Compromised BRCA1-PALB2 interaction is associated with breast cancer risk

Abstract

The major breast cancer suppressor proteins BRCA1 and BRCA2 play essential roles in homologous recombination (HR)-mediated DNA repair, which is thought to be critical for tumor suppression. The two BRCA proteins are linked by a third tumor suppressor, PALB2, in the HR pathway. While truncating mutations in these genes are generally pathogenic, interpretation of missense variants remains a challenge. To date, patient-derived missense variants that disrupt PALB2 binding have been identified in BRCA1 and BRCA2; however, there has not been sufficient evidence to prove their pathogenicity in humans, and no variants in PALB2 that disrupt either its BRCA1 or BRCA2 binding have been reported. Here we report on the identification of a novel PALB2 variant, c.104T>C (p.L35P), that segregates in a family with a strong history of breast cancer. Functional analyses showed that L35P abrogates the PALB2-BRCA1 interaction and completely disables its abilities to promote HR and confer resistance to platinum salts and PARP inhibitors. Whole-exome sequencing of a breast cancer from a c.104T>C carrier revealed a second, somatic, truncating mutation affecting PALB2, and the tumor displays hallmark genomic features of tumors with BRCA mutations and HR defects, cementing the pathogenicity of L35P. Parallel analyses of other germline variants in the PALB2 N-terminal BRCA1-binding domain identified multiple variants that affect HR function to varying degrees, suggesting their possible contribution to cancer development. Our findings establish L35P as the first pathogenic missense mutation in PALB2 and directly demonstrate the requirement of the PALB2-BRCA1 interaction for breast cancer suppression.

Figure 1

Characterization of a breast cancer from a family carrying the PALB2 c.104T>C [p.L35P] variant. (a) Pedigree of the family. The proband is marked by a filled triangle. Confirmed mutations carriers are indicated by a “+” sign. Obligate carriers are indicated by a [+] sign. Mutation status in all other person is unknown. (b) Presence of the L35P mutation in the germline DNA and tumor DNA of the affected grandmother. (c) Presence of the L35P and Q61* mutations in normal (blood) and tumor DNAs of the affected grandmother. (d) Circos plot depicting the mutations and copy number alterations across the genome. Mutations are shown along the outside, including annotations on cancer gene status and mutation type (color-coded according to the legend), with the chromosomal position arranged along the middle ring. The 96 substitution classifications defined by the substitution classes are shown, and clonal mutations are indicated with a golden mark. Copy number alterations are depicted along the center ring color-coded according to the legend. (e) Diagram depicting the somatic mutations identified affecting cancer genes. The LST status (top), mutation types (left) and cancer cell fractions (right) are shown, color-coded according to the legend. (f) Mutational signature of all somatic synonymous and non-synonymous SNVs identified. Variants are displayed according to the 96 substitution classification and the 5′ and 3′ sequence context, normalized to the trinucleotide frequency of the human genome. Indel, small insertion and deletion; LOH, loss of heterozygosity; LST, large-scale state transition; SNV, single nucleotide variant.

Figure 2

Effects of PALB2 N-terminal VUSs on BRCA1 binding, HR activity and PALB2 self-interaction. (a) Amino acid sequence alignment of the PALB2 N terminus. The alignment as generated by ClustalW. The VUSs studied are marked on top. (b) Predicted model of the interaction between the coiled-coil motifs of PALB2 and BRCA1. Hydrophobic residues at the interface are shown. Residues affected by VUSs are shown in red. (c) Effects of the PALB2 variants on BRCA1 and BRCA2 binding. The proteins were transiently expressed in 293T cells and IPed with anti-FLAG M2 beads. The amounts of relevant proteins in the input whole cell lysate (WCL) and IPed materials were determined by western blotting. (d) Effects of the variants on the HR activity of PALB2. U2OS/DR-GFP cells were first depleted of the endogenous PALB2 by an siRNA and then rescued with the wt and variant PALB2 proteins by transient transfection. Error bars represent standard deviations (SDs) from at least three independent experiments. Statistical significance was analyzed by one way ANOVA and Newman-Keuls post hoc analysis. ***, p<0.001; ns, not significant. (e–f) Effects of the variants on PALB2 self-interaction. The variants were introduced into PALB2Δ4 and the binding between these shortened PALB2 proteins and the endogenous PALB2 were assessed by IP-western following transient transfection into 293T cells (e). FLAG-HA-tagged full-length PALB2 variant proteins were transiently overexpressed in 293T cells and analyzed by gel filtration followed by western blotting using anti-PALB2. Fraction numbers are marked above the blots and the approximate corresponding molecular weights of the fractions below.

Figure 3

Effects of PALB2 N-terminal VUSs on PALB2 and RAD51 foci formation and cellular sensitivity to DNA damaging agents. (a–b) PALB2 and RAD51 IRIF formation in EUFA1341 stable cell lines. Cells were treated with 10 Gy of IR, fixed after 6 hr recovery and analyzed by immunofluorescence using PALB2 (a) and RAD51 (b) antibodies, respectively. BRCA1 was co-stained with each of PALB2 and RDA51 to indicate sites of DNA damage and level of co-localization. (c) Efficiency and size of RAD51 foci formation in the above stable cell lines. Left, the percentage of RAD51 foci positive cells among BRCA1 foci positive (S and G2 phase) cells; right, sizes of the RAD51 foci in EUFA1341 cells expressing wt PALB2 and PALB2-Y28C. Error bars represent standard deviations (SDs) from 3 independent experiments. Statistical significance was calculated with Student’s t-test. *, p<0.05; ***, p<0.001. (d) Representative western blots showing the levels of PALB2 and BRCA1 in the EUFA1341 stable cell lines. GAPDH was used a loading control. (e) Sensitivities of the EUFA1341 stable cell lines to cisplatin, olaparib and MMC. Cells were incubated with the drugs for 96 hr. Data presented are the means of at least 3 independent experiments. Error bars represent standard deviations.