Mary B. Olive

Cathepsin B Inhibition Limits Bone Metastasis in Breast Cancer

Metastasis to bone is a major cause of morbidity in breast cancer patients, emphasizing the importance of identifying molecular drivers of bone metastasis for new therapeutic targets. The endogenous cysteine cathepsin inhibitor stefin A is a suppressor of breast cancer metastasis to bone that is coexpressed with cathepsin B in bone metastases. In this study, we used the immunocompetent 4T1.2 model of breast cancer which exhibits spontaneous bone metastasis to evaluate the function and therapeutic targeting potential of cathepsin B in this setting of advanced disease. Cathepsin B abundancy in the model mimicked human disease, both at the level of primary tumors and matched spinal metastases. RNA interference-mediated knockdown of cathepsin B in tumor cells reduced collagen I degradation in vitro and bone metastasis in vivo. Similarly, intraperitoneal administration of the highly selective cathepsin B inhibitor CA-074 reduced metastasis in tumor-bearing animals, a reduction that was not reproduced by the broad spectrum cysteine cathepsin inhibitor JPM-OEt. Notably, metastasis suppression by CA-074 was maintained in a late treatment setting, pointing to a role in metastatic outgrowth. Together, our findings established a prometastatic role for cathepsin B in distant metastasis and illustrated the therapeutic benefits of its selective inhibition in vivo.

Bone marrow-derived cathepsin K cleaves SPARC in bone metastasis

Bone metastasis is a hallmark of advanced prostate and breast cancers, yet the critical factors behind attraction of tumors to the skeleton have not been validated. Here, we investigated the involvement of cathepsin K in the progression of prostate tumors in the bone, which occurs both by direct degradation of bone matrix collagen I and by cleavage of other factors in the bone microenvironment. Our results demonstrated that bone marrow-derived cathepsin K is capable of processing and thereby modulating SPARC, a protein implicated in bone metastasis and inflammation. The coincident up-regulation of SPARC and cathepsin K occurred both in vivo in experimental prostate bone tumors, and in vitro in co-cultures of bone marrow stromal cells with PC3 prostate carcinoma cells. PC3-bone marrow stromal cell interaction increased secretion and processing of SPARC, as did co-cultures of bone marrow stromal cells with two other cancer cell lines. In addition, bone marrow stromal cells that were either deficient in cathepsin K or treated with cathepsin K inhibitors had significantly reduced secretion and cleavage of SPARC. Increases in secretion of pro-inflammatory cytokines (ie, interleukin-6, -8) coincident with overexpression of cathepsin K suggest possible mechanisms by which this enzyme contributes to tumor progression in the bone. This is the first study implicating bone marrow cathepsin K in regulation of biological activity of SPARC in bone metastasis.

Chiral porphyrazine near-IR optical imaging agent exhibiting preferential tumor accumulation

A chiral porphyrazine (pz), H2[pz(trans-A2B2)] (247), has been prepared that exhibits preferential in vivo accumulation in the cells of tumors. Pz 247 exhibits near-infrared (NIR) emission with λ > 700 nm in the required wavelength range for maximum tissue penetration. When MDA-MB-231 breast tumor cells are treated with 247, the agent shows strong intracellular fluorescence with an emission maximum, 704 nm, which indicates that it localizes within a hydrophobic microenvironment. Pz 247 is shown to associate with the lipophilic core of LDL and undergo cellular entry primarily through receptor-mediated endocytosis accumulating in lysosomes. Preliminary in vivo studies show that 247 exhibits preferential accumulation and retention in the cells of MDA-MB-231 tumors subcutaneously implanted in mice, thereby enabling NIR optical imaging with excellent contrast between tumor and surrounding tissue. The intensity of fluorescence from 247 within the tumor increases over time up to 48 h after injection presumably due to the sequestration of circulating 247/LDL complex by the tumor tissue. As the need for cholesterol, and thus LDL, is elevated in highly proliferative tumor cells over nontumorigenic cells, 247 has potential application for all such tumors.

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. Curr. Protoc. Cell Biol. 39:4.20.1‐4.20.15.

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

Pre- and postnatal expressions of NMDA receptors 1 and 2B subunit proteins in the normal rat cortex.

The NMDA receptor is one of the ionotropic glutamate receptors essential for excitatory neurotransmission and synaptic plasticity. The present study examined the ontogenies of NMDAR1 and NMDAR2B during development (embryonic days: (E) 14, 15, 16, 17 and postnatal days: (P) 0, 5, 10, 20, and 60). The immunoreactivities of both NMDAR1 and NMDAR2B were visualized and semi- quantified simultaneously using batch-matched procedures during the developmental period. In addition, the heteromeric coassembly of NMDAR1 (NR1) and NMDAR2B (NR2B) was analyzed throughout development using immunocoprecipitation blots. Differential developmental expressions were observed between the two NMDA receptor subunits. Although both NR1 and NR2B were already expressed on cortical neurons at E14, NR1 immunoreactivity was lower than NR2B but increased up to P20 then was lower at the last sampling day (P60). By comparison, the high NR2B immunoreactivity at E14 increased until P5, and until the next sample day (P60). Despite the early embryonic protein expressions in prenatal cortex both NR1 and NR2B immunocoprecipitation analyses showed that NR1-NR2B coassembly was not initially detectable until birth (P0), and increased up to P20. This suggests that the physically interactive NMDAR1-2B complex may contribute to the plasticity of early postnatal development characterized by axonal dendritic synaptogenesis.

Identification and functional impact of homo-oligomers of the human proton-coupled folate transporter

Background: The human proton-coupled folate transporter (PCFT) may exist as homo-oligomers. Results: PCFT monomers form oligomers, and wild-type and inactive mutant PCFT monomers show dominant-positive activity when co-expressed. Conclusion: Wild-type PCFT may rescue the mutant phenotype via formation of hetero-oligomers. Significance: Better understanding of PCFT oligomerization may identify therapeutic applications for hereditary folate maladsorption and delivery of PCFT-targeted chemotherapy drugs for cancer. The proton-coupled folate transporter (PCFT; SLC46A1) is a proton-folate symporter that is abundantly expressed in solid tumors and normal tissues, such as duodenum. The acidic pH optimum for PCFT is relevant to intestinal absorption of folates and could afford a means of selectively targeting tumors with novel cytotoxic antifolates. PCFT is a member of the major facilitator superfamily of transporters. Because major facilitator superfamily members exist as homo-oligomers, we tested this for PCFT because such structures could be significant to PCFT mechanism and regulation. By transiently expressing PCFT in reduced folate carrier- and PCFT-null HeLa (R1-11) cells and chemical cross-linking with 1,1-methanediyl bismethanethiosulfonate and Western blotting, PCFT species with molecular masses approximating those of the PCFT dimer and higher order oligomers were detected. Blue native polyacrylamide gel electrophoresis identified PCFT dimer, trimer, and tetramer forms. PCFT monomers with hemagglutinin and His10 epitope tags were co-expressed in R1-11 cells, solubilized, and bound to nickel affinity columns, establishing their physical associations. Co-expressing YPet and ECFP*-tagged PCFT monomers enabled transport and fluorescence resonance energy transfer in plasma membranes of R1-11 cells. Combined wild-type (WT) and inactive mutant P425R PCFTs were targeted to the cell surface by surface biotinylation/Western blots and confocal microscopy and functionally exhibited a “dominant-positive” phenotype, implying positive cooperativity between PCFT monomers and functional rescue of mutant by WT PCFT. Our results demonstrate the existence of PCFT homo-oligomers and imply their functional and regulatory impact. Better understanding of these higher order PCFT structures may lead to therapeutic applications related to folate uptake in hereditary folate malabsorption, and delivery of PCFT-targeted chemotherapy drugs for cancer.

Maspin reprograms the gene expression profile of prostate carcinoma cells for differentiation.

Maspin is an epithelial-specific tumor suppressor gene. Previous data suggest that maspin expression may redirect poorly differentiated tumor cells to better differentiated phenotypes. Further, maspin is the first and only endogenous polypeptide inhibitor of histone deacetylase 1 (HDAC1) identified thus far. In the current study, to address what central program of tumor cell redifferentiation is regulated by maspin and how tumor microenvironments further define the effects of maspin, we conducted a systematic and extensive comparison of prostate tumor cells grown in 2-dimensional culture, in 3-dimensional collagen I culture, and as in vivo bone tumors. We showed that maspin was sufficient to drive prostate tumor cells through a spectrum of temporally and spatially polarized cellular processes of redifferentiation, a reversal of epithelial-to-mesenchymal transition (EMT). Genes commonly regulated by maspin were a small subset of HDAC target genes that are closely associated with epithelial differentiation and TGFβ signaling. These results suggest that a specific endogenous HDAC inhibitor may regulate one functionally related subset of HDAC target genes, although additional maspin-induced changes of gene expression may result from tumor interaction with its specific microenvironments. Currently, EMT is recognized as a critical step in tumor progression. To this end, our current study uncovered a link between maspin and a specific mechanism of prostate epithelial differentiation that can reverse EMT.

Full-length human placental sFlt-1-e15a isoform induces distinct maternal phenotypes of preeclampsia in mice

Objective Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring. Methods Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia. Results Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3±51.7μg/mg vs. 19.3±5.6μg/mg, p = 4.4x10-2; GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2x10-2). Placental and fetal weights did not differ between the groups. One mouse with liver disease developed early-onset preeclampsia-like symptoms with intrauterine growth restriction (IUGR). Conclusions A mouse model of late-onset preeclampsia was developed with the overexpression of hsFlt-1-e15a, verifying the in vivo pathologic effects of this primate-specific, predominant placental sFlt-1 isoform. HsFlt-1-e15a induced early-onset preeclampsia-like symptoms associated with IUGR in a mouse with a liver disease. Our findings support that hsFlt-1-e15a is central to the terminal pathway of preeclampsia, and it can induce the full spectrum of symptoms in this obstetrical syndrome.