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Development of Glycosylation-based Therapies for Breast Cancer: Targeting Sialic Acid-Mediated Metastasis with Metabolic Analogs that Modulate Sialoside Biosynthesis
Background: Sialic acid is an unusual 9-carbon monosaccharide that becomes dramatically altered as a mammary cell progresses from a healthy to malignant state. Changes in sialic acid alter a cell’s adhesive properties and contribute to a highly metastatic phenotype. In the past methods to manipulate glycosylation in living cells have been lacking thus hindering the development of drugs suitable for reversing the effects of cancer-associated forms of sialic acid. Now, however, sialic acid engineering methods where sugar metabolism is modulated through the use of N-acetylmannosamine (ManNAc) analogs provide the means to modify sialic acid expression in living cells and animals.
Objective/hypotheses: We propose that certain ManNAc analogs will modulate sialic acid display in breast cancer cells and thereby reduce their metastatic potential.
Specific Aims are to: 1) Screen ~20 ManNAc analogs in breast cancer cell lines to identify specific analogs that modulate the expression of four disease-related forms of sialic acid. 2) To analyze the effects of the analogs on gene expression to test if they alter cell adhesion by direct incorporation into surface sialic acids or indirectly by regulating the expression of biosynthetic enzymes or adhesion molecules.
Study Design: Aim 1) A panel of breast cell lines expressing each of the abnormal forms of sialic acid found in human breast carcinomas (specifically, polysialic acid, sialomucins, sLeX, and a-2,6-linked sialic acids) will be incubated with a panel of ManNAc analogs and appropriate assays to test adhesion to extracellular matrix components or selectins will then be done to predict the effect of each analog on metastasis. Aim 2) The expression of the genes that control sialic acid synthesis will be monitored by real-time PCR to test if the sugar analogs alter the “glycosylation machinery” of the cell. In addition, DNA microarray analysis will test if cell-wide gene expression is perturbed by the analogs.
Potential Outcomes: This proposal will demonstrate the feasibility of carbohydrate-based approaches to alter the metastatic potential of breast cancer cells and serve as an early, but important, step towards the development of a new class of anti-cancer therapeutics.
Breast cells are covered with dense layers of carbohydrates that play many roles in maintaining the health of a mammary gland. These sugars undergo dramatic changes during transformation to the cancerous state; in particular they aid the metastatic spread of malignant cells. The ability to “repair” faulty sugars would provide a new treatment strategy for breast cancer but efforts to correct defective carbohydrates in living cells and animals are at a very rudimentary stage. Recently-developed “glycosylation engineering” methods, however, now allow sugar biosynthesis to be manipulated in living cells and provide a potential strategy to reduce metastasis.
Glycosylation engineering is a technique where cells are supplied with non-natural monosaccharide analogs; metabolic pathways incorporate the analogs into cellular components thereby endowing cells with novel properties that alter their behavior. Sialic acid, an unusual sugar abnormally expressed on the surfaces of breast cancer cells, can be manipulated by this strategy. Interestingly, sialic acid can either increase or decrease the “stickiness” of a cell; its anti-adhesive form aids in the detachment of a cell from the original tumor, which is an early step in the metastatic process. The “sticky” forms help a previously-detached cell escape the bloodstream, attach to the underlying tissue, and grow into a second tumor. We propose that glycosylation engineering methods can be used to control the specific forms of sialic acid that contribute to the metastasis of breast cancer cells. This promising research is at an early stage and this proposal will provide a major step in propelling this work from a cell-based level to animal and clinical studies by:
(1) Identifying specific analogs that reduce metastasis. Over two dozen sugar analogs with potential anti-metastatic potential are now available. The objective of this proposal is to evaluate these compounds in cell-based assays to identify one or two specific analogs that best intervene at each step of the metastatic process to provide a manageable number of compounds for testing in follow-up animal studies.
(2) Determining the cellular basis of anti-metastatic effects. We expect that sugar analogs directly appear on the cell surface and it is their physical presence that alters the chemical properties and resulting metastatic behavior of the cell. Another possibility is that the analogs alter the cellular production of adhesion molecules; this proposal will test these two factors to better guide the subsequent development of sugar-based therapies.