Matrix Metalloprotease Inhibitor Research Articles

The role of the tumor microenvironment in papillary thyroid microcarcinoma nodal metastasis.

The genetic alterations currently identified in papillary thyroid microcarcinomas (PTMCs) are insufficient for distinguishing tumors with aggressive features. We aimed to identify candidate markers associated with lateral lymph node metastasis (LLNM, N1b disease) in patients with PTMC using transcriptomic analysis. RNA sequencing was performed on 26 matched tumor and normal thyroid tissue samples (N0, n = 14; N1b, n = 12), followed by functional enrichment analyses of differentially expressed genes (DEGs). EcoTyper was used to explore the distinct tumor microenvironment (TME). We identified 631 DEGs (213 upregulated and 418 downregulated) between N1b and N0 PTMCs. The most significantly upregulated genes in N1b were associated with tumorigenesis, adhesion, migration, and invasion. DEGs were mainly enriched in the pathways of idiopathic pulmonary fibrosis, TME, wound healing, and inhibition of matrix metalloproteases. We predicted the activation of these pathways in N1b PTMCs. N1b PTMCs had a unique TME with abundant fibroblasts and epithelial cells, associated with an increased risk of disease progression. Fibroblast marker genes, including POSTN, MMP11, TNFAIP6, and FN1, and epithelial cell marker genes, including NOX4, MFAP2, TGFVBI, and TNC, were selected. POSTN and FN1, fibroblast cell-specific genes, and NOX4 and TNC, epithelial cell-specific genes, were promising biomarkers for predicting LLNM development and recurrence in patients with PTMC. We delineated the cellular ecotypes within the TME of patients with N1b PTMC and revealed potential markers for predicting LLNM and the prognosis of PTMC. These findings provide valuable insights into the contributions of cancer-associated fibroblasts and epithelial cells to PTMC progression and metastasis.

Abstract A112: Mechanisms underlying platinum-induced ovarian cancer stem cell plasticity

Abstract Most women diagnosed with late-stage high grade serous ovarian cancer (HGSOC) develop recurrent, platinum-resistant tumors. Ovarian cancer stem cells (OCSCs) are hypothesized to contribute to the emergence of these resistant tumors. CSCs have been postulated to reside in a plastic state, which can allow for the conversion of non-CSC to CSC. This process of dedifferentiation continues during tumor development and chemotherapeutic agents like platinum can exaggerate CSC plasticity. We have previously demonstrated that acute platinum treatment enriched for OCSCs. However, whether platinum transforms non-OCSCs into OCSCs to contribute to this subpopulation of cells remains unclear, and the underlying mechanism remains incompletely understood. To examine OCSC plasticity, aldehyde dehydrogenase (ALDH; functional marker) and fluorescence activated cell sorting were used to isolate OCSCs (ALDH+) and non-OCSCs (ALDH-) from HGSOC cell lines, OVCAR5 and OVCAR3. To determine the stability of the non-OCSC phenotype, ALDH- cells were cultured for 3 and 5 days and ALDH activity was measured using flow cytometry. At both timepoints examined, ALDH- cells remained ALDH-, with approximately less than 1% being ALDH+ (p<0.05). To determine if platinum can induce conversion of non-OCSC to OCSC, ALDH- cells were treated with cisplatin (12µM or 15 µM for 16h), and the percent of ALDH+ cells was measured using flow cytometry. Treatment of ALDH- cells with cisplatin resulted in conversion of approximately 4% and 10% of ALDH- cells into ALDH+ cells (p<0.05) in OVCAR5 and OVCAR3 cells, respectively. Furthermore, both cell lines displayed increased expression (p<0.05) of stemness genes BMI1, NANOG, OCT4, and SOX2 and spheroid formation was observed in converted OCSCs compared to parental ALDH+ and whole cell populations treated with cisplatin, suggesting that platinum induced the observed differences in the stemness phenotype. With the goal of targeting key genes and pathways to inhibit platinum-induced OCSC conversion, RNA-sequencing was performed using converted OCSCs. Activated pathways in converted OCSCs were identified, including protein kinase signaling, inhibition of matrix metalloproteases, SPINK1 pathway, and VDR/RXR activation. We are further investigating if these pathways contribute to the mechanism involved in OCSC plasticity and whether targeting these pathways can reduce the OCSC population, which is critical to blocking the persistence of OCSCs and ultimately reduce mortality in patients. Citation Format: Tara X. Metcalfe, Shu Zhang, Christiane Hassel, Heather M. O'Hagan, Kenneth P. Nephew. Mechanisms underlying platinum-induced ovarian cancer stem cell plasticity [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A112.

Proteomic Associations of NT-proBNP (N-Terminal Pro-B-Type Natriuretic Peptide) in Heart Failure With Preserved Ejection Fraction.

NT-proBNP (N-terminal pro-B-type natriuretic peptide) levels are variably elevated in heart failure with preserved ejection fraction (HFpEF), even in the presence of increased left ventricular filling pressures. NT-proBNP levels are prognostic in HFpEF and have been used as an inclusion criterion for several recent randomized clinical trials. However, the underlying biologic differences between HFpEF participants with high and low NT-proBNP levels remain to be fully understood. We measured 4928 proteins using an aptamer-based proteomic assay (SOMAScan) in available plasma samples from 2 cohorts: (1) Participants with HFpEF enrolled in the PHFS (Penn Heart Failure Study; n=253); (2) TOPCAT (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist Trial) participants in the Americas (n=218). We assessed the relationship between SOMAScan-derived plasma NT-proBNP and levels of other proteins available in the SOMAScan assay version 4 using robust linear regression, with correction for multiple comparisons, followed by pathway analysis. NT-proBNP levels exhibited prominent proteome-wide associations in PHFS and TOPCAT cohorts. Proteins most strongly associated with NT-proBNP in both cohorts included SVEP1 (sushi, von Willebrand factor type-A, epidermal growth factor, and pentraxin domain containing 1; βTOPCAT=0.539; P<0.0001; βPHFS=0.516; P<0.0001) and ANGPT2 (angiopoietin 2; βTOPCAT=0.571; P<0.0001; βPHFS=0.459; P<0.0001). Canonical pathway analysis demonstrated consistent associations with multiple pathways related to fibrosis and inflammation. These included hepatic fibrosis and inhibition of matrix metalloproteases. Analyses using cut points corresponding to estimated quantitative concentrations of 360 pg/mL (and 480 pg/mL in atrial fibrillation) revealed similar proteomic associations. Circulating NT-proBNP levels exhibit prominent proteomic associations in HFpEF. Our findings suggest that higher NT-proBNP levels in HFpEF are a marker of fibrosis and inflammation. These findings will aid the interpretation of NT-proBNP levels in HFpEF and may guide the selection of participants in future HFpEF clinical trials.

Based on Clinical Research Matrix Metalloprotease (MMP) Inhibitors to Promote Diabetic Wound Healing.

Chronic inflammation is a common factor in obesity, diabetes mellitus, and the complications of diabetes, including diabetic wounds. These ulcers are characterized by persistent lesions that are challenging to heal, significantly decreasing patients' quality of life and imposing a substantial financial burden on society. MMP are zinc endopeptidases that play a role in wound healing in response to various stimuli, including diabetes mellitus. MMP levels fluctuate throughout the wound healing process in diabetic patients' serum, skin tissues, and wound fluid, indicating their potential as biomarkers for diabetic foot ulcers. Targeting MMP has emerged as a promising strategy for treating diabetic wounds, as these enzymes are involved in critical biological processes related to wound healing, including extracellular matrix secretion, angiogenesis, granulation tissue formation, collagen growth, re-epithelization, inflammatory response, and oxidative stress.

Peptide G-Protein-Coupled Receptors and ErbB Receptor Tyrosine Kinases in Cancer.

The ErbB RTKs (EGFR, HER2, HER3, and HER4) have been well-studied in cancer. EGFR, HER2, and HER3 stimulate cancer proliferation, principally by activating the phosphatidylinositol-3-kinase and extracellular signal-regulated kinase (ERK) pathways, resulting in increased cancer cell survival and proliferation. Cancer cells have high densities of the EGFR, HER2, and HER3 causing phosphorylation of tyrosine amino acids on protein substrates and tyrosine amino acids near the C-terminal of the RTKs. After transforming growth factor (TGF) α binds to the EGFR, homodimers or EGFR heterodimers form. HER2 forms heterodimers with the EGFR, HER3, and HER4. The EGFR, HER2, and HER3 are overexpressed in lung cancer patient tumors, and monoclonal antibodies (mAbs), such as Herceptin against HER2, are used to treat breast cancer patients. Patients with EGFR mutations are treated with tyrosine kinase inhibitors, such as gefitinib or osimertinib. Peptide GPCRs, such as NTSR1, are present in many cancers, and neurotensin (NTS) stimulates the growth of cancer cells. Lung cancer proliferation is impaired by SR48692, an NTSR1 antagonist. SR48692 is synergistic with gefitinib at inhibiting lung cancer growth. Adding NTS to lung cancer cells increases the shedding of TGFα, which activates the EGFR, or neuregulin-1, which activates HER3. The transactivation process is impaired by SRC, matrix metalloprotease, and reactive oxygen species inhibitors. While the transactivation process is complicated, it is fast and occurs within minutes after adding NTS to cancer cells. This review emphasizes the use of tyrosine kinase inhibitors and SR48692 to impair transactivation and cancer growth.

Inflammatory Mesenchymal Stem Cells Express Abundant Membrane-Bound and Soluble Forms of C-Type Lectin-like CD248.

CD248 (endosialin) belongs to a glycoprotein family that also includes thrombomodulin (CD141), CLEC14A, and CD93 (AA4) stem cell markers. We analyzed the regulated expression of CD248 in vitro using skin (HFFF) and synovial (FLS) mesenchymal stem cell lines, and in fluid and tissue samples of rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Cells were incubated with either rhVEGF165, bFGF, TGF-β1, IL1-β, TNF-α, TGFβ1, IFN-γ, or PMA (Phorbol ester). There was no statistically significant change in membrane expression. A soluble (s) form of cleaved CD248 (sCD248) was detected after cell treatment with IL1-β and PMA. Matrix metalloprotease (MMP) MMP-1 and MMP-3 mRNAs were significantly up-regulated by IL1-β and PMA. A broad MMP inhibitor blocked the release of soluble CD248. In RA synovial tissue, we identified CD90+ perivascular MSCs double-stained for CD248 and VEGF. High sCD248 levels were detected in synovial fluid from RA. In culture, subpopulations of CD90+ CD14- RA MSCs were either identified as CD248+ or CD141+ cells but CD93-. CD248 is abundantly expressed by inflammatory MSCs and shed in an MMP-dependent manner in response to cytokines and pro-angiogenic growth factors. Both membrane-bound and soluble CD248 (acting as a decoy receptor) may contribute to RA pathogenesis.

Evaluation of Matrix Metalloproteases by Artificial Intelligence Techniques in Negative Biopsies as New Diagnostic Strategy in Prostate Cancer.

Usually, after an abnormal level of serum prostate-specific antigen (PSA) or digital rectal exam, men undergo a prostate needle biopsy. However, the traditional sextant technique misses 15-46% of cancers. At present, there are problems regarding disease diagnosis/prognosis, especially in patients' classification, because the information to be handled is complex and challenging to process. Matrix metalloproteases (MMPs) have high expression by prostate cancer (PCa) compared with benign prostate tissues. To assess the possible contribution to the diagnosis of PCa, we evaluated the expression of several MMPs in prostate tissues before and after PCa diagnosis using machine learning, classifiers, and supervised algorithms. A retrospective study was conducted on 29 patients diagnosed with PCa with previous benign needle biopsies, 45 patients with benign prostatic hyperplasia (BHP), and 18 patients with high-grade prostatic intraepithelial neoplasia (HGPIN). An immunohistochemical study was performed on tissue samples from tumor and non-tumor areas using specific antibodies against MMP -2, 9, 11, and 13, and the tissue inhibitor of MMPs -3 (TIMP-3), and the protein expression by different cell types was analyzed to which several automatic learning techniques have been applied. Compared with BHP or HGPIN specimens, epithelial cells (ECs) and fibroblasts from benign prostate biopsies before the diagnosis of PCa showed a significantly higher expression of MMPs and TIMP-3. Machine learning techniques provide a differentiable classification between these patients, with greater than 95% accuracy, considering ECs, being slightly lower when considering fibroblasts. In addition, evolutionary changes were found in paired tissues from benign biopsy to prostatectomy specimens in the same patient. Thus, ECs from the tumor zone from prostatectomy showed higher expressions of MMPs and TIMP-3 compared to ECs of the corresponding zone from the benign biopsy. Similar differences were found for expressions of MMP-9 and TIMP-3, between fibroblasts from these zones. The classifiers have determined that patients with benign prostate biopsies before the diagnosis of PCa showed a high MMPs/TIMP-3 expression by ECs, so in the zone without future cancer development as in the zone with future tumor, compared with biopsy samples from patients with BPH or HGPIN. Expression of MMP -2, 9, 11, and 13, and TIMP-3 phenotypically define ECs associated with future tumor development. Also, the results suggest that MMPs/TIMPs expression in biopsy tissues may reflect evolutionary changes from prostate benign tissues to PCa. Thus, these findings in combination with other parameters might contribute to improving the suspicion of PCa diagnosis.

Peripheral Arterial Thrombosis following Russell's Viper Bites.

Envenomings by Russell's viper ( Daboia russelii ), a species of high medical importance in India and other Asian countries, commonly result in hemorrhage, coagulopathies, necrosis, and acute kidney injury. Although bleeding complications are frequently reported following viper envenomings, thrombotic events occur rarely (reported only in coronary and carotid arteries) with serious consequences. For the first time, we report three serious cases of peripheral arterial thrombosis following Russell's viper bites and their diagnostic, clinical management, and mechanistic insights. These patients developed occlusive thrombi in their peripheral arteries and symptoms despite antivenom treatment. In addition to clinical features, computed tomography angiography was used to diagnose arterial thrombosis and ascertain its precise locations. They were treated using thrombectomy or amputation in one case that presented with gangrenous digits. Mechanistic insights into the pathology through investigations revealed the procoagulant actions of Russell's viper venom in standard clotting tests as well as in rotational thromboelastometry analysis. Notably, Russell's viper venom inhibited agonist-induced platelet activation. The procoagulant effects of Russell's viper venom were inhibited by a matrix metalloprotease inhibitor, marimastat, although a phospholipase A 2 inhibitor (varespladib) did not show any inhibitory effects. Russell's viper venom induced pulmonary thrombosis when injected intravenously in mice and thrombi in the microvasculature and affected skeletal muscle when administered locally. These data emphasize the significance of peripheral arterial thrombosis in snakebite victims and provide awareness, mechanisms, and robust strategies for clinicians to tackle this issue in patients.

Prothymosin α Plays Role as a Brain Guardian through Ecto-F1 ATPase-P2Y12 Complex and TLR4/MD2.

Prothymosin alpha (ProTα) was discovered to be a necrosis inhibitor from the conditioned medium of a primary culture of rat cortical neurons under starved conditions. This protein carries out a neuronal cell-death-mode switch from necrosis to apoptosis, which is, in turn, suppressed by a variety of neurotrophic factors (NTFs). This type of NTF-assisted survival action of ProTα is reproduced in cerebral and retinal ischemia-reperfusion models. Further studies that used a retinal ischemia-reperfusion model revealed that ProTα protects retinal cells via ecto-F1 ATPase coupled with the Gi-coupled P2Y12 receptor and Toll-like receptor 4 (TLR4)/MD2 coupled with a Toll-IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF). In cerebral ischemia-reperfusion models, ProTα has additional survival mechanisms via an inhibition of matrix metalloproteases in microglia and vascular endothelial cells. Heterozygous or conditional ProTα knockout mice show phenotypes of anxiety, memory learning impairment, and a loss of neurogenesis. There are many reports that ProTα has multiple intracellular functions for cell survival and proliferation through a variety of protein-protein interactions. Overall, it is suggested that ProTα plays a key role as a brain guardian against ischemia stress through a cell-death-mode switch assisted by NTFs and a role of neurogenesis.

Reviving the use of inhibitors of matrix metalloproteases in spinal cord injury: a case for specificity.

At present, there are no restorative therapies in the clinic for spinal cord injury, with current treatments offering only palliative treatment options. The role of matrix metalloproteases is well established in spinal cord injury, however, translation into the clinical space was plagued by early designs of matrix metalloprotease inhibitors that lacked specificity and fears of musculoskeletal syndrome prevented their further development. Newer, much more specific matrix metalloprotease inhibitors have revived the possibility of using these inhibitors in the clinic since they are much more specific to their target matrix metalloproteases. Here, the evidence for use of matrix metalloproteases after spinal cord injury is reviewed and researchers are urged to overcome their old fears regarding matrix metalloprotease inhibition and possible side effects for the field to progress. Recently published work by us shows that inhibition of specific matrix metalloproteases after spinal cord injury holds promise since four key consequences of spinal cord injury could be alleviated by specific, next-generation matrix metalloprotease inhibitors. For example, specific inhibition of matrix metalloprotease-9 and matrix metalloprotease-12 within 24 hours after injury and for 3 days, alleviates spinal cord injury-induced edema, blood-spinal cord barrier breakdown, neuropathic pain and restores sensory and locomotor function. Attempts are now underway to translate this therapy into the clinic.

Multispectroscopic, virtual and in vivo insights into the photoaging defense mediated by the natural food colorant bixin.

An upsurge in early onset of photoaging due to repeated skin exposure to environmental stressors such as UV radiation is a challenge for pharmaceutical and cosmeceutical divisions. Current reports indicate severe side effects because of chemical or synthetic inhibitors of matrix metalloproteases (MMPs) in anti-skin aging cosmeceuticals. We evaluated the adequacy of bixin, a well-known FDA certified food additive, as a scavenger of free radicals and its inhibitory mechanism of action on MMP1, collagenase, elastase, and hyaluronidase. The anti-skin aging potential of bixin was evaluated by several biotechnological tools in silico, in vitro and in vivo. Molecular docking and simulation dynamics studies gave a virtual insight into the robust binding interaction between bixin and skin aging-related enzymes. Absorbance and fluorescence studies, enzyme inhibition assays, enzyme kinetics and in vitro bioassays of human dermal fibroblast (HDF) cells highlighted bixin's role as a potent antioxidant and inhibitor of skin aging-related enzymes. Furthermore, in vivo protocols were carried out to study the impact of bixin administration on UVA induced photoaging in C57BL/6 mice skin. Here, we uncover the UVA shielding effect of bixin and its efficacy as a novel anti-photoaging agent. Furthermore, the findings of this study provide a strong foundation to explore the pharmaceutical applications of bixin in several other biochemical pathways linked to MMP1, collagenase, elastase, and hyaluronidase.

Abstract 13724: Development of VS-041 as a Precision Medicine Targeting Cardiac-Relevant Matrix Metalloproteases (MMPs) in Heart Failure With Preserved Ejection Fraction (HFpEF)

Introduction. HFpEF is a heterogeneous syndrome, with several underlying etiologic and pathophysiologic factors. As “one size fits all” therapies are not equally efficacious across various HFpEF comorbidities, a precision medicine approach can offer a disease modifying treatment for HFpEF subpopulation stratified for specific molecular targets. Expression of MMP2, 9, and 13 in heart is elevated in HFpEF patients with hypertension, diabetes or coronary disease, and their levels in plasma correlate with survival, suggesting utility as predictive biomarkers. Genetic ablation of MMP2, 9, and 13 leads to a robust antifibrotic effect in the myocardium. Inhibition of MMP2, 9, and 13 directly regulates of Ca 2+ transients and protects sarcomere function. Hypothesis. We present preclinical profile of VS-041, a novel and narrow-spectrum MMP inhibitor, as a drug candidate for HFpEF patients with elevated levels of MMP2, 9, and 13. Methods and Results. VS-041, optimized with computer-aided drug design methods, shows single-digit IC 50 against MMP2 (1nM), MMP9 (2nM) and MMP13 (1nM), and maintains &gt;300-fold selectivity against MMP1 and TACE. In isolated cardiomyocytes VS-041 reduces contractility deficit due to fast pacing. With high predicted metabolic bioavailability (MF%=90), &gt;80% target occupancy by VS-041 is estimated after 50 mg/kg PO dose in rats. VS-041 demonstrated robust efficacy in Dahl Salt Sensitive rat model of hypertension and HFpEF after dosing at 25 or 50 mg/kg PO for 5 wks in a therapeutic mode (after 1-wk of high salt lead-in). VS-041 had no effect on blood pressure or ejection fraction confirming a direct effect on diastolic function. In Doppler mitral inflow, analysis of the isovolumic relaxation time and the E/A ratio of peak velocity of filling waves revealed 40% improvement in diastolic function. LV fibrosis was dose-proportionally decreased based on PSR morphometry. No adverse effects were observed after 5 weeks of dosing. Conclusions. Preclinical pharmacology and safety profile of VS-041 support its nomination as clinical candidate and initiation of IND-enabling studies. VS-041 has potential to be first-in-class precision medicine treatment for HFPEF patients with elevated levels of cardiac-relevant MMP2, 9 and 13.

How to place the duality of specific MMP-9 inhibition for treatment of inflammatory bowel diseases into clinical opportunities?

Crohn’s disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) with the involvement of immune cells and molecules, including cytokines, chemokines and proteases. A previous extensive review about the molecular biology of matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteases (TIMPs), related to intestinal barrier destruction and restoration functions in IBD, is here complemented with the literature from the last five years. We also compare IBD as a prototypic mucosal inflammation of an epithelial barrier against microorganisms with inflammatory retinopathy as a disease with a barrier dysfunction at the level of blood vessels. Multiple reasons are at the basis of halting clinical trials with monoclonal antibodies against MMP-9 for IBD treatment. These include (i) the absence of a causative role of MMP-9 in the pathology in animal models of IBD, (ii) the fact that endotoxins, crossing the intestinal barrier, induce massive local release of both neutrophil collagenase (MMP-8) and gelatinase B (MMP-9), (iii) insufficient recognition that MMPs modify the activities of cytokines, chemokines and their receptors, (iv) ignorance that MMPs exist as mixtures of proteoforms with different posttranslational modifications and with different specific activities and (v) the fact that MMPs and TIMPs act in an interactive network, possibly having also beneficial effects on IBD evolution. Nevertheless, inhibition of MMPs may be a useful therapeutic approach during specific IBD disease phases or in specific sub-phenotypes. This temporary “window of opportunity” for MMP-9 inhibition may be complemented by a locoregional one, provided that the pharmacological agents are targeted in time to affected tissues, as is achieved in ophthalmological inflammation. Thus, in order to discover spatial and temporal windows of opportunity for MMP inhibition as treatment of IBD, more preclinical work including well controlled animal studies will be further needed. In this respect, MMP-9/NGAL complex analysis in various body compartments is helpful for better stratification of IBD patients who may benefit from anti-MMP-9.

Plant Bioactives as Inhibitors of Matrix Metalloproteases and their Anti-skin Photoaging Potential

Plant Bioactives as Inhibitors of Matrix Metalloproteases and their Anti-skin Photoaging Potential

Abstract 3843: TIMP1 and TIMP2 re-educate TGFβ induced pro-inflammatory/pro angiogenic decidual-like NK cells phenotype

Abstract Background: Tissue inhibitors of metalloproteases (TIMPs) have been reported to exert dual roles in cancer, based on the different cancer type and interactions with the tumor microenvironments. As inhibitors of Matrix metalloproteases (MMP) they downregulate invasion, metastasis and angiogenesis. However, TIMP1 levels have been found increased in patients with different types of cancer. Natural Killer cells have been found to be phenotypically and functionally compromised in several solid and hematological cancers. We were the first in characterizing NK pro-angiogenic phenotype in the peripheral blood and tumor tissues of patients with Non-small Cell Lung cancer, colorectal cancers, prostate of patients. Also, we found that pro-angiogenic NK cells in the peripheral blood of colorectal cancer patients release of TIMP-1 and TIMP-2, in a STAT3/STAT5-independent manner. Here, we investigated the effects of TIMP-1 and TIMP-2 recombinant proteins, using a TGFβ-mediated NK cell polarization model, in vitro. Methods: We investigated the effects of TIMP1 and TIMP2 recombinant proteins in hindering decidual-like markers in NK cells, generated by polarizing cytolytic NK cells with TGFβ. The effects of TIMP1 or TIMP2 on NK cell surface antigens were determined by multicolor flow cytometry. Results: We found that TIMP1 and TIMP2 were effective in interfering with TGFβ induced NK cell polarization towards a decidual-like-phenotype. TIMP1 and TIMP2 counteracted the effect of TGFβ in increasing the percentage of CD56bright, CD16-, CD9+ and CD49a+, and restoring normal levels for TIMP 1 and 2 also inhibited decrease levels of the activation marker NKG2D induced by TGFβ and decreased the TGFβ upregulated exhaustion marker TIM-3. NK cell degranulation capabilities against K562 cells were also decreased by TGFβ and not by TIMP1 or TIMP2. TIMP1 treatment could partially restore degranulation marker CD107a expression. Conclusions: Our results suggest a potential role of TIMPs in controlling the tumor-associated cytokine TGFβ-induced NK cell polarization. TGFβ stimulation represents a model to prove TIMP's new properties, further studies will help defining TIMPs immunomodulatory activities of NK cells in cancer, and their possible future diagnostic-therapeutic roles. Citation Format: Adriana Albini, Matteo Gallazzi, Maria Teresa Palano, Valentina Carlini, Luana Calabrone, Riccardo Ricotta, William G. Stetler-Stevenson, Douglas M. Noonan. TIMP1 and TIMP2 re-educate TGFβ induced pro-inflammatory/pro angiogenic decidual-like NK cells phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3843.

Abstract 848: Inhibition of RTK fusion condensates enhances signal perception and promotes drug tolerance

Abstract Drug resistance remains a significant obstacle in the successful treatment of cancer, highlighting the critical need to understand how oncogenes and cancer drugs impact cell physiology and resistance development. EML4-ALK is a receptor tyrosine kinase (RTK) fusion oncogene that drives 3-7% of lung cancer. Despite potent ALK inhibitors, EML4-ALK+ cancers frequently develop resistance during therapy. Recently, it was discovered that EML4-ALK and other RTK fusions form cytoplasmic protein condensates, and that condensate formation was required for oncogenic signaling. However, whether oncogenic condensates play a role in drug responses is unclear. In this study, we applied an optogenetic technique called ‘functional profiling’ to understand how EML4-ALK condensates impact cell signal transmission and drug response. Using light-stimulated RTKs, we found that EML4-ALK condensates strongly suppress signaling through transmembrane RTKs, including through EGFR, a central receptor in resistance development. Strikingly, treatment with ALK inhibitors (ALKi) rapidly restored and hypersensitized RTK signaling. We found that EML4-ALK condensates suppress RTK signals through sequestration of the downstream adapter Grb2, which is essential for signaling through EGFR and other RTKs. The release of Grb2 from condensates resensitized RTKs within 10s of minutes of ALKi addition. Resensitized RTKs, in turn, caused sporadic RTK activation pulses throughout the cell population, and pulses originated from paracrine RTK signals released by apoptotic neighbors. We found that these paracrine signals counteracted ALK inhibitor therapy and promoted survival and drug tolerance. Blocking paracrine signals through co-treatment of ALKi with inhibitors of either EGFR or matrix metalloproteases enhanced cell killing and minimized long-term drug tolerance. Our study uncovers a role for oncogenic condensates in drug resistance signaling, reveals a novel mechanism for oncogene-induced suppression of RTK signaling, and suggests novel co-therapies to more effectively treat cancers driven by EML4-ALK and possibly other RTK fusions. Our work also demonstrates the potential of functional optogenetic profiling for drug discovery to promote cancer therapy. Citation Format: David Gonzalez-Martinez, Lee Roth, Thomas Mumford, Yael Mosse, Asmin Tulpule, Trever Bivona, Lukasz Jan Bugaj. Inhibition of RTK fusion condensates enhances signal perception and promotes drug tolerance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 848.

UVB Drives Metabolic Rewiring and Epigenetic Reprograming and Protection by Sulforaphane in Human Skin Keratinocytes.

Sulforaphane (SFN) is a potent anticancer agent which could protect the skin from ultraviolet (UV) radiation-induced insults. Currently, the metabolic rewiring and epigenetic reprograming induced by UVB and the role of SFN in UVB-mediated skin cell transformation remain largely unknown. Herein, we study the metabolome, epigenome, and transcriptome of human keratinocytes (HaCaT cells) exposed to UVB with or without SFN using liquid chromatography-mass spectroscopy, DNA methylation sequencing, and RNA sequencing. UVB increases intracellular reactive oxygen species (ROS) and SFN enhances ROS acutely in post-UVB-exposed HaCaT cells. UVB and SFN alter multiple metabolites and metabolism-related signaling pathways. Pathway analysis shows that UVB impacts numerous signaling pathways including STAT3, inhibition of matrix metalloproteases, and TGF-β, among others. DNA/CpG methylation analysis shows that SFN could partially reverse some of the alterations of UVB-induced CpG methylome. Integrating RNA-seq and Methyl-seq data, starburst plots show the correlation of mRNA expression and CpG methylation status. The potential linkages between the metabolome, CpG methylome, and transcriptome suggest that metabolites produced during metabolism act as cofactors or substrates for catalytic epigenetic modification and transcriptional regulation. These results indicate that UVB drives metabolic rewiring, epigenetic reprograming, and phenotypic transcriptomic alterations and SFN would block or attenuate many of these aberrations, potentially contributing to the overall protective effect of SFN against UVB-induced skin damage.

Genipin, an Inhibitor of UCP2 as a Promising New Anticancer Agent: A Review of the Literature.

Genipin is a protein cross-linking agent extracted from Gardenia (Gardenia jasminoides Ellis) fruits. This fruit has conventionally been used as a Chinese herbal medicine for the treatment of inflammation and jaundice and as an edible colorant in oriental countries. Uncoupling protein (UCP)-2 is a member of the family of uncoupling proteins, which are anion transporters positioned in the mitochondrial inner membrane. Genipin has been shown to have hepatoprotective activity, acting as an effective antioxidant and inhibitor of mitochondrial UCP2, and is also reported to exert significant anticancer effects. In this review, the author presents the latest progress of genipin as an anticancer agent and concisely describes its various mechanisms of action. In brief, genipin inhibits UCP2 to attenuate generation of reactive oxygen species (ROS), leading to ROS/c-Jun N-terminal kinase-dependent apoptosis of cancer cells. Genipin also increases the tissue inhibitors of matrix metalloproteases (MMP)-2, a kind of tumor promoter in a variety of cancers, as well as induces caspase-dependent apoptosis in in vitro and in vivo models. These findings suggest that genipin can serve as a promising novel antitumor agent that could be applicable for chemotherapy and/or chemoprevention for cancers.

Bacterial matrix metalloproteases and serine proteases contribute to the extra-host inactivation of enteroviruses in lake water

Enteroviruses are ubiquitous contaminants of surface waters, yet their fate in presence of microbial congeners is poorly understood. In this work, we investigated the inactivation of Echovirus-11 (E11) and Coxsackievirus-A9 (CVA9) by bacteria isolated from Lake Geneva. Incubation of E11 or CVA9 in biologically active lake water caused inactivation of 2- and 4-log10, respectively, within 48 h. To evaluate the antiviral action of individual bacterial species, we isolated 136 bacterial strains belonging to 31 genera from Lake Geneva. The majority of isolates (92) induced decay of at least 1.5-log10 of CVA9, whereas only 13 isolates induced a comparable inactivation on E11. The most extensive viral decay was induced by bacterial isolates producing matrix metalloproteases (MMPs). Correspondingly, the addition of a specific MMP inhibitor to lake water reduced the extent of inactivation for both viruses. A lesser, though significant protective effect was also observed with inhibitors of chymotrypsin-like or trypsin-like proteases, suggesting involvement of serine proteases in enterovirus inactivation in natural systems. Overall, we demonstrate the direct effect of bacterial proteases on the inactivation of enteroviruses and identify MMPs as effective controls on enteroviruses’ environmental persistence.

Evaluation of Small Molecule Inhibitors of Pseudomonas Virulence factor LasB as Non‐Traditional Immunotherapeutics

Pseudomonas aeruginosa can cause severe opportunistic pulmonary infections associated with mechanical ventilation and the genetic disease cystic fibrosis. Due to its multidrug resistance, it is a priority pathogen for therapeutic development. Inflammation during such infections, which causes destruction of lung architecture and function, is activated in part through the maturation of IL‐1β by the virulence factor LasB. Previous results in mouse models suggest that matrix metalloprotease inhibitor drugs also inhibit LasB, preventing cell death and inflammation caused by caused by P. aeruginosa. We hypothesized that other inhibitors of matrix metalloproteases, including tetracycline‐family antibiotics, could be used to target LasB. We evaluated small molecule inhibitors against LasB activation during infections of macrophages, neutrophils, and monocytes and the synergy of these inhibitors with conventional antibiotics and anti‐inflammatories. We first optimized the drug concentration by testing the ability of the various drugs to inhibit LasB at various dilutions. Using an effective concentration, we then infected the various cell types with P. aeruginosa and analyzed the impact of the inhibitors on cell death and maturation of IL‐1β in infected cells. The results indicate that several tetracycline‐family compounds inhibit LasB during P. aeruginosa infections and decrease cell death. This was true for compounds that had no antimicrobial activity yet still inhibited LasB. In addition, the results suggest that the drugs target different cell types. Together, we find that tetracyclines may have therapeutic benefits, even against resistant bacteria, due to their ability to decrease inflammation. This may serve as an effective adjunctive therapy during infection to limit tissue damage and give the immune system and antibiotics more time to respond.