RESUMO
Heparanase (Hpa1) is expressed by tumor cells and cells of the tumor microenvironment and functions to remodel the extracellular matrix (ECM) and regulate the bioavailability of ECM-bound factors that support tumor growth. Heparanase expression is upregulated in human carcinomas, sarcomas, and hematological malignancies, correlating with increased tumor metastasis, vascular density, and shorter postoperative survival of cancer patients, and encouraging the development of heparanase inhibitors as anti-cancer drugs. Among these are heparin/HS mimetics, the only heparanase-inhibiting compounds that are being evaluated in clinical trials. We have synthesized dicarboxylated oxy-heparins (DCoxHs) containing three carboxylate groups per split residue (DC-Hep). The resulting lead compound (termed XII) was upscaled, characterized, and examined for its effectiveness in tumor models. Potent anti-tumorigenic effects were obtained in models of pancreatic carcinoma, breast cancer, mesothelioma, and myeloma, yielding tumor growth inhibition (TGI) values ranging from 21 to 70% and extending the survival time of the mice. Of particular significance was the inhibition of spontaneous metastasis in an orthotopic model of breast carcinoma following resection of the primary tumor. It appears that apart from inhibition of heparanase enzymatic activity, compound XII reduces the levels of heparanase protein and inhibits its cellular uptake and activation. Heparanase-dependent and -independent effects of XII are being investigated. Collectively, our pre-clinical studies with compound XII strongly justify its examination in cancer patients.
Assuntos
Antineoplásicos , Neoplasias da Mama , Humanos , Animais , Camundongos , Feminino , Heparina/farmacologia , Heparina/química , Glucuronidase/metabolismo , Antineoplásicos/uso terapêutico , Carcinogênese , Neoplasias da Mama/tratamento farmacológico , Microambiente TumoralRESUMO
Heparanase 2 (Hpa2, HPSE2) is a close homolog of heparanase. Hpa2, however, lacks intrinsic heparan sulfate (HS)-degrading activity, the hallmark of heparanase enzymatic activity. Mutations of HPSE2 were identified in patients diagnosed with urofacial syndrome (UFS), a rare genetic disorder that exhibits abnormal facial expression and bladder voiding dysfunction, leading to renal damage and eventually renal failure. In order to reveal the role of HPSE2 in tissue homeostasis, we established a conditional Hpa2-KO mouse. Interestingly, the lack of Hpa2 was associated with a marked decrease in the expression of key pancreatic transcription factors such as PTF1, GATA6, and Mist1. This was associated with a two-fold decrease in pancreas weight, increased pancreatic inflammation, and profound morphological alterations of the pancreas. These include massive accumulation of fat cells, possibly a result of acinar-to-adipocyte transdifferentiation (AAT), as well as acinar-to-ductal metaplasia (ADM), both considered to be pro-tumorigenic. Furthermore, exposing Hpa2-KO but not wild-type mice to a carcinogen (AOM) and pancreatic inflammation (cerulein) resulted in the formation of pancreatic intraepithelial neoplasia (PanIN), lesions that are considered to be precursors of invasive ductal adenocarcinoma of the pancreas (PDAC). These results strongly support the notion that Hpa2 functions as a tumor suppressor. Moreover, Hpa2 is shown here for the first time to play a critical role in the exocrine aspect of the pancreas.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Pancreatite , Camundongos , Animais , Pâncreas/patologia , Células Acinares/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite/metabolismo , Diferenciação Celular , Inflamação/patologia , Carcinoma Ductal Pancreático/patologiaRESUMO
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.
Assuntos
Glucuronidase , Inibidores de Glicosídeo Hidrolases , Metástase Neoplásica , Animais , Proliferação de Células/efeitos dos fármacos , Glucuronidase/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/farmacologia , Inibidores de Glicosídeo Hidrolases/uso terapêutico , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Camundongos , Metástase Neoplásica/tratamento farmacológicoRESUMO
[This corrects the article DOI: 10.1039/D0CB00179A.].
RESUMO
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.
RESUMO
Cancer and other disease states can change the landscape of proteins post-translationally tagged with ubiquitin (Ub) chains. Molecules capable of modulating Ub chains are potential therapeutic agents, but their discovery represents a significant challenge. Recently, it was shown that de novo cyclic peptides, selected from trillion-member random libraries, are capable of binding particular Ub chains. However, these peptides were overwhelmingly proteinogenic, so the prospect of in vivo activity was uncertain. Here, we report the discovery of small, non-proteinogenic cyclic peptides, rich in non-canonical features like N-methylation, which can tightly and specifically bind Lys48-linked Ub chains. These peptides engage three Lys48-linked Ub units simultaneously, block the action of deubiquitinases and the proteasome, induce apoptosis in vitro, and attenuate tumor growth in vivo. This highlights the potential of non-proteinogenic cyclic peptide screening to rapidly find in vivo-active leads, and the targeting of ubiquitin chains as a promising anti-cancer mechanism of action.
RESUMO
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.
Assuntos
Glucuronidase , Neoplasias de Cabeça e Pescoço , Glucuronidase/genética , Neoplasias de Cabeça e Pescoço/genética , Heparitina Sulfato , Humanos , Fatores de Transcrição SOXB1/genéticaRESUMO
Defective amyloid-ß (Aß) clearance from the brain is a major contributing factor to the pathophysiology of Alzheimer's disease (AD). Aß clearance is mediated by macrophages, enzymatic degradation, perivascular drainage along the vascular basement membrane (VBM) and transcytosis across the blood-brain barrier (BBB). AD pathology is typically associated with cerebral amyloid angiopathy due to perivascular accumulation of Aß. Heparan sulfate (HS) is an important component of the VBM, thought to fulfill multiple roles in AD pathology. We previously showed that macrophage-mediated clearance of intracortically injected Aß was impaired in the brains of transgenic mice overexpressing heparanase (Hpa-tg). This study revealed that perivascular drainage was impeded in the Hpa-tg brain, evidenced by perivascular accumulation of the injected Aß in the thalamus of Hpa-tg mice. Furthermore, endogenous Aß accumulated at the perivasculature of Hpa-tg thalamus, but not in control thalamus. This perivascular clearance defect was confirmed following intracortical injection of dextran that was largely retained in the perivasculature of Hpa-tg brains, compared to control brains. Hpa-tg brains presented with thicker VBMs and swollen perivascular astrocyte endfeet, as well as elevated expression of the BBB-associated water-pump protein aquaporin 4 (AQP4). Elevated levels of both heparanase and AQP4 were also detected in human AD brain. These findings indicate that elevated heparanase levels alter the organization and composition of the BBB, likely through increased fragmentation of BBB-associated HS, resulting in defective perivascular drainage. This defect contributes to perivascular accumulation of Aß in the Hpa-tg brain, highlighting a potential role for heparanase in the pathogenesis of AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Regulação Enzimológica da Expressão Gênica , Glucuronidase/biossíntese , Sistema Glinfático/metabolismo , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/administração & dosagem , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/toxicidade , Animais , Encéfalo/patologia , Feminino , Glucuronidase/genética , Sistema Glinfático/patologia , Humanos , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/toxicidadeRESUMO
Cell surface proteoglycans are important constituents of the glycocalyx and participate in cell-cell and cell-extracellular matrix (ECM) interactions, enzyme activation and inhibition, and multiple signaling routes, thereby regulating cell proliferation, survival, adhesion, migration, and differentiation. Heparanase, the sole mammalian heparan sulfate degrading endoglycosidase, acts as an "activator" of HS proteoglycans, thus regulating tissue hemostasis. Heparanase is a multifaceted enzyme that together with heparan sulfate, primarily syndecan-1, drives signal transduction, immune cell activation, exosome formation, autophagy, and gene transcription via enzymatic and nonenzymatic activities. An important feature is the ability of heparanase to stimulate syndecan-1 shedding, thereby impacting cell behavior both locally and distally from its cell of origin. Heparanase releases a myriad of HS-bound growth factors, cytokines, and chemokines that are sequestered by heparan sulfate in the glycocalyx and ECM. Collectively, the heparan sulfate-heparanase axis plays pivotal roles in creating a permissive environment for cell proliferation, differentiation, and function, often resulting in the pathogenesis of diseases such as cancer, inflammation, endotheliitis, kidney dysfunction, tissue fibrosis, and viral infection.
Assuntos
Doença , Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , HumanosRESUMO
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.
Assuntos
Glucuronidase/metabolismo , Neoplasias , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Glucuronidase/antagonistas & inibidores , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Transdução de SinaisRESUMO
Tumor cell-platelet interactions are regarded as an initial crucial step in hematogenous metastasis. Platelets protect tumor cells from immune surveillance in the blood, mediate vascular arrest, facilitate tumor extravasation, growth, and finally angiogenesis in the metastatic foci. Tumor cells aggregate platelets in the bloodstream by activation of the plasmatic coagulation cascade and by direct contact formation. Antimetastatic activities of unfractionated or low molecular weight heparin (UFH/LMWH) can undoubtedly be related to attenuated platelet activation, but molecular mechanisms and contribution of contact formation vs. coagulation remain to be elucidated. Using a set of non-anticoagulant heparin derivatives varying in size or degree of sulfation as compared with UFH, we provide insight into the relevance of contact formation for platelet activation. Light transmission aggregometry and ATP release assays confirmed that only those heparin derivatives with P-selectin blocking capacities were able to attenuate breast cancer cell-induced platelet activation, while pentasaccharide fondaparinux was without effects. Furthermore, a role of P-selectin in platelet activation and signaling could be confirmed by proteome profiler arrays detecting platelet kinases. In this study, we demonstrate that heparin blocks tumor cell-induced coagulation. Moreover, we identify platelet P-selectin, which obviously acts as molecular switch and controls aggregation and secretion of procoagulant platelets.
Assuntos
Plaquetas/patologia , Comunicação Celular/efeitos dos fármacos , Glicosaminoglicanos/farmacologia , Neoplasias/patologia , Selectina-P/metabolismo , Plaquetas/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Heparina/farmacologia , Humanos , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Antineoplásicos/efeitos adversos , Carcinoma Pulmonar de Lewis/patologia , Glucuronidase/metabolismo , Macrófagos/imunologia , Microambiente Tumoral/imunologia , Animais , Carcinogênese/efeitos dos fármacos , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/imunologia , Linhagem Celular Tumoral , Cisplatino/efeitos adversos , Metilação de DNA/efeitos dos fármacos , Ensaios Enzimáticos , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucuronidase/genética , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Paclitaxel/efeitos adversos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Heparanase is regarded as a promising target for anticancer drugs and Ronepastat is one of the most promising heparanase inhibitors insert in clinical study for Multiple Myeloma Therapy. To improve its pharmacokinetic/pharmacodynamic profile, as well to have an antidote able to neutralize its activity in case of over dosages or intolerance, a new class of its derivatives was obtained inserting non-carbohydrate moieties of different length between the polysaccharide chain and biotin or its derivatives. In vitro these novel derivatives maintain the anti-heparanase activity without induced toxicity. The newly synthesized compounds retained the ability to attenuate the growth of CAG myeloma tumors in mice with potency similar, or in one case even higher than that of the reference compound Roneparstat as well as inhibited metastatic dissemination (lung colonization) of murine B16-F10 melanoma cells in vivo.
Assuntos
Antineoplásicos/farmacologia , Biotina/química , Glucuronidase/antagonistas & inibidores , Glicóis/farmacologia , Inibidores de Glicosídeo Hidrolases/farmacologia , Heparina/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Glucuronidase/metabolismo , Glicóis/síntese química , Glicóis/química , Inibidores de Glicosídeo Hidrolases/síntese química , Inibidores de Glicosídeo Hidrolases/química , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Imagem Óptica , Relação Estrutura-AtividadeRESUMO
Heparanase is an endoglycosidase involved in remodeling the extracellular matrix and thereby in regulating multiple cellular processes and biological activities. It cleaves heparan sulfate (HS) side chains of HS proteoglycans into smaller fragments and hence regulates tissue morphogenesis, differentiation, and homeostasis. Heparanase is overexpressed in various carcinomas, sarcomas, and hematological malignancies, and its upregulation correlates with increased tumor size, tumor angiogenesis, enhanced metastasis, and poor prognosis. In contrast, knockdown or inhibition of heparanase markedly attenuates tumor progression, further underscoring the potential of anti-heparanase therapy. Heparanase inhibitors were employed to interfere with tumor progression in preclinical studies, and selected heparin mimetics are being examined in clinical trials. However, despite tremendous efforts, the discovery of heparanase inhibitors with high clinical benefit and minimal adverse effects remains a therapeutic challenge. This review discusses the key roles of heparanase in cancer progression focusing on the status of natural, chemically modified, and synthetic heparanase inhibitors in various types of malignancies.
RESUMO
Heparanase is an endo-ß-d-glucuronidase that cleaves heparan sulfate (HS) side chains of heparan sulfate proteoglycans. Compelling evidence tie heparanase levels with all steps of tumor formation including tumor initiation, growth, metastasis and chemo-resistance, likely involving augmentation of signaling pathways and gene transcription. In order to reveal the molecular mechanism(s) underlying the protumorigenic properties of heparanase, we established an inducible (Tet-on) system in U87 human glioma cells and applied gene array methodology in order to identify genes associated with heparanase induction. We found that CD24, a mucin-like cell adhesion protein, is consistently upregulated by heparanase and by heparanase splice variant devoid of enzymatic activity, whereas heparanase gene silencing was associated with decreased CD24 expression. This finding was further substantiated by a similar pattern of heparanase and CD24 immunostaining in glioma patients (Pearson's correlation; R = 0.66, p = 0.00001). Noteworthy, overexpression of CD24 stimulated glioma cell migration, invasion, colony formation in soft agar and tumor growth in mice suggesting that CD24 functions promote tumor growth. Likewise, anti-CD24 neutralizing monoclonal antibody attenuated glioma tumor growth, and a similar inhibition was observed in mice treated with a neutralizing mAb directed against L1 cell adhesion molecule (L1CAM), a ligand for CD24. Importantly, significant shorter patient survival was found in heparanase-high/CD24-high tumors vs. heparanase-high/CD24-low tumors for both high-grade and low-grade glioma (p = 0.02). Our results thus uncover a novel heparanase-CD24-L1CAM axis that plays a significant role in glioma tumorigenesis.
Assuntos
Neoplasias Encefálicas/patologia , Antígeno CD24/metabolismo , Glioma/patologia , Glucuronidase/metabolismo , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Glioma/metabolismo , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Transdução de SinaisRESUMO
Heparanase, the heparan sulfate polysaccharide degrading endoglycosidase enzyme, has been correlated with tumor angiogenesis and metastasis and therefore has become a potential target for anticancer drug development. In this systematic study, the sulfation pattern of the pendant disaccharide moiety on synthetic glycopolymers was synthetically manipulated to achieve optimal heparanase inhibition. Upon evaluation, a glycopolymer with 12 repeating units was determined to be the most potent inhibitor of heparanase (IC50 = 0.10 ± 0.36 nM). This glycopolymer was further examined for cross-bioactivity using a solution-based competitive biolayer interferometry assay with other HS-binding proteins (growth factors, P-selectin, and platelet factor 4), which are responsible for mediating angiogenic activity, cell metastasis, and antibody-induced thrombocytopenia. The synthetic glycopolymer has low affinity for these HS-binding proteins in comparison to natural heparin. In addition, the glycopolymer possessed no proliferative properties toward human umbilical endothelial cells (HUVECs) and a potent antimetastatic effect against 4T1 mammary carcinoma cells. Thus, our study not only establishes a specific inhibitor of heparanase with high affinity but also illustrates the high effectiveness of this multivalent heparanase inhibitor in inhibiting experimental metastasis in vivo.
Assuntos
Inibidores Enzimáticos/farmacologia , Glucuronidase , Neoplasias Mamárias Experimentais , Proteínas de Neoplasias , Neovascularização Patológica , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Feminino , Glucuronidase/antagonistas & inibidores , Glucuronidase/metabolismo , Heparina/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Metástase Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/enzimologia , Neovascularização Patológica/patologia , Selectina-P/metabolismoRESUMO
Heparanase, the sole heparan sulfate (HS) degrading endoglycosidase, regulates multiple biological activities that enhance tumor growth, metastasis, angiogenesis, and inflammation. Heparanase accomplishes this by degrading HS and thereby facilitating cell invasion and regulating the bioavailability of heparin-binding proteins. HS mimicking compounds that inhibit heparanase enzymatic activity were examined in numerous preclinical cancer models. While these studies utilized established tumor cell lines, the current study utilized, for the first time, patient-derived xenografts (PDX) which better resemble the behavior and drug responsiveness of a given cancer patient. We have previously shown that heparanase levels are substantially elevated in lung cancer, correlating with reduced patients survival. Applying patient-derived lung cancer xenografts and a potent inhibitor of heparanase enzymatic activity (PG545) we investigated the significance of heparanase in the pathogenesis of lung cancer. PG545 was highly effective in lung cancer PDX, inhibiting tumor growth in >85% of the cases. Importantly, we show that PG545 was highly effective in PDX that did not respond to conventional chemotherapy (cisplatin) and vice versa. Moreover, we show that spontaneous metastasis to lymph nodes is markedly inhibited by PG545 but not by cisplatin. These results reflect the variability among patients and strongly imply that PG545 can be applied for lung cancer therapy in a personalized manner where conventional chemotherapy fails, thus highlighting the potential benefits of developing anti-heparanase treatment modalities for oncology.
RESUMO
Background: Mammalian cells express a single functional heparanase, an endoglycosidase that cleaves heparan sulfate and thereby promotes tumor metastasis, angiogenesis, and inflammation. Malignant mesothelioma is highly aggressive and has a poor prognosis because of the lack of markers for early diagnosis and resistance to conventional therapies. The purpose of this study was to elucidate the mode of action and biological significance of heparanase in mesothelioma and test the efficacy of heparanase inhibitors in the treatment of this malignancy. Methods: The involvement of heparanase in mesothelioma was investigated by applying mouse models of mesothelioma and testing the effect of heparanase gene silencing (n = 18 mice per experiment; two different models) and heparanase inhibitors (ie, PG545, defibrotide; n = 18 per experiment; six different models). Synchronous pleural effusion and plasma samples from patients with mesothelioma (n = 35), other malignancies (12 non-small cell lung cancer, two small cell lung carcinoma, four breast cancer, three gastrointestinal cancers, two lymphomas), and benign effusions (five patients) were collected and analyzed for heparanase content (enzyme-linked immunosorbent assay). Eighty-one mesothelioma biopsies were analyzed by H-Score for the prognostic impact of heparanase using immunohistochemistry. All statistical tests were two-sided. Results: Mesothelioma tumor growth, measured by bioluminescence or tumor weight at termination, was markedly attenuated by heparanase gene silencing (P = .02) and by heparanase inhibitors (PG545 and defibrotide; P < .001 and P = .01, respectively). A marked increase in survival of the mesothelioma-bearing mice (P < .001) was recorded. Heparanase inhibitors were more potent in vivo than conventional chemotherapy. Clinically, heparanase levels in patients' pleural effusions could distinguish between malignant and benign effusions, and a heparanase H-score above 90 was associated with reduced patient survival (hazard ratio = 1.89, 95% confidence interval = 1.09 to 3.27, P = .03). Conclusions: Our results imply that heparanase is clinically relevant in mesothelioma development. Given these preclinical and clinical data, heparanase appears to be an important mediator of mesothelioma, and heparanase inhibitors are worthy of investigation as a new therapeutic modality in mesothelioma clinical trials.
Assuntos
Glucuronidase/genética , Glucuronidase/metabolismo , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Mesotelioma/etiologia , Mesotelioma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Modelos Animais de Doenças , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Inativação Gênica , Glucuronidase/antagonistas & inibidores , Humanos , Inflamação/complicações , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Mesotelioma/mortalidade , Mesotelioma/patologia , Mesotelioma Maligno , Camundongos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neovascularização Patológica , Prognóstico , RNA Interferente Pequeno/genética , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Heparanase is an endoglucuronidase that uniquely cleaves the heparan sulfate side chains of heparan sulfate proteoglycans. This activity ultimately alters the structural integrity of the ECM and basement membrane that becomes more prone to cellular invasion by metastatic cancer cells and cells of the immune system. In addition, enzymatically inactive heparanase was found to facilitate the proliferation and survival of cancer cells by activation of signaling molecules such as Akt, Src, signal transducer and activation of transcription (Stat), and epidermal growth factor receptor. This function is thought to be executed by the C-terminal domain of heparanase (8c), because over expression of this domain in cancer cells accelerated signaling cascades and tumor growth. We have used the regulatory elements of the mouse mammary tumor virus (MMTV) to direct the expression heparanase and the C-domain (8c) to the mammary gland epithelium of transgenic mice. Here, we report that mammary gland branching morphogenesis is increased in MMTV-heparanase and MMTV-8c mice, associating with increased Akt, Stat5 and Src phosphorylation. Furthermore, we found that the growth of tumors generated by mouse breast cancer cells and the resulting lung metastases are enhanced in MMTV-heparanase mice, thus supporting the notion that heparanase contributed by the tumor microenvironment (i.e., normal mammary epithelium) plays a decisive role in tumorigenesis. Remarkably, MMTV-8c mice develop spontaneous tumors in their mammary and salivary glands. Although this occurs at low rates and requires long latency, it demonstrates decisively the pro-tumorigenic capacity of heparanase signaling.
Assuntos
Neoplasias da Mama/patologia , Glucuronidase/genética , Glucuronidase/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Glândulas Mamárias Animais/crescimento & desenvolvimento , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Glucuronidase/química , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Glândulas Mamárias Animais/metabolismo , Vírus do Tumor Mamário do Camundongo/fisiologia , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Domínios Proteicos , Transdução de SinaisRESUMO
Malignant mesothelioma is a highly aggressive form of cancer with poor prognosis due to lack of markers for early diagnosis and resistance to conventional therapies. Heparanase, the sole heparan sulfate (HS) degrading endoglycosidase, regulates multiple biological activities that enhance tumor growth, metastasis, angiogenesis, and inflammation. Heparanase accomplishes this by degrading HS and thereby facilitating cell invasion and regulating the bioavailability of heparin-binding proteins. Applying pre-clinical and clinical models of human mesothelioma and potent inhibitors of heparanase enzymatic activity (PG545, Defibrotide) we investigated the significance of heparanase in the pathogenesis of mesothelioma. We found that mesothelioma tumor growth was markedly attenuated by heparanase gene silencing and by heparanase inhibitors. Furthermore, heparanase inhibitors were more potent in vivo than conventional chemotherapy. Clinically, heparanase levels in patients' pleural effusions could distinguish between malignant and benign effusions, and heparanase H-score (immunostaining of tumor specimens) above 90 was associated with reduced patient survival. These results strongly imply that heparanase plays an important role in mesothelioma tumor progression, thus encouraging the use of heparanase inhibitors in combination with existing drugs as a new therapeutic modality in mesothelioma clinical trials.