Calreticulin regulates the expression of MMP14 and ADAR1 through EIF2AK2 signaling to promote the proliferation and progression of malignant melanoma cells.

It has been demonstrated that calreticulin (CALR) is expressed abnormally in various tumors and is involved in the occurrence and development of tumors. In this study, CALR and EIF2AK2 expression was measured in the clinical specimens of 39 patients with melanoma. Then, we constructed knockdown and overexpression cell models of CALR and EIF2AK2 and used wound healing and Transwell assays to observe cell migration and invasion. Apoptosis, EDU, and ROS assays were used to measure cell apoptosis and proliferation, as well as ROS levels. The effect of CALR on endoplasmic reticulum stress was detected using endoplasmic reticulum fluorescent probes. Western blotting was used to detect protein levels of CALR, EIF2AK2, ADAR1, and MMP14. The results indicated that CALR and EIF2AK2 expression levels were significantly higher in human melanoma tissues than in adjacent non-tumor tissue. In addition, we found a correlation between CALR and the expression of EIF2AK2 and MMP14, and the experimental results indicated that overexpression of CALR significantly upregulated the expression of EIF2AK2, MMP14, and ADAR1, while knockdown of CALR inhibited their expression. Notably, the knockdown of EIF2AK2 in the CALR overexpression group blocked the upregulation of MMP14 and ADAR1 expression by CALR, and the knockdown of both CALR and EIF2AK2 significantly inhibited MMP14 and ADAR1 expression. In conclusion, CALR and EIF2AK2 play a promoting role in melanoma progression, and knockdown of CALR and EIF2AK2 may be an effective anti-tumor target, and its mechanism may be through MMP14, ADAR1 signaling.

Time-course and muscle-specific gene expression of matrix metalloproteinases and inflammatory cytokines in response to acute treadmill exercise in rats.

BACKGROUND: The extracellular matrix (ECM) of skeletal muscle plays a pivotal role in tissue repair and growth, and its remodeling tightly regulated by matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and inflammatory cytokines. This study aimed to investigate changes in the mRNA expression of MMPs (Mmp-2 and Mmp-14), TIMPs (Timp-1 and Timp-2), and inflammatory cytokines (Il-1ß, Tnf-α, and Tgfß1) in the soleus (SOL) and extensor digitorum longus (EDL) muscles of rats following acute treadmill exercise. Additionally, muscle morphology was examined using hematoxylin and eosin (H&E) staining. METHODS AND RESULTS: Male rats were subjected to acute treadmill exercise at 25 m/min for 60 min with a %0 slope. The mRNA expression of ECM components and muscle morphology in the SOL and EDL were assessed in both sedentary and exercise groups at various time points (immediately (0) and 1, 3, 6, 12, and 24 h post-exercise). Our results revealed a muscle-specific response, with early upregulation of the mRNA expression of Mmp-2, Mmp-14, Timp-1, Timp-2, Il-1ß, and Tnf-α observed in the SOL compared to the EDL. A decrease in Tgfß1 mRNA expression was evident in the SOL at all post-exercise time points. Conversely, Tgfß1 mRNA expression increased at 0 and 3 h post-exercise in the EDL. Histological analysis also revealed earlier cell infiltration in the SOL than in the EDL following acute exercise. CONCLUSIONS: Our results highlight how acute exercise modulates ECM components and muscle structure differently in the SOL and EDL muscles, leading to distinct muscle-specific responses.

MCT4 and CD147 colocalize with MMP14 in invadopodia and support matrix degradation and invasion by breast cancer cells.

Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.

Intercellular transfer of cancer cell invasiveness via endosome-mediated protease shedding.

Overexpression of the transmembrane matrix metalloproteinase MT1-MMP/MMP14 promotes cancer cell invasion. Here we show that MT1-MMP-positive cancer cells turn MT1-MMP-negative cells invasive by transferring a soluble catalytic ectodomain of MT1-MMP. Surprisingly, this effect depends on the presence of TKS4 and TKS5 in the donor cell, adaptor proteins previously implicated in invadopodia formation. In endosomes of the donor cell, TKS4/5 promote ADAM-mediated cleavage of MT1-MMP by bridging the two proteases, and cleavage is stimulated by the low intraluminal pH of endosomes. The bridging depends on the PX domains of TKS4/5, which coincidently interact with the cytosolic tail of MT1-MMP and endosomal phosphatidylinositol 3-phosphate. MT1-MMP recruits TKS4/5 into multivesicular endosomes for their subsequent co-secretion in extracellular vesicles, together with the enzymatically active ectodomain. The shed ectodomain converts non-invasive recipient cells into an invasive phenotype. Thus, TKS4/5 promote intercellular transfer of cancer cell invasiveness by facilitating ADAM-mediated shedding of MT1-MMP in acidic endosomes.

Clinical significance and immune characteristics analysis of miR-221-3p and its key target genes related to epithelial-mesenchymal transition in breast cancer.

BACKGROUND: MicroRNA-221-3p (miR-221-3p) facilitates the advancement of breast cancer (BC) through the induction of epithelial-mesenchymal transition (EMT). Our research aimed to utilize bioinformatics to discover possible EMT-related target genes (ETGs) of miR-221-3p and examine their roles in breast cancer. METHODS: We employed bioinformatics techniques to identify ten key ETGs of miR-221-3p. Subsequently, we conducted an extensive analysis of both miR-221-3p and the ten ETGs, including clinical significance and immune characteristics. RESULTS: The expression of miR-221-3p was notably higher in Basal-like BC compared to other subtypes and adjacent normal tissue. Our pathway analysis suggested that miR-221-3p might regulate EMT through the MAPK signaling pathway by targeting its ETGs. Among the ETGs, seven core genes (EGFR, IGF1, KDR, FGF2, KIT, FGFR1, and FGF1) exhibited downregulation in BC. Conversely, ERBB2, SDC1, and MMP14 showed upregulation in BC and displayed potential diagnostic value. The analysis of prognostication indicated that increased levels of SDC1 and MMP14 were correlated with an unfavorable prognosis, whereas elevated expression of KIT was associated with a more favorable prognosis. The infiltration of various immune cells and the expression of immune checkpoint genes (ICGs) exhibited positive correlations with most ETGs and miR-221-3p. SDC1 exhibited a greater tumor mutational burden (TMB) score, while ERBB2, KDR, FGF2, KIT, FGFR1, and FGF1 showed lower TMB scores. Furthermore, decreased ERBB2 and KDR expression levels were correlated with elevated microsatellite instability (MSI) scores. Elevated expression of ETGs was linked to decreased mRNA stemness indices (mRNAsi), whereas miR-221-3p displayed the opposite pattern. Most ETGs and miR-221-3p expression exhibited a negative correlation with IC50 values for drugs. Among the ETGs, amplification was the most significant genetic alteration, except for IGF1. CONCLUSION: In conclusion, miR-221-3p acts as a unique indicator for Basal-like BC. The examination revealed ten essential ETGs of miR-221-3p, some of which show potential as diagnostic and prognostic markers. The in-depth examination of these ten ETGs and miR-221-3p indicates their participation in the development of BC, emphasizing their promise as innovative targets for therapy in BC patients.

MMP14high macrophages orchestrate progressive pulmonary fibrosis in SR-Ag-induced hypersensitivity pneumonitis.

Fibrotic hypersensitivity pneumonitis (FHP) is a fatal interstitial pulmonary disease with limited treatment options. Lung macrophages are a heterogeneous cell population that exhibit distinct subsets with divergent functions, playing pivotal roles in the progression of pulmonary fibrosis. However, the specific macrophage subpopulations and underlying mechanisms involved in the disease remain largely unexplored. In this study, a decision tree model showed that matrix metalloproteinase-14 (MMP14) had higher scores for important features in the up-regulated genes in macrophages from mice exposed to the Saccharopolyspora rectivirgula antigen (SR-Ag). Using single-cell RNA sequencing (scRNA-seq) analysis of hypersensitivity pneumonitis (HP) mice profiles, we identified MMP14high macrophage subcluster with a predominant M2 phenotype that exhibited higher activity in promoting fibroblast-to myofibroblast transition (FMT). We demonstrated that suppressing toll-like receptor 2 (TLR2) and nuclear factor kappa-B (NF-κB) could attenuate MMP14 expression and exosome secretion in macrophages stimulation with SR-Ag. The exosomes derived from MMP14-overexpressing macrophages were found to be more effective in regulating the transition of fibroblasts through exosomal MMP14. Importantly, it was observed that the transfer of MMP14-overexpressing macrophages into mice promoted lung inflammation and fibrosis induced by SR-Ag. NSC-405020 binding to the hemopexin domain (PEX) of MMP-14 ameliorated lung inflammation and fibrosis induced by SR-Ag in mice. Thus, MMP14-overexpressing macrophages may be an important mechanism contributing to the exacerbation of allergic reactions. Our results indicated that MMP14 in macrophages has the potential to be a therapeutic target for HP.

Association of MMP3, MMP14, and MMP25 gene polymorphisms with cerebral stroke risk: a case-control study.

BACKGROUND: Cerebral stroke (CS) is the leading cause of death in China, and a complex disease caused by both alterable risk factors and genetic factors. This study intended to investigate the association of MMP3, MMP14, and MMP25 single nucleotide polymorphisms (SNPs) with CS risk in a Chinese Han population. METHODS: A total of 1,348 Han Chinese were recruited in this case-control study. Four candidate loci including rs520540 A/G and rs679620 T/C of MMP3, rs2236302 G/C of MMP14, and rs10431961 T/C of MMP25 were successfully screened. The correlation between the four SNPs and CS risk was assessed by logistic regression analysis. The results were analyzed by false-positive report probability (FPRP) for chance or significance. The interactions between four SNPs associated with CS risk were assessed by multifactor dimensionality reduction (MDR). RESULTS: rs520540 A/G and rs679620 C/T SNP in MMP3 were associated with risk of CS in allele, codominant, dominant and log-additive models. Ischemic stroke risk were significantly lower in carriers with rs520540-A allele and rs679620-T allele than those with G/G or C/C genotypes. However, rs520540-A allele and rs679620-T allele were associated with higher risk of hemorrhagic stroke. Stratified analysis showed that these two SNPs were associated with reduced risk of CS in aged < 55 years, non-smoking and non-drinking participants, and rs679620 SNP also reduced CS risk in male participants. The levels of uric acid, high-density lipoprotein cholesterol, and eosinophil were different among patients with different genotypes of rs520540 and rs679620. No statistically significant association was found between MMP14 rs2236302 G/C or MMP25 rs10431961 T/C with CS even after stratification by stroke subtypes, age, gender as well as smoking and drinking conditions in all the genetic models. CONCLUSION: MMP3 rs520540 A/G and rs679620 C/T polymorphisms were associated with CS risk in the Chinese Han population, which provides useful information for the prevention and diagnosis of CS.

Assessment of MMP14, CAV2, CLU and SPARCL1 expression profiles in endometriosis.

Endometriotic cells exhibit a notable degree of invasiveness and some characteristics of tissue remodeling underlying lesion formation. In this regard, do matrix metalloproteinases 14 (MMP14) and other related genes such as SPARC-like protein 1 (SPARCL1), caveolin 2 (CAV2), and clusterin (CLU) exert any significant influence in the processes of endometriosis development and pathophysiology is not apparent. We aim to assess whether these genes could serve as potential diagnostic biomarkers in endometriosis. Microarray-based gene expression analysis was performed on total RNA extracted from endometriotic tissue samples treated with and without gonadotropin-releasing hormone agonist (GnRHa). The GnRHa untreated patients were considered the control group. The validation of genes was performed using quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR analysis showed significant downregulation in the expression of MMP14 (p = 0.024), CAV2 (p = 0.017), and upregulation of CLU (p = 0.005) in endometriosis patients treated with GnRHa. SPARCL1 did not show any significant (p = 0.30) change in the expression compared to the control group. These data have the potential to contribute to the comprehension of the molecular pathways implicated in the remodeling of the extracellular matrix, which is a vital step for the physiology of the endometrium. Based on the result, it is concluded that changes in the expression of MMP14, CAV2, and CLU post-treatment imply their role in the pathophysiology of endometriosis and may serve as a potential diagnostic biomarker of endometriosis in response to GnRHa treatment in patients with ovarian endometrioma.

KIF16B drives MT1-MMP recycling in macrophages and promotes co-invasion of cancer cells.

The matrix metalloproteinase MT1-MMP is a central effector of cellular proteolysis. Accordingly, regulation of the surface-localized pool of MT1-MMP is crucial for cell migration and invasion. Here, we identify the superprocessive kinesin KIF16B as a major driver of fast recycling of MT1-MMP to the surface of primary human macrophages. KIF16B associates with MT1-MMP on Rab14-positive vesicles, and its depletion results in strongly reduced MT1-MMP surface levels, as shown by microscopical, biochemical, and cell-sorting approaches. As a consequence, KIF16B-depleted macrophages exhibit strongly reduced matrix degradation and invasion. We further identify the cargo-binding C-terminus of KIF16B as a critical element of MT1-MMP transport, as its overexpression uncouples MT1-MMP vesicles from the endogenous motor, thus leading to a reduction of surface-associated MT1-MMP and to reduced matrix degradation and invasion. Importantly, depletion of KIF16B in primary macrophages also reduces the co-invasion of cancer cells from tumor spheroids, pointing to the KIF16B-driven recycling pathway in macrophages as an important regulatory element of the tumor microenvironment.

Degradomic Identification of Membrane Type 1-Matrix Metalloproteinase as an ADAMTS9 and ADAMTS20 Substrate.

The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative terminomics method, terminal amine isotopic labeling of substrates was used to compare the parental and gene-edited RPE-1 cells and their medium to identify ADAMTS9 substrates. Among differentially abundant neo-amino (N) terminal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease membrane type 1-matrix metalloproteinase (MT1-MMP), whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge, that is, between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by reexpression of ADAMTS9 and ADAMTS20 and was dependent on hinge O-glycosylation. A C-terminally semitryptic MT1-MMP peptide with greater abundance in WT RPE-1 medium identified a second ADAMTS9 cleavage site in the MT1-MMP hemopexin domain. Consistent with greater retention of MT1-MMP on the surface of gene-edited cells, pro-MMP2 activation, which requires cell surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell surface activity of MT1-MMP, and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.

Gallbladder cancer-associated genetic variants rs1003349 and rs1004030 regulate MMP14 expression by altering SOX10- and MYB-binding sites.

The pathogenesis of gallbladder cancer is complex, involving environmental and genetic risk factors. The matrix metallopeptidase 14 (MMP14) alters the tumor microenvironment and promotes tumorigenesis. In this study, we have characterized the role of the MMP14 promoter variants rs1004030 and rs1003049 in gallbladder cancer pathogenesis. Previously, we have shown the association of rs1004030 and rs1003049 with GBC and allele-specific differential expression of MMP14 in GBC patients. These variants reside within the cis-regulatory element (CRE) with high DNase and H3K4me3 signals, suggesting an active regulatory role in MMP14 expression. The luciferase-based reporter assay showed the role of promoter variants on expression levels in two GBC cell lines. Deleting the 119 bp promoter region surrounding the variants rs1004030 and rs1003049 by CRISPR-Cas9 genome editing resulted in reduced MMP14 expression in G415 cells. Electrophoretic mobility shift assay shows the presence of risk allele 'C'/'G' at rs1004030 and rs1003049 and create binding sites for transcription factors SOX10 and MYB, respectively. Further, stable knockdown of these transcription factors in G415 and TGBC1TKB cells showed reduced expression of MMP14. However, in both GBC cells, ectopic expression of these transcription factors increased the expression of MMP14. Rescue of MYB and SOX10 expression levels showed a significant increase in luciferase activity only in risk allele-carrying constructs. In conclusion, our study unveils a mechanistic role of the MMP14 promoter variants rs1004030 and rs1003049 in gallbladder cancer.

Designed Loop Extension Followed by Combinatorial Screening Confers High Specificity to a Broad Matrix MetalloproteinaseInhibitor.

Matrix metalloproteinases (MMPs) are key drivers of various diseases, including cancer. Development of probes and drugs capable of selectively inhibiting the individual members of the large MMP family remains a persistent challenge. The inhibitory N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP2), a natural broad MMP inhibitor, can provide a scaffold for protein engineering to create more selective MMP inhibitors. Here, we pursued a unique approach harnessing both computational design and combinatorial screening to confer high binding specificity toward a target MMP in preference to an anti-target MMP. We designed a loop extension of N-TIMP2 to allow new interactions with the non-conserved MMP surface and generated an efficient focused library for yeast surface display, which was then screened for high binding to the target MMP-14 and low binding to anti-target MMP-3. Deep sequencing analysis identified the most promising variants, which were expressed, purified, and tested for selectivity of inhibition. Our best N-TIMP2 variant exhibited 29 pM binding affinity to MMP-14 and 2.4 µM affinity to MMP-3, revealing 7500-fold greater specificity than WT N-TIMP2. High-confidence structural models were obtained by including NGS data in the AlphaFold multiple sequence alignment. The modeling together with experimental mutagenesis validated our design predictions, demonstrating that the loop extension packs tightly against non-conserved residues on MMP-14 and clashes with MMP-3. This study demonstrates how introduction of loop extensions in a manner guided by target protein conservation data and loop design can offer an attractive strategy to achieve specificity in design of protein ligands.

Tumour Suppressor Neuron Navigator 3 and Matrix Metalloproteinase 14 are Co-expressed in Most Melanomas but Downregulated in Thick Tumours.

Melanoma is a highly metastatic tumour originating from neural crest-derived melanocytes. The aim of this study was to analyse the expression of neuron navigator 3 (NAV3) in relation to membrane type-1 matrix metalloproteinase MMP14, a major regulator of invasion, in 40 primary melanomas, 15 benign naevi and 2 melanoma cell lines. NAV3 copy number changes were found in 18/27 (67%) primary melanomas, so that deletions dominated (16/27 of samples, 59%). NAV3 protein was found to be localized at the leading edge of migrating melanoma cells in vitro. Silencing of NAV3 reduced both melanoma cell migration in 2-dimensional conditions, as well as sprouting in 3-dimensional collagen I. NAV3 protein expression correlated with MMP14 in 26/37 (70%) primary melanomas. NAV3 and MMP14 were co-expressed in all tumours with Breslow thickness < 1 mm, in 11/23 of mid-thickness tumours (1-5 mm), but in only 1/6 samples of thick (> 5 mm) melanomas. Altogether, NAV3 number changes are frequent in melanomas, and NAV3 and MMP14, while expressed in all thin melanomas, are often downregulated in thicker tumours, suggesting that the lack of both NAV3 and MMP14 favours melanoma progression.

MT1-MMP and ADAM10/17 exhibit a remarkable overlap of shedding properties.

Membrane-type-I matrix metalloproteinase (MT1-MMP) is one of six human membrane-bound MMPs and is responsible for extracellular matrix remodelling by degrading several substrates like fibrillar collagens, including types I-III, or fibronectin. Moreover, MT1-MMP was described as a key player in cancer progression and it is involved in various inflammatory processes, as well as in the pathogenesis of Alzheimer's disease (AD). The membrane-tethered metalloprotease meprin ß as well as a disintegrin and metalloproteinase 10 (ADAM10) and ADAM17 are also associated with these diseases. Interestingly, meprin ß, ADAM10/17 and MT1-MMP also have a shared substrate pool including the interleukin-6 receptor and the amyloid precursor protein. We investigated the interaction of these proteases, focusing on a possible connection between MT1-MMP and meprin ß, to elucidate the potential mutual regulations of both enzymes. Herein, we show that besides ADAM10/17, MT1-MMP is also able to shed meprin ß from the plasma membrane, leading to the release of soluble meprin ß. Mass spectrometry-based cleavage site analysis revealed that the cleavage of meprin ß by all three proteases occurs between Pro602 and Ser603 , N-terminal of the EGF-like domain. Furthermore, only inactive human pro-meprin ß is shed by MT1-MMP, which is again in accordance with the shedding capability observed for ADAM10/17. Vice versa, meprin ß also appears to shed MT1-MMP, indicating a complex regulatory network. Further studies will elucidate this well-orchestrated proteolytic web under distinct conditions in health and disease and will possibly show whether the loss of one of the above-mentioned sheddases can be compensated by the other enzymes.

MicroRNA-149 Regulates Proliferation, Migration, and Invasion of Pituitary Adenoma Cells by Targeting ADAM12 and MMP14.

OBJECTIVE: Pituitary adenomas (PAs) can adapt an aggressive phenotype by invading adjacent brain structures with rapid cellular proliferation. Previous studies demonstrated that excessive expression of metalloproteases ADAM12 and MMP-14 is instrumental for the active proliferation and invasiveness of PA cells in vitro and of tumors in vivo. However, the mechanisms regulating ADAM12 and MMP-14 expression in PAs remain unclear. METHODS: Target gene prediction and transcriptomic profiling of invasive vs. noninvasive human PA samples were performed to identify miRNA species potentially involved in the regulation of ADAM12 and MMP14. For cellular analyses of miRNA functions, two mouse PA cell lines (AtT20 and TtT/GF) were transfected with miR-149-3p and miR-149-5p, respectively. The effects of miR-149 (3p and 5p) on expression levels of ADAM12 and MMP14 were determined by Western blotting followed by an analysis of proliferation and colony formation assays, scratch migration assays, and invasion assays. RESULTS: A significant downregulation of miRNA-149 was observed in invasive vs. noninvasive PA (0.32 vs. 0.09, P<0.0001). In AtT-20 and TtT/GF mouse PAs cells, transfection of mimic miRNA-149 (3p and 5p) caused a significantly reduced cell proliferation and matrigel invasion, whilst the effect on cell migration was less pronounced. Both strands of miRNA-149 (3p and 5p) markedly reduced protein levels of ADAM12 and MMP-14 by at least 40% in both cell lines. CONCLUSION: This study proved that the invasiveness of PA cells is, at least partly, regulated by miRNA-149-dependent expression of ADAM12 and MMP-14.

MT1-MMP Expression Levels and Catalytic Functions Dictate LDL Receptor-Related Protein-1 Ligand Internalization Capacity in U87 Glioblastoma Cells.

Modulations in cell surface receptor ectodomain proteolytic shedding impact on receptor function and cancer biomarker expression. As such, heavily pursued therapeutic avenues have exploited LDL receptor-related protein-1 (LRP-1)-mediated capacity in internalizing Angiopep-2 (An2), a brain-penetrating peptide that allows An2-drug conjugates to cross the blood-brain tumor barrier (BBTB). Given that LRP-1 is proteolytically shed from the cell surface through matrix metalloproteinase (MMP) activity, the balance between MMP expression/function and LRP-1-mediated An2 internalization is unknown. In this study, we found that membrane type-1 (MT1)-MMP expression increased from grade 1 to 4 brain tumors, while that of LRP-1 decreased inversely. MMP pharmacological inhibitors such as Ilomastat, Doxycycline and Actinonin increased in vitro An2 internalization by up to 2.5 fold within a human grade IV-derived U87 glioblastoma cell model. Transient siRNA-mediated MT1-MMP gene silencing resulted in increased basal An2 cell surface binding and intracellular uptake, while recombinant MT1-MMP overexpression reduced both cell surface LRP-1 expression as well as An2 internalization. The addition of Ilomastat to cells overexpressing recombinant MT1-MMP restored LRP-1 expression at the cell surface and An2 uptake to levels comparable to those observed in control cells. Collectively, our data suggest that MT1-MMP expression status dictates An2-mediated internalization processes in part by regulating cell surface LRP-1 functions. Such evidence prompts preclinical evaluations of combined MMP inhibitors/An2-drug conjugate administration to potentially increase the treatment of high-MT1-MMP-expressing brain tumors.

Progress on the role and mechanism of MT1-MMP in tumor metastasis.

The matrix metallopeptidase family (matrix metallopeptidase, MMPs) is a class of zinc-dependent endopeptidases that can degrade most extracellular matrices. MT1-MMP (membrane type 1 metalloprotease) is an important metallopeptidase, which is located on plasma membrane and highly expressed in most tumors. MT1-MMP promotes cancer metastasis through affecting the extracellular matrix remodeling, angiogenesis, lipid metabolism, and inflammation. However, the mechanisms of MT1-MMP in different tumors have not been fully elucidated. In this review, we summarize the latest progress and the metastasis-promoting regulatory mechanisms of MT1-MMP in different tumors, which will provide references for its in-depth research and application in the field of cancer biology.

Membrane-type 1 matrix metalloproteinase as predictor of survival and candidate therapeutic target in Ewing sarcoma.

BACKGROUND: Ewing sarcoma (ES) is the second most common primary bone malignancy, with an urgent need for new treatments. ES is associated with high rates of progression and relapse, driven by drug-resistant cells capable of migration, self-renewal and single-cell tumorigenesis, termed cancer stem-like cells (CSCs). Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound proteolytic enzyme, which, via direct and indirect mechanisms, digests four of the main types of collagen. This can be hijacked in malignancy for invasion and metastasis, with high expression predicting decreased survival in multiple cancers. In this study, we have examined the hypothesis that MT1-MMP is expressed by ES cells and explored the relationship between expression and outcomes. PROCEDURE: MT1-MMP expression in ES established cell lines, primary patient-derived cultures and daughter ES-CSCs was characterised by RNA sequencing, Western blotting, immunocytochemistry and flow cytometry. Immunohistochemistry was used to detect MT1-MMP in tumour biopsies, and the relationship between expression, event-free and overall survival examined. RESULTS: MT1-MMP was detected at both RNA and protein levels in five of six established cell lines, all primary cultures (n = 25) and all daughter ES-CSCs (n = 7). Immunohistochemistry of treatment-naïve biopsy tissue demonstrated that high MT1-MMP expression predicted decreased event-free and overall survival (p = .017 and .036, respectively; n = 47); this was not significant in multivariate analysis. CONCLUSIONS: MT1-MMP is expressed by ES cells, including ES-CSCs, making it a candidate therapeutic target. The level of MT1-MMP expression at diagnosis may be considered as a prognostic biomarker if validated by retrospective analysis of a larger cohort of clinical trial samples.

Atorvastatin attenuates NS1 (Non-structural protein-1) of dengue type-2 serotype-induced expressions of matrix metalloproteinases in HL-60 cells, differentiated to neutrophils: Implications for the immunopathogenesis of dengue viral disease.

BACKGROUND: The dengue is a vector borne viral infection in humans. Bite of mosquito infected with a dengue virus transmits the disease. The neutrophils support more to the innate immune response by switching to infected tissues and triggering immunomodulatory mechanisms including the release of proteases and host defence peptides. METHODS: Cell viability by MTT and trypan blue dye exclusion assay, bright field microscopy for assessment of cell morphology, cytokines measurements by ELISA, estimation of protein by Bradford assay were done. Assessments of matrix metalloproteinase genes mRNA expressions were done using real-time PCR. RESULTS: In the present study, we have for the first time unveiled that, NS1 antigen of dengue type-2 serotype, induce and stimulate the neutrophils cells to express high levels of matrix metalloproteases. NS1 exposure of HL-60 cells differentiated to neutrophils affected cell morphology and in 24 h of exposure. We have demonstrated that, the NS1 antigen has induced MMP-2, MMP-14 and MMP-9 expressions in neutrophils in a 24hrs exposure time. NS1 exposure has also further upregulated MMP-1, MMP-13, and MMP-8 expressions in neutrophils in a 24hrs exposure time. Notably, treatment with atorvastatin concentrations downregulated the expression profile of the all matrix metalloprotease significantly. Importantly, NS1 antigen has significantly increased the IL-6, IL-13 release by the HL,60 cells which was reversed by atorvastatin. On the other hand, NS1 exposure enhanced the mRNA expressions of VEGF-A and VEGF-D which was reversed by atorvastatin. However, we found that, NS1 exposure reduced the mRNA expressions profile of VEGF-C, which was reversed by atorvastatin. CONCLUSION: In conclusion, we report that, neutrophils associated matrix metalloprotease are involved in the pathogenesis of dengue viral disease. VEGF growth factors may also be released by the neutrophils which may subsequently participate in the endothelial dysfunctions leading to dengue shock syndrome.

[Matrix metalloproteinase 14 and plasma kallikrein 1 may be potential biomarkers in the diagnosis and treatment of sepsis: a proteomics and bioinformatics analysis].

OBJECTIVE: To analyze protein profiles in septic patients, and to find potential new targets for the diagnosis and treatment of sepsis. METHODS: A cross sectional observational study was conducted. From January to December 2019, 12 septic patients and 9 healthy volunteers were recruited in the emergency intensive care unit (EICU) of the emergency department of the Affiliated Hospital of Southwest Medical University. The peripheral blood of the two groups was collected for protein mass spectrometry analysis, and the data-independent acquisition technology was used to obtain the expression data of each protein. The obtained data was imported into the online network tool Integrated Differential Expression and Pathway analysis (IDEP2), the data underwent ID converted and were homogenized to verify their comparability, and then principal component analysis was used to eliminate outlier data. Then data with P < 0.05, log2fold change (FC) > 1 or log2FC < -1 were considered to have a statistically significant difference, and the differential proteins were screened out. On the DAVID website, the screened differential proteins would be analyzed by gene ontology (GO), and the biological process, cellular components, and molecular function of the proteins would be analyzed. Protein enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed. Protein-protein interaction (PPI) analysis was performed through the Search Tool for the Retrieval of Interacting Genes Database (STRING) website to find closely related proteins. RESULTS: The data in this study were shown to be comparable after normalization. A total of 125 differential proteins were screened, of which 99 were up-regulated and 26 were down-regulated. GO enrichment analysis discovered that these proteins were mainly extracellular, with cellular regulatory functions and catalytic functions involved in biological regulation, metabolic process and immune process. KEGG pathway analysis suggested that these proteins were involved in amino acid, carbohydrate metabolism and immune-related pathways. PPI analysis showed that key proteins included matrix metalloproteinase 14 (MMP14), fibulin 1 (FBLN1), plasma kallikrein 1 (KLKB1), etc., and finally screened out MMP14 and KLKB1, which were closely related to inflammation and immunity. Both might be potential new targets for early diagnosis and treatment of sepsis. CONCLUSIONS: MMP14 and KLKB1 may be potential biomarkers for the diagnosis, treatment and prognosis of sepsis.