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

Abstract

BackgroundThe 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.ResultsAnalyzing 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).ConclusionsAlthough rare, our findings suggest RB1 mutations to be of pathological importance potentially affecting sensitivity to mitomycin/anthracycline treatment in breast cancer.

Low expression levels of ATM may substitute for CHEK2 /TP53 mutations predicting resistance towards anthracycline and mitomycin chemotherapy in breast cancer

“…Peak areas of all MLPA products resulting from ATM specific probes were normalized and compared to references as previously described [24]. …”

Mapping genetic alterations causing chemoresistance in cancer: identifying the roads by tracking the drivers

“…Now, with improved therapy and better survival for their RBs it has become clear that these individuals have an increased risk of other cancers later in life as well. 107 Somatic alterations of RB1, including large deletions and promoter methylations, have been detected in different cancer forms, including breast cancer (see references in Berge et al 108 ). In a recent paper, Witkiewicz et al 109 found an RB-deficiency gene expression signature to be associated with increased chance of a pathological complete response among breast cancer patients receiving primary chemotherapy with 5-fluorouracil, adriamycin and cyclophosphamide, but also patients treated with combined anthracycline-and taxane-containing regimens.…”

“…In contrast, we found RB1 point mutations and intragenic deletions, albeit rare, to be associated with lack of response to anthracyclines and mitomycin in primary as well as metastatic breast cancers. 108,110 Interestingly, these mutations were located in the protein pocket domain and have never been reported as germline mutations in RB families (Table 2).…”

Screening RB1 Gene in Algerian Patients and Predicting the Pathogenicity of Variations by In Silico Analysis

“…This variation is located in the spacer region, previously assumed to be non-essential to peptide-binding activity of the pRb pocket by many authors [27]. Recent study employing Clustal X using default parameters [30], a multiple sequence alignment of the pRb spacer region including sequences from eight different species (cow, mouse, chicken, newt..) showed that the spacer region is fairly well conserved. This finding indicates that the spacer region is important.…”