Common variants in 94 loci have been associated with breast cancer including 15 loci with genome-wide significant associations (P<5 × 10(-8)) with oestrogen receptor (ER)-negative breast cancer and BRCA1-associated breast cancer risk. In this study, to identify new ER-negative susceptibility loci, we performed a meta-analysis of 11 genome-wide association studies (GWAS) consisting of 4,939 ER-negative cases and 14,352 controls, combined with 7,333 ER-negative cases and 42,468 controls and 15,252 BRCA1 mutation carriers genotyped on the iCOGS array. We identify four previously unidentified loci including two loci at 13q22 near KLF5, a 2p23.2 locus near WDR43 and a 2q33 locus near PPIL3 that display genome-wide significant associations with ER-negative breast cancer. In addition, 19 known breast cancer risk loci have genome-wide significant associations and 40 had moderate associations (P<0.05) with ER-negative disease. Using functional and eQTL studies we implicate TRMT61B and WDR43 at 2p23.2 and PPIL3 at 2q33 in ER-negative breast cancer aetiology. All ER-negative loci combined account for ∼11% of familial relative risk for ER-negative disease and may contribute to improved ER-negative and BRCA1 breast cancer risk prediction.

PARP1 is the main sensor of single- and double-strand breaks in DNA and, in building chains of poly(ADP-ribose), promotes the recruitment of many downstream signaling and effector proteins involved in the DNA damage response (DDR). We show a robust physical interaction between PARP1 and the replication fork protein TIMELESS, distinct from the known TIMELESS-TIPIN complex, which activates the intra-S phase checkpoint. TIMELESS recruitment to laser-induced sites of DNA damage is dependent on its binding to PARP1, but not PARP1 activity. We also find that the PARP1-TIMELESS complex contains a number of established PARP1 substrates, and TIMELESS mutants unable to bind PARP1 are impaired in their ability to bind PARP1 substrates. Further, PARP1 binding to certain substrates and their recruitment to DNA damage lesions is impaired by TIMELESS knockdown, and TIMELESS silencing significantly impairs DNA double-strand break repair. We hypothesize that TIMELESS cooperates in the PARP1-mediated DDR.

The endoplasmic reticulum (ER) chaperone protein, calreticulin (CRT), is essential for proper glycoprotein folding and maintaining cellular calcium homeostasis. During ER stress, CRT is overexpressed as part of the unfolded protein response (UPR). In addition, CRT can be released as a damage-associated molecular pattern (DAMP) molecule that may interact with pathogen-associated molecular patterns (PAMPs) during the innate immune response. One such PAMP is lipopolysaccharide (LPS), a component of the gram-negative bacterial cell wall. In this report, we show that recombinant and native human placental CRT strongly interacts with LPS in solution, solid phase, and the surface of gram-negative and gram-positive bacteria. Furthermore, LPS induces oilgomerization of CRT with a disappearance of the monomeric form. The application of recombinant CRT (rCRT) to size exclusion and anion exchange chromatography shows an atypical heterogeneous elution profile, indicating that LPS affects the conformation and ionic charge of CRT. Interestingly, LPS bound to CRT is detected in sera of bronchiectasis patients with chronic bacterial infections. By ELISA, rCRT dose-dependently bound to solid phase LPS via the N- and C-domain globular head region of CRT and the C-domain alone. The specific interaction of CRT with LPS may be important in PAMP innate immunity.

We introduce the Interaction Factor (IF), a measure for quantifying the interaction of molecular clusters in super-resolution microscopy images. The IF is robust in the sense that it is independent of cluster density, and it only depends on the extent of the pair-wise interaction between different types of molecular clusters in the image. The IF for a single or a collection of images is estimated by first using stochastic modelling where the locations of clusters in the images are repeatedly randomized to estimate the distribution of the overlaps between the clusters in the absence of interaction (IF = 0). Second, an analytical form of the relationship between IF and the overlap (which has the random overlap as its only parameter) is used to estimate the IF for the experimentally observed overlap. The advantage of IF compared to conventional methods to quantify interaction in microscopy images is that it is insensitive to changing cluster density and is an absolute measure of interaction, making the interpretation of experiments easier. We validate the IF method by using both simulated and experimental data and provide an ImageJ plugin for determining the IF of an image.

Studies have consistently indicated that the majority of individuals meeting the US Prevention Services Task Force guidelines for genetic testing have not had genetic counseling or testing. Despite increased availability and lower costs of multiplex cancer gene panels, there remains a gap in genetics services that has not been addressed by the current care delivery models. Lower cost of DNA sequencing with online patient-initiated ordering could increase test availability, but the ideal quantity and delivery method of patient education is not known. We hypothesized that online genetic education and testing with access to board certified genetic counselors could improve access to genetic testing while maintaining test quality and clinical utility. The MAGENTA (MAking GENetic Testing Accessible) trial is a nationwide randomized study designed to compare the effectiveness of online genetic education with pre- and post-test telephone genetic counseling to three potentially more accessible alternative approaches: online genetic education with optional telephone counseling, online genetic education with required pre-test telephone genetic counseling, and online genetic education with required post-test telephone genetic counseling.

Sporadic Alzheimer's disease (AD) exclusively affects elderly people. Using direct conversion of AD patient fibroblasts into induced neurons (iNs), we generated an age-equivalent neuronal model. AD patient-derived iNs exhibit strong neuronal transcriptome signatures characterized by downregulation of mature neuronal properties and upregulation of immature and progenitor-like signaling pathways. Mapping iNs to longitudinal neuronal differentiation trajectory data demonstrated that AD iNs reflect a hypo-mature neuronal identity characterized by markers of stress, cell cycle, and de-differentiation. Epigenetic landscape profiling revealed an underlying aberrant neuronal state that shares similarities with malignant transformation and age-dependent epigenetic erosion. To probe for the involvement of aging, we generated rejuvenated iPSC-derived neurons that showed no significant disease-related transcriptome signatures, a feature that is consistent with epigenetic clock and brain ontogenesis mapping, which indicate that fibroblast-derived iNs more closely reflect old adult brain stages. Our findings identify AD-related neuronal changes as age-dependent cellular programs that impair neuronal identity.

One of the most central questions about the repair of a double-strand DNA break (DSB) concerns how the two free DNA ends are brought together - a step called synapsis. Using single-molecule FRET (smFRET), we show here that both Ku plus XRCC4:DNA ligase IV are necessary and sufficient to achieve a flexible synapsis of blunt DNA ends, whereas either alone is not. Addition of XLF causes a transition to a close synaptic state, and maximum efficiency of close synapsis is achieved within 20 min. The promotion of close synapsis by XLF indicates a role that is independent of a filament structure, with action focused at the very ends of each duplex. DNA-PKcs is not required for the formation of either the flexible or close synaptic states. This model explains in biochemical terms the evolutionarily central synaptic role of Ku, X4L4, and XLF in NHEJ for all eukaryotes.

There is considerable interest in the clinical development of inhibitors of mTOR complexes mTORC1 and 2. Because mTORC1 and its downstream mRNA translation effectors may protect against genotoxic DNA damage, we investigated the inhibition of mTORC1 and mTORC1/2 in the ability to reverse platinum resistance in tissue culture and in animal tumor models of serous ovarian cancer. Cell survival, tumor growth, PI3K-AKT-mTOR pathway signaling, DNA damage and repair response (DDR) gene expression, and translational control were all investigated. We show that platinum-resistant OVCAR-3 ovarian cancer cells are resensitized to low levels of carboplatin in culture by mTOR inhibition, demonstrating reduced survival after treatment with either mTORC1 inhibitor everolimus or mTORC1/2 inhibitor PP242. Platinum resistance is shown to be associated with activating phosphorylation of AKT and CHK1, inactivating phosphorylation of 4E-BP1, the negative regulator of eIF4E, which promotes increased cap-dependent mRNA translation and increased levels of CHK1 and BRCA1 proteins. Animals with platinum-resistant OVCAR-3 tumors treated with carboplatin plus mTORC1/2 inhibition had significantly longer median survival and strikingly reduced metastasis compared with animals treated with carboplatin plus everolimus, which inhibits only mTORC1. Reduced tumor growth, metastasis, and increased survival by mTORC1/2 inhibition with carboplatin treatment was associated with reduced AKT-activating phosphorylation and increased 4E-BP1 hypophosphorylation (activation). We conclude that mTORC1/2 inhibition is superior to mTORC1 inhibition in reversing platinum resistance in tumors and strongly impairs AKT activation, DNA repair responses, and translation, promoting improved survival in the background of platinum resistance. Mol Cancer Ther; 15(7); 1557-67. ©2016 AACR.

We previously reported that aberrant TGF-β/Smad2/3 signaling in endometrial cancer (ECA) leads to continuous ubiquitylation of p27(kip1)(p27) by the E3 ligase SCF-Skp2/Cks1 causing its degradation, as a putative mechanism involved in the pathogenesis of this cancer. In contrast, normal intact TGF-β signaling prevents degradation of nuclear p27 by SCF-Skp2/Cks1 thereby accumulating p27 to block Cdk2 for growth arrest. Here we show that in ECA cell lines and normal primary endometrial epithelial cells, TGF-β increases Cdh1 and its binding to APC/C to form the E3 ligase complex that ubiquitylates Cks1 and Skp2 prompting their proteasomal degradation and thus, leaving p27 intact. Knocking-down Cdh1 in ECA cell lines increased Skp2/Cks1 E3 ligase activity, completely diminished nuclear and cytoplasmic p27, and obviated TGF-β-mediated inhibition of proliferation. Protein synthesis was not required for TGF-β-induced increase in nuclear p27 and decrease in Cks1 and Skp2. Moreover, half-lives of Cks1 and Skp2 were extended in the Cdh1-depleted cells. These results suggest that the levels of p27, Skp2 and Cks1 are strongly or solely regulated by proteasomal degradation. Finally, an inverse relationship of low p27 and high Cks1 in the nucleus was shown in patients in normal proliferative endometrium and grade I-III ECAs whereas differentiated secretory endometrium showed the reverse. These studies implicate Cdh1 as the master regulator of TGF-β-induced preservation of p27 tumor suppressor activity. Thus, Cdh1 is a potential therapeutic target for ECA and other human cancers showing an inverse relationship between Cks1/Skp2 and p27 and/or dysregulated TGF-β signaling.

Phase 2 trials suggest that prolonged intravenous (IV) infusion of the topoisomerase 1 inhibitor topotecan may be less toxic than when given by standard IV bolus 5-day administration. Oxaliplatin exhibits efficacy in platinum-pretreated disease and shows preclinical synergy with topoisomerase 1 inhibitors. We sought to determine the efficacy and safety of oxaliplatin plus infusion topotecan in recurrent platinum-pretreated ovarian cancer.

In many human cancers, the tumor suppressor, p27(kip1) (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization. Lack of nuclear p27 causes aberrant cell cycle progression, and cytoplasmic p27 mediates cell migration/metastasis. We previously showed that mitogenic 17-β-estradiol (E2) induces degradation of p27 by the E3 ligase Skp1-Cullin1-F-Box- S phase kinase-associated protein2/cyclin dependent kinase regulatory subunit 1 in primary endometrial epithelial cells and endometrial carcinoma (ECA) cell lines, suggesting a pathogenic mechanism for type I ECA, an E2-induced cancer. The current studies show that treatment of endometrial carcinoma cells-1 (ECC-1) with small molecule inhibitors of Skp2/Cks1 E3 ligase activity (Skp2E3LIs) stabilizes p27 in the nucleus, decreases p27 in the cytoplasm, and prevents E2-induced proliferation and degradation of p27 in endometrial carcinoma cells-1 and primary ECA cells. Furthermore, Skp2E3LIs increase p27 half-life by 6 hours, inhibit cell proliferation (IC50, 14.3μM), block retinoblastoma protein (pRB) phosphorylation, induce G1 phase block, and are not cytotoxic. Similarly, using super resolution fluorescence localization microscopy and quantification, Skp2E3LIs increase p27 protein in the nucleus by 1.8-fold. In vivo, injection of Skp2E3LIs significantly increases nuclear p27 and reduces proliferation of endometrial epithelial cells by 42%-62% in ovariectomized E2-primed mice. Skp2E3LIs are specific inhibitors of proteolytic degradation that pharmacologically target the binding interaction between the E3 ligase, SCF-Skp2/Cks1, and p27 to stabilize nuclear p27 and prevent cell cycle progression. These targeted inhibitors have the potential to be an important therapeutic advance over general proteasome inhibitors for cancers characterized by SCF-Skp2/Cks1-mediated destruction of nuclear p27.

Ventricular ATP-sensitive potassium (K(ATP)) channels link intracellular energy metabolism to membrane excitability and contractility. Our recent proteomics experiments identified plakoglobin and plakophilin-2 (PKP2) as putative K(ATP) channel-associated proteins. We investigated whether the association of K(ATP) channel subunits with junctional proteins translates to heterogeneous subcellular distribution within a cardiac myocyte. Co-immunoprecipitation experiments confirmed physical interaction between K(ATP) channels and PKP2 and plakoglobin in rat heart. Immunolocalization experiments demonstrated that K(ATP) channel subunits (Kir6.2 and SUR2A) are expressed at a higher density at the intercalated disk in mouse and rat hearts, where they co-localized with PKP2 and plakoglobin. Super-resolution microscopy demonstrate that K(ATP) channels are clustered within nanometer distances from junctional proteins. The local K(ATP) channel density, recorded in excised inside-out patches, was larger at the cell end when compared with local currents recorded from the cell center. The K(ATP) channel unitary conductance, block by MgATP and activation by MgADP, did not differ between these two locations. Whole cell K(ATP) channel current density (activated by metabolic inhibition) was ∼40% smaller in myocytes from mice haploinsufficient for PKP2. Experiments with excised patches demonstrated that the regional heterogeneity of K(ATP) channels was absent in the PKP2 deficient mice, but the K(ATP) channel unitary conductance and nucleotide sensitivities remained unaltered. Our data demonstrate heterogeneity of K(ATP) channel distribution within a cardiac myocyte. The higher K(ATP) channel density at the intercalated disk implies a possible role at the intercellular junctions during cardiac ischemia.

The levels of proteins that control the cell cycle are regulated by ubiquitin-mediated degradation via the ubiquitin-proteasome system (UPS) by substrate-specific E3 ubiquitin ligases. The cyclin-dependent kinase inhibitor, p27kip1 (p27), that blocks the cell cycle in G1, is ubiquitylated by the E3 ligase SCF-Skp2/Cks1 for degradation by the UPS. In turn, Skp2 and Cks1 are ubiquitylated by the E3 ligase complex APC/Cdh1 for destruction thereby maintaining abundant levels of nuclear p27. We previously showed that perpetual proteasomal degradation of p27 is an early event in Type I endometrial carcinogenesis (ECA), an estrogen (E2)-induced cancer. The present studies demonstrate that E2 stimulates growth of ECA cell lines and normal primary endometrial epithelial cells (EECs) and induces MAPK-ERK1/2-dependent phosphorylation of p27 on Thr187, a prerequisite for p27 ubiquitylation by nuclear SCF-Skp2/Cks1 and subsequent degradation. In addition, E2 decreases the E3 ligase [APC]Cdh1 leaving Skp2 and Cks1 intact to cause p27 degradation. Furthermore, knocking-down Skp2 prevents E2-induced p27 degradation and growth stimulation suggesting that the pathogenesis of E2-induced ECA is dependent on Skp2-mediated degradation of p27. Conversely, progesterone (Pg) as an inhibitor of endometrial proliferation increases nuclear p27 and Cdh1 in primary EECs and ECA cells. Pg, also increases Cdh1 binding to APC to form the active E3ligase. Knocking-down Cdh1 obviates Pg-induced stabilization of p27 and growth inhibition. Notably, neither E2 nor Pg affected transcription of Cdh1, Skp2, Cks1 nor p27. These studies provide new insights into hormone regulation of cell proliferation through the UPS. The data implicates that preventing nuclear p27 degradation by blocking Skp2/Cks1-mediated degradation of p27 or increasing Cdh1 to mediate degradation of Skp2-Cks1 are potential strategies for the prevention and treatment of ECA.

Nonhomologous end joining (NHEJ) must adapt to diverse end structures during repair of chromosome breaks. Here, we investigate the mechanistic basis for this flexibility. DNA ends are aligned in a paired-end complex (PEC) by Ku, XLF, XRCC4, and DNA ligase IV (LIG4); we show by single-molecule analysis how terminal mispairs lead to mobilization of ends within PECs and consequent sampling of more end-alignment configurations. This remodeling is essential for direct ligation of damaged and mispaired ends during cellular NHEJ, since remodeling and ligation of such ends both require a LIG4-specific structural motif, insert1. Insert1 is also required for PEC remodeling that enables nucleolytic processing when end structures block direct ligation. Accordingly, cells expressing LIG4 lacking insert1 are sensitive to ionizing radiation. Cellular NHEJ of diverse ends thus identifies the steps necessary for repair through LIG4-mediated sensing of differences in end structure and consequent dynamic remodeling of aligned ends.

Microsatellite instability-high (MSI-H) tumors are characterized by high tumor mutation burden and responsiveness to checkpoint blockade. We identified tumor-specific frameshifts encoding multiple epitopes that originated from indel mutations shared among patients with MSI-H endometrial, colorectal, and stomach cancers. Epitopes derived from these shared frameshifts have high population occurrence rates, wide presence in many tumor subclones, and are predicted to bind to the most frequent MHC alleles in MSI-H patient cohorts. Neoantigens arising from these mutations are distinctly unlike self and viral antigens, signifying novel groups of potentially highly immunogenic tumor antigens. We further confirmed the immunogenicity of frameshift peptides in T cell stimulation experiments using blood mononuclear cells isolated from both healthy donors and MSI-H cancer patients. Our study uncovers the widespread occurrence and strong immunogenicity of tumor-specific antigens derived from shared frameshift mutations in MSI-H cancer and Lynch syndrome patients, suitable for the design of common "off-the-shelf" cancer vaccines.

In recognition of the potential of calreticulin (CRT) protein in enhancing the rate and quality of wound healing in excisional animal wound models, this study was to incorporate CRT via polyblend electrospinning into polycaprolactone (PCL)/type 1 collagen (Col1) nanofibers (NFs; 334 ± 75 nm diameter) as biomimetic extracellular matrices to provide a novel mode of delivery and protection of CRT with enhanced synergistic activities for tissue regeneration. Release kinetic studies using fluoresceinated CRT (CRT-FITC) polyblend NFs showed a burst release within 4 h reaching a plateau at 72 h, with further intervals of release upon incubation with fresh phosphate buffered saline for up to 8 weeks. By measuring fluorescence during the first 4 h of release, CRT-FITC-containing NFs were shown to protect CRT from proteolytic digestion (e.g., by subtilisin) compared to CRT-FITC in solution. CRT incorporated into NFs (CRT-NFs) also showed retention of biological activities and potency for stimulating proliferation and migration of human keratinocytes and fibroblasts. Fibroblasts seeded on CRT-NFs, after 2 days, showed increased amounts of fibronectin, TGF-β1, and integrin β1 in cell lysates by immunoblotting. Compared to NFs without CRT, CRT-NFs supported cell responses consistent with greater cell polarization and increased laminin-5 deposition of keratinocytes and a more motile phenotype of fibroblasts, as suggested by vinculin-capping F-actin fibers nonuniformly located throughout the cell body and the secretion of phosphorylated focal adhesion kinase-enriched migrasomes. Altogether, CRT electrospun into PCL/Col1 NFs retained its structural integrity and biological functions while having additional benefits of customizable loading, protection of CRT from proteolytic degradation, and sustained release of CRT from NFs, coupled with innate physicochemical cues of biomimetic PCL/Col1 NFs. Such synergistic activities have potential for healing recalcitrant wounds such as diabetic foot ulcers.

Calreticulin (CRT) and citrullinated (citCRT) are implicated in rheumatoid arthritis (RA) pathology. citCRT binds to RA shared epitopes (SE) on HLA-DR molecules with high affinity and triggers pro-inflammatory events in adjacent cells. The aim of the study was to detect the presence of citCRT prior to developing RA and evaluate if citCT is a target for autoantibodies in RA cohorts with and without lung disease. Antibodies were assessed by ELISA against native CRT, citCRT and general protein citrullination, in sera from 50 RA patients without lung disease, 122 bronchiectasis (BR) patients, 52 bronchiectasis patients with RA (BRRA), 87 asthma patients and 77 healthy controls (HC). Serum citCRT was detected by immunoblotting and mass spectrometry. Genomic DNA was genotyped for HLA-DRB1 alleles. Patients were assessed for DAS28, rheumatoid factor, and anti-cyclic citrullinated peptide antibodies. Extracellular citCRT was detected in BR patients sera prior to them developing RA. A citCRT SE binding peptide GEWKPR261citQIDNPDYK was identified. Anti-CRT antibodies were observed in 18% of BR patients with or without RA. Anti-citCRT antibodies were observed in ∼35% of BR or RA patients, increasing to 58% in BRRA patients. In the RA alone patients 7/20 (35%) who were negative for RF and anti-CCP were anti-CRT antibody positive and had higher DAS28 scores than triple negative RA alone patients. Three of the four BR patients who developed RA over 18 months were anti-citCRT+ve SE+ve. The detection of citCRT in BR and development of anti-citCRT in BR patients suggests citCRT antigens are early targets of antigenicity in these patients, especially in SE+ve patients prior to the onset of RA.

Tamoxifen has long been the endocrine treatment of choice for women with breast cancer and is now employed for prophylactic use in women at high risk from breast cancer. Other selective estrogen receptor modulators (SERMs), such as raloxifene, mimic some of tamoxifen's beneficial effects and, like tamoxifen, exhibit a complex mixture of organ-specific estrogen agonist and antagonistic properties. However, accompanying the positive effects of tamoxifen has been the emergence of evidence for an increased risk of endometrial cancer associated with its use. A more complete understanding of the mechanism(s) of SERM carcinogenicity and endometrial effects is therefore required. We have sought to compare and characterise the transcript profile of tamoxifen, raloxifene and the agonist estradiol in human endometrial cells. Using primary cultures of human endometria, to best emulate the in vivo responses in a manageable in vitro system, we have shown 230 significant changes in gene expression for epithelial cultures and 83 in stromal cultures, either specific to 17beta-estradiol, tamoxifen or raloxifene, or changed across more than one of the treatments. Considering the transcriptome as a whole, the endometrial responses to raloxifene or tamoxifen were more similar than either drug was to 17beta-estradiol. Treatment of endometrial cultures with tamoxifen resulted in the largest number of gene changes relative to control cultures and a high proportion of genes associated with regulation of gene transcription, cell-cycle control and signal transduction. Tamoxifen-specific changes that might point towards mechanisms for its proliferative response in the endometrium included changes in retinoblastoma and c-myc binding proteins, the APCL, dihydrofolate reductase (DHFR) and E2F1 genes and other transcription factors. Tamoxifen was also found to give rise to the highest number of gene expression changes common to those that characterise malignant endometria. It is anticipated that this study will provide leads for further and more focused investigation into SERM carcinogenicity.

Despite significant increase in COVID-19 publications, characterization of COVID-19 infection in patients with gynecologic cancer remains limited. Here we present an update of COVID-19 outcomes among people with gynecologic cancer in New York City (NYC) during the initial surge of severe acute respiratory syndrome coronavirus 2 (coronavirus disease 2019 [COVID-19]).

Neurons are the longest-lived cells in our bodies and lack DNA replication, which makes them reliant on a limited repertoire of DNA repair mechanisms to maintain genome fidelity. These repair mechanisms decline with age, but we have limited knowledge of how genome instability emerges and what strategies neurons and other long-lived cells may have evolved to protect their genomes over the human life span. A targeted sequencing approach in human embryonic stem cell-induced neurons shows that, in neurons, DNA repair is enriched at well-defined hotspots that protect essential genes. These hotspots are enriched with histone H2A isoforms and RNA binding proteins and are associated with evolutionarily conserved elements of the human genome. These findings provide a basis for understanding genome integrity as it relates to aging and disease in the nervous system.