
cc
lus
ters
pe
r li
ve
r
A
B
Untreated 50mg/kg OP 30mg/kg OP
Confidential unpublished data
A 2 7 8 0
A 2 7 8 0 Slu
g KD
A 2 7 8 0 SN A IL
KD
Nu
mb
er
me
tas
tati
cc
lus
ters
pe
r lu
ng
A 2 7 8 0
A 2 7 8 0 Slu
g KD
A 2 7 8 0 SN A IL
KD
Nu
mb
er
me
tas
tati
cc
lus
ters
pe
r li
ve
r
A
B
Confidential unpublished data
A2780UntreatedA4 mouse
Tumor
TumorH&E
Bkg
VE-cad
A2780SLUG KD
B2 mouse
E-cad
N-cad
A2780SNAIL KDC2 mouse
A 2 7 8 0
A 2 7 8 0 SL U G
KD
A 2 7 8 0 SN A IL
KD
rela
tiv
e t
ota
ls
tain
ing
de
ns
ity
A
B
C
D
Signaling cascade
Tumor vasculaturesecreted cytokinesSNAIL KD
SLUG KD
Abdulkhalek et al 2013 Research and Reports in Biochemistry 3: 17-30 Novelty
?
Snail genes act primarily as survival factors and inducers of cell movement
SNAIL: a zinc fingertranscriptional repressor
ConclusionsTamiflu provides an unexpected therapeutic
benefit in cancer, by interfering with growth factor receptor-induced
survival signals and disabling cancer cell survival in acquire chemo-
resistance.
Tamiflu alone or in combination with chemotherapeutics against pancreatic and breast impedes the growth and metastatic spread, tumor neovascularization and chemo-resistance in heterotopic xenograft of tumors growing in RAGxCγ double mutant mice.
Priority #1: “Clinical trials”
Priority #2: To study the broad range efficacy of Tamiflu therapy in combination with standard chemotherapeutics.
Priority #3: To test this novel therapy on surgically obtained patient primary tumors in culture
