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.
Highlights
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
Sequencing the RB1 coding exons cDNA generated from 73 locally advanced breast cancer samples obtained prior to chemotherapy was analyzed by PCR and DNA sequencing for RB1 mutations
Based on our findings that all three point mutated pRb proteins expressed reduced pro-apoptotic effect in vitro, and three out of four tumors harboring RB1 mutations were resistant to chemotherapy, our data provide the first indication that RB1 alterations could influence breast cancer chemosensitivity in vivo
Summary
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. This study is part of a project investigating alterations in pivotal genes as predictive factors to chemotherapy sensitivity in breast cancer. PRb, the protein coded for by the RB1 gene, plays a pivotal role in cell cycle regulation, promoting G1/S arrest and growth restriction through inhibition of the E2F transcription factors [1]. Somatic alterations of the RB1 gene have been detected in different malignancies [3,4,5]. Previous studies have reported allelic imbalance (AI), loss of pRb protein expression [3], hypermethylation of the RB1 promoter [6] and, in some rare cases, large intragenic deletions [7] in the RB1 gene in primary breast cancer. To the best of our knowledge, no point mutations have previously been reported in biopsies from breast carcinomas
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