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A. Uninfected 4T1 tumor volume from mice treated 1 x weekly control diet-derived fecal transplant. B. Uninfected 4T1 tumor volume from mice treated 1 x weekly lard diet-derived fecal transplant. C. Tumor volume over time of uninfected 4T1 cells mixed with control diet-derived fecal conditioned media infected RAW 264.7 (macrophage) cells. D. Tumor volume over time of uninfected 4T1 cells mixed with lard diet-derived fecal conditioned media infected RAW 264.7 (macrophage) cells. E. Tumor proliferation as determined by Ki67 immunoreactivity. F. Gram-positive bacteria content in 4T1 breast tumors. G. LPS positive bacteria content in 4T1 tumors. H. Infiltrating tumor-associated macrophage content in 4T1 tumors was determined by F4/80 immunoreactivity. n=5-6.*p

The supplementary infomation containing Figure legends, seven supplemental figures and one supplemental table. Figure S1 XIST expression is preferentially correlated with brain metastasis in breast cancer. Figure S2 Knockout of XIST preferentially promotes brain metastases. Figure S3 Knockout of XIST enhances tumor growth and distant metastases. Figure S4 Knockdown of XIST promotes EMT and stemness. Figure S5 c-Met is regulated by X-chromosome gene, MSN. Figure S6 miR-503 promotes the conversion of M1 to M2 phenotype of microglia. Figure S7 Fludarabine selectively suppresses the growth of XISTlow cells. Table S1 reagent and resource.

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.

Three-dimensional (3D) imaging and object tracking is critical for medical and biological research and can be achieved by multifocal imaging with diffractive optical elements (DOEs) converting depth (To precisely track microscopic fluorescent objects in biological systems in 3D with a simple low-cost DOE system.We designed a multiring spiral phase plate (SPP) generating a single-spot rotating point spread function (SS-RPSF) in a microscope. Our simple, analytically transparent design process uses Bessel beams to avoid rotational ambiguities and achieve a significant depth range. The SPP was inserted into the Nomarski prism slider of a standard microscope. Performance was evaluated using fluorescent beads and in live cells expressing a fluorescent chromatin marker.Bead localization precision wasPrecise 3D localization and tracking can be achieved with a SS-RPSF SPP in a standard microscope with minor modifications.

Molecular links between breast cancer risk factors and pro-oncogenic tissue alterations are poorly understood. The goal of this study was to characterize the impact of overweight and obesity on tissue markers of risk, using normal breast biopsies, a mouse model of obesity, and cultured breast acini. Proliferation and disruption of epithelial polarity, both necessary for tumor initiation, were quantified by immunostaining. High BMI and elevated leptin were associated with compromised epithelial polarity whereas overweight was associated with proliferation in human and mice mammary glands. Human serum with unfavorable adipokine levels altered epithelial polarization of cultured acini, recapitulating the effect of leptin. Weight loss in mice led to metabolic improvements and restored epithelial polarity. In acini cultures, epithelial polarity loss was prevented by antioxidants and could be reverted by normalizing culture conditions. This study provides a framework to set target values for metabolic improvements and to assess the efficacy of interventional studies aimed at reducing breast cancer risk.

Chromatin motions depend on and may regulate genome functions, in particular the DNA damage response. In yeast, DNA double-strand breaks (DSBs) globally increase chromatin diffusion, whereas in higher eukaryotes the impact of DSBs on chromatin dynamics is more nuanced. We mapped the motions of chromatin microdomains in mammalian cells using diffractive optics and photoactivatable chromatin probes and found a high level of spatial heterogeneity. DNA damage reduces heterogeneity and imposes spatially defined shifts in motions: Distal to DNA breaks, chromatin motions are globally reduced, whereas chromatin retains higher mobility at break sites. These effects are driven by context-dependent changes in chromatin compaction. Photoactivated lattices of chromatin microdomains are ideal to quantify microscale coupling of chromatin motion. We measured correlation distances up to 2 µm in the cell nucleus, spanning chromosome territories, and speculate that this correlation distance between chromatin microdomains corresponds to the physical separation of A and B compartments identified in chromosome conformation capture experiments. After DNA damage, chromatin motions become less correlated, a phenomenon driven by phase separation at DSBs. Our data indicate tight spatial control of chromatin motions after genomic insults, which may facilitate repair at the break sites and prevent deleterious contacts of DSBs, thereby reducing the risk of genomic rearrangements.

The study of radiation effects on biological tissues is a diverse field of research with direct applications to improve human health, in particular in the contexts of radiation therapy and space exploration. Understanding the DNA damage response following radiation exposure, which is a key determinant for mutagenesis, requires reproducible methods for delivering known doses of ionizing radiation (IR) in a controlled environment. Multiple IR sources, including research X-ray and gamma-ray irradiators are routinely used in basic and translational research with cell and animal models. These systems are however not ideal when a high temporal resolution is needed, for example to study early DNA damage responses with live cell microscopy. Here, we characterize the dose rate and beam properties of a commercial, miniature, affordable, and versatile X-ray source (Mini-X). We describe how to use Mini-X on the stage of a fluorescence microscope to deliver high IR dose rates (up to 29 Gy/min) or lower dose rates (≤ 0.1 Gy/min) in live cell imaging experiments. This article provides a blueprint for radiation biology applications with high temporal resolution, with a step-by-step guide to implement a miniature X-ray system on an imaging platform, and the information needed to characterize the system.

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.

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

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 and poor diet often go hand-in-hand, altering metabolic signaling and thereby impacting breast cancer risk and outcomes. We have recently demonstrated that dietary patterns modulate mammary microbiota populations. An important and largely open question is whether the microbiome of the gut and mammary gland mediates the dietary effects on breast cancer. To address this, we performed fecal transplants between mice on control or high-fat diets (HFD) and recorded mammary tumor outcomes in a chemical carcinogenesis model. HFD induced protumorigenic effects, which could be mimicked in animals fed a control diet by transplanting HFD-derived microbiota. Fecal transplants altered both the gut and mammary tumor microbiota populations, suggesting a link between the gut and breast microbiomes. HFD increased serum levels of bacterial lipopolysaccharide (LPS), and control diet–derived fecal transplant reduced LPS bioavailability in HFD-fed animals. In vitro models of the normal breast epithelium showed that LPS disrupts tight junctions (TJ) and compromises epithelial permeability. In mice, HFD or fecal transplant from animals on HFD reduced expression of TJ-associated genes in the gut and mammary gland. Furthermore, infecting breast cancer cells with an HFD-derived microbiome increased proliferation, implicating tumor-associated bacteria in cancer signaling. In a double-blind placebo-controlled clinical trial of patients with breast cancer administered fish oil supplements before primary tumor resection, dietary intervention modulated the microbiota in tumors and normal breast tissue. This study demonstrates a link between the gut and breast that mediates the effect of diet on cancer. Significance: This study demonstrates that diet shifts the microbiome in the gut and the breast tumor microenvironment to affect tumorigenesis, and oral dietary interventions can modulate the tumor microbiota in patients with breast cancer.

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.

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.

Background: Mutant KRAS is a key stratification factor in determining colorectal cancer (CRC) treatment. Nearly 50% of CRC tumors have KRAS mutations and these patients are unlikely to respond to anti-EGFR therapy. 5-fluorouracil (5-FU)-based chemotherapy is the preferred treatment for CRC patients with KRAS mutations; however, response rates remain very low for patients with advanced disease. Further, a sub-set of patients treated with 5-FU-based regimens suffers from serious side effects resulting from RNA-directed effects that do not contribute to the anti-cancer activity. We are developing polymeric fluoropyrimidines, such as CF10, to treat CRC more effectively and with reduced side effects. CF10 is >300-fold more potent than 5-FU across the NCI 60 cell line screen with particularly strong potency to KRAS-mutant CRC cells. We investigate the role of KRAS mutation on CF10 and 5-FU response as part of an overall effort to determine if CF10-based regimens can more effectively treat CRC patients for whom 5-FU-based regimens are currently the preferred treatment option. Methods: HKe3-wtKRAS and HKe3-mtKRAS were generated from HCT-116 human CRC cells (ATCC) by transfection with the pMSCVpuro vector encoding HA-tagged mutant or wild-type KRAS. Cytotoxicity was determined using an Alamar blue assay. Thymidylate synthase (TS) inhibition was evaluated by detecting ternary complex formation by Western blot and using a TS activity assay. Topoisomerase 1 cleavage complex (Top1cc) was evaluated using a RADAR assay and by immunofluorescence. DNA double strand breaks were detected by IF for γH2AX. Results: CF10 is highly potent to CRC cells regardless of KRAS mutation status. In isogenic cell lines that differ only in KRAS, CF10 is more potent to KRAS-mutant relative to wild type cells. The improved potency of CF10 relative to 5-FU and increased sensitivity of KRAS-mutant cells is enhanced for cells grown in 3D culture. CF10 cytotoxicity is mediated by TS inhibition and Top1cc formation that induces DNA DSBs and activates apoptosis. In vivo, CF10 displays significantly improved anti-tumor activity relative to 5-FU in HCT-116 orthotopic tumors and causes minimal systemic toxicity. Conclusions. KRAS mutation confers increased sensitivity to the DNA-directed fluoropyrimidine CF10. The overall mechanism of CF10 cytotoxicity to CRC cells is similar in both KRAS-WT and KRAS-MT CRC cells and involves TS inhibition, and misincorporation of FdU into DNA followed by generation of Top1-mediated DNA DSBs. Our studies indicate FP polymers are likely to be effective in CRC regardless of KRAS-mutation. The increased sensitivity of KRAS-mutant CRC to CF10 together with the very low systemic toxicity indicates that CRC patients currently being treated with 5-FU-based therapy could more effectively be treated with CF10-based regimens. Citation Format: William H. Gmeiner, Mandira Manandhar, Amanda M. Hinds, Julija Holmes, Pierre Vidi, Toshiyuki Tsunoda, Senji Shirasawa. Enhanced potency of the polymeric fluoropyrimidine CF10 to KRAS-mutant colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2079.

Preventive actions for chronic diseases hold the promise of improving lives and reducing healthcare costs. For several diseases, including breast cancer, multiple risk and protective factors have been identified by epidemiologists. The impact of most of these factors has yet to be fully understood at the organism, tissue, cellular and molecular levels. Importantly, combinations of external and internal risk and protective factors involve cooperativity thus, synergizing or antagonizing disease onset. Models are needed to mechanistically decipher cancer risks under defined cellular and microenvironmental conditions. Here, we briefly review breast cancer risk models based on 3D cell culture and propose to improve risk modeling with lab-on-a-chip approaches. We suggest epithelial tissue polarity, DNA repair and epigenetic profiles as endpoints in risk assessment models and discuss the development of 'risks-on-chips' integrating biosensors of these endpoints and of general tissue homeostasis. Risks-on-chips will help identify biomarkers of risk, serve as screening platforms for cancer preventive agents, and provide a better understanding of risk mechanisms, hence resulting in novel developments in disease prevention.

Epithelial tissue morphogenesis is accompanied by the formation of a polarity axis – a feature of tissue architecture that is initiated by the binding of integrins to the basement membrane. Polarity plays a crucial role in tissue homeostasis, preserving differentiation, cell survival and resistance to chemotherapeutic drugs among others. An important aspect in the maintenance of tissue homeostasis is genome integrity. As normal tissues frequently experience DNA double-strand breaks (DSBs), we asked how tissue architecture might participate in the DNA damage response. Using 3D culture models that mimic mammary glandular morphogenesis and tumor formation, we show that DSB repair activity is higher in basally polarized tissues, regardless of the malignant status of cells, and is controlled by hemidesmosomal integrin signaling. In the absence of glandular morphogenesis, in 2D flat monolayer cultures, basal polarity does not affect DNA repair activity but enhances H2AX phosphorylation, an early chromatin response to DNA damage. The nuclear mitotic apparatus protein 1 (NuMA), which controls breast glandular morphogenesis by acting on the organization of chromatin, displays a polarity-dependent pattern and redistributes in the cell nucleus of basally polarized cells upon the induction of DSBs. This is shown using high-content analysis of nuclear morphometric descriptors. Furthermore, silencing NuMA impairs H2AX phosphorylation – thus, tissue polarity and NuMA cooperate to maintain genome integrity.

To probe the space at the floor of the orthosteric ligand binding site in the dopamine D(1) receptor, four methylated analogues of dihydrexidine (DHX) were synthesized with substitutions at the 7 and 8 positions. The 8α-axial, 8β-equatorial, and 7α-equatorial were synthesized by photochemical cyclization of appropriately substituted N-benzoyl enamines, and the 7β-axial analogue was prepared by an intramolecular Henry reaction. All of the methylated analogues displayed losses in affinity when compared to DHX (20 nM): 8β-Me(ax)-DHX (270 nM), 8α-Me(eq)-DHX (920 nM), 7β-Me(eq)-DHX (6540 nM), and 7α-Me(ax)-DHX (>10000 nM). Molecular modeling studies suggest that although the disruption of an aromatic interaction between Phe203(5.47) and Phe288(6.51) is the cause for the 14-fold loss in affinity associated with 8β-axial substitution, unfavorable steric interactions with Ser107(3.36) result in the more dramatic decreases in binding affinity suffered by the rest of the analogues.

The epithelial-to-mesenchymal transition (EMT) is an evolutionarily conserved process that is required for normal development. However, EMT is often dysregulated and hijacked by cancers during tumor progression. Cancer cells that have undergone EMT and acquired a mesenchymal phenotype harbor several distinct traits, including increased tumor initiating properties, augmented motility, increased metastatic properties, and a higher incidence of resistance to therapy. Therefore, treatment regimens specific to mesenchymal cancer cells would be advantageous. In this study, we discovered that a mesenchymal subpopulation of breast, ovarian, lung, colorectal, and prostate cancer cell lines with high expression of the transcription factor ZEB1 (zinc finger E-box binding homeobox 1) and low expression of epithelial genes repressed by ZEB1 including ESRP1 (epithelial splicing regulatory protein 1) and CDH1 (E-cadherin) are an order of magnitude (>10-fold) more sensitive to exposure to silver nanoparticles (AgNPs) than cells expressing only low levels of ZEB1. This finding is significant because: (i) This is the first time a biomarker has been identified that is predictive of a positive response to any form of engineered nanomaterial; (ii) ZEB1 expression is a marker of poor clinical prognosis, and therefore identification of improved therapeutics for this poor outcome patient population is desirable; (iii) this work establishes that there is a shared and exploitable vulnerability across multiple cancer types that is independent of tissue origin and identifiable based upon mRNA or protein quantification of ZEB1. We establish that the ZEB1high selective cytotoxic properties of biocompatible AgNPs are not achieved using ionic silver, and therefore is one of the first examples of a “new to nano” cytotoxic property. Mechanistically, we find that AgNPs are rapidly ionized in ZEB1high cells, leading to endoplasmic reticulum stress, DNA damage, and apoptotic cell death. Furthermore, we show that AgNPs do not disrupt the normal architecture of breast acini in 3D cell culture, nor do they cause DNA damage. In contrast, the same doses of AgNPs cause extensive DNA damage and apoptosis in ZEB1high breast tumor nodules produced in 3D culture. Induction of EMT using TGF-β increases sensitivity to AgNPs and, conversely, knockdown of ZEB1 desensitizes cells to AgNP therapy. Finally, we demonstrate that systemic administration of AgNPs is safe and effective for treatment of ZEB1high breast cancer orthotopic xenografts in mice. In conclusion, our work provides evidence that a therapeutic window exists for the safe use of AgNPs as a precision medicine for patients with ZEB1-expressing tumors regardless of tissue origin. Citation Format: Cale D. Fahrenholtz, Jessica Swanner, James Sears, Katherine Cook, Pierre-Alexandre Vidi, Ravi Singh. A mesenchymal subset of cancers with elevated ZEB1 expression is sensitive to low doses of silver nanoparticles [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B123.

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.

Most cellular functions, including signaling by G protein-coupled receptors (GPCRs), are mediated by protein-protein interactions, making the identification and localization of protein complexes key to the understanding of cellular processes. In complement to traditional biochemical techniques, noninvasive resonance energy transfer (RET) and protein-fragment complementation assays (PCAs) now allow protein interactions to be detected in the context of living cells. In this review, fluorescent and bioluminescent PCAs are discussed and their application illustrated with studies on GPCR signaling. Newly developed techniques combining PCA and RET assays for the detection of ternary and quaternary protein complexes are also presented.

Triple negative breast cancers (TNBC) are characterized by loss of expression of hormone receptors and decreased expression of the human epidermal growth factor receptor 2 (HER2). TNBC patients do not benefit from current targeted breast cancer (BC) treatments. Molecular profiling of breast cancer has found that TNBC is largely comprised of basal-like and claudin-low intrinsic molecular subtypes. Claudin-low breast cancer (CLBC) accounts for approximately one third of TNBCs, and early evidence suggests CLBC tumors may be more resistant to neoadjuvant anthracycline/taxane-based chemotherapy compared to basal-like tumors. For the development of novel breast cancer therapeutics, attention must be paid to therapeutic efficacy in specific sub-types of the disease. We discovered a type of silver nanoparticle (AgNP) that is selectively cytotoxic for treatment of CLBC. We find that the increased sensitivity of CLBC cells as compared to non-cancerous cells was independent of nanoparticle size, and CLBC cell lines (MDA-MB-231, BT-549, SUM-159) are more sensitive to AgNP exposure than luminal A BC (MCF-7), HER2 positive BC (SKBR3), and basal-like BC (MDA-MB-468, BT-20), or non-cancerous breast cells (MCF-10A, 184B5, HMT-3522 S1) via MTT assay. By treating CLBC cells and non-cancer breast cells with AgNPs or silver ions, in the form of silver nitrate, we demonstrated that intact AgNPs are necessary for selective cytotoxicity in CLBC. To determine the mechanism of cell death caused by AgNPs, annexin V (AnnV) and propidium iodide (PI) co-staining was performed on non-cancerous MCF-10A breast cells and MDA-MB-231 CLBC cells treated with AgNPs for 48 hours. AgNPs induced a dose-dependent increase in late-stage apoptosis and an increase in necrosis in MDA-MB-231 compared to vehicle control. Conversely, AgNPs had a minimal effect on late-stage apoptosis and necrosis in non-cancerous MCF-10A. Utilizing western blots, we showed that AgNPs induce oxidative damage to protein thiols and activate the unfolded protein response (UPR) in CLBC, but not in non-cancerous breast cells. To better recapitulate the tumor volume in an in vitro setting, multicellular tumor nodules from MDA-MB-231 TNBC cells or HMT-3522 S1 non-transformed mammary epithelial cells were formed by culturing the cells on basement membrane. When grown using 3D culture techniques, HMT-3522 S1 cells can develop growth arrested, polarized spherical structures containing lumens resembling a normal breast acinar structure characterized by a central lumen, cell-cell junctional complexes (including apical tight junctions, TJ), and basal expression of hemidesmosomal α6/β4 integrins ligating an endogenous basement membrane. 48h treatment with AgNPs did not prevent apical localization of the TJ marker ZO-1, alter the basal deposition of collagen-IV, induce proliferation of the growth arrested acini, or induce DNA damage. Conversely, significant amounts of DNA damage were observed in the MDA-MB-231 tumor nodules following 48h AgNP treatment. We conducted a 3 month in vivo safety/efficacy study in CLBC tumor-bearing mice where we showed that AgNPs can be delivered repeatedly at substantial doses intravenously and are effective for treatment of tumors without systemic toxicity. This is the first time any nanomaterial has been demonstrated subtype-specific therapeutic efficacy. This work demonstrates that AgNPs may provide a significant benefit for the CLBC patient population. Citation Format: Jessica L. Swanner, Iliana Tenvooren, Brian W. Bernish, Cale D. Fahrenholtz, Pierre A. Vidi, Katherine L. Cook, Ravi N. Singh. Silver nanoparticles exhibit subtype specific cytotoxic and therapeutic effects in claudin low breast cancer in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B47.

Individualized therapy is a noble anticipation in oncology. It would permit not only to adapt a treatment to an individual’s physiology and characteristics, but also to pinpoint therapeutic features for each neoplasia. The high degree of cell heterogeneity in cancer, with mixtures of cells of different origins and cell populations that evolve toward multiple genomic and epigenomic profiles, makes the precise targeting and treatment of each abnormal cell a priority. The phenotypic nature of heterogeneity in cancer is largely determined by changes in gene transcription requiring the identification of an increasing number of intracellular and even intranuclear markers. Such a requirement brings new challenges for state-of-the-art technology focusing on single-cell analysis and begs for the development of nanotools that can provide the multiplex approach necessary to target cancer locations and reach, detect, and appropriately treat subgroups of cells. In this chapter we introduce the reader to the origins of cell heterogeneity in cancer and discuss how this emerging knowledge has shed light on the difficulties encountered in the management of neoplasias over the past few decades. Our analysis illustrates how nanotheranostics might help effectively address the issue of cell heterogeneity to halt cancer progression, overcome resistance to treatment, and possibly decrease cancer risks among individuals.

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.

We present a disease-on-a-chip model in which cancer grows within phenotypically normal breast luminal epithelium on semicircular acrylic support mimicking portions of mammary ducts. The cells from tumor nodules developing within these hemichannels are morphologically distinct from their counterparts cultured on flat surfaces. Moreover, tumor nodules cocultured with the luminal epithelium in hemichannels display a different anticancer drug sensitivity compared to nodules cocultured with the luminal epithelium on a flat surface and to monocultures of tumor nodules. The mimicry of tumor development within the epithelial environment of mammary ducts provides a framework for the design and test of anticancer therapies.

Organs are made of the organized assembly of different cell types that contribute to the architecture necessary for functional differentiation. In those with exocrine function, such as the breast, cell-cell and cell-extracellular matrix (ECM) interactions establish mechanistic constraints and a complex biochemical signaling network essential for differentiation and homeostasis of the glandular epithelium. Such knowledge has been elegantly acquired for the mammary gland by placing epithelial cells under three-dimensional (3D) culture conditions.Three-dimensional cell culture aims at recapitulating normal and pathological tissue architectures, hence providing physiologically relevant models to study normal development and disease. The specific architecture of the breast epithelium consists of glandular structures (acini) connected to a branched ductal system. A single layer of basoapically polarized luminal cells delineates ductal or acinar lumena at the apical pole. Luminal cells make contact with myoepithelial cells and, in certain areas at the basal pole, also with basement membrane (BM) components. In this chapter, we describe how this exquisite organization as well as stages of disorganization pertaining to cancer progression can be reproduced in 3D cultures. Advantages and limitations of different culture settings are discussed. Technical designs for induction of phenotypic modulations, biochemical analyses, and state-of-the-art imaging are presented. We also explain how signaling is regulated differently in 3D cultures compared to traditional two-dimensional (2D) cultures. We believe that using 3D cultures is an indispensable method to unravel the intricacies of human mammary functions and would best serve the fight against breast cancer.

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.

G protein-coupled receptor (GPCR) signaling is mediated by protein-protein interactions at multiple levels. The characterization of the corresponding protein complexes is therefore paramount to the basic understanding of GPCR-mediated signal transduction. The number of documented interactions involving GPCRs is rapidly growing, and appreciating the functional significance of these complexes is clearly the next challenge. New experimental approaches including protein complementation assays (PCAs) have recently been used to examine the composition, plasma membrane targeting, and desensitization of protein complexes involved in GPCR signaling. These methods also hold promise for better understanding of drug-induced effects on GPCR interactions. This review focuses on the application of fluorescent PCAs for the study of GPCR signaling. Potential applications of PCAs in high-content screens are also presented. Non-fluorescent PCA techniques as well as combined assays for the detection of ternary and quaternary protein complexes are briefly discussed.

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'.

Adenosine A(2A) and dopamine D(2) receptors (A(2A) and D(2)) associate in homo- and heteromeric complexes in the striatum, providing a structural basis for their mutual antagonism. At the cellular level, the portion of receptors engaging in homo- and heteromers, as well as the effect of persistent receptor activation or antagonism on the cell oligomer repertoire, are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to visualize A(2A) and D(2) oligomerization in the Cath.a differentiated neuronal cell model. Receptor fusions to BiFC fluorescent protein fragments retained their function when expressed alone or in A(2A)/A(2A), D(2)/D(2), and A(2A)/D(2) BiFC pairs. Robust fluorescence complementation reflecting A(2A)/D(2) heteromers was detected at the cell membrane as well as in endosomes. In contrast, weaker BiFC signals, largely confined to intracellular domains, were detected with A(2A)/dopamine D(1) BiFC pairs. Multicolor BiFC was used to simultaneously visualize A(2A) and D(2) homo- and heteromers in living cells and to examine drug-induced changes in receptor oligomers. Prolonged D(2) stimulation with quinpirole lead to the internalization of D(2)/D(2) and A(2A)/D(2) oligomers and resulted in decreased A(2A)/D(2) relative to A(2A)/A(2A) oligomer formation. Opposing effects were observed in cells treated with D(2) antagonists or with the A(2A) agonist 5'-N-methylcarboxamidoadenosine (MECA). Subsequent radioreceptor binding analysis indicated that the drug-induced changes in oligomer formation were not readily explained by alterations in receptor density. These observations support the hypothesis that long-term drug exposure differentially alters A(2A)/D(2) receptor oligomerization and provide the first demonstration for the use of BiFC to monitor drug-modulated GPCR oligomerization.