Ignacio Gonzalez-Suarez

Novel roles for A-type lamins in telomere biology and the DNA damage response pathway

A‐type lamins are intermediate filament proteins that provide a scaffold for protein complexes regulating nuclear structure and function. Mutations in the LMNA gene are linked to a variety of degenerative disorders termed laminopathies, whereas changes in the expression of lamins are associated with tumourigenesis. The molecular pathways affected by alterations of A‐type lamins and how they contribute to disease are poorly understood. Here, we show that A‐type lamins have a key role in the maintenance of telomere structure, length and function, and in the stabilization of 53BP1, a component of the DNA damage response (DDR) pathway. Loss of A‐type lamins alters the nuclear distribution of telomeres and results in telomere shortening, defects in telomeric heterochromatin, and increased genomic instability. In addition, A‐type lamins are necessary for the processing of dysfunctional telomeres by non‐homologous end joining, putatively through stabilization of 53BP1. This study shows new functions for A‐type lamins in the maintenance of genomic integrity, and suggests that alterations of telomere biology and defects in DDR contribute to the pathogenesis of lamin‐related diseases.

Telomere recombination requires the MUS81 endonuclease

Telomerase-negative cancer cells maintain their telomeres through the alternative lengthening of telomeres (ALT) pathway. Although a growing body of evidence demonstrates that the ALT mechanism is a post-replicative telomere recombination process, molecular details of this pathway are largely unknown. Here we demonstrate that MUS81, a DNA structure specific recombination endonuclease, has a key role in the maintenance of telomeres in human ALT cells. We find that MUS81 specifically localizes to ALT-associated promyelocytic leukaemia (PML) nuclear bodies (APBs) and associates with telomeric DNA in ALT cells, which is enriched during the G2 phase of the cell cycle. Depletion of MUS81 results in the reduction of ALT-specific telomere recombination and leads to proliferation arrest of ALT cells. In addition, the endonuclease activity of MUS81 is required for recombination-based ALT cell survival, and the interaction of MUS81 with the telomeric repeat-binding factor TRF2 regulates this enzymatic activity, thereby maintaining telomere recombination. Thus, our results suggest that MUS81 is involved in the maintenance of ALT cell survival at least in part by homologous recombination of telomeres.

A new pathway that regulates 53BP1 stability implicates Cathepsin L and vitamin D in DNA repair

Genomic instability due to telomere dysfunction and defective repair of DNA double‐strand breaks (DSBs) is an underlying cause of ageing‐related diseases. 53BP1 is a key factor in DNA DSBs repair and its deficiency is associated with genomic instability and cancer progression. Here, we uncover a novel pathway regulating the stability of 53BP1. We demonstrate an unprecedented role for the cysteine protease Cathepsin L (CTSL) in the degradation of 53BP1. Overexpression of CTSL in wild‐type fibroblasts leads to decreased 53BP1 protein levels and changes in its cellular distribution, resulting in defective repair of DNA DSBs. Importantly, we show that the defects in DNA repair associated with 53BP1 deficiency upon loss of A‐type lamins are due to upregulation of CTSL. Furthermore, we demonstrate that treatment with vitamin D stabilizes 53BP1 and promotes DNA DSBs repair via inhibition of CTSL, providing an as yet unsuspected link between vitamin D action and DNA repair. Given that CTSL upregulation is a hallmark of cancer and progeria, regulation of this pathway could be of great therapeutic significance for these diseases.

The mTOR inhibitor rapamycin suppresses DNA double-strand break repair.

Abstract mTOR (mammalian target of rapamycin) signaling plays a key role in the development of many tumor types. Therefore, mTOR is an attractive target for cancer therapeutics. Although mTOR inhibitors are thought to have radiosensitization activity, the molecular bases remain largely unknown. Here we show that treating MCF7 breast cancer cells with rapamycin (an mTOR inhibitor) results in significant suppression of homologous recombination (HR) and nonhomologous end joining (NHEJ), two major mechanisms required for repairing ionizing radiation-induced DNA DSBs. We observed that rapamycin impaired recruitment of BRCA1 and Rad51 to DNA repair foci, both essential for HR. Moreover, consistent with the suppressive role of rapamycin on both HR and NHEJ, persistent radiation-induced DSBs were detected in cells pretreated with rapamycin. Furthermore, the frequency of chromosome and chromatid breaks was increased in cells treated with rapamycin before and after irradiation. Thus our results show that radiosensitization by mTOR inhibitors occurs via disruption of the major two DNA DSB repair pathways.

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

Cathepsin L degrades 53BP1 to overcome genomic instability and growth arrest in BRCA1-deficient and triple-negative breast cancers.

The role of RPA2 phosphorylation in homologous recombination in response to replication arrest.

Failure to reactivate stalled or collapsed DNA replication forks is a potential source of genomic instability. Homologous recombination (HR) is a major mechanism for repairing the DNA damage resulting from replication arrest. The single-strand DNA (ssDNA)-binding protein, replication protein A (RPA), plays a major role in multiple processes of DNA metabolism. However, the role of RPA2 hyperphosphorylation, which occurs in response to DNA damage, had been unclear. Here, we show that hyperphosphorylated RPA2 associates with ssDNA and recombinase protein Rad51 in response to replication arrest by hydroxyurea (HU) treatment. In addition, RPA2 hyperphosphorylation is critical for Rad51 recruitment and HR-mediated repair following HU. However, RPA2 hyperphosphorylation is not essential for both ionizing radiation (IR)-induced Rad51 foci formation and I-Sce-I endonuclease-stimulated HR. Moreover, we show that expression of a phosphorylation-deficient mutant of RPA2 leads to increased chromosomal aberrations following HU treatment but not after exposure to IR. Finally, we demonstrate that loss of RPA2 hyperphosphorylation results in a loss of viability when cells are confronted with replication stress whereas cells expressing hyperphosphorylation-defective RPA2 or wild-type RPA2 have a similar sensitivity to IR. Thus, our data suggest that RPA2 hyperphosphorylation plays a critical role in maintenance of genomic stability and cell survival after a DNA replication block via promotion of HR.

Loss of A-type lamins and genomic instability

Research performed in the last few years has revealed important roles for the spatial and temporal organization of the genome on genome function and integrity. A challenge in the field is to determine the molecular mechanisms involved in the organization of genome function. A-type lamins, key structural components of the nucleus, have been implicated in the maintenance of nuclear architecture and chromatin structure. Interestingly, alterations of A-type lamins lead to defects in DNA replication and repair as well as gene transcription and silencing. Elucidating the functions of these proteins is a topical subject since alterations of A-type lamins are associated with a variety of human diseases, ranging from muscular dystrophies and premature aging syndromes to cancer. Here, we discuss novels roles for A-type lamins in the maintenance of telomere structure, length and function as well as in the stabilization of a key DNA damage response factor. These studies support the notion that increased genomic instability due to defects in telomere biology and DNA repair contribute to the pathogenesis of lamin-related diseases.

EGFR Activation Increases Parathyroid Hyperplasia and Calcitriol Resistance in Kidney Disease

Calcitriol, acting through vitamin D receptors (VDR) in the parathyroid, suppresses parathyroid hormone synthesis and cell proliferation. In secondary hyperparathyroidism (SH), VDR content is reduced as hyperplasia becomes more severe, limiting the efficacy of calcitriol. In a rat model of SH, activation of the EGF receptor (EGFR) by TGF-alpha is required for the development of parathyroid hyperplasia, but the relationship between EGFR activation and reduced VDR content is unknown. With the use of the same rat model, it was found that pharmacologic inhibition of EGFR activation with erlotinib prevented the upregulation of parathyroid TGF-alpha, the progression of growth, and the reduction of VDR. Increased TGF-alpha/EGFR activation induced the synthesis of liver-enriched inhibitory protein, a potent mitogen and the dominant negative isoform of the transcription factor CCAAT enhancer binding protein-beta, in human hyperplastic parathyroid glands and in the human epidermoid carcinoma cell line A431, which mimics hyperplastic parathyroid cells. Increases in liver-enriched inhibitory protein directly correlated with proliferating activity and, in A431 cells, reduced VDR expression by antagonizing CCAAT enhancer binding protein-beta transactivation of the VDR gene. Similarly, in nodular hyperplasia, which is the most severe form of SH and the most resistant to calcitriol therapy, higher TGF-alpha activation of the EGFR was associated with an 80% reduction in VDR mRNA levels. Thus, in SH, EGFR activation is the cause of both hyperplastic growth and VDR reduction and therefore influences the efficacy of therapy with calcitriol.

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.