Multiple Matrix Metalloproteinases Research Articles

Abstract 462: Developing the miR-888 cluster as a therapeutic target for prostate cancer

Abstract Prostate Cancer (PCa) is a serious health crisis in the United States. 20% of men diagnosed with PCa will progress to fast-growing, advanced disease. There are no curative treatment options for PCa that has spread to distant sites and the 5-year survival rate for metastatic disease remains 30%. Recognizing the need for better PCa therapeutics, we are developing novel anti-microRNA delivery reagents targeted against the noncoding RNA miR-888 cluster to block prostate tumor progression. Our lab identified these oncomiRs in a biomarker discovery screen as elevated in human metastatic PCa cell lines and prostatic fluids from patients with high-grade PCa. We noted that over-expression of miR-888 cluster members (miR-888, miR-891a) promoted prostate cell growth and induced invasiveness in vitro. miR-888 and miR-891a also accelerated xenograft tumor growth in mice. Treatment of aggressive PCa cells in culture with synthetic antisense inhibitors against individual cluster members reversed these growth and invasion phenotypes, highlighting their clinical potential. We are testing pH Low Insertion Peptide (pHLIP)-Peptide Nucleic Acid (PNA)-based antimiR conjugates against miR-888 and miR-891a. The delivery vehicle pHLIP is a 36-amino acid peptide that at low pH (< pH 6.5) adopts an alpha-helical conformational change and facilitates the insertion of its C-terminus across lipid bilayers to transport conjugated anti-miRNA cargo into cells. When administered systemically, pHLIP effectively targets solid tumors with acidic microenvironments. We found that these reagents are readily internalized by human PCa cells cultured at pH 6.0 and in prostate xenograft tumors when delivered systemically in mice. Treatment significantly repressed miR-888 and miR-891a and acted to slow prostate cell growth in vitro. We are currently moving these studies into mouse prostate models. Furthermore, we are investigating the downstream signaling pathways for the miR-888 cluster. Our work indicates that the miR-888 cluster network coordinates the repression of the Tissue Inhibitors of Metalloproteinase (TIMP) and SMAD4 anti-metastatic pathways to drive extracellular matrix degradation and prostate cell invasion, and as a consequence, induces matrix metalloproteinase (MMP) activity. Expression profiling arrays and luciferase reporter assays indicated that multiple members of the miR-888 cluster co-suppress TIMPs in the prostate and similarly elevate multiple MMPs as well as cadherins, fibronectin and heparinase, and co-modulated several immune response genes. We are currently studying the consequence of various CRISPR-mediated miR-888 cluster deletions to understand how this non-coding RNA cluster functionally overlaps to promote aggressive PCa. This work will establish new clinical tools for aggressive PCa and optimize patient treatment management. Citation Format: Katherine Routon, Trevor Fachko, Vishal Kasina, Raman Bahal, Aurora Esquela Kerscher. Developing the miR-888 cluster as a therapeutic target for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 462.

Development of a peptide microarray-based metal-enhanced fluorescence assay for ultrasensitive detection of multiple matrix metalloproteinase activities by using a gold nanorod-polymer substrate

Matrix metalloproteinases (MMPs) are attractive biomarkers for cancer diagnosis and treatment, while it is still a challenge to precise analysis of MMP activities owing to their very low abundance in the biological samples, especially at the early stages of tumors. Herein, a peptide microarray-based metal-enhanced fluorescence assay (PMMEFA) is proposed to simultaneously detect MMP-1, -2, -3, -7, -9, and -13 activities. The assay involves immobilization of Förster resonance energy transfer dye pair decorated peptides (FRET-peptides) on a poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) coated gold nanorod modified glass slide (GNR@P(GMA-HEMA)). To fabricate the GNR@P(GMA-HEMA) slide, GNRs are self-assembled onto an aminated glass slide, and a polymer brush (P(GMA-HEMA)) is grown through a surface-initiated atom transfer radical polymerization reaction (SI-ATRP). Upon the addition of MMPs, the FRET pairs are broken due to the specific cleavage of FRET-peptides by enzymes, resulting in the recovery of fluorescence signals and further enhancement by the MEF of GNRs. The fluorescence recovery degree provides a direct indicator for MMP activity. The PMMEFA exhibits excellent sensitivity, which enables to detect MMP-1, -2, -3, -7, -9, and -13 activities, with low limits of detection (LODs) of 1.7 fg mL−1, 0.3 fg mL−1, 2.0 fg mL−1, 1.8 fg mL−1, 2.2 fg mL−1 and 14.0 fg mL−1, respectively. To substantiate the practicability of PMMEFA, MMP activities were measured in a range of matrices, encompassing cell culture medium, serum, and tumor tissue homogenate, and MMP activities can be detected only in 0.15 μL serum and 0.025 mg tumor tissue.

Abstract 3601: Tumor MK2 signaling regulates cell migration and invasion in head and neck squamous cell carcinoma

Abstract Poor 5-year overall survival in head and neck cancer (HNC) can be attributed to high rates of locoregional and distant metastasis. We have previously demonstrated that high phosphorylation/activation of MAPK-activated protein kinase 2 (MK2), a stress-activated kinase directly downstream of p38 MAPK, in head and neck squamous cell carcinoma (HNSCC) is associated with worse overall survival and inhibition of the MK2 pathway enhances in vivo HNSCC radiosensitivity. Our prior work also demonstrates that radiotherapy-induced epithelial-to-mesenchymal transition (EMT) gene expression can be suppressed in cells when MK2 is inhibited. Several studies have shown that EMT plays a prominent role in HNSCC tumor invasion, treatment resistance, and locoregional metastases to lymph nodes. In this study, we investigate the impact of MK2 activity on HNSCC tumor progression by examining its contribution to cancer cell morphology. We used lentiviral-shRNA and the CRISPR-Cas9 system to knock down/out the MK2 gene in multiple human and murine HNSCC cell lines. We measured tumor cell migration and invasion in both classic 2D in vitro culture as well as with 3D spheroids. We performed unbiased analysis via RNAseq in human HNSCC cell lines comparing MK2 shRNA vs scramble cell lines. Substantial gene and pathway alterations were examined using a combination of immunoblot and RT-qPCR. Our data shows knocking down/out MK2 in HNSCC cells significantly diminishes cell migration and invasion in 2D and 3D in vitro culture assays. RNAseq analysis demonstrated loss of MK2 leads to a significant increase in many cell adhesion molecules, including E-cadherin, and a decrease in N-cadherin, suggesting a shift from mesenchymal to more epithelial phenotype. Furthermore, we note expression of multiple matrix metalloproteinases and EMT markers (i.e., Snail) are substantially reduced in MK2 shRNA and KO cell lines. These results indicate that the MK2 pathway is involved in HNSCC motility and invasion. We also observe the loss of MK2 contributes to a shift in the epithelial:mesenchymal ratio potentially by suppressing cancer EMT plasticity. All in all, this study provides evidence suggesting tumor MK2 activation mediates HNC progression. Citation Format: Dakota D. Okwuone, Deri Morgan, Hannah M. Smith, Grace Millington, Kiersten L. Berggren, Christopher E. Lominska, Sufi M. Thomas, Gregory N. Gan. Tumor MK2 signaling regulates cell migration and invasion in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3601.

Utilizing genetic code expansion to modify N-TIMP2 specificity towards MMP-2, MMP-9, and MMP-14

Matrix metalloproteinases (MMPs) regulate the degradation of extracellular matrix (ECM) components in biological processes. MMP activity is controlled by natural tissue inhibitors of metalloproteinases (TIMPs) that non-selectively inhibit the function of multiple MMPs via interaction with the MMPs' Zn2+-containing catalytic pocket. Recent studies suggest that TIMPs engineered to confer MMP specificity could be exploited for therapeutic purposes, but obtaining specific TIMP-2 inhibitors has proved to be challenging. Here, in an effort to improve MMP specificity, we incorporated the metal-binding non-canonical amino acids (NCAAs), 3,4-dihydroxyphenylalanine (L-DOPA) and (8-hydroxyquinolin-3-yl)alanine (HqAla), into the MMP-inhibitory N-terminal domain of TIMP2 (N-TIMP2) at selected positions that interact with the catalytic Zn2+ ion (S2, S69, A70, L100) or with a structural Ca2+ ion (Y36). Evaluation of the inhibitory potency of the NCAA-containing variants towards MMP-2, MMP-9 and MMP-14 in vitro revealed that most showed a significant loss of inhibitory activity towards MMP-14, but not towards MMP-2 and MMP-9, resulting in increased specificity towards the latter proteases. Substitutions at S69 conferred the best improvement in selectivity for both L-DOPA and HqAla variants. Molecular modeling provided an indication of how MMP-2 and MMP-9 are better able to accommodate the bulky NCAA substituents at the intermolecular interface with N-TIMP2. The models also showed that, rather than coordinating to Zn2+, the NCAA side chains formed stabilizing polar interactions at the intermolecular interface with MMP-2 and MMP-9. Our findings illustrate how incorporation of NCAAs can be used to probe—and possibly exploit—differential tolerance for substitution within closely related protein–protein complexes as a means to improve specificity.

Abstract PR006: The racial disparity-linked gene CRYβB2 alters cell adhesion and tumor microenvironment to promote proliferation and invasion in triple-negative breast cancer

Abstract Background: Clinical and epidemiological data showed that African American (AA) women suffer higher incidence and mortality rates due to triple-negative breast cancer (TNBC) compared to European American (EA) women. These disparate disease outcomes are attributed to multiple factors including socioeconomic status, access to health care, and earlier disease onset. However, a growing body of evidence also shows the existence of different gene expression signatures and tumor microenvironments between these patient racial groups. We and others identified CRYβB2 as a cancer racial disparity gene that has higher expression in AA patients with breast, colon, and prostate cancers. We previously demonstrated, via in vitro and in vivo studies, that CRYβB2 promotes proliferation, metastasis, and IL-6 signaling. The objective of the current research is to identify the cellular and molecular mechanisms downstream of CRYβB2 leading to its tumor-promoting phenotypes. Methods: CRISPR/Cas9 and lentiviral knock-in gene editing tools were used to create TNBC cell lines with CRYβB2 knockout and overexpression respectively. The functional consequences of CRYβB2 were assessed in vitro using colony formation, tumor spheroid invasion, and collagenase activity assays. Additionally, tumor spheroids were grown and RNA sequencing (RNA-seq) was performed to assess the effect of CRYβB2 on gene expression. Results: Herein, functional assays showed that CRYβB2 promotes proliferation and invasion in vitro in both AA and EA cell lines. RNA-seq and Gene Ontology enrichment analyses revealed that CRYβB2 alters key pathways such as cell adhesion and extracellular matrix (ECM) organization. RT-qPCR validation also showed that CRYβB2 inhibits the expression of a specific class of adhesion molecules known as protocadherins (PCDHs). Downregulation of PCDHs has been shown to enhance tumor progression and is a predictive marker for low survival in many cancer types. Indeed, data mining of publicly available data shows that three of the identified PCDHs (PCDHGB5, PCDHGB6 and PCDHGB7) have significantly lower expression levels in breast cancer compared to normal tissue and their low expression leads to low overall survival. Furthermore, RNA-seq data showed that CRYβB2 promotes the expression of multiple matrix metalloproteinase (MMP) genes. This was functionally validated using collagenase activity assay where CRYβB2 enhanced collagen degradation in vitro. Conclusions: Our findings provide a mechanistic explanation for the role of CRYβB2 in driving disparate clinical outcome among AA and EA TNBC patients. We demonstrate a paradigm where high CRYβB2 expression promotes tumor progression by suppressing cell adhesion molecules and increasing ECM degradation to enhance tumor growth, invasion, and metastasis. Disclaimer: This work was prepared while Jodie Fleming was employed at NCCU. The opinions expressed in this article are the author's own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. Citation Format: Amr A. Waly, London Harper, Jodie M. Fleming, Lindsey M. Costantini. The racial disparity-linked gene CRYβB2 alters cell adhesion and tumor microenvironment to promote proliferation and invasion in triple-negative breast cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PR006.

Profiling of Multiple Matrix Metalloproteinases Activities in the Progression of Osteosarcoma by Peptide Microarray-Based Fluorescence Assay on Polymer Brush-Coated Zinc Oxide Nanorod Substrate.

Peptide microarray provides the ability to miniaturize, parallelize, and automate high-throughput screening substrate specificities of enzymes, profiling of multiple enzyme activities, discovery of disease biomarkers, and development of drugs. Matrix metalloproteinases (MMPs) are demonstrated as important biomarkers of tumor invasion and metastasis. Herein, a peptide microarray-based fluorescence assay is proposed to profile multiple MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13) activities in the culture medium of four human osteosarcoma (OS) cells and in the progression of OS by using the mouse-bearing xenograft OSs including U-2OS and Saos-2 human. This method has excellent selectivity and sensitivity, which enables to detect the activities of cellular secreted MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 with limit of detection downs to 10 pM, 30 pM, 113 pM, 13 pM, 93 pM, and 12 pM, respectively. Furthermore, it is demonstrated that the activity pattern of MMPs is serum closely relevant to the disease progression and type of tumor.

Patch grafting of organoids of stem/progenitors into solid organs can correct genetic-based disease states

Patch grafting, a novel strategy for transplantation of stem/progenitor organoids into porcine livers, has been found successful also for organoid transplantation into other normal or diseased solid organs in pigs and mice. Each organoid contained ∼100 cells comprised of biliary tree stem cells (BTSCs), co-hepato/pancreatic stem/progenitors, and partnered with early lineage stage mesenchymal cells (ELSMCs), angioblasts and precursors to endothelia and stellate cells. Patch grafting enabled transplantation into livers or pancreases of ≥108th (pigs) or ≥106th−7th (mice) organoids/patch. Graft conditions fostered expression of multiple matrix-metalloproteinases (MMPs), especially secretory isoforms, resulting in transient loss of the organ's matrix-dictated histological features, including organ capsules, and correlated with rapid integration within a week of organoids throughout the organs and without emboli or ectopic cell distribution. Secondarily, within another week, there was clearance of graft biomaterials, followed by muted expression of MMPs, restoration of matrix-dictated histology, and maturation of donor cells to functional adult fates.The ability of patch grafts of organoids to rescue hosts from genetic-based disease states was demonstrated with grafts of BTSC/ELSMC organoids on livers, able to rescue NRG/FAH-KO mice from type I tyrosinemia, a disease caused by absence of fumaryl acetoacetate hydrolase. With the same grafts, if on pancreas, they were able to rescue NRG/Akita mice from type I diabetes, caused by a mutation in the insulin 2 gene. The potential of patch grafting for cell therapies for solid organs now requires translational studies to enable its adaptation and uses for clinical programs.

Metalloproteinase inhibition reduces AML growth, prevents stem cell loss, and improves chemotherapy effectiveness

AbstractAcute myeloid leukemia (AML) is a blood cancer of the myeloid lineage. Its prognosis remains poor, highlighting the need for new therapeutic and precision medicine approaches. AML symptoms often include cytopenias linked to loss of healthy hematopoietic stem and progenitor cells (HSPCs). The mechanisms behind HSPC decline are complex and still poorly understood. Here, intravital microscopy (IVM) of a well-established experimental model of AML allows direct observation of the interactions between healthy and malignant cells in the bone marrow (BM), suggesting that physical dislodgment of healthy cells by AML through damaged vasculature may play an important role. Multiple matrix metalloproteinases (MMPs), known to remodel extracellular matrix, are expressed by AML cells and the BM microenvironment. We reason MMPs could be involved in cell displacement and vascular leakiness; therefore, we evaluate the therapeutic potential of MMP pharmacological inhibition using the broad-spectrum inhibitor prinomastat. IVM analyses of prinomastat-treated mice reveal reduced vascular permeability and healthy cell clusters in circulation and lower AML infiltration, proliferation, and cell migration. Furthermore, treated mice have increased retention of healthy HSPCs in the BM and increased survival following chemotherapy. Analysis of a human AML transcriptomic database reveals widespread MMP deregulation, and human AML cells show susceptibility to MMP inhibition. Overall, our results suggest that MMP inhibition could be a promising complementary therapy to reduce AML growth and limit HSPC loss and BM vascular damage caused by MLL-AF9 and possibly other AML subtypes.

E2F3 drives the epithelial-to-mesenchymal transition, cell invasion, and metastasis in breast cancer.

E2F3 is a transcription factor that may initiate tumorigenesis if overexpressed. Previously, we demonstrated that E2F3 mRNA is overexpressed in breast cancer and that E2F3 overexpression results in centrosome amplification and unregulated mitosis, which can promote aneuploidy and chromosome instability to initiate and sustain tumors. Further, we demonstrated that E2F3 leads to overexpression of the mitotic regulator Shugoshin-1, which until recently had unknown roles in cancer. This study aims to evaluate the roles of E2F3 and Shugoshin-1 in breast cancer metastatic potential. Here we demonstrated that E2F3 and Shugoshin-1 silencing leads to reduced cell invasion and migration in two mesenchymal triple-negative breast cancer (TNBC) cell lines (MDA-MB-231 and Hs578t). Moreover, E2F3 and Shugoshin-1 modulate the expression of epithelial-to-mesenchymal transition-associated genes such as Snail, E-Cadherin, and multiple matrix metalloproteinases. Furthermore, E2F3 depletion leads to reductions in tumor growth and metastasis in NOD-scid Gamma mice. Results from this study suggest a key role for E2F3 and a novel role for Shugoshin-1 in metastatic progression. These results can further help in the improvement of TNBC targeted therapies by interfering with pathways that intersect with the E2F3 and Shugoshin-1 signaling pathways.

Lung development, repair and cancer: A study on the role of MMP20 gene in adenocarcinoma.

Multiple matrix metalloproteinases have significant roles in tissue organization during lung development, and repair. Imbalance of proteinases may lead to chronic inflammation, changes in tissue structure, and are also highly associated to cancer development. The role of MMP20 is not well studied in lung organogenesis, however, it was previously shown to be present at high level in lung adenocarcinoma. The current study aimed to identify the functional properties of MMP20 on cell proliferation and motility in a lung adenocarcinoma in vitro cell model, and relate the interaction of MMP20 with other molecular signalling pathways in the lung cells after gaining tumoral properties. In this study, two different single guide RNA (sgRNAs) that specifically targeted on MMP20 sites were transfected into human lung adenocarcinoma A549 cells by using CRISPR-Cas method. Following that, the changes of PI3-K, survivin, and MAP-K mRNA gene expression were determined by Real-Time Polymerase Chain Reaction (RT-PCR). The occurrence of cell death was also examined by Acridine Orange/Propidium Iodide double staining. Meanwhile, the motility of the transfected cells was evaluated by wound healing assay. All the data were compared with non-transfected cells as a control group. Our results demonstrated that the transfection of the individual sgRNAs significantly disrupted the proliferation of the A549 cell line through suppression in the gene expression of PI3-K, survivin, and MAP-K. When compared to non-transfected cells, both experimental cell groups showed reduction in the migration rate, as reflected by the wider gaps in the wound healing assay. The current study provided preliminary evidence that MMP20 could have regulatory role on stemness and proliferative genes in the lung tissues and affect the cell motility. It also supports the notion that targeting MMP20 could be a potential treatment mode for halting cancer progression.

085 CCN1-induced age-related dermal microenvironment promotes skin cancer development

Aging is a major risk factor for keratinocyte skin cancer. Here we investigate the impact of Age-Related alterations in the Dermal Microenvironment (ARDM) on keratinocyte skin cancer initiation/development, using a transgenic mouse model of accelerated dermal aging. This model expresses CCN1, a secreted extracellular matrix (ECM) associated protein, driven by the fibroblast collagen1A2 promoter. CCN1 is significantly elevated in dermal fibroblasts in aged human skin and is a key driver of ARDM. By six months of age, Col1a2-CCN1 mice exhibited accelerated dermal aging including dermal thinning (reduced 39%, N=6, p<0.05), loss of collagen protein content and gene expression (reduced 51% and 63%, respectively, N=5, p<0.05), and increased degradation of collagen fibrils, measured by immunostaining and atomic force microscopy, compared to control littermates. Furthermore, Col1a2-CCN1 mice exhibited increased expression of multiple matrix metalloproteinases, hepatocyte growth factor, and cytokines and reduced expression of TGF-β type II receptor (N=5, p<0.05), as observed in aged human skin. Importantly, six-month-old Col1a2-CCN1 mice displayed dramatically increased susceptibility to skin papilloma/tumor formation in two different skin tumor models; two-stage chemical carcinogenesis (N=5) and keratinocyte-targeted inducible oncogenic HRas (N=4-5, p<0.05). In both of these models, multiple papillomas/tumors occurred only in Col1a2-CCN1 mice. No papillomas/tumors were formed in littermate controls. In stark contrast, two-month-old Col1a2-CCN1 mice, before the development of ARDM, were completely resistant to papilloma/tumor formation, similar to controls. These data demonstrate that ARDM creates a tissue milieu that actively promotes keratinocyte cancer. Col1a2-CCN1 mice provide a powerful model for investigating the mechanisms by which age-related changes in the stromal microenvironment contribute to cutaneous carcinoma development in the elderly.

Mycobacterium tuberculosis Peptidyl Prolyl Isomerase A Interacts With Host Integrin Receptor to Exacerbate Disease Progression.

Attenuated intracellular survival of Mycobacterium tuberculosis (Mtb) secretory gene mutants exemplifies their role as virulence factors. Mtb peptidyl prolyl isomerase A (PPiA) assists in protein folding through cis/trans isomerization of prolyl bonds. Here, we show that PPiA abets Mtb survival and aids in disease progression by exploiting host-associated factors. While the deletion of PPiA has no discernable effect on bacillary survival in a murine infection model, it compromises the formation of granuloma-like lesions and promotes host cell death through ferroptosis. Overexpression of PPiA enhances the bacillary load and exacerbates pathology in mice lungs. Importantly, PPiA interacts with the integrin α5β1 receptor through a conserved surface-exposed RGD motif. The secretion of PPiA as well as interaction with integrin contributes to disease progression by upregulating multiple host matrix metalloproteinases. Collectively, we identified a novel nonchaperone role of PPiA that is critical in facilitating host-pathogen interaction and ensuing disease progression.

Profiling of multiple matrix metalloproteinases activities in the progression of osteosarcoma by peptide microarray-based fluorescence assay on polymer brush coated zinc oxide nanorod substrate

Osteosarcoma (OS) is a primary malignant bone tumor with high rate of recurrence and lung metastasis. Matrix metalloproteinases (MMPs) are demonstrated as important biomarkers of tumor invasion and metastasis. Herein, a peptide microarray-based fluorescence assay is proposed to profile multiple MMPs (MMP-1, -2, -3, -7, -9 and -13) activities in the progression of OS by using the mouse-bearing xenograft U-2OS and Saos-2 human OSs. The peptide microarray containing different biotinylated peptide substrate spots is fabricated on the poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) brush coated zinc oxide nanorod ([email protected](GMA-HEMA) decorated glass slides, and labeled by cyanine 3 (Cy3) modified avidin (Avidin-Cy3) to produce fluorescence signal. Special cleavage of peptide substrate by MMP resulted the decrease of fluorescence signal. The MMPs activities are positively correlated with the change of fluorescence intensity. This method has excellent selectivity and sensitivity, which enables to detect the activities of cellular secreted MMP-1, -2, -3, -7, -9, and -13 with limit of detection downs to 10 pmol L−1, 30 pmol L−1, 113 pmol L−1, 13 pmol L−1, 93 pmol L−1 and 12 pmol L−1, respectively. Furthermore, it is demonstrated that the activity pattern of MMPs in serum closely relevant to the disease progression and type of tumor.

Diversity of Mechanisms Underlying Latent TGF-β Activation in Recessive Dystrophic Epidermolysis Bullosa

Injury- and inflammation-driven progressive dermal fibrosis is a severe manifestation of recessive dystrophic epidermolysis bullosa-a genetic skin blistering disease caused by mutations in COL7A1. TGF-β activation plays a prominent part in progressing dermal fibrosis. However, the underlying mechanisms are not fully elucidated. TGF-β is secreted in a latent form, which has to be activated for its biological functions. In this study, we determined that recessive dystrophic epidermolysis bullosa fibroblasts have an enhanced capacity to activate the latent form. Mechanistic and functional assessment demonstrated that this process depends on multiple latent TGF-β activators, including TSP-1, RGD-binding integrins, matrix metalloproteinases, and ROS, which act in concert, in a self-perpetuating feedback loop to progress fibrosis. Importantly, our study also disclosed keratinocytes as prominent facilitators of fibrosis in recessive dystrophic epidermolysis bullosa. They stimulate microenvironmental latent TGF-β activation through enhanced production of the above mediators. Collectively, our study provides data on the molecular mechanism behind dysregulated TGF-β signaling in recessive dystrophic epidermolysis bullosa, which are much needed for the development of evidence-based fibrosis-delaying treatments.

Construction of a sensitive protease sensor with DNA-peptide conjugates for single-molecule detection of multiple matrix metalloproteinases

Protease expression is closely linked to various pathological phenomena, and their accurate quantification is essential to clinical diagnosis and cancer therapy. Herein, we demonstrate for the first time the construction of a sensitive protease sensor by integrating protease-sensitive cleavage with nicking enzyme-assisted signal amplification (NESA) for single-molecule detection of multiple matrix metalloproteinases (MMPs). This protease sensor involves two DNA-peptide conjugates which contain both specific protease cleavage sites and trigger DNAs and two report DNAs which are modified with a fluorophore (Cy3 or Cy5) and a quencher (BHQ2). In the presence of specific MMPs, MMPs-mediated cleavage reactions lead to the release of specific trigger DNAs from the corresponding DNA-peptide conjugates. After the magnetic separation, the resultant trigger DNAs may hybridize with the corresponding report DNAs to initiate the cyclic NESA reaction, releasing large amounts of Cy3/Cy5 fluorescent molecules which can be simply quantified by using total internal reflection fluorescence-based single-molecule detection. Taking advantage of the high specificity of proteolytic cleavage, the high amplification efficiency of cyclic NESA, and the high sensitivity of single-molecule detection, this protease sensor can simultaneously detect multiple MMPs with a detection limit of 3.33 pM for MMP-2 and 1.71 pM for MMP-7, superior to the target peptide-based methods. Moreover, this protease sensor can be applied for the measurement of MMP-2 and MMP-7 in cancer cells and the screening of protease inhibitors, holding great promise in clinic diagnosis and drug discovery.

Intravenous antagomiR-494 lessens brain-infiltrating neutrophils by increasing HDAC2-mediated repression of multiple MMPs in experimental stroke.

Neutrophil infiltration and phenotypic transformation are believed to contribute to neuronal damage in ischemic stroke. Emerging evidence suggests that histone deacetylase 2 (HDAC2) is an epigenetic regulator of inflammatory cells. Here, we aimed to investigate whether microRNA-494 (miR-494) affects HDAC2-mediated neutrophil infiltration and phenotypic shift. MiR-494 levels in neutrophils from acute ischemic stroke (AIS) patients were detected by real-time PCR. Chromatin Immunoprecipitation (ChIP)-Seq was performed to clarify which genes are the binding targets of HDAC2. Endothelial cells and cortical neurons were subjected to oxygen-glucose deprivation (OGD), transwell assay was conducted to examine neutrophil migration through endothelial cells, and neuronal injury was examined after stimulating with supernatant from antagomiR-494-treated neutrophils. C57BL/6J mice were subjected to transient middle cerebral artery occlusion (MCAO) and antagomiR-494 was injected through tail vein immediately after reperfusion, and neutrophil infiltration and phenotypic shift was examined. We found that the expression of miR-494 in neutrophils was significantly increased in AIS patients. HDAC2 targeted multiple matrix metalloproteinases (MMPs) and Fc-gamma receptor III (CD16) genes in neutrophils of AIS patients. Furthermore, antagomiR-494 repressed expression of multiple MMPs genes, including MMP7, MMP10, MMP13, and MMP16, which reduced the number of brain-infiltrating neutrophils by regulating HDAC2. AntagomiR-494 could also exert its neuroprotective role through inhibiting the shift of neutrophils toward pro-inflammatory N1 phenotype in vivo and in vitro. Taken together, miR-494 may serve as an alternative predictive biomarker of the outcome of AIS patients, and antagomiR-494 treatment decreases the expression of multiple MMPs and the infiltration of neutrophils and inhibits the shift of neutrophils into N1 phenotype partly by targeting HDAC2.

Matrix metalloproteinases inactivate the proinflammatory functions of secreted moonlighting tryptophanyl-tRNA synthetase

Tryptophanyl-tRNA synthetase (WRS) is a cytosolic aminoacyl-tRNA synthetase essential for protein synthesis. WRS is also one of a growing number of intracellular proteins that are attributed distinct noncanonical "moonlighting" functions in the extracellular milieu. Moonlighting aminoacyl-tRNA synthetases regulate processes such as inflammation, but how these multifunctional enzymes are themselves regulated remains unclear. Here, we demonstrate that WRS is secreted from human macrophages, fibroblasts, and endothelial cells in response to the proinflammatory cytokine interferon γ (IFNγ). WRS signaled primarily through Toll-like receptor 2 (TLR2) in macrophages, leading to phosphorylation of the p65 subunit of NF-κB with associated loss of NF-κB inhibitor α (IκB-α) protein. This signaling initiated secretion of tumor necrosis factor α (TNFα) and CXCL8 (IL8) from macrophages. We also demonstrated that WRS is a potent monocyte chemoattractant. Of note, WRS increased matrix metalloproteinase (MMP) activity in the conditioned medium of macrophages in a TNFα-dependent manner. Using purified recombinant proteins and LC-MS/MS to identify proteolytic cleavage sites, we demonstrated that multiple MMPs, but primarily macrophage MMP7 and neutrophil MMP8, cleave secreted WRS at several sites. Loss of the WHEP domain following cleavage at Met48 generated a WRS proteoform that also results from alternative splicing, designated Δ1-47 WRS. The MMP-cleaved WRS lacked TLR signaling and proinflammatory activities. Thus, our results suggest that moonlighting WRS promotes IFNγ proinflammatory activities, and these responses can be dampened by MMPs.

Immunostimulatory plant polysaccharides impede cancer progression and metastasis by avoiding off-target effects

An unexploited homo-polysaccharide (PSM001) isolated from the seed kernel of Kottukonam variety of Mangifera indica, demonstrated selective cytotoxicity against cancer cells both in vitro and in murine models while maintaining the immunostimulatory potential. Galactoxyloglucan (PST001) isolated from the seeds of Tamarindus indica, was previously established to be an effective anticancer and immunomodulatory agent. Cancer metastasis, with key features including invasion, migration, increased angiogenesis and colony formation is only likely to accentuate in the coming decades, considering the ground realities of the modern lifestyle and environmental factors and hence both the polysaccharides were tested towards the management of malignancy. It was a startling observation with both the biopolymers in inhibiting various processes involved in the metastatic cascade. A quick perusal of the issue at hand would throw up the promising ability of both PSM001 and PST001 to inhibit lung metastatic nodules of C57BL/6 mice wherein the combinatorial treatment of these polysaccharides with vincristine delivered superior therapeutic output. Later, vascular endothelial growth factor and multiple matrix metalloproteinases were found to be the lead players in the polysaccharide mediated metastatic inhibition. Having considered the complexities associated with the chemotherapy in metastatic cancer in terms of palpable immunosuppression, the aftermaths with the co-administration of an immunostimulatory agent which itself possess unique anticancer and anti-metastatic potentials with a potent chemotherapeutic agent will be enormously consequential.

The Role of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Duchenne Muscular Dystrophy Cardiomyopathy

Cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD). Standard cardiac biomarkers are poor indicators of DMD cardiovascular disease. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate collagen turnover. Given the cardiac fibrosis seen in DMD, we hypothesized that MMPs and TIMPs correlate with severity of DMD cardiomyopathy. Prospectively enrolled DMD subjects (n = 42) underwent cardiac magnetic resonance imaging for function and late gadolinium enhancement (LGE), including LGE severity from 0 (no LGE) to 4 (severe). Serum from DMD and healthy male control subjects (n = 15) analyzed for MMPs 1, 2, 3, 7, 9, and 10 and TIMPs 1-4. MMP1, MMP7, and MMP10 were higher in DMD than in control (respectively, median 5080 pg/mL vs 2120 pg/mL [P = .007], 2170 pg/mL vs 1420 pg/mL [P < .001], and 216 pg/mL vs 140pg/mL [P = .040]); TIMP4 was lower in DMD (124 pg/mL vs 263 pg/mL; P = .046). Within DMD, MMP7 correlated inversely with left ventricular ejection fraction (r = -0.40; P = .012) and directly with strain (r = 0.54; P = .001) and LGE severity (r = 0.47; P = .003). MMP7 was higher in DMD patients with LGE compared with those without LGE and control subjects (P < .001). Multiple MMPs are elevated in DMD compared with control subjects. MMP7 is related to DMD cardiac dysfunction and myocardial fibrosis, possibly through remodeling of the extracellular matrix.

Blockade of ERK1/2 by U0126 alleviates uric acid-induced EMT and tubular cell injury in rats with hyperuricemic nephropathy.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are serine/threonine kinases and function as regulators of cellular proliferation and differentiation. Recently, we demonstrated that inhibition of ERK1/2 alleviates the development and progression of hyperuricemia nephropathy (HN). However, its potential roles in uric acid-induced tubular epithelial-mesenchymal transition (EMT) and tubular epithelial cell injury are unknown. In this study, we showed that hyperuricemic injury induced EMT as characterized by downregulation of E-cadherin and upregulation of vimentin and Snail1 in a rat model of HN. This was coincident with epithelial cells arrested at the G2/M phase of cell cycle, activation of Notch1/Jagged-1 and Wnt/β-catenin signaling pathways, and upregulation of matrix metalloproteinase-2 (MMP-2) and MMP-9. Administration of U0126, a selective inhibitor of ERK1/2, blocked all these responses. U0126 was also effective in inhibiting renal tubular cell injury, as shown by decreased expression of lipocalin-2 and kidney injury molecule-1 and active forms of caspase-3. U0126 or ERK1/2 siRNA can inhibit tubular cell EMT and cell apoptosis as characterized with decreased expression of cleaved caspase-3. Moreover, ERK1/2 inhibition suppressed hyperuricemic injury-induced oxidative stress as indicated by decreased malondialdehyde and increased superoxide dismutase. Collectively, ERK1/2 inhibition-elicited renal protection is associated with inhibition of EMT through inactivation of multiple signaling pathways and matrix metalloproteinases, as well as attenuation of renal tubule injury by enhancing cellular resistance to oxidative stress.