The Breast Tumor Suppressor Function of Hyaluronidase

Institution: University of California, San Francisco
Investigator(s): Robert Stern, M.D. -
Award Cycle: 1995 (Cycle I) Grant #: 1RB-0008 Award: $302,120
Award Type: Research Project Awards
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1995)
The enzyme hyaluronidase appears to interfere with metastasic spread of breast cancer through three mechanisms. Two of the requirements for cancer cells to metastasize are that they must become motile, and that they must have a space into which to move. Hyaluronan (HA) accomplishes both of these tasks. Hyaluronan is a carbohydrate polymer on the surface of cancer cells that hydrates, expands, and opens up spaces around cancer cells, enabling them to invade surrounding tissues. It is also involved in the development of motility. Hyaluronidase, the enzyme that breaks down HA, eliminates this mechanism for cancer cell spread. Hyaluronidase appears to have other direct effects on cancer cells. The receptor for HA, CD44, functions as an anchor to bind HA to the cancer cell surface. CD44 comes in a variety of forms, a short form found on normal cells, and longer variants that occur on motile cancer cells. A portion of CD44 protrudes through the surface membrane into the cell cytoplasm, where it interacts with the cytoskleleton, the machinery for cell movement. Experimentally, inserting this longer cancer form of CD44 into benign tumor cells changes them into cancer cells that aggressively metastasize. Hyaluronidase eliminates the cancer cells' ability to move, by converting the long cancer form of CD44 to the short "normal" immobile form. Remarkably, hyaluronidase also causes established breast cancers to shrink.

In mice with human breast cancer, inoculation of hyaluronidase i.v. shrinks the tumor to half its size in four days. If malignant cells cannot maintain motility and thus invasiveness, and if the tumor cannot progress or grow, it apparently must undergo regression. This third function of hyaluronidase is not understood, but may follow from the first two functions. Hyaluronidase may also prevent progression of human breast cancer, and it does not have the devastating toxic side effects of other anticancer agents. We will vary injection schedules, and modify the enzyme chemically, to stabilize activity, in order to maximize the anticancer effect in mice, in preparation for clinical trials. Simultaneously, we will examine breast cancer cells in culture using molecular and cell biology to determine precisely how hyaluronidase regulates this alternative splicing of CD44. We postulate that HA regulates its own receptor. When HA bound to CD44 is present outside the cell, tags are placed on the intracellular portion of CD44. We have identified these specific tagging enzymes or phosphokinases, and when they are inactivated by techniques involving antisense or mirror image DNA, CD44 converts to its short form, as CD44 does in response to hyaluronidase. Understanding these events in the regulation of CD44 will make it possible to develop a new generation of anticancer drugs that prevent the metastases associated with breast cancer progression.

Final Report (1998)
Levels of hyaluronic acid (HA), on the surface of breast cancer cells correlate with tumor aggressiveness. Hyaluronidase, the enzyme that degrades HA, interferes with the metastatic spread of breast cancer through three mechanisms. Two of the requirements for cancer cells to metastasize are that they must become motile, and that they must have a space into which to move. HA accomplishes both of these tasks. The water of hydration associated with HA expands the potential spaces around tumor cells creating places into which cells can move. HA, through its receptors on cancer cell surfaces, also endows these cells with the ability to move, by interacting with the cytoskeleton.

This laboratory has been studying the hyaluronidase family of enzymes, a previously unrecognized cancer therapeutic, and one that is non toxic. Hyaluronidase, following i.v. administration, not only degrades HA, but reduces the size of orthotopic xenografts of human breast cancers planted in the mammary fat pad of immunodeficient (SCID) mice. Simultaneously, it appears to reduce the levels of certain forms of the HA receptor (CD44) on cancer cell surfaces, isoforms that are associated with malignancy. Thess CD44 molecules function as an anchor to bind HA on the cancer cell surface, but also correlate with tumor progression and metastasis. HA thus has the ability to control the expression of isoforms of its own receptor. The third mechanism by which hyaluronidase impedes breast cancer progression and rnetastasis is to diminish expression of certain forms of CD44. The three mechanisms of tumor progression and spread related to the elimination of HA appear to function in concert that if a breast cancer cannot progress and metastasize it must regress.

Using CBCRP support, we have investigated the molecular mechanism of hyaluronidase action. In summary, the phosphorylation of serine residues in the cytoplasmic or intracellular portion of the CD44 molecule is required for the stabilization of intrinsically unstable forms of CD44. This requires the continuous presence of the HA ligand. The phosphorylation of these serines is catalyzed by casein kinase enzyme known to be present in breast cancers. Elimination of HA by hyaluronidase decreased casein kinase catalyzed phosphorylation. Inserting antisense RNA into cancer cells for casein K-kinase also eliminated this phosphorylation, and also decreased expression of the CD44 on cancer cell surfaces. We have also isolated hyaluronidase from human plasma, and are sequencing the protein, and establishing an expression system. Sequence analysis suggests hyaluronidase is related to a tumor suppressor, gene locus on chromosome 3p2l.

Purification and microsequencing of hyaluronidase isozymes from human urine.
Periodical:FEBS Journal
Index Medicus: FEBS Journal
Authors: Csoka TB, Frost GI, Wong T, and Stern R
Yr: 1997 Vol: 417 Nbr: 3 Abs: Pg:307-310

Phosphorylation stabilizes alternatively spliced CD44 mRNA transcripts in breast cancer cells: inhibition by antisense complementary to casein kinase II mRNA
Periodical:Molecular and Cellular Biochemistry
Index Medicus: Mol Cell Biochem
Authors: Formby B and Stern R
Yr: 1998 Vol: 187 Nbr: Abs: Pg:23-31

The hyaluronidase gene HYAL1 maps to chromosome 3p21.2-p21.3 in human and 9F1-F2 in mouse, a conserved candidate tumor supressor locus
Index Medicus: Genomics
Authors: Csoka TB, Frost GI, Heng HHQ, Sherer SW, Mohapatra G, and Stern R
Yr: 1998 Vol: 48 Nbr: Abs: Pg:63-70

Purification, cloning, and expression of human plasma hyaluronidase
Periodical:Biochemistry and Biophysics Research Communications
Index Medicus: Biochem Biophys Res Commun
Authors: Frost GI, Csoka TB, Wong T, and Stern R
Yr: 1997 Vol: 236 Nbr: Abs: Pg:10-15

Hyaluronidase in tissue invasion
Periodical:Invasion Metastasis
Index Medicus: Inv Metas
Authors: Csoka TB, Frost GI, and Stern R
Yr: 1997 Vol: 17 Nbr: Abs: Pg:297-311

A microtiter-based assay for hyaluronidase activity not requiring specialized reagents
Periodical:Analytical Biochemistry
Index Medicus: Anal Biochem
Authors: Frost GI and Stern R
Yr: 1997 Vol: 251 Nbr: 2 Abs: Pg:263-269

Patterns of hyaluronan staining are modified by fixation techniques
Periodical:Journal of Histochemistry and Cytochemistry
Index Medicus: J Histochem Cytochem
Authors: Lin W, Shuster S, Mailbach HI, and Stern R
Yr: 1997 Vol: 45 Nbr: 8 Abs: Pg:1157-1163

The overexpression of RHAMM, a hyaluron-binding protein that regulates ras signaling, correlates with overexpression of mitogen-activated protein kinase and is a significant parameter in breast cancer progression
Periodical:Clinical Cancer Research
Index Medicus: Clin Cancer Res
Authors: Wang C, Thor AD, Moore DH, Zhao Y, Kerchmann R, Stern R, Watson PH, and Turley EA
Yr: 1998 Vol: 4 Nbr: 3 Abs: Pg:567-576