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Supplementary Methods, Tables 1-3, Figure 1 from Common Breast Cancer Susceptibility Alleles and the Risk of Breast Cancer for BRCA1 and BRCA2 Mutation Carriers: Implications for Risk Prediction

Breast cancer is a heterogeneous genetic disease driven by the accumulation of individual mutations per tumor. Whole-genome sequencing approaches have identified numerous genes with recurrent mutations in primary tumors. Although mutations in well characterized tumor suppressors and oncogenes are overrepresented in these sets, the majority of the genetically altered genes have so far unknown roles in breast cancer progression. To improve the basic understanding of the complex disease breast cancer and to potentially identify novel drug targets or regulators of known cancer-driving pathways, we analyzed 86 wild-type genes and 94 mutated variants for their effect on cell growth using a serially constructed panel of MCF7 cell lines. We demonstrate in subsequent experiments that the metal cation transporter CNNM4 regulates growth by induction of apoptosis and identified a tumor suppressive role of complement factor properdin (CFP) in vitro and in vivo. CFP appears to induce the intracellular upregulation of the pro-apoptotic transcription factor DDIT3 which is associated with endoplasmic reticulum-stress response.

Nascent strand sequencing (NS-seq) is used to discover DNA replication origins genome-wide, allowing identification of features for their specification. NS-seq depends on the ability of lambda exonuclease (λ-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent strands intact. We used genomics and biochemical approaches to determine if λ-exo digests all parental DNA sequences equally. We report that λ-exo does not efficiently digest G-quadruplex (G4) structures in a plasmid. Moreover, λ-exo digestion of nonreplicating genomic DNA (LexoG0) enriches GC-rich DNA and G4 motifs genome-wide. We used LexoG0 data to control for nascent strand–independent λ-exo biases in NS-seq and validated this approach at the rDNA locus. The λ-exo–controlled NS-seq peaks are not GC-rich, and only 35.5% overlap with 6.8% of all G4s, suggesting that G4s are not general determinants for origin specification but may play a role for a subset. Interestingly, we observed a periodic spacing of G4 motifs and nucleosomes around the peak summits, suggesting that G4s may position nucleosomes at this subset of origins. Finally, we demonstrate that use of Na+ instead of K+ in the λ-exo digestion buffer reduced the effect of G4s on λ-exo digestion and discuss ways to increase both the sensitivity and specificity of NS-seq.

Small interfering RNA (siRNA) is a promising molecule for gene therapy, but its therapeutic administration remains problematic. Among the recently proposed vectors, cell-penetrating peptides show great promise in in vivo trials for siRNA delivery. Human protein DMBT1 (deleted in malignant brain tumor 1) is a pattern recognition molecule that interacts with polyanions and recognizes and aggregates bacteria. Taking advantage of these properties, we investigated whether specific synthetic DMBT1-derived peptides could be used to formulate nanoparticles for siRNA administration. Using an electrophoretic mobility shift assay and UV spectra, we identified two DMBT1 peptides that could encapsulate the siRNA with a self- and co-assembly mechanism. The complexes were stable for at least 2 hr in the presence of either fetal bovine serum (FBS) or RNase A, with peptide-dependent time span protection. ζ-potential, circular dichroism, dynamic light scattering, and transmission electron microscopy revealed negatively charged nanoparticles with an average diameter of 10–800 nm, depending on the reaction conditions, and a spherical or rice-shaped morphology, depending on the peptide and β-helix conformation. We successfully transfected human MCF7 cells with fluorescein isothiocyanate (FITC)-DMBT1-peptide-Cy3-siRNA complexes. Finally, DMBT1 peptides encapsulating an siRNA targeting a fluorescent reporter gene showed efficient gene silencing in MCF7-recombinant cells. These results lay the foundation for a new research line to exploit DMBT1-peptide nanocomplexes for therapeutic siRNA delivery., Graphical Abstract

Objective Determine mechanisms responsible for enhanced statin efficacy in a novel statin combination we name STOX (STatin–OXysterol). Methods Ovarian cancer cell lines were treated with combinations of statins and oxysterols. Cell viability was determined by a modified MTT assay. Apoptosis was evaluated by immunoblotting of PARP and DAPI-mediated visualization of apoptotic nuclei. STOX effects on the expression of genes of the mevalonate pathway were assessed by real-time qPCR and immunoblotting. siRNA-mediated gene silencing was used to test the involvement of oxysterol-mediated repression of SREBP-2 in STOX synergy. The impact of statin-mediated inhibition of protein prenylation and on cholesterol homeostasis was evaluated. Results Oxysterols dramatically enhance cytotoxicity of statins in ovarian cancer cells through increased apoptosis. Decreased expression of SREBP-2 down-regulates the mevalonate pathway and prevents the active statin-induced sterol feedback, enhancing statin toxicity. Comparison of two ovarian cancer cell lines reveals two distinct mechanisms of statin induced toxicity, namely, dependence on protein geranylgeranylation and/or perturbation of cellular cholesterol levels. Conclusions We provide evidence of statins' mechanisms of cytotoxicity in different ovarian cancer cells and discovered a new approach to significantly enhance the anti-tumor activity of statins. These observations provide a potential new path to improve statins as a treatment against ovarian cancer with obtainable dosages.

The CDKN2A locus encodes for two distinct tumor suppressor proteins, p16 INK4A and p14 ARF , involved in cell cycle regulation. CDKN2A germline mutations have been associated with familial predisposition to melanoma and other tumor types. Besides bona-fide pathogenic mutations, many sequence variants have been identified, but their effect is not well known. We detected the p.Gly23Asp missense mutation in one of the two tested melanoma patients of a family with three melanoma cases. Even though the mutated amino acid is located in a conserved domain that specifically binds to and blocks the function of CDK4/6, its lack of segregation with disease suggested a series of functional assays to discriminate between a pathogenic variant and a neutral polymorphism. The effect of this mutation has been investigated exploiting four p16 INK4A properties: its ability (i) to bind CDK4, (ii) to inhibit pRb phosphorylation, (iii) to evenly localize in the cell, and (iv) to cause cell cycle arrest. The mutant protein properties were evaluated transfecting three different cell lines (U2-OS and NM-39, both p16-null, and SaOS 2, p53 and pRb-null) with plasmids expressing either p16 wt , p16 23Asp , or the p16 32Pro pathogenic variant. We found that p16 23Asp was less efficient than p16 wt in CDK4 binding, in inhibiting pRb phosphorylation, in inducing G1 cell cycle arrest; moreover, its pattern of distribution throughout the cell was suggestive of protein aggregation, thus assessing a pathogenic role for p16 23Asp in familial melanoma.

Origins of DNA replication (ORIs) occur at defined regions in the genome. Although DNA sequence defines the position of ORIs in budding yeast, the factors for ORI specification remain elusive in metazoa. Several methods have been used recently to map ORIs in metazoan genomes with the hope that features for ORI specification might emerge. These methods are reviewed here with analysis of their advantages and shortcomings. The various factors that may influence ORI selection for initiation of DNA replication are discussed.

At epithelial barriers molecular pattern recognition mechanisms act as minesweepers against harmful environmental factors and thereby play a crucial role in the defense against invading bacterial and viral pathogens. However, it became evident that some of the proteins participating in these host defense processes may simultaneously function as regulators of tissue regeneration when in the extracellular matrix, thus coupling defense functions with regulation of stem cells. Although molecular pattern recognition has complex physiological roles and we just begin to understand its various functions, the simplicity of the underlying principles for recognition of specific classes of molecules may generate novel starting points for nanomedical approaches in drug delivery across epithelial barriers. The present article aims to provide an introduction into the biological context, processes, proteins, and general mechanisms of molecular pattern recognition in humans and, by using selected examples, to identify potential areas in nanomedicine for the exploitation of these mechanisms.

Which is the molecular mechanism that leads to DNA amplification and oncogenes activation in breast cancer cells? This project aims to understand the role of estrogen in inducing re-replication, thus leading to DNA amplifications. I worked on the establishment of cancer cell line models carrying an engineered replication origin to be tested for undergoing DNA amplification after estrogen treatment. Subsequent to some promising preliminary data obtained with MCF7 cells suggesting that estrogen might be able to elicit DNA amplification at this site, the in vitro system used was refined to have multiple possibilities on the selection of cells with DNA amplification. A new reporter construct was built and a second recombinant cell line model established from U2OS/ER-alpha cells was used. The efficiency of the Flp/FRT recombination system for the establishment of MCF7 cells carrying the new construct was too low and did not allow successful recombinants to be obtained during this time period. Moreover, the 17-beta estradiol (E2) treatment of U2OS/ER-alpha recombinant cells was toxic: upon E2 treatment of cells expressing estrogen receptor alpha irreversible cell death was apparent at 24 hours. Because of the limited time of E2 exposure allowed by this system, a new experimental approach of molecular visualization of DNA re-replication has been explored.

[Background]: Germline mutations in the BRCA1 and BRCA2 genes are associated with increased risks of breast and ovarian cancers. Although several common variants have been associated with breast cancer susceptibility in mutation carriers, none have been associated with ovarian cancer susceptibility. A genome-wide association study recently identified an association between the rare allele of the single-nucleotide polymorphism (SNP) rs3814113 (ie, the C allele) at 9p22.2 and decreased risk of ovarian cancer for women in the general population. We evaluated the association of this SNP with ovarian cancer risk among BRCA1 or BRCA2 mutation carriers by use of data from the Consortium of Investigators of Modifiers of BRCA1/2. [Methods]: We genotyped rs3814113 in 10 029 BRCA1 mutation carriers and 5837 BRCA2 mutation carriers. Associations with ovarian and breast cancer were assessed with a retrospective likelihood approach. All statistical tests were two-sided. [Results]: The minor allele of rs3814113 was associated with a reduced risk of ovarian cancer among BRCA1 mutation carriers (per-allele hazard ratio of ovarian cancer = 0.78, 95% confidence interval = 0.72 to 0.85; P = 4.8 × 10-9) and BRCA2 mutation carriers (hazard ratio of ovarian cancer = 0.78, 95% confidence interval = 0.67 to 0.90; P = 5.5 × 10-4). This SNP was not associated with breast cancer risk among either BRCA1 or BRCA2 mutation carriers. BRCA1 mutation carriers with the TT genotype at SNP rs3814113 were predicted to have an ovarian cancer risk to age 80 years of 48%, and those with the CC genotype were predicted to have a risk of 33%. [Conclusion]: Common genetic variation at the 9p22.2 locus was associated with decreased risk of ovarian cancer for carriers of a BRCA1 or BRCA2 mutation., Spanish National Cancer Center (CNIO) and the Spanish Consortium: Partially supported by Fundación Mutua Madrileña, Asociación Española Contra el Cáncer, and the Spanish Ministry of Science and Innovation (FIS PI08 1120).

Purpose A growing number of sequence changes of unknown clinical significance are being identified in the BRCA1 gene. However, these variants cannot be used for identification and surveillance of at-risk individuals unless their pathogenic role can be demonstrated. The frequency of these variants makes research on this subject a relevant topic in the field of predisposition to breast and ovarian cancers. Herein, we investigate the pathogenicity of the BRCA1 p.Val1688del (c.5181_5183delGTT) variant, which recurs in our population. Patients and Methods Recent studies have drawn attention to different strategies that, if considered singly, do not usually provide sufficient power to firmly state for or against causality, thus forcing to a re-evaluation of the literature on each specific variant. To increase the power of our study, we used a recently described strategy that integrates data from multiple independent evidences. By this approach, we analyzed data from the comprehensive study of 12 breast/ovarian cancer families carrying p.Val1688del. Results We succeeded in integrating five independent evidences of disease causality including segregation, tumor pathology, and evolutionary and epidemiologic data. Under this model, we obtained a final score of 349,000:1 in favor of disease causality. This result largely matches established cutoffs, and thus is readily translatable into a clear clinical message. Conclusion We show that p.Val1688del is a pathogenic mutation deriving from a common founder. Notably, this study alone increases by 15% the number of BRCA1-positive families in our patients’ cohort, thus substantially contributing to explain many of the families wherein prediction of a BRCA1 mutation contrasted with the absence of a molecular recognizable defect.

To support rapid growth, tumors need to synthesize lipids and cholesterol, and stopping production of these metabolites can inhibit tumors. Statins are among the most prescribed and well-tolerated drugs in the world to treat cardiovascular disease by inhibiting cholesterol synthesis. Statins are known to be toxic to cancer cells and have attracted renewed attention because of the increased appreciation of the importance of the regulation of metabolism in tumors. Statins have not progressed beyond clinical trials because some patients significantly benefit, while others do not in a range of cancers. Our strategy is to improve our understanding of the mechanisms of tumor responses to statins and thereby enhance statin toxicity to cancer cells. We have found that combining statins with oxysterols synergistically enhances statin toxicity by 10-fold. Oxysterols are oxidized cholesterol metabolites that naturally occur during cholesterol synthesis. We hypothesize that oxysterols inhibit the expression of cholesterol synthesis through a master regulator of metabolism, SREBP-2, making cells more sensitive to statins. We find that oxysterols enhance statin induced apoptosis and suppress the expression of SREBP-2 along with SREBP-2 targets including HMGCR. We have tested a number of oxysterols that work with a range of efficacies in seven representative ovarian cancer cell lines. P53 status is not a major factor in mediating oxysterol activity. Oxysterols inhibit expression of SREBP-2 leading to lower expression of the statin target, HMGCR, along with lower expression of most of the mevalonate and cholesterol biosynthesis pathways. These observations suggests that the cell's response to statins and not initial expression levels may be most critical to determine which patients may benefit from statin therapy. Current studies are testing the combination in mouse models and evaluating the function and regulation of SREBP-2 in ovarian cancer. In summary, we have discovered an approach to suppress cancer cell's response to statins leading to greatly improved statin efficacy highlighting the importance of the mevalonate and cholesterol pathways in mediating growth and survival. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C153. Citation Format: Alexander Brodsky, Cinzia Casella. New approaches to target mevalonate biosynthesis in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C153.

To support rapid growth, tumors need to synthesize lipids and cholesterol, and stopping production of these metabolites can inhibit tumors. Statins are among the most prescribed and well-tolerated drugs in the world to treat cardiovascular disease by inhibiting cholesterol synthesis. Statins are known to be toxic to cancer cells and have attracted renewed attention because of the increased appreciation of the importance of the regulation of metabolism in tumors. Statins have not progressed beyond clinical trials because some patients significantly benefit, while others do not in a range of cancers. Our strategy is to improve our understanding of the mechanisms of tumor responses to statins and thereby enhance statin toxicity to cancer cells. We have found that combining statins with oxysterols synergistically enhances statin toxicity by 10-fold. Oxysterols are oxidized cholesterol metabolites that naturally occur during cholesterol synthesis. We hypothesize that oxysterols inhibit the expression of cholesterol synthesis through a master regulator of metabolism, SREBP-2, making cells more sensitive to statins. We find that oxysterols enhance statin induced apoptosis and suppress the expression of SREBP-2 along with SREBP-2 targets including HMGCR. We have tested a number of oxysterols that work with a range of efficacies in seven representative ovarian cancer cell lines. P53 status is not a major factor in mediating oxysterol activity. Oxysterols inhibit expression of SREBP-2 leading to lower expression of the statin target, HMGCR, along with lower expression of most of the mevalonate and cholesterol biosynthesis pathways. These observations suggests that the cell's response to statins and not initial expression levels may be most critical to determine which patients may benefit from statin therapy. Current studies are testing the combination in mouse models and evaluating the function and regulation of SREBP-2 in ovarian cancer. In summary, we have discovered an approach to suppress cancer cell's response to statins leading to greatly improved statin efficacy highlighting the importance of the mevalonate and cholesterol pathways in mediating growth and survival. Citation Format: Cinzia Casella, Alexander S. Brodsky. New approaches to target mevalonate biosynthesis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A40.