TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis
Ramesh Singh, … , Bert W. O’Malley, Ping Yi
Ramesh Singh, … , Bert W. O’Malley, Ping Yi
Published May 1, 2018
Citation Information: J Clin Invest. 2018;128(7):3129-3143. https://doi.org/10.1172/JCI96060.
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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 2

TRAF4 plays a role in prostate cancer cell migration, invasion, and metastasis.

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TRAF4 plays a role in prostate cancer cell migration, invasion, and meta...
Knockdown of TRAF4 in PC3 cells inhibited cell migration (A) and invasion (B). Left panels: Cells migrated through migration chamber (A) or Matrigel (B) (n = 3). Images were obtained at ×100 magnification. Middle panels: Quantitation of cell numbers per field. Right panels: Levels of TRAF4 in control or TRAF4-knockdown cells as assessed by qRT-PCR. *P < 0.05 by Student’s t test versus siControl or siTRAF4 (A) and *P < 0.05 by 1-way ANOVA with Dunnett’s multiple comparisons test (B). Data are presented as mean ± SEM. (C) TRAF4 knockdown reduced prostate cancer cell colonization and metastasis in vivo. Top left: Representative bioluminescence images of SCID mice at different time periods after tail vein injection of PC3 shControl or shTRAF4 cells. Lower left: Quantitation of bioluminescence signals at the lung area in PC3 shControl- or shTRAF4-injected mice (n = 5). Right: Levels of TRAF4 in control or TRAF4-stable-knockdown cells as assessed by qRT-PCR. *P < 0.05 by 2-tailed Student’s t test. (D) TRAF4 knockdown reduced lung metastatic nodules. H&E-stained lung sections and graph showing the number of lung metastasis nodules after tail vein injection (n = 10). Data are presented as mean ± SEM. *P < 0.05 by 2-tailed Student’s t test. (E) H&E-stained bone sections (left) and immunohistochemistry using anti-luciferase antibody staining (right) confirming bone metastasis in the control group but not in the shTRAF4-knockdown group. Images were obtained at ×40 and ×100 (insets) magnification.