Cancer-causing mutations in the tumor suppressor PALB2 reveal a novel cancer mechanism using a hidden nuclear export signal in the WD40 repeat motif - PubMed (original) (raw)

Cancer-causing mutations in the tumor suppressor PALB2 reveal a novel cancer mechanism using a hidden nuclear export signal in the WD40 repeat motif

Joris Pauty et al. Nucleic Acids Res. 2017.

Abstract

One typical mechanism to promote genomic instability, a hallmark of cancer, is to inactivate tumor suppressors, such as PALB2. It has recently been reported that mutations in PALB2 increase the risk of breast cancer by 8-9-fold by age 40 and the life time risk is ∼3-4-fold. To date, predicting the functional consequences of PALB2 mutations has been challenging as they lead to different cancer risks. Here, we performed a structure-function analysis of PALB2, using PALB2 truncated mutants (R170fs, L531fs, Q775X and W1038X), and uncovered a new mechanism by which cancer cells could drive genomic instability. Remarkably, the PALB2 W1038X mutant, harboring a mutation in its C-terminal domain, is still proficient in stimulating RAD51-mediated recombination in vitro, although it is unusually localized to the cytoplasm. After further investigation, we identified a hidden NES within the WD40 domain of PALB2 and found that the W1038X truncation leads to the exposure of this NES to CRM1, an export protein. This concept was also confirmed with another WD40-containing protein, RBBP4. Consequently, our studies reveal an unreported mechanism linking the nucleocytoplasmic translocation of PALB2 mutants to cancer formation.

© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.

Figure 1.

PALB2 wild-type and mutant proteins. (A) Schematic representation of PALB2 domains and truncated variant proteins analyzed in this study. C.C.: coiled-coil domain, this domain also allows dimerization of PALB2; WD40: WD40-repeat domain. (B) SDS-PAGE of purified wild-type PALB2 (wt) and truncated variants of PALB2 (250 ng). The proteins were stained with Coomassie blue.

Figure 2.

Figure 2.

Competition EMSAs with five different DNA substrates and PALB2 mutant proteins. Left: Analysis of the preference of wild-type PALB2 (wt) and mutant proteins to bind specific DNA structures in competition. EMSAs were performed using single-strand DNA, double-strand DNA, splayed arms, Holliday junctions, and D-loops, altogether. Right: quantification of the data from three distinct experiments.

Figure 3.

Figure 3.

Cellular localization of PALB2 mutant proteins. (A) Immunofluorescence of mutant forms of Flag-tagged PALB2. DAPI (blue), anti-Flag (red), and the merge picture are shown. All mutants except W1038X were mainly nuclear. (B) Quantification of the cytoplasmic and nuclear accumulation of PALB2 mutants. Experiments were performed in quadruplicate. (C) Knockdown of endogenous PALB2 in HEK293T cells by expressing constitutively a shRNA against PALB2. (D) Analysis of the cellular localization of PALB2 mutant forms by cellular fractionation. The cytoplasmic (C), nuclear soluble (NS) and chromatin (N) fractions are shown. Blots against the GAPDH and histone H3 proteins are shown as controls.

Figure 4.

Figure 4.

Cellular localization of WD40-mutant PALB2 and WD40-truncated variant proteins. (A) Immunofluorescence of Flag-tagged PALB2 wt, Q988X (c.2962C>T) and W1038X (c.3113G>A). Both mutant forms present a similar mislocalization. DAPI (blue), anti-Flag (red), and the merge picture are shown. (B) Immunofluorescence of Flag-tagged PALB2 proteins truncated in the WD40-domain as indicated on the schematic representation. PALB2 1–852 corresponds to a complete deletion of the WD40 domain, while PALB2 1–1186 corresponds to the wild-type protein. All proteins truncated between position 1038 and 1162 present a similar mislocalization. DAPI (blue), anti-Flag (red) and the merge picture are shown.

Figure 5.

Figure 5.

A sequence contained in PALB2 WD40-domain acts as a CRM1-dependent nuclear export signal. (A, Top) The sequence predicted as a NES by ValidNESs is underlined. The hydrophobic amino acids that could constitute a NES are highlighted in red. Bottom: Immunofluorescence analysis of eGFP alone or fused to the NES sequence (amino acids 928–945). The analysis was performed with or without the CRM1-inhibitor leptomycin B. DAPI (blue), anti-Flag (red) and the merge picture are shown. (B) Co-immunoprecipitation of endogenous CRM1 with eGFP alone or fused to the putative NES of PALB2, or to BRCA2 NES (as a positive control).

Figure 6.

Figure 6.

Effect of PALB2 W1038X mutant proteins on RAD51 foci formation. (A) Representative immunofluorescence images of RAD51 staining (red) in HEK293T-shPALB2 cells expressing the empty vector or the indicated peYFP-c1-PALB2 constructs (green). DAPI (blue) and merge pictures are also shown. (B) Quantification of RAD51 foci in the PALB2 mutant cells with or without NCS treatment. HEK293T-shPALB2 cells were treated with 100 ng/ml neocarzinostatin for 1 hour, followed by a 3-hour release. Data are represented as mean percentage ± S.E. of cells with more than five foci (n > 100).

Figure 7.

Figure 7.

Effect of PALB2 W1038X mutant proteins on homologous recombination using a Cas9/mClover-LMNA1 homologous recombination assay. (A) Schematic representation of the mClover-LMNA1 homologous recombination assay. Following nucleofection, Cas9 creates a double-strand break in the LMNA locus leading to integration of the mClover gene by homologous recombination. Clover-labeled Lamin A/C proteins exhibit green fluorescence enriched at the nuclear periphery, which is indicative of successful gene targeting by homologous recombination. (B) Gene-targeting efficiency of siRNA PALB2 cells complemented with wild-type, W1038X and W1038XMutNES siRNA resistant constructs. mClover positive cells were quantified. ***P < 0.01 and ****P < 0.001. (C) Representative images of the quantifications shown in (B). iRFP positive cells are in purple and mClover integrated at the LMNA locus leads to green fluorescence in and around the nucleus.

Figure 8.

Figure 8.

RBBP4, another WD40 domain containing protein, becomes cytoplasmic due to a cancer causing mutation. (A) Schematic representation of wild-type RBBP4 and RBBP4 W382. (B) Immunofluorescence analysis of eGFP-RBBP4 or eGFP-RBBP4W382. DAPI (blue), eGFP fluorescence (green), and the merge picture are shown. (C) Modelling of the wild-type PALB2 and PALB2 W1038X WD40 domains. (D) Model. In normal cells, PALB2 is a nuclear protein that promotes homologous recombination and DNA repair. In cancer cells, mutations arise in the WD40 repeat (such as the Q988X and W1038X mutations), which leads to NES unmasking and accumulation of PALB2 mutants in the cytoplasm. In the cytoplasm, PALB2 mutants, can no longer promote DNA repair, leading to genomic instability.

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