Proteolytic Remodeling Research Articles

Impact of Hyperhomocysteinemia on Cytokine and Matrix Metalloproteinases Contents in Rat Skeletal Muscle

Abstract Background: High levels of homocysteine (Hcy) in the blood (hyperhomocysteinemia [HHcy]) are associated with many pathologies, including atherosclerosis, coronary heart disease, stroke, neurological disorders, and a decrease in muscle mass and strength. The present study aimed to evaluate the levels of pro- and anti-inflammatory cytokines and matrix metalloproteinases (MMPs) in skeletal muscle of rats with experimental HHcy. Methods: HHcy in rats of two age groups was induced by intragastric administration of D, L-thiolactone Hcy for 8 weeks. The criterion for the development of pathological conditions was a blood Hcy level above 15 mmol L−1. Cytokine and MMP levels in muscle homogenate were determined by enzyme-linked immunosorbent assay. Results: The findings of the study on the content of pro-inflammatory cytokines in skeletal muscles of rats with hyperhomocysteinemia indicated the following. For immature rats, the greatest reduction in tumor necrosis factor-α and interleukin-8 (IL-8) content was observed, whereas, for mature individuals, the most pronounced decline in IL-1b was evident in skeletal muscle tissue under the pathology state. The content of anti-inflammatory interferon-γ was also reduced in the muscles of both age groups by 30% compared to the control, besides a decrease in the content of collagenase MMP-1 was also observed. Conclusions: The presence of HHcy is associated with metabolic changes in skeletal muscles, which can result in a depletion of cytokines and a slowing down of proteolytic remodeling. This, in turn, can lead to the development of muscular abnormalities, including dysfunction, weakness, and muscle degeneration.

The β-arrestin1/endothelin axis bolsters ovarian fibroblast-dependent invadosome activity and cancer cell metastatic potential

Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix (ECM) remodeling, invasion, and metastasis, highlighting the need to investigate the molecular mechanisms driving CAF function. Endothelin-1 (ET-1) regulates the communication between cancer and stroma and facilitates the progression of serous ovarian cancer (SOC). By binding to Endothelin A (ETA) and B (ETB) receptors, ET-1 enables the recruitment of β-arrestin1 (β-arr1) and the formation of signaling complexes that coordinate tumor progression. However, how ET-1 receptors might “educate” human ovarian fibroblasts (HOFs) to produce altered ECM and promote metastasis remains to be elucidated. This study identifies ET-1 as a pivotal factor in the activation of CAFs capable of proteolytic ECM remodeling and the generation of heterotypic spheroids containing cancer cells with a propensity to metastasize. An autocrine/paracrine ET-1/ETA/BR/β-arr1 loop enhances HOF proliferation, upregulates CAF marker expression, secretes pro-inflammatory cytokines, and increases collagen contractility, and cell motility. Furthermore, ET-1 facilitates ECM remodeling by promoting the lytic activity of invadosome and activation of integrin β1. In addition, ET-1 signaling supports the formation of heterotypic HOF/SOC spheroids with enhanced ability to migrate through the mesothelial monolayer, and invade, representing metastatic units. The blockade of ETA/BR or β-arr1 silencing prevents CAF activation, invadosome function, mesothelial clearance, and the invasive ability of heterotypic spheroids. In vivo, therapeutic inhibition of ETA/BR using bosentan (BOS) significantly reduces the metastatic potential of combined HOFs/SOC cells, associated with enhanced apoptotic effects on tumor cells and stromal components. These findings support a model in which ET-1/β-arr1 reinforces tumor/stroma interaction through CAF activation and fosters the survival and metastatic properties of SOC cells, which could be counteracted by ETA/BR antagonists.

Mechanical forces across compartments coordinate cell shape and fate transitions to generate tissue architecture

Morphogenesis and cell state transitions must be coordinated in time and space to produce a functional tissue. An excellent paradigm to understand the coupling of these processes is mammalian hair follicle development, which is initiated by the formation of an epithelial invagination—termed placode—that coincides with the emergence of a designated hair follicle stem cell population. The mechanisms directing the deformation of the epithelium, cell state transitions and physical compartmentalization of the placode are unknown. Here we identify a key role for coordinated mechanical forces stemming from contractile, proliferative and proteolytic activities across the epithelial and mesenchymal compartments in generating the placode structure. A ring of fibroblast cells gradually wraps around the placode cells to generate centripetal contractile forces, which, in collaboration with polarized epithelial myosin activity, promote elongation and local tissue thickening. These mechanical stresses further enhance compartmentalization of Sox9 expression to promote stem cell positioning. Subsequently, proteolytic remodelling locally softens the basement membrane to facilitate a release of pressure on the placode, enabling localized cell divisions, tissue fluidification and epithelial invagination into the underlying mesenchyme. Together, our experiments and modelling identify dynamic cell shape transformations and tissue-scale mechanical cooperation as key factors for orchestrating organ formation.

Methionine aminopeptidase 2 and its autoproteolysis product have different binding sites on the ribosome

Excision of the initiator methionine is among the first co-translational processes that occur at the ribosome. While this crucial step in protein maturation is executed by two types of methionine aminopeptidases in eukaryotes (MAP1 and MAP2), additional roles in disease and translational regulation have drawn more attention to MAP2. Here, we report several cryo-EM structures of human and fungal MAP2 at the 80S ribosome. Irrespective of nascent chains, MAP2 can occupy the tunnel exit. On nascent chain displaying ribosomes, the MAP2-80S interaction is highly dynamic and the MAP2-specific N-terminal extension engages in stabilizing interactions with the long rRNA expansion segment ES27L. Loss of this extension by autoproteolytic cleavage impedes interactions at the tunnel, while promoting MAP2 to enter the ribosomal A-site, where it engages with crucial functional centers of translation. These findings reveal that proteolytic remodeling of MAP2 severely affects ribosome binding, and set the stage for targeted functional studies.

Proteolytic remodeling of 3D bioprinted tumor microenvironments

In native tissue, remodeling of the pericellular space is essential for cellular activities and is mediated by tightly regulated proteases. Protease activity is dysregulated in many diseases, including many forms of cancer. Increased proteolytic activity is directly linked to tumor invasion into stroma, metastasis, and angiogenesis as well as all other hallmarks of cancer. Here we show a strategy for 3D bioprinting of breast cancer models using well-defined protease degradable hydrogels that can facilitate exploration of the multifaceted roles of proteolytic extracellular matrix remodeling in tumor progression. We designed a set of bicyclo[6.1.0]nonyne functionalized hyaluronan (HA)-based bioinks cross-linked by azide-modified poly(ethylene glycol) (PEG) or matrix metalloproteinase (MMP) degradable azide-functionalized peptides. Bioprinted structures combining PEG and peptide-based hydrogels were proteolytically degraded with spatial selectivity, leaving non-degradable features intact. Bioprinting of tumor-mimicking microenvironments using bioinks comprising human breast cancer cells (MCF-7) and fibroblast in hydrogels with different susceptibilities to proteolytic degradation shows that MCF-7 proliferation and spheroid size were significantly increased in protease degradable hydrogel compartments, but only in the presence of fibroblasts. In the absence of fibroblasts in the stromal compartment, cancer cell proliferation was reduced and did not differ between degradable and nondegradable hydrogels. The interactions between spatially separated fibroblasts and MCF-7 cells consequently resulted in protease-mediated remodeling of the bioprinted structures and a significant increase in cancer cell spheroid size, highlighting the close interplay between cancer cells and stromal cells in the tumor microenvironment and the influence of proteases in tumor progression.

A mechanosensitive caveolae–invadosome interplay drives matrix remodelling for cancer cell invasion

Invadosomes and caveolae are mechanosensitive structures that are implicated in metastasis. Here, we describe a unique juxtaposition of caveola clusters and matrix degradative invadosomes at contact sites between the plasma membrane of cancer cells and constricting fibrils both in 2D and 3D type I collagen matrix environments. Preferential association between caveolae and straight segments of the fibrils, and between invadosomes and bent segments of the fibrils, was observed along with matrix remodelling. Caveola recruitment precedes and is required for invadosome formation and activity. Reciprocally, invadosome disruption results in the accumulation of fibril-associated caveolae. Moreover, caveolae and the collagen receptor β1 integrin co-localize at contact sites with the fibrils, and integrins control caveola recruitment to fibrils. In turn, caveolae mediate the clearance of β1 integrin and collagen uptake in an invadosome-dependent and collagen-cleavage-dependent mechanism. Our data reveal a reciprocal interplay between caveolae and invadosomes that coordinates adhesion to and proteolytic remodelling of confining fibrils to support tumour cell dissemination.

Versatile click alginate hydrogels with protease-sensitive domains as cell responsive/instructive 3D microenvironments

Alginate (ALG) is a widely used biomaterial to create artificial extracellular matrices (ECM) for tissue engineering. Since it does not degrade in the human body, imparting proteolytic sensitivity to ALG hydrogels leverages their properties as ECM-mimics. Herein, we explored the strain-promoted azide-alkyne cycloaddition (SPAAC) as a biocompatible and bio-orthogonal click-chemistry to graft cyclooctyne-modified alginate (ALG-K) with bi-azide-functionalized PVGLIG peptides. These are sensitive to matrix metalloproteinase (MMP) and may act as crosslinkers. The ALG-K-PVGLIG conjugates (50, 125, and 250 μM PVGLIG) were characterized for peptide incorporation, crosslinking ability (double-end grafting), and enzymatic liability. For producing cell-permissive multifunctional 3D matrices for dermal fibroblast culture, oxidized ALG-K was grafted with PVGLIG and with RGD peptides for cell-adhesion. SPAAC reactions were performed immediately before cell-laden hydrogel formation by secondary ionic-crosslinking, considerably reducing the steps and time of preparation. Hydrogels with intermediate PVGLIG concentration (125 μM) presented slightly higher stiffness while promoting extensive cell spreading and higher degree of cell-cell interconnections, likely favored by cell-driven proteolytic remodeling of the network. The hydrogel-embedded cells were able to produce their own pericellular ECM, expressed MMP-2 and 14, and secreted PVGLIG-degrading enzymes. By recapitulating key ECM-like features, these hydrogels provide biologically relevant 3D matrices for soft tissue regeneration.

Salivary peptidome analysis and protease prediction during orthodontic treatment with fixed appliances

Orthodontic tooth movement (OTM) occurs through proteolytic remodelling within the periodontium following the application of external force to the tooth. This study describes the first characterization of the salivary peptidome and protease profile during the alignment stage of fixed appliance orthodontic treatment. Unstimulated whole mouth saliva from 16 orthodontic patients (10 males, 6 females, mean (SD) age 15.2 (1.6) years) was collected prior to fixed appliance placement (T1), 1-h (T2), 1-week (T3) following fixed appliance placement and on completion of mandibular arch alignment (T4). Salivary peptides were extracted using filtration followed by mass spectrometry to identify amino acid sequences. Protease prediction was carried out in silico using Proteasix and validated with gelatin zymography and enzyme-linked immunosorbent assay. A total of 2852 naturally-occurring peptides were detected, originating from 436 different proteins. Both collagen and statherin-derived peptide levels were increased at T2. Proteasix predicted 73 proteases potentially involved in generating these peptides, including metalloproteinases, calpains and cathepsins. Changes in predicted activity of proteases over time were also observed, with most metalloproteinases showing increased predicted activity at T2–T3. Increased gelatinolytic activity and MMP8/MMP9 levels were detected at T3. Collectively, multiple protein targets and changes in protease-predicted activity during OTM have been identified.

Secreted proteolytic activity of vaginal prevotella species remodels structural components of cervical and uterine tissues

Elevated proteolysis is observed during bacterial vaginosis and has been linked to HIV acquisition and preterm labour. Proteolytic remodelling of structural components (collagens/elastin) within cervical/uterine tissues can impair cervical barrier function and promote pathogen invasion. During pregnancy, aberrant proteolysis can trigger premature cervical dilation and chorioamniotic membrane rupture. We sought to define the enzyme classes contributing to Prevotella species’ secreted proteolytic activities targeting structural components of cervical/uterine tissues.

Lysosomal damage drives mitochondrial proteome remodelling and reprograms macrophage immunometabolism

Transient lysosomal damage after infection with cytosolic pathogens or silica crystals uptake results in protease leakage. Whether limited leakage of lysosomal contents into the cytosol affects the function of cytoplasmic organelles is unknown. Here, we show that sterile and non-sterile lysosomal damage triggers a cell death independent proteolytic remodelling of the mitochondrial proteome in macrophages. Mitochondrial metabolic reprogramming required leakage of lysosomal cathepsins and was independent of mitophagy, mitoproteases and proteasome degradation. In an in vivo mouse model of endomembrane damage, live lung macrophages that internalised crystals displayed impaired mitochondrial function. Single-cell RNA-sequencing revealed that lysosomal damage skewed metabolic and immune responses in alveolar macrophages subsets with increased lysosomal content. Functionally, drug modulation of macrophage metabolism impacted host responses to Mycobacterium tuberculosis infection in an endomembrane damage dependent way. This work uncovers an inter-organelle communication pathway, providing a general mechanism by which macrophages undergo mitochondrial metabolic reprograming after endomembrane damage.

Proteolytic and mechanical remodeling of the extracellular matrix by invadopodia in cancer

Cancer invasion and metastasis require remodeling of the adjacent extracellular matrix (ECM). In this mini review, we will cover the mechanisms of proteolytic degradation and the mechanical remodeling of the ECM by cancer cells, with a focus on invadopodia. Invadopodia are membrane protrusions unique to cancer cells, characterized by an actin core and by the focal degradation of ECM via matrix metalloproteases (MMPs). While ECM can also be remodeled, at lower levels, by focal adhesions, or internal collagen digestion, invadopodia are now recognized as the major mechanism for MMP-dependent pericellular ECM degradation by cancer cells. Recent evidence suggests that the completion of epithelial-mesenchymal transition may be dispensable for invadopodia and metastasis, and that invadopodia are required not only for mesenchymal, single cell invasion, but also for collective invasion. During collective invasion, invadopodia was then shown to be located in leader cells, allowing follower cells to move via cooperation. Collectively, this suggests that invadopodia function may be a requirement not only for later steps of metastasis, but also for early invasion of epithelial cells into the stromal tissue. Over the last decade, invadopodia studies have transitioned into in 3D and in vivo settings, leading to the confirmation of their essential role in metastasis in preclinical animal models. In summary, invadopodia may hold a great potential for individual risk assessment as a prognostic marker for metastasis, as well as a therapeutic target.

Direct Identification of Proteolytic Cleavages on Living Cells Using a Glycan-Tethered Peptide Ligase.

Proteolytic cleavage of cell surface proteins triggers critical processes including cell–cell interactions, receptor activation, and shedding of signaling proteins. Consequently, dysregulated extracellular proteases contribute to malignant cell phenotypes including most cancers. To understand these effects, methods are needed that identify proteolyzed membrane proteins within diverse cellular contexts. Herein we report a proteomic approach, called cell surface N-terminomics, to broadly identify precise cleavage sites (neo-N-termini) on the surface of living cells. First, we functionalized the engineered peptide ligase, called stabiligase, with an N-terminal nucleophile that enables covalent attachment to naturally occurring glycans. Upon the addition of a biotinylated peptide ester, glycan-tethered stabiligase efficiently tags extracellular neo-N-termini for proteomic analysis. To demonstrate the versatility of this approach, we identified and characterized 1532 extracellular neo-N-termini across a panel of different cell types including primary immune cells. The vast majority of cleavages were not identified by previous proteomic studies. Lastly, we demonstrated that single oncogenes, KRAS(G12V) and HER2, induce extracellular proteolytic remodeling of proteins involved in cancerous cell growth, invasion, and migration. Cell surface N-terminomics is a generalizable platform that can reveal proteolyzed, neoepitopes to target using immunotherapies.

Abstract 11618: Serum and Glucocorticoid Inducible Kinase-1 (SGK-1) Activity is Upregulated in Hypertension and Promotes IL-6 Expression to Drive Aortic Macrophage Accumulation

Introduction: The serum and glucocorticoid inducible kinase-1 (SGK-1) is a mechanosensitive kinase and it is hypothesized that SGK-1 activity is upregulated with elevated aortic tension to augment pro-inflammatory cytokine expression and promote macrophage accumulation to drive hypertensive remodeling. Methods: Hypertension (HTN) was induced in C57Bl/6 mice with AngiotensinII (AngII) infusion (1.46mg/kg/day x21days, n=4). Terminal procedure included blood pressure (SBP) quantification, aortic diameter (AoD) assessment, and harvest of infrarenal aorta. Abundance of SGK-1, pSGK-1, and the mature macrophage marker F4/80 was determined by immunoblot. The spontaneously hypertensive BPH/2 mice were also utilized and assessed for SBP, AoD, and target proteins (n=4). A subset of animals had systemic infusion of EMD638683, a selective SGK-1 inhibitor (2.5mg/kg/day x 21 days, n=4). Aortic VSMCs were subjected to 12% biaxial cyclic stretch for 3 and 12 hours +/- EMD638683 (10μM) with subsequent QPCR for IL-6 and MCP-1 expression. Statistical analysis included two-way ANOVA across treatment groups with significance at p<0.05. Results: C57Bl/6+AngII mice and BPH/2 mice had significant elevation in SBP (p<0.05) without change in AoD. The pSGK-1/SGK-1 and abundance F4/80 were increased in both models of HTN (p<0.05) and decreased with systemic EMD638683 (p<0.05), with no change in SBP. IL-6 expression was upregulated in VSMC that had Stretch for 3hrs (1.64 + 0.22 fold; p<0.05) or 12hrs (1.99 + 0.33 fold; p<0.05), and at both timepoints EMD638683 significantly inhibited IL-6 expression (p<0.05 vs respective Stretch). MCP-1 expression was unchanged after 3hr Stretch, but it was significantly elevated following 12hr Stretch (2.05 + 0.38 fold; p<0.05), and inhibited by EMD638683 (p<0.05). This data suggests a biomechanical link between aortic VSMC mechanotransduction and cytokine production to promote macrophage accumulation, mediated in-part by SGK-1 activation. Conclusions: Mechanical stimuli can elevate SGK-1 activity and increase pro-inflammatory cytokine production to accumulate macrophages, which may lead to proteolytic aortic remodeling. Therefore, SGK-1 may represent a novel target to abrogate hypertensive aortic inflammation.

Extracellular Vesicles as Central Mediators of COPD Pathophysiology.

Chronic obstructive pulmonary disease (COPD) is a complex, heterogeneous, smoking-related disease of significant global impact. The complex biology of COPD is ultimately driven by a few interrelated processes, including proteolytic tissue remodeling, innate immune inflammation, derangements of the host-pathogen response, aberrant cellular phenotype switching, and cellular senescence, among others. Each of these processes are engendered and perpetuated by cells modulating their environment or each other. Extracellular vesicles (EVs) are powerful effectors that allow cells to perform a diverse array of functions on both adjacent and distant tissues, and their pleiotropic nature is only beginning to be appreciated. As such, EVs are candidates to play major roles in these fundamental mechanisms of disease behind COPD. Furthermore, some such roles for EVs are already established, and EVs are implicated in significant aspects of COPD pathogenesis. Here, we discuss known and potential ways that EVs modulate the environment of their originating cells to contribute to the processes that underlie COPD.

Mapping proteolytic neo-N termini at the surface of living cells

N terminomics is a powerful strategy for profiling proteolytic neo-N termini, but its application to cell surface proteolysis has been limited by the low relative abundance of plasma membrane proteins. Here we apply plasma membrane-targeted subtiligase variants (subtiligase-TM) to efficiently and specifically capture cell surface N termini in live cells. Using this approach, we sequenced 807 cell surface N termini and quantified changes in their abundance in response to stimuli that induce proteolytic remodeling of the cell surface proteome. To facilitate exploration of our datasets, we developed a web-accessible Atlas of Subtiligase-Captured Extracellular N Termini (ASCENT; http://wellslab.org/ascent). This technology will facilitate greater understanding of extracellular protease biology and reveal neo-N termini biomarkers and targets in disease.

IL‐4 Stimulated Macrophages Induce a Pro‐Inflammatory Secretome in Fibroblasts

The precis continuum manifold of cell state of macrophages and fibroblast cells during skin fibrosis, described as a chronic inflammatory and tissue remodeling microenvironment, is not well understood. Both cell populations respond directly to damage and present a coordinated chain of activation states that dictates the reparative outcome. The spectrum change in phenotype during late remodeling is important, because it is by both cells that autocrine and paracrine communication contribute to the deposition of extracellular matrix (ECM) and chronic inflammation. Here, we provide evidence that the anti‐inflammatory M2‐like (IL‐4 induced) macrophage cell state drives activated fibroblasts to secrete important pro‐inflammatory mediators and thus changing the fibroblast cell state to one that promotes a continuous fibrotic milieu. Indirect and direct co‐culture systems were used to elevate the effects paracrine and autocrine feedback of secreted factors and cell‐contact dependent factors. These studies showed the exposure of TGF‐β‐activated fibroblasts to M2‐like macrophages altered the cytokine/chemokine secretion profile of fibroblasts to express IL‐1β, TNFα, and CXCL10 and enhanced its expression of profibrotic matrix production by using transcriptomic analysis. To understand the necessity of fibroblast‐macrophage crosstalk in the fibrotic process a gain of function approach was used by reconstituting the mice with IL‐4 polarized macrophage in an in vivo sterile wound/fibrosis bleomycin model. Tissue fibroblasts increased expression of pro‐inflammatory cytokines included IL‐10, INF‐γ, and IL‐13, as confirmed by transcriptomic analysis. These analyses additionally identified an imbalance in proteolytic remodeling actions of TIMPs and MMPs. These findings identify an important role of macrophage‐specific cell state and phenotype‐directed crosstalk with tissue remodeling activated fibroblast in signaling to orchestrate a chronic inflammatory and fibrotic microenvironment.Support or Funding InformationThis work was supported by NIH grants R01AR68317, R01AG055564, and R01GM121558.

An evaluation of patisiran: a viable treatment option for transthyretin-related hereditary amyloidosis

ABSTRACTIntroduction: Hereditary transthyretin-mediated amyloidosis (ATTRv; v for variant) is a rare, progressive, fatal multi-systemic disease, autosomal dominantly inherited with heterogeneous clinical phenotype caused by mutations in the TTR gene. Mutations promoting proteolytic remodeling and tetramer dissociation result in fragmented and full-length TTR monomers that misfold, aggregate and deposit at multiple sites (mainly nerves and heart) causing peripheral neuropathy and/or cardiomyopathy.Areas covered: The authors discuss patisiran, the first approved RNA interference-based therapeutic agent that suppresses the circulating levels of the amyloidogenic protein TTR both wild-type and mutant. This compound demonstrated a safe clinical profile in phase I and II studies and showed a significant clinical effect in a phase III (APOLLO) trial in ATTRv patients. An open-label-extension study is still underway but, based on the positive results, the regulatory agencies granted approval for the treatment of ATTRv with polyneuropathy in Stage I and II.Expert opinion: The patisiran program has demonstrated that substantial TTR concentration reduction is associated with significant and sustained improvement in polyneuropathy scores, quality-of-life profile and several outcome measures that capture the systemic burden of the disease. The drug resulted safe also in long term follow-up studies while its efficacy for ATTR with cardiomyopathy is under investigation.

Demystifying the extracellular matrix and its proteolytic remodeling in the brain: structural and functional insights.

The extracellular matrix (ECM) plays diverse roles in several physiological and pathological conditions. In the brain, the ECM is unique both in its composition and in functions. Furthermore, almost all the cells in the central nervous system contribute to different aspects of this intricate structure. Brain ECM, enriched with proteoglycans and other small proteins, aggregate into distinct structures around neurons and oligodendrocytes. These special structures have cardinal functions in the normal functioning of the brain, such as learning, memory, and synapse regulation. In this review, we have compiled the current knowledge about the structure and function of important ECM molecules in the brain and their proteolytic remodeling by matrix metalloproteinases and other enzymes, highlighting the special structures they form. In particular, the proteoglycans in brain ECM, which are essential for several vital functions, are emphasized in detail.

Strong association of tissue inhibitor of metalloproteinase (TIMP)-2 and -3 promoter single nucleotide polymorphisms with risk of colorectal cancer in ethnic Kashmiri population - a case control study.

Background: The tissue inhibitors of metalloproteinases (TIMPs) including TIMP2 and TIMP3 are the key physiological inhibitors of matrix metalloproteinases (MMPs) and along with MMPs, TIMPs play a vital role in the coordinated proteolytic breakdown and remodeling of the extracellular matrix (ECM) and the basement membrane that represent the barriers to any malignant tumor invasion and progression. These enzymes are vital for tumor invasion and metastasis and also play a critical role in several other stages of tumor development and progression. The studies on the association of various polymorphisms in human TIMP2 and TIMP3 genes including TIMP2-418G/C and TIMP3-1296T/C single nucleotide polymorphisms (SNPs) and CRC risk are limited, mixed, and inconclusive.Materials and methods: The aim of the present study was to analyze the association of TIMP2-418G/C and TIMP3-1296T/C promoter SNPs with colorectal cancer (CRC) susceptibility and development risk and also to evaluate the modifying effects of possible TIMP2-418G/C and TIMP3-1296T/C SNPs’ genotypes on different risk factors of CRC or the reciprocal effect in ethnic population of Kashmir, India through a case–control setup. The genotype frequencies of TIMP2-418G/C and TIMP3-1296T/C promoter SNPs were compared between 142 CRC patients and 184 individually matched healthy controls by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The associations between the TIMP2-418G/C and TIMP3-1296T/C SNPs and CRC risk were examined through conditional logistic regression models adjusted for multiple possible confounding (third) variables. The possible effect measure modification of the association between the relevant SNP genotypes and CRC risk by various CRC risk factors including age, gender, and smoking status was also analyzed. Further, the associations between these SNPs and various clinico-pathological parameters, demographic variables, and environmental factors within the case group subjects with regard to CRC risk were also evaluated.Results: The overall association between the TIMP2-418G/C and TIMP3-1296T/C SNPs and the modulation of CRC risk was found to be highly significant (P=0.019 and P=0.000 for TIMP2 and TIMP3 SNPs, respectively). The heterozygous genotype (GC) of TIMP2-418G/C was significantly associated with an increased risk of colorectal cancer [OR, 1.87 (95%CI, 1.07–3.27); P=0.027] whereas the heterozygous genotype (TC) of TIMP3-1296T/C SNP was significantly associated with a decreased risk of colorectal cancer [OR, 0.53 (95%CI, 0.32–0.86); P=0.011]. The variant genotype (CC) of TIMP3-1296T/C SNP was also significantly associated with a decreased risk of colorectal cancer [OR, 0.18 (95%CI, 0.05–0.65); P=0.009].Conclusion: The present study demonstrates that there is a strong and highly significant association between the TIMP2-418G/C and TIMP3-1296T/C promoter SNPs and the risk of developing CRC in ethnic Kashmiri population. However, in order to substantiate our findings, the present study needs to be replicated with bigger sample size and should involve other ethnically defined populations with high CRC risk.

Oxalomalate suppresses metastatic melanoma through IDH-targeted stress response to ROS

Melanoma is the most aggressive skin cancer due to a high propensity for metastasis, with a 10-year survival rate of less than 10%. The devastating clinical outcome and lack of effective preventative therapeutics for metastatic melanoma necessitate the development of new therapeutic strategies targeted to inhibit the regulatory circuits underlying the progression and metastasis of melanoma. Melanoma metastasis requires migration and invasion of the malignant tumour cells driven by proteolytic remodelling of the extracellular matrix (ECM) executed by matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9. Inhibiting components of these circuits defines new therapeutic opportunities for melanoma with metastatic malignancy. Oxalomalate (OMA) is a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase (IDH), which plays an important role in cellular signalling pathways regulated by reactive oxygen species (ROS). In this study, we investigated the therapeutic role of OMA in metastatic melanoma and the associated underlying mechanism of action. We report that OMA-mediated inhibition of IDH enzymes suppresses metastatic melanoma through inhibition of invasive cell migration based on MMP-9-mediated proteolytic remodelling of the ECM. In particular, our study provides the mechanistic foundation that OMA reduces the expression and secretion of MMP-9 through LKB1-mediated PEA3 degradation via the ROS-dependent ATM-Chk2-p53 signalling axis, resulting from inhibition of IDH enzymes. These results provide evidence that OMA targeting of the stress response to ROS by IDH inhibition is a promising therapy for the treatment of metastatic melanoma.