Seron Eaton

Single cells and intracellular processes studied by a plasmonic-based electrochemical impedance microscopy

Electrochemical impedance spectroscopy is a crucial tool for the detection and study of various biological substances, from DNA and proteins to viruses and bacteria. It does not require any labelling species, and methods based on it have been developed to study cellular processes (such as cell spreading, adhesion, invasion, toxicology and mobility). However, data have so far lacked spatial information, which is essential for investigating heterogeneous processes and imaging high-throughput microarrays. Here, we report an electrochemical impedance microscope based on surface plasmon resonance that resolves local impedance with submicrometre spatial resolution. We have used an electrochemical impedance microscope to monitor the dynamics of cellular processes (apoptosis and electroporation of individual cells) with millisecond time resolution. The high spatial and temporal resolution makes it possible to study individual cells, but also resolve subcellular structures and processes without labels, and with excellent detection sensitivity (~2 pS). We also describe a model that simulates cellular and electrochemical impedance microscope images based on local dielectric constant and conductivity.

Piezoelectric printing and probing of Lectin NanoProbeArrays for glycosylation analysis

Glycans have great potential as disease biomarkers and therapeutic targets. However, the major challenge for glycan biomarker identification from clinical samples is the low abundance of key glycosylated proteins. To demonstrate the potential for glycan analysis with nanoliter amounts of glycoprotein, we have developed a new technology (Lectin NanoProbeArray) based on piezoelectric liquid dispensing for non-contact printing and probing of a lectin array. Instead of flooding the glycoprotein probe on the lectin array surface, as in conventional microarray screening, a piezoelectric printer is used to dispense nanoliters of fluorescently labeled glycoprotein probe over the lectin spots on the array. As a proof-of-concept, the ability of Lectin NanoProbeArrays to precisely identify and reliably distinguish between the closely related glycoforms of fetuin is illustrated here. Sensitivity levels comparable to lectin arrays that use evanescent-field scanners was achieved along with several orders of magnitude reduction in the amount of probe required for glycosylation analysis.

In situ drug-receptor binding kinetics in single cells: a quantitative label-free study of anti-tumor drug resistance

Many drugs are effective in the early stage of treatment, but patients develop drug resistance after a certain period of treatment, causing failure of the therapy. An important example is Herceptin, a popular monoclonal antibody drug for breast cancer by specifically targeting human epidermal growth factor receptor 2 (Her2). Here we demonstrate a quantitative binding kinetics analysis of drug-target interactions to investigate the molecular scale origin of drug resistance. Using a surface plasmon resonance imaging, we measured the in situ Herceptin-Her2 binding kinetics in single intact cancer cells for the first time, and observed significantly weakened Herceptin-Her2 interactions in Herceptin-resistant cells, compared to those in Herceptin-sensitive cells. We further showed that the steric hindrance of Mucin-4, a membrane protein, was responsible for the altered drug-receptor binding. This effect of a third molecule on drug-receptor interactions cannot be studied using traditional purified protein methods, demonstrating the importance of the present intact cell-based binding kinetics analysis.

NanoProbeArrays for the Analysis of Ultra-Low-Volume Protein Samples Using Piezoelectric Liquid Dispensing Technology

Antibody microarrays are gaining popularity as a high-throughput technology to investigate the proteome. However, protein extracts from most body fluid or biopsy samples are available in very small volumes and are often unsuitable for large-scale antibody microarray studies. To demonstrate the potential for protein analysis with as little as a few nanoliters of sample, we have developed a new technology called NanoProbeArrays based on piezoelectric liquid dispensing for non-contact printing and probing of antibody arrays. Instead of flooding the protein sample on the antibody microarray surface, as in conventional microarray screening, a piezoelectric inkjet printer is used to dispense nanoliters of fluorescently labeled proteins over the antibody spots on the array. The ability of NanoProbeArrays to precisely identify and reliably distinguish between test proteins from different sources, without any loss of sensitivity and specificity as compared with conventional antibody microarrays, is illustrated here. The utility of NanoProbeArrays for biomarker identification in a complex biological sample was tested by detecting the cytokine interleukin-4 in serum. The significant reduction in volume of sample during NanoProbeArray analysis, as compared with conventional antibody microarrays, offers new opportunities for basic and applied proteomic research.

Kinome-level screening identifies inhibition of polo-like kinase-1 (PLK1) as a target for enhancing non-viral transgene expression.

Human cells contain hundreds of kinase enzymes that regulate several cellular processes, which likely include transgene delivery and expression. We identified several kinases that influence gene delivery and/or expression by performing a kinome-level screen in which, we identified small-molecule kinase inhibitors that significantly enhanced non-viral (polymer-mediated) transgene (luciferase) expression in cancer cells. The strongest enhancement was observed with several small-molecule inhibitors of Polo-like Kinase 1 (PLK 1) (e.g., HMN-214 and BI 2536), which enhanced luciferase expression up to 30-fold by arresting cells in the G2/M phase of the cell cycle and influencing intracellular trafficking of plasmid DNA. Knockdown of PLK 1 using an shRNA-expressing lentivirus further confirmed the enhancement of polymer-mediated transgene expression. In addition, pairwise and three-way combinations of PLK1 inhibitors with the histone deacetylase-1 (HDAC-1) inhibitor Entinostat and the JAK/STAT inhibitor AG-490 enhanced luciferase expression to levels significantly higher than individual drug treatments acting alone. These findings indicate that inhibition of specific intracellular kinases (e.g., PLK1) can significantly enhance non-viral transgene expression for applications in biotechnology and medicine.

Ibrutinib inhibition of ERBB4 reduces cell growth in a WNT5A-dependent manner

Alterations in ERBB family members have been associated with many tumor malignancies. EGFR and ERBB2 have been extensively explored in clinical oncology and several drugs currently target them therapeutically. However, the significance of ERBB4 as a potential therapeutic target remains mostly unexplored, even though ERBB4 is overexpressed or mutated in many solid tumors. Using a unique functional protein microarray platform, we found that ibrutinib inhibits ERBB4 activity in the same nM range as its canonical target, BTK. Cell-based assays revealed that ibrutinib treatment inhibited cell growth and decreased phosphorylation of ERBB4 and downstream targets MEK and ERK in cancer cell lines with high levels of endogenous ERBB4. In vivo, ibrutinib-responsive mouse xenograft tumors showed decreased tumor volumes with ibrutinib treatment. Interestingly, global gene expression comparisons between responsive and non-responsive cells identified a signature featuring the WNT pathway that predicts growth responsiveness to ibrutinib. Non-responsive ERBB4-expressing cell lines featured elevated activity of the WNT pathway, through the overexpression of WNT5A. Moreover, inhibition of WNT5A expression led to an ibrutinib response in non-responsive cell lines. Our data show that inhibiting ERBB4 reduces cell growth in cells that have low WNT5A expression and reveal a link between the ERBB4 and WNT pathways.

Abstract P4-05-07: Functional genomics of TP53 mutations and its impact in breast cancer progression

Somatic TP53 mutations are prevalent in basal-like breast cancer (BLBC) tumors. Patients with BLBC tumors have fewer treatment options and respond poorly to current therapies. The majority of TP53 point mutations occurs in the DNA binding domain and can be categorized as either DNA contact or structural mutations. TP53 mutation results in a dominant negative phenotype with neomorphic activity. We predicted that different p53 mutations may lead to different phenotypic characteristics. To investigate this, we generated MCF10A stable transduced cell lines over-expressing the ten most frequent TP53 point mutations associated with breast cancer located in the DNA binding domain of TP53. Ectopic expression of TP53 in these stable cells has been confirmed by qRT-PCR and immunoblot. To assess the impact of mutation on carcinogenesis, we developed a series of high-throughput quantitative assays that measure several hallmarks of cancer, including proliferation, escape from apoptosis, epithelial to mesenchymal transition (EMT), cell migration and invasion, anoikis and morphology in 3D. We observed that one DNA contact mutation with the substitution of a positively charged amino acid with hydrophobic side chains such as R248W, and two structural mutants Y234C and H179R are resistant to apoptosis in presence of doxorubicin, are the most invasive displaying a mesenchymal phenotype characterized by the presence of disrupted B-catenin and E-cadherin staining, with reported worst clinical outcome, suggesting that these are the most aggressive phenotypes. Interestingly, the DNA contact mutants (R248Q, R273H, R248W, and R273C) had a growth advantage in absence of growth factors while structural mutants (R175H, H179R, Y220C, Y234C and Y163C) were more resistant to apoptosis after the cells were challenged with doxorubicin. G245S is comparable to the MCF10Ap53wt and is less proliferative, sensitive to apoptosis, and neither migratory nor invasive. In comparison, R248W which is one of the most aggressive mutants, together with R273C, and H179R resist anoikis; but Y234C, requires matrix for attachment in order to be invasive. In conjunction, these results confirmed our hypothesis that different TP53 point mutants have distinct phenotypes and functional effects on hallmarks of cancer due to distinct underlying cellular programs. Citation Format: Anasuya Pal, Laura Gonzalez-Malerva, Seron Eaton, Mayra Guzman, Donald Chow, Hongwei Yin, Jin Park, Karen Anderson, Joshua LaBaer. Functional genomics of TP53 mutations and its impact in breast cancer progression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-07.

JAK2 and PD-L1 Amplification Enhance the Dynamic Expression of PD-L1 in Triple-negative Breast Cancer

Background Activation of the JAK/STAT pathway is common in triple‐negative breast cancer (TNBC) and affects the expression of genes controlling immune signaling. A subset of TNBC cases will have somatic amplification of chromosome 9p24.1, encoding PD‐L1, PD‐L2, and JAK2, which has been associated with decreased survival. Materials and Methods Eleven TNBC cell lines were evaluated using array comparative genomic hybridization. A copy number gain was defined as an array comparative genomic hybridization log2 ratio of ≥ 1. Cell surface expression of programmed cell death ligand 1 (PD‐L1) was detected using flow cytometry and compared with the median fluorescence intensity of isotype control immunoglobulin. To selectively inhibit JAK2, lentiviral vectors encoding 2 different short hairpin RNA (shRNA) were generated. JAK2, STAT1, STAT3, phosphorylated (p) STAT1, and pSTAT3 expression were measured by immunoblot. Statistical significance was defined as P < .05. Results The cell line HCC70 had 9p24.1 copy number amplification that was associated with both increased JAK2 and pSTAT3; however, knockdown of JAK2 inhibited cell growth independently of 9p24.1 copy number status. In TNBC cell lines with 9p24.1 gain or amplification, PD‐L1 expression rapidly and strikingly increased 5‐ to 38‐fold with interferon‐&ggr; (P < .05), and inducible PD‐L1 expression was completely blocked by JAK2 knockdown and the JAK1/2 inhibitor ruxolitinib. In tumor tissue, expression of interferon‐&ggr;–related genes correlated with 9p24.1 copy number status. Conclusion These data suggest that the JAK2/STAT1 pathway in TNBC might regulate the dynamic expression of PD‐L1 that is induced in the setting of an inflammatory response. Inhibition of JAK2 might provide a synergistic therapy when combined with other immunotherapies in the subset of TNBC with 9p24.1 amplification. Micro‐Abstract The present study evaluated the interaction between the JAK2 and programmed cell death ligand 1 (PD‐L1) pathways in triple‐negative breast cancer (TNBC). A subset of TNBC tumors had a 9p24.1 amplification encoding JAK2 and PD‐L1. We observed a synergy between the JAK2/interferon‐&ggr; pathway and 9p24.1 amplification, leading to increased PD‐L1 expression.

Abstract 1218: Synthetic lethal targeting of p53 mutant cells with prenylation inhibitors

Introduction: TP53 is mutated in 30-40% of breast cancers and is associated with poor prognosis. Mutation of TP53 causes increased cellular proliferation, migration and invasion, and downstream activation of multiple pathways. HMG CoA reductase inhibitors, such as simvastatin, inhibit tumorigenic properties induced by TP53 mutation. Mechanism of this response is an important question in targeted cancer therapy. It is well known that HMG CoA reductase inhibitors block cholesterol production and prenylation. Therefore, we hypothesized that prenylation inhibitors would target p53 mutant cells primarily by inhibiting activation of the ras family induced by TP53 mutation. Method: We generated 5 MCF10A stably transduced cell lines over-expressing each of the common TP53 point mutations, R273H, G245S, R248Q, Y234C, or wild type p53. Total Ras, Ras-GTP, total RhoA and RhoA-GTP was measured by immunoprecipitation and immunoblot. Proliferation was measured using CellTiterGlo. The functional effects of a panel of prenylation inhibitors was measured using a caspase3 reporter assay, and migration was measured with a scratch assay. Illumina mRNA sequencing was performed to measure gene expression of mutant and wild type cells before and after simvastatin treatment (2.5µl, 48 h). The RNAseq was analyzed with the DAVID Enrichment tool to evaluate ras pathway activation induced by TP53 mutation. Results: Mutation in TP53 was associated with significant activation of both wild-type Ras and RhoA. Proliferation of cells expressing mutant TP53 was sensitive to a panel of prenylation inhibitors, including simvastatin. Growth inhibition by simvastatin correlated with induction of apoptosis, and could be fully rescued by addition of farnesylpyrophosphate or geranylgeranylpyrophosphate, suggesting that simvastatin functionally blocks both prenylation pathways which are activated in the presence of mutant p53 (IC50(wt/mut) = 4). RNAseq analysis confirms that Ras signaling pathways, including GAPs and GEFs are enriched in the presence of mutant TP53, and FOXO signaling is significantly targeted post statin treatment (P Conclusion: p53 mutant breast cancer cells are highly sensitive to prenylation inhibition due to activation of both ras and rhoA. Simvastatin inhibits both farnesylation and geranylgeranylation, effectively blocking ras pathway activation and induction of proliferation in p53 mutant cell lines. A gene expression panel associated with ras pathway activation was identified and may be predictive biomarkers for sensitivity to statin therapy. Citation Format: Shay R. Ferdosi, Benjamin Katchman, Jia Loo, Harneet Grewal, Seron Eaton, Shanshan Yang, Jin Park, Joshua Labaer, Karen S. Anderson. Synthetic lethal targeting of p53 mutant cells with prenylation inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1218. doi:10.1158/1538-7445.AM2017-1218

Abstract P6-03-15: Inhibition of breast cancer tumor cell lines by simvastatin is dependent on specific p53 mutation

Background: Simvastatin is an HMG-CoA reductase inhibitor widely used to treat cardiovascular diseases. In retrospective studies, statin treatment has been associated with a modest decrease in overall cancer incidence, including breast cancer (10-15%). Simvastatin Inhibited the PI3K/Akt/mTOR pathway in a pilot window-of-opportunity trial in neoadjuvant breast cancer. Mutation in TP53 is correlated with elevated expression of genes regulating cholesterol biosynthesis and sensitivity to statin treatment in vitro. We hypothesized that specific mutations in TP53 would be associated with differential sensitivity to simvastatin. Method: We generated MCF10A stably transduced cell lines over-expressing ten frequent TP53 missense point mutations. We assessed the impact of TP53 mutation on growth inhibition induced by simvastatin treatment (range of 1-30 µM for 96 hrs). In parallel, induction of apoptosis was measured by caspase3 reporter assay. Illumina 4-plexed 1x50bp RNA sequencing was performed on cells with R273H, G245S, R248Q, Y234C and wt TP53 before and after exposure to 2.5 µM simvastatin. Results: We confirmed that mutation in TP53 was markedly associated with growth inhibition by simvastatin treatment in vitro. We now demonstrate that TP53 mutation in R273C, G245S and R273H are highly sensitive to simvastatin with IC50 values ≤ 2.1 µM. In contrast, TP53 mutations in Y234C and R248Q were approximately five-fold more resistant. The resistant mutations were similar to MCF10A overexpressing wild type TP53. Growth inhibition was correlated with induction of apoptosis, and could be rescued by addition of farnesylpyrpphosphate and geranylgeranylpyrophosphate. HMGCR upregulation was observed across all treated cell lines. RNA-seq analysis confirms down regulation of the PIK3CA, PIK3CB, and Akt in the sensitive cell lines, but paradoxical upregulation in the resistant cells. Further gene expression analysis will be presented. Conclusion: Specific TP53 mutation status may impact sensitivity of breast cancer to simvastatin treatment. Citation Format: Ferdosi SR, Grewal H, Gonzalez-Malerva L, Eaton S, Briones N, LaBaer J, Anderson KS. Inhibition of breast cancer tumor cell lines by simvastatin is dependent on specific p53 mutation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-03-15.