Reverse Phase Protein Microarray Research Articles

Combining the "Sibling Technologies" of Laser Capture Microdissection and Reverse Phase Protein Microarrays.

Reverse phase protein microarrays (RPPA) and laser capture microdissection (LCM) are "sibling" technologies that originated from the same laboratory to overcome the challenge of quantifying low-abundance proteins in heterogeneous tissues. Combining both technologies provides both unique opportunities and unique challenges. Enabling the unprecedented resolution of the activation state of labile biomarkers, such as phosphorylated cell signaling proteins, has had a substantial impact on our understanding of diseases and is playing a significant role in clinical trials. At the same time, quantifying proteins at this sensitivity in very small amounts of material requires cognizance of pre-analytical variability and the limits of downstream detection technologies. Here, we discuss both the potential that the combination of both technologies presents and the potential pitfalls that must be navigated.

Utilization of Proteomic Technologies for Precision Oncology Applications.

Genomic analysis of tumor specimens has revealed that cancer is fundamentally a proteomic disease at the functional level: driven by genomically defined derangements, but selected for in the proteins that are encoded and the aberrant activation of signaling and biochemical networks. This activation is measured by posttranslational modifications such as phosphorylation and other modifications that modulate cellular signaling, and these events cannot be effectively measured by genomic analysis alone. Moreover, these signaling networks by and large represent the targets for many FDA-approved and experimental molecularly targeted therapeutics. Consequently, it is important that we consider new classification schemas for oncology based not on tumor site of origin or histology under the microscope but on the functional protein signaling architecture. There are numerous proteomic technologies that could be discussed from a purely technological standpoint, but this chapter will concentrate on an overview of the main proteomic technologies available for conducting protein pathway activation analysis of clinical specimens such as multiplex immunoassays, phospho-specific flow cytometry, reverse phase protein microarrays, quantitative immunohistochemistry, and mass spectrometry. This chapter will focus on the application of these technologies to cancer-based clinical studies evaluating prognostic/predictive markers or for stratifying patients to personalized treatments.

Network proteomics of human dermal wound healing

Objective: The healing of wounds is critical in protecting the human body against environmental factors. The mechanisms involving protein expression during this complex physiological process have not been fully elucidated. Approach: Here, we use reverse-phase protein microarrays (RPPA) involving 94 phosphoproteins to study tissue samples from tubes implanted in healing dermal wounds in seven human subjects tracked over two weeks. We compare the proteomic profiles to proteomes of controls obtained from skin biopsies from the same subjects. Main results: Compared to previous proteomic studies of wound healing, our approach focuses on wound tissue instead of wound fluid, and has the sensitivity to go beyond measuring only highly abundant proteins. To study the temporal dynamics of networks involved in wound healing, we applied two network analysis methods that integrate the experimental results with prior knowledge about protein–protein physical and regulatory interactions, as well as higher-level biological processes and associated pathways. Significance: We uncovered densely connected networks of proteins that are up- or down-regulated during human wound healing, as well as their relationships to microRNAs and to proteins outside of our set of targets that we measured with proteomic microarrays.

Three Different Jak2/Stat3-Related Pathways Favor the Survival of Chronic Lymphocytic Leukemia Neoplastic Clone

INTRODUCTIONChronic lymphoproliferative disorders are characterized by the expansion of malignant lymphocytes, the most common form being Chronic Lymphocytic Leukemia (CLL). Besides intrinsic abnormalities, the acquisition of the transformed phenotype and the diffusion of disease are related to the favorable cross-talking tumor cell-microenvironment. Furthermore, it has been demonstrated that CLL cells own an amplified mitochondrial respiration leading to an increased Reactive Oxygen Species (ROS) production and intrinsic oxidative stress. Several molecules released by microenvironmental partners signal through JAK-STAT pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells. We previously analyzed STAT3 and JAK2 expression, phosphorylation and localization in normal and leukemic B cells, demonstrating an abnormal activation of this pathway in the neoplastic clone with respect to normal B lymphocytes. We focused on STAT3 constitutive phosphorylation at serine (Ser) 727 residue since it has been recently observed that the presence of mt-STAT3 Ser727 promotes mitochondrial respiration and, generally, cell viability. We hypothesized the involvement of JAK2/STAT3 axis in 3 different pathways: (i) canonical “IL-6 related pathway”; (ii) JAK2/STAT3 - BCR/Lyn crosstalk; (iii) STAT3 effects on mitochondrial regulation.METHODSSTAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC). Purified cells (2x106 cells/ml) were cultured, and treated with the JAK2 inhibitor AG490 (10, 50 and 100μM) and the STAT3 inhibitor Stattic (5, 7.5, and 10μM) for 24, 48 and 72h. Experiments with AG490 and Stattic were performed with/without MSCs and with/without Ibrutinib (2.5μM) and Venetoclax (1nM). CLL and normal B cell viability was tested with Annexin V/PI test by FC. P-STAT3 Ser727 expression has been correlated with p44/42, SAPK-JNK, NF-kB p65 and p38 MAPK activation by Reverse Phase Protein Microarrays (RPPA) in 57 therapy-free patients presenting good (mutated IGHV, absence of CD38 or normal karyotype) and poor (unmutated IGHV, CD38 expression or 17p deletion) prognostic factors.RESULTSWe found that STAT3 was highly expressed in malignant B cells with respect to normal B lymphocytes. We demonstrated that STAT3 and JAK2 were similarly overexpressed in both good and poor prognosis CLL patients. However, a significant correlation between STAT3 expression levels and their overall survival was observed. Considering STAT3 over-expression and its correlation with the clinical outcome in CLL and, according to our hypothesis, we moved forward with the analysis of the three different JAK2/STAT3 pathways. We demonstrated that AG490 and Stattic were able to induce a dose-dependent apoptosis in CLL cells, also bypassing environmental protection. AG490, targeting JAK2, inhibited the phosphorylation of SHP-1 at Ser591, activating the phosphatase. In turn, SHP-1 activation led to Lyn Tyr396 dephosphorylation/inactivation. Treatment with Stattic did not affect Lyn and SHP-1 phosphorylation since this inhibitor acts downstream to AG490. In fact, simultaneous administration of Ibrutinib led to an increase of apoptosis only in Stattic, but not in AG490 treated cells. This confirms a possible dual role of JAK2 inhibition. Venetoclax/AG490 and Venetoclax/Stattic co-treatment did not show an increased cell death rate, consistent with a downstream effect of Venetoclax to both AG490 and Stattic. RPPA analysis demonstrated a correlation between STAT3, Ser727 constitutively phosphorylated in CLL, and P-p44/42 Thr202/Tyr204, P-SAPK-JNK Thr-183/Tyr185, NF-kB p65 Ser-536 and p38 MAPK Thr-180/Tyr-182 expression. All these proteins are described as involved in an alternative pathway that allows STAT3 Ser727 to exert a pro-survival effect thorough the regulation of mitochondrial activity.CONCLUSIONSThe analysis of JAK2 and STAT3 expression, activation and inhibition lets us to highlight the importance of JAK2/STAT3 axis in the maintenance of 3 different survival pathways in CLL B cells. Furthermore, the correlation we demonstrated with clinical outcome of patients and the strengthening of Ibrutinib effect when we targeted this axis could represent a starting point for the development of new therapeutic strategies in CLL. DisclosuresTrentin:Abbvie: Honoraria; Gilead: Research Funding; Janssen: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees.

Tobacco smoke and nicotine suppress expression of activating signaling molecules in human dendritic cells

Cigarette smoke has significant toxic effects on the immune system, and increases the risk of developing autoimmune diseases; one immunosuppressive effect of cigarette smoke is that it inhibits the T cell-stimulating, immunogenic properties of myeloid dendritic cells (DCs). As the functions of DCs are regulated by intra-cellular signaling pathways, we investigated the effects of cigarette smoke extract (CSE) and nicotine on multiple signaling molecules and other regulatory proteins in human DCs to elucidate the molecular basis of the inhibition of DC maturation and function by CSE and nicotine. Maturation of monocyte-derived DCs was induced with the TLR3-agonist poly I:C or with the TLR4-agonist lipopolysaccharide, in the absence or presence of CSE or nicotine. Reverse-phase protein microarray was used to quantify multiple signaling molecules and other proteins in cell lysates. Particularly in poly I:C-matured DCs, cigarette smoke constituents and nicotine suppressed the expression of signaling molecules associated with DC maturation and T cell stimulation, cell survival and cell migration. In conclusion, constituents of tobacco smoke suppress the immunogenic potential of DCs at the signaling pathway level.

An exploratory study examining how nano-liquid chromatography\u2013mass spectrometry and phosphoproteomics can differentiate patients with advanced fibrosis and higher percentage collagen in non-alcoholic fatty liver disease

BackgroundNon-alcoholic steatohepatitis (NASH) is among the leading causes of liver disease worldwide. It is increasingly recognized that the phenotype of NASH may involve a number of different pathways, of which each could become important therapeutic targets. The aim of this study is to use high resolution mass spectrometry (MS) and phosphoproteomics techniques to assess the serum proteome and hepatic phosphoproteome in subjects with NASH-related fibrosis.MethodsSixty-seven biopsy-proven NAFLD subjects with frozen sera and liver tissue were included. Reverse phase protein microarray was used to quantify the phosphorylation of key signaling proteins in liver and nano-liquid chromatography (LC)-MS was used to sequence target biomarkers in the serum. An image analysis algorithm was used to quantify the percentage of collagen (% collagen) using computer-assisted morphometry. Using multiple regression models, serum proteomes and phosphorylated hepatic proteins that were independently (p ≤ 0.05) associated with advanced fibrosis (stage ≥ 2) and higher % collagen were assessed.ResultsPhosphorylated signaling pathways in the liver revealed that apoptosis signal-regulating kinase 1, mitogen-activated protein kinase (ASK1-MAPK pathway involving ASK1 S38 (p < 0.02) and p38 MAPK (p = 0.0002)) activated by the inflammatory cytokine interleukin (IL-10) (p < 0.001), were independently associated with higher % collagen. LC-MS data revealed that serum alpha-2 macroglobulin (α2M) (p = 0.0004) and coagulation factor V (p = 0.0127) were independently associated with higher % hepatic collagen.ConclusionsSimultaneous profiling of serum proteome and hepatic phosphoproteome reveals that the activation of ASK1 S38, p38 MAPK in the liver, and serum α2M and coagulation factor V are independently associated with hepatic collagen deposition in patients with NASH. These data suggest the role of these pathways in the pathogenesis of NASH-related fibrosis as a potential therapeutic target.

Evaluation of the HER/PI3K/AKT Family Signaling Network as a Predictive Biomarker of Pathologic Complete Response for Patients With Breast Cancer Treated With Neratinib in the I-SPY 2 TRIAL.

In the I-SPY 2 TRIAL (Investigation of Serial Studies to Predict Your Therapeutic Response With Imaging and Molecular Analysis 2), the pan-erythroblastic oncogene B inhibitor neratinib was available to all hormone receptor (HR)/human epidermal growth factor receptor 2 (HER2) subtypes and graduated in the HR-negative/HER2-positive signature. We hypothesized that neratinib response may be predicted by baseline HER2 epidermal growth factor receptor (EGFR) signaling activation/phosphorylation levels independent of total levels of HER2 or EGFR proteins. Complete experimental and response data were available for between 130 and 193 patients. In qualifying analyses, which used logistic regression and treatment interaction analysis, 18 protein/phosphoprotein, 10 mRNA, and 12 DNA biomarkers that related to HER family signaling were evaluated. Exploratory analyses used Wilcoxon rank sum and t tests without multiple comparison correction. HER pathway DNA biomarkers were either low prevalence or nonpredictive. In expression biomarker analysis, only one gene (STMN1) was specifically associated with response to neratinib in the HER2-negative subset. In qualifying protein/phosphoprotein analyses that used reverse phase protein microarrays, six HER family markers were associated with neratinib response. After analysis was adjusted for HR/HER2 status, EGFR Y1173 (pEGFR) showed a significant biomarker-by-treatment interaction (P = .049). Exploratory analysis of HER family signaling in patients with triple-negative (TN) disease found that activation of EGFR Y1173 (P = .005) and HER2 Y1248 (pHER2) (P = .019) were positively associated with pathologic complete response. Exploratory analysis in this pEGFR/pHER2-activated TN subgroup identified elevated levels of estrogen receptor α (P < .006) in these patients. Activation of HER family phosphoproteins associates with response to neratinib, but only EGFR Y1173 and STMN1 appear to add value to the graduating signature. Activation of HER2 and EGFR in TN tumors may identify patients whose diseases respond to neratinib and implies that there is a subset of patients with TN disease who paradoxically exhibit HER family signaling activation and may achieve clinical benefit with neratinib; this concept must be validated in future studies.

Mouse Hepatomas with Ha-ras and B-raf Mutations Differ in Mitogen-Activated Protein Kinase Signaling and Response to Constitutive Androstane Receptor Activation.

Nuclear receptors mediate the hepatic induction of drug-metabolizing enzymes by xenobiotics. Not much is known about enzyme induction in liver tumors. Here, we treated tumor-bearing mice with phenobarbital, an activator of the constitutive androstane receptor (CAR), to analyze the response of chemically induced Ha-ras- and B-raf-mutated mouse liver adenoma to CAR activation in vivo. Both tumor subpopulations possess almost identical gene expression profiles. CAR target gene induction in the tumors was studied at the mRNA and protein levels, and a reverse-phase protein microarray approach was chosen to characterize important signaling cascades. CAR target gene induction was pronounced in B-raf-mutated but not in Ha-ras-mutated tumors. Phosphoproteomic profiling revealed that phosphorylation-activated extracellular signal-regulated kinase (ERK) 1/2 was more abundant in Ha-ras-mutated than in B-raf-mutated tumors. ERK activation in tumor tissue was negatively correlated with CAR target induction. ERK activation is known to inhibit CAR-dependent transcription. In summary, profound differences exist between the two closely related tumor subpopulations with respect to the activation of mitogenic signaling cascades, and these dissimilarities might explain the differences in xenobiotic induction of CAR target genes.

Abstract 1829: PRAS40 as a mediator of insulin-like growth factor-1 receptor-induced resistance to epidermal growth factor receptor inhibition in head and neck cancer

Abstract BACKGROUND: Despite the known growth-promoting role of the epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinoma (HNSCC), EGFR tyrosine kinase inhibitors (TKIs) have shown low efficacy in this disease. The insulin-like growth factor-1 receptor (IGF-1R) has been shown to induce resistance to EGFR TKIs in HNSCC predominantly through anti-apoptotic pathways. PRAS40 is an inhibitor of mTOR that ceases inhibition upon phosphorylation by Akt. Phosphorylated PRAS40 in turn inhibits FOXO3a, contributing to an overall pro-survival state. This study evaluates the role of PRAS40 in IGF-1R mediated resistance to EGFR TKIs. METHODS: In vitro experiments using alamarBlue, CyQuant, reverse phase protein microarray (RPPA), and immunoblot techniques to evaluate protein expression/phosphorylation and correlate with cell physiologic behavior. RESULTS: Proliferation assays were used to separate 6 HNSCC cell lines into 2 groups: those rescued from EGFR inhibition by IGF-1R activation and those not rescued. RPPA analysis identified a correlation between PRAS40 phosphorylation and rescue status. Immunoblot analysis confirmed the RPPA findings: in rescued cell lines, PRAS40 phosphorylation decreases with EGFR inhibition, but phosphorylation is restored by IGF-1R activation. However, these treatments have little effect on PRAS40 phosphorylation in non-rescued cell lines. In a representative cell line from each group, p70S6K phosphorylation was found to follow this pattern as well, suggesting possible involvement of mTOR in the rescue mechanism. However the addition of temsirolimus, an mTORC1 inhibitor, to treatment with an EGFR TKI was not sufficient to overcome IGF-induced rescue. CONCLUSIONS: PRAS40 phosphorylation is tightly correlated with IGF1R activation in HNSCC cells that exhibit IGF1R-induced rescue from EGFR TKI treatment. This phenomenon is absent in non-rescued cells, suggesting a potential role for pPRAS40 in IGF1R-based therapeutic resistance. While PRAS40 phosphorylation results in mTOR activation, the inability of mTOR inhibition to overcome IGF-induced rescue from EGFR antagonism suggests an important alternative downstream pathway. One possible mechanism is through inhibition of FOXO3a, a function of pPRAS40 that has been previously reported in other cell types. Citation Format: Michael I. Dougherty, Christine E. Lehman, Rolando E. Mendez, Linnea E. Taniguchi, Julie Wulfkuhle, Emanuel F. Petricoin, Daniel G. Gioeli, Mark J. Jameson. PRAS40 as a mediator of insulin-like growth factor-1 receptor-induced resistance to epidermal growth factor receptor inhibition in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1829.

Abstract 2254: Dynamic network topologies analysis in proteomics data of NSCLC cell lines using a new pipeline based on machine learning tools

Abstract Background: Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer, characterizing ~80% of lung cancer diagnoses. Thus, the development of novel therapies for NSCLC is of primary importance in oncology. The aim of this study was to develop a novel cancer systems biology approach to understand the complex interactions in NSCLC and predict the response to target treatments. We combined proteomic experimental data with a machine learning tool to discover the diverse signaling networks of both KRAS mutant (MUT) and wild-type (WT) NSCLC cell lines (CL). Material and Methods: Eight NSCLC CL (5 KRAS MUT and 3 KRAS WT) were subjected to reverse phase protein microarray (RPPA) to explore the activation level of 183 proteins treated with selumetinib (SE) and its combination with everolimus (EV) or tamoxifen (TA). Measures were taken at 6 time points: 5 minutes (min), 1, 2 , 6 and 24 hours (h). RPPA was performed also on the baseline and on CL with only dimethyl sulfoxide. Recursive Feature Elimination with Support Vector Machine (RFE-SVM) was used to identify the subset of proteins/features that optimally separated samples in 2 groups. In order to build the interaction network from this subset and browse the main pathways involved, we used Reactome FI and the Pathway Enrichment tool, two Cytoscape plugins for network-based data analysis. Results: We applied RFE-SVM to discover the most divergent proteins between control and treated samples in the whole CL panel. For all the 3 drug combinations, the network in output contained not only the target of the corresponding drug, but also showed the activation of other parallel pathways. The analysis revealed also that all drugs were mainly effective in a short time, since the number of significant proteins was considerably higher at 5 min and 1 h. For instance, SE treatment activated NGFR, mTOR, PI3K-Akt and proteoglycans pathways in addition to the MAPK pathway. Network at 1 h contained 25 proteins linked by 76 interactions, while at 6 and 24 h the distinctive subsets of features included only 2 and 4 proteins. Then, we applied RFE-SVM to analyze KRAS MUT vs WT CLs. The algorithm showed that treated KRAS MUT CL had a more complex signaling network than the WT ones. Indeed, in SE WT samples MER and ERK were the main relevant proteins to distinguish from the control. In treated KRAS MUT samples, a complex network of 70 proteins and 278 interactions was active during the first hour. Conclusion: The proposed framework processes high-throughput data and identifies new possible molecular targets in cancer research. This approach is highly flexible for a multitude of data and purposes. We applied it to a RPPA dataset of NSCLC CL and it revealed that all drug combinations had fast dynamics and that KRAS MUT CL had a more complicated response due to a higher number of active pathways. Citation Format: Chiara Antonini, Lorenzo Tomassoni, Elisa Baldelli, Vienna Ludovini, Sara Baglivo, Mariaelena Pierobon, Emanuel F Petricoin, Lucio Crinò, Paolo Valigi, Fortunato Bianconi. Dynamic network topologies analysis in proteomics data of NSCLC cell lines using a new pipeline based on machine learning tools [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2254.

Abstract 3290: The Side-Out Foundation Metastatic Breast Cancer Database, an open-access portal for multi-omic molecular data and more

Abstract Introduction: With the high volume of molecular data being generated daily from human malignancies, there is an increasing need to create novel internet-based portals where this information is readily and easily accessible to physicians, scientists, and the general public. Although numerous databases have been created to capture the molecular characteristics of primary tumors, limited resources are available when it comes to the metastatic lesions. We have developed a novel open-access database to capture demographic, clinical, and pathological information, outcome data, and multi-omic based molecular profiles from metastatic breast cancer (MBC) patients. Materials and Methods: The portal was created using the open-source relational database management system MySQL and the custom-codes were written using the PHP server-side scripting language. User interface, management, and authentication were created in WordPress. The database is primarily used to record information collected through the Side-Out clinical trials, a series of prospective Phase II trials targeting refractory MBC (NCT01074814, NCT01919749, NCT03195192). De-identified patients' information includes patients' demographics, treatment history, pathological characteristics of the primary tumor, outcome data, and multi-omic molecular profiles. Molecular information collected from each lesion include genomic (NGS-based whole/targeted exome sequencing), transcriptomic (RNA microarray/RNA Seq), proteomic (protein expression by IHC), and phosphoproteomic (protein pathway activation mapping by Reverse Phase Protein Microarray) data. Over 700 data fields are collected for each patient. A higher level of security for the recorded information is achieved by using a secondary database along with custom-codes during the data entry process. Investigators are exploring how Block-chain database design can be used to make portion of the data public while encrypting patient identifiers and key variables. Results: We present an overview of the portal, its usability, how access can be requested by interested third party, the user-friendly interface for downloading clinical, pathological and molecular data, and a few examples of how these data can be used to explore different aspects of metastatic breast cancers. Conclusions: To our knowledge, this is the first web-based publicly accessible database portal where broad multi-omic profiles are captured from the MBC patients along with demographic, clinical and pathological data. This open-access portal represents a unique and highly valuable tool as it integrates different aspects of the disease and can be used for correlative analyses and hypothesis-generating studies. Finally, this web-based portal allows free-of-charge dissemination of data from existing or upcoming clinical and translational studies targeting breast cancer patients. Citation Format: Elisa Baldelli, Shiva Ghaemi, Sanja Avramovic, Janusz Wojtusiak, Lance A. Liotta, Farrokh Alemi, Emanuel F. Petricoin, Bryant Dunetz, Mariaelena Pierobon. The Side-Out Foundation Metastatic Breast Cancer Database, an open-access portal for multi-omic molecular data and more [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3290.

Abstract 3129: PD-L1 expression differs across cancer metastatic sites from breast tumors

Abstract Background: Although the efficacy of different immunotherapeutic agents varies greatly across tumor types, little is known about the role of different metastatic tumor microenvironments in modulating the expression of immune-checkpoints like PD-L1. Using metastatic breast cancer as a model, this work explored whether metastatic lesions derived from different patients, but colonizing the same host target organ, showed organ-specific immune signatures. Methods: Snap frozen metastatic lesions collected from 30 breast cancer patients enrolled in a prospective phase II multi-omic precision medicine clinical trial (“Side-Out II”) were used for this analysis. Sites of metastasis were: liver (LI) (n=10), skin/chest wall (SC) (n=10), and other lesions (OL) (n=10) [including lymph nodes (LN) (n=4), lung (LU) (n=3), and intra-abdominal lesions (IA) (n=3)]. Pure tumor epithelia were isolated from each specimen via Laser Capture Microdissection (LCM). PD-L1 expression (Antibody Clone E1L3N) was quantitatively measured using the Reverse Phase Protein Microarray (RPPA), a high-throughput, multiplex immunoassay that provides operator-independent quantitative data starting from a small amount of biological material. Proportion of patients with PD-L1 expression above the median and the 75th percentile of the population was compared across metastatic sites. Results: PD-L1 expression had a &amp;gt;5 fold dynamic range across breast cancer-derived metastatic lesions with different distribution across metastatic sites. Specifically, PD-L1 expression of 6/10 LIs, 5/10 OLs (including 2 LU and 3 LN positive lesions), and 4/10 SC metastases was greater than the population median of all metastases combined. LI metastases were the most represented group above the 75th percentile of the population (4/10), followed by OS (2/10) (including 1 LU and 1 LN positive lesions), and SC (1/10). Of the 15 lesions above the population median, 6 were LI (40%), 5 were OS (33%), and 4 were SC (27%); of the 7 metastases above the 75th percentile, 4 were LI (57%), 2 were OS (29%), and 1 was SC (14%). Conclusions: The LCM-RPPA workflow can capture PD-L1 protein expression on a continuous quantitative scale and provide a broader understanding of the dynamic range of expression. Our data suggest that organ specific microenvironments in which metastatic lesions develop may strongly influence overall PD-L1 tumor cell expression. If different metastatic host organs can modulate PD-L1 expression, location of the metastatic lesion should be kept in consideration when selecting patients that are candidates for immunotherapy. Such hypotheses should be further validated in prospective clinical trial where quantitative PD-L1 expression of different metastatic lesions is evaluated along with response to targeted immuno-oncology compounds. Citation Format: Mariaelena Pierobon, K Alex Hodge, Elisa Baldelli, Steven Anthony, Nicholas J. Robert, Donald W. Northfelt, Mohammad J. Jahanzeb, Linda Vocila, Lance A. Liotta, Bryant Dunetz, Emanuel F. Petricoin. PD-L1 expression differs across cancer metastatic sites from breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3129.

Abstract 3476: Chemotherapy modulates proteomic pathways and neuronal metabolites in the brain

Abstract Chemotherapy modulates proteomic pathways and neuronal metabolites in the brainChemotherapy saves lives but diminishes the quality of life and functional ability of cancer patients by impairing cognitive function in a phenomenon called “chemobrain”. Elucidating the neural substrates modulated by chemotherapy is critical for deciphering the molecular underpinnings of cognitive dysfunction. Animal models have been used to investigate the impact of numerous chemotherapy drugs on cognitive ability and revealed negative effects of nearly all drugs tested, with oxidative stress, inflammation, apoptosis, and a reduction in hippocampal neurogenesis observed after treatment. Still, the underlying proteomic and metabolic mechanisms of chemotherapy-related cognitive dysfunction is not fully understood. We developed an animal model to assess the effects of cyclophosphamide (CTX) and docetaxel (DTX), two chemotherapy agents commonly delivered to breast cancer patients. Our study goals were to determine the proteomic effects of chemotherapy in specific brain regions and the associated global metabolomic shifts in the brain. Healthy, female Long Evans rats were treated with CTX and/or DTX, in four weekly doses. Controls received injections of vehicle alone. Following chemotherapy, fresh brain tissue was dissected medially. The right brain region was frozen for global metabolomic analysis via tandem mass spectrometry. The left brain was fixed in TheraLin for proteomic analysis of post-translationally modified proteins using reverse phase protein microarrays and immunohistochemistry. Fixed brain tissue was sectioned, and the hippocampus, cerebellum, olfactory bulb, and cerebral cortex were procured using laser capture microdissection. We identified biochemical shifts associated with chemotherapy that impact neurobiological processes underlying memory dysfunction. Hierarchical clustering of signal transduction proteins revealed that chemotherapy resulted in elevated levels of Calpain 1 and 2 in the cerebellum, Calpain 2 in the cerebral cortex, and autophagy proteins in the hippocampus and cerebellum. Doublecortin, a neurogenesis protein, was elevated in the cerebellum. Docetaxel raised the abundance of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), while all chemotherapies diminished levels of glutathione, a neuromodulator of excitatory NMDA receptors. This animal study provides molecular information that offers empirical support for future intervention research that may help prevent cognitive decline and preserve cognitive health in cancer survivors. Citation Format: Justin B. Davis, Grace N. Lawrence, Michael G. Harpole, Robin D. Couch, Lance A. Liotta, Theodore C. Dumas, Virginia A. Espina. Chemotherapy modulates proteomic pathways and neuronal metabolites in the brain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3476.

Extracellular Vesicles in Oncology: Progress and Pitfalls in the Methods of Isolation and Analysis.

The possibility to study solid tumors through the analysis of extracellular vesicles in biological fluids is one of the most exciting and rapidly advancing field in cancer research. The extracellular vesicles are tiny sacs released in both physiological and pathological conditions and can be used to monitor the evolution of several pathological states, including neoplastic diseases. Indeed, these vesicles carry biological informations and can affect the behavior of recipient cells by transferring proteins, DNA, RNA, and microRNA. In this review the authors analyze the methods to collect biological fluid samples (urine, plasma/serum, and cell supernatant), and to isolate and quantify extracellular vesicles highlighting advantages and drawbacks. Moreover, the authors provide an overview on the adoption and the advantages of the methods (such as digital PCR, next generation sequencing, reverse-phase protein microarrays, flow-cytometry, etc.) most frequently used to analyze the molecular content of extracellular vesicles. Despite the great scientific interest on this topic, there is still a great uncertainty about which is the best method for the collection, isolation, quantification, and molecular evaluation of these vesicles and a standardization is needed. The features of EVs make them ideal candidates for liquid biopsy-based biomarkers. However, the small size of EVs makes their analysis very difficult and requires multiple advanced technologies, being therefore a limitation.

Induction of nerve growth factor by phorbol 12-myristate 13-acetate is dependent upon the mitogen activated protein kinase pathway

Several small molecules have been identified that induce glial cells to synthesize and secrete nerve growth factor (NGF), a critical neurotrophin that supports neuronal growth and survival, and as such show promise in the development of drugs for the chemoprevention of Alzheimer's disease. To map the signal transduction cascade leading to NGF synthesis and secretion, cultured human glial cells were stimulated by phorbol 12-myristate 13-acetate (PMA), an agonist of Protein Kinase C. Changes in intracellular protein phosphorylation states were evaluated by reverse phase protein microarrays (RPPA), selectively screening over 130 protein endpoints. Of these, 55 proteins showed statistically significant changes in phosphorylation state due to cellular exposure to PMA. A critical signal transduction pathway was identified, and subsequent validation by ELISA and qPCR revealed that the signaling proteins Raf, MEK, ERK, and the signal transduction factor CREB are all essential to the upregulation of NGF gene expression by PMA. Additionally, members of the RSK family of kinases appear to be involved in glial secretion (exocytosis) of the NGF protein. Furthermore, through RPPA, the effects of PMA on apoptosis signaling events and cell proliferation were differentiated from the pathway to NGF upregulation. Overall, this study reveals potential protein targets for the rational design of Alzheimer's therapeutics.

Pharmacological TLR4 Antagonism Using Topical Resatorvid Blocks Solar UV-Induced Skin Tumorigenesis in SKH-1 Mice.

An urgent need exists for the development of more efficacious molecular strategies targeting nonmelanoma skin cancer (NMSC), the most common malignancy worldwide. Inflammatory signaling downstream of Toll-like receptor 4 (TLR4) has been implicated in several forms of tumorigenesis, yet its role in solar UV-induced skin carcinogenesis remains undefined. We have previously shown in keratinocyte cell culture and SKH-1 mouse epidermis that topical application of the specific TLR4 antagonist resatorvid (TAK-242) blocks acute UV-induced AP-1 and NF-κB signaling, associated with downregulation of inflammatory mediators and MAP kinase phosphorylation. We therefore explored TLR4 as a novel target for chemoprevention of UV-induced NMSC. We selected the clinical TLR4 antagonist resatorvid based upon target specificity, potency, and physicochemical properties. Here, we confirm using ex vivo permeability assays that topical resatorvid can be effectively delivered to skin, and using in vivo studies that topical resatorvid can block UV-induced AP-1 activation in mouse epidermis. We also report that in a UV-induced skin tumorigenesis model, topical resatorvid displays potent photochemopreventive activity, significantly suppressing tumor area and multiplicity. Tumors harvested from resatorvid-treated mice display reduced activity of UV-associated signaling pathways and a corresponding increase in apoptosis compared with tumors from control animals. Further mechanistic insight on resatorvid-based photochemoprevention was obtained from unsupervised hierarchical clustering analysis of protein readouts via reverse-phase protein microarray revealing a significant attenuation of key UV-induced proteomic changes by resatorvid in chronically treated high-risk SKH-1 skin prior to tumorigenesis. Taken together, our data identify TLR4 as a novel molecular target for topical photochemoprevention of NMSC. Cancer Prev Res; 11(5); 265-78. ©2018 AACRSee related editorial by Sfanos, p. 251.

Associations between expression levels of nucleotide excision repair proteins in lymphoblastoid cells and risk of squamous cell carcinoma of the head and neck

Squamous cell carcinoma of head and neck (SCCHN) is one of the most common malignancies worldwide, and nucleotide excision repair (NER) is involved in SCCHN susceptibility. In this analysis of 349 newly diagnosed SCCHN patients and 295 cancer-free controls, we investigated whether expression levels of eight core NER proteins were associated with risk of SCCHN. We quantified NER protein expression levels in cultured peripheral lymphocytes using a reverse-phase protein microarray. Compared with the controls, SCCHN patients had statistically significantly lower expression levels of ERCC3 and XPA (P = 0.001 and 0.001, respectively). After dividing the subjects by controls' median values of expression levels, we found a dose-dependent association between an increased risk of SCCHN and low expression levels of ERCC3 (adjusted OR, 1.75, and 95% CI: 1.26-2.42; Ptrend = 0.008) and XPA (adjusted OR, 1.88; 95% CI, 1.35-2.60; Ptrend = 0.001). We also identified a significant multiplicative interaction between smoking status and ERCC3 expression levels (P = 0.014). Finally, after integrating demographic and clinical variables, we found that the addition of ERCC3 and XPA expression levels to the model significantly improved the sensitivity of the expanded model on SCCHN risk. In conclusion, reduced protein expression levels of ERCC3 and XPA were associated with an increased risk of SCCHN. However, these results need to be confirmed in additional large studies.

Development of a Sol-gel-assisted Reverse-phase Microarray Platform for Simple and Rapid Detection of Prostate-specific Antigen from Serum

A sol-gel-based reverse-phase microarray was developed with improved sensitivity for prostatespecific antigen (PSA) from serum. The pore-sizecontrolled 3D sol-gel matrix was created with a large surface area to capture target molecules densely. Using the optically active sol-gel nanocomposites, human female serum was spiked with PSA and assessed using the reverse-phase protein microarray. The reverse-phase assay exhibited a limit of detection of 1 pg/mL for PSA and a dynamic range of 103 orders of magnitude. Notably, the platform matched the demand of the immunoassay in a simple and feasible manner. Moreover, the platform shortened the total assay time with an increased accuracy of diagnosis.

Abstract P4-04-08: Therapeutic strategy for ERα mutation driven-endocrine resistance in ER-positive breast cancers

Abstract Background: Although estrogen receptor (ER)-positive breast cancers are treated with endocrine therapy, 25% of these patients are at risk of relapse and the development of acquired endocrine resistance. Recently mutations in the ER gene (ESR1) have been identified which induce resistance to endocrine therapy. The most frequent ESR1 mutation, Y537S, promotes ligand-independent ER activity. It is known that ER regulates the cell cycle in a ligand-dependent manner. In this study, we examined the effects of the Y537S ESR1 mutation on cell cycle signaling and therapeutic response to checkpoint inhibitor. Material and Methods: MCF-7 cells expressing the Y537S ESR1 mutation were generated by CRISPR-Cas9 knock-in techniques. Cells were incubated in phenol red minus medium containing 5% charcoal-dextran treated serum for 5 days to remove exogenous hormones. Cell cycle and apoptosis were examined by Flow cytometry and Annexin-V assays. Proliferation was analyzed by BrdU incorporation. Cell senescence was determined using beta-galactosidase assays. Cell cycle checkpoint kinases were examined by western blot analysis. Cell growth was analyzed using soft agar or MTT assays. Results: Levels of p53 and apoptosis pathway proteins were significantly elevated in Y537S ESR1 mutant cells using RNA expression and reverse-phase protein microarrays. The ATM/ATR and Chk1/Chk2 mediated checkpoint signaling, the upstream pathway of p53, was activated in ESR1 Y537S mutation, which was repressed with fulvestrant treatment. ESR1 Y537 mutant cells accumulated about 5 fold in S phase and 1.7 fold in G2/M phase compared to control cells in estrogen deprived (ED) condition. BrdU incorporation was also increased about 2.5 fold compared with parental cells in estrogen-free medium. In addition, ESR1 Y537 mutant cells expressed a DNA double-strand break marker, gamma-H2AX protein in ED condition. Apoptosis and senescence were observed in ESR1 Y537S mutant cells in regular medium, however, apoptosis was not shown in ED medium. Chk1 inhibitor, PF477736 sensitized MCF-7 expressing ESR1 Y537S mutation to endocrine treatments such as fulvestrant, tamoxifen and AZD9496. Conclusion: Combination therapy with cell cycle checkpoint kinase inhibitor may lead better prognosis in ER mutation driven-endocrine resistance in postmenopausal breast cancers. Citation Format: Kim J-A, Fuqua SAW. Therapeutic strategy for ERα mutation driven-endocrine resistance in ER-positive breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-08.

Characterization of Behavioral, Signaling and Cytokine Alterations in a Rat Neurodevelopmental Model for Schizophrenia, and Their Reversal by the 5-HT6 Receptor Antagonist SB-399885

Post-weaning social isolation of rats produces neuroanatomical, neurochemical and behavioral alterations resembling some core features of schizophrenia. This study examined the ability of the 5-HT6 receptor antagonist SB-399885 to reverse isolation-induced cognitive deficits, then investigated alterations in hippocampal cell proliferation and hippocampal and frontal cortical expression of selected intracellular signaling molecules and cytokines. Male Lister hooded rats (weaned on post-natal days 21–24 and housed individually or in groups of 3–4) received six i.p. injections of vehicle (1% Tween 80, 1 mL/kg) or SB-399885 (5 or 10 mg/kg) over a 2-week period starting 40 days post-weaning, on the days that locomotor activity, novel object discrimination (NOD), pre-pulse inhibition of acoustic startle and acquisition, retention and extinction of a conditioned freezing response (CFR) were assessed. Tissue was collected 24 h after the final injection for immunohistochemistry, reverse-phase protein microarray and western blotting. Isolation rearing impaired NOD and cue-mediated CFR, decreased cell proliferation within the dentate gyrus, and elevated hippocampal TNFα levels and Cdc42 expression. SB-399885 reversed the NOD deficit and partially normalized CFR and cell proliferation. These effects were accompanied by altered expression of several members of the c-Jun N-terminal Kinase (JNK) and p38 MAPK signaling pathways (including TAK1, MKK4 and STAT3). Although JNK and p38 themselves were unaltered at this time point hippocampal TAK1 expression and phosphorylation correlated with visual recognition memory in the NOD task. Continued use of this neurodevelopmental model could further elucidate the neurobiology of schizophrenia and aid assessment of novel therapies for drug-resistant cognitive symptoms.