TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis
Ramesh Singh, Dileep Karri, Hong Shen, Jiangyong Shao, Subhamoy Dasgupta, Shixia Huang, Dean P. Edwards, Michael M. Ittmann, Bert W. O’Malley, Ping Yi
Ramesh Singh, Dileep Karri, Hong Shen, Jiangyong Shao, Subhamoy Dasgupta, Shixia Huang, Dean P. Edwards, Michael M. Ittmann, Bert W. O’Malley, Ping Yi
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Research Article Cell biology Genetics

TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis

Abstract

Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of WT RTKs plays an important role in driving cancer progression. However, the mechanisms underlying how RTKs drive prostate cancer remain incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, an E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK-mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin RTK, as a TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and found that inhibition of TrkA activity abolished TRAF4-dependent cell invasion. TRAF4 promoted K27- and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine autophosphorylation and its interaction with downstream proteins. TRAF4 knockdown also suppressed nerve growth factor (NGF) stimulated TrkA downstream p38 MAPK activation and invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion–associated gene expression in prostate cancer patients, indicating that this signaling axis is significantly activated during oncogenesis. Our results revealed a posttranslational modification mechanism contributing to aberrant non-mutated RTK activation in cancer cells.

Authors

Ramesh Singh, Dileep Karri, Hong Shen, Jiangyong Shao, Subhamoy Dasgupta, Shixia Huang, Dean P. Edwards, Michael M. Ittmann, Bert W. O’Malley, Ping Yi

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Figure 5

TrkA plays an important role in TRAF4-promoted cell invasion.

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TrkA plays an important role in TRAF4-promoted cell invasion. (A) Valida...
(A) Validation of the RPPA data for some of the invasion-related genes after TRAF4 knockdown using qRT-PCR. Right: Levels of TRAF4 in control or TRAF4-knockdown cells as assessed by qRT-PCR. *P < 0.05 by 1-way ANOVA with Dunnett’s multiple comparisons test. n = 3. (B) Expression of TRAF4-regulated genes at different time points following NGF stimulation as assessed by qRT-PCR. n = 3. (C) TrkA knockdown also downregulates TRAF4-regulated invasion-related genes. *P < 0.05 by 1-way ANOVA with Dunnett’s multiple comparisons test. n = 3. (D) Knockdown of TRAF4 inhibited NGF-stimulated gene expression. n = 3. (E) TrkA-selective inhibitor treatment abolished TRAF4-stimulated cell invasion (n = 3). Images were obtained at ×100 magnification. PC3 cells were infected with GFP or TRAF4 adenovirus and then treated with or without 0.5 µM GW441756 for 2 days before seeding in an invasion chamber. *P < 0.05 by 1-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SEM.