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Role of Novel Sulfatases in Breast Cancer
a) Background: Cancers develop through a multistep process that involves aberrant gene expression in the cancer cell and the mobilization/activation of host cells. One example of such host cells are capillary endothelial cells which can be activated to form new blood vessels (i.e., angiogenesis) during early stages of tumor development, allowing for growth of the tumor mass and for metastasis. A large body of evidence has established the importance of new vessel generation for the progression of tumors, including breast carcinomas. Thus, in the absence of a sufficient blood supply, the size of the tumor mass is restrained as the tumor cells die from necrosis or apoptosis. Regulation of tumor vascularization involves a balance between the production of molecules that positively or negatively regulate angiogenesis. Angiogenesis is induced by angiogenic factors like VEGF or FGF. These factors are known to bind to heparan-sulfate proteoglycans (HSPGs) which are found on cell surfaces and in the extracellular matrix. The specific sulfation pattern and, in particular the 6-O-sulfation status of HSPGs, plays a critical role in the binding of angiogenic factors. Recent findings indicate that HSPGs are responsible for the sequestering of growth factors (e.g., angiogenic factors) and other signaling molecules in the extracellular matrix. Mobilization of these factors by heparan sulfate degrading enzymes (HSDEs) provides a potential mechanism for the activation of angiogenesis. This research project will examine two recently identified proteins called Sulf-1 and a closely related molecule called Sulf-2 which were identified in humans and mice in the laboratory of Dr. Steven D. Rosen. Both proteins encode novel extracellular sulfatases with endoglucosamine-6-sulfatase activity on heparin and HSPGs. Notably, mRNA transcripts of both sulfatases are significantly upregulated in different cancers. Protein expression in cancer was shown in MMTV-Wnt mouse model of breast cancer. Additionally, Sulf-2 induces angiogenesis in a chick chorioallantoic membrane (CAM) assay. Treatment of heparin with Sulf-2 inhibits binding of the angiogenic factor VEGF to heparin and Sulf-2 can mobilize VEGF from its heparin bound state into medium. These results strongly indicate involvement of Sulfs in tumor angiogenesis.
b) Objective/Hypothesis: In our working hypothesis, the induction of tumor growth is initiated by increasing the bioavailability of growth factors like VEGF or FGF bound to HSPGs by the enzymatic action of HSDEs. We will evaluate both Sulfs in respect to their role as an HSDE involved in tumor angiogenesis.
c) Specific Aims: 1) Determine expression of the Sulfs in breast cancer and in mouse models for breast cancer (MMTV-Wnt, MMTV-neu); 2) Determine the effects of the Sulfs on the in vitro and in vivo binding of angiogenic factors and growth factors to heparin and heparan sulfate proteoglycans; 3) Determine the contribution of Sulfs expressed by breast carcinoma cells in angiogenesis and 4) Determine the angiogenic activity of the Sulfs in mouse models of cancer.
d) Study Design: We will analyse expression and activity of Sulfs using newly developed antibodies directed against the proteins. Using an ELISA based on growth factor/signaling molecule binding to immobilized heparin-BSA, we will determine the ability of the two Sulfs to reverse the binding of the factors. We will perform loss-of-function and gain-of-function approaches to study the effect of Sulfs in vitro and in vivo in order to study Sulf activity in tumor angiogenesis and tumor progression. For these experiments we will use CHO cells transfected with Sulf-DNA constructs and breast carcinoma cells that endogenously express Sulf proteins. We will utilize the CAM angiogenesis assay and mouse models of tumorigenesis.
e) Potential Outcomes and Benefits of the Research: New insights into the process of tumor development may result, motivating the development of novel therapies based on the antagonism of the Sulfs. Furthermore, new prognostic and diagnostic tests for human cancer and in particular breast cancer can be envisioned.
A crucial step in the development of tumors is the formation of new vessels (i.e., angiogenesis) during early stages of tumor development, allowing for growth of the tumor mass and for metastasis. Thus, in the absence of a sufficient blood supply, the size of the tumor mass is restrained and the tumor cells die. In this research project, I will examine two novel proteins regarding their potential role in inducing tumor angiogenesis.
The regulation of cell growth and differentiation is controlled by a large number of growth factors and signaling molecules that are present in the surrounding tissue. Accumulating evidence indicates that these growth factors and signaling molecules can be controlled in their bioavailability by heparan sulfate proteoglycans (HSPGs) which are ubiquitous constituents of both cell surfaces and the extracellular matrix (ECM). HSPGs consists of Heparan-Sulfate polysaccharide chains that are bound to different core proteins. These polysaccharide chains are modified specifically by negative charged sulfate groups. The position of those sulfate groups mediates the specificity of interactions with other proteins. Recent findings led to the hypothesis that growth factors and signaling molecules can be mobilized from their sequestered state with HSPGs by activity of heparan sulfate degrading enzymes. Increases in the level or activity of these enzymes could raise the concentration of growth factors, leading to cancer formation and tumor angiogenesis.
In the Laboratory of Dr. Steven D. Rosen, two novel enzymes called Sulf-1 and Sulf-2 were identified in both humans and mice. Both enzymes are secreted and have 6-O-endosulfatase activity which means that they remove sulfate from the 6-O position within heparan sulfate chains. Interestingly, both molecules are expressed in cancer specimens at higher levels particularly in breast cancer. Our experiments have shown a potential role of Sulfs in mobilizing HSPG bound growth factors in vitro. Additionally it was shown, that Sulfs can induce angiogenesis in a standard assay system. In this research project, the activity of both proteins will be examined with respect to their ability to stimulate angiogenesis during tumor development. If this study supports a critical role for a Sulf in regulating formation of breast cancer carcinogenesis, then it would become an excellent target for therapy. An entirely new approach for treating breast cancer may emerge from these investigations. For example, inactivation of the enzymatic activity of the Sulf using antibodies or small molecule inhibitors might provide new therapeutic approaches. Also, one can envision diagnostic and prognostic tests for the early detection of breast cancer based on the level of Sulf protein or enzymatic activity in bodily fluids.