*4.2. Enzymatic Modulation of HS–Protein Interactions*

Heparanase, the only mammalian enzyme responsible for HS/Hep cleavage, is a strict endo-β-glucuronidase, favoring the fission of a GlcA linked to 6O-sulfated GlcN, which can either be N-sulfated or N-acetylated [56]. However, advances have implicated the potential controlling role of the surrounding saccharide sequences and their sulfation pattern in regulating the extent of substrate degradation [56].This plasticity of substrate specificity enhances the execution of various heparanases' roles [215]. The cleavage of HS chains bound into PGs releases latent growth factors, including FGF2, hepatocyte growth factor (HGF), keratinocyte growth factor (FGF4), and TGF-β, which are sequestered to the matrix and cell surface, but also inherently modulates the protein-GAG interactions and downstream signaling [216]. Indeed, trimming of HS can enhance the binding of

growth factors to their respective receptors, as in the case of FGF-2 where the creation of tertiary FGF2-FGFR-HS complex is increased by moderate heparanase activity [217]. Moreover, heparanase was found to reside and accumulate in lysosomes suggesting that it also exhibits intracellular functions [218].

Heparanase strongly affects protein–HS interactions, whereas tumor-associated activated fibroblasts, endothelial cells, and immune cells exhibit increased heparanase activity [219]. The overexpression of heparanase results in vivo in increased tumor metastasis, whereas downregulating heparanase markedly decreases cancer cells' ability to metastasize [220].

Heparanase expression was shown to be upregulated in all cancer types, including sarcomas, carcinomas, and hematological neoplasms [221]. Notably, heparanase activity has been correlated to various human cancers' metastatic potential. Thus, the examination of the Cancer Genome Atlas (TCGA) data on heparanase expression in breast cancer clinical samples showed its upregulation in the majority of specimens. Furthermore, increased heparanase expression was correlated with poor patient survival [222]. Similar results have been obtained for other cancer types, including multiple myeloma [223] and bladder cancer [224]. Moreover, heparanase has been shown to affect cancer angiogenesis [225], invasion, and autophagy [226] and partly through syndecan-1-dependent mechanisms to modulate inflammation-associated tumorigenesis [227].

Heparanase can affect the response to chemotherapy. Thus, anti-myeloma chemotherapeutic agents, including bortezomib (proteasome inhibitor) or melphalan (alkylating agent), were shown to increase the expression and secretion of heparanase in an in vitro myeloma model. The subsequent uptake of soluble heparanase by tumor cells initiated ERK and Akt signaling pathways, stimulated the expression of vascular endothelial growth factor (VEGF), HGF, and MMP-9, and was correlated with an aggressive tumor phenotype [228].

An essential mechanism of heparanase action is promoting exosome secretion, which affects both tumor and host cells' biological behavior and finally drives tumor progression [229]. In a myeloma model, it was shown that chemotherapeutic drugs increase exosome secretion. Notably, chemoexosomes have an increased heparanase load, enhancing cell HS's cleaving activity and initiating ERK signaling and syndecan-1 shedding. These authors suggest that anti-myeloma therapy stimulates the secretion of high heparanase content exosomes, facilitates ECM remodeling, changes tumor and stroma cell behavior, and contributes to chemoresistance [230].

Several therapeutic approaches have been tested to develop efficient inhibitors of heparanase activity. Non-anticoagulant heparin derivatives such as SST0001 or roneparstat significantly downregulated heparanase-dependent cleavage of syndecan-1 HS chains, attenuated HGF, VEGF, and MMP-9 expression resulting in decreased tumor growth and angiogenesisinvivo [231,232]. Preclinical evidence resulted in the first human study (NCT01764880) assessing the safety and tolerability of roneparstat in patients with relapsed or refractory multiple myeloma (MM). Patients treated with Roneparstat exhibited acceptable tolerance at clinically significant doses [233].

PI-88 is an inhibitor of heparanase, in addition to its antagonist of angiogenic growth factors function [234]. Even though it exerted adjuvant properties in hepatocellular carcinoma and melanoma patients [235,236], PI88 has been correlated with bleeding events, and thus, did not progress to clinical practice [237].

A series of PI-88 analogs have been synthesized, exhibiting superior performance. The improved analogs comprise single, characterized oligosaccharides with discrete functionalizations and exhibit more efficient antagonism of angiogenic growth factors and respective receptors binding with HS. These properties are translated into potent inhibition of growth factor-dependent endothelial cell growth and strong downregulation of the endothelial tube formation [234]. PG545 is the outstanding member of the PI88 analogs series exhibiting significant anti-angiogenic, anti-proliferation, and antimetastatic effects through potent heparanase inhibitory and angiogenic growth factor antagonist effects [238]. Moreover, PG545 was shown to exert anti-tumor effects discrete from heparanase inhibition as it induces lymphoma cell apoptosis in a non-heparanase-dependent

manner [239]. PG545 (pixatimod) is currently being tested in clinical trials [238]. However, despite promising breakthroughs, the development of heparanase inhibitors with beneficial clinical performance and acceptable adverse effects is still elusive. Therapeutics targeting HS are summarized in Table 3.


**Table 3.** Therapeutics targeting HS.

However, some studies targeting heparanase demonstrated contradictory results. In some model systems, inactive heparanase facilitated adhesion and migration of endothelial cells and induced factors that promote angiogenesis, such as vascular endothelial growth factor [240]. The enzyme has a C-terminus domain involved in the molecule's signaling capacity. The human heparanase variant (T5) lacking enzymatic activity has protumorigenic properties, indicating the enzyme's complex role in cancer pathogenesis [240].
