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IMI CONFIDENTIAL
Cancer research, 2013;73:6938-6950, IF= 8.65
You chunyan2014.02.20
TRAF4 Is a Critical Molecule for Akt Activation in Lung Cancer
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Introduction
Lung cancer
a. Lung cancer is the leading cause of cancer-
related deaths in the world.
b. A major challenge in treating lung cancer
is to identify novel therapeutic targets that can
complement current chemotherapy.
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Akt signaling a. Akt signaling plays a major role in tumorigenesis by regulating cell proliferation, cell cycle, cell survival, and metabolism
b. Akt is constitutively activated in lung cancer, in which it can regulate cancer cell growth and survival and confer resistance to chemo-or radiotherapy
c. Akt activated by various stimuli, including insulin and EGF, and Akt phosphorylation at Thr308 by PDK1 and at Ser473 by mTORC2 is required for full activation. Lys63-mediated ubiquitination is essential for Akt activity.
Introduction
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Introduction
TRAF4
a. TRAF4 is involved in embryogenesis and central nervous
system myelin homeostasis.
b. TRAF4 is widely and highly expressed during development
and TRAF4 deficiency results in embryonic lethal, severe
developmental changes in the respiratory system, axial
skeleton, and nervous system.
c. TRAF4 was involved in the initiation and progression of
primary breast cancers and metastases.
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Introduction
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In this report:TRAF4 plays an important role in lung tumorigenesis
a. The knockdown of TRAF4 dramatically attenuated the malignant phenotype in lung cancer, including proliferation, anchorage-independent growth, and tumor formation ability in nude mice.
b. TRAF4, but not Skp2, is required for EGF-induced Akt activation through its ubiquitination in lung cancer.
c. TRAF4 deficiency markedly impaired the activity of Akt signaling and Akt-mediated lung cancer glycolysis
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Materials and Methods
Mice
Athymic nude mice （无胸腺裸鼠）Cell lines
293T 、 HaCaT cells ， H460 、 H1650 、 H1975 、 H1299 、 H5
20 、 HCC827 、 H358
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Materials and Methods Reagents and antibodies
Construction of expression vectors
Cell culture and transfection
Lentiviral Infection
Immunoblotting and immunoprecipitation
Membrane fractionation
MTS assay
Immunofluorescence staining
Anchorage-independent growth
Immunohistochemical analysis of tissue array
In vivo tumor growth
Glucose uptake and lactate production measurement
Ubiquitination assays
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Results
FIGURE 1. A, TRAF4 is highly expressed in lung cancer cells.B, TRAF4 is highly expressed in lung cancer tissues.
1. TRAF4 is overexpressed in human lung cancer
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Results
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2. TRAF4 has a critical role in cell transformation
Figure 2.TRAF4 plays a critical role in cell transformation. A, knockdown of TRAF4 blocks EGF-induced transformation. B, overexpression of TRAF4 promotes proliferation.
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C, Overexpression of TRAF4 increases anchorage-independent growth.
2. TRAF4 has a critical role in cell transformation
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2. TRAF4 has a critical role in cell transformation
D, TRAF4 facilitates H-RasG12V–induced cell transformation.
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Results
Figure 3. Knocking down TRAF4 expression in human lungcancer cells reduces tumorigenic properties. A and C, knockdown TRAF4 attenuates A549 anchorage-dependent and -independent growth.
3.Knockdown TRAF4 in human lung cancer cells reduces tumorigenic properties
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B and D, knockdown TRAF4 attenuates anchorage-dependent and anchorage-independent growth of H460 cells.
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E, knockdown of TRAF4 reduces tumorigenic properties of A549 human lung cancer cells.
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Figure 4. TRAF4 regulates EGF-induced Akt activation. A, TRAF4 regulates Akt activation in HaCaT cells.
Results4.TRAF4 is required for Akt activation and recruitment to the membrane
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Results
4.TRAF4 is required for Akt activation and recruitment to the membrane
FIGURE 4. B ， TRAF4 regulates Akt activation in A549 lung cancer cells.
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FIGURE 4. B ， TRAF4 regulates Akt activation in A549 lung cancer cells.
Results4.TRAF4 is required for Akt activation and recruitment to the membrane
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FIGURE 4. D, TRAF4 regulates recruitment of Akt to the membrane.
Results
4.TRAF4 is required for Akt activation and recruitment to the membrane
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5. TRAF4 interacts with Akt1
Results
Figure 5.TRAF4 binds to Akt1. A, TRAF4 interacts with Akt1.
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Results
Figure 5. TRAF4 binds to Akt1. B, TRAF4 interacts with endogenous Akt1. C, TRAF4 interacts with Akt1 in HaCaT cells stably overexpressing TRAF4.
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Results
FIGURE 5. D, the TRAF domain mediates the interaction between TRAF4 and Akt1
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Results
Figure 6. A, TRAF4 induces Lys63-linked Akt1 polyubiquitination. B, TRAF4 affects endogenous Akt ubiquitination upon EGF treatment
6. TRAF4 is required for Akt1 Ub-Lys63 ubiquitination
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Results
FIGURE 6. C, TRAF6 and Ubc13 mediate Akt1 ubiquitination. D, TRAF4 and UbcH5a mediate Akt1 ubiquitination.
6. TRAF4 is required for Akt1 Ub-Lys63 ubiquitination
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Figure 7. A ， Akt activation is required for TRAF4-regulated glycolysis in human lung cancer cell A549.
Results7. TRAF4 regulates lung cancer cellular glycolysis that is dependent on Akt activity
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Figure 7. B ， Akt activation is required for TRAF4-regulated glycolysis in human lung cancer cell H460.
7. TRAF4 regulates lung cancer cellular glycolysis that is dependent on Akt activity
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Figure 7. C ， Overexpression of constitutively active Akt (CA-Akt1) rescues glycolysis in knockdown TRAF4-A549 lung cancer cells.
7. TRAF4 regulates lung cancer cellular glycolysis that is dependent on Akt activity
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Figure 7. D, Overexpression of constitutively active Akt (CA-Akt1) rescues glycolysis in knockdown TRAF4-H460 lung cancer cells.
7. TRAF4 regulates lung cancer cellular glycolysis that is dependent on Akt activity
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Discussion
TRAF4 was initially identified in human breast carcinoma. TRAF4 is engaged in several signaling pathways.
TRAF4 is overexpressed in human lung cancer cell lines and tissues. TRAF4 promoted cell transformation.
TRAF4 is involved in regulating the Akt signaling pathway.
This is the first report that TRAF4 is required for Akt ubiquitination.
This report first demonstrated that TRAF4 mediates glycolysis in human lung cancer by regulating the expression of Glut1 and HK2 through Akt signaling
Overall, TRAF4 regulates Akt ubiquitination and activation and plays a critical role in human lung carcinogenesis
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