The Role of Serine and Metallo-Hydrolases in Breast Cancer

Institution: Scripps Research Institute
Investigator(s): Sherry Niessen, M.S. -
Award Cycle: 2006 (Cycle 12) Grant #: 12GB-0026 Award: $72,275
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2006)

Like most solid tumors, it is metastasis from the primary tumor to secondary organs that is the leading cause of mortality in breast cancer. As such, the continued discovery of molecules underlying breast cancer development and metastasis and their molecular contribution to such processes remains an important and essential challenge for researchers. Extracellular and cell-surface enzymes from the serine and metallo-hydrolase family are known to play important roles during development and normal tissue homeostasis. In addition, these enzymes are believed to be important during primary tumor progression, invasion, and metastasis. For example, proteases are able to remodel the extracellular matrix and the basement membrane creating a pro-migratory and pro-invasive environment. This can occur through the degradation of the basement membrane to permit the passage of tumor cells to the blood and lymphatic vessels, or through the proteolysis and activation or inactivation of growth factors, growth factor receptors, cell-associated molecules and cytokines.

To identify serine and metallo-hydrolase enzyme activities that could play a potential role in tumor progression and metastasis we propose the use of newly developed functional proteomic technologies. Our three major aims are to:
• Identify serine and metallo-hydrolase enzyme activities that are elevated or decreased in an in vivo-derived breast cancer cell line that displays enhanced pathogenic properties compared to its parental cell line.
• Test the contribution that identified enzyme activities provide to the aggressive properties of the in vivo-derived breast cancer line.
• Profile serine and metallo-hydrolases activities across a panel of ductal and lobular primary human breast tumors.

The identification of serine and metallo- hydrolase enzyme activities will involve a novel technology called activity-based protein profiling (ABPP), that utilizes active site-directed probes to measure changes in enzyme activity directly in native proteomes, in combination with an LC-MS based platform called multidimensional protein identification technology (MudPIT). ABPP-MudPIT will be used to profile enzyme activities in an in vivo derived breast cancer cell line that displays enhanced tumorigenic potential compared to its parental cell line. The contribution that identified protease activities provide to the aggressive properties of this breast cancer cell line will be tested by disrupting their expression using RNA interference. Finally, the potential contribution of these enzyme activities to the progression of primary human breast cancer will be examined by profiling a panel of ductal and lobular primary human breast tumors.

Final Report (2008)

The goal of this research project is to utilize advanced proteomic technologies to identify and functionally characterize novel serine and metallo-hydrolase enzymes that participate in breast cancer biology. To this end, we have utilized activity based protein profiling (ABPP) in combination with multi-dimensional protein profiling technology (MudPIT) to characterize enzyme activities in a panel of non-aggressive versus aggressive breast cancer cell lines and human breast tumors. These advanced proteomic technologies are unified to provide a sensitive and quantitative assessment of the activities of many enzymes in parallel within native proteomes. Cancer relevant serine and metallo¬hydrolase enzymes were defined as those with elevated or decreased activity in aggressive (MDA-MB 231 and 231MFP) verses non-aggressive (MCF7) cancer cell lines and in human breast tumor specimens.

From these studies we identified that the serine hydrolase KIAA1363 was highly upregulated (increased) in tumors and aggressive cell lines. To understand the biological consequence of an up-regulation of KIAA1363 we investigated its endogenous biochemical function. We determined that this enzyme regulates the endogenous levels of a family of ether lipids known as monoalkylglycerol ethers (MAGEs). Further studies demonstrated that KIAA1363 impacted a larger ether lipid signaling network which included the bioactive lipids lysophosphatidylcholine (alkyl-LPC) and lysophosphatidic acid (alkyl-LPA). To investigate the role of KIAA1363 in pathogenesis we used two approaches. First, we reduced KIAA1363 levels in aggressive cancer cells and observed a decrease in the cellular levels of the MAGEs, alkyl-LPC, and alkyl-LPA. Moreover, these engineered lines displayed a decrease in their cell tumorigenic and migration potential in animal models. Second, we overexpressed KIAA1363 in non-aggressive cancer cells and observed an increase in the aforementioned ether lipids with a corresponding increase in their tumorigenic and migration potential. These data suggest that the ability of KIAA1363 to regulate pathogenesis is through modulating the endogenous levels of these ether lipids. Indeed, using both a pharmacological and genetic approach we were able to define that an alkyl-LPA-LPAR1 pathways is essential for KIAA1363-induced cell migration. To more globally explore pathways activated by the KIAA1363-ether lipid network, we performed gene expression profiling on cancer cells overexpressing KIAA1363 as well as a panel of cell lines that contain low or high levels of this enzyme. Using this approach, we defined an 'aggressive gene signature' regulated by KIAA1363 that included the oncogenic transcription factor Fra-1. Further studies demonstrated that Fra-1 was an essential component of KIAA1363-regulated pathogenesis in cancer cells and is regulated by both alkyl-LPC and alkyl-LPA.

Symposium Abstract (2007)

Many metabolic enzymes, including lipases, oxidoreductases, and glutathione S-transferases, are believed to make key contributions to primary tumor progression, invasion, and metastasis. As such, there is an urgent need for elucidating the precise roles that metabolic enzymes play in cancer biology, so as to uncover new potential therapeutic targets and biomarkers. Using activity based protein profiling (ABPP), an advanced proteomic technology that assesses the activity of many enzymes in parallel within native proteomes, we have identified the serine hydrolase KIAA1363 as being highly elevated in numerous pathogenic human cancer cell lines. These included cell lines from several tumorigenic origins including the breast, ovary, and skin. Moreover, this enzyme was found to be raised in a number of primary human tumors compared to their corresponding normal tissues. Together, these data suggest an important role for KIAA1363 in human tumorigenesis.

Using retroviral gene transfer in combination with either RNAi or overexpression technology we have demonstrated that KIAA1363 is not only necessary but also sufficient to support several of the in vitro and in vivo pathogenic properties of both breast and ovarian human cancer cell lines. The overexpression of KIAA1363 leads to an augmentation of the in vitro proliferation and migration potential and the in vivo tumor growth of cancer cells. Moreover, a reduction of KIAA1363 activity decreased both the migration potential and the in vivo tumor growth of cancer cells. Importantly, KIAA1363’s ability to regulate these properties was entirely dependent upon its catalytic activity. The molecular mechanism by which KIAA1363 regulates the pathogenic properties of human cancer cell lines was addressed using a global metabolite profiling strategy in combination with metabolic labeling studies to determine that KIAA1363 serves as a central node in an ether lipid signaling pathway that bridges the platelet-activating factor (PAF) and lysophosphatidic acid (LPA). Biochemical studies revealed that KIAA1363 regulates this network by hydrolyzing the metabolic intermediate 2-acetyl monoalkylglycerol to monoalkylglycerol (MAGE). Perturbation of KIAA1363 levels or catalytic activity in human cancer cell lines directly correlates with changes in the concentration of these metabolites.

The significance of ether lipids, such as LPA, in cancer biology is demonstrated by the fact that LPA is an established biomarker of ovarian cancer being elevated nearly 10 fold in ascites fluid and LPA has been shown to regulate many of the pathogenic properities of cancer cell lines. Significantly, we have found that KIAA1363-regulated cellular migration is dependent on the bioactive lipid LPA, as the addition of as little as 10 nm alkyl-LPA rescued the migratory defect induced by an inhibition of KIAA1363 activity. These findings indicate that KIAA1363 is an important molecule in human cancer biology.