Nathan L. Avaritt

A quantitative proteomic analysis of FFPE melanoma.

To the Editor, Currently, melanoma is diagnosed based on microscopic features, and some of these attributes, including tumor thickness, ulceration, mitotic index, and extent of lymph node involvement, have prognostic significance (1). Patients with melanoma detected at an early stage undergo surgery to remove the primary tumor, but some patients progress to advanced stage disease despite treatment (2). Thus, there is a major need for the identification of prognostic biomarkers of melanoma. Unfortunately, biomarker studies using frozen tissue from primary human tumors are problematic, due to the inherent instability and tissue heterogeneity of the samples. In contrast, formalin-fixed paraffin embedded (FFPE) tissue is very stable and can be coupled with laser microdissection for targeted sample isolation. However, harvesting enough cells and extracting the cross-linked proteins has been challenging. We describe an approach that successfully extracts sufficient amounts of protein from FFPE tissue for mass spectrometric analysis and for free quantification of identified proteins. Similar approaches have proven successful for the analysis of other FFPE samples (3,4,5). Our approach is the first described for the comprehensive analysis of melanoma and melanocytic nevi using a coupled method with gel electrophoresis and spectral counting. For this proof-of-principle analysis, FFPE patient samples were collected from a single melanocytic nevus and single example of metastatic melanoma. Approximately 100,000 cells of melanocytic nevus and metastatic melanoma lesions were harvested with a Leica AS LMD laser microdissector. Proteins were uncross-linked and extracted with the Liquid Tissue MS Protein Prep Kit (Expression Pathology). Equal amounts of the proteins were split into 3 gel lanes and were analyzed by Coomassie/SDS-PAGE, which revealed the extraction of micrograms of protein (Figure 1). Each gel lane was sliced into 17 bands of 2 mm each and digested with trypsin. Tryptic peptides from the 102 gel bands were analyzed by LC-MS/MS with a Thermo LTQ-XL mass spectrometer coupled to an Eksigent nanoLC-2D (6). We identified a total of 888 proteins (0.45% false discovery rate using a decoy database from 56,013 spectra). Relaxing the stringency of the protein identification to a false discovery rate of 1.7% provided for the identification of 1,167 unique proteins from 88,180 spectra. Figure 1 Technical triplicate analyses of metastatic melanoma and nevus samples demonstrate the reproducibility of the quantitative mass spectrometric approach for the analysis of FFPE tissue samples In order to determine whether a protein was differentially expressed between nevus and metastatic melanoma samples, a label-free approach based on spectral counting was used (7,8,9,10). Spectral count is the number of tandem mass spectra assigned to a given protein in all bands from a single gel lane. To determine the relative amount of a protein in a given gel lane, a normalized spectral abundance factor (NSAF) was calculated (7). The NSAF was calculated as follows: (NSAF)k=(SpCL)k∑i=1N(SpCL)i where k is a given protein, SpC is the spectral count, L is the length of the protein, and N is all proteins identified in the gel lane. Plotting the frequency distribution of ln(NSAF) values clearly showed that the data followed a normal distribution as indicated by the fitting of a Gaussian curve with an R2 value of 0.99 (Figure 2A). In accordance to t-test, there were 390 proteins out of 888 total proteins that were found differentially expressed (p<0.05) between metastatic melanoma and nevus lesions. The distribution of the p-values from the t-test was then divided into bins of size 0.025 and the number of proteins for each bin plotted in a bar graph (Figure 2B). The 32 most significant proteins, according to the lowest p-value from the t-test and adjusted with Bonferroni correction, were visually inspected by hierarchical clustering (Figure 2C). Two proteins of particular interest, silver and fatty acid synthase (SILV and FASN), were found in the top 10 most significant proteins and appeared as up-regulated in metastatic melanoma as compared to melanocytic nevus. SILV and FASN both have been shown to be up-regulated in cancers, which makes them appropriate validation tools for this proof-of-principle study (11,12,13). Figure 2 Label-free quantification of proteins identified from FFPE nevus and metastatic melanoma tissues For validation, melanocytic nevus and melanoma samples were stained with either SILV or FASN antibodies and the samples were scored based on the intensity of the staining and the percentage of extent (Figures 3 & 4). The intensity of staining was scored as nil (0), low (1), medium (2), or high (3). The percentage of melanocyte or melanoma cell staining was scored as 0 (no staining), 1 ( 50% staining). The intensity was multiplied by the percentage of extent and the following product is then categorized as such: 0–1 is 0; 2–3 is 1+; 4–5 is 2+; 6–9 is 3+. In general, most of benign lesions (benign nevi or dysplastic nevi) have low or no expression (0 or 1+) of SILV or FASN. Many of the melanomas have higher expression (2+ or 3+) of SILV or FAS. Table 1 shows the number of cases with each score for SILV and FASN indicating higher staining in melanoma compared to benign. Both SILV and FASN were found to be significantly different by Chi square analysis between melanoma and benign with p-values of >0.0001 and 0.0015, respectively. Figure 3 SILV is up-regulated in melanoma Figure 4 Fatty acid synthase is up-regulated in melanoma Table 1 Scoring of SILV and FASN staining In conclusion, we present an unbiased, high throughput and quantitative approach for the identification of proteins that are differentially expressed in metastatic melanoma. Using quantitative label-free mass spectrometry of laser microdissected samples, we have identified 390 proteins differentially expressed in melanoma. Two of these proteins, silver and fatty acid synthase, were validated as being up-regulated in melanoma. Our proof-of-principle analysis lays the foundation for an extensive examination of archived human melanoma tissues for the discovery of biomarkers that will help clinicians with diagnosis, prognosis and treatment of this cancer.

MassSQUIRM: An assay for quantitative measurement of lysine demethylase activity.

In eukaryotes, DNA is wrapped around proteins called histones and is condensed into chromatin. Post-translational modification of histones can result in changes in gene expression. One of the most well-studied histone modifications is the methylation of lysine 4 on histone H3 (H3K4). This residue can be mono-, di- or tri-methylated and these varying methylation states have been associated with different levels of gene expression. Understanding exactly what the purpose of these methylation states is, in terms of gene expression, has been a topic of much research in recent years. Enzymes that can add (methyltransferases) and remove (demethylases) these modifications are of particular interest. The first demethylase discovered, LSD1, is the most well-classified and has been implicated in contributing to human cancers and to DNA damage response pathways. Currently, there are limited methods for accurately studying the activity of demethylases in vitro or in vivo. In this work, we present MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation), a quantitative method for studying the activity of demethylases capable of removing mono- and di-methyl marks from lysine residues. We focus specifically on LSD1 due to its potential as a prime therapeutic target for human disease. This quantitative approach will enable better characterization of the activity of LSD1 and other chromatin modifying enzymes in vitro, in vivo or in response to inhibitors.

Proteomics-Based Identification of Differentially Abundant Proteins from Human Keratinocytes Exposed to Arsenic Trioxide

Introduction Arsenic is a widely distributed environmental toxicant that can cause multi-tissue pathologies. Proteomic assays allow for the identification of biological processes modulated by arsenic in diverse tissue types. Method The altered abundance of proteins from HaCaT human keratinocyte cell line exposed to arsenic was quantified using a label-free LC-MS/MS mass spectrometry workflow. Selected proteomics results were validated using western blot and RT-PCR. A functional annotation analytics strategy that included visual analytical integration of heterogeneous data sets was developed to elucidate functional categories. The annotations integrated were mainly tissue localization, biological process and gene family. Result The abundance of 173 proteins was altered in keratinocytes exposed to arsenic; in which 96 proteins had increased abundance while 77 proteins had decreased abundance. These proteins were also classified into 69 Gene Ontology biological process terms. The increased abundance of transferrin receptor protein (TFRC) was validated and also annotated to participate in response to hypoxia. A total of 33 proteins (11 increased abundance and 22 decreased abundance) were associated with 18 metabolic process terms. The Glutamate--cysteine ligase catalytic subunit (GCLC), the only protein annotated with the term sulfur amino acid metabolism process, had increased abundance while succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial precursor (SDHB), a tumor suppressor, had decreased abundance. Conclusion A list of 173 differentially abundant proteins in response to arsenic trioxide was grouped using three major functional annotations covering tissue localization, biological process and protein families. A possible explanation for hyperpigmentation pathologies observed in arsenic toxicity is that arsenic exposure leads to increased iron uptake in the normally hypoxic human skin. The proteins mapped to metabolic process terms and differentially abundant are candidates for evaluating metabolic pathways perturbed by arsenicals.

Quantitative Histone Mass Spectrometry Identifies Elevated Histone H3 Lysine 27 (Lys27) Trimethylation in Melanoma

Normal cell growth is characterized by a regulated epigenetic program that drives cellular activities such as gene transcription, DNA replication, and DNA damage repair. Perturbation of this epigenetic program can lead to events such as mis-regulation of gene transcription and diseases such as cancer. To begin to understand the epigenetic program correlated to the development of melanoma, we performed a novel quantitative mass spectrometric analysis of histone post-translational modifications mis-regulated in melanoma cell culture as well as patient tumors. Aggressive melanoma cell lines as well as metastatic melanoma were found to have elevated histone H3 Lys27 trimethylation (H3K27me3) accompanied by overexpressed methyltransferase EZH2 that adds the specific modification. The altered epigenetic program that led to elevated H3K27me3 in melanoma cell culture was found to directly silence transcription of the tumor suppressor genes RUNX3 and E-cadherin. The EZH2-mediated silencing of RUNX3 and E-cadherin transcription was also validated in advanced stage human melanoma tissues. This is the first study focusing on the detailed epigenetic mechanisms leading to EZH2-mediated silencing of RUNX3 and E-cadherin tumor suppressors in melanoma. This study underscores the utility of using high resolution mass spectrometry to identify mis-regulated epigenetic programs in diseases such as cancer, which could ultimately lead to the identification of biological markers for diagnostic and prognostic applications.

Do checkpoint inhibitors rely on gut microbiota to fight cancer

The field of gut microbiota is of growing interest, especially in the recent discoveries of its interaction with host immune responses, which when disrupted, can further alter immunity. It also plays a role in cancer development, its microenvironment and response to anticancer therapeutics. Several recently published experimental studies had explored the efficacy of modifying microbiota to enhance the response of checkpoint inhibitors, suggesting its beneficial function in cancer management and potential to be targeted as a therapeutic agent to enhance efficacy of checkpoint inhibitors. Here we review available evidence, mechanisms and hypotheses of its use to enhance cancer response.

Abstract B15: Combination chemotherapy in melanoma using EZH2 inhibitor

Melanoma is the deadliest variety of skin cancer, which according to the American Cancer Society (ACS) will account for around 9710 deaths and diagnosis of around 76,100 new melanoma cases in the current year. According to the ACS, melanoma is also the fastest growing cancer, and hence it is vital to investigate the pathogenesis involved in the advanced stage of melanoma, and design effective therapeutics for its treatment. Aberrant activity of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) has been reported to be associated with different types of cancer. The EZH2 catalyzes the deposition of the repressive mark histone H3 lysine 27 trimethylation (H3K27me3) on the promoter of the target genes using the catalytic SET domain, thereby epigenetically silencing the expression of the target genes. This is because the H3K27me3 mark results in chromatin compaction which reduces the accessibility of various transcription factors to the gene, and hence adversely affects transcription. Several of such EZH2 target genes have been found to be tumor suppressor genes like RUNX3, DAB2IP, and E-cadherin, and silencing of such tumor suppressor genes is a chief mechanism by which EZH2 leads to cancer progression. Hence derepression of the tumor suppressor genes by inhibition of EZH2 could be an effective treatment strategy in different types of cancer. There has been no study till date investigating the role of EZH2 in melanoma. Our laboratory is investigating the role of EZH2 in the pathogenesis involved in the advanced stage of melanoma, to enable the design of effective therapeutic strategies for the treatment of melanoma. Drugs targeting epigenetic modifiers are becoming increasingly popular because of the fact that most epigenetic modifications and their effects can be reversed using these drugs. Our lab has strong evidence that metastatic (or advanced) melanoma is associated with upregulation of EZH2 and H3K27me3. We have also observed that EZH2 inhibition inhibits proliferation and invasion, and also induces apoptosis in metastatic melanoma cells. Recently our lab is performing combination chemotherapeutic studies in vitro, where EZH2 inhibitor GSK126 (S-adenosyl-methionine competitive inhibitor) is being used in combination with conventional chemotherapeutics like cisplatin and Bcl-2 inhibitors. Our results strongly suggest that when EZH2 inhibitor is used in combination with cisplatin or Bcl-2 inhibitor in metastatic melanoma cell lines, the inhibition is synergistic. This synergistic effect of GSK126 and cisplatin or Bcl-2 inhibitor has been found to be caspase-mediated. Citation Format: Deepanwita Sengupta, Nathan L. Avaritt, Alan J. Tackett. Combination chemotherapy in melanoma using EZH2 inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B15.

Misregulation of Rad50 expression in melanoma cells.

DNA double‐strand breaks are increased in human melanoma tissue as detected by histone H2AX phosphorylation. 1–3 We investigated two of the downstream effectors of DNA double‐strand breaks, Rad50 and 53BP1 (tumor suppressor p53 binding protein 1), to determine if they are altered in human primary melanoma cells. Melanoma cases showed high Rad50 staining (81.8%; 9/11) significantly more frequently than conventional or atypical melanocytic nevi (0%; 0/18). In contrast, the staining pattern for 53BP1 appears similar between melanoma and nevi. This is the first study that shows activation and misregulation of the DNA repair pathway in human melanoma cells. The staining features of Rad50, a component of an essential DNA double‐strand break repair complex, are clearly increased in melanoma cells with regards to both staining intensity and the number of positive melanoma cells. Interestingly, among the melanoma cases with increased Rad50 staining, most demonstrated cytoplasmic rather than nuclear staining (88.9%, 8/9). Further studies are needed to determine the cause of this mislocalization and its affects, if any, on DNA double‐strand break repair in melanoma.

Immune surveillance in melanoma: From immune attack to melanoma escape and even counterattack

ABSTRACT Pharmacologic inhibition of the cytotoxic T lymphocyte antigen 4 (CTLA4) and the programmed death receptor-1 (PD1) has resulted in unprecedented durable responses in metastatic melanoma. However, resistance to immunotherapy remains a major challenge. Effective immune surveillance against melanoma requires 4 essential steps: activation of the T lymphocytes, homing of the activated T lymphocytes to the melanoma microenvironment, identification and episode of melanoma cells by activated T lymphocytes, and the sensitivity of melanoma cells to apoptosis. At each of these steps, there are multiple factors that may interfere with the immune surveillance machinery, thus allowing melanoma cells to escape immune attack and develop resistance to immunotherapy. We provide a comprehensive review of the complex immune surveillance mechanisms at play in melanoma, and a detailed discussion of how these mechanisms may allow for the development of intrinsic or acquired resistance to immunotherapeutic modalities, and potential avenues for overcoming this resistance.

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases (1-3). As a result, these enzymes are prime targets for small molecule studies and drug development( 4). Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity. Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate (5). This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format (5).