*4.2. Role of Heparan Sulfate and Heparan Sulfate Proteoglycans in Cancer Cellular Features and Extracellular Matrix Remodelling*

Cancer cells undergo relevant morphological changes, such as the epithelial to mesenchymal transition (EMT), to increase motility capacity. HS chains play a key role in this transition, due to their binding affinity to key growth factors secreted into the tumour microenvironment [117,118]. Particularly, upregulation of SDC4, in lung adenocarcinoma, was shown to stimulate cell's migration and invasion via TGF-β1, accompanied by induction of EMT [119]. Cell proliferation is also a crucial characteristic of malignant transformation. The HSPGs GPC1 and SDC1, overexpressed in a high percentage of breast cancer pathologies, enhance the mitogen effects associated with heparin-binding growth factors like Basic Fibroblast Growth Factor (FGF2), HBEGF and Hepatocyte growth factor (HGF), promoting cell proliferation [120]. Some studies have shown that HS and HSPGs can translocate to the nucleus and contribute to gene expression regulation [121,122]. Although the role of nuclear HSPGs is still not fully uncovered, another important role of nuclear HSPGs is the translocation of specific cargo to the nucleus. Nuclear translocation of SDC1 was shown to regulate tumour signalling by shuttling growth factors to the nucleus and by altering histone acetylation [123].

Tumour progression is accompanied by the development of new blood vessels [124]. HS chains, by binding to angiogenic growth factors, namely, FGFs, platelet-derived growth factors and VEGFs, dictate HSPGs relevant roles in angiogenesis [91,125,126]. It has been shown that HS presence on endothelial-cells' surface can serve as a binding site for the potent antiangiogenic factor endostatin. Several studies have indicated that the HS binding site for endostatin is distinct from that of pro-angiogenic factors, such as FGF. This raises the possibility that endothelial cells modulate their HS cell-surface profile to become either more or less sensitive to angiogenic signals from a growing tumour [127]. As referred earlier, SDC1 overexpression in multiple myeloma was also shown to promote angiogenesis by its ability to physically interact with VEGFR2 and prevent the receptor recycling [128]. Perlecan is also an important player in angiogenesis, since its expression is abnormally high in the basement membranes of highly metastatic human melanoma tumour cells [129], promoting the binding of pro-angiogenic FGF2 to its receptors, and consequently increasing angiogenesis [130]. In addition, SDC3 expression is positively associated with angiogenesis in neuronal and brain tissues [131,132].

Beyond the structural modifications of HS and pattern of sulfation, mentioned in Section 4.1, alterations in the HS levels can compromise the stiffness of the ECM, thus modulating cell adhesion and migration. A steady ECM does not offer the best conditions for cell migration, preventing or delaying cell motility. In this light, the ability of cancer cells to invade the surrounding tissues is modulated by changes in the expression of HS and HSPGs, which mediate several events of cell-matrix interaction, and the secretion of HPSE and metalloproteinases that allow cells to penetrate the basement membrane and ECM to invade surrounding tissues [118,133,134]. In hepatocellular carcinoma, SDC1 and SDC4 are key for migration, invasion and increased motility mediated by chemokine-SDC interactions [135]. SDC1 abnormal expression, for example, is determinant in tumour cell growth, invasion and migration in different types of cancer, such as colorectal, gallbladder and oesophageal carcinomas [136–139]. SDC2 overexpression in breast [133,140], colon [141] and pancreatic [142] tumour cells, is associated with altered cell morphology, focal adhesion formation, spreading, enhanced migration and invasion capabilities, and overall to a more aggressive tumour cell behaviour and disease progression [143]. GPCs are also frequently reported to play a part in cancer progression. GPC1, for instance, when upregulated, increases tumour angiogenesis and metastasis in pancreatic cancer [144,145]. In addition, GPC1 modulates heparin-binding growth factors and plays a role in tumour progression in breast cancer [46,120]. In esophageal squamous cell carcinoma and glioblastomas, GPC1 is also upregulated and associated with tumour angiogenesis and patients' poor prognosis [146–148]. GPC3 is overexpressed in hepatocellular carcinomas tissues [149], and associates with higher invasion and migration [150]. Similar to GPC1, an increased expression of GPC6 has been reported in breast cancer. GPC6 overexpression stimulates cancer invasion through NFAT (nuclear factor of activated T-cells) signalling pathway– previously reported as an inducer of pro-invasion and migration gene expression [151].

Cancer cells ability to penetrate blood vessels is preponderant for metastatic spread and is followed by circulation through the intravascular stream and establishment in other sites [152]. During the process of metastasis formation, the reorganisation of HSPGs in the ECM, creates an opportunity for new partners to bind to tumour stroma. This process also involves interactions between cancer cells and platelets, endothelial cells and host organ cells, being HS implicated in the formation of tumour metastasis in sites, such as the liver, lungs or spleen [91,153]. Moreover, SDC1 expression was shown to decrease in hepatocellular [154] and colorectal [155] carcinomas, resulting in more invasive phenotypes, with higher metastatic potential.

HS can also contribute for the immune system deceiving to either disregard or promote the tumour growth [156]. In breast cancer for example, SDC1 has been suggested to act both as a regulator of cancer stem cell (CSC) phenotype and as a modulator of lymphocytes, in particular of T helper cells, depending on the subtype of the disease [157].

A schematic representation of HSPG functional implications in cancer progression and their clinical potential is illustrated in Figure 2B,C.
