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Silver nanoparticles (AgNPs) show promise for treatment of aggressive cancers including triple‐negative breast cancer (TNBC) in preclinical cancer models. For clinical development of AgNP‐based therapeutics, it will be necessary to clearly define the specific physicochemical features of the nanoparticles that will be used, and to tie these properties to biological outcomes. To fill this knowledge gap, we performed thorough structure/function, mechanistic, safety, and efficacy studies to assess the potential for AgNPs to treat TNBC. We establish that AgNPs, regardless of size, shape, or stabilizing agent, are highly cytotoxic to TNBC cells at doses that are not cytotoxic to non‐malignant breast epithelial cells. In contrast, TNBC cells and non‐malignant breast epithelial cells are similarly sensitive to exposure to silver cation (Ag+), indicating that the nanoparticle formulation is essential for the TNBC‐specific cytotoxicity. Mechanistically, AgNPs are internalized by both TNBC and non‐malignant breast cells, but are rapidly degraded only in TNBC cells. Exposure to AgNPs depletes cellular antioxidants and causes endoplasmic reticulum stress in TNBC cells without causing similar damage in non‐malignant breast epithelial cells. AgNPs also cause extensive DNA damage in 3D TNBC tumor nodules in vitro, but do not disrupt the normal architecture of breast acini in 3D cell culture, nor cause DNA damage or induce apoptosis in these structures. Lastly, we show that systemically administered AgNPs are effective at non‐toxic doses for reducing the growth of TNBC tumor xenografts in mice. This work provides a rationale for development of AgNPs as a safe and specific TNBC treatment.

P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53BP1 outside repair foci. 53BP1 movements are constrained throughout the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma NuMA prevents 53BP1 accumulation at DNA breaks, and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a mechanism involving the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such a mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.

Image-based particle tracking is an essential tool to answer research questions in cell biology and beyond. A major challenge of particle tracking in living systems is that low light exposure is required to avoid phototoxicity and photobleaching. In addition, high-speed imaging used to fully capture particle motion dictates fast image acquisition rates. Short exposure times come at the expense of tracking accuracy. This is generally true for quantitative microscopy approaches and particularly relevant to single molecule tracking where the number of photons emitted from a single chromophore is limited. Image restoration methods based on deep learning dramatically improve the signal-to-noise ratio in low-exposure datasets. However, it is not clear whether images generated by these methods yield accurate quantitative measurements such as diffusion parameters in (single) particle tracking experiments. Here, we evaluate the performance of two popular deep learning denoising software packages for particle tracking, using synthetic datasets and movies of diffusing chromatin as biological examples. With synthetic data, both supervised and unsupervised deep learning restored particle motions with high accuracy in two-dimensional datasets, whereas artifacts were introduced by the denoisers in 3D datasets. Experimentally, we found that, while both supervised and unsupervised approaches improved the number of trackable particles and tracking accuracy, supervised learning generally outperformed the unsupervised approach, as expected. We also highlight that with extremely noisy image sequences, deep learning algorithms produce deceiving artifacts, which underscores the need to carefully evaluate the results. Finally, we address the challenge of selecting hyper-parameters to train convolutional neural networks by implementing a frugal Bayesian optimizer that rapidly explores multidimensional parameter spaces, identifying networks yielding optional particle tracking accuracy. Our study provides quantitative outcome measures of image restoration using deep learning. We anticipate broad application of the approaches presented here to critically evaluate artificial intelligence solutions for quantitative microscopy.

Obesity is a highly prevalent and modifiable breast cancer risk factor. While the role of obesity in fueling breast cancer progression is well established, the mechanisms linking obesity to breast cancer initiation are poorly understood. A hallmark of breast cancer initiation is the disruption of apical polarity in mammary glands. Here we show that mice with diet-induced obesity display mislocalization of Par3, a regulator of cellular junctional complexes defining mammary epithelial polarity. We found that epithelial polarity loss also occurs in a 3D coculture system that combines acini with human mammary adipose tissue, and establish that a paracrine effect of the tissue adipokine leptin causes loss of polarity by overactivation of the PI3K/Akt pathway. Leptin sensitizes non-neoplastic cells to proliferative stimuli, causes mitotic spindle misalignment, and expands the pool of cells with stem/progenitor characteristics, which are early steps for cancer initiation. We also found that normal breast tissue samples with high leptin/adiponectin transcript ratio characteristic of obesity have an altered distribution of apical polarity markers. This effect is associated with increased epithelial cell layers. Our results provide a molecular basis for early alterations in epithelial architecture during obesity-mediated cancer initiation. ispartof: ONCOGENE vol:38 issue:20 pages:3855-3870 ispartof: location:England status: published

The nuclear mitotic apparatus protein, NuMA, is involved in major cellular events such as DNA damage response, apoptosis and p53-mediated growth-arrest, all of which are under the control of the nucleolus upon stress. Proteomic investigation has identified NuMA among hundreds of nucleolar proteins. Yet, the precise link between NuMA and nucleolar function remains undetermined. We confirm that NuMA is present in the nucleolus and reveal redistribution of NuMA upon actinomycin D or doxorubicin-induced nucleolar stress. NuMA coimmunoprecipitates with RNA polymerase I, with ribosomal proteins RPL26 and RPL24, and with components of B-WICH, an ATP-dependent chromatin remodeling complex associated with rDNA transcription. NuMA also binds to 18S and 28S rRNAs and localizes to rDNA promoter regions. Downregulation of NuMA expression triggers nucleolar stress, as shown by decreased nascent pre-rRNA synthesis, fibrillarin perinucleolar cap formation and upregulation of p27kip1, but not p53. Physiologically relevant nucleolar stress induction with reactive oxygen species reaffirms a p53-independent p27kip1 response pathway and leads to nascent pre-rRNA reduction. It also promotes the decrease in the amount of NuMA. This previously uncharacterized function of NuMA in rDNA transcription and p53-independent nucleolar stress response supports a central role for this nuclear structural protein in cellular homeostasis.

Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative "imaging-derived" parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions.

Preventing cancer is vastly better than treating the disease in terms of a patient's quality of life and healthcare costs. Yet, to screen for chemopreventative drugs or evaluate interventions aimed at lowering cancer risk, quantitative readouts of risk are needed. In the breast and in other organs of epithelial origin, apical-basal polarity is key to homeostasis and is one of the first tissue characteristics lost during cancer initiation. Therefore, apical-basal polarity may be leveraged as an "architectural" determinant of cancer risk. A classic approach to quantify the localization of epithelial polarity markers is visual scoring at the microscope by trained investigators. This approach is time-intensive and limited to low throughput. To increase the speed, accuracy, and scoring volume, we developed an algorithm that essentially replaces the human eye to objectively quantify epithelial polarity in microscopy images of breast glandular units (acini). Acini in culture are identified based on a nuclear stain and the corresponding masks are divided into concentric terraces of equal width. This positional information is used to calculate radial intensity profiles (RP) of polarity markers. Profiles with a steep slope represent polarized structures, whereas more horizontal curves are indicative of non-polarized acini. To compare treatment effects, RP curves are integrated into summary values of polarity. We envision applications of this method for primary cancer prevention research with acini organoids, specifically (1) to screen for chemoprevention drugs, (2) for toxicological assessment of suspected carcinogens and pharmacological hit compounds, and (3) for personalized evaluation of cancer risk and risk-reducing interventions. The RadialProfiler algorithm developed for the MATLAB computing environment and for users without prior informatics knowledge is publicly available on the Open Science Framework (OSF).

Cell–cell communication is essential for tissue homeostasis, but its contribution to disease prevention remains to be understood. We demonstrate the involvement of connexin 43 (Cx43, also known as GJA1) and related gap junction in epithelial homeostasis, illustrated by polarity-mediated cell cycle entry and mitotic spindle orientation (MSO). Cx43 localization is restricted to the apicolateral membrane of phenotypically normal breast luminal epithelial cells in 3D culture and in vivo . Chemically induced blockade of gap junction intercellular communication (GJIC), as well as the absence of Cx43, disrupt the apicolateral distribution of polarity determinant tight junction marker ZO-1 (also known as TJP1) and lead to random MSO and cell multilayering. Induced expression of Cx43 in cells that normally lack this protein reestablishes polarity and proper MSO in 3D culture. Cx43-directed MSO implicates PI3K–aPKC signaling, and Cx43 co-precipitates with signaling node proteins β-catenin (CTNNB1) and ZO-2 (also known as TJP2) in the polarized epithelium. The distribution of Cx43 is altered by pro-inflammatory breast cancer risk factors such as leptin and high-fat diet, as shown in cell culture and on tissue biopsy sections. The control of polarity-mediated quiescence and MSO may contribute to the tumor-suppressive role of Cx43.

Up to 30% of patients with metastatic breast cancer eventually develop brain metastasis, yet the pathologic mechanism behind this development remains poorly understood. Here, we profiled long noncoding RNAs in brain metastatic tumors from patients with breast cancer and found that the X-inactive–specific transcript (XIST) was significantly downregulated in these tissues. XIST expression levels inversely correlated with brain metastasis, but not with bone metastasis in patients. Silencing of XIST preferentially promoted brain metastatic growth of XISThigh cells in our xenograft models. Moreover, knockout of XIST in mice mammary glands accelerated primary tumor growth as well as metastases in the brain. Decreased expression of XIST stimulated epithelial–mesenchymal transition and activated c-Met via MSN-mediated protein stabilization, which resulted in the promotion of stemness in the tumor cells. Loss of XIST also augmented secretion of exosomal miRNA-503, which triggered M1–M2 polarization of microglia. This M1–M2 conversion upregulated immune suppressive cytokines in microglia that suppressed T-cell proliferation. Furthermore, we screened an FDA-approved drug library and identified fludarabine as a synthetic lethal drug for XISTlow breast tumor cells and found that fludarabine blocked brain metastasis in our animal model. Our results indicate that XIST plays a critical role in brain metastasis in breast cancer by affecting both tumor cells and the tumor microenvironment and that the XIST-mediated pathway may serve as an effective target for treating brain metastasis. Significance: These findings describe mechanisms of how loss of the lncRNA XIST promotes brain metastasis in breast cancer and identify fludarabine as a potential therapeutic agent that specifically eliminates XISTlow tumor cells in the brain. Cancer Res; 78(15); 4316–30. ©2018 AACR.

Accumulation of 53BP1 at DNA breaks determines DNA repair pathway choice and promotes checkpoint activation. Here, we show regulation of 53BP1 beyond repair foci. 53BP1 movements are constrained in the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma, NuMA prevents 53BP1 accumulation at DNA breaks and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a new mechanism that involves the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.

We describe a simple optical method that creates structured illumination of a photoactivatable probe and apply this method to characterize chromatin motions in nuclei of live cells. A laser beam coupled to a diffractive optical element at the back focal plane of an excitation objective generates an array of near diffraction-limited beamlets with FWHM of [Formula: see text] , which simultaneously photoactivate a [Formula: see text] matrix pattern of GFP-labeled histones, with spots [Formula: see text] apart. From the movements of the photoactivated spots, we map chromatin diffusion coefficients at multiple microdomains of the cell nucleus. The results show correlated motions of nearest chromatin microdomain neighbors, whereas chromatin movements are uncorrelated at the global scale of the nucleus. The method also reveals a DNA damage-dependent decrease in chromatin diffusion. The diffractive optical element instrumentation can be easily and cheaply implemented on commercial inverted fluorescence microscopes to analyze adherent cell culture models. A protocol to measure chromatin motions in nonadherent human hematopoietic stem and progenitor cells is also described. We anticipate that the method will contribute to the identification of the mechanisms regulating chromatin mobility, which influences most genomic processes and may underlie the biogenesis of genomic translocations associated with hematologic malignancies.

Agriculture in the United States employs youth ages ten and older in work environments with high pesticide levels. Younger children in rural areas may also be affected by indirect pesticide exposures. The long-term effects of pesticides on health and development are difficult to assess and poorly understood. Yet, epidemiologic studies suggest associations with cancer as well as cognitive deficits. We report a practical and cost-effective approach to assess environmental pesticide exposures and their biological consequences in children. Our approach combines silicone wristband personal samplers and DNA damage quantification from hair follicles, and was tested as part of a community-based participatory research (CBPR) project involving ten Latino children from farmworker households in North Carolina. Our study documents high acceptance among Latino children and their caregivers of these noninvasive sampling methods. The personal samplers detected organophosphates, organochlorines, and pyrethroids in the majority of the participants (70%, 90%, 80%, respectively). Pesticides were detected in all participant samplers, with an average of 6.2 ± 2.4 detections/participant sampler. DNA damage in epithelial cells from the sheath and bulb of plucked hairs follicles was quantified by immunostaining 53BP1-labled DNA repair foci. This method is sensitive, as shown by dose response analyses to γ radiations where the lowest dose tested (0.1 Gy) led to significant increased 53BP1 foci density. Immunolabeling of DNA repair foci has significant advantages over the comet assay in that specific regions of the follicles can be analyzed. In this cohort of child participants, significant association was found between the number of pesticide detections and DNA damage in the papilla region of the hairs. We anticipate that this monitoring approach of bioavailable pesticides and genotoxicity will enhance our knowledge of the biological effects of pesticides to guide education programs and safety policies.

Chromatin remodeling factors play an active role in the DNA damage response by shaping chromatin to facilitate the repair process. The spatiotemporal regulation of these factors is key to their function, yet poorly understood. We report that the structural nuclear protein NuMA accumulates at sites of DNA damage in a poly[ADP-ribose]ylation-dependent manner and functionally interacts with the ISWI ATPase SNF2h/SMARCA5, a chromatin remodeler that facilitates DNA repair. NuMA coimmunoprecipitates with SNF2h, regulates its diffusion in the nucleoplasm and controls its accumulation at DNA breaks. Consistent with NuMA enabling SNF2h function, cells with silenced NuMA exhibit reduced chromatin decompaction after DNA cleavage, lesser focal recruitment of homologous recombination repair factors, impaired DNA double-strand break repair in chromosomal (but not in episomal) contexts and increased sensitivity to DNA cross-linking agents. These findings reveal a structural basis for the orchestration of chromatin remodeling whereby a scaffold protein promotes genome maintenance by directing a remodeler to DNA breaks.

International audience; Plastoglobules, lipid-protein bodies in the stroma of plant chloroplasts, are enriched in non-polar lipids, in particular prenyl quinols. In the present study we show that, in addition to the thylakoids, plastoglobules also contain a considerable proportion of the plastidial PQ-9 (plastoquinol-9), the redox component of photosystem II, and of the cyclized product of PQ-9, PC-8 (plastochromanol-8), a tocochromanol with a structure similar to γ-tocopherol and γ-tocotrienol, but with a C-40 prenyl side chain. PC-8 formation was abolished in the Arabidopsis thaliana tocopherol cyclase mutant vte1, but accumulated in VTE1-overexpressing plants, in agreement with a role of tocopherol cyclase (VTE1) in PC-8 synthesis. VTE1 overexpression resulted in the proliferation of the number of plastoglobules which occurred in the form of clusters in the transgenic lines. Simultaneous overexpression of VTE1 and of the methyltransferase VTE4 resulted in the accumulation of a compound tentatively identified as 5-methyl-PC-8, the methylated form of PC-8. The results of the present study suggest that the existence of a plastoglobular pool of PQ-9, along with the partial conversion of PQ-9 into PC-8, might represent a mechanism for the regulation of the antioxidant content in thylakoids and of the PQ-9 pool that is available for photosynthesis.

Toll-like receptor 9 (TLR9) activates the innate immune system in response to oligonucleotides rich in CpG whereas DNA lacking CpG could inhibit its activation. However, the mechanism of how TLR9 interacts with nucleic acid and becomes activated in live cells is not well understood. Here, we report on the successful implementation of single molecule tools, constituting fluorescence correlation/cross-correlation spectroscopy (FCS and FCCS) and photon count histogram (PCH) with fluorescence lifetime imaging (FLIM) to study the interaction of TLR9-GFP with Cy5 labeled oligonucleotide containing CpG or lacking CpG in live HEK 293 cells. Our findings show that i) TLR9 predominantly forms homodimers (80%) before binding to a ligand and further addition of CpG or non CpG DNA does not necessarily increase the proportion of TLR9 dimers, ii) CpG DNA has a lower dissociation constant (62 nM +/- 9 nM) compared to non CpG DNA (153 nM +/- 26 nM) upon binding to TLR9, suggesting that a motif specific binding affinity of TLR9 could be an important factor in instituting a conformational change-dependant activation, and iii) both CpG and non CpG DNA binds to TLR9 with a 1: 2 stoichiometry in vivo. Collectively, through our findings we establish an in vivo model of TLR9 binding and activation by CpG DNA using single molecule fluorescence techniques for single cell studies.

The conditional flu mutant of Arabidopsis thaliana generates singlet oxygen ((1)O(2)) in plastids during a dark-to-light shift. Seedlings of flu bleach and die, whereas mature plants stop growing and develop macroscopic necrotic lesions. Several suppressor mutants, dubbed singlet oxygen-linked death activator (soldat), were identified that abrogate (1)O(2)-mediated cell death of flu seedlings. One of the soldat mutations, soldat10, affects a gene encoding a plastid-localized protein related to the human mitochondrial transcription termination factor mTERF. As a consequence of this mutation, plastid-specific rRNA levels decrease and protein synthesis in plastids of soldat10 is attenuated. This disruption of chloroplast homeostasis in soldat10 seedlings affects communication between chloroplasts and the nucleus and leads to changes in the steady-state concentration of nuclear gene transcripts. The soldat10 seedlings suffer from mild photo-oxidative stress, as indicated by the constitutive up-regulation of stress-related genes. Even though soldat10/flu seedlings overaccumulate the photosensitizer protochlorophyllide in the dark and activate the expression of (1)O(2)-responsive genes after a dark-to-light shift they do not show a (1)O(2)-dependent cell death response. Disturbance of chloroplast homeostasis in emerging soldat10/flu seedlings seems to antagonize a subsequent (1)O(2)-mediated cell death response without suppressing (1)O(2)-dependent retrograde signaling. The results of this work reveal the unexpected complexity of what is commonly referred to as 'plastid signaling'.

International audience; Chloroplasts contain lipoprotein particles termed plastoglobules. Plastoglobules are generally believed to have little function beyond lipid storage. Here we report on the identification of plastoglobule proteins using mass spectrometry methods in Arabidopsis thaliana. We demonstrate specific plastoglobule association of members of the plastid lipid-associated proteins/fibrillin family as well as known metabolic enzymes, including the tocopherol cyclase (VTE1), a key enzyme of tocopherol (vitamin E) synthesis. Moreover, comparative analysis of chloroplast membrane fractions shows that plastoglobules are a site of vitamin E accumulation in chloroplasts. Thus, in addition to their lipid storage function, we propose that plastoglobules are metabolically active, taking part in tocopherol synthesis and likely other pathways.

Two homologous GTP-binding proteins, atToc33 and atToc159, control access of cytosolic precursor proteins to the chloroplast. atToc33 is a constitutive outer chloroplast membrane protein, whereas the precursor receptor atToc159 also exists in a soluble, cytosolic form. This suggests that atToc159 may be able to switch between a soluble and an integral membrane form. By transient expression of GFP fusion proteins, mutant analysis, and biochemical experimentation, we demonstrate that the GTP-binding domain regulates the targeting of cytosolic atToc159 to the chloroplast and mediates the switch between cytosolic and integral membrane forms. Mutant atToc159, unable to bind GTP, does not reinstate a green phenotype in an albino mutant (ppi2) lacking endogenous atToc159, remaining trapped in the cytosol. Thus, the function of atToc159 in chloroplast biogenesis is dependent on an intrinsic GTP-regulated switch that controls localization of the receptor to the chloroplast envelope.

Oligomerization of G protein-coupled receptors (GPCRs) is known to play important roles in regulating receptor pharmacology and function. Whereas many bivalent GPCR interactions have been described, the stoichiometry and localiza- tion of GPCR oligomers are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to study adenosine A2A receptor (A2AR) oligomerization. The data sug- gest specificity of the A2AR/A2AR interaction monitored by BiFC and proper sub-cellular localization of tagged receptors. More- over, using a novel approach combining fluorescence resonance energy transfer and BiFC, we found that at least three A2A receptors assemble into higher-order oligomers at the plasma membrane in Cath.A differentiated neuronal cells. Structured summary: MINT-6797156, MINT-6797142: A2AR (uniprotkb:P29274) physically interacts (MI:0218) with A2AR (uniprotkb:P29274) by bimolecular fluorescence comple- mentation (MI:0809) MINT-6797129: A2AR (uniprotkb:P29274) physically interacts (MI:0218) with A2AR (uniprotkb:P29274) by fluorescent resonance energy transfer (MI:0055)