J Korean Med Sci.  2017 Aug;32(8):1327-1336. 10.3346/jkms.2017.32.8.1327.

Identification of Downstream Genes of the mTOR Pathway that Predict Recurrence and Progression in Non-Muscle Invasive High-Grade Urothelial Carcinoma of the Bladder

• Jin S
• Chang IH
• Kim JW
• Whang YM
• Kim HJ
• Hong SA
• Lee TJ

• KMID: 2439468
• DOI: http://doi.org/10.3346/jkms.2017.32.8.1327

Abstract

Microarray analysis was used to investigate the lack of identified mammalian target of rapamycin (mTOR) pathway downstream genes to overcome cross-talk at non-muscle invasive high-grade (HG)-urothelial carcinoma (UC) of the bladder, gene expression patterns, gene ontology, and gene clustering by triple (p70S6K, S6K, and eIF4E) small interfering RNAs (siRNAs) or rapamycin in 5637 and T24 cell lines. We selected mTOR pathway downstream genes that were suppressed by siRNAs more than 2-fold, or were up-regulated or down-regulated by rapamycin more than 2-fold. We validated mTOR downstream genes with immunohistochemistry using a tissue microarray (TMA) of 125 non-muscle invasive HG-UC patients and knockout study to evaluate the synergistic effect with rapamycin. The microarray analysis selected mTOR pathway downstream genes consisting of 4 rapamycin up-regulated genes (FABP4, H19, ANXA10, and UPK3A) and 4 rapamycin down-regulated genes (FOXD3, ATP7A, plexin D1, and ADAMTS5). In the TMA, FABP4, and ATP7A were more expressed at T1 and FOXD3 was at Ta. ANXA10 and ADAMTS5 were more expressed in tumors ≤ 3 cm in diameter. In a multivariate Cox regression model, ANXA10 was a significant predictor of recurrence and ATP7A was a significant predictor of progression in non-muscle invasive HG-UC of the bladder. In an ATP7A knock-out model, rapamycin treatment synergistically inhibited cell viability, wound healing, and invasion ability compared to rapamycin only. Activity of the ANXA10 and ATP7A mTOR pathway downstream genes might predict recurrence and progression in non-muscle invasive HG-UC of the bladder. ATP7A knockout overcomes rapamycin cross-talk.

Keyword

Bladder Cancer; mTOR; Biomarker; Microarray; Recurrence; Progression

MeSH Terms

Cell Line
Cell Survival
Gene Expression
Gene Ontology
Humans
Immunohistochemistry
Microarray Analysis
Recurrence*
RNA, Small Interfering
Sirolimus
Urinary Bladder Neoplasms
Urinary Bladder*
Wound Healing
RNA, Small Interfering
Sirolimus

Figure

• 

  Fig. 1 Protein-level validation of p70S6K and eIF4E suppression after RPS6KB1 and eIF4E siRNA treatments, gene filtering, and gene expression profiling in HG-UC cell lines. (A) Reduced gene expression was observed in 5637 and T24 cells after transfection with siRNAs against p70S6K and eIF4E. Inhibited gene expression of both p70S6K and eIF4E was evident after treatment with triple siRNAs (p70S6K, S6K, and eIF4E). (B) Gene filtering process in 5637 and T24 cells. (C) Hierarchical clustering analysis of 5637 and T24 cells; red spots indicate up-regulation, green spots indicate down-regulation, black spots indicate an absence of modulation, and gray spots indicate the absence of values. siRNA = small interfering RNA, HG = high-grade, UC = urothelial carcinoma, PI3K = phosphoinositide 3-kinase, mTOR = mammalian target of rapamycin.

• 

  Fig. 2 Expression of downstream genes of the mTOR pathway in HG-UC of bladder tissues, and Kaplan-Meier curves based on staining results in HG-UC patients. (A) Immunohistochemical staining of ANXA10 and ATP7A in paraffin-embedded sections of HG-UC tissues (× 40, × 200). (B) In RFS curves, HG-UC with no or weak ANXA10 immunohistochemical staining exhibited decreased RFS (P = 0.037). In PFS curves, HG-UC with strong ATP7A immunohistochemical staining exhibited decreased PFS (P = 0.004). mTOR = mammalian target of rapamycin, HG = high-grade, UC = urothelial carcinoma, RFS = recurrence-free survival, PFS = progression-free survival.

• 

  Fig. 3 Down-regulation of up-regulated ATP7A expression after siRNAs or rapamycin treatment in HG-UC 5637 cell line. (A) Western blots of whole-cell lysates from ATP7A stable knockdown and non-targeted shRNA control 5637 cell line after puromycin selection: control, pLKO.1 control vector; shATP7A#1 and shATP7A#2, ATP7A shRNA vectors. (B) Cell viability determined by MTT assay. Data are mean ± SD (n = 6). (C) ATP7A stable knockdown 5637 cell lines exhibited reduced cell migration to rapamycin treatment. (D) ATP7A stable knockdown 5637 cell lines exhibited significantly reduced cell invasion compared to that of the control. siRNA = small interfering RNA, HG = high-grade, UC = urothelial carcinoma, shRNA = short-hairpin RNA, MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, SD = standard deviation. *P < 0.050, †P < 0.010.

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