Palb2 synergizes with Trp53 to suppress mammary tumor formation in a model of inherited breast cancer

Abstract

Germ-line mutations in PALB2 lead to a familial predisposition to breast and pancreatic cancer or to Fanconi Anemia subtype N. PALB2 performs its tumor suppressor role, at least in part, by supporting homologous recombination-type double strand break repair (HR-DSBR) through physical interactions with BRCA1, BRCA2, and RAD51. To further understand the mechanisms underlying PALB2-mediated DNA repair and tumor suppression functions, we targeted Palb2 in the mouse. Palb2-deficient murine ES cells recapitulated DNA damage defects caused by PALB2 depletion in human cells, and germ-line deletion of Palb2 led to early embryonic lethality. Somatic deletion of Palb2 driven by K14-Cre led to mammary tumor formation with long latency. Codeletion of both Palb2 and Tumor protein 53 (Trp53) accelerated mammary tumor formation. Like BRCA1 and BRCA2 mutant breast cancers, these tumors were defective in RAD51 focus formation, reflecting a defect in Palb2 HR-DSBR function, a strongly suspected contributor to Brca1, Brca2, and Palb2 mammary tumor development. However, unlike the case of Brca1-mutant cells, Trp53bp1 deletion failed to rescue the genomic instability of Palb2- or Brca2-mutant primary lymphocytes. Therefore, Palb2-driven DNA damage control is, in part, distinct from that executed by Brca1 and more similar to that of Brca2. The mechanisms underlying Palb2 mammary tumor suppression functions can now be explored genetically in vivo.

Keywords: familial breast cancer; mouse model.

Fig. 1.

Conditional gene targeting of mouse Palb2. (A) Schematic representation of Palb2 domains and the exons from which they are encoded. The yellow area corresponds to the frameshifted ORF that results from recombination of the inserted loxP recombination sites. (B) Western blot analysis for PALB2 isolated in chromatin-enriched extracts (S420) of three independent ES cell lines. The full-length mouse PALB2 protein is ∼120 kDa. A nonspecific background band is indicated by an asterisk and can be used as an internal loading control. (C) Recruitment of RAD51 to DSBs marked by γH2AX IRIF 2 h after exposure of Palb2^fl/fl and Palb2^−/− ES cells to to 5 Gy of ionizing radiation (IR). (D) Western blot analysis of chromatin-bound (S420) RAD51 in Palb2^fl/fl and Palb2^−/− ES cells that received 10 Gy of IR and the respective unirradiated control cells. Histone H3 was used as a loading control. (E) Dose–response curves of Palb2^fl/fl and Palb2^−/− ES cells after exposure to increasing concentrations of neocarzinostatin.

Fig. 2.

Tumor formation in Palb2^fl/fl conditional mice. (A–C) Kaplan–Meier curves display that Palb2 loss accelerates tumor formation both on a Trp53-conditional null background (A), on a Trp53-conditional heterozygous background (B), and on a Trp53 WT background (C). (D) Gene dosages of Palb2 (Upper) and Trp53 (Lower) in mammary tumors derived from Palb2/Trp53 double conditional mouse cohorts. The germ-line Palb2 and Trp53 genotypes of the mice are indicated in red below the graphs. (E) Spectrum of tumors arising in mouse cohorts with different combinations of Palb2 and Trp53 alleles. The genotypes of the mice are shown above the graphs.

Fig. 3.

CGH analysis of Palb2 tumors. (A) Control (spleen) and tumor DNAs were hybridized to whole genome arrays to determine regions of loss or gains in mouse breast tumor samples. Representative rainbow graphs for each tumor genotype showing log₂ mean DNA relative dose ratio (tumor/spleen) across the entire genome are presented. Each dot represents the average signal from 10 or more consecutive probes (or a segment of ∼40 kb of genomic DNA). The amplitude of the data points above or below the midline indicates the extent of loss/gain in each segment, respectively. (B and C) Scatter plot graphs indicating the relative dose of the deleted segments (log₂ < −0.5, B) and amplified segments (log₂ > 0.5, C), each dot representing one segment. The number of dots represents the number of segments for all tumors of the relevant genotype, and the number of tumors analyzed (n) for Brca1/Trp53, Brca2/Trp53, Trp53-only, and Palb2/Trp53 tumors were 4, 5, 7, and 8, respectively. The horizontal lines are the average segment dose per genotype, and the P values displayed correspond to the result of the nonparametric Mann–Whitney–Wilcoxon signed-rank test, which followed the Kruskal–Wallis one-way analysis of variance (P < 0.0001).

Fig. 4.

The HR defect in Palb2-deficient cells and tumors. (A) qRT-PCR for Trp53bp1 mRNA in freshly isolated tumor samples that are either Palb2-proficient (+/+ and +/−, n = 8) or Palb2-deficient (−/−, n = 14). Horizontal lines represent the average relative expression value and the P value associated to this comparison (Mann–Whitney U test) is indicated. (B) Acute chromosomal damage and genome instability observed in chromosome spreads following PARPi treatment that are not rescued by Trp53bp1 deletion in Palb2^fl/fl;CD19-Cre B lymphocytes. (C) Established Palb2/Trp53-deficient breast tumor cell lines (example shown in the Bottom panels) reveal a defect in the recruitment of RAD51 to IRIF whereas Palb2 heterozygosity does not impair the proper IRIF localization of RAD51 in breast tumor lines (Middle panels). Both should be compared with a Palb2 WT control breast tumor line (Top panels).