1,4-dihydroxy quininib activates ferroptosis pathways in metastatic uveal melanoma and reveals a novel prognostic biomarker signature.

Uveal melanoma (UM) is an ocular cancer, with propensity for lethal liver metastases. When metastatic UM (MUM) occurs, as few as 8% of patients survive beyond two years. Efficacious treatments for MUM are urgently needed. 1,4-dihydroxy quininib, a cysteinyl leukotriene receptor 1 (CysLT1) antagonist, alters UM cancer hallmarks in vitro, ex vivo and in vivo. Here, we investigated the 1,4-dihydroxy quininib mechanism of action and its translational potential in MUM. Proteomic profiling of OMM2.5 cells identified proteins differentially expressed after 1,4-dihydroxy quininib treatment. Glutathione peroxidase 4 (GPX4), glutamate-cysteine ligase modifier subunit (GCLM), heme oxygenase 1 (HO-1) and 4 hydroxynonenal (4-HNE) expression were assessed by immunoblots. Biliverdin, glutathione and lipid hydroperoxide were measured biochemically. Association between the expression of a specific ferroptosis signature and UM patient survival was performed using public databases. Our data revealed that 1,4-dihydroxy quininib modulates the expression of ferroptosis markers in OMM2.5 cells. Biochemical assays validated that GPX4, biliverdin, GCLM, glutathione and lipid hydroperoxide were significantly altered. HO-1 and 4-HNE levels were significantly increased in MUM tumor explants from orthotopic patient-derived xenografts (OPDX). Expression of genes inhibiting ferroptosis is significantly increased in UM patients with chromosome 3 monosomy. We identified IFerr, a novel ferroptosis signature correlating with UM patient survival. Altogether, we demontrated that in MUM cells and tissues, 1,4-dihydroxy quininib modulates key markers that induce ferroptosis, a relatively new type of cell death driven by iron-dependent peroxidation of phospholipids. Furthermore, we showed that high expression of specific genes inhibiting ferroptosis is associated with a worse UM prognosis, thus, the IFerr signature is a potential prognosticator for which patients develop MUM. All in all, ferroptosis has potential as a clinical biomarker and therapeutic target for MUM.

cFLIPL acts as a suppressor of TRAIL- and Fas-initiated inflammation by inhibiting assembly of caspase-8/FADD/RIPK1 NF-κB-activating complexes.

TRAIL and FasL are potent inducers of apoptosis but can also promote inflammation through assembly of cytoplasmic caspase-8/FADD/RIPK1 (FADDosome) complexes, wherein caspase-8 acts as a scaffold to drive FADD/RIPK1-mediated nuclear factor κB (NF-κB) activation. cFLIP is also recruited to FADDosomes and restricts caspase-8 activity and apoptosis, but whether cFLIP also regulates death receptor-initiated inflammation is unclear. Here, we show that silencing or deletion of cFLIP leads to robustly enhanced Fas-, TRAIL-, or TLR3-induced inflammatory cytokine production, which can be uncoupled from the effects of cFLIP on caspase-8 activation and apoptosis. Mechanistically, cFLIPL suppresses Fas- or TRAIL-initiated NF-κB activation through inhibiting the assembly of caspase-8/FADD/RIPK1 FADDosome complexes, due to the low affinity of cFLIPL for FADD. Consequently, increased cFLIPL occupancy of FADDosomes diminishes recruitment of FADD/RIPK1 to caspase-8, thereby suppressing NF-κB activation and inflammatory cytokine production downstream. Thus, cFLIP acts as a dual suppressor of apoptosis and inflammation via distinct modes of action.

Synthesis of mono-nitroxides and of bis-nitroxides with varying electronic through-bond communication.

Nitroxides are a unique class of persistent radicals finding a wide range of applications, from spin probes to polarizing agents, and recently bis-nitroxides have been used as proof-of-concept molecules for quantum information processing. Here we present the syntheses of pyrroline-based nitroxide (NO) radicals and give a comparision of two possible synthetic routes to form two key intermediates, namely 2,2,5,5-tetramethylpyrroline-1-oxyl-3-acetylene (TPA) and 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxylic acid (TPC). TPC and TPA were then used as precursors for the synthesis of three model compounds featuring two distant NO groups with a variable degree of conjugation and thus electronic communication between them. Using relatively facile synthetic routes, we produced a number of mono- and bis-nitroxides with the structures of multiple compounds unambiguously characterized by X-ray crystallography, while Continuous Wave Electron Paramagnetic Resonance (CW-EPR) allowed us to quantify the electronic communication in the bis-nitroxides. Our study expands the repertoire of mono- and bis-nitroxides with possibilities of exploiting them for studying quantum coherence effects and as polarizing agents.

Correction: Hamilton et al. Therapeutics Targeting the Core Apoptotic Machinery. Cancers 2021, 13, 2618.

There was an error in the original publication [...].

Therapeutics Targeting the Core Apoptotic Machinery.

Therapeutic targeting of the apoptotic pathways for the treatment of cancer is emerging as a valid and exciting approach in anti-cancer therapeutics. Accumulating evidence demonstrates that cancer cells are typically "addicted" to a small number of anti-apoptotic proteins for their survival, and direct targeting of these proteins could provide valuable approaches for directly killing cancer cells. Several approaches and agents are in clinical development targeting either the intrinsic mitochondrial apoptotic pathway or the extrinsic death receptor mediated pathways. In this review, we discuss the main apoptosis pathways and the key molecular targets which are the subject of several drug development approaches, the clinical development of these agents and the emerging resistance factors and combinatorial treatment approaches for this class of agents with existing and emerging novel targeted anti-cancer therapeutics.

Effect of Chitosan Microparticles on the Uterine Microbiome of Dairy Cows with Metritis.

The objective of this study was to evaluate the effect of chitosan microparticles on the uterine microbiome of cows with metritis. Dairy cows with metritis (n = 89) were assigned to 1 of 3 treatments: chitosan microparticles (n = 21), in which the cows received an intrauterine infusion of chitosan microparticles at metritis diagnosis (day 0), day 2, and day 4; ceftiofur (n = 25), in which the cows received a subcutaneous injection of ceftiofur on day 0 and day 3; and no intrauterine or subcutaneous treatment (n = 23). Nonmetritic cows (n = 20) were healthy cows matched with cows with metritis by the number of days postpartum at metritis diagnosis. Uterine swab samples collected on days 0, 3, 6, 9, and 12 were used for 16S rRNA gene sequencing and 16S RNA gene copy number quantification by quantitative PCR. Principal-coordinate analysis showed that the microbiome of the ceftiofur-treated and metritic untreated groups progressed toward that of the nonmetritic group by day 3, whereas that of the chitosan microparticle-treated group remained unchanged. The differences on day 3 were mainly due to a greater relative abundance of Fusobacteria, particularly Fusobacterium, in the chitosan microparticle-treated group than in the ceftiofur-treated and metritic untreated groups. Furthermore, the microbiome of the ceftiofur-treated group became similar to that of the nonmetritic group by day 9, whereas the microbiome of the chitosan microparticle-treated and metritic untreated groups became similar to that of the nonmetritic group only by day 12. The total bacterial 16S rRNA gene counts in the chitosan microparticle-treated group were greater than those in the metritic untreated controls on days 6 and 9, whereas the ceftiofur treatment group was the only group in which the total bacterial 16S rRNA gene count became similar to that in the nonmetritic group by day 12. In summary, chitosan microparticles slowed the progression of the uterine microbiome toward a healthy state, whereas ceftiofur hastened the progression toward a healthy state.IMPORTANCE Third-generation cephalosporins, such as ceftiofur, are commonly used to treat metritis in dairy cows. Chitosan microparticles has been shown to have a broad spectrum of activity in vitro and to be effective against uterine pathogens in vivo; therefore, they have been hailed as a possible alternative to traditional antibiotics. Nonetheless, in the present study, we saw that chitosan microparticle treatment slowed the progression of the uterine microbiome of cows with metritis toward a healthy state, whereas ceftiofur treatment hastened the progression toward a healthy state. Given the lack of an effective alternative to traditional antibiotics and an increased concern about antimicrobial resistance, a greater effort should be devoted to the prevention of metritis in dairy cows.

The SCFSkp2 ubiquitin ligase complex modulates TRAIL-R2-induced apoptosis by regulating FLIP(L).

TRAIL-R2 (DR5) is a clinically-relevant therapeutic target and a key target for immune effector cells. Herein, we identify a novel interaction between TRAIL-R2 and the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2). We find that SCFSkp2 can interact with both TRAIL-R2's pre-ligand association complex (PLAC) and ligand-activated death-inducing signalling complex (DISC). Moreover, Cullin-1 interacts with TRAIL-R2 in its active NEDDylated form. Inhibiting Cullin-1's DISC recruitment using the NEDDylation inhibitor MLN4924 (Pevonedistat) or siRNA increased apoptosis induction in response to TRAIL. This correlated with enhanced levels of the caspase-8 regulator FLIP at the TRAIL-R2 DISC, particularly the long splice form, FLIP(L). We subsequently found that FLIP(L) (but not FLIP(S), caspase-8, nor the other core DISC component FADD) interacts with Cullin-1 and Skp2. Importantly, this interaction is enhanced when FLIP(L) is in its DISC-associated, C-terminally truncated p43-form. Prevention of FLIP(L) processing to its p43-form stabilises the protein, suggesting that by enhancing its interaction with SCFSkp2, cleavage to the p43-form is a critical step in FLIP(L) turnover. In support of this, we found that silencing any of the components of the SCFSkp2 complex inhibits FLIP ubiquitination, while overexpressing Cullin-1/Skp2 enhances its ubiquitination in a NEDDylation-dependent manner. DISC recruitment of TRAF2, previously identified as an E3 ligase for caspase-8 at the DISC, was also enhanced when Cullin-1's recruitment was inhibited, although its interaction with Cullin-1 was found to be mediated indirectly via FLIP(L). Notably, the interaction of p43-FLIP(L) with Cullin-1 disrupts its ability to interact with FADD, caspase-8 and TRAF2. Collectively, our results suggest that processing of FLIP(L) to p43-FLIP(L) at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP(L)'s interactions with other DISC components and promoting its ubiquitination and degradation, thereby modulating TRAIL-R2-mediated apoptosis.

A revised model of TRAIL-R2 DISC assembly explains how FLIP(L) can inhibit or promote apoptosis.

The long FLIP splice form FLIP(L) can act as both an inhibitor and promoter of caspase-8 at death-inducing signalling complexes (DISCs) formed by death receptors such as TRAIL-R2 and related intracellular complexes such as the ripoptosome. Herein, we describe a revised DISC assembly model that explains how FLIP(L) can have these opposite effects by defining the stoichiometry (with respect to caspase-8) at which it converts from being anti- to pro-apoptotic at the DISC. We also show that in the complete absence of FLIP(L), procaspase-8 activation at the TRAIL-R2 DISC has significantly slower kinetics, although ultimately the extent of apoptosis is significantly greater. This revised model of DISC assembly also explains why FLIP's recruitment to the TRAIL-R2 DISC is impaired in the absence of caspase-8 despite showing that it can interact with the DISC adaptor protein FADD and why the short FLIP splice form FLIP(S) is the more potent inhibitor of DISC-mediated apoptosis.

Analysis of head and neck carcinoma progression reveals novel and relevant stage-specific changes associated with immortalisation and malignancy.

We report changes in the genomic landscape in the development of head and neck squamous cell carcinomas HNSCC from potentially premalignant lesions (PPOLS) to malignancy and lymph node metastases. Likely pathological mutations predominantly involved a relatively small set of genes reported previously (TP53, KMT2D, CDKN2A, PIK3CA, NOTCH1 and FAT1) but also other predicted cancer drivers (MGA, PABPC3, NR4A2, NCOR1 and MACF1). Notably, all these mutations arise early and are present in PPOLs. The most frequent genetic changes, which follow acquisition of immortality and loss of senescence, are of consistent somatic copy number alterations (SCNAs) involving chromosomal regions enriched for genes in known and previously unreported cancer-related pathways. We mapped the evolution of SCNAs in HNSCC progression. One of the earliest SCNAs involved deletions of CSMD1 (8p23.2). CSMD1 deletions or promoter hypermethylation were present in all of the immortal PPOLs and occurred at high frequency in the immortal HNSCC cell lines. Modulation of CSMD1 in cell lines revealed significant suppression of proliferation and invasion by forced expression, and significant stimulation of invasion by knockdown of expression. Known cancer drivers NOTCH1, PPP6C, RAC1, EIF4G1, PIK3CA showed significant increase in frequency of SCNA in transition from PPOLs to HNSCC that correlated with their expression. In the later stages of progression, HNSCC with and without nodal metastases showed some clear differences including high copy number gains of CCND1, hsa-miR-548k and TP63 in the metastases group.

The microbial metagenome and bone tissue composition in mice with microbiome-induced reductions in bone strength.

The genetic components of microbial species that inhabit the body are known collectively as the microbiome. Modifications to the microbiome have been implicated in disease processes throughout the body and have recently been shown to influence bone. Prior work has associated changes in the microbial taxonomy (phyla, class, species, etc.) in the gut with bone phenotypes but has provided limited information regarding mechanisms. With the goal of achieving a more mechanistic understanding of the effects of the microbiome on bone, we perform a metagenomic analysis of the gut microbiome that provides information on the functional capacity of the microbes (all microbial genes present) rather than only characterizing the microbial taxa. Male C57Bl/6 mice were subjected to disruption of the gut microbiota (ΔMicrobiome) using oral antibiotics (from 4 to 16 weeks of age) or remained untreated (n = 6-7/group). Disruption of the gut microbiome in this manner has been shown to lead to reductions in tissue mechanical properties and whole bone strength in adulthood with only minor changes in bone geometry and density. ΔMicrobiome led to modifications in the abundance of microbial genes responsible for the synthesis of the bacterial cell wall and capsule; bacterially synthesized carbohydrates; and bacterially synthesized vitamins (B and K) (p < 0.01). Follow up analysis focused on vitamin K, a factor that has previously been associated with bone health. The vitamin K content of the cecum, liver and kidneys was primarily microbe-derived forms of vitamin K (menaquinones) and was decreased by 32-66% in ∆Microbiome mice compared to untreated animals (p < 0.01). Bone mineral crystallinity determined using Raman spectroscopy was decreased in ∆Microbiome mice (p = 0.01). This study illustrates the use of metagenomic analysis to link the microbiome to bone phenotypes and provides preliminary findings implicating microbially synthesized vitamin-K as a regulator of bone matrix quality.

Genomic Diversity, Virulence, and Antimicrobial Resistance of Klebsiella pneumoniae Strains from Cows and Humans.

Klebsiella pneumoniae is a leading cause of severe infections in humans and dairy cows, and these infections are rapidly becoming untreatable due to the emergence of multidrug-resistant (MDR) strains. However, little is known about the relationship between bovine and human K. pneumoniae isolates at the genome population level. Here, we investigated the genomic structures, pangenomic profiles, virulence determinants, and resistomes of 308 K. pneumoniae isolates from humans and dairy cows, including 96 newly sequenced cow isolates. We identified 177 functional protein families that were significantly different across human and bovine isolates; genes expressing proteins related to metal ion (iron, zinc, and calcium) metabolism were significantly more prevalent among the bovine isolates. Siderophore systems were found to be prevalent in both the bovine and the human isolates. In addition, we found that the Klebsiella ferric uptake operon kfuABC was significantly more prevalent in clinical mastitis cases than in healthy cows. Furthermore, on two dairy farms, we identified a unique IncN-type plasmid, pC5, coharboring blaCTX-M-1 and mph(A) genes, which confer resistance to cephalosporins and macrolides, respectively. We provide here the complete annotated sequence of this plasmid.IMPORTANCE We demonstrate here the genetic diversity of K. pneumoniae isolates from dairy cows and the mixed phylogenetic lineages between bovine and human isolates. The ferric uptake operon kfuABC genes were more prevalent in strains from clinical mastitis cows. Furthermore, we report the emergence of an IncN-type plasmid carrying the blaCTX-M-1 and mph(A) genes among dairy farms in the United States. Our study evaluated the genomic diversity of the bovine and human isolates, and the findings uncovered different profiles of virulence determinants among bovine and human K. pneumoniae isolates at the genome population level.

The impact of obesity and bariatric surgery on circulating and tissue biomarkers of endometrial cancer risk.

Obesity is the strongest risk factor for endometrial cancer (EC). To inform targeted screening and prevention strategies, we assessed the impact of obesity and subsequent bariatric surgery-induced weight loss on endometrial morphology and molecular pathways implicated in endometrial carcinogenesis. Blood and endometrial tissue were obtained from women with class III-IV obesity (body mass index ≥40 and ≥50 kg/m2 , respectively) immediately prior to gastric bypass or sleeve gastrectomy, and at two and 12 months' follow up. The endometrium underwent pathological examination and immunohistochemistry was used to quantify proliferation (Ki-67), oncogenic signaling (PTEN, pAKT, pERK) and hormone receptor (ER, PR) expression status. Circulating biomarkers of insulin resistance, reproductive function and inflammation were also measured at each time point. Seventy-two women underwent bariatric surgery. At 12 months, the mean change in total and excess body weight was -32.7 and -62.8%, respectively. Baseline endometrial biopsies revealed neoplastic change in 10 women (14%): four had EC, six had atypical hyperplasia (AH). After bariatric surgery, most cases of AH resolved (5/6) without intervention (3/6) or with intrauterine progestin (2/6). Biomarkers of endometrial proliferation (Ki-67), oncogenic signaling (pAKT) and hormone receptor status (ER, PR) were significantly reduced, with restoration of glandular PTEN expression, at 2 and 12 months. There were reductions in circulating biomarkers of insulin resistance (HbA1c, HOMA-IR) and inflammation (hsCRP, IL-6), and increases in reproductive biomarkers (LH, FSH, SHBG). We found an unexpectedly high prevalence of occult neoplastic changes in the endometrium of women undergoing bariatric surgery. Their spontaneous reversal and accompanying down-regulation of PI3K-AKT-mTOR signaling with weight loss may have implications for screening, prevention and treatment of this disease.

The bovine colostrum microbiome and its association with clinical mastitis.

In an effort to characterize colostrum microbial diversity and its potential associations with early-lactation clinical mastitis, we used high-throughput sequencing of the 16S rRNA gene to investigate the bovine colostrum microbiome. A prospective observational study was conducted that included 70 Holstein cows; colostrum samples were collected from all 4 mammary gland quarters. Colostrum samples were categorized according to whether the quarter was diagnosed (CMC) or not diagnosed (NCMC) with clinical mastitis during the first 30 d postpartum. Colostrum samples were dominated by Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Fusobacteria, and Tenericutes phyla, with the 6 most common taxa [order (o), family (f), and genus (g)] being g_Staphylococcus, g_Prevotella, f_Ruminococcaceae, o_Bacteroidales, o_Clostridiales, and g_Pseudomonas. The colostrum microbiota of primiparous cows was significantly richer (higher number of bacterial species) than that of multiparous cows, and differences in colostrum taxonomic structure between parities were also observed. The microbial community of NCMC samples of primiparous cows was significantly more diverse than that of CMC samples, and the relative abundances of the Tenericutes and Fusobacteria phyla as well as the Mycoplasma and Fusobacterium genera were significantly higher in NCMC than in CMC samples of primiparous cows. The colostrum core microbiome, defined as the bacterial taxa common to all colostrum samples examined, was composed of 20 taxa and included bacterial genera already known to be associated with mastitis (e.g., Staphylococcus, Mycoplasma, and Streptococcus spp.). Our results indicate that the colostrum microbiome of primiparous cows differs from that of multiparous cows, and it harbors some diversity and taxonomic markers of mammary gland health specific to primiparous cows only.

Clinical utility of a nasal swab methicillin-resistant Staphylococcus aureus polymerase chain reaction test in intensive and intermediate care unit patients with pneumonia.

This retrospective study aimed to validate the concordance between nasal swab methicillin-resistant Staphylococcus aureus (MRSA) polymerase chain reaction (PCR) test and respiratory culture and to determine the number of potentially preventable days of anti-MRSA therapy in patients with pneumonia. Two hundred adult inpatients in the intensive and intermediate care units were included. The nasal swab MRSA PCR test was positive in 55 (27.5%) patients. MRSA was isolated from respiratory culture in 21 (10.5%) patients. The nasal swab MRSA PCR test demonstrated 90.5% sensitivity, 79.9% specificity, 34.5% positive predictive value, and 98.6% negative predictive value. Anti-MRSA therapy was initiated in 168 (84%) patients. Patients in the study received a combined 782days of anti-MRSA therapy; 300days were considered potentially preventable. This study suggests that the nasal swab MRSA PCR test may be used to guide discontinuation of anti-MRSA antibiotics in patients with clinically confirmed pneumonia in the intensive or intermediate care units.

The upper respiratory tract microbiome and its potential role in bovine respiratory disease and otitis media.

The upper respiratory tract (URT) hosts a complex microbial community of commensal microorganisms and potential pathogens. Analyzing the composition and nature of the healthy URT microbiota and how it changes over time will contribute to a better understanding of the pathogenesis of pneumonia and otitis. A longitudinal study was conducted including 174 Holstein calves that were divided in four groups: healthy calves, calves diagnosed with pneumonia, otitis or both diseases. Deep pharyngeal swabs were collected on days 3, 14, 28, and 35 of life, and next-generation sequencing of the 16S rRNA gene as well as quantitative PCR was performed. The URT of Holstein dairy calves aged 3 to 35 days revealed to host a highly diverse bacterial community. The relative abundances of the bacterial genera Mannheimia, Moraxella, and Mycoplasma were significantly higher in diseased versus healthy animals, and the total bacterial load of newborn calves at day 3 was higher for animals that developed pneumonia than for healthy animals. Our results corroborate the existing knowledge that species of Mannheimia and Mycoplasma are important pathogens in pneumonia and otitis. Furthermore, they suggest that species of Moraxella can potentially cause the same disorders (pneumonia and otitis), and that high neonatal bacterial load is a key contributor to the development of pneumonia.

FLIP: A Targetable Mediator of Resistance to Radiation in Non-Small Cell Lung Cancer.

Resistance to radiotherapy due to insufficient cancer cell death is a significant cause of treatment failure in non-small cell lung cancer (NSCLC). The endogenous caspase-8 inhibitor FLIP is a critical regulator of cell death that is frequently overexpressed in NSCLC and is an established inhibitor of apoptotic cell death induced via the extrinsic death receptor pathway. Apoptosis induced by ionizing radiation (IR) has been considered to be mediated predominantly via the intrinsic apoptotic pathway; however, we found that IR-induced apoptosis was significantly attenuated in NSCLC cells when caspase-8 was depleted using RNA interference (RNAi), suggesting involvement of the extrinsic apoptosis pathway. Moreover, overexpression of wild-type FLIP, but not a mutant form that cannot bind the critical death receptor adaptor protein FADD, also attenuated IR-induced apoptosis, confirming the importance of the extrinsic apoptotic pathway as a determinant of response to IR in NSCLC. Importantly, when FLIP protein levels were downregulated by RNAi, IR-induced cell death was significantly enhanced. The clinically relevant histone deacetylase (HDAC) inhibitors vorinostat and entinostat were subsequently found to sensitize a subset of NSCLC cell lines to IR in a manner that was dependent on their ability to suppress FLIP expression and promote activation of caspase-8. Entinostat also enhanced the antitumor activity of IR in vivo Therefore, FLIP downregulation induced by HDAC inhibitors is a potential clinical strategy to radiosensitize NSCLC and thereby improve response to radiotherapy. Overall, this study provides the first evidence that pharmacological inhibition of FLIP may improve response of NCSLC to IR. Mol Cancer Ther; 15(10); 2432-41. ©2016 AACR.

Development of a potent and selective cell penetrant Legumain inhibitor.

This Letter describes the continued SAR exploration of small molecule Legumain inhibitors with the aim of developing a potent and selective in vitro tool compound. Work continued in this Letter explores the use of alternative P2-P3 linker units and the P3 group SAR which led to the identification of 10t, a potent, selective and cellularly active Legumain inhibitor. We also demonstrate that 10t has activity in both cancer cell viability and colony formation assays.

Distribution of alkaline phosphatase, osteopontin, RANK ligand and osteoprotegerin in calcified human carotid atheroma.

Ectopic vascular calcification is a significant component of atherosclerotic disease. Osteopontin (OPN), Osteoprotegerin (OPG), Receptor Activator of NFκB Ligand (RANKL), and alkaline phosphatase (ALP) are each thought to play central roles in the calcification or demineralization of atherosclerotic lesions. Abnormalities in the balance of these proteins may lead to perturbations in bone remodeling and arterial calcification. The purpose of this study was to measure the distribution of these proteins in human carotid lesions and to elucidate possible mechanism(s) whereby they control the deposition or depletion of arterial calcification. Thirty-three patients who had undergone carotid endarterectomy (CEA) within the previous 18 months and 11 control patients were enrolled. CEA specimens were analyzed by EBCT for calcification content in terms of Agatston (AGAT) and Volume scores. CEA specimens were then cut into 5 mm segments which were homogenized and extracted. Extracts were analyzed for tissue levels of calcium, phosphorus, ALP, OPN, RANKL, and OPG. Fasting blood samples were analyzed for the same components. In CEA tissue segments, the calcification levels (CHA AGAT) were inversely associated with the levels of OPG (r = -0.432/-0.579, p < 0.05) and positively associated with the levels of RANKL (r = 0.332/0.415, p < 0.05). In turn, the tissue levels of OPG were associated with homologous serum levels of OPG (r = 0.820/0.389, p < 0.001), and the tissue levels of RANKL were associated with the serum levels of homologous RANKL (r = 0.739/0.666, p < 0.0001). This study suggests that serum levels of OPG and RANKL may be useful biomarkers for estimating the degree of calcification in carotid atherosclerotic lesions.

P3 SAR exploration of biphenyl carbamate based Legumain inhibitors.

This Letter describes the further development and SAR exploration of a novel series of Legumain inhibitors. Based upon a previously identified Legumain inhibitor from our group, we explored the SAR of the carbamate phenyl ring system to probe the P3 pocket of the enzyme. This led to the identification of a sub-nanomolar inhibitor of Legumain.

TBX2 represses CST6 resulting in uncontrolled legumain activity to sustain breast cancer proliferation: a novel cancer-selective target pathway with therapeutic opportunities.

TBX2 is an oncogenic transcription factor known to drive breast cancer proliferation. We have identified the cysteine protease inhibitor Cystatin 6 (CST6) as a consistently repressed TBX2 target gene, co-repressed through a mechanism involving Early Growth Response 1 (EGR1). Exogenous expression of CST6 in TBX2-expressing breast cancer cells resulted in significant apoptosis whilst non-tumorigenic breast cells remained unaffected. CST6 is an important tumor suppressor in multiple tissues, acting as a dual protease inhibitor of both papain-like cathepsins and asparaginyl endopeptidases (AEPs) such as Legumain (LGMN). Mutation of the CST6 LGMN-inhibitory domain completely abrogated its ability to induce apoptosis in TBX2-expressing breast cancer cells, whilst mutation of the cathepsin-inhibitory domain or treatment with a pan-cathepsin inhibitor had no effect, suggesting that LGMN is the key oncogenic driver enzyme. LGMN activity assays confirmed the observed growth inhibitory effects were consistent with CST6 inhibition of LGMN. Knockdown of LGMN and the only other known AEP enzyme (GPI8) by siRNA confirmed that LGMN was the enzyme responsible for maintaining breast cancer proliferation. CST6 did not require secretion or glycosylation to elicit its cell killing effects, suggesting an intracellular mode of action. Finally, we show that TBX2 and CST6 displayed reciprocal expression in a cohort of primary breast cancers with increased TBX2 expression associating with increased metastases. We have also noted that tumors with altered TBX2/CST6 expression show poor overall survival. This novel TBX2-CST6-LGMN signaling pathway, therefore, represents an exciting opportunity for the development of novel therapies to target TBX2 driven breast cancers.