RB1 deficiency in triple-negative breast cancer induces mitochondrial protein translation
Robert A. Jones, … , Aaron Schimmer, Eldad Zacksenhaus
Robert A. Jones, … , Aaron Schimmer, Eldad Zacksenhaus
Published August 29, 2016
Citation Information: J Clin Invest. 2016;126(10):3739-3757. https://doi.org/10.1172/JCI81568.
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Research Article Oncology

RB1 deficiency in triple-negative breast cancer induces mitochondrial protein translation

Abstract

Triple-negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which no specific treatment is currently available. Although the retinoblastoma tumor-suppressor gene (RB1) is frequently lost together with TP53 in TNBC, it is not directly targetable. There is thus great interest in identifying vulnerabilities downstream of RB1 that can be therapeutically exploited. Here, we determined that combined inactivation of murine Rb and p53 in diverse mammary epithelial cells induced claudin-low–like TNBC with Met, Birc2/3-Mmp13-Yap1, and Pvt1-Myc amplifications. Gene set enrichment analysis revealed that Rb/p53-deficient tumors showed elevated expression of the mitochondrial protein translation (MPT) gene pathway relative to tumors harboring p53 deletion alone. Accordingly, bioinformatic, functional, and biochemical analyses showed that RB1-E2F complexes bind to MPT gene promoters to regulate transcription and control MPT. Additionally, a screen of US Food and Drug Administration–approved (FDA-approved) drugs identified the MPT antagonist tigecycline (TIG) as a potent inhibitor of Rb/p53-deficient tumor cell proliferation. TIG preferentially suppressed RB1-deficient TNBC cell proliferation, targeted both the bulk and cancer stem cell fraction, and strongly attenuated xenograft growth. It also cooperated with sulfasalazine, an FDA-approved inhibitor of cystine xCT antiporter, in culture and xenograft assays. Our results suggest that RB1 deficiency promotes cancer cell proliferation in part by enhancing mitochondrial function and identify TIG as a clinically approved drug for RB1-deficient TNBC.

Authors

Robert A. Jones, Tyler J. Robinson, Jeff C. Liu, Mariusz Shrestha, Veronique Voisin, YoungJun Ju, Philip E.D. Chung, Giovanna Pellecchia, Victoria L. Fell, SooIn Bae, Lakshmi Muthuswamy, Alessandro Datti, Sean E. Egan, Zhe Jiang, Gustavo Leone, Gary D. Bader, Aaron Schimmer, Eldad Zacksenhaus

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Figure 8

FDA-approved drug screens of Rb/p53-deficient tumors identify TIG, an MPT inhibitor.

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FDA-approved drug screens of Rb/p53-deficient tumors identify TIG, an MP...
(A) Western blot analysis of pRb and p53 in 3 primary mammary tumor cell lines (lines 1 and 2 RbΔp53Δ; line 3 RbΔp53C139R/+). 293T human cells served as a positive control; tubulin was used as a loading control. (B) Heatmap showing expression levels of Rb, p53, and genes associated with the claudin-low subtype in the 3 mouse Rb/p53 lines. (C) Scatter plot depicting the inhibitory effects of 312 FDA-approved drugs at a dose of 10 μM in RbΔp53Δ and RbΔp53mut/+ lines and schematic of the molecular structure of TIG. (D) TIG dose-response curves for 3 RB1-null and 3 RB1-proficient human claudin-low TNBC cell lines by MTT assay. P < 0.0001, by nonlinear regression analysis using GraphPad Prism 6.0. n = 3 assays, each performed in sextuplicate. (E) TIG dose-response curves for 2 RB1-null and 3 RB1-proficient human basal-like TNBC cell lines. P < 0.0001, by nonlinear regression analysis using GraphPad Prism 6.0. n = 3, each performed in sextuplicate. (F) Western blot for COX I and COX II (mitochondrially translated) and COX IV (cytosolically translated) proteins following TIG treatment. Relative protein levels were normalized to the tubulin loading control. (G) Luciferase assay measuring ATP content in TNBC lines after treatment with 2 μM TIG. n = 3, performed with 4 to 10 replicates per experiment. *P < 0.05, by 2-tailed t test. (H) MTT growth assay following combined TIG plus doxorubicin (Dox) on MDA-MB-231 cells. n = 2, each performed in triplicate. ctrl, control.