Inhibition of tumor growthin vivo by hyaluronan oligomers

128

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that interacts with cell-surface receptors, including CD44. Although HA usually exists as a high molecular mass polymer, HA of a much lower molecular mass that shows a variety of biological activities can be detected under certain pathological conditions, particularly in tumors. We previously reported that low molecular weight HAs (LMW-HAs) of a certain size range induce the proteolytic cleavage of CD44 from the surface of tumor cells and promote tumor cell migration in a CD44-dependent manner. Here, we show that MIA PaCa-2, a human pancreatic carcinoma cell line, secreted hyaluronidases abundantly and generated readily detectable levels of LMW-HAs ranging from ϳ10-to 40-mers. This occurred in the absence of any exogenous stimulation. The tumor-derived HA oligosaccharides were able to enhance CD44 cleavage and tumor cell motility. Inhibition of the CD44-HA interaction resulted in the complete abrogation of these cellular events. These results are consistent with the concept that tumor cells generate HA oligosaccharides that bind to tumor cell CD44 through the expression of their own constitutive hyaluronidases. This enhances their own CD44 cleavage and cell motility, which would subsequently promote tumor progression. Such an autocrine/paracrine-like process may represent a novel activation mechanism that would facilitate and promote the malignant potential of tumor cells.Hyaluronan (HA), 2 one of the major components of the extracellular matrix, is a high molecular weight linear glycosaminoglycan consisting of repeating disaccharide units of D-glucuronic acid and N-acetyl-Dglucosamine, whose molecular size can reach 10 7 kDa (1, 2). Despite its simple composition, HA possesses a number of functions. It influences the hydration and physical properties of tissues (1, 3), interacts with other extracellular matrix macromolecules such as aggrecan (4) and versican (5), and interacts with cell-surface receptors, notably CD44 (6). Through these functions, HA forms a pericellular coat around cells (2, 7) and participates in physiological events such as cell adhesion, migration, and proliferation (8). It is also involved in pathological conditions, including cancer (7). An increased synthesis of HA has been reported in various malignant tumors such as breast and ovarian carcinomas, and predicts a poor prognosis (9, 10). HA enhances tumor cell adhesion and migration (11) and influences several signaling pathways, including some that promote tumor cell growth and survival, such as ErbB2, Ras, mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/ Akt (12-14).Hyaluronidases play critical roles in HA metabolism (15). Six human hyaluronidase-like sequences are present in the human genome. These are HYAL1, HYAL2, HYAL3, HYAL4, SPAM1, and PHYAL1, which, respectively, encode Hyal-1, Hyal-2, Hyal-3, Hyal-4, PH-20, and a pseudogene that is transcribed but not translated (16). Of these hyaluronidases, only Hyal-1 and Hyal-2 have been characterized well. Hyal...

Section: Discussionmentioning

confidence: 99%

Tumor Cells Enhance Their Own CD44 Cleavage and Motility by Generating Hyaluronan Fragments

Sugahara

Hirata

Hayasaka

et al. 2006

128

“…Materials-Hyaluronan oligomers used in this study are a highly purified, mixed fraction of average molecular weight ϳ2.5 ϫ 10 3 and are composed of 3-10 disaccharide units (19,21). Plasmids for constitutively active PI3K and Akt, and dominant-negative Akt, were obtained from Upstate Biotechnology, Lake Placid, NY.…”

Section: Methodsmentioning

confidence: 99%

Regulation of MDR1 Expression and Drug Resistance by a Positive Feedback Loop Involving Hyaluronan, Phosphoinositide 3-Kinase, and ErbB2*♦

Misra¹,

Ghatak²,

Toole

2005

Self Cite

230

222

Multidrug resistance is a potent barrier to effective, long term therapy in cancer patients. It is frequently attributed to enhanced expression of multidrug transporters or to the action of receptor kinases, such as ErbB2, and downstream anti-apoptotic signaling pathways, such as the phosphoinositide 3-kinase/Akt pathway. However, very few connections have been made between receptor kinases or anti-apoptotic pathways and multidrug transporter expression or function. Data presented herein show that constitutive interaction of the pericellular polysaccharide, hyaluronan, with its receptor, CD44, regulates assembly and activation of an ErbB2-containing signaling complex, which in turn stimulates phosphoinositide 3-kinase activity in multidrug-resistant MCF-7/Adr human breast carcinoma cells. Phosphoinositide 3-kinase activates Akt and downstream anti-apoptotic events, which contribute to drug resistance. However, hyaluronan and phosphoinositide 3-kinase stimulate expression of the multidrug transporter, MDR1 (P-glycoprotein), in an interdependent, but Akt-independent, manner. Furthermore, constitutively active phosphoinositide 3-kinase, but not Akt, stimulates hyaluronan production. These Akt-independent effects are dominant over the effects of Akt on doxorubicin resistance in MCF-7/Adr cells. Thus hyaluronan, phosphoinositide 3-kinase, and ErbB2 form a positive feedback loop that strongly amplifies MDR1 expression and regulates drug resistance in these cells. This pathway may also be important in progression of other malignant characteristics. These results illustrate the potential importance of hyaluronan as a therapeutic target in multidrug-resistant carcinomas.

“…These small sugar molecules induce a variety of cancer cells to undergo programmed cell death or apoptosis (6,7). This intriguing finding immediately suggests the use of HA oligosaccharides as an aid to chemotherapy or radiation treatments to reduce tumor load or limit spread (metastasis).…”

mentioning

confidence: 98%

“…HA has numerous essential roles in mammals, including modulation of cellular adhesion, signaling, and motility (1,2). The polymer is thought to be synthesized initially as a chain of ϳ10 [5][6] Da (ϳ10 [3][4] sugars), which then degrades during turnover in the body to generate a variety of smaller polymers. Although the mechanisms are not completely understood, it has been observed that the size of the HA polymer dictates its effect on cells.…”

mentioning

confidence: 99%

Rapid Chemoenzymatic Synthesis of Monodisperse Hyaluronan Oligosaccharides with Immobilized Enzyme Reactors

DeAngelis

Oatman

Gay

2003

We describe the chemoenzymatic synthesis of a variety of monodisperse hyaluronan (␤4-glucuronic acid-␤3-N-acetylglucosamine (HA)) oligosaccharides. Potential medical applications for HA oligosaccharides (ϳ10 -20 sugars in length) include killing cancerous tumors and enhancing wound vascularization. Previously, the lack of defined oligosaccharides has limited the exploration of these sugars as components of new therapeutics. The Pasteurella multocida HA synthase, pmHAS, a polymerizing enzyme that normally elongates HA chains rapidly (ϳ1-100 sugars/s), was converted by mutagenesis into two single-action glycosyltransferases (glucuronic acid transferase and N-acetylglucosamine transferase). The two resulting enzymes were purified and immobilized individually onto solid supports. The two types of enzyme reactors were used in an alternating fashion to produce extremely pure sugar polymers of a single length (up to HA20) in a controlled, stepwise fashion without purification of the intermediates. These molecules are the longest, non-block, monodisperse synthetic oligosaccharides hitherto reported. This technology platform is also amenable to the synthesis of medicanttagged or radioactive oligosaccharides for biomedical testing. Furthermore, these experiments with immobilized mutant enzymes prove both that pmHAS-catalyzed polymerization is non-processive and that a monomer of enzyme is the functional catalytic unit.Complex carbohydrates play many essential roles in vertebrates, but these molecules are often difficult to isolate or to synthesize in pure form and in large amounts for study. HA, 1 a member of the glycosaminoglycan polysaccharide family that includes heparin and chondroitin, is a repeating ␤4-glucuronic acid-␤3-N-acetylglucosamine polymer that is prevalent in the vertebrate body. HA has numerous essential roles in mammals, including modulation of cellular adhesion, signaling, and motility (1, 2). The polymer is thought to be synthesized initially as a chain of ϳ10 5-6 Da (ϳ10 3-4 sugars), which then degrades during turnover in the body to generate a variety of smaller polymers. Although the mechanisms are not completely understood, it has been observed that the size of the HA polymer dictates its effect on cells. Various cell-surface receptors or binding proteins, termed hyaladherins, are postulated to sense the presence and the size of HA (3-5). Hyaladherins thought to play important roles include CD44 and RHAMM. Multivalent binding and/or multimerization of receptors may potentially occur with large polymers but not with small chains, resulting in the alteration of various signaling events.Recently, several groups have reported that HA oligosaccharides (ϳ2-3.5 kDa or ϳ10 -17 sugars) have extremely interesting effects on cellular behavior. These small sugar molecules induce a variety of cancer cells to undergo programmed cell death or apoptosis (6, 7). This intriguing finding immediately suggests the use of HA oligosaccharides as an aid to chemotherapy or radiation treatments to reduce tumor load or limit...