Mutant TP53 modulates metastasis of triple negative breast cancer through adenosine A2b receptor signaling

Eisuke Horigome, Michiru Fujieda, Tadashi Handa, Ayaka Katayama, Masashi Ito, Ami Ichihara, Daiki Tanaka, Navchaa Gombodorj, Shinji Yoshiyama, Arito Yamane, Keiichi Yamada, Jun Horiguchi, Kazuo Shinozuka, Tetsunari Oyama, Masahiko Nishiyama, Susumu Rokudai, Eisuke Horigome, Michiru Fujieda, Tadashi Handa, Ayaka Katayama, Masashi Ito, Ami Ichihara, Daiki Tanaka, Navchaa Gombodorj, Shinji Yoshiyama, Arito Yamane, Keiichi Yamada, Jun Horiguchi, Kazuo Shinozuka, Tetsunari Oyama, Masahiko Nishiyama, Susumu Rokudai

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.

Keywords: ADORA2B; NF-kB; TP53; breast cancer; synthetic lethality.

Conflict of interest statement

CONFLICTS OF INTEREST The authors have declared no conflicts of interests.

Figures

Figure 1. Gene expression profiling to identify…
Figure 1. Gene expression profiling to identify synthetic lethal genes in TP53-mutated TNBC
(A) Schematic of the method used to identify synthetic lethal genes in TP53-mutated (R248Q) cell lines. A total of 117 significantly up-regulated genes (P < 0.05 and FDR < 0.5) were identified by comparison with mutant TP53-R248Q cells (HCC-70 and HCC-1143) and wild-type TP53 cells (MCF-7). (B) Subsequently, 12 synthetic lethal candidate genes were identified by shRNA lentiplex screening. The genes are listed in descending order in regards to the relative fold-change in the level of mutant TP53 (R248Q).
Figure 2. ADORA2B expression is correlated with…
Figure 2. ADORA2B expression is correlated with p53 expression and poor prognosis in TNBC samples
(A) Representative immunohistochemical staining of a breast cancer sample. ADORA2B immunostaining demonstrates a nuclear and cytoplasmic pattern, with a score of 5. Scale bars are 200 μm. (B) Stratification of TNBC patient samples (n = 66) based on the expression of ADORA2B and p53. The chi-square test was performed for the association (P = 0.013). (C, D) Kaplan–Meier analysis of survival in TNBC patients grouped by ADORA2B expression (n = 66). A statistically significant difference in OS and PFS was observed between high ADORA2B expression patients and those with low ADORA2B expression (OS, P = 0.025; PFS, P = 0.042). (E, F) Kaplan–Meier analysis of survival in breast cancer patients grouped by ADORA2B expression (n = 232). No statistically significant difference in OS and PFS was observed between those with high or low ADORA2B expression (OS, P = 0.54; PFS, P = 0.53).
Figure 3. Mutant TP53 (R248Q) induces ADORA2B…
Figure 3. Mutant TP53 (R248Q) induces ADORA2B expression and tumorigenesis in breast cancer cells
(A) MCF-7 cells were retrovirally transduced with empty vector control (Mock), or plasmids expressing wild-type or mutant TP53 (R248Q, R175H, R273H and G245F). The mRNA levels of ADORA2B were subsequently determined by real-time RT-PCR. (B) The cells transduced as in (A), were subjected to immunoblotting using antibodies against ADORA2B, p53, and β-actin (as a loading control). (C) Breast cancer tumorigenesis is increased by mutant TP53 (R248Q) expression. HCC70 cells were retrovirally transduced with empty vector control (Mock), mutant TP53 (R248Q), or ADORA2B expression plasmids. Anchorage-independent growth of the cells after transduction was monitored by soft agar colony formation assay. (D) Breast cancer metastasis is increased by mutant TP53 (R248Q). The cells transduced as in (C), were evaluated by invasion assay. Data in bar graphs represent mean ± SD; *P < 0.05.
Figure 4. Mutant TP53 depletion inhibits breast…
Figure 4. Mutant TP53 depletion inhibits breast cancer tumorigenesis and modulates ADORA2B induction through a gain-of function of mutant TP53
(A) The breast cancer cell line HCC-70 was treated with siRNAs for either luciferase (siLUC) as a control, TP53, NF-kB or ADORA2B (A2B#1 and A2B#2). Induction of ADORA2B expression was determined by real-time RT-PCR. (B) HCC-70 cells were treated with siRNAs as in (A), and cultured under either normoxic or hypoxic condition for 24 h. Cell lysates were subsequently analyzed by immunoblotting analysis using anti-ADORA2B, TP53, or β-actin antibodies. (C) Breast cancer tumorigenesis is decreased by the shRNA-mediated depletion of mutant TP53. HCC-70 cells were treated with shRNAs for either luciferase (siLUC) as a control, TP53, or ADORA2B. Anchorage-independent growth after depletion was monitored by soft agar colony formation. (D) p53-depletion induces tumorigenesis through ADORA2B in breast cancer cells. The growth of p53-depleted HCC-70 cells after induction of ADORA2B was monitored by xenograft assay. The growth of ADORA2B-expressing HCC-70 cells after depletion of luciferase as a control (shLUC) or mutant p53, was monitored by xenograft assays. Representative images of xenografts from subcutaneously transplanted are shown. The results of six independent injections of knockdown cells are shown. Twenty days after implantation, the length (L) and width (W) of the tumor mass were measured, and the tumor volume (TV) was calculated using the equation: TV = (L × W2)/2. *P < 0.05. (E) The cyclic peptide SA-I decreases breast cancer tumorigenesis via ADORA2B. HCC-70 cells were treated with 10 μM of PSB-1115, 10 μM of Bay 11-7082, or 10 μM of cyclic peptide SA-I for 24 h. Cell lysates were subsequently immunoblotted with anti-ADORA2B, HIF1, or β-actin antibodies. (F) HCC-70 cells treated with 10 uM of PSB-1115, 10 uM of Bat11-7082, or 10 uM of SA-I were monitored for growth by soft agar colony formation assay. Data in bar graphs represent mean ± SD; *P < 0.05.

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