The Mre11 complex suppresses oncogene-driven breast tumorigenesis and metastasis

Abstract

The DNA damage response (DDR) is activated by oncogenic stress, but the mechanisms by which this occurs, and the particular DDR functions that constitute barriers to tumorigenesis, remain unclear. We established a mouse model of sporadic oncogene-driven breast tumorigenesis in a series of mutant mouse strains with specific DDR deficiencies to reveal a role for the Mre11 complex in the response to oncogene activation. We demonstrate that an Mre11-mediated DDR restrains mammary hyperplasia by effecting an oncogene-induced G2 arrest. Impairment of Mre11 complex functions promotes the progression of mammary hyperplasias into invasive and metastatic breast cancers, which are often associated with secondary inactivation of the Ink4a-Arf (CDKN2a) locus. These findings provide insight into the mechanism of DDR engagement by activated oncogenes and highlight genetic interactions between the DDR and Ink4a-Arf pathways in suppression of oncogene-driven tumorigenesis and metastasis.

Figure 1. NeuT-Induced Mammary Hyperplasia in DDR Mutant Animals

(A) The experimental timeline for mammary intraductal RCAS injection and subsequent analyses. (B) Representative H&E staining of mammary glands from (Bi and Bii) WT, (Biii). Chk2^−/−, (Biv) p53^515C/515C, (Bv) p53^−/−, (Bvi) Nbs1^ΔB/ΔB, (Bvii) Mre11^ATLD1/ATLD1, or (Bviii) 53BP1^−/− mice, 3 weeks after injection with (Bi) 2 × 3 10⁶ IU RCAS-HA-β-actin or (Bii–Bviii) 2 × 3 10⁶ IU RCAS-HA-NeuT. Scale bar = 50 μm. (C) Percent glandular area in the injected mammary gland #4 relative to the percent glandular area in the noninjected mammary gland #5, with at least three injected glands per genotype analyzed. Error bars indicate SEM. *p < 0.05 in comparison to WT control, and **p < 0.05 in comparison to WT + NeuT, using an unpaired t test. (D) Summary of NeuT-induced qualitative changes in mammary gland morphology across the various genotypes.

Figure 2. The Oncogene-Induced DDR Is Mre11 Dependent

(A) Confocal imaging of γH2AX immunofluorescence (IF) in (Ai) WT control, (Aii) WT + NeuT, (Aiii) Mre11^ATLD1/ATLD1 + NeuT, and (Aiv) p53^−/− + NeuT mammary glands. Scale bar = 20 μm. (Av) depicts volumetric analysis of γH2AX signal per nuclear volume in the various genotypes, with each data point representing a distinct nucleus. Error bars indicate the interquartile range around the median value. ****p < 0.0001, as per Kolmogorov-Smirnov (K-S) test. (B) 53BP1 IF in (Bi) WT control, (Bii) WT + NeuT, (Biii) Mre11^ATLD1/ATLD1 + NeuT, and (Biv) 53BP1^−/− + NeuT mammary glands. Scale bar = 20 μm. The inset in (Bii) shows a higher magnification image of the nuclear 53BP1 nuclear signal. (Bv) depicts volumetric quantification of 53BP1 signal per nuclear volume from at least three glands for each genotype. Error bars represent the interquartile range relative to the median value. ****p < 0.0001, as per K-S test. (C) Confocal imaging of pKAP1-Ser824 IF in (Ci) WT control, (Cii) WT + NeuT, and (Ciii) WT + 5Gy IR (4 hr). Scale bar = 20 μm.

Figure 3. The Mre11 Complex Mediates Oncogene-Induced pHH3-S10 Accumulation

Immunohistochemistry (IHC) for (A) Ki67, (B) TUNEL reactivity, and (C) pHH3-S10 in (Ci) WT + NeuT and (Cii) Mre11^ATLD1/ATLD1 mammary glands 3 weeks after oncogene introduction. Scale bar = 50 μm. Quantification of Ki67 (Aiii) and TUNEL (Biii) IHC signal per mammary glandular area from at least five distinct high-powered fields (HPFs) from three independent experimental animals for WT control, WT + NeuT, and Mre11^ATLD1/ATLD1 + NeuT. Error bars indicate SEM, and p values were calculated using a two-tailed t test. The inset in (Ci) depicts a higher-magnification image of the pHH3-S10 nuclear signal observed, which is distinct from a mitotic pattern.

Figure 4. The Mre11 Complex Induces an Oncogene-Driven G2 Arrest

(A) Representative confocal z slices of DAPI-stained (Ai) pHH3-S10 and (Aii) H3K9me3 IF in WT + NeuT mammary glands. Scale bar = 10 μm. Lower panels depict linescan plots for the indicated lines in (Ai) and (Aii). (B) Confocal imaging of pHH3-S10 (red), centrin (green), and γ-tubulin (magenta) coIF in a representative pHH3-S10-negative (Bi) or pHH3-S10-positive cell (Bii) in a WT + NeuT hyperplastic mammary gland. Scale bar = 2 μm. (Biii) Quantification of proportion of postduplication (S/G2) and preduplication (G1) centrioles in pHH3-S10-negative (n = 45) or pHH3-S10-positive (n = 32) cells. p value calculated using Fisher's exact test. (C) Confocal imaging of macroH2A IF in (Ci) WT control, (Cii) WT + NeuT, and (Ciii) Mre11^ATLD1/ATLD1 + NeuT mammary glands, 3 weeks after injection. Scale bar = 20 μm. (Civ) Volumetric analysis of macro-H2A signal per nuclear volume. Error bars represent the interquartile range relative to the median value. p values were calculated according to the K-S nonparametric test (****p < 0.0001).

Figure 5. Mre11^ATLD1/ATLD1 Animals Are Predisposed to NeuT-Driven Mammary Tumorigenesis

(A) Kaplan-Meier mammary tumor-free survival curves after introduction of RCAS-HA-NeuT in the various genotypes indicated. p values were calculated using the two-tailed log rank test, relative to the WT genotype. (B) Mammary tumor growth doubling times for WT* (n = 5) or Mre11^ATLD1/ATLD1 (n = 10) mammary tumors. Due to the limited number of tumors in non-Mre11^ATLD1/ATLD1 genotypes, they were analyzed collectively under the designation WT*. Error bars indicate SEM, and p value was calculated using an unpaired t test. (C) Representative H&E-stained sections from Mre11^ATLD1/ATLD1 mammary tumors demonstrating aggressive histopathological features. Scale bar = 50 μm. (D) Representative IHC staining of Mre11^ATLD1/ATLD1 mammary tumors for (Di) ER, (Dii) EGFR, and (Diii) cytokeratin 5/6. Scale bars = 50 μm.

Figure 6. Oncogene-Associated p19Arf Induction in Mammary Hyperplasias and Tumors

(A) IHC for p19Arf in (Ai) WT control, (Aii) WT + NeuT, and (Aiii) Mre11^ATLD1/ATLD1 + NeuT mammary glands, 3 weeks after intraductal injection. Scale bar = 50 μm. (Aiv) Quantification of p19Arf signal relative to glandular area from at least five HPFs in three independent mammary glands for each genotype. (B) IHC for p19Arf in NeuT-induced mammary tumors from (Bi) WT*, (Bii) Ink4a-Arf nonmutated Mre11^ATLD1/ATLD1, and (Biii) Ink4a-Arf mutated Mre11^ATLD1/ATLD1 genotypes. Scale bars = 50 μm. (Biv) Digital quantification of p19Arf IHC relative to total area from at least three independent tumors for each genotype. Error bars indicate SEM, and p values were calculated using an unpaired t test.

Figure 7. Mre11^ATLD1/ATLD1 Mammary Tumors Are Highly Metastatic to Lung

(A) Gross pathology of lungs from a tumor-bearing Mre11^ATLD1/ATLD1 animal, depicting lung metastases. (B) H&E staining of a representative NeuT-driven Mre11^ATLD1/ATLD1 lung metastasis. Scale bar = 100 μm, and the inset shows a higher magnification of metastatic breast cancer cells within the lesion. (C) Frequency of histologically confirmed lung metastases observed in Mre11^ATLD1/ATLD1 and WT* mammary tumors-bearing animals. p value calculated using Fisher's exact test. (D) Quantification of pulmonary bioluminescence (Di) at different time points in mouse cohorts injected via tail vein inoculation with 5 × 10⁴ WT (n = 5) or Mre11^ATLD1/ATLD1 (n = 8) mammary tumor cells. Error bars indicate SEM, and p value was calculated using a Wilcoxon rank sum test. Representative bioluminescence images (Dii) at different time points after cell inoculation, demonstrating lung metastatic outgrowth. Also shown are gross necropsy images of macroscopic lung metastases, corresponding to the associated bioluminescence images. (E) Oncogene expression induces an Mre11-dependent DDR that constitutes an inducible barrier to tumorigenesis. In Mre11^ATLD1/ATLD1 mice, oncogene expression no longer activates the DDR, and there is more extensive oncogene-induced hyperplasia. These hyperplastic lesions are notable for p19Arf upregulation. Upon selection for Ink4a-Arf pathway inactivation, the oncogene-induced lesions progress to genomically unstable breast tumors that metastasize to the lungs.