Hyaluronan Metabolism in Urologic Cancers

Hyaluronan (HA) is one of the major components of the extracellular matrix in tumor tissue. Recent reports have made it clear that the balance of HA synthesis and degradation is critical for tumor progression. HA is synthesized on the cytoplasmic surface of the plasma membrane by hyaluronan synthases (HAS) and extruded into the extracellular space. Excessive HA production in cancer is associated with enhanced HA degradation in the tumor microenvironment, leading to the accumulation of HA fragments with small molecular weight. These perturbations in both HA synthesis and degradation may play important roles in tumor progression. Recently, it has become increasingly clear that small HA fragments can induce a variety of biological events, such as angiogenesis, cancer‐promoting inflammation, and tumor‐associated immune suppression. Progression of urologic malignancies, particularly of prostate and bladder cancers, as well as of certain types of kidney cancer show markedly perturbed metabolism of tumor‐associated HA. This review highlights the recent research findings regarding HA metabolism in tumor microenvironments with a special focus on urologic cancers. It also will discuss the potential implications of these findings for the development of novel therapeutic interventions for the treatment of prostate, bladder, and kidney cancers.


DOI: 10.1002/adbi.202300168
Hyaluronan (HA) is a major component of both the extracellular and pericellular matrix, which supports normal tissue homeostasis.HA is present in almost every tissue of all vertebrates but is most abundant in the extracellular matrix of soft connective tissues.HA is a very large molecule, with a typical molecular weight between ˜2 × 10 5 to 10 7 Da and an extended length of 2-25 μm. [1,2]The HA interacts in paracrine and autocrine manners with its receptors on neighboring cells.Such interactions have been shown essential for the structure and assembly of multiple tissues. [1,2]10][11][12][13][14] The biosynthesis of HA is regulated by the three transmembrane glucosyltransferase enzymes: hyaluronan synthase 1, 2, and 3 (HAS1-HAS3).The major HA source in the tumor microenvironment is epithelial tumor cells as well as cancerassociated fibroblasts (CAFs). [15,16]In addition to increased HA synthesis, tumor progression is associated with enhanced HA degradation. [17,18]25] Hyal2 is a rate-limiting membrane-bound enzyme that is involved in the degradation of extracellular HA to the fragments with MW 20 kDa, whereas Hyal1 is an intracellular lysosomal enzyme that breaks the engulfed HA fragments into smaller proinflammatory fragments.Accumulating evidence suggests that enhanced HA degradation is associated with various types of diseases including cancer, arthritis, lung diseases, diabetes, etc.However, our current mechanistic understanding of these processes is limited.
[27] Based on our experience in cancer biology, we accept that HA fragments with molecular weight <20 kDa belong to low molecular weight (LMW-HA).Whereas, HA with molecular weight 20-200 kDa can be considered as intermediate size, and >200kDA as a high molecular weight (HMW-HA).We have to acknowledge that this HA classification in the future could be more specified, particularly for HA with intermediate and high molecular weight.
The elevated hyaluronidase expression and activity in the tumor microenvironment results in the accumulation of small HA fragments with LMW-HA.The LMW-HA and HMW-HA have opposite biologic functions.HMW-HA is anti-oncogenic and shows anti-inflammatory and wound-healing activities. [28,29]In contrast, the LMW-HA is pro-oncogenic.It was recently shown that small HA fragments produced in a hyaluronidase-dependent manner, inhibit Hippo signaling by competing with HMW-HA for CD44 binding and promoting the proliferation of tumor cells. [29][32][33][34][35][36][37][38][39][40] Lokeshwar et al. [15] proposed that cross-talk between cancer cells and tumor stroma stimulates HA production and fragmentation through HAS/Hyal1, leading to the accumulation of small angiogenic tumor-promoting HA fragments.It appears that in addition to stromal fibroblasts and tumor cells, the tumorinfiltrating myeloid cells are also involved in that cross-talk.The tumor-associated myeloid cells, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), represent a major component of solid tumors that are involved in the regulation of cancer immunity, inflammation, and angiogenesis. [41,42]Furthermore, tumors mobilize those immunosuppressive and pro-inflammatory myeloid cells from bone marrow through a chemokine network to protect the tumor from attack by immune cells. [43,44]Upon recruitment in tumor tissue, those cells quickly integrate into the HA-rich stroma and up-regulate the immunosuppressive ligand PD-L1, thus protecting tumors from immune cells such as cytolytic T lymphocytes (CTLs). [45]We recently demonstrated that tumorassociated myeloid cells express high levels of enzymes Hyal2 and Hyal1. [16]This observation has been confirmed by another research group. [46]The increased presence in tumor tissue of Hyal1/Hyal2-expressing myeloid cells, which can degrade extracellular HA, supports the idea that extensive HA fragmentation in the tumor microenvironment could be a product of close cooperation between HA-producing tumor cells, CAFS, and tumorrecruited Hyal1 + /Hyal2 + myeloid cells.

Therapeutic Applications for Targeting Hyaluronan in Cancer
Considering the supporting role of HA in cancer, targeting HAassociated metabolic, and signaling pathways could be potentially used to treat cancer.Thus, multiple studies demonstrated the anti-tumor therapeutic activity of 4-methyl umbelliferone (4-MU) through inhibition synthesis, which is its main mechanism of action.More specifically, this agent reduces the availability of HA substrates by diminishing the cellular content of the HA precursor UDP-glucuronic acid, and inhibits the activity of several HAS enzymes. [47,48]4-MU is a natural compound of the coumarins group and abundantly exists in many edible plants, such as broccoli and celery. [49]Several studies demonstrated that 4-MU inhibits the proliferation, migration, and invasion of multiple cancer types through the inhibition of HA synthesis.4-MU also exerts its anti-tumor effects in vivo in various animal tumor models, including melanoma, [50,51] ovarian cancer, [52] breast cancer, [13,53,54] esophageal cancer, [55] lung cancer, [56] and hepatocellular carcinoma. [57]However, there is uncertainty regarding how 4-MU treatment provides benefits in these animal models and the potential long-term consequences of HA inhibition. [58]he ability of an enriched layer of pericellular HA to physically cover the molecular targets on cancer cells such as receptors, and tumor antigen epitopes, suggests that excessive production of HA in the tumor microenvironment could protect tumors from immune attack.HA has also been identified as an obstacle to cancer chemotherapies hampering drugs' extravasation and intra-tumoral spreading. [59,60]Therefore, breaching the HA barrier from the tumor could improve the delivery of anticancer drugs, monoclonal antibodies (mAbs), or therapeutic efficacy of adoptively transferred T cells.Indeed, the enzymatic depletion of tumor-associated HA augmented anti-tumor immune responses in several preclinical animal models.For the depletion of HA, Thompson and co-authors [61] utilized a recombinant human HA-degrading enzyme PEGPH20 (PH20), which efficiently removed the pericellular HA coating produced by tumor cells.Furthermore, PH20 enhanced both docetaxel, and liposomal doxorubicin anti-tumor activity.
Systemic administration of an enzymatic agent PH20 in combination with gemcitabine in an autochthonous murine model with pancreatic ductal adenocarcinomas permanently remodeled the tumor microenvironment and achieved objective tumor responses resulting in a near doubling of overall survival. [62]ore recent studies demonstrate that targeting extracellular HA with PH20 sensitizes pancreatic adenocarcinoma for anti-PD-1 therapy. [63]Thus, PH20-mediated HA removal in combination with anti-PD-1 antibody and FAK inhibitor treatments reduced granulocytes, decreased metastatic spreading, and reduced numbers of MDSCs.It also has been shown that PH20 could potentiate the antitumor activity of mesothelin-specific CAR-T cells against gastric cancer. [64]Taken together, it appears that observed therapeutic effects of PH20 are mediated not by biochemical or metabolic changes but rather by physical "loosening" of the tumor ECM to enable better penetration of drugs or migration of immune cells.However, the complexity of the therapeutic effects of PH20 and potential side effects associated with HA degradation and possible accumulation of pro-oncogenic small HA fragments remains to be investigated.
Major HA receptor CD44 is also involved in cancer progression, metastasis development, and resistance to therapy.CD44 is an integral HA receptor that can promote or inhibit mitogenic signaling in tumor cells. [65]The proximal extracellular domain of CD44 is the site of alternative splicing for CD44 mRNA that produces different variant isoforms of CD44.While the standard isoform of CD44 (CD44s) is expressed predominantly in normal epithelial cells, CD44 variant isoforms (CD44v), which contain additional insertions, are highly expressed in many epithelialtype carcinomas. [66][69] Collectively, presented data suggest that targeting tumor-associated HA could result in the development of novel approaches for the treatment of solid cancers as well as improving the efficacy of existing cancer therapeutic modalities.

Prostate Cancer
After skin cancer, prostate cancer is the most widely diagnosed cancer in men, with nearly one in eight males diagnosed during their lifetime. [70]Prostate cancer represents a heterogeneous disease state, with some men able to survive cancer and others requiring a combination of systemic and local therapy (NCCN Guidelines).Typically, prostate cancer is relatively slow growing and mostly localized.However, cancerous cells may spread to other areas of the body, particularly the bones and lymph nodes.The prostate tumor microenvironment is likely a significant driver in developing aggressive disease phenotypes. [71]e provide evidence that HA metabolism influences the tumor microenvironment leading to prostate cancer progression and metastasis.

HA Metabolism in Prostate Cancer Progression
Elevated levels of HA in prostate tissue obtained from patients with prostate cancer have been reported by De Klerk. [72,73]He found that tissue-associated HA was correlated with the dedifferentiation of epithelial prostate cells.The increased production of glycosaminoglycans extracted from prostate tissues of patients with prostatic hyperplasia and prostate cancer was confirmed later by Iida et al. [74] utilizing high-performance liquid chromatography.Indeed, data presented in Figure 1 demonstrate that the expansion of human primary prostate cancer cells in vitro is associated with increased HA production.
Lokeshwar and co-authors reported that the majority of HA in prostate tissues (75-80%) was found to exist in the free form. [75]ancer-associated fibroblasts and cancerous epithelial cells secreted 3-8-fold more HA than respective normal adult prostate and benign prostate hyperplasia cultures.The association of high stromal HA levels with poor differentiation and metastasis in prostate cancer was documented by Lipponen et al. [76] Analysis of human prostate cancer cell lines revealed the up-regulation of HA synthase isoforms HAS2 and HAS3 relative to levels expressed by normal prostate, which corresponded to elevated HA synthesis and increased cell adhesion. [77]Simpson et al. [78] reported that aggressive PC3M-LN4 prostate tumor cells synthesize excessive HA relative to less aggressive cells, and they express correspondingly higher levels of the HA-synthesizing enzymes HAS2 and HAS3.
Importantly, prostate cancer progression is associated with elevated expression and activity of the HA-degrading enzyme Hyal1.Lokeshwar et al. demonstrated a significant elevation (3-10-fold) of this enzyme in prostate cancer tissues compared to that in normal adult prostate and benign prostate hyperplasia tissues. [79]Furthermore, the hyaluronidase levels in tissues correlate well with the tumor grade.Analysis of human prostate cancer cell lines showed that DU145 cells secrete four-fold higher hyaluronidase levels than those secreted by LNCaP cells.Interestingly, the primary epithelial explant culture set up from a metastatic prostate cancer lesion secreted more Hyal1 than LNCaP, but lower than DU145. [78]In a separate study, Kovar and co-authors reported that forced overexpression of Hyal1 in non-metastatic human prostate cancer cell line 22Rv1 cells promoted the development of metastasis in lymph nodes once tumor cells were orthotopically injected in mice. [80]

Clinical and Therapeutic Implications
Since the tissues from patients with high-grade prostate cancer showed both elevated HA and Hyal1 levels and correlated with recurrence of elevated PSA after tumor resection, it has been proposed that both the stromal-epithelial HA and Hyal1 may serve as prognostic markers for prostate cancer. [15,81,82]However, later investigations with a minimum five-year follow-up found that high HA levels in radical prostatectomy specimens are not an independent predictor of PSA biochemical recurrence that serves as a predictor of local or distant metastasis.Nonetheless, HA appears to increase the accuracy of Hyal1 in predicting biochemical recurrence.
Perturbations of tumor-associated HA metabolism in prostate cancer suggest that targeting HA could have therapeutic implications.Lokeshwar and co-authors studied the effects of 4-MU, an inhibitor of HA production, on human prostate cancer cell lines. [83]In vitro, 4-MU inhibited proliferation, motility, and invasion of various cell lines including DU145, PC3-ML, LNCaP, C4-2B, and LAPC-4 cells.Oral administration of 4-MU resulted in the inhibition of PC3-ML tumor growth (>3-fold) without toxicity.Tumors from 4-MU-treated animals showed reduced microvessel density, HA production, and increased expression of apoptosis-related molecules.Yates et al. evaluated the efficiency of 4-MU in preventive and therapeutic action using three different experimental animal models of prostate cancer. [84]In the DU145 subcutaneous xenograft model, daily gavage of 4-MU inhibited tumor growth by 85% to 90%.Furthermore, 70% of animals did not form tumors at the endpoint (46 days), although the treatment was terminated on day 28.In the PC3-ML/Luc intra-cardiac bone metastasis model, no animals in any treatment group developed skeletal metastasis.In transgenic adenocarcinoma of the prostate (TRAMP) mice, administration of 4-MU prevented the development of prostate tumors in most of the treated mice.Taken together, these studies demonstrate that dietary supplement 4-MU shows strong preventive and therapeutic efficacy against localized and metastatic prostate tumors in preclinical models.
HA, and its fragments interact with specific HA receptors such as CD44 and RHAMM.RHAMM, a multifunctional protein that regulates cancer progression, [85,86] is frequently up-regulated in prostate cancer and associated with poor prognosis. [87,88]HAMM-mediated signaling activates and regulates multiple cellular functions including cell motility, cytokine production, microtubule dynamics, etc.It appears that RHAMM contributes to motility and invasion of prostate cancer cells by interacting with HA. [89] A recent study demonstrates that genetic modulation or pharmacologic inhibition of RHAMM activity was sufficient and necessary for metastatic phenotypes in prostate cancer induced by retinoblastoma tumor suppressor protein (RB) loss. [90]It was shown that RHAMM stabilizes the F-actin polymerization in epithelial cells by controlling ROCK signaling.This study highlights RHAMM as a potential candidate therapeutic target for treating advanced prostate cancer.Taken together, it ap-pears that targeting HA metabolism in prostate cancer could provide an effective approach to the treatment of prostate cancer.

Bladder Cancer
Bladder cancer is most common in men, with a male-to-female ratio of 4:1.Treatment and prognosis are heavily influenced by the depth of invasion in patients with clinically localized disease.This leads to patients being classified as having non-muscle invasive versus muscle-invasive disease.Modest advances in treatment have been made across all stages of the disease.Despite these advances, frequent surveillance is still required due to high recurrence rates with current treatment options.This is especially critical for patients with locally advanced and metastatic bladder cancer, with the average life expectancy in patients with metastatic disease being less than two years. [91]

Hyaluronan Metabolism in Bladder Cancer Progression
94][95][96] Thus, analysis of bladder tissue-associated HA using polyacrylamide gel electrophoresis revealed (Figure 2) that bladder cancer tissues are enriched with small HA fragments (< 20 kDa), whereas normal, non-malignant bladder tissues predominantly produce HA with high molecular weight (>150 kDa).
Expression of HA synthases and Hyal1 are elevated in bladder cancer tissue at both transcriptional and protein levels.It appears that in addition to Hyal1, Hyal2 also contributes to enhanced degradation of tumor-associated HA in bladder cancer. [97]yal2 expression was detected in tumor-infiltrating CD11b + myeloid cells, which are abundant in bladder cancer tissue.Hyal2 + CD11b + myeloid cells were detected in tumor tissue as well as in the peripheral blood of cancer patients.Preclinical studies indicate that Hyal2 + MDSCs directly contribute to the development of immunosuppressive PD-L1-expressing macrophages in tumor tissue and tumor-draining lymph nodes. [16]Accumulation of immunosuppressive PD-L1 + cells in tumors, particularly in the HA-enriched tumor stroma, confers immune suppression and resistance to cancer immunotherapy.

Clinical and Therapeutic Implications
Several members of HA-metabolic/catabolic and signaling pathways are associated with the prognosis of bladder cancer.Hyal1 has been proposed as a potential prognostic indicator for progression to muscle invasion and recurrence. [98]A combination of Hyal1 and HAS1 expression predicted bladder cancer metastasis and expression of Hyal1 alone predicted disease-specific survival. [99]Aboughalia et al. [100] have shown that Hyal1 expression in tumor cells exfoliated in urine correlates with tumor invasion into the bladder muscle and beyond.In turn, Hyal2 was found to be associated with the progression of bladder cancer. [101]urthermore, multivariate analysis revealed that RHAMM expression in bladder cancer tissue was associated with poor prognosis independent from other survival factors. [102]igure 2. Enhanced HA degradation in human bladder cancer tissue.Precision-cut tissue slices were prepared from freshly obtained normal and tumor human bladder tissue pieces and cultured in 24-well plates in a complete culture medium.Cell-free supernatants were collected on days 5-7, and stored at 80 °C until analysis of tumor-produced HA using polyacrylamide gel electrophoresis.A) Normal bladder tissue: 1000, 700, 500, and 200k -control commercial HA samples with high molecular weight; N1, N2, N3 -normal bladder tissue samples from three patients.B) 500k, 200k-control HA samples with high molecular weight; 20 and 5k -low molecular weight; T1, T2, T3 -human cancer bladder tissue samples from three patients.Adapted with permission. [97]2021, American Association for Cancer Research.
Targeting HA production with HAS inhibitor 4-MU has shown potential benefits in preclinical studies.4-MU significantly inhibited in vivo growth of HT1376 bladder tumor cells in a xenograft model at doses 200 and 400 mg kg −1 [103] without impacting animal weight.In addition, 4-MU has been reported to complement the cytotoxic effects of chemotherapeutic drugs cisplatin or doxorubicin by enhancing the sensitivity of bladder cancer cells.

Kidney Cancer
Kidney cancer, also known as renal cancer, is a group of cancers that is one of the ten most commonly diagnosed cancers.There are several types of kidney cancers, such as renal cell carcinomas (RCC), transitional cell carcinomas (TCC), Wilms tumors, and renal sarcomas.In turn, there are three main histological subtypes of RCC including clear cell RCC (70%), papillary RCC (10-15%), and chromophobe RCC.RCC is the most lethal urological malignancy; it contributes to 175,000 deaths per year globally.It is often incidentally diagnosed because it develops and progresses asymptomatically.For localized cancers that are confined to the kidney, the five-year survival rate is 93%; if it has spread to the surrounding lymph nodes, it is 70%.When it has metastasized widely, the rate of five-year survival is only 12%.Metastases are present in ˜30% of RCC cases at initial diagnosis, which leads to poor clinical outcomes. [104]Existing targeted immunotherapies and other therapeutic strategies against metastatic RCCs have limited efficacy, which has prompted interest in the development of alternative strategies.

Hyaluronan Metabolism in Kidney Cancer Progression
In contrast to prostate and bladder cancers, there are considerably fewer publications focused on HA metabolism in kidney cancer.
Using immunohistochemistry, Jokelainen et al. [105] reported that in RCC tissue, HA showed mainly a focal distribution and was associated mostly with stroma.Lower stromal HA staining was associated with high tumor grades, large tumor sizes, and reduced metastasis-free survival.According to a study by Chi et al., [106] the median HAS1, CD44s, and RHAMM transcript levels were 3-25fold elevated in clear cell RCC (ccRCC), papillary, and chromophobe tumors when compared to normal tissues.Expression of Hyal4, CD44s, and RHAMM l were 4-12-fold elevated in ccRCC and papillary tumors when compared to oncocytomas.In addition, increased numbers of Hyal2+ myeloid cells, capable of HA degradation, have been observed in RCC patients. [107]It appears that the recruitment of those cells to the tumor tissue promotes the degradation of tumor-associated HA.

Clinical and Therapeutic Implications
Wilms' tumor is a renal neoplasm that is histologically similar to fetal kidney tissue.Both Wilms' tumor and the fetal kidney have high levels of HA in the extracellular matrix. [108]To test the utility of urinary HA as a Wilms' tumor marker, the authors compared HA levels in urine specimens from 105 Wilms' tumor patients with those of age-matched controls.The authors conclude that measurement of urinary HA levels is effective in the initial diagnosis of Wilms' tumor, as well as the detection of its relapse after surgery.In a recent study, Wang and co-authors reported increased expression of HAS3 in metastatic RCC. [109]In the clinical and TCGA-KIRC/TCGA-KIRP cohorts, high expression HAS3 predicted metastasis and shorter survival of cancer patients.The combination of 4-MU with the chemotherapeutic drug sorafenib inhibited in vitro growth of RCC by inducing apoptosis.This combination also suppressed the in vivo tumor growth and development of metastasis in mice with implanted RCC.

Conclusion
Excessive HA production in cancer is associated with enhanced HA degradation in the tumor microenvironment, leading to the accumulation of HA fragments with low molecular weight.These perturbations in HA synthesis and degradation play important roles in tumor progression.Thus, it has become increasingly clear that small HA fragments can induce a variety of biological events, such as angiogenesis, cancer-promoting inflammation, and tumor-associated immune suppression.The enhanced degradation of HA in tumor tissue occurs with the help of tumor-recruited myeloid cells.Specifically, the close interaction and extensive cross-talk between tumor cells, CAFs, and tumorrecruited myeloid cells leads to enhanced HA degradation.Thus, disruption of these interactions between HA-producing cells and HA-degrading cells could be beneficial for the tumor host and may potentially lead to the reversal of tumor growth.Multiple successful preclinical studies with 4-MU [13,[50][51][52][53][54][55][56][57][58] indicate that the metabolism of tumor-associated HA could be a central mechanism that drives tumor progression.Therefore, further advancements in understanding the complex mechanisms involved in abnormal HA metabolism in solid cancers are required.Wayne Brisbane, an assistant professor of urology, received his medical degree from Loma Linda University School of Medicine.He completed a General Surgery internship and Urology residency at the University of Washington, followed by a Urologic Oncology fellowship at UCLA focusing on prostate cancer.His research and clinical care focus on image-guided biopsy, surgery, and focal therapy.UCLA uniquely offers both clinical and research programs in Micro-Ultrasound, Magnetic Resonance Imaging (MRI), and PSMA PET CT/MRI.Brisbane provides diverse options for prostate cancer treatment, assisting patients in identifying the most suitable approach for achieving cancer remission while minimizing adverse effects.

Figure 1 .
Figure1.HA production in human prostate tissue culture slices.Precision-cut tissue slices were prepared using fresh prostate tissue obtained from a patient diagnosed with prostate cancer (Gleason score 6).On day nine, tissue slice cultures were fixed with 4% formaldehyde solution and stained for HA (red).For visualization of cell nuclei, samples we stained with DAPI (blue).Representative images are shown.
Donelan received his Ph.D. in molecular cell biology from the College of Medicine Interdisciplinary Program at the University of Florida, followed by a postdoctoral fellowship in biochemistry and molecular biology at the UF College of Medicine.Currently, a Research Assistant Scientist in the Department of Urology at the University of Florida, his work focuses on urologic cancers, specifically examining the molecular events and immune responses involved in hyaluronic acid metabolism in the tumor microenvironment.Donelan is actively pursuing the development of antibody-drug conjugates and chimeric antigen receptor T cells as therapies for urologic cancers.