Altered regulation of BRCA1 exon 11 splicing is associated with breast cancer risk in carriers of BRCA1 pathogenic variants.

Germline pathogenic variants in BRCA1 confer a high risk of developing breast and ovarian cancer. The BRCA1 exon 11 (formally exon 10) is one of the largest exons and codes for the nuclear localization signals of the corresponding gene product. This exon can be partially or entirely skipped during pre-mRNA splicing, leading to three major in-frame isoforms that are detectable in most cell types and tissue, and in normal and cancer settings. However, it is unclear whether the splicing imbalance of this exon is associated with cancer risk. Here we identify a common genetic variant in intron 10, rs5820483 (NC_000017.11:g.43095106_43095108dup), which is associated with exon 11 isoform expression and alternative splicing, and with the risk of breast cancer, but not ovarian cancer, in BRCA1 pathogenic variant carriers. The identification of this genetic effect was confirmed by analogous observations in mouse cells and tissue in which a loxP sequence was inserted in the syntenic intronic region. The prediction that the rs5820483 minor allele variant would create a binding site for the splicing silencer hnRNP A1 was confirmed by pull-down assays. Our data suggest that perturbation of BRCA1 exon 11 splicing modifies the breast cancer risk conferred by pathogenic variants of this gene.

ALK1 Loss Results in Vascular Hyperplasia in Mice and Humans Through PI3K Activation.

OBJECTIVE: ALK1 (activin-receptor like kinase 1) is an endothelial cell-restricted receptor with high affinity for BMP (bone morphogenetic protein) 9 TGF-ß (transforming growth factor-ß) family member. Loss-of-function mutations in ALK1 cause a subtype of hereditary hemorrhagic telangiectasia-a rare disease characterized by vasculature malformations. Therapeutic strategies are aimed at reducing potential complications because of vascular malformations, but currently, there is no curative treatment for hereditary hemorrhagic telangiectasia. APPROACH AND RESULTS: In this work, we report that a reduction in ALK1 gene dosage (heterozygous ALK1+/- mice) results in enhanced retinal endothelial cell proliferation and vascular hyperplasia at the sprouting front. We found that BMP9/ALK1 represses VEGF (vascular endothelial growth factor)-mediated PI3K (phosphatidylinositol 3-kinase) by promoting the activity of the PTEN (phosphatase and tensin homolog). Consequently, loss of ALK1 function in endothelial cells results in increased activity of the PI3K pathway. These results were confirmed in cutaneous telangiectasia biopsies of patients with hereditary hemorrhagic telangiectasia 2, in which we also detected an increase in endothelial cell proliferation linked to an increase on the PI3K pathway. In mice, genetic and pharmacological inhibition of PI3K is sufficient to abolish the vascular hyperplasia of ALK1+/- retinas and in turn normalize the vasculature. CONCLUSIONS: Overall, our results indicate that the BMP9/ALK1 hub critically mediates vascular quiescence by limiting PI3K signaling and suggest that PI3K inhibitors could be used as novel therapeutic agents to treat hereditary hemorrhagic telangiectasia.

DNA double-strand break-capturing nuclear envelope tubules drive DNA repair.

Current models suggest that DNA double-strand breaks (DSBs) can move to the nuclear periphery for repair. It is unclear to what extent human DSBs display such repositioning. Here we show that the human nuclear envelope localizes to DSBs in a manner depending on DNA damage response (DDR) kinases and cytoplasmic microtubules acetylated by α-tubulin acetyltransferase-1 (ATAT1). These factors collaborate with the linker of nucleoskeleton and cytoskeleton complex (LINC), nuclear pore complex (NPC) protein NUP153, nuclear lamina and kinesins KIF5B and KIF13B to generate DSB-capturing nuclear envelope tubules (dsbNETs). dsbNETs are partly supported by nuclear actin filaments and the circadian factor PER1 and reversed by kinesin KIFC3. Although dsbNETs promote repair and survival, they are also co-opted during poly(ADP-ribose) polymerase (PARP) inhibition to restrain BRCA1-deficient breast cancer cells and are hyper-induced in cells expressing the aging-linked lamin A mutant progerin. In summary, our results advance understanding of nuclear structure-function relationships, uncover a nuclear-cytoplasmic DDR and identify dsbNETs as critical factors in genome organization and stability.

Differential DNA damage repair and PARP inhibitor vulnerability of the mammary epithelial lineages.

It is widely assumed that all normal somatic cells can equally perform homologous recombination (HR) and non-homologous end joining in the DNA damage response (DDR). Here, we show that the DDR in normal mammary gland inherently depends on the epithelial cell lineage identity. Bioinformatics, post-irradiation DNA damage repair kinetics, and clonogenic assays demonstrated luminal lineage exhibiting a more pronounced DDR and HR repair compared to the basal lineage. Consequently, basal progenitors were far more sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis) in both mouse and human mammary epithelium. Furthermore, PARPi sensitivity of murine and human breast cancer cell lines as well as patient-derived xenografts correlated with their molecular resemblance to the mammary progenitor lineages. Thus, mammary epithelial cells are intrinsically divergent in their DNA damage repair capacity and PARPi vulnerability, potentially influencing the clinical utility of this targeted therapy.

AURKA Overexpression Is Driven by FOXM1 and MAPK/ERK Activation in Melanoma Cells Harboring BRAF or NRAS Mutations: Impact on Melanoma Prognosis and Therapy.

The cell cycle-related genes AURKA and FOXM1 are overexpressed in melanoma. We show here that AURKA overexpression is associated with poor prognosis in three independent cohorts of melanoma patients and correlates with the presence of genomic amplification of AURKA locus and BRAFV600E mutation. AURKA overexpression may also be driven by increased promoter activation through elements such as ETS and FOXM1 found within the 5' proximal promoter region. Activated MAPK/ERK signaling pathway mediates robust AURKA promoter activation, thereby knockdown of BRAFV600E and ERK inhibition results in reduced AURKA transcription and expression. We show a positive correlation between FOXM1 and AURKA expression in three independent cohorts of melanoma patients. FOXM1 silencing decreases expression of AURKA and late cell cycle genes in melanoma cells. We further found that FOXM1 expression levels are significantly higher in tumors carrying the BRAFV600E mutation compared with the wild-type BRAF (BRAFwt). Accordingly, the knockdown of BRAFV600E also reduces the expression of FOXM1 in BRAFV600E cells. Moreover, Aurora kinase A and FOXM1 inhibition by either genetic knockdown or pharmacologic inhibitors impair melanoma growth and survival both in culture and in vivo, underscoring their therapeutic value for melanoma patients who fail to benefit from BRAF/MEK signaling inhibition.

Evaluation of PAX3 genetic variants and nevus number.

The presence of a high nevus number is the strongest phenotypic predictor of melanoma risk. Here, we describe the results of a three-stage study directed at identifying risk variants for the high nevus phenotype. At the first stage, 263 melanoma cases from Barcelona were genotyped for 223 single-nucleotide polymorphisms (SNPs) in 39 candidate genes. Seven SNPs in the PAX3 gene were found to be significantly associated with nevus number under the additive model. Next, the associations for seven PAX3 variants were evaluated in 1217 melanoma cases and 475 controls from Leeds; and in 3054 healthy twins from TwinsUK. Associations with high nevus number were detected for rs6754024 (P values < 0.01) in the Barcelona and Leeds datasets and for rs2855268 (P values < 0.01) in the Barcelona and the TwinsUK sets. Associations (P values < 0.001) in the opposite direction were detected for rs7600206 and rs12995399 in the Barcelona and TwinsUK sets. This study suggests that SNPs in PAX3 are associated with nevus number, providing support for PAX3 as a candidate nevus gene. Further studies are needed to examine the role of PAX3 in melanoma susceptibility.