Targeting the tumor microenvironment in cancer: why hyaluronidase deserves a second look

Abstract

Increased extracellular matrix (ECM) deposition is a characteristic observed in many solid tumors. Increased levels of one ECM component, namely hyaluronan (HA), leads to reduced elasticity of tumor tissue and increased interstitial fluid pressure. Multiple initial reports demonstrated that the addition of hyaluronidase to chemotherapeutic regimens could significantly improve efficacy. Unfortunately, the bovine hyaluronidase used in those studies was limited therapeutically by immunologic responses to treatment. Newly developed recombinant human hyaluronidase has recently been introduced into clinical trials. In this article, we describe the role of HA in cancer, methods of targeting HA, clinical studies performed to date, and propose that targeting HA could now be an effective treatment option for patients with many different types of solid tumors.

INTRODUCTION

Many solid tumors develop extensive fibroses, a result of what is termed the desmoplastic reaction [reviewed in ¹]. Desmoplasia leads to a significant increase in the production of extracellular matrix proteins, as well as extensive proliferation of myofibroblast-like cells. The result is the formation of a dense and fibrous connective tissue that is comprised of multiple extracellular matrix (ECM) components including collagen types I, III, and IV, fibronectin, laminin, hyaluronan, as well as the glycoprotein osteonectin (also known as secreted protein, acidic and rich in cysteine, or SPARC) (Figure 1). This fibroinflammatory component of the tumor (sometimes called stroma) contributes to an increase in tumor interstitial fluid pressure, blocking perfusion of anticancer therapies to the tumor cells, and contributes generally to chemoresistance [2] (see Table 1 for individual components and their proposed mechanism of chemoresistance). Consequently, targeting the components of the stromal compartment, in conjunction with cytotoxic agents directed against the tumor cells, is gaining traction as a potential approach for treating patients and overcoming chemoresistance.

Figure 1.

Illustration of invading epithelial tumor cells and the associated tumor microenvironment. Dissolution of the basement membrane is accompanied by the production and secretion of numerous extracellular matrix components, including the collagens, fibronectin, laminin, and hyaluronan, as part of the myofibroblast-mediated desmoplastic reaction. Infiltrating immune cells also contribute to the signaling involved in the desmoplastic reaction. The expansion of the stromal compartment and the production of the extracellular matrix proteins are thought to result in greater intratumoral pressure and contribute to a reduction in effective drug delivery.

Table 1.

Extracellular matrix components which may contribute to chemoresistance

ECM component	Functional role in chemoresistance
Collagen I, III, IV	Enhances tumor cell proliferation, structural support of ECM
Decorin	Binds TGF-β, tightens collagen fibrils
Hyaluronan	Synergizes with collagen network, increases interstitial fluid pressure
Versican	Enhances tumor cell proliferation, confers resistance to apoptosis
Fibronectin	Confers resistance to apoptosis
Laminin	Confers resistance to apoptosis
Osteonectin/SPARC	Enhances tumor cell proliferation and metastasis

The concept of directing therapies towards the stromal compartment as a means to enhance drug perfusion is supported by a recent report demonstrating that stromal depletion by nab-paclitaxel (which accumulates in tumor tissues high in SPARC) resulted in improved gemcitabine delivery in a primary human xenograft model for pancreatic cancer [3]. In addition, using a genetically engineered mouse model of pancreatic cancer, Olive and colleagues demonstrated that stromal depletion by hedgehog pathway inhibitors enhanced the intratumor concentration of gemcitabine and resulted in significantly increased survival of tumor bearing mice [4].

Enzymes that degrade the extracellular matrix (ECM) have also been proposed as stroma-targeting agents. However, the immunoreactivity and pH sensitivity of ECM targeting agents such as the collagenases has been a problem that has limited their study in vivo. Another ECM component that may be targeted by a degrading enzyme is hyaluronan. Hyaluronan (HA) is a linear polysaccharide comprised of glucuronic acid and N-acetylglucosamine and plays an important role in a diverse range of cellular processes (Figure 2). Elements of HA and HA metabolism are thought to be involved in biological functions related to cell proliferation, tissue hydration, cell motility, inflammation, angiogenesis, and malignancy. Hyaluronan is distributed universally in the extracellular spaces of most tissues, with especially high concentrations found within connective tissues. Unlike the other protein bound and sulfated glycosaminoglycans (GAGs), HA is a unique polyanionic and protein-free polysaccharide that has an exceptional ability to increase viscosity, expand volume, and provide structural support in various locations and contexts within the body. While steady-state levels of HA are generally quite low in most normal tissues, HA levels dramatically increase with many disease states such as vascular disease (e.g. atherosclerosis) and cancer. Furthermore, high HA levels have been correlated with poor prognosis in many different cancer types, including gastric, colorectal, breast, ovarian, and bladder cancer. Because HA functions in ion exchange, and may also act as a molecular sieve that prevents the penetration of drugs, it is thought that treatment with agents that degrade HA, such as a hyaluronidase, may have the potential to increase penetration of drugs through the stromal compartment and ultimately into tumor cells. Given the important role of HA and the ECM in solid tumors, it is possible that targeting the ECM with agents such as hyaluronidase could prove effective in improving therapeutic outcomes in patients with solid tumors. This has led us to consider “why aren’t we now targeting the tumor stroma with hyaluronidase in our treatment of cancer?”

Figure 2.

Chemical structure of hyaluronan. Hyaluronan is a linear polysaccharide composed of repeating units of glucuronic acid and N-acetylglucosamine. Hyaluronan is a nonsulfated glycosaminoglycan and participates as an integral component of the extracellular matrix.

HYALURONAN AND ITS ROLE IN CANCER

Hyaluronan is a protein-free, acetone insoluble polysaccharide first isolated from hyaloid (or vitreous) matter and reported to contain uronic acid. It is ubiquitously distributed throughout the human body, with particularly high concentrations seen in the skin, eyes, in cartilage, and in synovial fluid. HA’s structure gives it great capacity to interact with water molecules, resulting in a vastly increased volume, as well as increased viscosity of HA containing solutions. These properties have also led many to infer that HA was largely an inert molecule functioning to maintain the physical volume and rigidity of connective tissue. However, with the discovery of the HA binding proteins, or hyaladherins, it became clear that their functional reach was much greater. The discovery of the proteoglycans, aggrecan and link protein, as well as the discovery of the HA-binding surface receptors, CD44 and RHAMM, revealed that HA is involved in the direct signaling of many biological processes, including cell proliferation, migration, adhesion, and even the recruitment of leucocytes such as the neutrophils.

Studies of many cancer types, including pancreatic ductal adenocarcinoma (PDAC), indicate that an accumulation of HA occurs in neoplastic tissues. Indeed, it appears that most epithelial tumors exhibit high levels of HA localizing to their peritumoral, or stromal, compartments. Interestingly, high HA levels have also been detected at the invasive front of growing tumors, suggesting that HA may be involved not only in cell proliferation, but possibly in invasion as well. Indeed, Bertrand and colleagues observed a 4.4-fold (± 0.4) increase in HA staining relative to adjacent normal tissue at the invasive edges in breast tumors, whereas only a 3.3-fold (± 0.4) increase was seen in central locations within the tumor (P<0.05) [5]. HA interaction with CD44 facilitates colon tumor cell migration, as well as migration in other tumor models, including breast and brain cancer cells. The level of HA itself correlates with overall tumor aggressiveness and increased cell migration and proliferation in breast and ovarian cancer. Tumor cells that overexpress the hyaluronan synthase, HAS1, to varying degrees experience increased proliferation rates. While it remains to be seen whether the HAS may serve as a definitive tumor biomarker, urine HA and hyaluronidase levels may serve as suitable markers for bladder cancer, including assessment of tumor grade. In addition, we now know that HA levels correlate with malignancy in mesothelioma, and may serve as a potential diagnostic marker. It seems clear that a balance of the activity of the hyaluronan synthases with hyaluronidase activity is necessary for normal tissue function.

TARGETING HYALURONAN

In normal tissues, HA levels are maintained through a balance of synthesis by hyaluronan synthase (HAS) and degradation by the enzyme hyaluronidase. HA is synthesized in mammals via the expression of three related hyaluronan synthases, HAS1, HAS2, and HAS3. Corticosteroids can inhibit the synthesis of HA by HAS. Indeed, the addition of cortisol to cultured aortic smooth muscle cells can reduce the production of HA by as much as 50%. This effect was also observed in a recent study by Gebhardt et al, wherein they report a rapid decrease of approximately 50% of HA levels, as well as a decrease in HAS2 expression, in the skin following topical treatment with dexamethasone [6]. Except in the treatment of patients with hematologic malignancies, clinical research into the addition of the corticosteroids to anticancer therapies for patients with solid tumors has been limited (except to prevent nausea and vomiting).

In addition to corticosteroids, the HAS inhibitor, 4-methylumbelliferone (MU), has been developed and proposed as an alternative approach to lowering HA levels. MU has been shown to increase the efficacy of gemcitabine by inhibiting the growth of cancer cell lines over gemcitabine alone, without significant growth inhibition itself. Furthermore, Yoshihara et al. have shown that MU decreases liver metastases in a mouse model for melanoma [7]. In addition, MU also reduces tumorigenicity, including reduced proliferation and motility, in esophageal squamous cell carcinoma or prostate cancer cells. MU is currently being investigated in clinical trials for the treatment of patients with chronic hepatitis infection (www.clinicaltrials.gov, NCT00225537), though there are no reports to determine if the effects of MU will enhance anticancer therapies in patients. With several very recent reports of the efficacy of MU in breast or prostate cancer xenograft mouse models showing significant reduction in tumor growth, studies of MU’s efficacy in human trials are likely to be forthcoming [8].

Altering the breakdown of HA has also been proposed as a means to target HA accumulation. Catabolism of HA, and balance of HA levels, are mainly mediated by the hyaluronidases. Six genes have been identified that encode for the different hyaluronidases (HYALs), including HYAL1, -2, -3, -4, PHYAL1, and PH20. The HYALs catalyze the hydrolysis of HA and function as endo-β-acetyl-hexosaminidases. HYAL1 and -2 maintain the highest enzymatic activity in mammals, turning over as much as a third of the total HA each day. The addition of hyaluronidase to chemotherapeutics enhances the catabolism of HA, as well as significantly increases the efficacy of the chemotherapeutics even in tumors previously deemed chemoresistant. The effectiveness of hyaluronidase in improving chemotherapies has been explored in multiple tumor types including breast, brain, melanoma, and sarcoma (Table 2). The synergistic effect of adding hyaluronidase to chemotherapeutics is thought to aid in cancer treatment by reducing intratumoral pressure, or by breaking down hyaluronan’s ability to function as a molecular sieve [2]. Alternatively, the synergistic benefit may instead occur by means of a chemosensitizing effect.

Table 2.

Early clinical trials investigating the coadministration of bovine hyaluronidase with chemotherapy. References: [12–14, 16, 20]

Study	Trial type	Tumor type	Chemotherapy	Number of patients	Endpoint	Results
Klocker et al., 1998	phase II	Adv. SCC - H&N	Cisplatin/Vindesine	48	Response	CR in 84%, 47% survival over 5 yrs
Baumgartner et al., 1998	phase III	Bladder Cancer	Mitomycin C	56	Recurrence	27% vs 59% recurrence in HYAL treated vs untreated
Pillwein et al., 1998	phase II	Malignant Brain	Carboplatin/Etoposide	40	Survival	3 yr survival, 84% vs 50% in HYAL treated vs untreated
Smith et al., 1997	phase I	Kaposi’s sarcoma	Vinblastine	6	Toxicity/Recurrence	0% vs 50% recurrence in HYAL treated lesions, no added toxicity
Baumgartner et al., 1988	phase I	Gastrointestinal and others	Adriamycin and others	12	Toxicity/Recurrence	PR/MR in 5 of 12 resistant, no added toxicity

“Adv. SCC - H&N” = Advanced squamous cell carcinoma of the head and neck, “CR” = complete response, “PR” = partial response, “MR” = minimal response

In cell culture models, the addition of hyaluronidase decreases intrinsic chemoresistance in spheroid models of cancer, resulting in significantly disaggregated spheroids, increased drug penetration, and increased cell death [9]. Using a breast cancer xenograft model, however, Beckenlehner and colleagues (1992) demonstrated an increased susceptibility to doxorubicin when animals were pretreated with hyaluronidase prior to doxorubicin [10]. Other investigators have shown that hyaluronidase pretreatment can result in increased intratumor drug concentrations. Indeed, Muckenschabel et al., (1996) observed increases as high as 16- to 32- fold in tumor-specific melphalan concentrations in a melanoma study [11]. Mounting evidence suggests that drugs may fail due to an inability to attain significant intratumor concentrations [2]. Thus, the finding that hyaluronidase pretreatment increased intratumor drug concentration is particularly exciting, as it may increase the efficacy of current therapies in patients.

CLINICAL STUDIES INCORPORATING HYALURONIDASE

Multiple preliminary clinical studies have demonstrated increased efficacy with bovine hyaluronidase pretreatment in cancer patients (Table 2). Baumgartner et al (1998) have reviewed the early pilot clinical trials involving bovine hyaluronidase [12]. In a small study of six patients the addition of intralesional bovine hyaluronidase to intralesional vinblastine treatment was shown to be more effective at treating cutaneous lesions of Kaposi’s sarcoma than vinblastine alone and resulted in reduced recurrence [13]. This reduced recurrence was also seen in a study reporting on the addition of bovine hyaluronidase to standard carboplatin and etoposide treatment for malignant brain tumors [14]. In this study, which included 40 pediatric brain cancer patients, both event free survival and overall survival at 36 months were significantly improved with the addition of a 30 minute infusion of bovine hyaluronidase prior to chemotherapy treatment (Table 2). Similarly, in a study looking at the effects of mitomycin C in combination with bovine hyaluronidase administered intravesically, recurrence in bladder cancer patients was reduced from 32% in mitomycin c only treated patients to 7% with the addition of hyaluronidase (P<0.05) [15]. In two studies, which enrolled a total of 80 patients, bovine hyaluronidase delivered intravenously was added to chemotherapy or chemotherapy plus radiation therapy for advanced squamous cell carcinoma of the head and neck. In these studies, a complete response was achieved in 84% of those treated, with a 47% survival observed over five years [16]. In a study of 43 patients with high-grade astrocytoma, it was reported that the addition of hyaluronidase did not produce a significant improvement in tumor regression [12]. However, the authors suggest that any synergy of hyaluronidase may have been obscured after being compared to a different, more effective agent later in the study. More specifically, the drug used in the first segment of the astrocytoma study, Lomustine (CCNU), is only slightly effective against astrocytoma as a single agent and thus hyaluronidase did not produce a significant improvement relative to the more effective agent used in segment two, Carmustine (BCNU) [12].

Considering these results, one might ask why hyaluronidase has not been studied more fully or gained broader acceptance. One particular limitation of hyaluronidase as a therapeutic agent in prior studies has been the development of allergic reactions by patients to this enzyme due to its bovine origin. Indeed, multiple studies report that as many as 32% of patients harbor reactive IgE antibodies to the bovine hyaluronidase preparation, prior to therapy, inducing various reactions from urticaria, tachycardia, to shock [12]. Bovine hyaluronidase treatment has resulted in allergic reactions, even anaphylaxis. Furthermore, the development of anti-hyaluronidase antibodies following treatment limits its usefulness in any subsequent treatment, with elevated antibodies present for six weeks or more following intravenous or intramuscular treatment.

Recombinant human hyaluronidase (Hylenex™) has been developed in recent years. The recombinant human material eliminates the risk of disease transmission via contaminants found in animal derived hyaluronidase. The recombinant human material did not induce allergic reactions in a cohort of 100 volunteer subjects who were injected intradermally [17]. The recombinant human molecule is now used subcutaneously, consistent with the FDA approved label to help with dispersion and absorption of various injected drugs [17]. Recombinant hyaluronidase is currently being investigated under different formulations for both superficial bladder cancer (phase I/II, NCT00318643) where it is being used as purified recombinant material and in patients with solid tumors (phase I, NCT00834704) where is being utilized in the pegylated form.

Considering the biological role of HA, and the many locations in which it is found, side effects of repeated hyaluronidase treatment as part of an anticancer regimen might induce inflammation or pain in the joints. It appears that some of the side effects of enhanced hyaluronidase activity in normal tissue observed in earlier studies, however, were controlled by the administration of corticosteroids [12]. The ongoing phase I trial used pegylated material, PEGPH20, because of the improved half life of the recombinant enzyme. In this study, 50µg/kg induced grade 3 muscle/joint pain, while doses of 0.5µg/kg and 0.75µg/kg of hyaluronidase were generally well tolerated [18]. Additional work in canine models suggesting amelioration of the musculoskeletal events by use of dexamethasone is also being examined in the ongoing phase I trial.

Given the role of the stromal compartment in pancreatic ductal adenocarcinoma and other cancers, it is likely that targeting HA in pancreatic cancer has potential for improving current therapies [1]. With the clinical availability of recombinant hyaluronidase, there are improved prospects for targeting HA for the treatment of cancer, particularly in cancer types known to be fibrotic. In a recent conference report, the authors Thompson and colleagues observed a 50% increase in median survival time in mice bearing pancreatic cancer xenograft tumors treated with gemcitabine plus hyaluronidase [19]. Taken together, these results warrant further clinical investigation of targeting HA in a variety of tumors, including pancreatic ductal adenocarcinoma, in which the stroma is thought to play a key role in limiting drug delivery.

CONCLUDING REMARKS

With such promising preliminary clinical results following the addition of early forms (e.g., bovine) of hyaluronidase to chemotherapeutic treatments, we should again reconsider the power in targeting components of the tumor microenvironment, and especially hyaluronan. Based on current and past clinical studies, future therapeutic regimens for patients with cancer may significantly benefit from agents targeting critical pathways in the development, progression, and perpetuation of the tumor stroma, particularly hyaluronan. Certainly, one must remain cognizant of HA’s function in many other parts of the body. HA’s role in synovial fluid or the vitreous humor could become problematic following long term treatment with hyaluronidase. However, with the development of a recombinant hyaluronidase, some of the significant limitations (i.e., immune reactions) to targeting hyaluronan with bovine hyaluronidase have been addressed. These developments will allow for greater utility in studying hyaluronidase as part of an anticancer therapy regimen by yielding greater flexibility in route of administration as well as treatment schedule in clinical trials. Even using bovine hyaluronidase, targeting hyaluronan as part of a combination regimen has shown promise in the clinic. Utilizing recombinant human hyaluronidase as part of an anticancer regimen is now possible. It also has one key advantage over other ECM targeting alternatives in that it is available now. Pegylated recombinant hyaluronidase is in ongoing phase I trials. Although it is likely the enzyme will cause some musculoskeletal events, and may also present challenges in wound healing, inhibiting a key stromal component such as HA with recombinant human hyaluronidase could improve the clinical outcomes in individuals with the most deadly types of cancer, such as pancreatic ductal adenocarcinoma. In such a disease, any potential improvement in the effective delivery of therapeutics should be cause for serious consideration.