Several ABPs have been developed to target enzymes implicated in cancer progression and tumorigenesis, including metalloproteases, cysteine cathepsins, and esterases [11
]. These ABPs have been used to profile human tumors and tumor cell lines and identify novel enzyme activities for the diagnosis and treatment of cancer (). In a typical experiment, normal and disease proteomes are labeled with an ABP and the proteins are separated and analyzed by gel electrophoresis (). Enzymes that differ in their activity levels can then be identified using selective antibody pulldown or MS methods. In one application of ABPs to cancer biomarker discovery, FP-rhodamine, an ABP that targets the serine hydrolase superfamily of enzymes, was used to profile the activities of these enzymes in a set of human breast and melanoma cancer cell lines [18
]. This study confirmed that highly invasive cancer cells from several different tumor types upregulate a distinct set of serine hydrolase activities, including the protease urokinase and a novel integral membrane hydrolase, KIAA1363. Although urokinase was known to be involved in tumor progression, KIAA1363 had never been implicated in cancer and therefore represents a potentially novel cancer biomarker [18
]. In a related study, a panel of primary human breast cancer tissues was probed with the biotin-labeled version of FP-rhodamine [19
]. Probe-labeled proteins were enriched using avidin-conjugated beads, digested by trypsin, and subjected to semi-quantitative MS analysis. A set of enzymes, including KIAA1363, with elevated activities in the most aggressive tumor tissues were identified as potential breast cancer biomarkers. Recently, both FP-rhodamine and FP-biotin were used to identify enzymes that are involved in cancer cell intravasation, the process by which tumor cells enter into the vasculature [20
]. The activity level of the serine protease urokinase-type plasminogen activator (uPA) was substantially elevated in the high intravasating (HT-hi/diss) variants of the human fibrosarcoma cell line HT-1080. Inhibition of uPA activity significantly reduced the rate of intravasation and metastasis of HT-hi/diss cells, suggesting that active uPA is a key determinant of these processes [20
Enzyme classes characterized using ABPs.
Figure 2 Tumor biomarker discovery using ABPs. Enzymes from cancer cells and normal cells are reacted with a biotin-containing ABP and are then separated and analyzed by gel electrophoresis. Probe-labeled enzymes are visualized, and enzymes with altered activities (more ...)
Metalloproteases are another family of enzymes that play key roles in cancer progression events such as angiogenesis and metastasis [4
]. Several metalloprotease genes are overexpressed in metastatic cancers, and inhibitors of these enzymes reduce tumor angiogenesis in animal models of cancer [21
]. Most enzymes from the metalloprotease family use a zinc-activated water molecule for catalysis and do not covalently bind to their substrates, thus complicating probe design. However, ABPs for these enzymes have been constructed that contain a zinc-chelating moiety and a photocrosslinking group that allows for covalent labeling of the target enzymes. A library of metalloprotease probes was used to profile the activities of metalloproteases in both breast carcinoma and melanoma cell lines [17
]. Neprilysin, alanyl aminopeptidase, and ADAM10 activities were found to be elevated in invasive cells. Although neprilysin has historically been considered a negative regulator of tumorigenesis, its high activity in invasive melanoma cells suggests that this enzyme may contribute to cancer progression.
Histone deacetylases (HDACs) are enzymes that remove acetyl groups from lysine residues on histone tails and therefore are important regulators of gene expression. These enzymes have also been implicated in tumor growth and development [23
]. HDACs hydrolyze amide bonds using an active-site zinc cation; therefore, ABPs that target these enzymes are similar to probes that target metalloproteases. An HDAC-selective ABP has been designed based on a hydroxamic acid zinc-chelating moiety and a photoactivatible benzophenone group [24
]. This probe was used to analyze HDAC activity in melanoma and ovarian cacncer cell proteomes [24
]. Differences in the composition and activity of HDACs were found among cancer cells with distinct biological properties, indicating that the members of the HDAC enzyme family may have a variety of functional roles in cancer.
The ubiquitin-specific proteases (USPs) are a large family of proteolytic enzymes that regulate the production and recycling of ubiquitin and are involved in cell growth and differentiation [25
]. ABPs containing a warhead conjugated to the full-length ubiquitin protein are highly selective probes of the USPs. A number of these probes were used to identify unique and tumor-specific activities in a variety of human tumor cell lines [27
]. One specific USP, UCH-L1, was highly active in numerous malignant tumor cell lines. UCH-L1 activity was also found to be upregulated in normal B cells after in vitro
Epstein-Barr virus infection. This upregulation correlated with a transition from slow to rapid proliferation of the cells, implicating UCH-L1 in this adaptation. USP-specific ABPs have been used to profile USP activity in human cervical cancer biopsies [25
]. The activities of two USPs, UCH-L3 and UCH37, were elevated in tumor tissue when compared to normal tissue. Additionally, the activities of four USPs were upregulated in primary keratinocytes upon infection with human papilloma virus oncogenes, providing further evidence that the USPs are involved in growth transformation [25
ABPs have also been applied to functionally characterize enzyme activities in mouse models of cancer. The biotinylated ABP DCG-04 that targets the papain family of cysteine proteases was used to evaluate cysteine cathepsin activity in mammary tumor cells from PyMT;ctsb−/−
mice, a mouse mammary cancer model deficient in cathepsin B [28
]. Although cathepsin B is the most active cysteine cathepsin on the surface of PyMT;ctsb+/+
mammary cells, tumor cells lacking this protease (from PyMT;ctsb−/−
mice) show an upregulation of active cathepsin X on their cell surfaces. Cathepsin X activity partially compensates for the deficiency of cathepsin B in these tumor cells. Data from these experiments suggest that proteases can dynamically compensate for each other, thus complicating the analysis of knock-out data.
In an effort to more fully characterize the enzyme activity profiles of xenografted mouse tumors, ABPs such as FP-rhodamine have been used to characterize enzyme activities in MDA-MB-231 breast cancer cells both before and after growth as tumors in the mammary fat pad of immune-deficient mice [29
]. Many serine hydrolase activities, such as uPA and tissue plasminogen activator (tPA), were highly elevated in the in vivo
-derived lines of MDA-MB-231 and correlated with increased tumor growth rates and metastasis upon reintroduction into mice.
Most of the ABPs developed to date are specific for an individual class of enzymes; however, broad-spectrum probes have also been designed to target enzymes for which no covalent mechanism-based inhibitors exist [30
]. Adam and coworkers synthesized a variety of ABPs based on the reactive sulfonate ester (SE) warhead. These probes were found to covalently modify a wide range of mechanistically distinct enzymes including sugar kinases and thiolases [30
]. Profiling estrogen receptor-positive (ER+
) and –negative (ER−
) breast cancer cells with SE-containing ABPs identified omega glutathione S-transferase (GSTO 1-1) as having elevated activity in ER−
]. GSTO 1-1 had no previous association with invasive breast cancer and therefore represents a potentially new cancer biomarker. SE-based ABPs have also been used to evaluate enzyme activities in live human breast cancer cells [32
]. These efforts led to the identification of a novel enoyl-CoA hydratase whose activity was highly upregulated in the ER+
cell line T-47D.