Ubiquitin ligase RNF8 suppresses Notch signaling to regulate mammary development and tumorigenesis
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
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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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 4

Aberrant expansion of the luminal lineage in the mammary glands of Rnf8-mutant females.

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Aberrant expansion of the luminal lineage in the mammary glands of Rnf8-...
(A) Representative FACS plots showing Lin– (CD45–CD31–TER119–) luminal (L: CD29loCD24+) and basal (B: CD29hiCD24+) cells of inguinal mammary glands from 10- to 12-week-old WT and Rnf8–/– females. Data are representative of 3 independent experiments. (B) Dot plots depicting percentages of mammary luminal and basal populations from inguinal mammary glands from females in A (n = 3; mean ± SEM). (C) Dot plots depicting the absolute numbers of MECs and luminal and basal cells per inguinal mammary gland from WT and Rnf8–/– females (n = 3; mean ± SEM). (D) CD24+ MECs from A were analyzed for luminal progenitor (LP; CD29loCD24+CD61+) and luminal differentiated (LD; CD29loCD24+CD61–) cells. Representative FACS images of 3 independent experiments are shown. (E and F) Dot plots depicting percentages (E) and absolute numbers (F) of LP and LD subpopulations from inguinal mammary glands of 10- to 12-week-old WT and Rnf8–/– females (n = 3; mean ± SEM). (G) Dot plots depicting the clonogenic potentials (mean ± SEM) in Matrigel of MECs from WT and Rnf8–/– littermate females (n = 4 each). (H) Representative images of 3 independent experiments showing Ki67/DAPI staining of mammary glands from 6.5-month-old WT and Rnf8–/– female littermates (scale bar: 100 μm), and dot plots depicting quantification of Ki67+ cells from mammary glands of WT and Rnf8–/– females (n = 3; mean ± SEM). A minimum of 1,000 cells per gland were counted. (I) Representative FACS plots showing luminal and basal cells of mammary glands from 6.5-month-old females as indicated (n = 4 each) that had undergone 4 pregnancies and involution to induce Cre-mediated deletion of Trp53. All females were at estrus phase. Two-sided Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001.