Transcript PowerPoint **

NF-κB-driven suppression of FOXO3a contributes to
EGFR mutation-independent gefitinib resistance
PNAS (2016) Published online E2526-E2535
Speaker: Huang Ya-Ling
Adviser: Kao Jung-Yie , Way Tzong-Der
Date:
2016/12/13
Types of Lung Cancer
Nature Vol.513 No.7517
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OncoTargets and Therapy 2014:7 841–852
Epithelial growth factor receptor (EGFR)
EGFR tyrosine kinase inhibitors(EGFR-TKIs)
Gefitinib
Erlotinib
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EGFR mutation
• EGFR-TKI sensitive
-mutations of L858R
-exon 19 deletions
• EGFR-TKI resistant
-T790M site mutation
-exon 20 insertion
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FOXO family
• The human FOXO family includes FOXO1, FOXO3a, FOXO4,and FOXO6.
• FOXO3a is a transcription factor that acts as a tumor suppressor.
FoxO3a and disease progression.
Nho RS, Hergert P (2014) World J Biol Chem 5(3):346–354.
FOXO3a has been found to increase several target genes, such as
TRAIL, PUMA, FasL, and BIM, which is essential for the gefitinibinduced killing of NSCLC cells.
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The transcription factor FOXO3a is a crucial cellular target of gefitinib
(Iressa) in breast cancer cells.
Krol J, et al. (2007) Mol Cancer Ther 6(12 Pt 1):3169–3179.
Knockdown of FOXO3a in breast cancer results in a reduction of
gefitinib-induced cell cycle arrest and cell death.
FOXO3a (1)might be involved in resistance to EGFR-TKIs and the CSC properties
Reprogramming
ovarian
and
breast
cancer
cells
into
non-cancerous
cells
of lung cancer. (2)could be another pathway for cancer cells to survive by resisting
by low-dose
metformin or SN-38 through FOXO3 activation.
gefitinib
agents.
Theodore Hu, Young Min Chung, Michelle Guan, Michael Ma, Jessica Ma, Jonathan S. Berek & Mickey C-T.
Hu(2014)
FOXO3a nuclear localization induced by metformin or SN-38 would
down-regulate the properties of stem-like cells in breast and ovarian
cancer cells.
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Cancer Stem cell
Aldehyde dehydrogenase
ALDH
activity
assay
SOX2
Nanog
OCT4
CD133
KLF4
CD133+
CD44+
CSC
Hoechst 33342
Side
population
(SP)
Sphere
formation
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NF- κB
mir-155
FOXO3a
Gefitinib resistance
Clinical cancer specimens
Cancer stem cell properties
Animal model
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FOXO3a Expression Is Associated with EGFR-TKI
Resistance and Cancer Stemness
80 cases of Patients
Immunohistochemical
(IHC) staining
RECIST1.1 criteria
EGFR-TKIs
Responder
(n=42)
EGFR-TKIs
non-responder
(n=38)
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Lung tumor tissue
High Pure FFRE RNA Micro Kit
Real-time RT-PCR
Lower ΔCt values indicate higher expression of gene.
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Online PrognoScan database
Progression-free survival (PFS):
The time elapsed between
treatment initiation and tumor
progression or death from any
cause.
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EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like
side-population cells in non-small cell lung cancer.
Singh S, et al. (2012) Mol Cancer 11:73.
SOX2 expression maintained the self-renewal of lung cancer cells and was
positively correlated with the metastatic progression of NSCLC.
CD133 is indicative for a resistance phenotype but does not represent a prognostic
marker for survival of non-small cell lung cancer patients.
Salnikov AV, et al. (2010) Int J Cancer 126(4):950–958.
CD133 expression is linked to a resistant phenotype in NSCLC.
Suggesting that SOX2 andCD133 expression plays an important role in
studying the properties of cancer stem cells and the therapeutic response in
lung cancer.
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Association between cancer stem cell
markers and FOXO3a expression in lung
cancer datasets. (Oncomine database)
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The expression of FOXO3a between wild-type and mutant EGFR in lung cancer
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Analyzed the relationship of FOXO3a and
gefitinib sensitivity in lung cancer cell lines
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The expression of FOXO3a was positively correlated with gefitinib sensitivity
and CSC suppression of lung cancer in an EGFR mutation-independent manner.
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Effects of FOXO3a Involved in Gefitinib Resistance
and CSC Properties
To elucidate whether FOXO3a affects sensitivity to gefitinib
Western blot
MTT assay
Flow cytometric analysis
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To elucidate whether FOXO3a-knockdown affects
sensitivity to gefitinib
MTT assay
Flow cytometric analysis
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Effects of FOXO3a Involved in CSC Properties
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4-6 weeks NOD/SCID mice
Tumor growth
(A549 cells)
+Gefitinib 2 weeks
Tumor volume
Western blot
A549:
Lung adenocarcinoma
FOXO3a
involved in the gefitinib response and the CSC characteristics
(EGFR was
Wild-type)
of lung
cancer
cells.
IC50 of
Gefitinib=4.859
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miR-155 Targeting of FOXO3a Is Correlated with
Gefitinib Sensitivity
mir-155
Neurotensin signaling activates microRNAs-21 and -155 and Akt,
promotes tumor growth in mice, and is increased in human colon tumors.
Bakirtzi K, et al. (2011) Gastroenterology 141(5):1749–1761.e1.
MiR-155 at the heart of oncogenic pathways.
Czyzyk-Krzeska MF, Zhang X (2014) Oncogene 33(6):677–678.
miR-155 has been considered an “oncomicroRNA “by targeting several
tumor suppressors, including FOXO3a.
It has been found to promote the epithelial-mesenchymal transition
(EMT), invasion,metastasis, growth, and angiogenesis of cancer cells.
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MicroRNA
biogenesis and function
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To further identify whether FOXO3a was a direct target of miR-155
Luciferase reporter assay
Cotransfection with miR-155 and the WT-3′UTR reporter resulted in a
significant inhibition of luciferase activity in a dose-dependent manner, but
not in the MT-3′UTR reporter
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miR-155 directly hinders FOXO3a expression and is involved in FOXO3amediated gefitinib sensitivity and cancer stemness.
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miR-155 Represses FOXO3a-Mediated Gefitinib Sensitivity
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FOXO3a-expressing construct lacking the FOXO3a 3 ′ UTR
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The expression of miR-155 is increased in patients who are considered to
have poor response to EGFR-TKIs, and its expression is inversely
correlated with FOXO3a expression.
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NF-κB Is Involved in the Regulation of miR-155 Expression
NF- κB
is a transcription factor that are involved in
immune and inflammatory responses, evelopmental
processes, cellular growth, and apoptosis.
MicroRNAs: New regulators of immune cell development and function.
Baltimore D, Boldin MP, O’Connell RM, Rao DS, Taganov KD (2008) Nat Immunol 9(8):839–845.
A previous study indicated an important role for NF- κB in regulating the
miR-155 expression of immune cell development and function.
However, the role of NF- κB mediated miR-155 in cancer progression
and gefitinib resistance and cancer stemness were not investigated.
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We found that primary, precursor, and mature forms of miR-155 were
all up-regulated in PC9/GR cells compared with PC9/WT cells.
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Neurotensin signaling activates microRNAs-21 and -155 and Akt,
promotes tumor growth in mice, and is increased in human colon tumors.
Bakirtzi K, et al. (2011) Gastroenterology 141(5):1749–1761.e1.
Previous study indicated that NF-κB could bind to a region of the miR155 promoter.
Luciferase reporter assay
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Chromatin immunoprecipitation
(ChIP) assay
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NF-κB binds to the miR-155 promoter region and induces miR-155 expression
and subsequently decreases FOXO3a expression, resulting in the increased
tumorigenicity of lung cancer.
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Inhibition of NF-κB Activity Enhances Gefitinib Sensitivity
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To further investigate whether NF-κB inactivation was associated
with a decrease in the transcriptional activity of miR-155
BIM:
Bcl-2-like protein 11(proapoptotic protein)
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H292:
Mucoepidermoid carcinoma
(EGFR Wild-type)
Suppression of p50 in H292 cells obviously increased gefitinib sensitivity.
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NF-κB activity is responsible for generating gefitinib resistance and CSC
properties through the suppression of FOXO3a via miR-155 in lung cancer.
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Physiological Effects of NF-κB/miR-155/FOXO3a Is Related to the
Gefitinib Response
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TRAIL:
Tumor necrosis factorrelated apoptosis
inducing ligand
PUMA:
P53 up-regulated
modulator of apoptosis
FasL:
Cell death surface
receptor Fas ligand
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These data demonstrate that the NF-κB /miR-155/FOXO3a axis is critical for
gefitinib sensitivity and the suppression of the cancer stemness of lung cancer.
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Conclusion
(1)FOXO3a could be an independent prognostic marker of lung
cancer.
(2)NF-κB /miR-155/FOXO3a axis plays an important role in lung
cancer patients with acquired resistance to EGFR-TKIs.
(3)Combination of both the NF-κB and EGFR inhibitors might be
a potential therapeutic strategy to improve tumors with resistance
to EGFR-TKIs.
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Chromatin ImmunoPrecipitation assay(ChIP)
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