Investigation of Phosphate Pro-Drug Led Inhibition of ENO2 in ENO1-Deleted Cells

https://openworks.mdanderson.org/sumexp22/1075/thumbnail.jp

An enolase inhibitor for the targeted treatment of ENO1-deleted cancers

https://openworks.mdanderson.org/sumexp21/1197/thumbnail.jp

Transcriptional Activation by NFκB Increases Perlecan/HSPG2 Expression in the Desmoplastic Prostate Tumor Microenvironment

Perlecan/HSPG2, a heparan sulfate proteoglycan typically found at tissue borders including those separating epithelia and connective tissue, increases near sites of invasion of primary prostatic tumors as previously shown for other proteins involved in desmoplastic tissue reaction. Studies of prostate cancer cells and stromal cells from both prostate and bone, the major site for prostate cancer metastasis, showed that cancer cells and a subset of stromal cells increased production of perlecan in response to cytokines present in the tumor microenvironment. In silico analysis of the HSPG2 promoter revealed two conserved NFκB binding sites, in addition to the previously reported SMAD3 binding sites. By systematically transfecting cells with a variety of reporter constructs including sequences up to 2.6 kb from the start site of transcription, we identified an active cis element in the distal region of the HSPG2 promoter, and showed that it functions in regulating transcription of HSPG2. Treatment with TNF-α and/or TGFβ1 identified TNF-α as a major cytokine regulator of perlecan production. TNF-α treatment also triggered p65 nuclear translocation and binding to the HSPG2 regulatory region in stromal cells and cancer cells. In addition to stromal induction of perlecan production in the prostate, we identified a matrix-secreting bone marrow stromal cell type that may represent the source for increases in perlecan in the metastatic bone marrow environment. These studies implicate perlecan in cytokine-mediated, innate tissue responses to cancer cell invasion, a process we suggest reflects a modified wound healing tissue response co-opted by prostate cancer cells

Single molecule force measurements of perlecan/HSPG2: A key component of the osteocyte pericellular matrix

Perlecan/HSPG2, a large, monomeric heparan sulfate proteoglycan (HSPG), is a key component of the lacunar canalicular system (LCS) of cortical bone, where it is part of the mechanosensing pericellular matrix (PCM) surrounding the osteocytic processes and serves as a tethering element that connects the osteocyte cell body to the bone matrix. Within the pericellular space surrounding the osteocyte cell body, perlecan can experience physiological fluid flow drag force and in that capacity function as a sensor to relay external stimuli to the osteocyte cell membrane. We previously showed that a reduction in perlecan secretion alters the PCM fiber composition and interferes with bone's response to a mechanical loading in vivo. To test our hypothesis that perlecan core protein can sustain tensile forces without unfolding under physiological loading conditions, atomic force microscopy (AFM) was used to capture images of perlecan monomers at nanoscale resolution and to perform single molecule force measurement (SMFMs). We found that the core protein of purified full-length human perlecan is of suitable size to span the pericellular space of the LCS, with a measured end-to-end length of 170 ± 20 nm and a diameter of 2–4 nm. Force pulling revealed a strong protein core that can withstand over 100 pN of tension well over the drag forces that are estimated to be exerted on the individual osteocyte tethers. Data fitting with an extensible worm-like chain model showed that the perlecan protein core has a mean elastic constant of 890 pN and a corresponding Young's modulus of 71 MPa. We conclude that perlecan has physical properties that would allow it to act as a strong but elastic tether in the LCS

Interplay of Perlecan and MMP-7/Matrilysin Regulates Metastatic Prostate Cancer Cell Behavior: Basic and Clinical Implications

Perlecan/HSPG2 is a large extracellular heparan sulfate proteoglycan concentrated at tissue borders and separating epithelium and stroma. Along with its proteolytic consumers, the matrix metalloproteinases (MMPs), perlecan helps orchestrate development and homeostasis in nearly all studied multicellular organisms. However, both molecule classes can be coopted by prostate cancer (PCa) to advance the disease to its most deadly metastatic form. This work aimed to understand that relationship both at the basic and clinical level. Perlecan with its HS chains and tight domain structure is generally resistant to proteolysis, but a PCa cell must produce an associated enzyme to cleave the border proteoglycan in order to metastasize. This work was the first to identify an active protease produced by PCa cells that can completely digest intact perlecan. Following in silico proteolytic analysis, matrilysin/MMP-7, was identified as a likely candidate for in vitro assays. MMP-7, unlike other enzymes tested, cleaved perlecan when presented in multiple contexts. Perlecan and a subdomain, domain IV-3 (Dm IV-3), but not other subdomains (Dm I, IV-1 and IV-2), induced a striking clustering phenotype. MMP-7 incubation completely reversed this effect to favor cell dispersion and adhesion. Proteomic signaling arrays point towards global Src kinase activation as a major influence of perlecan DmIV-3 effects. To determine if this perlecan/MMP-7 relationship exists in PCa subjects, I performed a tissue microarray, along with β2-microglobulin (β2M), a GF that binds perlecan and induces MMP secretion. Besides increased levels of the two proteins within the patients (cancer/normal), MMP-7 and perlecan levels statistically correlated in multiple grades and localized at tissue interfaces. Additionally, I developed a new assay probing the perlecan fragment signature in the same PCa subjects’ serum. Perlecan fragments were largely increased in PCa and some of the fragments were associated with MMP-7 expression in the subjects. Overall, this work demonstrates a unique interplay between perlecan and its efficient proteolyzer, MMP-7, a relationship that is relevant from the cell and tissue to the clinic and which is likely to contribute to PCa progression to metastatic lethal disease

Sustained delivery of recombinant human bone morphogenetic protein-2 from perlecan domain I - functionalized electrospun poly (ε-caprolactone) scaffolds for bone regeneration

Background: Biomaterial scaffolds that deliver growth factors such as recombinant human bone morphogenetic proteins-2 (rhBMP-2) have improved clinical bone tissue engineering by enhancing bone tissue regeneration. This approach could be further improved if the controlled delivery of bioactive rhBMP-2 were sustained throughout the duration of osteogenesis from fibrous scaffolds that provide control over dose and bioactivity of rhBMP-2. In nature, heparan sulfate attached to core proteoglycans serves as the co-receptor that delivers growth factors to support tissue morphogenesis. Methods: To mimic this behavior, we conjugated heparan sulfate decorated recombinant domain I of perlecan/HSPG2 onto an electrospun poly(ε-caprolactone) (PCL) scaffold, hypothesizing that the heparan sulfate chains will enhance rhBMP-2 loading onto the scaffold and preserve delivered rhBMP-2 bioactivity. Results: In this study, we demonstrated that covalently conjugated perlecan domain I increased loading capacity of rhBMP-2 onto PCL scaffolds when compared to control unconjugated scaffolds. Additionally, rhBMP-2 released from the modified scaffolds enhanced alkaline phosphatase activity in W20–17 mouse bone marrow stromal cells, indicating the preservation of rhBMP-2 bioactivity indicative of osteogenesis. Conclusions: We conclude that this platform provides a sophisticated and efficient approach to deliver bioactive rhBMP-2 for bone tissue regeneration applications

Additional file 1: Figure S1. of Sustained delivery of recombinant human bone morphogenetic protein-2 from perlecan domain I - functionalized electrospun poly (ε-caprolactone) scaffolds for bone regeneration

Perlecan domain I (Dm1) purification and glycosaminoglycan characterization. Perlecan Dm1 purified from HEK293 cells was incubated alone (lanes 2, 9) or with heparitinases 1, 2, 3 and chondroitinase ABC either together (lanes 3, 10) or separately (lanes 5–8). Lane 1 is the molecular weight marker and Lane 4 is all enzymes without Dm1. On the left is a Coomassie stain (lanes 1–8) and on the right is a western blot (lanes 9–10) using a domain I specific antibody (N-20). The arrow head indicates the glycosylated form of Dm I and the arrow indicates the protein core. All enzymes were incubated at 0.1 Units per 10 μg of Dm 1 in a 20 μL of reaction at 37 °C for 4 h. The buffer was 20 mM Tris-HCl, 10 mM NaCl, and 3 mM calcium acetate at pH 8.0. Figure S2. PInDI modified scaffolds controlled rhBMP-2 cumulative release. The absolute amount of rhBMP-2 released from PlnD1-conjugated or unmodified PCL scaffolds over 23 days. (n = 3) Error bars correspond to standard deviation. Figure S3. DNA concentration of W20–17 cells after exposing to rhBMP-2 released from either PlnD1-conjugated or unmodified PCL scaffolds. Fresh W20–17 cultures were used for each time-point (n = 5). (DOCX 227 kb

Matrilysin/MMP-7 Cleavage of Perlecan/HSPG2 Complexed with Semaphorin 3A Supports FAK-Mediated Stromal Invasion by Prostate Cancer Cells

Interrupting the interplay between cancer cells and extracellular matrix (ECM) is a strategy to halt tumor progression and stromal invasion. Perlecan/heparan sulfate proteoglycan 2 (HSPG2) is an extracellular proteoglycan that orchestrates tumor angiogenesis, proliferation, differentiation and invasion. Metastatic prostate cancer (PCa) cells degrade perlecan-rich tissue borders to reach bone, including the basement membrane, vasculature, reactive stromal matrix and bone marrow. Domain IV-3, perlecan's last 7 immunoglobulin repeats, mimics native proteoglycan by promoting tumoroid formation. This is reversed by matrilysin/matrix metalloproteinase-7 (MMP-7) cleavage to favor cell dispersion and tumoroid dyscohesion. Both perlecan and Domain IV-3 induced a strong focal adhesion kinase (FAK) dephosphorylation/deactivation. MMP-7 cleavage of perlecan reversed this, with FAK in dispersed tumoroids becoming phosphorylated/activated with metastatic phenotype. We demonstrated Domain IV-3 interacts with the axon guidance protein semaphorin 3A (Sema3A) on PCa cells to deactivate pro-metastatic FAK. Sema3A antibody mimicked the Domain IV-3 clustering activity. Direct binding experiments showed Domain IV-3 binds Sema3A. Knockdown of Sema3A prevented Domain IV-3-induced tumoroid formation and Sema3A was sensitive to MMP-7 proteolysis. The perlecan-Sema3A complex abrogates FAK activity and stabilizes PCa cell interactions. MMP-7 expressing cells destroy the complex to initiate metastasis, destroy perlecan-rich borders, and favor invasion and progression to lethal bone disease