The Ubiquitin Ligase UBE4A Inhibits Prostate Cancer Progression by Targeting Interleukin-Like EMT Inducer (ILEI)

Epithelial to mesenchymal transition (EMT) is an important prerequisite for metastasis to secondary organs. Interleukin-like EMT inducer (ILEI) protein has been shown to translationally upregulated during EMT and metastatic progression as a consequence of aberrant TGF-beta signaling. Our initial evaluation of FAM3C (encoding ILEI) and ILEI expression in normal prostate (PCS-440-010) and prostate cancer cell lines (DU145, LNCaP, and PC3) revealed detectable protein expression in only LNCaP cell line even though all cell lines tested had comparable FAM3C expression. Given that PC3 and DU145 cell lines did not have detectable ILEI expression hinted at additional level of regulation of ILEI expression. Treatment with MG-132 resulted in robust detection of ILEI in the PCS-440-010, PC3 and DU145 cell lines, suggesting that at least in these cell lines, ILEI is actively degraded by the proteasome. Mass spectrometric analysis of FLAG immunoprecipitates of untreated and MG-132 treated FLAG-ILEI transfected cells indicated that UBE4A and UBE3C ubiquitin ligases were interacting with ILEI. Ectopic overexpression of UBE4A, but not UBE3C, resulted in destabilization of ILEI in LNCaP cells, whereas RNAi-mediated silencing of UBE4A in PCS-440-010, PC3 and DU145 cell lines resulted in robust accumulation of ILEI, indicating UBE4A as the cognate ubiquitin ligase for ILEI. Co-immunoprecipitation experiments established direct interaction of endogenous ILEI and UBE4A. Furthermore, co-immunoprecipitation of FLAG-tagged ILEI in cells co-transfected with either HA-UBE4A or HA-UBE3C revealed robust polyubiquitinated smear of ILEI in cells transfected with UBE4A, but not UBE3C, thus confirming UBE4A as the ubiquitin ligase for ILEI degradation. Ectopic overexpression of UBE4A, but not UBE3C, in cells was downregulated in vitro migration and invasion in these cells. Cumulatively, our data reveals a novel post-translational regulatory mechanism of regulating ILEI1 expression, a protein required for metastatic progression in prostate cancer cells. (C) 2016