FBXO15 regulates P‐glycoprotein/ABCB1 expression through the ubiquitin–proteasome pathway in cancer cells

• Published in: Cancer science Vol. 104; no. 6; pp. 694 - 702
• Main Authors: Katayama, Kazuhiro, Noguchi, Kohji, Sugimoto, Yoshikazu
• Format: Journal Article
• Language: English
• Published: England John Wiley and Sons Inc 01.06.2013
• Subjects: Anaphase-Promoting Complex-Cyclosome; ATP Binding Cassette Transporter, Sub-Family B; ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism; Blotting, Western; Cell Line, Tumor; Colonic Neoplasms - metabolism; F-Box Proteins - metabolism; Flow Cytometry; Gene Knockdown Techniques; Humans; Immunoprecipitation; Original; Proteasome Endopeptidase Complex - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transfection; Ubiquitin - metabolism; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligase Complexes - metabolism
• ISSN: 1347-9032; 1349-7006
• DOI: 10.1111/cas.12145

Expression of P‐glycoprotein (P‐gp)/ABCB1 on cancer cell surfaces is a critical determinant of anticancer drug resistance. Regulators of P‐gp expression and function are key molecules controlling drug resistance. Here we report the mechanism underlying the ubiquitin–proteasome pathway‐mediated degradation of P‐gp. The proteasome inhibitor MG132 increased the P‐gp level, enhanced its ubiquitination, and delayed the disappearance of the ubiquitinated P‐gp. To search for regulators of P‐gp ubiquitination, MALDI–time of flight mass spectrometry analyses were carried out, and 22 candidates were identified as P‐gp binding partners. Among them, FBXO15/Fbx15 is known as an F‐box protein in the ubiquitin E3 ligase complex, Skp1‐Cullin1‐FBXO15 (SCFFbx15); therefore, we further studied the involvement of FBXO15 on P‐gp degradation. Coprecipitation assays revealed that FBXO15 bound to P‐gp. We screened ubiquitin‐conjugating enzyme E2s that bind to FBXO15 and P‐gp; Ube2r1/Cdc34/Ubc3 was found to be a binding partner. Exogenous FBXO15 expression enhanced P‐gp ubiquitination, but FBXO15 knockdown suppressed it. FBXO15 knockdown increased P‐gp expression without affecting its mRNA level. Ube2r1 knockdown decreased P‐gp ubiquitination, and simultaneous knockdown of Ube2r1 with FBXO15 further suppressed the ubiquitination. Ube2r1 knockdown increased P‐gp expression, suggesting that Ube2r1 is a partner of FBXO15 in P‐gp ubiquitination. FBXO15 knockdown enhanced vincristine resistance and lowered intracellular levels of rhodamine 123. These data suggest that FBXO15 and Ube2r1 regulate P‐gp expression through the ubiquitin–proteasome pathway.