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

Berge EO, Knappskog S, Geisler S, Staalesen V, Pacal M, Børresen-Dale AL, Puntervoll P, Lillehaug JR, Lønning PE - Mol. Cancer (2010)

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

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Oncology, Institute of Medicine, University of Bergen, Norway.

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.

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Related in: MedlinePlus

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.
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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.

Mentions: The Arg698Trp mutation is located in the B box of the pRb pocket (Figure 4a). In silico structural analysis of the pRb pocket [23] revealed the Arg698 residue to form a hydrogen-bond network (Figure 4b) and predicted Arg698Trp to disrupt this intramolecular hydrogen bond network with a possible structural and functional consequence on the pRb protein.


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

Berge EO, Knappskog S, Geisler S, Staalesen V, Pacal M, Børresen-Dale AL, Puntervoll P, Lillehaug JR, Lønning PE - Mol. Cancer (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908580&req=5

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.
Mentions: The Arg698Trp mutation is located in the B box of the pRb pocket (Figure 4a). In silico structural analysis of the pRb pocket [23] revealed the Arg698 residue to form a hydrogen-bond network (Figure 4b) and predicted Arg698Trp to disrupt this intramolecular hydrogen bond network with a possible structural and functional consequence on the pRb protein.

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

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Oncology, Institute of Medicine, University of Bergen, Norway.

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.

Show MeSH
Related in: MedlinePlus