Identification of retinal oligomeric, citrullinated, and other tau isoforms in early and advanced AD and relations to disease status.

This study investigates various pathological tau isoforms in the retina of individuals with early and advanced Alzheimer's disease (AD), exploring their connection with disease status. Retinal cross-sections from predefined superior-temporal and inferior-temporal subregions and corresponding brains from neuropathologically confirmed AD patients with a clinical diagnosis of either mild cognitive impairment (MCI) or dementia (n = 45) were compared with retinas from age- and sex-matched individuals with normal cognition (n = 30) and non-AD dementia (n = 4). Retinal tau isoforms, including tau tangles, paired helical filament of tau (PHF-tau), oligomeric-tau (Oligo-tau), hyperphosphorylated-tau (p-tau), and citrullinated-tau (Cit-tau), were stereologically analyzed by immunohistochemistry and Nanostring GeoMx digital spatial profiling, and correlated with clinical and neuropathological outcomes. Our data indicated significant increases in various AD-related pretangle tau isoforms, especially p-tau (AT8, 2.9-fold, pS396-tau, 2.6-fold), Cit-tau at arginine residue 209 (CitR209-tau; 4.1-fold), and Oligo-tau (T22+, 9.2-fold), as well as pretangle and mature tau tangle forms like MC-1-positive (1.8-fold) and PHF-tau (2.3-fold), in AD compared to control retinas. MCI retinas also exhibited substantial increases in Oligo-tau (5.2-fold), CitR209-tau (3.5-fold), and pS396-tau (2.2-fold). Nanostring GeoMx analysis confirmed elevated retinal p-tau at epitopes: Ser214 (2.3-fold), Ser396 (2.6-fold), Ser404 (2.4-fold), and Thr231 (1.8-fold), particularly in MCI patients. Strong associations were found between retinal tau isoforms versus brain pathology and cognitive status: a) retinal Oligo-tau vs. Braak stage, neurofibrillary tangles (NFTs), and CDR cognitive scores (ρ = 0.63-0.71), b) retinal PHF-tau vs. neuropil threads (NTs) and ABC scores (ρ = 0.69-0.71), and c) retinal pS396-tau vs. NTs, NFTs, and ABC scores (ρ = 0.67-0.74). Notably, retinal Oligo-tau strongly correlated with retinal Aß42 and arterial Aß40 forms (r = 0.76-0.86). Overall, this study identifies and quantifies diverse retinal tau isoforms in MCI and AD patients, underscoring their link to brain pathology and cognition. These findings advocate for further exploration of retinal tauopathy biomarkers to facilitate AD detection and monitoring via noninvasive retinal imaging.

Hippo Signaling Pathway Has a Critical Role in Zika Virus Replication and in the Pathogenesis of Neuroinflammation.

Zika virus (ZIKV) is a reemerging human pathogen that causes congenital abnormalities, including microcephaly and eye disease. The cellular/molecular basis of ZIKV and host interactions inducing ocular and neuronal pathogenesis are unclear. Herein, we noted that the Hippo/Salvador-Warts-Hippo signaling pathway, which controls organ size through progenitor cell proliferation and differentiation, is dysregulated after ZIKV infection. In human fetal retinal pigment epithelial cells, there is an early induction of transcriptional coactivator, Yes-associated protein (YAP), which is later degraded with a corresponding activation of the TANK binding kinase 1/interferon regulatory factor 3 type I interferon pathway. YAP/transcriptional co-activator with a PDZ-binding domain (TAZ) silencing results in reduced ZIKV replication, indicating a direct role of Hippo pathway in regulating ZIKV infection. Using an in vivo Ifnar1-/- knockout mouse model, ZIKV infection was found to reduce YAP/TAZ protein levels while increasing phosphorylated YAP Ser127 in the retina and brain. Hippo pathway is activated in major cellular components of the blood-brain barrier, including endothelial cells and astrocytes. In addition, this result suggests AMP-activated protein kinase signaling pathway's role in regulating YAP/TAZ in ZIKV-infected cells. These data demonstrate that ZIKV infection might initiate a cross talk among AMP-activated protein kinase-Hippo-TBK1 pathways, which could regulate antiviral and energy stress responses during oculoneuronal inflammation.

Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis.

We report that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome was activated by mitochondrial apoptotic signaling that licensed production of interleukin-1ß (IL-1ß). NLRP3 secondary signal activators such as ATP induced mitochondrial dysfunction and apoptosis, resulting in release of oxidized mitochondrial DNA (mtDNA) into the cytosol, where it bound to and activated the NLRP3 inflammasome. The antiapoptotic protein Bcl-2 inversely regulated mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induced NLRP3 inflammasome activation, because macrophages lacking mtDNA had severely attenuated IL-1ß production, yet still underwent apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-1ß secretion could be competitively inhibited by the oxidized nucleoside 8-OH-dG. Thus, our data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome.

An Inhibitor of GSK3B and HDACs Kills Pancreatic Cancer Cells and Slows Pancreatic Tumor Growth and Metastasis in Mice.

BACKGROUND & AIMS: Growth, progression, and drug resistance of pancreatic ductal adenocarcinomas (PDACs) have been associated with increased levels and activity of glycogen synthase kinase 3 beta (GSK3B) and histone deacetylases (HDACs). We designed and synthesized molecules that simultaneously inhibit the activities of both enzymes. We tested the effects of one of these molecules, Metavert, in pancreatic cancer cells and mice with pancreatic tumors. METHODS: We tested the ability of Metavert to bind GSK3B and HDACs using surface plasmon resonance. MIA PaCa-2, Bx-PC3, HPAF-II, and HPDE6 cell lines were incubated with different concentrations of Metavert, with or without paclitaxel or gemcitabine, or with other inhibitors of GSK3B and HDACs; cells were analyzed for apoptosis and migration and by immunoblotting, immunofluorescence, and real-time polymerase chain reaction. Krasþ/LSLG12D;Trp53þ/LSLR172H;Pdx-1-Cre (KPC) mice (2 months old) were given injections of Metavert (5 mg/kg, 3 times/week) or vehicle (control). B6.129J mice with tumors grown from UN-KPC961-Luc cells were given injections of Metavert or vehicle. Tumors and metastases were counted and pancreata were analyzed by immunohistochemistry. Glucose metabolism was measured using 13C-glucose tracer and mass spectroscopy and flow cytometry. Cytokine levels in blood samples were measured using multiplexing enzyme-linked immunosorbent assay. RESULTS: Metavert significantly reduced survival of PDAC cells but not nontransformed cells; the agent reduced markers of the epithelial-to-mesenchymal transition and stem cells in PDAC cell lines. Cells incubated with Metavert in combination with irradiation and paclitaxel or gemcitabine had reduced survival compared with cells incubated with either agent alone; Metavert increased killing of drug-resistant PDAC cells by paclitaxel and gemcitabine. PDAC cells incubated with Metavert acquired normalized glucose metabolism. Administration of Metavert (alone or in combination with gemcitibine) to KPC mice or mice with syngeneic tumors significantly increased their survival times, slowed tumor growth, prevented tumor metastasis, decreased tumor infiltration by tumor-associated macrophages, and decreased blood levels of cytokines. CONCLUSIONS: In studies of PDAC cells and 2 mouse models of PDAC, we found a dual inhibitor of GSK3B and HDACs (Metavert) to induce cancer cell apoptosis, reduce migration and expression of stem cell markers, and slow growth of tumors and metastases. Metavert had synergistic effects with gemcitabine.

Rapid Assessment of Mitochondrial Complex I Activity and Metabolic Phenotyping of Breast Cancer Cells by NAD(p)H Cytometry.

Cancer cells are known to display a variety of metabolic reprogramming strategies to fulfill their own growth and proliferative agenda. With the advent of high resolution imaging strategies, metabolomics techniques, and so forth, there is an increasing appreciation of critical role that tumor cell metabolism plays in the overall breast cancer (BC) growth. In this report, we demonstrate a sensitive, flow-cytometry-based assay for rapidly assessing the metabolic phenotypes in isolated suspensions of breast cancer cells. By measuring the temporal variation of NAD(p)H signals in unlabeled, living cancer cells, and by measuring mitochondrial membrane potential {Δψm } in fluorescently labeled cells, we demonstrate that these signals can reliably distinguish the metabolic phenotype of human breast cancer cells and can track the cellular sensitivity to drug candidates. We further show the utility of this metabolic ratio {Δψm /NAD(p)H} in monitoring mitochondrial functional improvement as well as metabolic heterogeneity in primary murine tumor cells isolated from tumor biopsies. Together, these results demonstrate a novel possibility for rapid metabolic functional screening applications as well as a metabolic phenotyping tool for determining drug sensitivity in living cancer cells. © 2018 International Society for Advancement of Cytometry.

Direct measurement of catalase activity in living cells and tissue biopsies.

Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies - can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 µg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings.

Targeting metabolic plasticity in breast cancer cells via mitochondrial complex I modulation.

Heterogeneity commonly observed in clinical tumors stems both from the genetic diversity as well as from the differential metabolic adaptation of multiple cancer types during their struggle to maintain uncontrolled proliferation and invasion in vivo. This study aims to identify a potential metabolic window of such adaptation in aggressive human breast cancer cell lines. With a multidisciplinary approach using high-resolution imaging, cell metabolism assays, proteomic profiling and animal models of human tumor xenografts and via clinically-relevant pharmacological approach for modulating mitochondrial complex I function in human breast cancer cell lines, we report a novel route to target metabolic plasticity in human breast cancer cells. By a systematic modulation of mitochondrial function and by mitigating metabolic switch phenotype in aggressive human breast cancer cells, we demonstrate that the resulting metabolic adaptation signatures can predictably decrease tumorigenic potential in vivo. Proteomic profiling of the metabolic adaptation in these cells further revealed novel protein-pathway interactograms highlighting the importance of antioxidant machinery in the observed metabolic adaptation. Improved metabolic adaptation potential in aggressive human breast cancer cells contribute to improving mitochondrial function and reducing metabolic switch phenotype-which may be vital for targeting primary tumor growth in vivo.

Metabolic imaging in multiple time scales.

We report here a novel combination of time-resolved imaging methods for probing mitochondrial metabolism in multiple time scales at the level of single cells. By exploiting a mitochondrial membrane potential reporter fluorescence we demonstrate the single cell metabolic dynamics in time scales ranging from microseconds to seconds to minutes in response to glucose metabolism and mitochondrial perturbations in real time. Our results show that in comparison with normal human mammary epithelial cells, the breast cancer cells display significant alterations in metabolic responses at all measured time scales by single cell kinetics, fluorescence recovery after photobleaching and by scaling analysis of time-series data obtained from mitochondrial fluorescence fluctuations. Furthermore scaling analysis of time-series data in living cells with distinct mitochondrial dysfunction also revealed significant metabolic differences thereby suggesting the broader applicability (e.g. in mitochondrial myopathies and other metabolic disorders) of the proposed strategies beyond the scope of cancer metabolism. We discuss the scope of these findings in the context of developing portable, real-time metabolic measurement systems that can find applications in preclinical and clinical diagnostics.

Upregulated Matrisomal Proteins and Extracellular Matrix Mechanosignaling Underlie Obesity-Associated Promotion of Pancreatic Ductal Adenocarcinoma.

Diet-induced obesity (DIO) promotes pancreatic ductal adenocarcinoma (PDAC) in mice expressing KRasG12D in the pancreas (KC mice), but the precise mechanisms remain unclear. Here, we performed multiplex quantitative proteomic and phosphoproteomic analysis by liquid chromatography-tandem mass spectrometry and further bioinformatic and spatial analysis of pancreas tissues from control-fed versus DIO KC mice after 3, 6, and 9 months. Normal pancreatic parenchyma and associated proteins were steadily eliminated and the novel proteins, phosphoproteins, and signaling pathways associated with PDAC tumorigenesis increased until 6 months, when most males exhibited cancer, but females did not. Differentially expressed proteins and phosphoproteins induced by DIO revealed the crucial functional role of matrisomal proteins, which implies the roles of upstream regulation by TGFß, extracellular matrix-receptor signaling to downstream PI3K-Akt-mTOR-, MAPK-, and Yap/Taz activation, and crucial effects in the tumor microenvironment such as metabolic alterations and signaling crosstalk between immune cells, cancer-associated fibroblasts (CAFs), and tumor cells. Staining tissues from KC mice localized the expression of several prognostic PDAC biomarkers and elucidated tumorigenic features, such as robust macrophage infiltration, acinar-ductal metaplasia, mucinous PanIN, distinct nonmucinous atypical flat lesions (AFLs) surrounded by smooth muscle actin-positive CAFs, invasive tumors with epithelial-mesenchymal transition arising close to AFLs, and expanding deserted areas by 9 months. We next used Nanostring GeoMX to characterize the early spatial distribution of specific immune cell subtypes in distinct normal, stromal, and PanIN areas. Taken together, these data richly contextualize DIO promotion of Kras-driven PDAC tumorigenesis and provide many novel insights into the signaling pathways and processes involved.

Acoustic ejection mass spectrometry empowers ultra-fast protein biomarker quantification.

The global scientific response to COVID 19 highlighted the urgent need for increased throughput and capacity in bioanalytical laboratories, especially for the precise quantification of proteins that pertain to health and disease. Acoustic ejection mass spectrometry (AEMS) represents a much-needed paradigm shift for ultra-fast biomarker screening. Here, a quantitative AEMS assays is presented, employing peptide immunocapture to enrich (i) 10 acute phase response (APR) protein markers from plasma, and (ii) SARS-CoV-2 NCAP peptides from nasopharyngeal swabs. The APR proteins were quantified in 267 plasma samples, in triplicate in 4.8 h, with %CV from 4.2% to 10.5%. SARS-CoV-2 peptides were quantified in triplicate from 145 viral swabs in 10 min. This assay represents a 15-fold speed improvement over LC-MS, with instrument stability demonstrated across 10,000 peptide measurements. The combination of speed from AEMS and selectivity from peptide immunocapture enables ultra-high throughput, reproducible quantitative biomarker screening in very large cohorts.

Identification of retinal tau oligomers, citrullinated tau, and other tau isoforms in early and advanced AD and relations to disease status.

Importance: This study identifies and quantifies diverse pathological tau isoforms in the retina of both early and advanced-stage Alzheimer's disease (AD) and determines their relationship with disease status. Objective: A case-control study was conducted to investigate the accumulation of retinal neurofibrillary tangles (NFTs), paired helical filament (PHF)-tau, oligomeric tau (oligo-tau), hyperphosphorylated tau (p-tau), and citrullinated tau (Cit-tau) in relation to the respective brain pathology and cognitive dysfunction in mild cognitively impaired (MCI) and AD dementia patients versus normal cognition (NC) controls. Design setting and participants: Eyes and brains from donors diagnosed with AD, MCI (due to AD), and NC were collected (n=75 in total), along with clinical and neuropathological data. Brain and retinal cross-sections-in predefined superior-temporal and inferior-temporal (ST/IT) subregions-were subjected to histopathology analysis or Nanostring GeoMx digital spatial profiling. Main outcomes and measure: Retinal burden of NFTs (pretangles and mature tangles), PHF-tau, p-tau, oligo-tau, and Cit-tau was assessed in MCI and AD versus NC retinas. Pairwise correlations revealed associations between retinal and brain parameters and cognitive status. Results: Increased retinal NFTs (1.8-fold, p=0.0494), PHF-tau (2.3-fold, p<0.0001), oligo-tau (9.1-fold, p<0.0001), CitR 209 -tau (4.3-fold, p<0.0001), pSer202/Thr205-tau (AT8; 4.1-fold, p<0.0001), and pSer396-tau (2.8-fold, p=0.0015) were detected in AD patients. Retinas from MCI patients showed significant increases in NFTs (2.0-fold, p=0.0444), CitR 209 -tau (3.5-fold, p=0.0201), pSer396-tau (2.6-fold, p=0.0409), and, moreover, oligo-tau (5.8-fold, p=0.0045). Nanostring GeoMx quantification demonstrated upregulated retinal p-tau levels in MCI patients at phosphorylation sites of Ser214 (2.3-fold, p=0.0060), Ser396 (1.8-fold, p=0.0052), Ser404 (2.4-fold, p=0.0018), and Thr231 (3.3-fold, p=0.0028). Strong correlations were found between retinal tau forms to paired-brain pathology and cognitive status: a) retinal oligo-tau vs. Braak stage (r=0.60, P=0.0002), b) retinal PHF-tau vs. ABC average score (r=0.64, P=0.0043), c) retinal pSer396-tau vs. brain NFTs (r=0.68, P<0.0001), and d) retinal pSer202/Thr205-tau vs. MMSE scores (r= -0.77, P=0.0089). Conclusions and Relevance: This study reveals increases in immature and mature retinal tau isoforms in MCI and AD patients, highlighting their relationship with brain pathology and cognition. The data provide strong incentive to further explore retinal tauopathy markers that may be useful for early detection and monitoring of AD staging through noninvasive retinal imaging.

Hexokinase dissociation from mitochondria promotes oligomerization of VDAC that facilitates NLRP3 inflammasome assembly and activation.

NLRP3 inflammasome activation is a highly regulated process for controlling secretion of the potent inflammatory cytokines IL-1ß and IL-18 that are essential during bacterial infection, sterile inflammation, and disease, including colitis, diabetes, Alzheimer's disease, and atherosclerosis. Diverse stimuli activate the NLRP3 inflammasome, and unifying upstream signals has been challenging to identify. Here, we report that a common upstream step in NLRP3 inflammasome activation is the dissociation of the glycolytic enzyme hexokinase 2 from the voltage-dependent anion channel (VDAC) in the outer membrane of mitochondria. Hexokinase 2 dissociation from VDAC triggers activation of inositol triphosphate receptors, leading to release of calcium from the ER, which is taken up by mitochondria. This influx of calcium into mitochondria leads to oligomerization of VDAC, which is known to form a macromolecule-sized pore in the outer membranes of mitochondria that allows proteins and mitochondrial DNA (mtDNA), often associated with apoptosis and inflammation, respectively, to exit the mitochondria. We observe that VDAC oligomers aggregate with NLRP3 during initial assembly of the multiprotein oligomeric NLRP3 inflammasome complex. We also find that mtDNA is necessary for NLRP3 association with VDAC oligomers. These data, together with other recent work, help to paint a more complete picture of the pathway leading to NLRP3 inflammasome activation.

Thyroid hormone differentially modulates Warburg phenotype in breast cancer cells.

Sustenance of cancer cells in vivo critically depends on a variety of genetic and metabolic adaptations. Aerobic glycolysis or Warburg effect has been a defining biochemical hallmark of transformed cells for more than five decades although a clear molecular basis of this observation is emerging only in recent years. In this study, we present our findings that thyroid hormone exerts its non-genomic and genomic actions in two model human breast cancer cell lines differentially. By laying a clear foundation for experimentally monitoring the Warburg phenotype in living cancer cells, we demonstrate that thyroid hormone-induced modulation of bioenergetic profiles in these two model cell lines depends on the degree of Warburg phenotype that they display. Further we also show that thyroid hormone can sensitize mitochondria in aggressive, triple-negative breast cancer cells favorably to increase the chemotherapeutic efficacy in these cells. Even though the role of thyroid hormone in modulating mitochondrial metabolism has been known, the current study accentuates the critical role it plays in modulating Warburg phenotype in breast cancer cells. The clinical significance of this finding is the possibility to devise strategies for metabolically modulating aggressive triple-negative tumors so as to enhance their chemosensitivity in vivo.

Biomarker signatures of mitochondrial NDUFS3 in invasive breast carcinoma.

We present evidence for potential biomarker utility of a mitochondrial complex I subunit, (NDUFS3) in discriminating normal and highly invasive breast carcinoma specimens obtained from clinical patients. Besides being a robust indicator of breast cancer aggressiveness, NDUFS3 also shows clear signatures of a hypoxia/necrosis marker in invasive ductal carcinoma specimens. Statistically significant positive correlation was observed between nuclear grade and NDUFS3 expression level in the tumor specimens analyzed. We support these findings with a plausible mechanism involving mitochondrial complex I assembly defects and/or redox buffering induced mitochondrial dysfunction during the process of cancer cell transformation. From a clinical standpoint, this novel observation adds value in augmenting the current receptor-based biomarkers for better accuracy in diagnosis and predicting survival rate in patients with breast carcinoma.

Preclinical evaluation of nuclear morphometry and tissue topology for breast carcinoma detection and margin assessment.

Prevention and early detection of breast cancer are the major prophylactic measures taken to reduce the breast cancer related mortality and morbidity. Clinical management of breast cancer largely relies on the efficacy of the breast-conserving surgeries and the subsequent radiation therapy. A key problem that limits the success of these surgeries is the lack of accurate, real-time knowledge about the positive tumor margins in the surgically excised tumors in the operating room. This leads to tumor recurrence and, hence, the need for repeated surgeries. Current intraoperative techniques such as frozen section pathology or touch imprint cytology severely suffer from poor sampling and non-optimal detection sensitivity. Even though histopathology analysis can provide information on positive tumor margins post-operatively (~2-3 days), this information is of no immediate utility in the operating rooms. In this article, we propose a novel image analysis method for tumor margin assessment based on nuclear morphometry and tissue topology and demonstrate its high sensitivity/specificity in preclinical animal model of breast carcinoma. The method relies on imaging nuclear-specific fluorescence in the excised surgical specimen and on extracting nuclear morphometric parameters (size, number, and area fraction) from the spatial distribution of the observed fluorescence in the tissue. We also report the utility of tissue topology in tumor margin assessment by measuring the fractal dimension in the same set of images. By a systematic analysis of multiple breast tissues specimens, we show here that the proposed method is not only accurate (~97% sensitivity and 96% specificity) in thin sections, but also in three-dimensional (3D) thick tissues that mimic the realistic lumpectomy specimens. Our data clearly precludes the utility of nuclear size as a reliable diagnostic criterion for tumor margin assessment. On the other hand, nuclear area fraction addresses this issue very effectively since it is a combination of both nuclear size and count in any given region of the analyzed image, and thus yields high sensitivity and specificity (~97%) in tumor detection. This is further substantiated by an independent parameter, fractal dimension, based on the tissue topology. Although the basic definition of cancer as an uncontrolled cell growth entails a high nuclear density in tumor regions, a simple but systematic exploration of nuclear distribution in thick tissues by nuclear morphometry and tissue topology as performed in this study has never been carried out, to the best of our knowledge. We discuss the practical aspects of implementing this imaging approach in automated tissue sampling scenario where the accuracy of tumor margin assessment can be significantly increased by scanning the entire surgical specimen rather than sampling only a few sections as in current histopathology analysis.

Disabling the Nuclear Translocalization of RelA/NF-κB by a Small Molecule Inhibits Triple-Negative Breast Cancer Growth.

INTRODUCTION: Constitutive activation of NF-κB has been implicated as being contributive to cancer cell growth, drug resistance, and tumor recurrence in many cancers including breast cancer. Activation of NF-κB leads to nuclear translocation of RelA, a critical component of the NF-κB transcription factor complex, which subsequently binds to specific DNA sites and activates a multitude of genes involved in diverse cell functions. Studies show that triple-negative breast cancer (TNBC) cells possess constitutively active NF-κB and concomitantly have higher levels of nuclear localization of RelA than cytoplasmic RelA. This feature is considered to be associated with the response to chemotherapy. However, currently, there is no specific inhibitor to block nuclear translocation of RelA. METHODS: A structure-based approach was used to develop a small-molecule inhibitor of RelA nuclear translocation. The interaction between this molecule and RelA was verified biophysically through isothermal titration calorimetry and microscale thermophoresis. TNBC cell lines MDA-MB-231 and MDA-MB-468 and a human TNBC xenograft model were used to verify in vitro and in vivo efficacy of the small molecule, respectively. RESULTS: We found that the small molecule, CRL1101, bound specifically to RelA as indicated by the biophysical assays. Further, CRL1101 blocked RelA nuclear translocation in breast cancer cells in vitro, and markedly reduced breast tumor growth in a triple-negative breast cancer xenograft model. CONCLUSION: Our study demonstrates that CRL1101 may lead to new NF-κB-targeted therapeutics for TNBC. Further, blocking of nuclear translocation of shuttling transcription factors may be a useful general strategy in cancer drug development.

Quantitative Imaging of Morphometric and Metabolic Signatures Reveals Heterogeneity in Drug Response of Three-Dimensional Mammary Tumor Spheroids.

PURPOSE: In order to monitor the drug responses of three-dimensional mammary tumor spheroids and to elucidate the role of inter- and intra-spheroid heterogeneity in determining drug sensitivity in the spheroids, an integrated image analysis framework was developed for morphometric and metabolic characterization of the three-dimensional tumor spheroids. PROCEDURE: Three-dimensional spheroid cultures of primary mammary tumor epithelial cells isolated from freshly excised tumors from a transgenic mouse model of adenocarcinoma (MMTV-PyMT) were imaged by using vital dyes and mitochondrial membrane potential markers. Custom-developed java and python program codes facilitated image processing, numerical computation, and graphical analysis of large datasets generated from the experiments. A panel of cancer drugs (rapamycin, BEZ235, MK2206, and flavopiridol) was tested to determine the degree of drug sensitivity as well as heterogeneity in drug response. RESULTS: A new quantitative metric (growth/toxicity) was developed based on morphometric parameters that were found to track the growth and apoptotic cell populations. Further, this study identified two parameters, namely, skew and kurtosis-which report the spatial heterogeneity in mitochondrial metabolism within the spheroids. The results of this study show that three-dimensional tumor spheroids selectively respond to cancer drugs depending on the specific metabolic pathways (AKT inhibition pathway in the present study), and there exists significant heterogeneity in the untreated tumor spheroids. Drug sensitivity of the spheroids was found to be associated with significant alterations in mitochondrial heterogeneity within the spheroids. CONCLUSIONS: In conclusion, the quantitative imaging of morphometric and metabolic analysis in large image datasets can serve as an excellent tool box for characterizing tumor heterogeneity in three-dimensional tumor spheroids and potentially, in intact tumors as well.

Nonlinear scaling analysis of glucose metabolism in normal and cancer cells.

Cancer progression is commonly accompanied by an altered glucose metabolism. In general, spatially resolved imaging of glucose metabolism and its subtle alterations might provide valuable diagnostic information in vivo. A classical example is positron emission tomography that exploits this feature in obtaining preferential accumulation of fluorescent analog of glucose in tumors, thereby achieving an imaging contrast. We report a novel scaling analysis of glucose metabolism in mammary epithelial (NMuMG) cells by detrended fluctuation analysis of Cerulean (cyan fluorescent protein variant) fluorescence. Fluorescence fluctuations of Cerulean are reasoned to be indicative of dynamic pH changes associated with glucose metabolism. Normal parental cells and the spontaneously transformed (cancerous) NMuMG cells displayed robust scaling exponent that reflects nonrandom fluctuations in Cerulean fluorescence. Acute dependence of cancer cells on glycolysis as compared with normal cells is exploited to yield a statistically significant difference in scaling exponent, thereby providing discrimination between normal and cancer cells in vitro. By careful design of experiments in vivo, the proposed scaling approach might even have diagnostic potential for early detection of cancer lesions in small animal models.

Spatio-temporal kinetics of growth hormone receptor signaling in single cells using FRET microscopy.

The growth hormone (GH) receptor (R)-mediated JAK2 (Janus kinase-2)-STAT5 (signaling transducer and activator of transcription-5) pathway involves a cascade of protein-protein interactions and tyrosine phosphorylations that occur in a spatially and temporally sensitive manner in cells. To study GHR dimerization or GH-induced conformational change of predimerized GHRs and STAT5 activation kinetics in intact cells, fluorescence resonance energy transfer (FRET) and live-cell imaging methods were employed. FRET measurements at the membrane of HEK-293T cells co-expressing GHRs tagged at the C-terminus with cyan (C) and yellow (Y) fluorescent proteins (FPs) revealed transient GHR dimerization lasting 2-3 min, with a maximum at 3 min after GH stimulation, which was sufficient to induce STAT5 activation. The transient nature of the dimerization or GH-induced conformational change of predimerized GHRs kinetics was not a result of GHR internalization, as neither potassium- nor cholesterol-depletion treatments prolonged the FRET signal. YFP-tagged STAT5 recruitment to the membrane, binding to GHR-CFP, and phosphorylation, occurred within minutes of GH stimulation. Activated STAT5a-YFP did not show nuclear accumulation, despite nuclear pSTAT5 increase, suggesting high turnover of STAT5 nuclear shuttling. Although GHR dimerization and STAT5 activation have been reported previously, this is the first spatially resolved demonstration of GHR-signaling kinetics in intact cells.

Trastuzumab-Resistant Luminal B Breast Cancer Cells Show Basal-Like Cell Growth Features Through NF-κB-Activation.

A major clinical problem in the treatment of breast cancer is mortality due to metastasis. Understanding the molecular mechanisms associated with metastasis should aid in designing new therapeutic approaches for breast cancer. Trastuzumab is the main therapeutic option for HER2+ breast cancer patients; however, the molecular basis for trastuzumab resistance (TZR) and subsequent metastasis is not known. Earlier, we found expression of basal-like molecular markers in TZR tissues from patients with invasive breast cancer.(( 1 )) The basal-like phenotype is a particularly aggressive form of breast cancer. This observation suggests that TZR might contribute to an aggressive phenotype. To understand if resistance to TZR can lead to basal-like phenotype, we generated a trastuzumab-resistant human breast cancer cell line (BT-474-R) that maintained human epidermal growth factor receptor 2 (HER2) overexpression and HER2 mediated signaling. Analysis showed that nuclear factor-kappa B (NF-κB) was constitutively activated in the BT-474-R cells, a feature similar to the basal-like tumor phenotype. Pharmacologic inhibition of NF-κB improved sensitivity of BT-474-R cells to trastuzumab. Interestingly, activation of HER2 independent NF-κB is not shown in luminal B breast cancer cells. Our study suggests that by activating the NF-κB pathway, luminal B cells may acquire a HER2+ basal-like phenotype in which NF-κB is constitutively activated; this notion is consistent with the recently proposed "progression through grade" or "evolution of resistance" hypothesis. Furthermore, we identified IKK-α/IKK-ß and nuclear accumulation of RelA/p65 as the major determinants in the resistant cells. Thus our study additionally suggests that the nuclear accumulation of p65 may be a useful marker for identifying metastasis-initiating tumor cells and targeting RelA/p65 may limit metastasis of breast and other cancers associated with NF-κB activation.