The BRCA1 Variant p.Ser36Tyr Abrogates BRCA1 Protein Function and Potentially Confers a Moderate Risk of Breast Cancer

<div><p>The identification of variants of unknown clinical significance (VUS) in the <i>BRCA1</i> gene complicates genetic counselling and causes additional anxiety to carriers. <i>In silico</i> approaches currently used for VUS pathogenicity assessment are predictive and often produce conflicting data. Furthermore, functional assays are either domain or function specific, thus they do not examine the entire spectrum of BRCA1 functions and interpretation of individual assay results can be misleading. PolyPhen algorithm predicted that the BRCA1 p.Ser36Tyr VUS identified in the Cypriot population was damaging, whereas Align-GVGD predicted that it was possibly of no significance. In addition the BRCA1 p.Ser36Tyr variant was found to be associated with increased risk (OR = 3.47, 95% CI 1.13-10.67, P = 0.02) in a single case-control series of 1174 cases and 1109 controls. We describe a cellular system for examining the function of exogenous full-length BRCA1 and for classifying VUS. We achieved strong protein expression of full-length BRCA1 in transiently transfected HEK293T cells. The p.Ser36Tyr VUS exhibited low protein expression similar to the known pathogenic variant p.Cys61Gly. Co-precipitation analysis further demonstrated that it has a reduced ability to interact with BARD1. Further, co-precipitation analysis of nuclear and cytosolic extracts as well as immunofluorescence studies showed that a high proportion of the p.Ser36Tyr variant is withheld in the cytoplasm contrary to wild type protein. In addition the ability of p.Ser36Tyr to co-localize with conjugated ubiquitin foci in the nuclei of S-phase synchronized cells following genotoxic stress with hydroxyurea is impaired at more pronounced levels than that of the p.Cys61Gly pathogenic variant. The p.Ser36Tyr variant demonstrates abrogated function, and based on epidemiological, genetic, and clinical data we conclude that the p.Ser36Tyr variant is probably associated with a moderate breast cancer risk.</p></div

Image1_Amygdalin as a chemoprotective agent in co-treatment with cisplatin.PNG

Amygdalin is a naturally occurring glycoside used in traditional Chinese medicine and is known to have anti-cancer properties. Even though the anti-cancer properties of amygdalin are well known, its effect on normal cells has not been thoroughly investigated. The aim of the present study was to investigate a possible chemo-protective role of amygdalin against the cytotoxic effects of chemotherapy for normal human cells. Specifically, it was tested in combination with a strong chemotherapeutic drug cisplatin. Human non-tumorigenic MCF12F epithelial cell line, human fibroblasts cells, human breast cancer MCF7 and MDA-MB-231 cells were treated with cisplatin in a dose- and time-depended manner in the absence or presence of amygdalin. When MCF12F cells and fibroblasts underwent pre-treatment with amygdalin followed by cisplatin treatment (24 h amygdalin + 24 h cisplatin), the cell viability was increased (22%, p < 0.001) as indicated using MTT assay. As attested by flow cytometry, combination treatment was associated with decreased the percentage of late apoptotic cells compared with monotherapy (fold-change of decrease = 1.6 and 4.5 for 15 and 20 μΜ, respectively). Also, the proteins expression of PUMA, p53, phospho-p53 and Bax decreased, when a combination treatment was used vs. cisplatin alone, while the proapoptotic proteins Bcl-2 and Bcl-xL exhibited an increased tendency in the presence of amygdalin. Moreover, the levels of pro-apoptotic genes PUMA, p53, and BAX mRNA were significantly downregulated (∼83%, ∼66%, and ∼44%, respectively) vs. cisplatin alone, while the mRNA levels of anti-apoptotic genes BCl-2 and Bcl-XL were upregulated (∼44.5% and ∼51%, respectively), vs. cisplatin alone after 24 h of combination treatment. The study on the Combination index (CI) assay indicated that amygdalin could be possibly considered as an antagonist to cisplatin (2.2 and 2.3) for MCF12F and fibroblast cells, respectively. In contrast, for the breast cancer MCF7 and MDA-MB-231 cells, amygdalin and cisplatin indicated a synergistic effect (0.8 and 0.65), respectively. Our present findings suggest that amygdalin has chemo-modulatory effect when used in co-treatment with cisplatin and is able to protect normal breast cells as well as the fibroblasts during chemotherapy treatment, indicating a strong selective chemoprotective ability and may contribute to a better quality of life for cancer patients.</p

Image2_Amygdalin as a chemoprotective agent in co-treatment with cisplatin.PNG

Amygdalin is a naturally occurring glycoside used in traditional Chinese medicine and is known to have anti-cancer properties. Even though the anti-cancer properties of amygdalin are well known, its effect on normal cells has not been thoroughly investigated. The aim of the present study was to investigate a possible chemo-protective role of amygdalin against the cytotoxic effects of chemotherapy for normal human cells. Specifically, it was tested in combination with a strong chemotherapeutic drug cisplatin. Human non-tumorigenic MCF12F epithelial cell line, human fibroblasts cells, human breast cancer MCF7 and MDA-MB-231 cells were treated with cisplatin in a dose- and time-depended manner in the absence or presence of amygdalin. When MCF12F cells and fibroblasts underwent pre-treatment with amygdalin followed by cisplatin treatment (24 h amygdalin + 24 h cisplatin), the cell viability was increased (22%, p < 0.001) as indicated using MTT assay. As attested by flow cytometry, combination treatment was associated with decreased the percentage of late apoptotic cells compared with monotherapy (fold-change of decrease = 1.6 and 4.5 for 15 and 20 μΜ, respectively). Also, the proteins expression of PUMA, p53, phospho-p53 and Bax decreased, when a combination treatment was used vs. cisplatin alone, while the proapoptotic proteins Bcl-2 and Bcl-xL exhibited an increased tendency in the presence of amygdalin. Moreover, the levels of pro-apoptotic genes PUMA, p53, and BAX mRNA were significantly downregulated (∼83%, ∼66%, and ∼44%, respectively) vs. cisplatin alone, while the mRNA levels of anti-apoptotic genes BCl-2 and Bcl-XL were upregulated (∼44.5% and ∼51%, respectively), vs. cisplatin alone after 24 h of combination treatment. The study on the Combination index (CI) assay indicated that amygdalin could be possibly considered as an antagonist to cisplatin (2.2 and 2.3) for MCF12F and fibroblast cells, respectively. In contrast, for the breast cancer MCF7 and MDA-MB-231 cells, amygdalin and cisplatin indicated a synergistic effect (0.8 and 0.65), respectively. Our present findings suggest that amygdalin has chemo-modulatory effect when used in co-treatment with cisplatin and is able to protect normal breast cells as well as the fibroblasts during chemotherapy treatment, indicating a strong selective chemoprotective ability and may contribute to a better quality of life for cancer patients.</p

Ribbon representation of the BARD1:BRCA1 complex.

<p>A. The RING domains of BRCA1 and BARD1 (PDB 1JM7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093400#pone.0093400-Brzovic3" target="_blank">[63]</a>) are represented in magenta and blue respectively and the residues isoleucine 26, serine 36 and C61G are indicated in yellow. Orange spheres represent the Zn²⁺ ions. This model demonstrates that serine 36 lies within the second β-sheet of the RING structure. B. and C. represent the complex in a different orientation revealing the position of serine 36 (B.) and its substitution by tyrosine (C.) may distort the structure. The model was modified using PyMOL software.</p

p.Ser36Tyr (S36Y) BRCA1:BARD1 complex is withheld within the cytoplasm following HU treatment.

<p>Lysates of transfected cells were fractionated into nuclear and cytosolic extracts following S-phase synchronization and treatment with HU. BARD1 was co-precipitated with the ectopically expressed BRCA1 in the A. Nuclear and B. Cytosolic extracts with the anti-DYKDDDDK tag antibody which recognizes only the exogenous BRCA1. A. Immunoblot analysis of the pull-downs demonstrated that unlike wild type BRCA1, both the p.Ser36Tyr and p.Cys61Gly (C61G) variants exhibit reduced ability to co-precipitate BARD1 in the nuclear extracts of stressed cells. B. However, in the cytosolic extracts the p.Ser36Tyr variant, but not p.Cys61Gly, co-precipitated a higher proportion of BARD1 in treated cells compared to wild type transfected cells. In untreated cells, both variants, including wild type BRCA1, co-precipitated larger amounts of BARD1 compared to treated cells. Bar charts showing that C. in the nuclei of treated cells, both the p.Ser36Tyr and p.Cys61Gly variants exhibit reduced ability to co-precipitate with BARD1, whereas D. in the cytosol the p.Ser36Tyr variant co-precipitates similar levels of BARD1 compared to wild type BRCA1, but it is not statistically significant. Immunoblot analysis of nuclear and cytoplasmic extracts demonstrating their purity is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093400#pone.0093400.s002" target="_blank">Fig. S2</a>. The results are representative of 2 experiments.</p

Expression of full-length, epitope-tagged BRCA1 in HEK293T cells.

<p>Protein expression analysis of wild type BRCA1, p.Ser36Tyr (S36Y) VUS and of the known pathogenic p.Cys61Gly (C61G) variant following transient transfection in HEK293T cells was confirmed with the anti-DYKDDDDK tag antibody that recognizes ectopically expressed BRCA1. Immunoblot analysis detected a 220 KDa band which corresponds to full-length BRCA1 protein. Both p.Ser36Tyr and p.Cys61Gly variants demonstrated reduced protein expression compared to wild type BRCA1. Co-transfection with wild type BRCA1 and BARD1 induced an increase in BARD1 protein expression (Supp Fig.1). EV corresponds to empty vector and WT to wild type protein. β-actin served as loading control. The results are representative of between 3 and 5 experiments.</p

The mutational spectrum of Lynch syndrome in cyprus.

Lynch syndrome is the most common form of hereditary colorectal cancer and is caused by germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. Mutation carriers have an increased lifetime risk of developing colorectal cancer as well as other extracolonic tumours. The aim of the current study was to evaluate the frequency and distribution of mutations in the MLH1, MSH2 and MSH6 genes within a cohort of Cypriot families that fulfilled the revised Bethesda guidelines. The study cohort included 77 patients who fulfilled at least one of the revised Bethesda guidelines. Mutational analysis revealed the presence of 4 pathogenic mutations, 3 in the MLH1 gene and 1 in the MSH2 gene, in 5 unrelated individuals. It is noted that out of the 4 pathogenic mutations detected, one is novel (c.1610delG in exon 14 of the MLH1) and has been detected for the first time in the Cypriot population. Overall, the pathogenic mutation detection rate in our patient cohort was 7%. This percentage is relatively low but could be explained by the fact that the sole criterion for genetic screening was compliance to the revised Bethesda guidelines. Larger numbers of Lynch syndrome families and screening of the two additional predisposition genes, PMS2 and EPCAM, are needed in order to decipher the full spectrum of mutations associated with Lynch syndrome predisposition in Cyprus

Summary of the pathogenic mutations detected and family history of the probands.

+<p>Mutation nomenclature according to NM_000249.3 (<i>MLH1</i>), NM_000251.2 (<i>MSH2</i>) and NM_000179.2 (<i>MSH6</i>). For the nomenclature of mutations nucleotide 1 is the A of the ATG-translation initiation codon.</p>++<p>BrC – brain cancer.</p><p>CRC – colorectal cancer.</p><p>EC – endometrial cancer.</p><p>RC – renal cancer.</p