Absolute Proteome Analysis of Colorectal Mucosa, Adenoma, and Cancer Reveals Drastic Changes in Fatty Acid Metabolism and Plasma Membrane Transporters.

Colorectal cancer is a leading cause of cancer-related death. It develops from normal enterocytes, through a benign adenoma stage, into the cancer and finally into the metastatic form. We previously compared the proteomes of normal colorectal enterocytes, cancer and nodal metastasis to a depth of 8100 proteins and found extensive quantitative remodeling between normal and cancer tissues but not cancer and metastasis (Wisniewski et al. PMID 22968445). Here we utilize advances in the proteomic workflow to perform an in depth analysis of the normal tissue (N), the adenoma (A), and the cancer (C). Absolute proteomics of 10 000 proteins per patient from microdissected formalin-fixed and paraffin-embedded clinical material established a quantitative protein repository of the disease. Between N and A, 23% of all proteins changed significantly, 17.8% from A to C and 21.6% from N to C. Together with principal component analysis of the patient groups, this suggests that N, A, and C are equidistant but not on one developmental line. Our proteomics approach allowed us to assess changes in varied cell size, the composition of different subcellular components, and alterations in basic biological processes including the energy metabolism, plasma membrane transport, DNA replication, and transcription. This revealed several-fold higher concentrations of enzymes in fatty acid metabolism in C compared with N, and unexpectedly, the same held true of plasma membrane transporters.

SILAC-based proteomics of human primary endothelial cell morphogenesis unveils tumor angiogenic markers.

Proteomics has been successfully used for cell culture on dishes, but more complex cellular systems have proven to be challenging and so far poorly approached with proteomics. Because of the complexity of the angiogenic program, we still do not have a complete understanding of the molecular mechanisms involved in this process, and there have been no in depth quantitative proteomic studies. Plating endothelial cells on matrigel recapitulates aspects of vessel growth, and here we investigate this mechanism by using a spike-in SILAC quantitative proteomic approach. By comparing proteomic changes in primary human endothelial cells morphogenesis on matrigel to general adhesion mechanisms in cells spreading on culture dish, we pinpoint pathways and proteins modulated by endothelial cells. The cell-extracellular matrix adhesion proteome depends on the adhesion substrate, and a detailed proteomic profile of the extracellular matrix secreted by endothelial cells identified CLEC14A as a matrix component, which binds to MMRN2. We verify deregulated levels of these proteins during tumor angiogenesis in models of multistage carcinogenesis. This is the most in depth quantitative proteomic study of endothelial cell morphogenesis, which shows the potential of applying high accuracy quantitative proteomics to in vitro models of vessel growth to shed new light on mechanisms that accompany pathological angiogenesis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000359.

[The application of cell cultures, body fluids and tissues in oncoproteomics]. / Zastosowanie hodowli komórkowych, plynów ustrojowych i tkanek w onkoproteomice.

Mass spectrometry (MS)-based proteomics is a rapidly developing technology for the large scale analysis of proteins, their interactions and subcellular localization. In recent years proteomics has attracted much attention in medicine. Since a single biomarker might not have sufficient sensitivity and specificity in clinical practice, the identification of biomarker panels that comprise several proteins would improve the detection and clinical management of cancer patients. Additionally, the characteristics of protein profiles of most severe human malignancies certainly contribute to the understanding of the biology of cancer and fill the gap in our knowledge of carcinogenesis. This knowledge also is likely to result in the discovery of novel potential cancer markers and targets for molecular therapeutics. It is believed that the novel biomarkers will help in the development of personalized therapy tailored to the individual patient and will thereby reduce the mortality rate from cancer. In this review, the use of different types of human clinical samples (cell cultures, tissues and body fluids) in oncoproteomics is explained and the latest advances in mass spectrometry-based proteomics biomarker discovery are discussed.

Super-SILAC mix for quantitative proteomics of human tumor tissue.

We describe a method to accurately quantify human tumor proteomes by combining a mixture of five stable-isotope labeling by amino acids in cell culture (SILAC)-labeled cell lines with human carcinoma tissue. This generated hundreds of thousands of isotopically labeled peptides in appropriate amounts to serve as internal standards for mass spectrometry-based analysis. By decoupling the labeling from the measurement, this super-SILAC method broadens the scope of SILAC-based proteomics.

Extensive quantitative remodeling of the proteome between normal colon tissue and adenocarcinoma.

We report a proteomic analysis of microdissected material from formalin-fixed and paraffin-embedded colorectal cancer, quantifying > 7500 proteins between patient matched normal mucosa, primary carcinoma, and nodal metastases. Expression levels of 1808 proteins changed significantly between normal and cancer tissues, a much larger fraction than that reported in transcript-based studies. Tumor cells exhibit extensive alterations in the cell-surface and nuclear proteomes. Functionally similar changes in the proteome were observed comparing rapidly growing and differentiated CaCo-2 cells. In contrast, there was minimal proteomic remodeling between primary cancer and metastases, suggesting that no drastic proteome changes are necessary for the tumor to propagate in a different tissue context. Additionally, we introduce a new way to determine protein copy numbers per cell without protein standards. Copy numbers estimated in enterocytes and cancer cells are in good agreement with CaCo-2 and HeLa cells and with the literature data. Our proteomic data set furthermore allows mapping quantitative changes of functional protein classes, enabling novel insights into the biology of colon cancer.

Combined small cell and squamous cell carcinoma of the larynx.

A case of combined small cell and squamous cell carcinoma of the larynx in a male patient in the sixth decade of life is reported. The etiopathogenesis of this combined tumor remains unclear; however, a number of hypotheses were proposed in the past including the pivotal role of Kulchitsky, squamous cells and the glandular cells. The gene mutations may also play an important role in laryngeal carcinogenesis. This unusual type of laryngeal combined carcinoma has previously been reported worldwide in only 17 cases. This is an extremely rare tumor the histological nature of which makes the diagnosis more complicated than in other types of laryngeal cancers. The diagnosis of this carcinoma is based on light microscopy and should be supported by immunohistochemical studies. In our case, the tumor was growing in the left pyriform sinus. Metastatic neck lymph nodes were found on the left side, but no distant metastases were observed. Microscopic sections revealed a combined tumor composed of small cell carcinoma neuroendocrine type and non-keratinizing squamous cell carcinoma. Positive reaction to p16, bcl-2, thyroid transcription factor 1, synaptophysin and chromogranin A in the small cell neuroendocrine type carcinoma component was observed. The cells from squamous cell carcinoma component showed positive reaction to p63, high-molecular-weight cytokeratin and cytokeratin 5/6.

High recovery FASP applied to the proteomic analysis of microdissected formalin fixed paraffin embedded cancer tissues retrieves known colon cancer markers.

Proteomic analysis of samples isolated by laser capture microdissection from clinical specimens requires sample preparation and fractionation methods suitable for small amounts of protein. Here we describe a streamlined filter-aided sample preparation (FASP) workflow that allows efficient analysis of lysates from low numbers of cells. Addition of carrier substances such as polyethylene glycol or dextran to the processed samples improves the peptide yields in the low to submicrogram range. In a single LC-MS/MS run, analyses of 500, 1000, and 3000 cells allowed identification of 905, 1536, and 2055 proteins, respectively. Incorporation of an additional SAX fractionation step at somewhat higher amounts enabled the analysis of formalin fixed and paraffin embedded human tissues prepared by LCM to a depth of 3600-4400 proteins per single experiment. We applied this workflow to compare archival neoplastic and matched normal colonic mucosa cancer specimens for three patients. Label-free quantification of more than 6000 proteins verified this technology through the differential expression of 30 known colon cancer markers. These included Carcino-Embryonic Antigen (CEA), the most widely used colon cancer marker, complement decay accelerating factor (DAF, CD55) and Metastasis-associated in colon cancer protein 1 (MACC1). Concordant with literature knowledge, mucin 1 was overexpressed and mucin 2 underexpressed in all three patients. These results show that FASP is suitable for the low level analysis of microdissected tissue and that it has the potential for exploration of clinical samples for biomarker and drug target discovery.

Vacata- and divacataporphyrin: new photosensitizers for application in photodynamic therapy-an in vitro study.

BACKGROUND AND OBJECTIVE: The photodynamic therapy is a well-known method of treatment of both malignant tumors and non-tumor lesions in human patients. In the present study, we aimed at evaluating the in vitro efficacy of the new photosensitizing agents, vacataporphyrin (VP), and divacataporphyrin (DVP). MATERIALS AND METHODS: The effectiveness of VP and DVP was compared to well-known photosensitizers, that is, hematoporphyrin derivative (HPD) and chlorin e6 (Ce6) in identical in vitro conditions. The experiment was performed on a well-established breast cancer cell line, MCF-7 and compared to HCV 29T11-2-D1 cell line. Cells were incubated in standard conditions and they were exposed to different concentrations of VP, DVP, HPD, and Ce6, that is, 180, 90, 45, 22.5, and 10 µg/ml. After incubation with photosensitizers, the cells were washed, medium was exchanged and the cells were subjected to irradiation at the proper wavelengths, light intensity (100 mW/sq cm), and total light doses 4.5 and 9 J/sq cm. RESULTS: Our results showed that the VP and DVP are potent photosensitizers and the photocytotoxic effect after the incubation with DVP was much better than that of VP. The cytotoxic effects of VP and DVP were less intensive than these of HPD and Ce6. VP and DVP also accumulated well in the tumor cells. Our results also indicated that the VP and DVP effectiveness on MCF-7 cells was photosensitizer dose and light dose dependent. CONCLUSION: The overall properties revealed by both new porphyrins and particularly a possibility for excitation at a higher wavelength and thus a deeper tissue penetration, make them promising candidates for further in vivo experiments.

Proteome, phosphoproteome, and N-glycoproteome are quantitatively preserved in formalin-fixed paraffin-embedded tissue and analyzable by high-resolution mass spectrometry.

Tissue samples in biobanks are typically formalin-fixed and paraffin-embedded (FFPE), in which form they are preserved for decades. It has only recently been shown that proteins in FFPE tissues can be identified by mass spectrometry-based proteomics but analysis of post-translational modifications is thought to be difficult or impossible. The filter aided sample preparation (FASP) method can analyze proteomic samples solubilized in high concentrations of SDS and we use this feature to develop a simple protocol for FFPE analysis. Combination with simple pipet-tip based peptide fractionation identified about 5000 mouse liver proteins in 24 h measurement time-the same as in fresh tissue. Results from the FFPE-FASP procedure do not indicate any discernible changes due to storage time, hematoxylin staining or laser capture microdissection. We compared fresh against FFPE tissue using the SILAC mouse and found no significant qualitative or quantitative differences between these samples either at the protein or the peptide level. Application of our FFPE-FASP protocol to phosphorylation and N-glycosylation pinpointed nearly 5000 phosphosites and 1500 N-glycosylation sites. Analysis of FFPE tissue of the SILAC mouse revealed that these post-translational modifications were quantitatively preserved. Thus, FFPE biobank material can be analyzed by quantitative proteomics at the level of proteins and post-translational modifications.

In vitro and in vivo matrix metalloproteinase expression after photodynamic therapy with a liposomal formulation of aminolevulinic acid and its methyl ester.

Photodynamic therapy (PDT) is a well-known method for the treatment of malignant tumors, and its principles have been well established over the past 30 years. This therapy involves the application of a chemical called a photosensitizer and its subsequent excitation with light at the appropriate wavelength and energy. Topical photodynamic therapy with aminolevulinic acid (5-ALA) is an alternative therapy for many malignant processes, including nonmelanoma skin cancers such as basal-cell carcinoma (BCC). Our novel approach for this study was to use a liposomal formulation of 5-ALA and its methyl ester (commercially available as metvix) both in vitro and in vivo, and to check whether the liposome-entrapped precursors of photosensitizers can induce the expression of metalloproteinases (MMPs) in animal tumor cells and in other tissues from tumor-bearing rats and in selected cell lines in vitro. We also checked whether the application of tissue inhibitors of matrix metalloproteinases (TIMPs) has any effect on MMPs in the above-mentioned experimental models, and if they can cause complete inhibition of MMP expression. Immunohistochemical studies revealed that after the PDT, the intensity of expression of MMPs in healthy animals was very low and seen in single cells only. After the PDT in tumor-bearing rats, MMP-3 was expressed in the tumor cells with the highest intensity of staining in the tissues directly adjacent to the tumors, while MMP-2 and -9 were not found. In the control groups, there was no observed expression of MMPs. In vitro studies showed that MMP-3 was expressed in MCF-7 cells after PDT, but MMP-9 was not observed and MMP-2 was only seen in single cases. Our studies confirmed that the application of an MMP-3 inhibitor may block an induction of MMP-3 expression which had previously been initiated by PDT. The preliminary data obtained from cancer patients revealed that new precursors are effective in terms of PDT, and that using MMP inhibitors should be considered as a potential enhancing factor in clinical PDT.

Quantitative analysis of gene expression in fixed colorectal carcinoma samples as a method for biomarker validation.

Biomarkers have been described as the future of oncology. Modern proteomics provide an invaluable tool for the near­whole proteome screening for proteins expressed differently in neoplastic vs. healthy tissues. However, in order to select the most promising biomarkers, an independent method of validation is required. The aim of the current study was to propose a methodology for the validation of biomarkers. Due to material availability the majority of large scale biomarker studies are performed using formalin­fixed paraffin­embedded (FFPE) tissues, therefore these were selected for use in the current study. A total of 10 genes were selected from what have been previously described as the most promising candidate biomarkers, and the expression levels were analyzed with reverse transcription­quantitative polymerase chain reaction (RT­qPCR) using calibrator normalized relative quantification with the efficiency correction. For 6/10 analyzed genes, the results were consistent with the proteomic data; for the remaining four genes, the results were inconclusive. The upregulation of karyopherin α 2 (KPNA2) and chromosome segregation 1­like (CSE1L) in colorectal carcinoma, in addition to downregulation of chloride channel accessory 1 (CLCA1), fatty acid binding protein 1 (FABP1), sodium channel, voltage gated, type VII α subunit (SCN7A) and solute carrier family 26 (anion exchanger), member 3 (SLC26A3) was confirmed. With the combined use of proteomic and genetic tools, it was reported, for the first time to the best of our knowledge, that SCN7A was downregulated in colorectal carcinoma at mRNA and protein levels. It had been previously suggested that the remaining five genes served an important role in colorectal carcinogenesis, however the current study provided strong evidence to support their use as biomarkers. Thus, it was concluded that combination of RT­qPCR with proteomics offers a powerful methodology for biomarker identification, which can be used to analyze FFPE samples.

Pattern of Melanotransferrin Expression in Human Colorectal Tissues: An Immunohistochemical Study on Potential Clinical Application.

BACKGROUND: Our previous liquid chromatography-tandem mass spectrometry (LC-MS/MS) study on colorectal cancer proteome resulted in identification of 10,000 differentially expressed proteins. We observed a significantly changed expression of 25% of all identified proteins between patient and matched adjacent normal mucosa, carcinoma and colorectal adenoma, including melanotransferrin. Herein, we consider this protein as a potential biomarker of colorectal cancer. MATERIALS AND METHODS: Immunohistochemical detection of melanotransferrin was carried-out to localize its expression pattern within the colorectal tissues by tissue microarray. The diagnostic utility of melanotransferrin was evaluated in patient serum by enzyme-linked immunosorbent assay (ELISA). RESULTS: Strong melanotransferrin expression was found to be related to clinicopathological characteristics, lymph node involvement (p=0.008), tumor localization in colon (p=0.001), presence of mucin (p<0.013) and increasing tumor grade (p<0.001). Melanotransferrin level in serum from patients with colorectal cancer was significantly higher than that in healthy controls (p<0.001). CONCLUSION: We provide novel evidence that melanotransferrin may be involved in transformation from benign tumor to malignancy and is a marker of an invasive tumor phenotype.

Aberrant methylation of ERBB pathway genes in sporadic colorectal cancer.

The ErbB signalling network plays a crucial role in the growth and progression of several cancers, including colorectal cancer (CRC), and includes potentially drug-targetable genes. Oncogenic activation of the ErbB pathway by mutations and focal amplifications have emerged recently as an important predictive marker of the prognosis of CRC patients. However, in contrast to genetic events, little is known about epigenetic alternations of ErbB-associated genes and their impact on gene expression. Genome-wide methylation in sporadic CRCs (n = 12) paired with adjacent normal tissues have been previously analysed by Illumina Infinium HumanMethylation27 (HM27) at 27,578 CpG sites. For confirmation of our initial genome-wide analysis, we used a published HM27 dataset (GSE25062). Subsequently, CpG island methylation of selected ErbB pathway-associated genes was assessed on 233 CRC samples using methylation-sensitive polymerase chain reaction (MS-PCR) and analysed along with various genetic factors associated with CRC [epigenotype, BRAF and KRAS mutations, microsatellite instability (MSI)]. Methylation and expression integration was performed using published datasets including 25 pairs of CRC and normal colon tissues (GSE25062 and GSE25070), and confirmed with real-time PCR. Our previous microarray-based genome-wide DNA methylation analysis of 12 CRCs revealed that four ErbB-associated genes (PIK3CD, PKCΒ, ERBB4, ) were differentially methylated in CRCs. This was further confirmed by statistical re-analysis of an HM27 dataset (GSE25062). Frequent methylation at these loci in tumours was subsequently confirmed by MS-PCR (63%, 43%, 43% and 92%, respectively). Hypermethylation of PKCΒ associated with KRAS mutation (p = 0.04), whereas hypermethylation of ERBB4 associated with high-methylation epigenotypes (HME), BRAF mutation and MSI (p = 0.001, 0.002 and 0.0002, respectively). One of the four analysed genes (PKCΒ) was significantly downregulated in CRC tissue, as revealed by real-time PCR and re-analysis of the GSE25062 and GSE25070 datasets. After careful re-analysis of published methylation and expression data, we conclude that methylation of ERBB4, PAK7 and PIK3CD has no functional role in CRC carcinogenesis. In contrast, methylation seems to have a potential impact on the biology of colorectal tumours by negatively modulating the expression of PKCΒ. Importantly, the relationship between DNA methylation of PKCΒ and gene expression may warrant further attention in the context of colon cancer chemoprevention and anti-cancer therapy.