Evaluation of FGFR targeting in breast cancer through interrogation of patient-derived models.

BACKGROUND: Particular breast cancer subtypes pose a clinical challenge due to limited targeted therapeutic options and/or poor responses to the existing targeted therapies. While cell lines provide useful pre-clinical models, patient-derived xenografts (PDX) and organoids (PDO) provide significant advantages, including maintenance of genetic and phenotypic heterogeneity, 3D architecture and for PDX, tumor-stroma interactions. In this study, we applied an integrated multi-omic approach across panels of breast cancer PDXs and PDOs in order to identify candidate therapeutic targets, with a major focus on specific FGFRs. METHODS: MS-based phosphoproteomics, RNAseq, WES and Western blotting were used to characterize aberrantly activated protein kinases and effects of specific FGFR inhibitors. PDX and PDO were treated with the selective tyrosine kinase inhibitors AZD4547 (FGFR1-3) and BLU9931 (FGFR4). FGFR4 expression in cancer tissue samples and PDOs was assessed by immunohistochemistry. METABRIC and TCGA datasets were interrogated to identify specific FGFR alterations and their association with breast cancer subtype and patient survival. RESULTS: Phosphoproteomic profiling across 18 triple-negative breast cancers (TNBC) and 1 luminal B PDX revealed considerable heterogeneity in kinase activation, but 1/3 of PDX exhibited enhanced phosphorylation of FGFR1, FGFR2 or FGFR4. One TNBC PDX with high FGFR2 activation was exquisitely sensitive to AZD4547. Integrated 'omic analysis revealed a novel FGFR2-SKI fusion that comprised the majority of FGFR2 joined to the C-terminal region of SKI containing the coiled-coil domains. High FGFR4 phosphorylation characterized a luminal B PDX model and treatment with BLU9931 significantly decreased tumor growth. Phosphoproteomic and transcriptomic analyses confirmed on-target action of the two anti-FGFR drugs and also revealed novel effects on the spliceosome, metabolism and extracellular matrix (AZD4547) and RIG-I-like and NOD-like receptor signaling (BLU9931). Interrogation of public datasets revealed FGFR2 amplification, fusion or mutation in TNBC and other breast cancer subtypes, while FGFR4 overexpression and amplification occurred in all breast cancer subtypes and were associated with poor prognosis. Characterization of a PDO panel identified a luminal A PDO with high FGFR4 expression that was sensitive to BLU9931 treatment, further highlighting FGFR4 as a potential therapeutic target. CONCLUSIONS: This work highlights how patient-derived models of human breast cancer provide powerful platforms for therapeutic target identification and analysis of drug action, and also the potential of specific FGFRs, including FGFR4, as targets for precision treatment.

Glucocorticoid-induced leucine zipper modulates macrophage polarization and apoptotic cell clearance.

Macrophages are professional phagocytes that display remarkable plasticity, with a range of phenotypes that can be broadly characterized by the M1/M2 dichotomy. Glucocorticoid (GC)-induced leucine zipper (GILZ) is a protein known to mediate anti-inflammatory and some pro-resolving actions, including as neutrophil apoptosis. However, the role of GILZ in key macrophage function is not well understood. Here, we investigated the role of GILZ on macrophage reprogramming and efferocytosis. Using murine bone-marrow-derived macrophages (BMDMs), we found that GILZ was expressed in naive BMDMs and exhibited increased expression in M2-like macrophages (IL4-differentiated). M1-like macrophages (IFN/LPS-differentiated) from GILZ-/- mice showed higher expression of the M1 markers CD86, MHC class II, iNOS, IL-6 and TNF-α, associated with increased levels of phosphorylated STAT1 and lower IL-10 levels, compared to M1-differentiated cells from WT mice. There were no changes in the M2 markers CD206 and arginase-1 in macrophages from GILZ-/- mice differentiated with IL-4, compared to cells from WT animals. Treatment of M1-like macrophages with TAT-GILZ, a cell-permeable GILZ fusion protein, decreased the levels of CD86 and MHC class II in M1-like macrophages without modifying CD206 levels in M2-like macrophages. In line with the in vitro data, increased numbers of M1-like macrophages were found into the pleural cavity of GILZ-/- mice after LPS-injection, compared to WT mice. Moreover, efferocytosis was defective in the context of GILZ deficiency, both in vitro and in vivo. Conversely, treatment of LPS-injected mice with TAT-GILZ promoted inflammation resolution, associated with lower numbers of M1-like macrophages and increased efferocytosis. Collectively, these data indicate that GILZ is a regulator of important macrophage functions, contributing to macrophage reprogramming and efferocytosis, both key steps for the resolution of inflammation.

Uropathogenic Escherichia coli modulates innate immunity to suppress Th1-mediated inflammatory responses during infectious epididymitis.

Infectious epididymitis in men, a frequent entity in urological outpatient settings, is commonly caused by bacteria originating from the anal region ascending the genitourinary tract. One of the most prevalent pathogens associated with epididymitis is Escherichia coli. In our previous study, we showed that semen quality is compromised in men following epididymitis associated with specific E. coli pathovars. Thus, our aim was to investigate possible differences in immune responses elicited during epididymitis following infection with the uropathogenic E. coli (UPEC) strain CFT073 and the nonpathogenic enteric E. coli (NPEC) strain 470. Employing an in vivo experimental epididymitis model, C57BL/6 mice were infected with UPEC CFT073, NPEC 470, or phosphate-buffered saline (PBS) as a sham control for up to 7 days. After infection with NPEC 470, the expression of proinflammatory cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha in the epididymis was significantly increased. Conversely, UPEC CFT073-challenged mice displayed inflammatory gene expression at levels comparable to sham PBS-treated animals. Moreover, by day 7 only NPEC-infected animals showed activation of adaptive immunity evident by a substantial influx of CD3+ and F4/80+ cells in the epididymal interstitium. This correlated with enhanced production of Th1-associated cytokines IL-2 and gamma interferon (IFN-γ). Furthermore, splenocytes isolated from UPEC-infected mice exhibited diminished T-cell responses with significantly reduced secretion of IL-2 and IFN-γ in contrast to NPEC-infected animals. Overall, these findings provide new insights into understanding pathogen-specific modulation of host immunity during acute phases of epididymitis, which may influence severity of disease and clinical outcomes.

Structural and functional integrity of spermatozoa is compromised as a consequence of acute uropathogenic E. coli-associated epididymitis.

Uropathogenic Escherichia coli (UPEC)-associated epididymitis is commonly diagnosed in outpatient settings. Although the infection can be successfully cleared using antimicrobial medications, 40% of patients unexplainably show persistent impaired semen parameters even after treatment. Our aim was to investigate whether pathogenic UPEC and its associated virulence factor hemolysin (hlyA) perturb the structural and functional integrity of both the epididymis and sperm, actions that may be responsible for the observed impairment and possibly a reduction of fertilization capabilities. Semen collected from patients diagnosed with E. coli-only related epididymitis showed that sperm counts were low 14 days postantimicrobial treatment regardless of hlyA status. At Day 84 following treatment, hlyA production correlated with approximately 4-fold lower sperm concentrations than in men with hlyA-negative strains. In vivo experiments with the hlyA-producing UPEC CFT073 strain in a murine epididymitis model showed that just 3 days postinfection, structural damage to the epididymis (epithelial damage, leukocyte infiltration, and edema formation) was present. This was more severe in UPEC CFT073 compared to nonpathogenic E. coli (NPEC 470) infection. Moreover, pathogenic UPEC strains prematurely activated the acrosome in vivo and in vitro. Raman microspectroscopy revealed that UPEC CFT073 undermined sperm integrity by inducing nuclear DNA damage. Consistent with these observations, the in vitro fertilization capability of hlyA-treated mouse sperm was completely abolished, although sperm were motile. These findings provide new insights into understanding the possible processes underlying clinical manifestations of acute epididymitis.

Is There a Relationship Between Frequency of Port-Care Maintenance and Related Complications in Patients With Cancer?

PURPOSE: Totally implantable ports require regular maintenance to prevent port-related complications. Manufacturers recommend monthly maintenance port flushes for patients for the life of the port. Previous studies show that extending intervals between maintenance port flushes up to 16 weeks does not increase incidence of port-related complications. To date, no prospective study has been conducted to evaluate the medical safety of extending flush intervals from monthly to every 12 weeks within a heterogeneous disease cohort. Research Question: Is it feasible and medically safe to extend intervals between maintenance port flushes to every 12 weeks in patients with cancer not on active treatment? PATIENTS AND METHODS: This study enrolled oncology and hematology patients who had retained their port following completion of anticancer treatment. Clinical data were extracted for 1,059 participants. The primary end points of this study were the overall number of ports removed and incidence of port-related complications reported between cohorts 1 and 2 (flushes every 4-8 weeks), and cohort 3 (flushes every 12 weeks). RESULTS: Data were allocated into three study cohorts on the basis of year and duration between port flushes. No difference was observed in the overall percentage of ports removed because of physician-reported complications across all cohorts (25%-30%). No change in the incidence of port-related complications including suspected infection and malfunction was observed between cohorts 1 and 2 (8%), or cohort 3 (5%). CONCLUSION: Our findings show that extending maintenance port flush intervals to 12 weeks does not increase the incidence of port-related adverse events and is medically safe.

The hepatitis B e antigen (HBeAg) targets and suppresses activation of the toll-like receptor signaling pathway.

BACKGROUND & AIMS: Viruses target innate immune pathways to evade host antiviral responses. Recent studies demonstrate a relationship between hepatitis B disease states and the host's innate immune response, although the mechanism of immunomodulation is unknown. In humans, the innate immune system recognizes pathogens via pattern recognition receptors such as the Toll-like receptors (TLR), initiating anti-inflammatory responses. TLR expression and pro-inflammatory cytokine production is reduced in hepatitis B e antigen (HBeAg)-positive patients following TLR stimulation. The aim of this study was to investigate interactions between TLR signaling pathways and the mature HBeAg protein localized in the cytosol. METHODS: The ability of HBeAg to inhibit TLR signaling and association with TLR adapters was evaluated by immunoprecipitation, immunostaining, and reporter studies. RESULTS: Our findings show that HBeAg co-localizes with Toll/IL-1 receptor (TIR)-containing proteins TRAM, Mal, and TLR2 at the sub-cellular level, which was not observed for Hepatitis B core antigen. Co-immunoprecipitation analysis demonstrated HBeAg interacted with TIR proteins Mal and TRAM, while a mutated HBeAg ablated interaction between Mal and MyD88. Importantly, HBeAg also disrupted homotypic TIR:TIR interaction critical for TLR-mediated signaling. Finally, HBeAg suppressed TIR-mediated activation of the inflammatory transcription factors, NF-κB and Interferon-ß promoter activity. CONCLUSIONS: Our study provides the first molecular mechanism describing HBeAg immunomodulation of innate immune signal transduction pathways via interaction and targeting of TLR-mediated signaling pathways. These finding suggest the mechanism as to how HBeAg evades innate immune responses contributing to the pathogenesis of chronic hepatitis B infection and the establishment of viral persistence.

Analysis of Secreted MIF from Cultured Murine Bone Marrow-Derived Immune Cells.

Macrophage migration inhibitory factor (MIF) is expressed and released ubiquitously by numerous cell types and tissues. MIF is detected and constitutively expressed at the protein level both intra- and extracellularly. This chapter outlines methods for cultivating, purifying, detecting, and quantifying concentrations of MIF from murine primary derived macrophages and dendritic cell culture supernatants.

Measuring MIF in Biological Fluids.

MIF is a key regulator of host immune responses and increased levels secreted from cells, or found circulating systemically, have been implicated in the pathogenesis of many inflammatory and autoimmune disorders. Here, we describe methods for detecting and quantifying extracellular concentrations of MIF in both human- and murine-derived biological samples.

Analysis of serum interleukin(IL)-1α, IL-1ß and IL-18 in patients with systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune disease characterised by fibrosis, vascular dysfunction and immune dysregulation. The pathogenesis of SSc remains poorly understood, although studies have indicated a role for the innate immune response. METHODS: Here, we measured serum interleukin (IL)-1α, IL-1ß and IL-18 levels in 105 SSc patients and 47 healthy controls (HC) and analysed them with respect to multiple clinical parameters. RESULTS: Serum IL-18 concentrations were significantly higher in SSc patients than in HC, while no significant differences in concentrations of IL-1α and IL-1ß were observed between SSc and HC. In both SSc and HC serum, IL-1α and IL-1ß were positively correlated, while in SSc, both cytokines negatively correlated with IL-18. Serum IL-18 was significantly negatively correlated with both carbon monoxide transfer coefficient (KCO) and diffusing capacity of the lungs for carbon monoxide (DLCO). Serum IL-1ß was positively correlated with the modified Rodnan skin score (mRSS), particularly in patients with limited subtype. DLCO, KCO and tricuspid regurgitation (TR) velocity were significantly higher in patients with high serum IL-1ß. Serum IL-1α was significantly lower in SSc patients with low KCO and positively correlated with KCO. SSc patients with high serum IL-1α concentrations were more likely to have digital ulcers. CONCLUSIONS: Our data suggest that these IL-1 family cytokines may have different roles in the pathogenesis of SSc fibrotic complications.

Analysis of serum macrophage migration inhibitory factor and D-dopachrome tautomerase in systemic sclerosis.

OBJECTIVES: Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (DDT), members of the same cytokine superfamily, are linked to the pathogenesis of a number of inflammatory diseases. The aim of this study was to investigate their clinical relevance in systemic sclerosis (SSc). METHODS: Serum MIF and DDT were quantified in 105 SSc patients by ELISA and levels compared to healthy controls (HC) (47) and patients with systemic lupus erythematosus (SLE) (184). Clinical parameters included organ involvement, serum laboratory markers and results of pulmonary function tests, and overall disease activity assessed using the European Scleroderma Trials and Research group (EUSTAR) activity index. RESULTS: There was no significant difference in serum DDT concentrations between patients with SSc and HC. However, serum MIF was significantly increased in SSc compared to both HC and SLE cohorts. Serum MIF was increased in SSc patients with low forced vital capacity (FVC) and was also associated with the use of angiotensin II receptor blockers and beta blockers in SSc, confirmed after adjusting for the presence of systemic hypertension and low FVC. Serum DDT was significantly higher in SSc patients with low FEV1 and negatively correlated with EUSTAR score, particularly in patients with limited disease. CONCLUSION: Although not significantly linked to specific clinical parameters, serum MIF was significantly higher in SSc patients than in HC and SLE patients, suggesting a fundamental role for MIF in SSc. DDT, while closely related to MIF, did not show a similar expression profile, suggesting functional differences between these molecules.

Analysis of Serum Interleukin (IL)-1ß and IL-18 in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disease characterized by biological and clinical heterogeneity. The interleukin (IL)-1 superfamily is a group of innate cytokines that contribute to pathogenesis in many autoimmune diseases. IL-1ß and IL-18 are two members that have been shown to play a role in murine lupus-like models, but their role in human SLE remains poorly understood. Here, IL-1ß and IL-18 were quantified by enzyme-linked immunosorbent assay in the serum of healthy controls (HCs) and SLE patients from a prospectively followed cohort. Disease activity and organ damage were assessed using SLE disease activity index 2000 (SLEDAI-2K) and SLE damage index scores (SDI), respectively. 184 SLE patients (mean age 44.9 years, 91% female, 56% double-stranded deoxyribonucleic acid positive) were compared to 52 HC. SLE patients had median [IQR] SLEDAI-2K of 4 [2,6], and SDI of 1 [0-2]. Serum IL-18 levels were statistically significantly higher in SLE patients compared to HCs. Univariable linear regression analyses showed that patients with active renal disease or irreversible organ damage had statistically significantly elevated serum IL-18 levels. The association between serum IL-18 and active renal disease was confirmed in multivariable analysis after adjusting for ethnicity and organ damage. High baseline serum IL-18 levels were associated with organ damage at the subsequent visit. Serum IL-1ß levels were not significantly elevated in SLE patients when compared to HCs and had no association with overall or organ-specific disease activity or organ damage in cross-sectional and longitudinal analyses. Our data suggest that serum IL-18 and IL-1ß have different clinical implications in SLE, with IL-18 being potentially associated with active renal disease.

Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation.

Macrophage migration inhibitory factor (MIF) exerts multiple effects on immune cells, as well as having functions outside the immune system. MIF can promote inflammation through the induction of other cytokines, including TNF, IL-6, and IL-1 family cytokines. Here, we show that inhibition of MIF regulates the release of IL-1α, IL-1ß, and IL-18, not by affecting transcription or translation of these cytokines, but via activation of the NLRP3 inflammasome. MIF is required for the interaction between NLRP3 and the intermediate filament protein vimentin, which is critical for NLRP3 activation. Further, we demonstrate that MIF interacts with NLRP3, indicating a role for MIF in inflammasome activation independent of its role as a cytokine. These data advance our understanding of how MIF regulates inflammation and identify it as a factor critical for NLRP3 inflammasome activation.

Autophagy and inflammasomes.

Autophagy is a ubiquitous cellular mechanism for the targeted lysosomal degradation of various cytosolic constituents, from proteins to organelles. As an essential homeostatic mechanism, autophagy is upregulated in response to numerous environmental and pharmacological stimuli, including starvation, where it facilitates the recycling of essential amino acids. In addition, autophagy plays specific roles within the immune system; it serves as a source of peptides for antigen presentation, a mechanism for the engulfment and degradation of intracellular pathogens and as a key regulator of inflammatory cytokines. In particular, autophagy has been shown to play a number of roles in regulating inflammasome activation, from the removal of inflammasome-activating endogenous signals, to the sequestration and degradation of inflammasome components. Autophagy also plays a role in determining the fate of IL-1ß, which is concentrated in autophagosomes. This review discusses a growing body of literature that suggests autophagy is a critical regulator of inflammasome activation and the subsequent release of IL-1 family cytokines.

Loss of autophagy enhances MIF/macrophage migration inhibitory factor release by macrophages.

MIF (macrophage migration inhibitory factor [glycosylation-inhibiting factor]) is a pro-inflammatory cytokine expressed in multiple cells types, including macrophages. MIF plays a pathogenic role in a number of inflammatory diseases and has been linked to tumor progression in some cancers. Previous work has demonstrated that loss of autophagy in macrophages enhances secretion of IL1 family cytokines. Here, we demonstrate that loss of autophagy, by pharmacological inhibition or siRNA silencing of Atg5, enhances MIF secretion by monocytes and macrophages. We further demonstrate that this is dependent on mitochondrial reactive oxygen species (ROS). Induction of autophagy with MTOR inhibitors had no effect on MIF secretion, but amino acid starvation increased secretion. This was unaffected by Atg5 siRNA but was again dependent on mitochondrial ROS. Our data demonstrate that autophagic regulation of mitochondrial ROS plays a pivotal role in the regulation of inflammatory cytokine secretion in macrophages, with potential implications for the pathogenesis of inflammatory diseases and cancers.

MIF: Implications in the Pathoetiology of Systemic Lupus Erythematosus.

Macrophage migration Inhibitory factor (MIF) was one of the earliest pro-inflammatory cytokines to be identified. Increasing interest in this cytokine in recent decades has followed the cloning of human MIF and the generation of Mif(-/-) mice. Deepening understanding of signaling pathways utilized by MIF and putative receptor mechanisms have followed. MIF is distinct from all other cytokines by virtue of its unique induction by and counter regulation of glucocorticoids (GCs). MIF is further differentiated from other cytokines by its structural homology to specific tautomerase and isomerase enzymes and correlative in vitro enzymatic functions. The role of MIF in immune and inflammatory states, including a range of human autoimmune diseases, is now well established, as are the relationships between MIF polymorphisms and a number of inflammatory diseases. Here, we review the known pleiotropic activities of MIF, in addition to novel functions of MIF in processes including autophagy and autophagic cell death. In addition, recent developments in the understanding of the role of MIF in systemic lupus erythematosus (SLE) are reviewed. Finally, we discuss the potential application of anti-MIF strategies to treat human diseases such as SLE, which will require a comprehensive understanding of the unique and complex activities of this ubiquitously expressed cytokine.

Acute epididymitis induces alterations in sperm protein composition.

OBJECTIVE: To use a proteomic approach to evaluate possible postinflammatory alterations in the protein composition of motile sperm in patients 3 months after acute epididymitis. DESIGN: Prospective case-control study. SETTING: University medical school research laboratory. PATIENT(S): Eight patients 3 months after acute unilateral epididymitis and 10 healthy controls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Proteome analysis of sperm samples collected by swim-up from control and acute epididymitis patients analyzed by two-dimensional gel electrophoresis and subsequent protein identification by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry; immunofluorescence staining for mitochondrial ATP synthase subunit ß (ATP5B), α-tubulin (TUBA1A), and tubulin-ß2c (TUBB4B) for validation purposes. RESULT(S): Proteome analysis identified 35 proteins in sperm from epididymitis patients that were down-regulated, irrespective of subcellular localization and biologic function. Furthermore, immunofluorescence microscopy confirmed ATP5B, TUBA1A, and TUBB4B were less abundantly expressed in epididymitis samples compared with controls. CONCLUSION(S): Despite normal semen parameters observed by conventional semen analysis in patients after epididymitis, significant changes to sperm protein composition were observed. These changes may be implicated as additional factors contributing to subfertility/infertility in men after episodes of epididymitis.

The negative regulation of Toll-like receptor and associated pathways.

Toll-like receptors (TLRs) are essential mediators of both innate and adaptive immunity by recognizing and eliciting responses upon invasion of pathogens. The response of TLRs must be stringently regulated as exaggerated expression of signalling components as well as pro-inflammatory cytokines can have devastating effects on the host, resulting in chronic inflammatory diseases, autoimmune disorders and aid in the pathogenesis of TLR-associated human diseases. Therefore, it is essential that negative regulators act at multiple levels within TLR signalling cascades, as well as through eliciting negative-feedback mechanisms in order to synchronize the positive activation and negative regulation of signal transduction to avert potentially harmful immunological consequences. This review explores the various mechanisms employed by negative regulators to ensure the appropriate modulation of both immune and inflammatory responses.