Identification and characterization of retinoblastoma gene mutations disturbing apoptosis in human breast cancers

Abstract

Background: The tumor suppressor pRb plays a key role regulating cell cycle arrest, and disturbances in the RB1 gene have been reported in different cancer forms. However, the literature reports contradictory findings with respect to a pro--versus anti--apoptotic role of pRb, and the consequence of alterations in RB1 to chemotherapy sensitivity remains unclear. This study is part of a project investigating alterations in pivotal genes as predictive factors to chemotherapy sensitivity in breast cancer.

Results: Analyzing 73 locally advanced (stage III) breast cancers, we identified two somatic and one germline single nucleotide changes, each leading to amino acid substitution in the pRb protein (Leu607Ile, Arg698Trp, and Arg621Cys, respectively). This is the first study reporting point mutations affecting RB1 in breast cancer tissue. In addition, MLPA analysis revealed two large multiexon deletions (exons 13 to 27 and exons 21 to 23) with the exons 21-23 deletion occurring in the tumor also harboring the Leu607Ile mutation. Interestingly, Leu607Ile and Arg621Cys point mutations both localize to the spacer region of the pRb protein, a region previously shown to harbor somatic and germline mutations. Multiple sequence alignment across species indicates the spacer to be evolutionary conserved. All three RB1 point mutations encoded nuclear proteins with impaired ability to induce apoptosis compared to wild-type pRb in vitro. Notably, three out of four tumors harboring RB1 mutations displayed primary resistance to treatment with either 5-FU/mitomycin or doxorubicin while only 14 out of 64 tumors without mutations were resistant (p = 0.046).

Conclusions: Although rare, our findings suggest RB1 mutations to be of pathological importance potentially affecting sensitivity to mitomycin/anthracycline treatment in breast cancer.

Figure 1

Observed RB1 mutations. (a) Schematic representation of the pRb protein. (b) We observed three novel point mutations in RB1: The C1819A mutation (Leu607Ile), the C1861T mutation (Arg621Cys), and the A2092T mutation (Arg698Trp), all leading to amino acid substitutions inside the pRb pocket domain.

Figure 2

Methylation of the RB1 promoter. RB1 promoter methylation status was analyzed by methylation-specific PCR in 71 patients. Genomic DNA from patients was bisulfite converted. Figure shows products from MSP and USP of a representative selection of patients. Uctr: DNA from healthy donor; Mctr: Universally Methylated Control DNA.

Figure 3

Multiple sequence alignment of the pRb spacer region. A multiple sequence alignment of the spacer region between the A and B boxes of the pRb pocket was generated with ClustalX using default parameters [24]. The locations of two of the mutations reported here are marked with black arrows, and two previously reported mutations are shown in grey [32,33]. Sequences from eight different species were included in the alignment: Human [UNIPROT: P06400 RB_HUMAN], cow [UNIPROT: Q08E68_BOVIN], mouse [UNIPROT: P13405 RB_MOUSE], chicken [UNIPROT: Q90600 RB_CHICK], newt [UNIPROT: Q98966_NOTVI], salmon [UNIPROT: C0H9R0_SALSA], killifish [UNIPROT: Q5J3Q9_FUNHE], and zebrafish [UNIPROT: A0JMQ4_DANRE].

Figure 4

Structural model of the pRb pocket domain. (a) The cartoon shows the pRb pocket in complex with peptides from the transcription factor E2F (magenta) and the human papilloma virus protein E7 (yellow). The A and B boxes are colored green and cyan, respectively. The model was made from two structures: PDB: 1GUX (Rb pocket and E7 peptide) and PDB: 1O9K (E2F peptide). (b) Close-up view of the arginine 698 (R698) and amino acids that have at least one atom within a 4Å distance to the side chain atoms of R698. The R698 that is mutated to tryptophan in one of the tumors is located in the B box of the pRb pocket, and forms a hydrogen bond network with three backbone carbonyls. Hydrogen bonds to backbone carbonyls of residues L694, L743 and I744 are shown by yellow dotted lines. The structures were visualized using PyMOL http://www.pymol.org.

Figure 5

Subcellular localization of pRb mutants. Immunofluorescence staining of RB1-deficient C-33 A cells transfected with the pcDNA3.1/V5-His-TOPO vector or one of the four plasmids RB1wild-type-V5, RB1Arg621Cys-V5, RB1Leu607Ile-V5, and RB1Arg698Trp-V5. All mutants display nuclear localization. Left: DAPI, Centre: Anti-V5 (Texas Red), Right: Overlay.

Figure 6

Mutant pRb proteins display reduced apoptotic function. (a) Diagram shows percentage of apoptotic (TUNEL positive) cells following doxorubicin treatment of RB1-deficient C-33 A and (b) Saos-2 cells, transfected with vectors expressing the different pRb mutants, relative to pRb wild-type (100%). For each mutant, a minimum of 1000 cells were counted in each of three independent experiments. Error bars indicate standard deviations between the three experiments. p-values given are based on analyses of variance (ANOVA) between the individual mutants and pRb wild-type. All over, each mutants revealed reduced apoptotic function in both cell lines (p ≤ 0.01). (c) Western blot controls for TUNEL assay. Panels displaying expression of pRb wild-type and mutants with corresponding actin expression (loading control) from transfections performed in parallel to those used for TUNEL assays in C-33 A and (d) Saos-2.

Figure 7

Stability of pRb wild-type and mutant proteins. Panels show the protein levels of pRb wild-type and mutants including corresponding actin levels as loading control. The samples were harvested 0-6 h after addition of cycloheximide (50 ug/ml) to transfected C-33 A and Saos-2 cells and examined by SDS-PAGE and western blot analyses.