ABSTRACT
PURPOSE: The identification of genes with synthetic lethality in the context of mutant TP53 is a promising strategy for the treatment of basal-like triple negative breast cancer (TNBC). This study investigated regulators of mutant TP53 (R248Q) in basal-like TNBC and their impact on tumorigenesis. EXPERIMENTAL DESIGN: TNBC cells were analyzed by RNA-seq, and synthetic-lethal shRNA knock-down screening, to identify genes related to the expression of mutant TP53. A tissue microarray of 232 breast cancer samples, that included 66 TNBC cases, was used to assess clinicopathological correlates of tumor protein expression. Functional assays were performed in vitro and in vivo to assess the role of ADORA2B in TNBC. RESULTS: Transcriptome profiling identified ADORA2B as up-regulated in basal-like TNBC cell lines with R248Q-mutated TP53, with shRNA-screening suggesting the potential for a synthetic-lethal interaction between these genes. In clinical samples, ADORA2B was highly expressed in 39.4% (26/66) of TNBC patients. ADORA2B-expression was significantly correlated with ER (P < 0.01), PgR (P = 0.027), EGFR (P < 0.01), and tumor size (P = 0.037), and was an independent prognostic factor for outcome (P = 0.036). In line with this, ADORA2B-transduced TNBC cells showed increased tumorigenesis, and ADORA2B knockdown, along with mutant p53 knockdown, decreased metastasis both in vitro and in vivo. Notably, the cytotoxic cyclic peptide SA-I suppressed ADORA2B expression and tumorigenesis in TNBC cell lines. CONCLUSIONS: ADORA2B expression increases the oncogenic potential of basal-like TNBC and is an independent factor for poor outcome. These data suggest that ADORA2B could serve as a prognostic biomarker and a potential therapeutic target for basal-like TNBC.
ABSTRACT
Levels of the N-terminally truncated isoform of p63 (ΔN p63), well documented to play a pivotal role in basal epidermal gene expression and epithelial maintenance, need to be strictly regulated. We demonstrate here that the anaphase-promoting complex/cyclosome (APC/C) complex plays an essential role in the ubiquitin-mediated turnover of ΔNp63α through the M-G1 phase. In addition, syntaxin-binding protein 4 (Stxbp4), which we previously discovered to bind to ΔNp63, can suppress the APC/C-mediated proteolysis of ΔNp63. Supporting the physiological relevance, of these interactions, both Stxbp4 and an APC/C-resistant version of ΔNp63α (RL7-ΔNp63α) inhibit the terminal differentiation process in 3D organotypic cultures. In line with this, both the stable RL7-ΔNp63α variant and Stxbp4 have oncogenic activity in soft agar and xenograft tumor assays. Notably as well, higher levels of Stxbp4 expression are correlated with the accumulation of ΔNp63 in human squamous cell carcinoma (SCC). Our study reveals that Stxbp4 drives the oncogenic potential of ΔNp63α and may provide a relevant therapeutic target for SCC.
Subject(s)
Anaphase-Promoting Complex-Cyclosome/metabolism , Carcinogenesis/pathology , Carcinoma, Squamous Cell/pathology , Gene Expression Regulation, Neoplastic , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Vesicular Transport Proteins/metabolism , Anaphase-Promoting Complex-Cyclosome/genetics , Animals , Apoptosis , Carcinogenesis/genetics , Carcinogenesis/metabolism , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Cell Cycle , Cell Differentiation , Cell Proliferation , Cells, Cultured , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Mice , Mice, SCID , Proteolysis , Transcription Factors/genetics , Tumor Suppressor Proteins/genetics , Vesicular Transport Proteins/genetics , Xenograft Model Antitumor AssaysABSTRACT
Purpose: Expression of the ΔN isoform of p63 (ΔNp63) is a diagnostic marker highly specific for lung squamous cell carcinoma (SCC). We previously found that Syntaxin Binding Protein 4 (STXBP4) regulates ΔNp63 ubiquitination, suggesting that STXBP4 may also be an SCC biomarker. To address this issue, we investigated the role of STXBP4 expression in SCC biology and the impact of STXBP4 expression on SCC prognosis.Experimental Design: We carried out a clinicopathologic analysis of STXBP4 expression in 87 lung SCC patients. Whole transcriptome analysis using RNA-seq was performed in STXBP4-positive and STXBP4-negative tumors of lung SCC. Soft-agar assay and xenograft assay were performed using overexpressing or knockdown SCC cells.Results: Significantly higher levels of STXBP4 expression were correlated with accumulations of ΔNp63 in clinical lung SCC specimens (Spearman rank correlation ρ = 0.219). Notably, STXBP4-positive tumors correlated with three important clinical parameters: T factor (P < 0.001), disease stage (P = 0.030), and pleural involvement (P = 0.028). Whole transcriptome sequencing followed by pathway analysis indicated that STXBP4 is involved in functional gene networks that regulate cell growth, proliferation, cell death, and survival in cancer. Platelet-derived growth factor receptor alpha (PDGFRα) was a key downstream mediator of STXBP4 function. In line with this, shRNA mediated STXBP4 and PDGFRA knockdown suppressed tumor growth in soft-agar and xenograft assays.Conclusions: STXBP4 plays a crucial role in driving SCC growth and is an independent prognostic factor for predicting worse outcome in lung SCC. These data suggest that STXBP4 is a relevant therapeutic target for patients with lung SCC. Clin Cancer Res; 23(13); 3442-52. ©2017 AACR.
