Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness - PubMed (original) (raw)

Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness

Nadim Jessani et al. Proc Natl Acad Sci U S A. 2002.

Abstract

By primarily measuring changes in transcript and protein abundance, conventional genomics and proteomics methods may fail to detect significant posttranslational events that regulate protein activity and, ultimately, cell behavior. To address these limitations, activity-based proteomic technologies that measure dynamics in protein function on a global scale would be of particular value. Here, we describe the application of a chemical proteomics strategy to quantitatively compare enzyme activities across a panel of human breast and melanoma cancer cell lines. A global analysis of the activity, subcellular distribution, and glycosylation state for the serine hydrolase superfamily resulted in the identification of a cluster of proteases, lipases, and esterases that distinguished cancer lines based on tissue of origin. Strikingly, nearly all of these enzyme activities were down-regulated in the most invasive cancer lines examined, which instead up-regulated a distinct set of secreted and membrane-associated enzyme activities. These invasiveness-associated enzymes included urokinase, a secreted serine protease with a recognized role in tumor progression, and a membrane-associated hydrolase KIAA1363, for which no previous link to cancer had been made. Collectively, these results suggest that invasive cancer cells share discrete proteomic signatures that are more reflective of their biological phenotype than cellular heritage, highlighting that a common set of enzymes may support the progression of tumors from a variety of origins and thus represent attractive targets for the diagnosis and treatment of cancer.

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Figures

Fig 1.

Fig 1.

Serine hydrolase activity profiles of the secreted proteomes of human cancer cell lines. (A) Representative in-gel fluorescence analysis of secreted serine hydrolase activity profiles obtained from reactions between cancer cell line conditioned media and a rhodamine-tagged FP. Enzyme activities are identified on either side of the gel (arrowheads point to the deglycosylated form of each enzyme; see Fig. 3_A_ for complete names of proteins). Deglycosylation was accomplished by treatment of a portion of the FP-labeled proteomes with PNGaseF before analysis. APH* refers to acyl peptide hydrolase, a cytosolic protein detected in the conditioned media. (B) Expanded view of FP-labeled secreted MDA-MB-435 proteome highlights the increased resolution that is achieved for highly glycosylated enzyme activities (e.g., SAE) after treatment with PNGaseF. (C) Levels of active urokinase secreted by cancer cell lines as measured by ABPP (left panel) and urokinase mRNA levels as measured by Northern analysis (n = 3 or 4). mRNA levels are expressed in arbitrary units relative to an internal control. (D) Inhibition of urokinase (uPA) activity by PA-I. Pretreatment of each proteome with PA-I (20 μg/ml) blocked the labeling of uPA by FP-rhodamine, but did not affect the labeling of other serine proteases (e.g., Comp 1s and cat. A).

Fig 2.

Fig 2.

Serine hydrolase activity profiles of the membrane proteomes of human cancer cell lines. (A) Representative in-gel fluorescence analyses of the serine hydrolase activity profiles of cancer cell membrane proteomes. Enzyme activities are identified on either side of the gels (arrowheads point to the deglycosylated form of each enzyme; see Fig. 3_A_ for full names of proteins). Proteins marked with an asterisk represent soluble hydrolases also detected in the membrane proteome. DG, deglycosylated. (B) The activity of FAAH in breast cancer membranes as measured by ABPP (Left) and FAAH substrate (Right) assays. (C) Relative activity levels for upper and lower glycosylated forms of the membrane hydrolase KIAA1363 in MDA-MB-231 and MDA-MB-435 lines. Shown are a representative in-gel fluorescence analysis (Left) and the ratio of upper to lower glycosylated forms, expressed as ratio-1 (Right).

Fig 3.

Fig 3.

Cluster analysis of the serine hydrolase activity profiles of human cancer cell lines. (A_–_C) A hierarchical clustering algorithm was applied to the cell lines by average linkage clustering using the Pearson correlation coefficient as the measure of similarity (CLUSTER computer package). Bars to the left of the dendrograms represent similarity scores. Shown are the results of cluster analyses conducted on total (A), membrane/secreted (B), and soluble (C) serine hydrolase activity profiles. The intensity of blue color scales directly with the relative activity of each hydrolase among the cell lines (0–100%, where for each enzyme, 100% represents the cell line with the highest activity, and the rest of the cell lines are expressed as a percentage of this highest activity to normalize the data sets). Gray, not measured. Red, breast cancer lines. Green, melanoma cancer lines. Black, NCI/ADR is of unknown origin.

Fig 4.

Fig 4.

Correlation between the activity of the membrane-associated hydrolase KIAA1363 and the invasiveness of human cancer cell lines. (A_–_C) Levels of active KIAA1363 present in cancer cell membrane proteomes as measured by ABPP (Left), and cancer cell invasiveness as measured by matrigel invasion assays (Right). Results expressed as number of invading cells refers to average number of invading cells per 8 fields counted (n = 3–4 for each cell line). (A) Breast carcinoma lines. (B) Melanoma lines. (C) Ovarian carcinoma lines.

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