Regulating the levels of key factors in cell cycle and DNA repair: New pathways revealed by lamins

Spatial and temporal organization of the genome represents an additional step in the regulation of nuclear functions. The nuclear lamina, a polymeric meshwork formed by lamins (A/C and B type) and lamin-associated proteins, plays a key role in the maintenance of genome localization, structure and function. Specifically, mutations in the LMNA gene encoding lamins A/C or changes in its expression, either upregulation or silencing, are associated with defects in DNA replication, transcription and repair, as well as alterations in epigenetic modifications of chromatin. These data, together with the fact that defects in A-type lamins are associated with a whole variety of degenerative disorders, premature aging syndromes and cancer, support the notion that these proteins operate as caretakers of the genome. However, our understanding of their functions is limited due to the lack of well-defined mechanisms behind the genomic instability observed in lamin-related diseases. Here, we summarize our recent discovery of new pathways that are affected by the loss of A-type lamins. In particular, we found that A-type lamins control transcription and degradation of proteins with key roles in cell cycle regulation and DNA double-strand breaks (DSBs) repair by nonhomologous end-joining (NHEJ) and homologous-recombination (HR). Importantly, the proteins regulated by A-type lamins—Rb family members, 53BP1, BRCA1 and RAD51— exert tumor suppressor functions, with their loss being associated with cancer susceptibility. Moreover, our studies revealed novel pathways that contribute to genomic instability and that can be activated in disease states independent of the status of A-type lamins

Lamin A Δexon9 mutation leads to telomere and chromatin defects but not genomic instability

Over 300 mutations in the LMNA gene, encoding A-type lamins, are associated with 15 human degenerative disorders and premature aging syndromes. Although genomic instability seems to contribute to the pathophysiology of some laminopathies, there is limited information about what mutations cause genomic instability and by which molecular mechanisms. Mouse embryonic fibroblasts depleted of A-type lamins or expressing mutants lacking exons 8–11 (Lmna(Δ8–11/Δ8–11)) exhibit alterations in telomere biology and DNA repair caused by cathepsin L-mediated degradation of 53BP1 and reduced expression of BRCA1 and RAD51. Thus, a region encompassing exons 8–11 seems essential for genome integrity. Given that deletion of lamin A exon 9 in the mouse (Lmna(Δ9/Δ9)) results in a progeria phenotype, we tested if this domain is important for genome integrity. Lmna(Δ9/Δ9) MEFs exhibit telomere shortening and heterochromatin alterations but do not activate cathepsin L-mediated degradation of 53BP1 and maintain expression of BRCA1 and RAD51. Accordingly, Lmna(Δ9/Δ9) MEFs do not present genomic instability, and expression of mutant lamin A Δexon9 in lamin-depleted cells restores DNA repair factors levels and partially rescues nuclear abnormalities. These data reveal that the domain encoded by exon 9 is important to maintain telomere homeostasis and heterochromatin structure but does not play a role in DNA repair, thus pointing to other exons in the lamin A tail as responsible for the genomic instability phenotype in Lmna(Δ8–11/Δ8–11) mice. Our study also suggests that the levels of DNA repair factors 53BP1, BRCA1 and RAD51 could potentially serve as biomarkers to identify laminopathies that present with genomic instability

A dual role for A-type lamins in DNA double-strand break repair

A-type lamins are emerging as regulators of nuclear organization and function. Changes in their expression are associated with cancer and mutations are linked to degenerative diseases—laminopathies. Although a correlation exists between alterations in lamins and genomic instability, the molecular mechanisms remain largely unknown. We previously found that loss of A-type lamins leads to degradation of 53BP1 protein and defective long-range non-homologous end-joining (NHEJ) of dysfunctional telomeres. Here, we determined how loss of A-type lamins affects the repair of short-range DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). We find that lamins deficiency allows activation of the DNA damage response, but compromises the accumulation of 53BP1 at IR-induced foci (IRIF), hindering the fast phase of repair corresponding to classical-NHEJ. Importantly, reconstitution of 53BP1 is sufficient to rescue long-range and short-range NHEJ. Moreover, we demonstrate an unprecedented role for A-type lamins in the maintenance of homologous recombination (HR). Depletion of lamins compromises HR by a mechanism involving transcriptional downregulation of BRCA1 and RAD51 by the repressor complex formed by the Rb family member p130 and E2F4. In line with the DNA repair defects, lamins-deficient cells exhibit increased radiosensitivity. This study demonstrates that A-type lamins promote genomic stability by maintaining the levels of proteins with key roles in DNA DSBs repair by NHEJ and HR. Our results suggest that silencing of A-type lamins by DNA methylation in some cancers could contribute to the genomic instability that drives malignancy. In addition, lamins-deficient tumor cells could represent a good target for radiation therapy

BRCA1 loss activates cathepsin L–mediated degradation of 53BP1 in breast cancer cells

Loss of 53BP1 rescues BRCA1 deficiency and is associated with BRCA1-deficient and triple-negative breast cancers (TNBC) and with resistance to genotoxic drugs. The mechanisms responsible for decreased 53BP1 transcript and protein levels in tumors remain unknown. Here, we demonstrate that BRCA1 loss activates cathepsin L (CTSL)–mediated degradation of 53BP1. Activation of this pathway rescued homologous recombination repair and allowed BRCA1-deficient cells to bypass growth arrest. Importantly, depletion or inhibition of CTSL with vitamin D or specific inhibitors stabilized 53BP1 and increased genomic instability in response to radiation and poly(adenosine diphosphate–ribose) polymerase inhibitors, compromising proliferation. Analysis of human breast tumors identified nuclear CTSL as a positive biomarker for TNBC, which correlated inversely with 53BP1. Importantly, nuclear levels of CTSL, vitamin D receptor, and 53BP1 emerged as a novel triple biomarker signature for stratification of patients with BRCA1-mutated tumors and TNBC, with potential predictive value for drug response. We identify here a novel pathway with prospective relevance for diagnosis and customization of breast cancer therapy