Cancer-selective tetra-branched peptides, named NT4, can be coupled to different functional units for cancer cell imaging or therapy. NT4 peptides specifically bind to lipoprotein receptor-related proteins (LRP) receptors and to heparan sulfate chains on membrane proteoglycans and can be efficiently internalized by cancer cells expressing these membrane targets. Since binding and internalization of NT4 peptides is mediated by specific NT4 receptors on cancer cell membranes and this may allow drug resistance produced by drug membrane transporters to be by-passed, we tested the ability of drug-armed NT4 to by-pass drug resistance in cancer cell lines. We found that MTX-conjugated NT4 allows drug resistance to be by-passed in MTX-resistant human breast cancer cells lacking expression of folate reduced carrier. NT4 peptides appear to be extremely promising cancer-selective targeting agents that can be exploited as theranostics in personalized oncological applications.

Glioblastoma (GBM), a highly malignant brain tumor of adults and children, diffusely invades within the non-neoplastic brain. Despite aggressive current therapeutic interventions, improved therapeutic strategies are greatly needed. Interactions between the tumor and constituents of its microenvironment are known to regulate malignancy, and heparan sulfate proteoglycans (HSPGs) are important as they bind diverse extracellular proteins, including growth factors and cell adhesion molecules, regulating the activity of several ligand-mediated signaling pathways. Recent work from our group described a mechanism by which GBM regulates PDGFR-alpha signaling via enzymatic alteration of heparan sulfate proteoglycans (HSPGs) in the extracellular microenvironment. Blocking tumor-induced alterations of HSPGs, which can be achieved by pharmacological strategies, would potentially inhibit multiple oncogenic signaling pathways in tumor cells and disrupt critical tumormicroenvironment interactions. Here we examine HSPGs and the enzymes that modify them in GBM. We compare their expression across tumor subtypes, their potential roles in oncogenesis, and their potential as novel therapeutic targets in GBM.

Autotaxin (ATX) promotes cancer cell metastasis through the production of lysophosphatidic acid (LPA). ATX binds to αvβ3 integrins controlling metastasis of breast cancer cells. We screened a series of cancer cell lines derived from diverse human and mouse solid tumors for the capacity of binding to ATX and found only a modest correlation with their level of αvβ3 integrin expression. These results strongly suggested the existence of another cell surface ATX-interacting factor. Indeed, ATXα has been shown to bind heparan-sulfate chains because of its unique polybasic insertion sequence, although the biological significance is unknown. We demonstrated here, that among all cell surface heparan-sulfate proteoglycans, syndecan-4 (SDC4) was essential for cancer cell interaction with ATXβ but was restrained by heparan-sulfate chains. In addition, exogenous ATXβ-induced MG63 osteosarcoma cell proliferation required physical interaction of ATXβ with the cell surface via an SDC4-dependent mechanism. In a preclininal mouse model, targeting SDC4 on 4T1 mouse breast cancer cells inhibited early bone metastasis formation. Furthermore, SDC4-prometastatic activity was totally abolished in absence of ATX expression. In conclusion our results determined that ATX and SDC4 are engaged in a reciprocal collaboration for cancer cell metastasis providing the rational for the development of novel anti-metastasis therapies.

Heparanase (HPSE), a heparan sulfate-specific endo-β-D-glucuronidase, plays an important role in tumor cell metastasis through the degradation of extracellular matrix heparan sulfate proteoglycans. Suramin, a polysulfonated naphthylurea, is an inhibitor of HPSE with suramin analogues. Our objective was to analyze the HPSE involvement in gastric signet ring cell adenocarcinoma (SRCA) invasion. High expression of HPSE mRNA and protein was found in the tumor and in ascites of SRCA as well as in KATO-III cell line. Beside of collagen-I, growth factors (TGF-β1 and VEGF-A, except FGF-2) and epithelial mesenchymal transition (EMT) markers (Snail, Slug, Vimentin, α-SMA and Fibronectin, except E-cadherin) were found higher in main nodules of SRCA as compared to peritumoral sites. Among MDR proteins, MDR-1 and LRP (lung resistance protein) were highly expressed in tumor cells. The formation of 3D cell spheroids was found to be correlated with their origin (adherent or non-adherent KATO-III). After treatment of KATO-III cells with a HPSE inhibitor (suramin), cell proliferation and EMT-related markers, besides collagen-1 expression, were down regulated. In conclusion, in SRCA, HPSE via an autocrine secretion is involved in acquisition of mesenchymal phenotype and tumor cell malignancy. Therefore, HPSE could be an interesting pharmacological target for the treatment of SRCA.

Glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs) are the first enzymes that mediate the initiation of heparan sulfate sulfation. We previously identified NDST4 as a putative tumor suppressor in human colorectal cancer. In the study, we generated an Ndst4 knockout (Ndst4-/-) mouse strain and explored its phenotypic characteristics, particularly in the development of colonic epithelial homeostasis. The Ndst4-deficient mice were viable and fertile, and their life spans were similar to those of wild-type littermates. No gross behavioral or morphological differences were observed between the Ndst4-/- and wild-type mice, and no significant changes were determined in the hematological or serum biochemical parameters of the Ndst4-/- mice. Ndst4 RNA transcripts were expressed in the brain, lung, gastrointestinal tract, pancreas, and ovary. However, Ndst4-null mice exhibited no gross or histological abnormalities in the studied organs, except for the colon. Although no alterations were observed in the crypt length or number of proliferating cells, the Ndst4-/- mice exhibited an increased number of goblet cells and a decreased number of colonocytes in the proximal colon compared with the wild-type mice. Moreover, Ndst4 deficiency increased the basal level of apoptosis in the colonic epithelium. Taken together, we established, for the first time, an Ndst4-/- mouse strain and revealed the involvement of Ndst4 in the development and homeostasis of colonic epithelium. Accordingly, NDST4 in human colon might direct the biosynthesis of specific heparan sulfate proteoglycans that are essential for the maintenance of colonic epithelial homeostasis. Thus, the loss of its function may result in the tumorigenesis and progression of colorectal cancer.

The human sulfatase 1 (hSulf-1) gene encodes an endosulfatase that functions to inhibit the heparin-binding growth factor signaling, including the basic fibroblast growth factor (bFGF)-mediated pathway, by desulfating the cell surface heparan sulfate proteoglycans (HSPGs). bFGF could stimulate cell cycle progression and inhibit cell apoptosis, this biological effect can be reversed by hSulf-1. However, molecular mechanisms have not been fully reported. In the current study, by reactivation of hSulf-1 expression and function in the hSulf-1-negative hepatocellular carcinoma (HCC) cell lines and HCC xenograft tumors, we found that hSulf-1 blocked the bFGF effect on the promotion of cell cycle and inhibition of apoptosis. The bFGF-stimulated activation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) pathways was suppressed by hSulf-1, which led to a decreased expression of the target genes Cyclin D1 and Survivin, then finally induced cell cycle arrest and apoptosis in HCC cells. Our data suggested that hSulf-1 may be a suitable target for cancer therapy.

Using syngeneic BALB/c mouse breast cancer models, we show that the chromatin remodeling subunit bromodomain PHD finger transcription factor (BPTF) suppresses natural killer (NK) cell antitumor activity in the tumor microenvironment (TME). In culture, BPTF suppresses direct natural cytotoxicity receptor (NCR) mediated NK cell cytolytic activity to mouse and human cancer cell lines, demonstrating conserved functions. Blocking mouse NCR1 in vivo rescues BPTF KD tumor weights, demonstrating its importance for the control of tumor growth. We discovered that BPTF occupies heparanase (Hpse) regulatory elements, activating its expression. Increased heparanase activity results in reduced cell surface abundance of the NCR co-ligands: heparan sulfate proteoglycans (HSPGs). Using gain and loss of function approaches we show that elevated heparanase levels suppress NK cell cytolytic activity to tumor cells in culture. These results suggest that BPTF activates heparanase expression, which in turn reduces cell surface HSPGs and NCR co-ligands, inhibiting NK cell activity. Furthermore, gene expression data from human breast cancer tumors shows that elevated BPTF expression correlates with reduced antitumor immune cell signatures, supporting conserved roles for BPTF in suppressing antitumor immunity. Conditional BPTF depletion in established mouse breast tumors enhances antitumor immunity, suggesting that inhibiting BPTF could provide a novel immunotherapy.

Sulfatase 2 (SULF2), an extracellular sulfatase that alters sulfation on heparan sulfate proteoglycans, is involved in the tumorigenesis and progression of several carcinomas. SULF2 expression has not been evaluated in squamous cell carcinoma of the head and neck (HNSCC). Here we report results of IHC of SULF2 expression in HNSCC tissue. SULF2 was detected in 57% of tumors (n = 40) with a significant increase in intensity and number of stained cells compared to adjacent cancer-free tissue (p-value < 0.01), increasing with cancer stage when comparing stages 1 and 2 to stages 3 and 4 (p-value 0.01). SULF2 was not detected in epithelial cells of cancer-free controls, and expression was independent of patient demographics, tumor location and etiological factors, smoking and HPV infection by p16 IHC analysis. Sandwich ELISA was performed on serum of HNSCC patients (n = 28) and controls (n = 35), and although SULF2 was detectable, no change was observed in HNSCC. Saliva, collected by mouthwash, from HNSCC patients (n = 8) and controls (n = 8) was also tested by ELISA in a preliminary investigation and an increase in SULF2 was observed in HNSCC (p-value 0.041). Overall, this study shows that SULF2 is increased in HNSCC independent of tissue location (oral cavity, oropharynx, larynx and hypopharynx), patient demographics and etiology. Although no change in SULF2 was detected in HNSCC serum, its detection in saliva makes it worthy of further investigation as a potential HNSCC biomarker.

Syndecan family proteins are heparan sulfate proteoglycans, which involved in various cellular activities and associating with metastatic potential and chemosensitivity of tumor cells. Melanoma is one of malignant tumors with poor prognosis upon metastasis. Previously, we had shown that melanoma cells remained survived under cell detachment, which was similar to the initial steps of tumor metastasis. Downregulation of syndecan-1 and upregulation of syndecan-2 in melanoma A375 cells were observed by different suspension conditions. Specific gene alterations also increased melanoma malignancy under anchorage independency. Thus, we would like to investigate in further the role of specific gene alteration, so that it could be used to develop novel strategy to treat melanoma. In this paper, we found that syndecan-2 expression level as well the kinase phosphorylation levels increased upon anchorage independency. The pathway to regulate syndecan-2 expression shifted from PKCα/β-dependent under adhesion into PKCδ-dependent under cell suspension. Manipulation of syndecan-2 expression showed that PI3K and ERK phosphorylation as well the migratory ability increased with increased syndecan-2 expression level. In addition, suspended melanoma cells were more sensitive to chemoagents, which correlated with syndecan-2 overexpression, PI3K and ERK activations, serum level, and the presence of glycosaminoglycans. In conclusion, we showed upregulation of syndecan-2 in anoikis-resistant melanoma cells enhanced chemosensitivity through PI3K and ERK activation. This observation would support and refine the strategy of adjuvant chemotherapy to overcome metastatic melanoma.

Urothelial carcinoma (UC), arising from the urothelium of the urinary tract, can occur in the upper (UTUC) and the urinary bladder (UBUC). A representative molecular aberration for UC characteristics and prognosis remains unclear. Data mining of Gene Expression Omnibus focusing on UBUC, we identified sulfatase-1 (SULF1) upregulation is associated with UC progression. SULF1 controls the sulfation status of heparan sulfate proteoglycans and plays a role in tumor growth and metastasis, while its role is unexplored in UC. To first elucidate the clinical significance of SULF1 transcript expression, real-time quantitative RT-PCR was performed in a pilot study of 24 UTUC and 24 UBUC fresh samples. We identified that increased SULF1 transcript abundance was associated with higher primary tumor (pT) status. By testing SULF1 immunoexpression in independent UTUC and UBUC cohorts consisted of 340 and 295 cases, respectively, high SULF1 expression was significantly associated with advanced pT and nodal status, higher histological grade and presence of vascular invasion in both UTUC and UBUC. In multivariate survival analyses, high SULF1 expression was independently associated with worse DSS (UTUC hazard ratio [HR] = 3.574, P < 0.001; UBUC HR = 2.523, P = 0.011) and MeFS (UTUC HR = 3.233, P < 0.001; UBUC HR = 1.851, P = 0.021). Furthermore, depletion of SULF1 expression by using RNA interference leaded to impaired cell proliferative, migratory, and invasive abilities in vitro. In addition, we further confirmed oncogenic role of SULF1 with gain-of function experiments. In conclusion, our findings implicate the oncogenic role of SULF1 expression in UC, suggesting SULF1 as a prognostic and therapeutic target of UC.