The proinflammatory cytokines interleukin-1α and tumor necrosis factor α promote the expression and secretion of proteolytically active cathepsin S from human chondrocytes.

Osteoarthritis and rheumatoid arthritis are destructive joint diseases that involve the loss of articular cartilage. Degradation of cartilage extracellular matrix is believed to occur due to imbalance between the catabolic and anabolic processes of resident chondrocytes. Previous work has suggested that various lysosomal cysteine cathepsins participate in cartilage degeneration; however, their exact roles in disease development and progression have not been elucidated. In order to study degradation processes under conditions resembling the in vivo milieu of the cartilage, we cultivated chondrocytes on a type II collagen-containing matrix. Stimulation of the cultivated chondrocytes with interleukin-1α and/or tumor necrosis factor α resulted in a time-dependent increase in cathepsin S expression and induced its secretion into the conditioned media. Using a novel bioluminescent activity-based probe, we were able to demonstrate a significant increase in proteolytic activity of cathepsin S in the conditioned media of proinflammatory cytokine-stimulated chondrocytes. For the first time, cathepsin S was demonstrated to be secreted from chondrocytes upon stimulation with the proinflammatory cytokines, and displayed proteolytic activity in culture supernatants. Its stability at neutral pH and potent proteolytic activity on extracellular matrix components mean that cathepsin S may contribute significantly to cartilage degradation and may thus be considered a potential drug target in joint diseases.

Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics.

The worldwide COVID-19 pandemic caused by the SARS-CoV-2 betacoronavirus has highlighted the need for a synthetic biology approach to create reliable and scalable sources of viral antigen for uses in diagnostics, therapeutics and basic biomedical research. Here, we adapt plasmid-based systems in the eukaryotic microalgae Phaeodactylum tricornutum to develop an inducible overexpression system for SARS-CoV-2 proteins. Limiting phosphate and iron in growth media induced expression of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein from the P. tricornutum HASP1 promoter in the wild-type strain and in a histidine auxotrophic strain that alleviates the requirement for antibiotic selection of expression plasmids. The RBD was purified from whole cell extracts (algae-RBD) with yield compromised by the finding that 90-95% of expressed RBD lacked the genetically encoded C-terminal 6X-histidine tag. Constructs that lacked the TEV protease site between the RBD and C-terminal 6X-histidine tag retained the tag, increasing yield. Purified algae-RBD was found to be N-linked glycosylated by treatment with endoglycosidases, was cross-reactive with anti-RBD polyclonal antibodies, and inhibited binding of recombinant RBD purified from mammalian cell lines to the human ACE2 receptor. We also show that the algae-RBD can be used in a lateral flow assay device to detect SARS-CoV-2 specific IgG antibodies from donor serum at sensitivity equivalent to assays performed with RBD made in mammalian cell lines. Our study shows that P. tricornutum is a scalable system with minimal biocontainment requirements for the inducible production of SARS-CoV-2 or other coronavirus antigens for pandemic diagnostics.

Bone microenvironment modulates expression and activity of cathepsin B in prostate cancer.

Prostate cancers metastasize to bone leading to osteolysis. Here we assessed proteolysis of DQ-collagen I (a bone matrix protein) and, for comparison, DQ-collagen IV, by living human prostate carcinoma cells in vitro. Both collagens were degraded, and this degradation was reduced by inhibitors of matrix metallo, serine, and cysteine proteases. Because secretion of the cysteine protease cathepsin B is increased in human breast fibroblasts grown on collagen I gels, we analyzed cathepsin B levels and secretion in prostate cells grown on collagen I gels. Levels and secretion were increased only in DU145 cells--cells that expressed the highest baseline levels of cathepsin B. Secretion of cathepsin B was also elevated in DU145 cells grown in vitro on human bone fragments. We further investigated the effect of the bone microenvironment on cathepsin B expression and activity in vivo in a SCID-human model of prostate bone metastasis. High levels of cathepsin B protein and activity were found in DU145, PC3, and LNCaP bone tumors, although the PC3 and LNCaP cells had exhibited low cathepsin B expression in vitro. Our results suggest that tumor-stromal interactions in the context of the bone microenvironment can modulate the expression of the cysteine protease cathepsin B.

Postsurgical adjuvant or metastatic renal cell carcinoma therapy models reveal potent antitumor activity of metronomic oral topotecan with pazopanib.

Renal cell carcinoma (RCC), normally considered an intrinsically chemotherapy-resistant cancer, is currently treated with targeted biologic therapies, including antiangiogenic tyrosine kinase inhibitors (TKIs), such as pazopanib. The efficacy of these agents is limited by both intrinsic and acquired resistance. Death is almost always due to advanced metastatic disease, a treatment circumstance seldom modeled in preclinical (mouse) drug testing. Similarly, therapy results using postsurgical adjuvant therapy models of microscopic disease have not been reported. Using in vivo selection and transfection of established human RCC cell lines (786-0 and SN12-PM6), we derived clonal luciferase-expressing variants capable of spontaneous metastasis from an orthotopic primary tumor to organs typical of clinical RCC, including bone, lungs, and brain. The bioluminescence and consistent metastatic spread of von Hippel-Lindau-wild type SN12-PM6-1 cells allowed for the establishment of perioperative therapy models of RCC. We report that the combination of daily low-dose metronomic topotecan with pazopanib has highly potent antiprimary tumor as well as both postsurgical adjuvant and metastatic therapy efficacy despite lack of an antimetastatic effect of pazopanib monotherapy. The combination therapy resulted in sustained metastatic tumor cell dormancy, but tumor progression occurred upon treatment cessation. We also obtained evidence for a direct effect of pazopanib on RCC cells, resulting in increased intracellular concentration of topotecan. Our results suggest that this type of treatment combination should be considered for clinical evaluation in early- or late-stage metastatic disease, even for tumors seemingly intrinsically "resistant" to antiangiogenic TKIs or chemotherapy.

Neoadjuvant antiangiogenic therapy reveals contrasts in primary and metastatic tumor efficacy.

Thousands of cancer patients are currently in clinical trials evaluating antiangiogenic therapy in the neoadjuvant setting, which is the treatment of localized primary tumors prior to surgical intervention. The rationale is that shrinking a tumor will improve surgical outcomes and minimize growth of occult micrometastatic disease-thus delaying post-surgical recurrence and improving survival. But approved VEGF pathway inhibitors have not been tested in clinically relevant neoadjuvant models that compare pre- and post-surgical treatment effects. Using mouse models of breast, kidney, and melanoma metastasis, we demonstrate that primary tumor responses to neoadjuvant VEGFR TKI treatment do not consistently correlate with improved post-surgical survival, with survival worsened in certain settings. Similar negative effects did not extend to protein-based VEGF pathway inhibitors and could be reversed with altered dose, surgical timing, and treatment duration, or when VEGFR TKIs are combined with metronomic 'anti-metastatic' chemotherapy regimens. These studies represent the first attempt to recapitulate the complex clinical parameters of neoadjuvant therapy in mice and identify a novel tool to compare systemic antiangiogenic treatment effects on localized and disseminated disease.

Fibroblast hepatocyte growth factor promotes invasion of human mammary ductal carcinoma in situ.

Stromal-derived hepatocyte growth factor (HGF) acting through its specific proto-oncogene receptor c-Met has been suggested to play a paracrine role in the regulation of tumor cell migration and invasion. The transition from preinvasive ductal carcinoma in situ (DCIS) to invasive breast carcinoma is marked by infiltration of stromal fibroblasts and the loss of basement membrane. We hypothesized that HGF produced by the infiltrating fibroblasts may alter proteolytic pathways in DCIS cells, and, to study this hypothesis, established three-dimensional reconstituted basement membrane overlay cocultures with two human DCIS cell lines, MCF10.DCIS and SUM102. Both cell lines formed large dysplastic structures in three-dimensional cultures that resembled DCIS in vivo and occasionally developed invasive outgrowths. In coculture with HGF-secreting mammary fibroblasts, the percentage of DCIS structures with invasive outgrowths was increased. Activation of c-Met with conditioned medium from HGF-secreting fibroblasts or with recombinant HGF increased the percentage of DCIS structures with invasive outgrowths, their degradation of collagen IV, and their secretion of urokinase-type plasminogen activator and its receptor. In agreement with the in vitro findings, coinjection with HGF-secreting fibroblasts increased invasiveness of MCF10.DCIS xenografts in severe combined immunodeficient mice. Our study shows that paracrine HGF/c-Met signaling between fibroblasts and preinvasive DCIS cells enhances the transition to invasive carcinomas and suggests that three-dimensional cocultures are appropriate models for testing therapeutics that target tumor microenvironment-enhanced invasiveness.

Imaging and quantifying the dynamics of tumor-associated proteolysis.

The roles of proteases in cancer are dynamic. Furthermore, the roles or functions of any one protease may differ from one stage of cancer to another. Proteases from tumor-associated cells (e.g., fibroblasts, inflammatory cells, endothelial cells) as well as from tumor cells make important contributions to 'tumor proteolysis'. Many tumors exhibit increases in expression of proteases at the level of transcripts and protein; however, whether those proteases play causal roles in malignant progression is known for only a handful of proteases. What the critical substrate or substrates that are cleaved in vivo by any given protease is also known for only a few proteases. Therefore, the recent development of techniques and reagents for live cell imaging of protease activity, in conjunction with informed knowledge of critical natural substrates, should help to define protease functions. Here we describe live cell assays for imaging proteolysis, protocols for quantifying proteolysis and the use of such assays to follow the dynamics of proteolysis by tumor cells alone and tumor cells interacting with other cells found in the tumor microenvironment. In addition, we describe an in vitro model that recapitulates the architecture of the mammary gland, a model designed to determine the effects of dynamic interactions with the surrounding microenvironment on 'tumor proteolysis' and the respective contributions of various cell types to 'tumor proteolysis'. The assays and models described here could serve as screening platforms for the identification of proteolytic pathways that are potential therapeutic targets and for further development of technologies and imaging probes for in vivo use.

Visualizing protease activity in living cells: from two dimensions to four dimensions.

Proteolytic degradation of extracellular matrix (ECM) components by cells is an important metabolic activity as cells grow, remodel, and migrate through the ECM. The ability to analyze ECM degradation can be valuable in the study of developmental processes as well as pathologies, such as cancer. In this unit we describe an in vitro live cell-based method to image and quantitatively measure the degradation of ECM components by live cells. Cells are grown in the presence of fluorescent dye-quenched protein substrates (DQ-gelatin, DQ-collagen I, and DQ-collagen IV) that are mixed with protein matrices. Upon proteolytic cleavage, fluorescence is released that directly reflects the level of proteolysis by the cells. Using confocal microscopy and advanced imaging software, the fluorescence is detected and accurate measurements of proteolytic degradation in three and four dimensions can be assessed.

Requirement for microtubules and dynein motors in the earliest stages of peroxisome biogenesis.

Our aim was to determine the role of microtubules in the biogenesis of peroxisomes. Fusion experiments between human PEX16- and PEX1-mutant cells in the presence of nocodazol implied that microtubules were not required for import of proteins into the peroxisomal matrix after cell fusion complementation. We further studied the importance of microtubules in the early stages of peroxisome biogenesis following the microinjection complementation of PEX16-mutant cells. In the absence of nocodazol, nuclear microinjection of plasmids expressing EGFP-SKL and Pex16p in PEX16-mutant cells resulted in the accumulation of EGFP-SKL into newly formed peroxisomes. However, pretreatment of the cells with nocodazol, prior to microinjection, resulted in the inhibition of complementation of the PEX16 mutant and the cytosolic location of the EGFP-SKL. In addition, coexpression of a dominant-negative CC1 subunit of the dynein/dynactin motor complex resulted in the inability to complement PEX16-mutant cells. Both of these treatments resulted in the cytosolic localization of expressed Pex16p. Our results demonstrate that the formation of peroxisomes via the preperoxisomal compartment is dependent upon microtubules and minus-end-directed motor proteins and that the inhibition described above occurs at a step that precedes the association of Pex16p with the vesicles that would otherwise become the peroxisomes.

Dynamic imaging of protease activity with fluorescently quenched activity-based probes.

Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.

Cysteine cathepsins in human cancer.

Proteases play causal roles in the malignant progression of human tumors. This review centers on the roles in this process of cysteine cathepsins, i.e., peptidases belonging to the papain family (C1) of the CA clan of cysteine proteases. Cysteine cathepsins, most likely along with matrix metalloproteases (MMPs) and serine proteases, degrade the extracellular matrix, thereby facilitating growth and invasion into surrounding tissue and vasculature. Studies on tumor tissues and cell lines have shown changes in expression, activity and distribution of cysteine cathepsins in numerous human cancers. Molecular, immunologic and pharmacological strategies to modulate expression and activity of cysteine cathepsins have provided evidence for a causal role for these enzymes in tumor progression and invasion. Clinically, the levels, activities and localization of cysteine cathepsins and their endogenous inhibitors have been shown to be of diagnostic and prognostic value. Understanding the roles that cysteine proteases play in cancer could lead to the development of more efficacious therapies.

Protein structure and import into the peroxisomal matrix.

Proteins destined for the peroxisomal matrix are synthesized in the cytosol, and imported post-translationally. It has been previously demonstrated that stably folded proteins are substrates for peroxisomal import. Mammalian peroxisomes do not contain endogenous chaperone molecules. Therefore, it is possible that proteins are required to fold into their stable, tertiary conformation in order to be imported into the peroxisome. These investigations were undertaken to determine whether proteins rendered incapable of folding were also substrates for import into peroxisomes. Reduction of albumin resulted in a less compact tertiary structure as measured by analytical centrifugation. Microinjection of unfolded albumin molecules bearing the PTS1 targeting signal resulted in their import into peroxisomes. Kinetic analysis indicated that native and unfolded molecules were imported into peroxisomes at comparable rates. While import was unaffected by treatment with cycloheximide, hsc70 molecules were observed to be imported along with the unfolded albumin molecules. These results indicate that proteins, which are incapable of assuming their native conformation, are substrates for peroxisomal import. When combined with previous observations demonstrating the import of stably folded proteins, these results support the model that tertiary structure has no effect on protein import into the peroxisomal matrix.