Acquired platinum resistance involves epithelial to mesenchymal transition through ubiquitin ligase FBXO32 dysregulation
Nobuyuki Tanaka, … , Makoto Suematsu, Mototsugu Oya
Nobuyuki Tanaka, … , Makoto Suematsu, Mototsugu Oya
Published November 3, 2016
Citation Information: JCI Insight. 2016;1(18):e83654. https://doi.org/10.1172/jci.insight.83654.
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Research Article Oncology

Acquired platinum resistance involves epithelial to mesenchymal transition through ubiquitin ligase FBXO32 dysregulation

Abstract

To identify the molecules involved in epithelial to mesenchymal transition (EMT) in urothelial carcinoma (UC) after acquisition of platinum resistance, here we examined the changes in global gene expression before and after platinum treatment. Four invasive UC cell lines, T24, 5637, and their corresponding sublines T24PR and 5637PR with acquired platinum resistance, were assessed by microarray, and the ubiquitin E3 ligase FBXO32 was newly identified as a negative regulator of EMT in UC tumors after acquisition of platinum resistance. In vitro and in vivo studies showed an intimate relationship between FBXO32 expression and EMT, demonstrating that FBXO32 dysregulation in T24PR cells results in elevated expression of the mesenchymal molecules SNAIL and vimentin and decreased expression of the epithelial molecule E-cadherin. The association between FBXO32 expression and EMT was further validated using clinical samples. Knockdown of MyoD expression, a specific target of FBXO32 polyubiquitination, revealed upregulation of E-cadherin expression and downregulation of SNAIL and vimentin expression in T24PR cells. Comparative genomic hybridization array analysis demonstrated loss of heterozygosity at 8q24.13 in T24PR cells, which harbors FBXO32. Our findings suggest the importance of the association between EMT and ubiquitin-proteasome regulation when tumors develop acquired platinum resistance.

Authors

Nobuyuki Tanaka, Takeo Kosaka, Yasumasa Miyazaki, Shuji Mikami, Naoya Niwa, Yutaro Otsuka, Yoji Andrew Minamishima, Ryuichi Mizuno, Eiji Kikuchi, Akira Miyajima, Hisataka Sabe, Yasunori Okada, Per Uhlén, Makoto Suematsu, Mototsugu Oya

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Figure 6

Knockdown and overexpression of FBXO32 and its substrate MyoD.

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Knockdown and overexpression of FBXO32 and its substrate MyoD. (A) Effec...
(A) Effect of FBXO32 knockdown on the expression of E-cadherin, vimentin, and SNAIL in T24 cells. (B) Matrigel invasion assay of T24 cells transfected with FBXO32-specific siRNA (si-FBXO32) at 24 hours (n = 6). The invading cells were stained with Diff-Quik. Box-and-whiskers plots show median (line within box), upper and lower quartile (bounds of box), and minimum and maximum values (bars). *P < 0.05, **P < 0.01, compared with mock-transfected cells. (C) Effect of FBXO32 knockdown on the sensitivity to CDDP in T24 cells. Box-and-whiskers plots show median (line within box), upper and lower quartile (bounds of box), and minimum and maximum values (bars). *P < 0.05, **P < 0.01, compared with mock-transfected cells. (D) Effect of FBXO32 overexpression on the expression of E-cadherin, vimentin, and SNAIL in T24PR cells. (E) Matrigel invasion assay of T24PR cells transfected with FBXO32 at 24 hours (n = 6). The invading cells were stained with Diff-Quik. Box-and-whiskers plots show median (line within box), upper and lower quartile (bounds of box), and minimum and maximum values (bars). *P < 0.05, compared with control cells. (F) Effect of FBXO32 overexpression on the sensitivity to CDDP in T24PR cells. Box-and-whiskers plots show median (line within box), upper and lower quartile (bounds of box), and minimum and maximum values (bars). *P < 0.05, compared with control cells. (G) Western blot analysis of MyoD in T24PR and T24 cells. (H) Immunofluorescence staining for MyoD in T24PR and T24 cells. (I) Effect of FBXO32 overexpression on the expression of MyoD in T24PR cells. (J) Effect of MyoD knockdown on the expression of E-cadherin, vimentin, and SNAIL in T24PR cells. (K) Matrigel invasion assay of T24PR cells transfected with si-MYOD at 24 hours (n = 6). The invading cells were stained with Diff-Quik. Box-and-whiskers plots show median (line within box), upper and lower quartile (bounds of box), and minimum and maximum values (bars). *P < 0.05, **P < 0.01, compared with mock-transfected cells. HPF, high-power field.