Human DNA ligases I and III, but not ligase IV, are required for microhomology-mediated end joining of DNA double-strand breaks

DNA nonhomologous end-joining (NHEJ) and homologous recombination are two distinct pathways of DNA double-strand break repair in mammalian cells. Biochemical and genetic studies showed that DNA ends can also be joined via microhomology-mediated end joining (MHEJ), especially when proteins responsible for NHEJ, such as Ku, are reduced or absent. While it has been known that Ku-dependent NHEJ requires DNA ligase IV, it is unclear which DNA ligase(s) is required for Ku-independent MHEJ. In this study, we used a cell-free assay to determine the roles of DNA ligases I, III and IV in MHEJ and NHEJ. We found that siRNA mediated down-regulation of DNA ligase I or ligase III in human HTD114 cells led to impaired end joining that was mediated by 2-, 3- or 10-bp microhomology. In addition, nuclear extract from human fibroblasts harboring a mutation in DNA ligase I displayed reduced MHEJ activity. Furthermore, treatment of HTD114 nuclear extracts with an antibody against DNA ligase I or III also significantly reduced MHEJ. These data indicate that DNA ligases I and III are required in MHEJ. DNA ligase IV, on the contrary, is not required in MHEJ but facilitates Ku-dependent NHEJ. Therefore, MHEJ and NHEJ require different DNA ligases.     

Tumor-suppressive effects of psoriasin (S100A7) are mediated through the β-catenin/T cell factor 4 protein pathway in estrogen receptor-positive breast cancer cells

Psoriasin (S100A7) is expressed in several epithelial malignancies including breast cancer. Although S100A7 is associated with the worst prognosis in estrogen receptor α-negative (ERα(-)) invasive breast cancers, its role in ERα-positive (ERα(+)) breast cancers is relatively unknown. We investigated the significance of S100A7 in ERα(+) breast cancer cells and observed that S100A7 overexpression in ERα(+) breast cancer cells, MCF7 and T47D, exhibited decreased migration, proliferation, and wound healing. These results were confirmed in vivo in nude mouse model system. Mice injected with S100A7-overexpressing MCF7 cells showed significant reduction in tumor size compared with mice injected with vector control cells. Further mechanistic studies revealed that S100A7 mediates the tumor-suppressive effects via a coordinated regulation of the β-catenin/TCF4 pathway and an enhanced interaction of β-catenin and E-cadherin in S100A7-overexpressing ERα(+) breast cancer cells. We observed down-regulation of β-catenin, p-GSK3β, TCF4, cyclin D1, and c-myc in S100A7-overexpressing ERα(+) breast cancer cells. In addition, we observed increased expression of GSK3β. Treatment with GSK3β inhibitor CHIR 99021 increased the expression of β-catenin and its downstream target c-myc in S100A7-overexpressing cells. Tumors derived from mice injected with S100A7-overexpressing MCF7 cells also showed reduced activation of the β-catenin/TCF4 pathway. Therefore, our studies reveal for the first time that S100A7-overexpressing ERα(+) breast cancer cells exhibit tumor suppressor capabilities through down-modulation of the β-catenin/TCF4 pathway both in vitro and in vivo. Because S100A7 has been shown to enhance tumorigenicity in ERα(-) cells, our studies suggest that S100A7 may possess differential activities in ERα(+) compared with ERα(-) cells.     

The nucleoside analog sangivamycin induces apoptotic cell death in breast carcinoma MCF7/adriamycin-resistant cells via protein kinase Cdelta and JNK activation

Sangivamycin has shown a potent antiproliferative activity against a variety of human cancers. However, little is known about the mechanism of action underlying its antitumor activity. Here we demonstrate that sangivamycin has differential antitumor effects in drug-sensitive MCF7/wild type (WT) cells, causing growth arrest, and in multidrug-resistant MCF7/adriamycin-resistant (ADR) human breast carcinoma cells, causing massive apoptotic cell death. Comparisons between the effects of sangivamycin on these two cell lines allowed us to identify the mechanism underlying the apoptotic antitumor effect. Fluorescence-activated cell sorter analysis indicated that sangivamycin induced cell cycle arrest in the G(2)/M phase in MCF7/ADR cells. A marked induction of c-Jun expression as well as phosphorylation of c-Jun and JNK was observed after sangivamycin treatment of MCF7/ADR cells but not MCF7/WT cells. Sangivamycin also induced cleavage of lamin A and poly(ADP-ribose) polymerase (PARP) in MCF7/ADR cells, probably via activation of caspase-6, -7, and -9. Pretreatment with a caspase-9-specific inhibitor or pan-caspase inhibitor abolished sangivamycin-induced cleavage of lamin A and PARP but not sangivamycin induction of c-Jun expression and phosphorylation. Pretreatment of MCF7/ADR cells with SP600125, a specific inhibitor of JNK, or with rottlerin, a specific inhibitor of protein kinase Cdelta (PKCdelta), significantly reduced the sangivamycin-induced apoptosis and almost completely abolished sangivamycin-induced phosphorylation of c-Jun and cleavage of lamin A and PARP. Transfection of MCF7/ADR cells with PKCdelta small interfering RNAs or PKCdelta antibody or rottlerin pretreatment significantly suppressed the phosphorylation of JNK. Taken together, our data suggest that sangivamycin induces mitochondria-mediated apoptotic cell death of MCF7/ADR cells via activation of JNK in a protein kinase Cdelta-dependent manner.     

Differential Effect of Phosphorylation-Defective Survivin on Radiation Response in Estrogen Receptor-Positive and -Negative Breast Cancer

Survivin is a key member of the inhibitor of apoptosis protein family, and is considered a promising therapeutic target due to its universal overexpression in cancers. Survivin is implicated in cellular radiation response through its role in apoptosis, cell division, and DNA damage response. In the present study, analysis of publically available data sets showed that survivin gene expression increased with breast cancer stage (p < 0.00001) and was significantly higher in estrogen receptor-negative cancers as compared to estrogen receptor-positive cancers (p = 9e-46). However, survivin was prognostic in estrogen receptor-positive tumors (p = 0.03) but not in estrogen receptor-negative tumors (p = 0.28). We assessed the effect of a survivin dominant-negative mutant on colony-formation (2D) and mammosphere-formation (3D) efficiency, and radiation response in the estrogen receptor-positive MCF7 and estrogen receptor-negative SUM149 breast cancer cell lines. The colony-formation efficiency was significantly lower in the dominant-negative survivin-transduced cells versus control MCF7 cells (0.42 vs. 0.58, p < 0.01), but it was significantly higher in dominant-negative population versus control-transduced SUM149 cells (0.29 vs. 0.20, p < 0.01). A similar, non-significant, trend in mammosphere-formation efficiency was observed. We compared the radiosensitivity of cells stably expressing dominant-negative survivin with their controls in both cell lines under 2D and 3D culture conditions following exposure to increasing doses of radiation. We found that the dominant-negative populations were radioprotective in MCF7 cells but radiosensitive in SUM149 cells compared to the control-transduced population; further, Taxol was synergistic with the survivin mutant in SUM149 but not MCF7. Our data suggests that survivin modulation influences radiation response differently in estrogen receptor-positive and estrogen receptor-negative breast cancer subtypes, warranting further investigation.     

Histone H1 functions as a stimulatory factor in backup pathways of NHEJ

DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ and B-NHEJ. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4/XLF, B-NHEJ utilizes, at least partly, DNA ligase III/XRCC1 and PARP-1. Using in vitro end-joining assays and protein fractionation protocols similar to those previously applied for the characterization of DNA ligase III as an end-joining factor, we identify here histone H1 as an additional putative NHEJ factor. H1 strongly enhances DNA-end joining and shifts the product spectrum from circles to multimers. While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger. Histone H1 also enhances the activity of PARP-1. Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ.     

Structure and function of the DNA ligases encoded by the mammalian LIG3 gene

Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target.     

Low levels of DNA ligases III and IV sufficient for effective NHEJ

Cells of higher eukaryotes rejoin double strand breaks (DSBs) in their DNA predominantly by a non-homologous DNA end joining (NHEJ) pathway that utilizes the products of DNA-PKcs, Ku, LIG4, XRCC4, XLF/Cernunnos, Artemis as well as DNA polymerase lambda (termed D-NHEJ). Mutants with defects in these proteins remove a large proportion of DSBs from their genome utilizing an alternative pathway of NHEJ that operates as a backup (B-NHEJ). While D-NHEJ relies exclusively on DNA ligase IV, recent work points to DNA ligase III as a component of B-NHEJ. Here, we use RNA interference (RNAi) to further investigate the activity requirements for DNA ligase III and IV in the pathways of NHEJ. We report that 70-80% knock down of LIG3 expression has no detectable effect on DSB rejoining, either in D-NHEJ proficient cells, or in cells where D-NHEJ has been chemically or genetically compromised. Surprisingly, also LIG4 knock down has no effect on repair proficient cells, but inhibits DSB rejoining in a radiosensitive cell line with a hypomorphic LIG4 mutation that severely compromises its activity. The results suggest that complete coverage for D-NHEJ or B-NHEJ is afforded by very low ligase levels and demonstrate residual end joining by DNA ligase IV in cells of patients with mutations in LIG4.     

Prolylcarboxypeptidase regulates proliferation, autophagy, and resistance to 4-hydroxytamoxifen-induced cytotoxicity in estrogen receptor-positive breast cancer cells

Endocrine therapy with tamoxifen (TAM) significantly improves outcomes for patients with estrogen receptor-positive breast cancer. However, intrinsic (de novo) or acquired resistance to TAM occurs in a significant proportion of treated patients. To identify genes involved in resistance to TAM, we introduced full-length cDNA expression library into estrogen receptor-positive MCF7 cells and exposed them to a cytotoxic dose of 4-hydroxytamoxifen (4OHTAM). Four different library inserts were isolated from surviving clones. Re-introduction of the genes individually into naive MCF7 cells made them resistant to 4OHTAM. Cells overexpressing these genes had an increase in acidic autophagic vacuoles induced by 4OHTAM, suggesting their role in autophagy. One of them, prolylcarboxypeptidase (PRCP), was investigated further. Overexpression of PRCP increased cell proliferation, boosted several established markers of autophagy, including expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA in an ER-α dependent manner, and increased resistance to 4OHTAM. Conversely, knockdown of endogenous PRCP in MCF7 cells increased cell sensitivity to 4OHTAM and at the same time decreased cell proliferation and expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA. Inhibition of enzymatic activity of PRCP enhanced 4OHTAM-induced cytotoxicity in MCF7 cells. Cells with acquired resistance to 4OHTAM exhibited increased PRCP activity, although inhibition of PRCP prevented development of 4OHTAM resistance in parental MCF7 cells and restored response to 4OHTAM in MCF7 cells with acquired resistance to 4OHTAM. Thus, we have for the first time identified PRCP as a resistance factor for 4OHTAM resistance in estrogen receptor-positive breast cancer cells.     

Estrogen-responsive RING finger protein controls breast cancer growth

Most of the breast cancers initially respond to endocrine therapy that reduces the levels of estrogens or competes with estrogen for binding to its receptor. Most of the patients, however, acquire resistance to endocrine therapy with tamoxifen and aromatase inhibitors later. We assumed that identification of estrogen-responsive genes those regulate the growth of breast cancer is indispensable to develop new strategies targeting the genes and overcome the resistance to current endocrine therapy. Estrogen-responsive finger protein (Efp) is one of the estrogen receptor (ER)-target genes we have cloned using genomic binding site cloning. Efp features a structure of the RING-finger B-box coiled-coil (RBCC) motif. We postulated that Efp is a critical factor in proliferation of breast tumors. In a model system using MCF7 cells grown in xenografts, we showed that inhibition of Efp expression by antisense oligonucleotide reduced the tumor growth. MCF7 cells overexpressing Efp formed tumors in xenografts even in estrogen deprivation environment. By yeast two-hybrid screen, we identified that Efp interacts with 14-3-3σ, which is known as a cell cycle brake that causes G2 arrest and expressed in normal mammary glands. In vitro studies have revealed that Efp functions as a ubiquitin-protein ligase (E3) that targets 14-3-3σ. These data suggest that Efp controls breast cancer growth through ubiquitin-dependent proteolysis of 14-3-3σ. Future studies may provide a new therapy to block breast tumor proliferation by targeting Efp.     

The nonhomologous DNA end joining pathway is important for chromosome stability in primary fibroblasts

There are two types of chromosome instability, structural and numerical, and these are important in cancer. Many structural abnormalities are likely to involve double-strand DNA (dsDNA) breaks. Nonhomologous DNA end joining (NHEJ) and homologous recombination are the major pathways for repairing dsDNA breaks. NHEJ is the primary pathway for repairing dsDNA breaks throughout the G0, G1 and early S phases of the cell cycle [1]. Ku86 and DNA ligase IV are two major proteins in the NHEJ pathway. We examined primary dermal fibroblasts from mice (wild type, Ku86(+/-), Ku86(-/-), and DNA ligase IV(+/-)) for chromosome breaks. Fibroblasts from Ku86(+/-) or DNA ligase IV(+/-) mice have elevated frequencies of chromosome breaks compared with those from wild-type mice. Fibroblasts from Ku86(-/-) mice have even higher levels of chromosome breaks. Primary pre-B cells from the same animals did not show significant accumulation of chromosome breaks. Rather the pre-B cells showed increased cell death. These studies demonstrate that chromosome breaks arise frequently and that NHEJ is required to repair this constant spontaneous damage.     

Elevation of SIPL1 (SHARPIN) Increases Breast Cancer Risk

SIPL1 (Sharpin) or Sharpin plays a role in tumorigenesis. However, its involvement in breast cancer tumorigenesis remains largely unknown. To investigate this issue, we have systemically analyzed SIPL1 gene amplification and expression data available from Oncomine datasets, which were derived from 17 studies and contained approximately 20,000 genes, 3438 breast cancer cases, and 228 normal individuals. We found a SIPL1 gene amplification in invasive ductal breast cancers compared to normal breast tissues and a significant elevation of SIPL1 mRNA in breast cancers in comparison to non-tumor breast tissues. These results collectively reveal that increases in SIPL1 expression occur during breast cancer tumorigenesis. To further investigate this association, we observed increases in the SIPL1 gene and mRNA in the breast cancer subtypes of estrogen receptor (ER)+, progesterone receptor (PR)+, HER2+, or triple negative. Additionally, a gain of the SIPL1 gene correlated with breast cancer grade and the levels of SIPL1 mRNA associated with both breast cancer stages and grades. Elevation of SIPL1 gene copy and mRNA is linked to a decrease in patient survival, especially for those with PR+, ER+, or HER2- breast cancers. These results are supported by our analysis of SIPL1 protein expression using a tissue microarray containing 224 breast cancer cases, in which higher levels of SIPL1 relate to ER+ and PR+ tumors and AKT activation. Furthermore, we were able to show that progesterone significantly reduced SIPL1 mRNA and protein expression in MCF7 cells. As progesterone enhances breast cancer tumorigenesis in a context dependent manner, inhibition of SIPL1 expression may contribute to progesterone''s non-tumorigenic function which might be countered by SIPL1 upregulation. Taken together, we demonstrate a positive correlation of SIPL1 with BC tumorigenesis.     

Analysis of defective protein ubiquitylation associated to adriamycin resistant cells

DNA damage activated by Adriamycin (ADR) promotes ubiquitin–proteasome system-mediated proteolysis by stimulating both the activity of ubiquitylating enzymes and the proteasome. In ADR-resistant breast cancer MCF7 (MCF7^ADR) cells, protein ubiquitylation is significantly reduced compared to the parental MCF7 cells. Here, we used tandem ubiquitin-binding entities (TUBEs) to analyze the ubiquitylation pattern observed in MCF7 or MCF7^ADR cells. While in MCF7, the level of total ubiquitylation increased up to six-fold in response to ADR, in MCF7^ADR cells only a two-fold response was found. To further explore these differences, we looked for cellular factors presenting ubiquitylation defects in MCF7^ADR cells. Among them, we found the tumor suppressor p53 and its ubiquitin ligase, Mdm2. We also observed a drastic decrease of proteins known to integrate the TUBE-associated ubiquitin proteome after ADR treatment of MCF7 cells, like histone H2AX, HMGB1 or β-tubulin. Only the proteasome inhibitor MG132, but not the autophagy inhibitor chloroquine partially recovers the levels of total protein ubiquitylation in MCF7^ADR cells. p53 ubiquitylation is markedly increased in MCF7^ADR cells after proteasome inhibition or a short treatment with the isopeptidase inhibitor PR619, suggesting an active role of these enzymes in the regulation of this tumor suppressor. Notably, MG132 alone increases apoptosis of MCF7^ADR and multidrug resistant ovarian cancer A2780DR1 and A2780DR2 cells. Altogether, our results highlight the use of ubiquitylation defects to predict resistance to ADR and underline the potential of proteasome inhibitors to treat these chemoresistant cells.     

Antiproliferative effect of adiponectin on MCF7 breast cancer cells: a potential hormonal link between obesity and cancer.

Adiponectin, a hormone secreted by adipose tissue, circulates at high concentrations in human plasma. Paradoxically, plasma levels of adiponectin are approximately 50% lower in obese than in lean subjects. An association between low plasma levels of adiponectin and higher risk of developing breast and other cancers was recently reported. Obesity and overweight have also been associated with increased mortality from cancer. To test the hypothesis that adiponectin exerts direct antiproliferative and/or pro-apoptotic effects on cancer cells, we used the MCF7 human breast adenocarcinoma cell line. The proliferation rate of the MCF7 cells was measured using the MTT method, while apoptosis was examined by quantifying the DNA fragmentation using an ELISA assay. In addition, adiponectin receptor 1 (AdipoR1) and AdipoR2 mRNA expression was detected using RT-PCR. Adiponectin diminished the proliferation rate of MCF7 cells; this effect was significant after 48-96 hours of treatment. The presence of receptor expression suggested that the effect of adiponectin on cell proliferation was most likely specific and adiponectin receptor-mediated. Adiponectin induced no apoptosis of MCF7 cells over 48 hours. We conclude that adiponectin inhibits proliferation but causes no apoptosis of MCF7 breast cancer cells. These data suggest that adiponectin may represent a direct hormonal link between obesity and cancer.     

DNA Ligase IV and Artemis Act Cooperatively to Suppress Homologous Recombination in Human Cells: Implications for DNA Double-Strand Break Repair

Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.