Ubiquitin ligase RNF8 suppresses Notch signaling to regulate mammary development and tumorigenesis
Li Li, … , Anne Hakem, Razq Hakem
Li Li, … , Anne Hakem, Razq Hakem
Published September 17, 2018
Citation Information: J Clin Invest. 2018;128(10):4525-4542. https://doi.org/10.1172/JCI120401.
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Research Article Cell biology Oncology

Ubiquitin ligase RNF8 suppresses Notch signaling to regulate mammary development and tumorigenesis

Abstract

The E3 ubiquitin ligase RNF8 plays critical roles in maintaining genomic stability by promoting the repair of DNA double-strand breaks (DSBs) through ubiquitin signaling. Abnormal activation of Notch signaling and defective repair of DSBs promote breast cancer risk. Here, we found that low expression of the full-length RNF8 correlated with poor prognosis for breast cancer patients. Our data revealed that in addition to its role in the repair of DSBs, RNF8 regulated Notch1 signaling and cell-fate determination of mammary luminal progenitors. Mechanistically, RNF8 acted as a negative regulator of Notch signaling by ubiquitylating the active NOTCH1 protein (N1ICD), leading to its degradation. Consistent with abnormal activation of Notch signaling and impaired repair of DSBs in Rnf8-mutant mammary epithelial cells, we observed increased risk of mammary tumorigenesis in mouse models for RNF8 deficiency. Notably, deficiency of RNF8 sensitized breast cancer cells to combination of pharmacological inhibitors of Notch signaling and poly(ADP-ribose) polymerase (PARP), suggesting implications for treatment of breast cancer associated with impaired RNF8 expression or function.

Authors

Li Li, Kiran Kumar Naidu Guturi, Brandon Gautreau, Parasvi S. Patel, Amine Saad, Mayako Morii, Francesca Mateo, Luis Palomero, Haithem Barbour, Antonio Gomez, Deborah Ng, Max Kotlyar, Chiara Pastrello, Hartland W. Jackson, Rama Khokha, Igor Jurisica, El Bachir Affar, Brian Raught, Otto Sanchez, Moulay Alaoui-Jamali, Miguel A. Pujana, Anne Hakem, Razq Hakem

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Figure 5

RNF8 negatively regulates Notch signaling in murine mammary luminal progenitors and mammary tumors.

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RNF8 negatively regulates Notch signaling in murine mammary luminal prog...
(A) Quantitative reverse transcriptase PCR (RT-qPCR) showing the expression changes of Notch targets and other genes in luminal progenitors from mammary glands of Rnf8–/– females at estrus phase compared with WT littermates. (B) Immunoblot analysis of N1ICD and HES1 in the indicated cells. (C) Representative immunohistochemical analysis of N1ICD levels in the indicated mammary adenocarcinomas (n = 3 each; scale bar: 25 μm). (D) Immunoblot showing expression of the indicated proteins in Rnf8–/– Trp53Δ/Δ mammary tumor cells reconstituted with mock or RNF8-FLAG. (E) RT-qPCR showing increased expression of indicated Notch1 targets in mock-reconstituted, compared with RNF8WT-reconstituted, Rnf8–/– Trp53Δ/Δ mammary tumor cells engrafted for 40 days in NSG mice (see Figure 1C). (F) Notch reporter 10xCBF1-Luc showing relative activation of Notch signaling in mock-reconstituted Rnf8–/– Trp53Δ/Δ mammary tumor cells compared with RNF8WT-reconstituted controls. *P < 0.05, ****P < 0.0001, 2-way ANOVA followed by Tukey’s test. (G) Heatmap showing the standardized expression of Notch targets and/or Notch pathway elements in Rnf8–/– Trp53Δ/Δ mammary tumor cells reconstituted with mock or RNF8-FLAG. (H) Notch targets differentially expressed in RNA-Seq analysis in G were validated by RT-qPCR in 3 different Rnf8–/– Trp53Δ/Δ mammary tumor cell lines and their RNF8-reconstituted controls. Data in B and D are representative of at least 3 experiments. A, E, F, and H: At least 3 experiments were performed, and the means ± SEM are shown. A, E, and H: 2-sided Student’s t test, Rnf8–/– Trp53Δ/Δ mammary tumor cells mock-reconstituted compared with RNF8WT-reconstituted controls. *P < 0.05, **P < 0.01, ***P < 0.001. H: All P values were less than 0.05.