This genetic association study provides new data on a prior case report of persistent neonatal pemphigus, reclassifying it as genetic rather than autoimmune in origin.

ABSTRACT Background Bullous pemphigoid (BP) is the most common autoimmune blistering skin disease, primarily affecting the elderly. The role of interleukin‐13 (IL‐13) in BP pathogenesis remains unclear, particularly regarding its potential as a therapeutic target. Objectives To evaluate differences in serum IL‐13 levels between idiopathic and drug‐induced BP patients and healthy controls, and to investigate correlations between IL‐13 levels, disease severity, mucosal involvement, and prognosis. Methods This retrospective cohort study included 42 BP patients diagnosed between 2008 and 2023 at a dermatology referral center, along with 12 healthy controls and four pemphigus vulgaris patients. Serum IL‐13 levels were measured using ELISA. The primary outcomes were IL‐13 levels and their association with disease etiology, eosinophil counts, mucosal involvement, and the need for adjuvant therapy. Results Serum IL‐13 levels were lower in BP patients ( n = 42, mean 62.46 pg/mL ± 16.53) compared to healthy controls ( n = 12, mean 87.83 pg/mL ±8.87, p < 0.0001) and pemphigus patients ( n = 4, mean 87.6 pg/mL ± 5.16, p = 0.013). Idiopathic BP patients exhibited higher IL‐13 levels than Dipeptidyl peptidase‐4 (DPP4) ‐induced BP patients (67.01 pg/mL ± 14.3 vs. 57.36 pg/mL ± 21, p = 0.0104). No significant correlation was found between IL‐13 levels and mucosal involvement ( p = 0.338), eosinophil counts (Pearson correlation coefficient = 0.18, p = 0.253), the need for adjuvant therapy ( p = 0.32), or prognosis ( p = 0.45). Higher eosinophil levels correlated with the need for adjuvant therapy ( p = 0.027). Conclusions Serum IL‐13 levels are lower in BP patients compared to healthy controls, and in DPP4‐induced BP compared to idiopathic BP, suggesting a complex role of IL‐13 in BP pathogenesis. IL‐13 levels did not correlate with disease severity or prognosis, indicating that IL‐13 may not be a reliable marker for BP severity or outcome. Further research is needed to clarify IL‐13's role in BP and to guide therapeutic strategies.

miR-184-knockout mice display perturbed epidermal stem cell differentiation. However, the potential role of miR-184 in skin pathology is unclear. Here, we report that miR-184 controls epidermal stem cell dynamics and that miR-184 ablation enhances skin carcinogenesis in mice. In agreement, repression of miR-184 in human squamous cell carcinoma (SCC) enhances neoplastic hallmarks of human SCC cells in vitro and tumor development in vivo . Characterization of miR-184-regulatory network, suggests that miR-184 inhibits pro-oncogenic pathways, cell proliferation, and epithelial to mesenchymal transformation. Of note, depletion of miR-184 enhances the levels of β-catenin under homeostasis and following experimental skin carcinogenesis. Finally, the repression of β-catenin by miR-184, inhibits the neoplastic phenotype of SCC cells. Taken together, miR-184 behaves as an epidermal tumor suppressor, and may provide a potentially useful target for skin SCC therapy.

Unlike the intense research effort devoted to exploring the significance of heparanase in cancer, very little attention was given to Hpa2, a close homolog of heparanase. Here, we explored the role of Hpa2 in breast cancer. Unexpectedly, we found that patients endowed with high levels of Hpa2 exhibited a higher incidence of tumor metastasis and survived less than patients with low levels of Hpa2. Immunohistochemical examination revealed that in normal breast tissue, Hpa2 localizes primarily in the cell nucleus. In striking contrast, in breast carcinoma, Hpa2 expression is not only decreased but also loses its nuclear localization and appears diffuse in the cell cytoplasm. Importantly, breast cancer patients in which nuclear localization of Hpa2 is retained exhibited reduced lymph-node metastasis, suggesting that nuclear localization of Hpa2 plays a protective role in breast cancer progression. To examine this possibility, we engineered a gene construct that directs Hpa2 to the cell nucleus (Hpa2-Nuc). Notably, overexpression of Hpa2 in breast carcinoma cells resulted in bigger tumors, whereas targeting Hpa2 to the cell nucleus attenuated tumor growth and tumor metastasis. RNAseq analysis was performed to reveal differentially expressed genes (DEG) in Hpa2-Nuc tumors vs. control. The analysis revealed, among others, decreased expression of genes associated with the hallmark of Kras, beta-catenin, and TNF-alpha (via NFkB) signaling. Our results imply that nuclear localization of Hpa2 prominently regulates gene transcription, resulting in attenuation of breast tumorigenesis. Thus, nuclear Hpa2 may be used as a predictive parameter in personalized medicine for breast cancer patients.

Supplementary Figure Legends. Legend for Supplementary Figures S1-S3.

Suppl. Figure legends Suppl. Fig. 1. The structure of H1001, a small molecule heparanase inhibitor. H1001 inhibit heparanase activity inside and outside the cell, and attenuates cellular invasion Suppl. Fig. 2. Medium conditioned by PCT-treated macrophages promote cell migration; Matrigel containing PCT-treated macrophages recruits far more host macrophages into the plug. Suppl. Fig. 3. Injecting LLC cells with PCT-treated macrophages modestly increase tumor growth in WT C57Bl/6 mice Suppl. Fig. 4. Differential cytokine induction in WT vs KO macrophages is not mediated by TLR2 and TLR4 or MLL1-4, SET1A or RBBP5 Suppl. Fig. 5. Cytokine induction by PCT is attenuated by OICR-9429, a WDR5 inhibitor Suppl. Fig. 6. WDR5 silencing attenuates while WDR5 over expression promote cytokine induction. Cytokine induction by PCT is attenuated by CCG-203971, a MKL1 inhibitor Suppl. Fig. 7. PCT enhances the phosphorylation of JNK in WT but not KO macrophages, and cytokine induction by PCT is attenuated by inhibitor of JNK Suppl. Table 1. Mouse primers sets utilized in this study Suppl. Table 2. Antibodies utilized for FACS analyses

Supplementary Figure S1. Heparanase enhances autophagy.

Supplementary Figure S3. PTEN and TSC do not localizes to acidic vesicles.

Supplementary Figure S2. Heparanase provide cells with chemo-resistance.

Supplementary Figure 2: Over-expression of heparanase/8C in MCF10AT1 cells.

Purpose Heparanase is an endo-beta-glucuronidase that cleaves side chains of heparan-sulfate proteoglycans, an integral constituent of the extra cellular matrix. The abundance of heparanase in placental trophoblast cells implies its role in the processes of placentation and trophoblast invasion. This study aims to explore the involvement of heparanase in parturition and preterm deliveries (PTD). Methods Sixteen human placentas were collected following singleton spontaneous onset term vaginal deliveries (n = 6), spontaneous onset preterm vaginal deliveries (n = 7) and term elective cesarean sections (n = 3). Placentas were excluded in case of any maternal chronic illness, pregnancy or delivery complications apart from PTD. Placental tissue samples were dissected, homogenized and proteins were extracted. Additionally, cryosections were prepared from the placental tissues. Heparanase expression was evaluated utilizing western blot analysis and immunofluorescence staining using heparanase specific antibodies. Heparanase expression was compared between the study groups qualitatively and quantitatively. Results Western blot analysis results demonstrated higher expression of both pro-heparanase and heparanase in PTD placentas compared to term vaginal placentas. Accordingly, immunofluorescence staining shows elevated heparanase expression in PTD placentas compared to term vaginal placentas (5.1 +/- 0.92 vs. 1.2 +/- 0.18, p < .005). Expression level of heparanase was higher in term cesarean section placentas as compared to term vaginal deliveries placentas, but did not reach statistical significance (1.8 +/- 0.39 vs. 1.2 +/- 0.18, p = .06). Conclusion This study demonstrates for the first time that preterm vaginal deliveries are associated with higher expression of heparanase in placental tissue. This may imply a direct effect of heparanase on preterm labor. Further studies should evaluate the functional role by which heparanase influence preterm delivery.

Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.

The pro-tumorigenic properties of heparanase are well documented, and heparanase inhibitors are being evaluated clinically as anti-cancer therapeutics. In contrast, the role of heparanase 2 (Hpa2), a close homolog of heparanase, in cancer is largely unknown. Previously, we have reported that in head and neck cancer, high levels of Hpa2 are associated with prolonged patient survival and decreased tumor cell dissemination to regional lymph nodes, suggesting that Hpa2 functions to restrain tumorigenesis. Also, patients with high levels of Hpa2 were diagnosed as low grade and exhibited increased expression of cytokeratins, an indication that Hpa2 promotes or maintains epithelial cell differentiation and identity. To reveal the molecular mechanism underlying the tumor suppressor properties of Hpa2, and its ability to induce the expression of cytokeratin, we employed overexpression as well as gene editing (Crispr) approaches, combined with gene array and RNAseq methodologies. At the top of the list of many genes found to be affected by Hpa2 was Sox2. Here we provide evidence that silencing of Sox2 resulted in bigger tumors endowed with reduced cytokeratin levels, whereas smaller tumors were developed by cells overexpressing Sox2, suggesting that in head and neck carcinoma, Sox2 functions to inhibit tumor growth. Notably, Hpa2-null cells engineered by Crispr/Cas 9, produced bigger tumors vs control cells, and rescue of Hpa2 attenuated tumor growth. These results strongly imply that Hpa2 functions as a tumor suppressor in head and neck cancer, involving Sox2 upregulation mediated, in part, by the high-affinity interaction of Hpa2 with heparan sulfate.

Heparan sulfate (HS) is a ubiquitous glycosaminoglycan component of the extracellular matrix (ECM), which facilitates important structural and signalling interactions between cells and their surroundings.The principal enzyme responsible for extracellular HS breakdown is heparanase (HPSE), an endo-glucuronidase of the CAZy GH79 family.Whilst normal HPSE activity is essential for HS processing, excessive HPSE overexpression weakens HS networks in the ECM, leading to increased cell mobility and release of growth factors stored by HS.Thus HPSE is an oncogene whose overexpression promotes metastasis in a range of cancers.In this talk, I will give an overview of our work in this area over the last few years, covering our initial structural investigations into the molecular basis of HPSE activity, the development of probes to visualize HPSE in tissues, and most recently, the structure guided rational design of HPSE inhibitors as anti-metastatic agents.

Compelling evidence ties heparanase, an endoglycosidase that cleaves heparan sulfate side (HS) chains of proteoglycans, with all steps of tumor development, including tumor initiation, angiogenesis, growth, metastasis, and chemoresistance. Moreover, heparanase levels correlate with shorter postoperative survival of cancer patients, encouraging the development of heparanase inhibitors as anti-cancer drugs. Heparanase-inhibiting heparin/heparan sulfate-mimicking compounds and neutralizing antibodies are highly effective in animal models of cancer progression, yet none of the compounds reached the stage of approval for clinical use. The present study focused on newly synthesized triazolo–thiadiazoles, of which compound 4-iodo-2-(3-(p-tolyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)phenol (4-MMI) was identified as a potent inhibitor of heparanase enzymatic activity, cell invasion, experimental metastasis, and tumor growth in mouse models. To the best of our knowledge, this is the first report showing a marked decrease in primary tumor growth in mice treated with small molecules that inhibit heparanase enzymatic activity. This result encourages the optimization of 4-MMI for preclinical and clinical studies primarily in cancer but also other indications (i.e., colitis, pancreatitis, diabetic nephropathy, tissue fibrosis) involving heparanase, including viral infection and COVID-19.

Two decades following the cloning of the heparanase gene, the significance of this enzyme for tumor growth and metastasis cannot be ignored. Compelling pre-clinical and clinical evidence tie heparanase with all steps of tumor formation namely, initiation, growth, metastasis, and chemo resistance, thus confirming and significantly expanding earlier observations that coupled heparanase activity with the metastatic capacity of tumor cells. This collective effort has turned heparanase from an obscure enzyme to a valid target for the development of anti-cancer drugs, and led basic researchers and biotech companies to develop heparanase inhibitors as anti-cancer therapeutics, some of which are currently examined clinically. As expected, the intense research effort devoted to understanding the biology of heparanase significantly expanded the functional repertoire of this enzyme, but some principle questions are still left unanswered or are controversial. For example, many publications describe increased heparanase levels in human tumors, but the mechanism underlying heparanase induction is not sufficiently understood. Moreover, heparanase is hardly found to be increased in many studies utilizing methodologies (i.e., gene arrays) that compare tumors vs (adjacent) normal tissue. The finding that heparanase exert also enzymatic activity-independent function significantly expands the mode by which heparanase can function outside, but also inside the cell. Signaling aspects, and a role of heparanase in modulating autophagy are possibly as important as its enzymatic aspect, but these properties are not targeted by heparanase inhibitors, possibly compromising their efficacy. This Book chapter review heparanase function in oncology, suggesting a somewhat different interpretation of the results.

Abstract The emerging role of heparanase in tumor initiation, growth, metastasis, and chemoresistance is well recognized, encouraging the development of heparanase inhibitors as anticancer drugs. Unlike the function of heparanase in cancer cells, little attention has been given to heparanase contributed by cells composing the tumor microenvironment. Here, we focused on the cross-talk between macrophages, chemotherapy, and heparanase and the combined effect on tumor progression. Macrophages were markedly activated by chemotherapeutics paclitaxel and cisplatin, evidenced by increased expression of proinflammatory cytokines, supporting recent studies indicating that chemotherapy may promote rather than suppress tumor regrowth and spread. Strikingly, cytokine induction by chemotherapy was not observed in macrophages isolated from heparanase-knockout mice, suggesting macrophage activation by chemotherapy is heparanase dependent. paclitaxel-treated macrophages enhanced the growth of Lewis lung carcinoma tumors that was attenuated by a CXCR2 inhibitor. Mechanistically, paclitaxel and cisplatin activated methylation of histone H3 on lysine 4 (H3K4) in wild-type but not in heparanase-knockout macrophages. Furthermore, the H3K4 presenter WDR5 functioned as a molecular determinant that mediated cytokine induction by paclitaxel. This epigenetic, heparanase-dependent host-response mechanism adds a new perspective to the tumor-promoting functions of chemotherapy, and offers new treatment modalities to optimize chemotherapeutics. Significance: Chemotherapy-treated macrophages are activated to produce proinflammatory cytokines, which are blunted in the absence of heparanase.

L-arginine CP only L-arginine CP + Pir-Immune cells p value (n=5) fenidone (n=5) Macrophages(CD45+F4/ 3.26 ± 0.4 % 1.09 ± 0.18 % 0.001** 80+/ total events#) Activated macrophages (CD45+ F4/ 2.35 ± 0.3 % 0.69 ± 0.1 % 0.007** 80+CD11b+/total events#) M2 macrophages 2.8 ± 0.2 % 1.5 ± 0.1 % 0.007** (F4/80+IL4+) M1 macrophages(F4/ 60.1 ± 7.7 % 52.8 ± 7.2 % >0.99(ns) 80+MHCII+) Neutrophils (CD 45+ Ly6G+/ total 1.4 ± 0.3 % 0.32 ± 0.6 % 0.007** events#) Activated neutrophils (CD45+ 1.36 ± 0.3 % 0.306 ± 0.06 % 0.007** Ly6G+CD11b+/ total events#) #Total events=100000/sample.;*p value<0.05(significant).;(ns)-notsignificant.