STEM-15. SerpinB3 DRIVES CANCER STEM CELL SURVIVAL IN GLIOBLASTOMA

Abstract Despite therapeutic interventions for glioblastoma (GBM), self-renewing, therapy-resistant populations of cells referred to as cancer stem cells (CSCs) drive recurrence. Previously, we identified the unique expression of junctional adhesion molecule-A (JAM-A) on CSCs and demonstrated that JAM-A is both necessary and sufficient for self-renewal and tumor growth. Moreover, we determined that JAM-A signals via Akt in GBM CSCs to sustain pluripotency transcription factor activity; however, the entire signaling network has yet to be fully elucidated. To further delineate this pathway, we immunoprecipitated JAM-A from patient-derived GBM CSCs and performed mass spectrometry to determine JAM-A binding proteins. This led to the identification of the cysteine protease inhibitor SerpinB3 as a putative JAM-A binding partner. Using in vitro CSC functional assays, we show that SerpinB3 is necessary for CSC maintenance and survival. In an in vivo orthotopic xenograft model, knockdown of SerpinB3 extended survival. Mechanistically, knockdown of SerpinB3 led to decreased expression of TGF-β, Myc, WNT, and Notch signaling, known regulators of the CSC state. Additionally, knockdown of SerpinB3 increases susceptibility to radiation therapy. SerpinB3 is essential for buffering cells against cathepsin-mediated cell death, and we found that elevated lysosomal membrane permeability after radiation leads to cathepsin release into the cytoplasm. As a result, SerpinB3 knockdown cells have a diminished capacity to inhibit cathepsin-driven cell death after radiation. The addition of the cathepsin inhibitor E64D partially rescues the SerpinB3 knockdown, however, SerpinB3 mutants that are unable to inhibit cathepsins fail to do the same. Taken together, our findings, identify a novel GBM CSC-specific survival mechanism involving a previously uninvestigated cysteine protease inhibitor, SerpinB3, and provide a potential target to increase the efficacy of standard of care GBM therapies against therapy-resistant CSCs.

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SerpinB3 drives cancer stem cell survival in glioblastoma

10.1101/2021.12.21.473663 ◽

2021 ◽

Author(s):

Adam Lauko ◽

Josephine Volovetz ◽

Soumya M Turaga ◽

Defne Bayik ◽

Dennis C Watson ◽

...

Keyword(s):

Cell Death ◽

Protease Inhibitor ◽

Cysteine Protease ◽

Cathepsin L ◽

Standard Of Care ◽

Surface Expression ◽

Therapeutic Interventions ◽

Cysteine Protease Inhibitor ◽

Cell Surface Expression ◽

Downstream Signaling

Despite therapeutic interventions for glioblastoma (GBM), cancer stem cells (CSCs) drive recurrence. The precise mechanisms underlying CSC therapeutic resistance, namely inhibition of cell death, are unclear. We built on previous observations that the high cell surface expression of junctional adhesion molecule-A drives CSC maintenance and identified downstream signaling networks, including the cysteine protease inhibitor SerpinB3. Using genetic depletion approaches, we found that SerpinB3 is necessary for CSC maintenance, survival, and tumor growth, as well as CSC pathway activation. The knockdown of SerpinB3 also increased apoptosis and susceptibility to radiation therapy. Mechanistically, SerpinB3 was essential to buffer cathepsin L-mediated cell death, which was enhanced with radiation. Finally, we found that SerpinB3 knockdown dramatically increased the efficacy of radiation in pre-clinical models. Taken together, our findings identify a novel GBM CSC-specific survival mechanism involving a previously uninvestigated cysteine protease inhibitor, SerpinB3, and provide a potential target to improve the efficacy of standard-of-care GBM therapies against therapeutically resistant CSCs.

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Effect of phenyl vinyl sulphone cysteine protease inhibitor on Schistosoma mansoni: in vitro and in vivo experimental studies

Journal of Parasitic Diseases ◽

10.1007/s12639-017-0933-3 ◽

2017 ◽

Vol 41 (4) ◽

pp. 1049-1058

Author(s):

Manal Salah El-Din Mahmoud ◽

Ayman Nabil Ibrahim ◽

Abeer Fathy Badawy ◽

Nourhan Mohamed Abdelmoniem

Keyword(s):

Protease Inhibitor ◽

Schistosoma Mansoni ◽

Cysteine Protease ◽

Experimental Studies ◽

Cysteine Protease Inhibitor ◽

Phenyl Vinyl

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Nippocystatin, a Cysteine Protease Inhibitor fromNippostrongylus brasiliensis, Inhibits Antigen Processing and Modulates Antigen-Specific Immune Response

Infection and Immunity ◽

10.1128/iai.69.12.7380-7386.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7380-7386 ◽

Cited By ~ 78

Author(s):

Teruki Dainichi ◽

Yoichi Maekawa ◽

Kazunari Ishii ◽

Tianqian Zhang ◽

Baher Fawzy Nashed ◽

...

Keyword(s):

Immune System ◽

Protease Inhibitor ◽

Cysteine Protease ◽

Antigen Processing ◽

Cysteine Proteases ◽

Cysteine Protease Inhibitor ◽

Host Immune System ◽

Nippostrongylus Brasiliensis

ABSTRACT During infection, parasites evade the host immune system by modulating or exploiting the immune system; e.g., they suppress expression of major histocompatibility complex class II molecules or secrete cytokine-like molecules. However, it is not clear whether helminths disturb the immune responses of their hosts by controlling the antigen-processing pathways of the hosts. In this study, we identified a new cysteine protease inhibitor, nippocystatin, derived from excretory-secretory (ES) products of an intestinal nematode,Nippostrongylus brasiliensis. Nippocystatin, which belongs to cystatin family 2, consists of 144 amino acids and is secreted as a 14-kDa mature form. In vivo treatment of ovalbumin (OVA)-immunized mice with recombinant nippocystatin (rNbCys) profoundly suppressed OVA-specific proliferation of splenocytes but not non-antigen-specific proliferation of splenocytes. OVA-specific cytokine production was also greatly suppressed in rNbCys-treated mice. Although the serum levels of both OVA-specific immunoglobulin G1 (IgG1) and IgG2a were not affected by rNbCys treatment, OVA-specific IgE was preferentially downregulated in rNbCys-treated mice. In vitro rNbCys inhibited processing of OVA by lysosomal cysteine proteases from the spleens of mice. Mice with anti-nippocystatin antibodies became partially resistant to infection with N. brasiliensis. Based on these findings, N. brasiliensis appears to skillfully evade host immune systems by secreting nippocystatin, which modulates antigen processing in antigen-presenting cells of hosts.

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Antimalarial Activity of Allicin, a Biologically Active Compound from Garlic Cloves

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.5.1731-1737.2006 ◽

2006 ◽

Vol 50 (5) ◽

pp. 1731-1737 ◽

Cited By ~ 75

Author(s):

Alida Coppi ◽

Melissa Cabinian ◽

David Mirelman ◽

Photini Sinnis

Keyword(s):

Life Cycle ◽

Protease Inhibitor ◽

Cysteine Protease ◽

Malaria Infection ◽

Drug Targets ◽

Host Cells ◽

Cysteine Protease Inhibitor ◽

New Drug

ABSTRACT The incidence of malaria is increasing, and there is an urgent need to identify new drug targets for both prophylaxis and chemotherapy. Potential new drug targets include Plasmodium proteases that play critical roles in the parasite life cycle. We have previously shown that the major surface protein of Plasmodium sporozoites, the circumsporozoite protein (CSP), is proteolytically processed by a parasite-derived cysteine protease, and this processing event is temporally associated with sporozoite invasion of host cells. E-64, a cysteine protease inhibitor, inhibits CSP processing and prevents invasion of host cells in vitro and in vivo. Here we tested allicin, a cysteine protease inhibitor found in garlic extracts, for its ability to inhibit malaria infection. At low concentrations, allicin was not toxic to either sporozoites or mammalian cells. At these concentrations, allicin inhibited CSP processing and prevented sporozoite invasion of host cells in vitro. In vivo, mice injected with allicin had decreased Plasmodium infections compared to controls. When sporozoites were treated with allicin before injection into mice, malaria infection was completely prevented. We also tested allicin on erythrocytic stages and found that a 4-day regimen of allicin administered either orally or intravenously significantly decreased parasitemias and increased the survival of infected mice by 10 days. Together, these experiments demonstrate that the same cysteine protease inhibitor can target two different life cycle stages in the vertebrate host.

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A protein extract and a cysteine protease inhibitor enriched fraction from Jatropha curcas seed cake have in vitro anti-Toxoplasma gondii activity

Experimental Parasitology ◽

10.1016/j.exppara.2015.03.011 ◽

2015 ◽

Vol 153 ◽

pp. 111-117 ◽

Cited By ~ 4

Author(s):

A.M.S. Soares ◽

L.P. Carvalho ◽

E.J.T. Melo ◽

H.P.S. Costa ◽

I.M. Vasconcelos ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Protease Inhibitor ◽

Cysteine Protease ◽

Jatropha Curcas ◽

Cysteine Protease Inhibitor ◽

Protein Extract ◽

Seed Cake

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Dkk-3, a Secreted Wnt Antagonist, Suppresses Tumorigenic Potential and Pulmonary Metastasis in Osteosarcoma

Sarcoma ◽

10.1155/2013/147541 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 23

Author(s):

Carol H. Lin ◽

Yi Guo ◽

Samia Ghaffar ◽

Peter McQueen ◽

Jonathan Pourmorady ◽

...

Keyword(s):

Cell Lines ◽

Negative Impact ◽

Xenograft Model ◽

Pulmonary Nodules ◽

Cellular Motility ◽

Control Vector ◽

Orthotopic Xenograft Model ◽

Tumorigenic Potential

Osteosarcoma (OS) is the most common primary bone malignancy with a high propensity for local invasion and distant metastasis. Despite current multidisciplinary treatments, there has not been a drastic change in overall prognosis within the past 2 decades. Dickkopf-3 protein (Dkk-3/REIC) has been known to inhibit canonical Wnt/β-catenin pathway, and its expression has been shown to be downregulated in OS cell lines. Usingin vivoandin vitrostudies, we demonstrated that Dkk-3-transfected 143B cells inhibited tumorigenesis and metastasis in an orthotopic xenograft model of OS. Inoculation of Dkk-3-transfected 143B cell lines into nude mice showed significant decreased tumor growth and less metastatic pulmonary nodules (88.7%) compared to the control vector.In vitroexperiments examining cellular motility and viability demonstrated less anchorage-independent growth and decreased cellular motility for Dkk-3-transfected 143B and SaOS2 cell lines compared to the control vector. Downstream expressions of Met, MAPK, ALK, and S1004A were also downregulated in Dkk-3-transfected SaOS2 cells, suggesting the ability of Dkk-3 to inhibit tumorigenic potential of OS. Together, these data suggest that Dkk-3 has a negative impact on the progression of osteosarcoma. Reexpressing Dkk-3 in Dkk-3-deficient OS tumors may prove to be of benefit as a preventive or therapeutic strategy.

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In vivo inhibition of cyclin B degradation and induction of cell-cycle arrest in mammalian cells by the neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.8.3353 ◽

1993 ◽

Vol 90 (8) ◽

pp. 3353-3357 ◽

Cited By ~ 57

Author(s):

S. W. Sherwood ◽

A. L. Kung ◽

J. Roitelman ◽

R. D. Simoni ◽

R. T. Schimke

Keyword(s):

Cell Cycle ◽

Protease Inhibitor ◽

Cell Cycle Arrest ◽

Cysteine Protease ◽

Mammalian Cells ◽

Cyclin B ◽

Cysteine Protease Inhibitor ◽

Cycle Arrest

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A Phytophthora palmivora Extracellular Cystatin-Like Protease Inhibitor Targets Papain to Contribute to Virulence on Papaya

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-06-17-0131-fi ◽

2018 ◽

Vol 31 (3) ◽

pp. 363-373 ◽

Cited By ~ 22

Author(s):

Rebecca Gumtow ◽

Dongliang Wu ◽

Janice Uchida ◽

Miaoying Tian

Keyword(s):

Protease Inhibitor ◽

Protease Inhibitors ◽

Cysteine Protease ◽

Gene Editing ◽

Sequence Data ◽

Cysteine Protease Inhibitor ◽

Phytophthora Palmivora ◽

Cysteine Protease Inhibitors ◽

Successful Infection

Papaya fruits, stems, and leaves are rich in papain, a cysteine protease that has been shown to mediate plant defense against pathogens and insects. Yet the oomycete Phytophthora palmivora is a destructive pathogen that infects all parts of papaya plants, suggesting that it has evolved cysteine protease inhibitors to inhibit papain to enable successful infection. Out of five putative extracellular cystatin-like cysteine protease inhibitors (PpalEPICs) from P. palmivora transcriptomic sequence data, PpalEPIC8 appeared to be unique to P. palmivora and was highly induced during infection of papaya. Purified recombinant PpalEPIC8 strongly inhibited papain enzyme activity, suggesting that it is a functional cysteine protease inhibitor. Homozygous PpalEPIC8 mutants were generated using CRISPR/Cas9-mediated gene editing via Agrobacterium-mediated transformation (AMT). Increased papain sensitivity of in-vitro growth and reduced pathogenicity during infection of papaya fruits were observed for the mutants compared with the wild-type strain, suggesting that PpalEPIC8, indeed, plays a role in P. palmivora virulence by inhibiting papain. This study provided genetic evidence demonstrating that plant-pathogenic oomycetes secrete cystatins as important weapons to invade plants. It also established an effective gene-editing system for P. palmivora by the combined use of CRISPR/Cas9 and AMT, which is expected to be applicable to other oomycetes.

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