**4. Heparan Sulfate and Heparan Sulfate Proteoglycans Aberrant Expression in Cancer**

*4.1. Heparan Sulfate Changes in Cancer*

Several hallmarks of cancer, such as continuous growth signalling, abrogation of apoptosis, deregulated metabolism, immune evasion and angiogenesis are boosted through pathological alterations of normal physiological processes [83–85]. There is cumulative evidence that changes in cellular glycosylation are concomitant with the acquisition of cellular features involved in tumour growth and progression and ECM remodelling. The glycosylation alterations described in cancer include the expression of truncated *O*-glycan structures, increased expression of branched *N*-glycans, de novo expression of sialylated glycans, altered fucosylation and aberrant PGs expression and modification [86,87].

HS chains are key modulators of cancer cell proliferation events, intervening in altered signalling by interacting with growth factor receptors, promoting their dimerisation and consequent activation, leading to overstimulation of downstream signalling cascades [88]. As an example, in multiple myeloma cells, SDC1 was shown to interact with HGF via HS chains, promoting enhanced activation of Met and consequent activation of the PI3K/protein kinase B and RAS-Raf MAPK pathways, which are related to cell proliferation and survival [89]. In addition, the activation of the Wnt/β-catenin cascade in multiple myeloma is also promoted by HS chains of SDC1, leading to cancer cells proliferation [90].

Besides the altered expression of HSPGs, the abnormal activity of HS biosynthetic and post-synthetic enzymatic machinery, which determines HS chains' length, epimerisation, acetylation and sulfation patterns, is also known as a major event behind HS deregulation in cancer [91]. Comparative studies demonstrated that the expression of genes coding for HS biosynthetic machinery is deregulated in several types of cancer, weighing on its role in carcinogenic events [92]. In colorectal cancer, it was shown the aberrant expression of enzymes that catalyse uronic acid structural changes (epimerisation and 2-*O* sulfation), and enzymes that impact glucosamine residues sulfation pattern (NDST1 and 2, 6OST isoforms 3B, 5 and 6), depending on the anatomical location and the metastatic nature of the tumours [93,94]. HS modifying enzymes were also shown to present deregulated expression on breast cancer tissue samples [46]. As for glycoenzymes that intervene in HS polymerisation, the analysis performed using estrogen receptor positive and triple negative breast cancer cell lines revealed altered expression of EXT1, EXT2, EXTL2 and EXTL3 [95].

HS 6-*O*-sulfation levels, determined by the expression of 6OSTs, have been reported as critical for the activation of epidermal growth factor receptor (EGFR) by heparin biding-EGF (HBEGF), and the consequent increase in the expression of angiogenic cytokines on ovarian tumour cells [96]. In lung cancer, *3OST2* hypermethylation and consequent deregulation, was associated with lung tumourigenesis and poor overall patient survival, possibly resulting from the altered HSPGs ability to interact with proteins participating in cell growth and adhesion [97]. Likewise, the hypermethylation in HS 6-*O*-endosulfatase *Sulf-1* promoter region downregulates its expression in gastric cancer cell lines and tissue samples [98], and the decreased levels of HS 6-O-endosulfatase associate with gastric cancer progression [99]. Conversely, the sulfatase Sulf-2 is described to be overexpressed in hepatocellular carcinomas and associated with worse prognosis [100].

Heparanase (HPSE), a β-D-endoglucuronidase, is the only mammalian enzyme known to cleave HS and is one of the most studied glycosylation-related enzymes in cancer [101,102]. This enzyme is known to be a tumour inducer acting in several signalling pathways modulating angiogenesis, cell proliferation, migration and metastasis [103–105]. HER2- and EGFR-positive breast cancer cells resistant to lapatinib, a tyrosine kinase inhibitor that blocks the activation of the EGFR and HER2 pathways, revealed increased activity of HPSE. This feature was associated with enhanced activation of EGFR, FAK and ERK1/2 signalling pathways and subsequent cell growth. HPSE inhibition, was shown to sensitise these cells to lapatinib and inhibit formation of brain metastases [106]. More recently, vascular endothelial growth factor receptor 3 (VEGFR3) expressing macrophages and cathepsin release, both playing a significant role in metastasis formation in chemotherapy-treated tumours, were found to promote lymph angiogenesis as a result of VEGF-C upregulation by HPSE [107]. Autophagy is another cellular attribute modulated by HPSE. This catabolic pathway maintains homeostasis in normal cells, while it is completely hijacked in several tumours, promoting cancer cell survival. Autophagy induced by lysosomal HPSE has been shown to enhance tumour development and chemoresistance [108–110]. Although HPSE activity has been mainly described extracellularly or within the cytoplasm, nuclear HPSE has also been reported [111–113]. In melanoma, nuclear HPSE was shown to suppress tumour progression by competing for DNA binding and inhibiting the transcription of genes, such as those coding for ECMdegrading enzymes that promote metastasis formation [112]. In multiple myeloma disease context, HSPE was recently associated with chromatin opening and transcriptional activity concomitant with downregulation of PTEN tumour suppressor activity [111]. Also supporting the role of HPSE in tumour progression and metastasis formation, HPSE has been shown to promote EV secretion by tumour cells, affecting its protein cargo [114,115], and modulating HS structure on recipient cells to facilitate EVs internalisation [9,115,116].
