Download The PI3K/AKT/mTOR Pathway

The PI3-Kinase Pathway
Thomas W. Grunt
Signaling Networks Program
Division of Oncology
Department of Medicine I
Comprehensive Cancer Center
Medical University Vienna &
Ludwig Boltzmann Cluster Oncology
The PI3K/AKT/mTOR Pathway (incl.
neg. Feedback Loops)
Manning, Cantley. Cell. 2007;129:1261-74
1
The PI3K/AKT/mTOR Pathway (incl.
neg. Feedback Loops)
Carnero. Curr Pharm Design. 2010;16:34-44
Growth Factors
IGF-1, EGF, TGFα, VEGF,
etc
Growth Factors and
the mTOR Pathway
PI3-K
•
–
–
PTEN
Oxygen, energy,
and nutrients
Ras/Raf
Akt/PKB
mTOR
Abl
ER
TSC2 TSC1
Ras/Raf
pathway
kinases
mTOR
S6K1
4E-BP1
S6
Protein Production
Cell Growth
and Proliferation
4
elF-4E
Intracellular protein
Central controller of
cell growth and
proliferation
•
mTOR signaling is often
deregulated in cancer
•
Downstream inhibition of mTOR
has potential for
–
Antiproliferative effects on
tumor cells
–
–
Angiogenesis inhibition
Enhancement of the effects
of chemotherapy
Angiogenesis
This slide shows only a few of the many kinases involved in these signaling pathways
2
Signaling Through
mTOR
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5
The PI3K family and generation of
phosphatidylinositol-3,4,5-trisphosphate
Liu et al. Nature Rev Drug Discovery. 2009;8:627-44
3
Feedback Regulation of Akt by Rictor-mTOR
(mTORC2)
Cheng et al. Oncogene. 2005;24:7482-92
The PI3K/AKT/mTOR Pathway and Cancer
¾ PI3K–Akt pathway inhibitors are in clinical development for cancer (e.g. dual PI3K–mTOR
inhibitors, PI3K inhibitors, Akt inhibitors and mTOR inhibitors).
¾ PI3K–Akt pathway activated in many cancers (e.g. by receptor tyrosine kinases, mutation,
amplification).
¾ Most effective drug used to inhibit this pathway depends on mechanism of PI3K–Akt
activation.
¾ PI3K–Akt pathway inhibitors might have single-agent activity in breast cancers with
ERBB2 amplifications or PIK3CA mutations. These drugs might also be effective in
overcoming acquired resistance to therapies that target receptor tyrosine kinases.
¾ PI3K–Akt pathway inhibitors probably most effectivein combination with other targeted
drugs, such as MEK inhibitors.
Engelman. Nature Rev. Cancer. 2009;9:550-62
4
The mTOR Pathway and Cancer
• mTOR is a central controller of cell growth
and proliferation in normal cells
• mTOR pathway deregulation causes loss of growth
control in cancer
• mTOR controls responses essential to cancer cells
– mTOR regulates cell growth and division
– mTOR influences responses to DNA damage
– mTOR regulates angiogenesis
– mTOR responds to antiestrogens
– mTOR controls translation
9
Somatic genetic mutations activating the
PI3K-Akt pathway
Engelman. Nature Rev. Cancer. 2009;9:550-62
5
Incidence of genetic
alterations in the
PI3K pathway in
cancer
Liu et al. Nature Rev Drug Discovery. 2009;8:627-44
The mTOR Pathway Is Deregulated
in Many Cancers
Brain
Thyroid
Oral
SCC
Breast
Lung
Blood
Kidney
Ovary
Prostate
Pancreas
Colon
Uterus
Skin
Sarcoma
12
6
mTOR-Linked Pathway Deregulations in
Selected Cancers
EGFR, 32%–60%1
p-Akt, 23%–50%18
Ras, 30%12
PTEN, 24%22
HER2, 5%30
PI3-K, 4%13
TGFα/TGFβ1,
60%–100%35
VHL, 30%–50%36,37
IGF-1/IGF-IR,
39%-69%9
p-Akt, 38%38
PTEN, 31%39
TSC1/TSC240
Lung
Breast
NET
Kidney
Colon
p-Akt, 42%16
PTEN, 15%–41%25
HER2, 30%–36%26,27
PI3-K, 18%–26%27,28
EGFR, 6%29
TSC1/TSC231,32
IGF-1/IGF-1R33
VHL34
Ras, 50%12
p-Akt, 46%15
PTEN, 35%41
PI3-K, 20%–32%13,41
EGFR, 8%42
HER2, 3%42
13
The PI3K/AKT/mTOR Pathway and Cancer:
Main Targets for Therapeutic Intervention
van der Heijden, Bernards. Clin Cancer Res. 2010;16:3094-9
7
Targeting the
PI3K/AKT/mTOR
Pathway in Cancer
a | Inhibitors that target key nodes in the
phosphoinositide 3-kinase (PI3K) signalling pathway,
including receptor tyrosine kinases (RTKs), PI3K,
AKT and mammalian target of rapamycin (mTOR),
have reached clinical trials. Dual inhibitors that target
both PI3K and RTK or PI3K and mTOR may provide
more potent therapeutic effects in suppressing the
PI3K signalling. Combinations of PI3K and RAF–
mitogen-activated protein kinase (MAPK) inhibitors
may achieve more effective clinical results.
b | Inhibitors in clinical development that
target the PI3K or related pathways are shown.
EGFR, epidermal growth factor receptor; ERK,
extracellular signal-regulated kinase; HER2, human
epidermal growth factor receptor 2 (also known as
ERBB2); MEK, mitogen-activated protein kinase
kinase; VEGFR, vascular endothelial growth factor
receptor. *Bevacizumab targets VEGFA instead of
VEGFR directly. ‡Both AZD8055 and OSI-027 are
ATP-competitive mTOR inhibitors that target the
mTOR complexes mTORC1 and mTORC2.
Liu et al. Nature Rev Drug Discovery. 2009;8:627-44
Summary of Drugs
Targeting the PI3K
Pathway in Clinical
Trials for Cancer
Treatment
Liu et al. Nature Rev Drug Discovery. 2009;8:627-44
8
mTOR Inhibition May Enhance the Antitumor
Effects of Other Therapies
Radiation
Chemotherapy
mTOR
Inhibition
ErbB Inhibitors
Antiestrogens
Antiangiogenics
17
mTOR Inhibition May Enhance the Antitumor
Effects of Other Therapies (cont)
Agent
Rationale
ErbB inhibitors
Defects in the mTOR signaling pathway may counter the effects
of ErbB inhibitors on cell growth and proliferation. Combined
treatment has been beneficial in preclinical studies1
Cytotoxic
chemotherapy
Cytotoxic drugs such as the platinum derivatives, taxanes,
anthracyclines, and gemcitabine have shown improved
antitumor effects in preclinical models when used in
combination with mTOR inhibitors2-4
Antiangiogenic
agents
mTOR inhibition affects angiogenesis through mechanisms that
enhance and complement those of anti-VEGF/anti-VEGFR
signaling inhibitors5
Antiestrogens
Defects in the mTOR signaling pathway may render estrogendependent tumor cells resistant to antiestrogens and
aromatase inhibitors. Combinations effective preclinically6-8
Radiation
In preclinical studies, mTOR inhibition enhances cell killing
induced by radiation, possibly by interfering with repair of
damage to DNA9
18
9
mTOR Inhibition Blocks Cell Cycle Progression
at the G1–S Restriction Point
M
G2
mTOR
G1
S
Restriction
point
19 From Israels and Israels. Oncologist. 2000;5:510-513, with permission.
mTOR Inhibition Enhances the Activity
of Many Chemotherapeutic Agents
• Preclinical evidence suggests that mTOR inhibition could
also enhance the activity of
– Paclitaxel, carboplatin, vinorelbine, doxorubicin, or
gemcitabine in breast cancer1
– Immunomodulatory agents in multiple myeloma2
– Gemcitabine in pancreatic cancer3
– Doxorubicin in PTEN-negative prostate cancer4
20
1. Mondesire et al. Clin Cancer Res. 2004;10:7031-7042.
2. Raje et al. Blood. 2004;104:4188-4193.
3. Bruns et al. Clin Cancer Res. 2004;10:2109-2119.
4. Grünwald et al. Cancer Res. 2002;62:6141-6145.
10
mTOR Inhibition May Enhance the
Cytotoxicity of DNA-Damaging Agents
• DNA damage caused by agents
Cisplatin
such as cisplatin activates p53
DNA
• p53 triggers DNA repair, which
Cross-linked DNA
Cycle Arrest
DNA Repair
Cell Death
Cell
Death
Cell
Death
21
allows the cell to survive, or,
failing that, p53 initiates cell death
• mTOR regulates production of
p21, a cell cycle inhibitor that
allows DNA repair
• mTOR inhibition blocks p21
translation, forcing cell death
even when the DNA damage is
otherwise nonlethal
• mTOR inhibition can enhance
the activity of certain drugs
such as cisplatin and other
platinum derivatives
Survival
Beuvink et al. Cell. 2005;120:747-759.
mTOR Inhibition Decreases Angiogenesis
• mTOR regulates HIF-1α
and HIF-2α expression
• HIF-1 and HIF-2 are
transcription factors for
hypoxic stress-related
genes
• HIF-1α/2α are normally
degraded by VHL protein
• HIF-1 and HIF-2
condition the tumor to
adapt to growth under
hypoxic conditions and
promote angiogenesis
and metastasis
22
HIF = hypoxia-inducible factor; VHL = von Hippel-Lindau protein.
11
mTOR
Tumor Cell Growth and Angiogenesis
Growth factors
Endothelial cell
Smooth muscle cell (pericyte)
Cancer cell
PI3-K
PI3-K
PTEN
PI3-K
Akt/
PKB
TSC2
TSC1
Akt/
PKB
Akt/
PKB
mTOR
mTOR
mTOR
Protein production
HIF-1α
HIF-2α
VHL
Cell growth
and proliferation
Cell growth
and proliferation
Angiogenic
growth factors
Cell growth
and proliferation
23
Angiogenesis Inhibitors and mTOR Inhibitors
May Act Synergistically
Primary orthotopic (ear) B16 melanoma
Fractional Tumor Volume
(V/Vo, mean ± SEM)
8
Vehicle, n = 6
1 mg/kg mTOR inhibitor, po q24h, n = 6
100 mg/kg VEGFR inhibitor, po q24h, n = 6
1 mg/kg mTOR inhibitor + VEGFR inhibitor, n = 6
5 mg/kg mTOR inhibitor + VEGFR inhibitor, n = 6
6
4
*
*
2
*P < .05 vs vehicle controls
and single agents.
0
7
14
21
Days Post Tumor Cell Injection
*O’Reilly et al. Proc Am Assoc Cancer Res. 2005;46:715. Abstract 3038.
24
12
mTOR Inhibition Enhances
Effects of Antiestrogenic Agents
• Akt activation predicts a worse outcome for breast cancer
patients treated with endocrine therapy1
• Activated Akt mediates resistance to antiestrogen therapy
related to HER2 overexpression2,3
• mTOR inhibition restores responses to tamoxifen in breast
cancer cells with high levels of Akt activity4
• Synergistic in vitro and in vivo effects have been seen
with combined antiestrogen therapy and mTOR inhibition5
25
1. Perez-Tenorio et al. Br J Cancer. 2002;86:540-545.
2. Campbell et al. J Biol Chem. 2001;276:9817-9824..
3. Kurokawa and Arteaga. Clin Cancer Res. 2003;9(suppl):511s-515s.
4. de Graffenried et al. Clin Cancer Res. 2004;10:8059-8067.
5. Zhang et al. Proc Am Assoc Cancer Res. 2003;44(2nd ed):739. Abstract 3715.
Dual mTOR and Aromatase Inhibition Induces
Apoptosis in Breast Cancer Models
Apoptotic Cells, %
25
20
15
10
5
0
0
2
0
100 nM AI
2
0
2
500 nM AI
mTOR Inhibitor
(nM)
The combination of an mTOR inhibitor and an aromatase inhibitor (AI)
increases induction of apoptosis, compared with 2 nM of the
mTOR inhibitor alone or 100 nM or 500 nM of the AI alone
P < .05 (Friedman test).
26
Modified from Boulay et al. Clin Cancer Res. 2005;11:5319-5328, with permission.
13
Summary
Rationale for Targeting mTOR
• mTOR, an intracellular protein, acts as a central regulator of
multiple signaling pathways that may mediate abnormal growth
and proliferation
• mTOR provides a stable genetic target
• mTOR activity is influenced by nutrient and energy levels and
by signaling through pathways often deregulated or
overexpressed in cancer, such as
– Cell surface receptors, such as EGFR/HER, IGFR, VEGFR
– The PI3-K/Akt survival pathway, in which the PTEN tumor
suppressor gene is often lost
– Ras-Raf
– Intracellular receptors, such as ER and PR
27
Summary
Rationale for Targeting mTOR (cont)
• Aberrant signaling through upstream pathways can
activate mTOR inappropriately, promoting
– Abnormal cell growth, proliferation, and angiogenesis
– Survival of cancer cells in the nutrient- and oxygendepleted tumor environment
• Targeting deregulated pathways has been a successful
clinical strategy in cancer
• Combination therapy targeting mTOR and deregulated
pathways may provide enhanced anticancer activity
28
14
Phospho-AKT correlates with ErbB drug resistance
Breast cancer
Pelitinib
sensitive
SKBR3
µM Pelitinib
resistant
T47D
0 0.1 1 2 4 8
0 0.1 1 2 4 8
pAKT(Ser473)
pAKT(Thr308)
AKT
pERK1,2
ERK1,2
Actin
• Resistant cells reveal drugrefractory PI3K signaling
• MAPK signaling is
uncoupled from resistance
Canertinib
sensitive
SKBR3
µM Canertinib
resistant
T47D
0 0.1 1 2 4 6 10 0 0.1 1 2 4 6 10
pAKT(Ser473)
pAKT(Thr308)
AKT
pERK1,2
ERK1,2
Actin
thway
MAPK pa
Ovarian cancer
PI3K
/AKT
path
wa y
Pelitinib
sensitive
HOC7
µM Pelitinib 0 0.1 1 2 4 8
pAKT(Ser473)
pAKT(Thr308)
AKT
pERK1,2
ERK1,2
Actin
intermediate
SKOV3
resistant
CAOV3
0 0.1 1 2 4 8
0 0.1 1 2 4 8
sensitive
resistant
Brünner-Kubath et al., BCRT, Nov 2010
Constitutively active AKT induces ErbB drug resistance
ErbB drug sensitive SKBR3 breast cancer cells
Signaling pathways
MAPK pathway
Cell growth
MEK1,2
Pelitinib IC50 (µM)
pERK1,2
ERK1,2
PI3K/AKT pathway
pAKT(Ser473)
AKT
pGSK3β
mTOR
Controls
pS6
ca
ca
MEK AKT
Transfections
ErbB drug sensitive
ErbB drug resistant
α,β-Tubulin
Pelitinib
***
SK
BR
SK
3
BR
SK
3L
BR
TX
3p
cD
N
A3
SK
BR
3G
SK
FP
BR
3M
E
SK
K1
BR
3A
KT
1
pmTOR
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
-
+
-
Controls
+
-
+
-
+
caMEK caAKT
thway
MAPK pa
Transfections
PI3K
/AKT
path
w ay
Brünner-Kubath et al., BCRT, Nov 2010
15
Dominant negative AKT induces ErbB drug sensitivity
ErbB drug resistant T47D breast cancer cells
PI3K/AKT signaling pathway
Cell growth
pAKT(Ser473)
Pelitinib IC50 (µM)
AKT
pS6
α,β-Tubulin
Pelitinib
-
-
+
+
Controls
-
+
dnAKT
Transfections
ErbB drug sensitive
ErbB drug resistant
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
***
Controls
dnAKT
Transfections
thway
MAPK pa
PI3K
/AKT
path
w ay
Brünner-Kubath et al., BCRT, Nov 2010
PI3K inhibitors overcome ErbB drug resistance - I
120
110
100
90
80
70
60
50
40
30
20
10
0
Cell Number (% of Control)
PI3K inhibitors
sensitize the cells
against ErbB drugs
Cell Number (% of Control)
ErbB drug resistant T47D breast cancer cells
Pelitinib
Pelitinib+BEZ235
Pelitinib+Rapamycin
Pelitinib+Akti-1/2
0
1
2
3
4
5
6
7
120
110
100
90
80
70
60
50
40
30
20
10
0
8
Canertinib
Canertinib+BEZ235
Canertinib+Rapamycin
Canertinib+Akti-1/2
0
1
2
thway
MAPK pa
120
110
100
90
80
70
60
50
40
30
20
10
0
Pelitinib
Pelitinib+AZD6244
Pelitinib+UO126
0
1
2
3
4
5
Pelitinib (µM)
3
4
5
6
7
8
6
7
8
Canertinib (µM)
Cell Number (% of Control)
MAPK inhibitors do
NOT sensitize the cells
against ErbB drugs
Cell Number (% of Control)
Pelitinib (µM)
6
7
8
120
110
100
90
80
70
60
50
40
30
20
10
0
Canertinib
Canertinib+AZD6244
Canertinib+UO126
0
1
2
3
4
5
Canertinib (µM)
PI3K
/AKT
path
w ay
Brünner-Kubath et al., BCRT, Nov 2010
16
PI3K inhibitors overcome ErbB drug resistance - II
ErbB drug resistant T47D breast cancer cells
0,07
IC50
0,06
NVP-BEZ235 (µM)
The PI3K/mTOR
inhibitor NVP-BEZ235
synergizes with ErbB
drugs
NVP-BEZ235 (µM)
0,07
0,05
0,04
0,03
0,02
0,01
0,00
IC50
0,06
0,05
0,04
0,03
0,02
0,01
0,00
0
2
4
6
8
10
12
14
16
0
2
Pelitinib (µM)
IC50
8
10
12
14
16
IC50
60,0
U0126 (µM)
50
40
30
20
10
thway
MAPK pa
6
Canertinib (µM)
60
U0126 (µM)
The MEK inhibitor
U0126 does not
synergize with ErbB
drugs
4
50,0
40,0
30,0
20,0
10,0
0
0,0
0
2
4
6
8
10
12
14
16
Pelitinib (µM)
0
2
4
6
8
10
12
Canertinib (µM)
PI3K
/AKT
path
w ay
Brünner-Kubath et al., BCRT, Nov 2010
Signaling and metabolism: Interaction
between the ErbB/PI3K/AKT/mTOR
pathway and fatty acid synthase
• ErbB membrane receptor tyrosine kinases are upstream
activators of PI3K/AKT/mTOR
• Fatty acid synthase (FASN) is key enzyme in fatty acid
production
– Overexpressed in many tumors
– FASN inhibitors block tumor growth
• ErbB- and FASN-pathways cross-talk
- ErbB drugs inhibit FASN and vice versa
- FASN drugs silence PI3K/AKT/mTOR
• Combination of both drug types yield strong antitumor effects
17
FASN inhibitor downregulates PI3K signaling and
cooperates with ErbB blockers
C75
OVCAR-3
+
SKOV-3
+
HEY
-
+
p-AKT
FASN inhibitor C75
• Dephosphorylates/downregulates AKT
and S6
• Stimulates protein
ubiquitination/proteasomal degradation
• Has less effect on ERK signaling
AKT
p-S6
S6
p-ERK1/2
ERK1/2
Ubiquitin
Actin
Cell Number (% of Control)
40 µM C75 alone
10µM ErbB inhibitor alone
C75 and ErbB inhibitor together
3 days
40
SKOV3
5 days
Combination of FASN inhibitor C75 with
• ErbB drugs (e.g. pelitinib, canertinib)
reveal strongly improved antitumor effects
35
30
25
20
15
10
5
1
2
1
2
1
2
1
2
C
75 an C7
+C er 5
an tini
er b
tin
ib
C
P C
75 eli 75
+P tin
el ib
iti
ni
b
C
75 Can C7
+C er 5
an tin
er ib
tin
ib
C
C
C
75 Pel 75
+P itin
el ib
iti
ni
b
0
Grunt et al. BBRC. 2009;385:454-9
Tomek et al. Submitted, 2010
Conclusions
• Drug-refractory phosphorylation of AKT is a
biomarker for ErbB drug resistance of breast
and ovarian cancer cells
• PI3K/AKT is more important than MAPK for
breast and ovarian cancer growth and
survival
• Silencing of PI3K/AKT can overcome ErbB
drug resistance in breast and ovarian
cancer
18