Novel Enzymes Associated with Breast Cancer Angiogensis

Institution: University of California, San Francisco
Investigator(s): Steven Rosen, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7IB-0036 Award: $100,000
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2001)
Tumors need a blood supply in order to increase in size and for their spread (metastasis) throughout the body. Tumors are able cause surrounding normal tissue to supply a increased number of blood vessels for growth and spread in the body. A variety of approaches and novel therapeutics are being investigated to block of new blood vessel formation. However, there remain many undiscovered aspects of angiogenesis, since the underlying biology is so complex. A key element that links tumor cells, blood vessels, and normal tissues is the extracellular matrix. It is composed of a variety of proteins and protein-sugar macromolecules (proteoglycans) that serve as barriers to angiogenesis and cell movements. Proteoglycans also can bind soluble factors that regulate the growth of new blood vessels. One of the key proteoglycans is heparan sulfate (HS). We have discovered two new enzymes that could potentially degrade HS, and represent potential factors produced by breast cancer cells that may be involved in the regulation of angiogenesis.

Based on a virtual northern analysis, our preliminary evidence is that two novel HS degrading enzymes are increased in human breast cancer samples as compared to normal breast tissue We will use recombinant DNA techniques to provide us with the tools to measure the presence of the genes, and the amount protein products from these genes. Finally, we will use mouse models of breast cancer to test whether the HS degrading enzymes are really required for the growth of new blood vessels in tumors.

We are studying new genes that have not previously been considered in the context of cancer biology. We have a plausible theory of how this type of gene could contribute to the formation of new blood vessels in tumors. If our hypothesis is correct, then these enzymes could be studied with the aim of developing a new class of anti-angiogenic drug.

Final Report (2002)
Our original aim was to study two novel proteins, called Sulf-1 and Sulf-2, which we had inferred to exist based upon our analysis of mouse and human expressed sequence tags (ESTs) in NCBI public database. We had predicted potential full-length cDNAs from this database analysis. In addition, we further predicted that these cDNAs would encode extracellular sulfatases with activity against heparan sulfate proteoglycans. Furthermore, we speculated that these enzymes might be involved in releasing proangiogenic factors from the extracellular matrix and thereby could contribute to tumor angiogenesis.

Using CBCRP support, we cloned and sequenced cDNAs for both Sulf-1 and Sulf-2 in mouse and human. We expressed the proteins in CHO cells and showed they were secreted after processing by a furin enzyme in the secretory pathway. We directly confirmed that the Sulfs are both sulfatases with activity against an arylsulfatase substrate (4-MUS). More significantly, they both exhibit endosulfatase activity against heparin, removing sulfate from the C-6 position of glucosamine residues. This modification is known to be critical for the binding of heparan sulfate to various angiogenic factors and growth factors. The Sulfs are optimally active at neutral pH, consistent with a function in the extracellular milieu. The results of our cloning of these enzymes and their characterization as novel extracellular sulfatases have been published in the Journal of Biological Chemistry. Following up preliminary SAGE (serial analysis of gene expression) expression data indicating upregulation of Sulf-2 mRNA in human breast cancer, we went on to show that Sulf-2 is strongly expressed at the mRNA level in mouse models of breast cancer and in human breast cancers. Additionally, we obtained evidence that Sulf-2 is pro-angiogenic in the chick embryo CAM assay: it induces the formation of new blood vessels when introduced into 9 day chick embryos.

Our results are compatible with the possibility that the Sulfs have a role in regulating the association of angiogenic factors with heparan sulfate in the extracellular matrix and can promote the formation of new blood vessels in tumor settings. Future work will be directed at testing the validity of this important possibility in mouse models of mammary tumors.

Symposium Abstract (2003)
We are exploring the possibility that novel extracel-lular sulfatases are involved in the regulation of angiogenesis in breast cancer. Angiogenesis is clearly important for breast cancer, yet the regulation of angiogenesis in this process is poorly understood. One potential mechanism for controlling angiogenesis is through regulation of the bioavailability of angiogenic factors such as VEGF. This factor and other angiogenic factors bind to heparan sulfate proteoglycans (HSPG), sequestering these factors away from their receptors on endothe-lial cells. We are interested in the molecular events that control the release of angiogenic factors from HSPG. Bergers et al. showed that treatment of normal pancreatic islets with bacterial heparitinases, which releases VEGF from heparan sulfate, caused the islets to become angiogenic. There are two potential types of heparan sulfate degrading enzymes: mammalian heparanases and extracellular sulfatases. At a previous CBCREP Symposium, we described the cloning of two novel extracellular sulfatases (Sulf-1 and Sulf-2) in both human and mouse, which are highly homologous to Qsulf1 and share the same overall structural organization.

Sulf-1 and Sulf-2 showed both arylsulfatase and endosulfatase activity removing sulfate from the C-6 position of glucosamine from intact heparin. We performed RT-PCR analysis using cDNAs prepared from independent normal human breast tissues and independent breast cancer patients. We found that nine out of 12 of the breast cancer specimens expressed human Sulf-2 (HSulf-2) whereas none (0 out of 12) of the normal breast tissue samples were positive. HSulf-1 is positive in the all breast cancer and normal breast tissue samples. HSulf-2 mRNA was also detected in the breast cancer cell lines, MCF-7, BT549, and BT20. HSulf-2 protein was detected in the conditioned media of MCF-7 cells by the anti HSulf-2 antibody. The conditioned media of MCF-7 cells showed both arylsulfatase and endosulfatase activity on heparin. To determine whether HSulf-2 is involved in angiogenesis, we performed the CAM (chick chorioallantoic membrane) assay using the conditioned media from CHO cells transiently transfected with HSulf-2 or using HSulf-2 stably transfected CHO cells. Preliminary results suggest that HSulf-2 protein may promote angiogenesis.

Our results establish that the mammalian Sulfs are extracellular endosulfatases with strong potential for regulating angiogenesis through the modulation of the interactions between HSPG and angiogenic factors in the extracellular microenvironment. Our aim is to show whether or not HSulf-2 from MCF-7 cells also promotes angiogenesis. For these studies we will employ MCF-7 cells to determine how HSulf-2 participates in the formation of new blood vessels in breast cancers.

Cloning and characterization of two extracellular heparin-degrading endosulfatases in mice and humans
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Morimoto-Tomita M, Uchimura K, Werb Z, Hemmerich S, Rosen SD
Yr: 2002 Vol: 277 Nbr: 51 Abs: Pg:49175-85