Paracrine and autocrine signals induce and maintain mesenchymal and stem cell states in the breast.

The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of motility, invasiveness, and self-renewal traits. During both normal development and tumor pathogenesis, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment. The signals that are responsible for inducing an EMT and maintaining the resulting cellular state have been unclear. We describe three signaling pathways, involving transforming growth factor (TGF)-ß and canonical and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal state. Downregulation of endogenously synthesized inhibitors of autocrine signals in epithelial cells enables the induction of the EMT program. Conversely, disruption of autocrine signaling by added inhibitors of these pathways inhibits migration and self-renewal in primary mammary epithelial cells and reduces tumorigenicity and metastasis by their transformed derivatives.

FSP1 is a glutathione-independent ferroptosis suppressor.

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ10-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.

The Hippo kinases LATS1 and 2 control human breast cell fate via crosstalk with ERα.

Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.

Atypical pediatric presentation of alopecic and aseptic nodules of the scalp with features of dissecting cellulitis.

Alopecic and aseptic nodules of the scalp (AANS) and dissecting cellulitis of the scalp (DCS) are rare, closely related conditions of young men that exclusively affect the hair-bearing scalp. We describe a 9-year-old boy who presented with a 6-year history of chronically relapsing, sterile, partially scarring nodules of the scalp and facial skin. Histopathology revealed mixed inflammatory infiltrates consisting of neutrophils, macrophages, lymphocytes, and plasma cells in the deep dermis, consistent with the morphological pattern of suppurative, partly granulomatous dermatitis. The present atypical case is characterized by prepubertal onset and facial involvement which, to our knowledge, has not yet been described before, may be included in the spectrum of "typical" AANS and "typical" DCS.

Screening for Diabetes Mellitus in Patients with Hidradenitis Suppurativa-A Monocentric Study in Germany.

Hidradenitis suppurativa (HS) is a chronic skin disease that is often associated with metabolic disorders. Diabetes mellitus (DM) is a frequent comorbidity in HS. There is currently no established screening for DM in HS patients. The aim of our study was to identify high-risk groups of HS patients that develop DM and to assess the frequency of different types of DM present in HS patients. To do so, we conducted a monocentric study in 99 patients with HS. All patients underwent detailed clinical and laboratory assessments, including the determination of glycated hemoglobin. Among the 20.2% of patients that presented with DM, type 2 was by far the most prevalent (19 out of 20 patients). Moreover, male gender, age, BMI, Hurley stage, modified Hidradenitis Suppurativa Score (mHSS), DLQI and hypertension all correlated with the glycated hemoglobin levels in the HS patients. In the multivariable analysis, Hurley stage III, older age, and higher BMI were significantly associated with DM. Specifically, patients at Hurley stage III were at a 5.3-fold increased risk of having DM type II compared to patients at earlier Hurley stages. Since many of the HS patients had not been diagnosed, our study reveals shortcomings in the screening for DM and suggest that this should be routinely performed in HS patients at high risk to avoid secondary complications.

Generation of ductal organoids from normal mammary luminal cells reveals invasive potential.

Here we present an experimental model for human luminal progenitor cells that enables single, primary cells isolated from normal tissue to generate complex branched structures resembling the ductal morphology of low-grade carcinoma of no special type. Thereby, we find that ductal structures are generated through invasive branching morphogenesis via matrix remodeling and identify reduced actomyosin contractility as a prerequisite for invasion. In addition, we show that knockout of E-cadherin causes a dissolution of duct formation as observed in invasive lobular carcinoma, a subtype of invasive carcinomas where E-cadherin function is frequently lost. Thus, our model shows that invasive capacity can be elicited from normal luminal cells in specific environments, which results in low-grade no special type morphology. This assay offers a platform to investigate the dynamics of luminal cell invasion and unravel the impact of genetic and non-genetic aberrations on invasive morphology. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Outcome of extracorporeal photopheresis in mycosis fungoides patients is not predicted by quotients of systemic immune-inflammatory biomarkers.

BACKGROUND: Systemic immune-inflammatory biomarkers (SIIBs) have not been studied in mycosis fungoides (MF) patients undergoing extracorporeal photopheresis (ECP). OBJECTIVE: The objective was to determine whether recently proposed SIIBs are suitable to predict ECP treatment outcome and overall prognosis of patients with MF. METHODS: Twenty-nine MF patients were retrospectively evaluated who had undergone ECP. SIIBs included neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, lymphocyte-to-monocyte ratio, and pan-immune-inflammation value. RESULTS: Lymphocyte count (P = .021), CD4+/CD8+ cells (P = .00006), CD4+/CD56+ NK cells (P = .00008), and LDH levels (P = .0041) significantly declined after 6-month ECP. We could not detect significant cutoff values for baseline SIIBs capable of predicting advanced disease, overall response to 6-month ECP, or 5-year lymphoma-specific (LS) survival (P > .05). Circulating baseline counts of CD4+/CD7- cells (cutoff: ≤ 12.2; P = .010) and CD4+/CD26- cells (cutoff: ≤ 19.5; P < .0001) significantly predicted ECP treatment response after 6 months. Moreover, CD4+/CD8+ ratio (cutoff: > 1.34; P = .045) and increased thrombocyte counts (cutoff: >259 000; P = .010) were baseline predictors for 5-year LS death. CONCLUSION: ECP appears to be beneficial in early-stage CTCL as well. Lower percentages of circulating CD4+/CD7- and CD4+/CD26- lymphocytes at baseline correlate with response to ECP. In this study, however, baseline SIIBs did not appear to serve as suitable biomarkers for the prediction of treatment outcome and LS survival.

Morphological Analysis of Human Milk Membrane Enclosed Structures Reveals Diverse Cells and Cell-like Milk Fat Globules.

Over the past decade, the cellular content of human milk has been a focus in lactation research due to the benefit a potential non-invasive stem cell compartment could provide either to the infant or for therapeutic applications. Despite an increase in the number of studies in this field, fundamental knowledge in regard to milk cell identification and characterisation is still lacking. In this project, we investigated the nature, morphology and content of membrane enclosed structures (MESs) and explored different methods to enrich human milk cells (HMCs) whilst reducing milk fat globule (MFG) content. Using both flow cytometry and immunofluorescence imaging, we confirmed previous reports and showed that nucleated HMCs make up a minority of milk-isolated MESs and are indistinguishable from MFGs without the use of a nuclear stain. HMC heterogeneity was demonstrated by differential uptake of nuclear stains Hoechst 33258 and DRAQ5™ using a novel technique of imaging milk MESs (by embedding them in agar), that enabled examination of both extracellular and intracellular markers. We found that MESs often contain multiple lipid droplets of various sizes and for the first time report that late post-partum human milk contains secretory luminal binucleated cells found across a number of participants. After investigation of different techniques, we found that viably freezing milk cells is an easy and effective method to substantially reduce MFG content of samples. Alternatively, milk MESs can be filtered using a MACS® filter and return a highly viable, though reduced population of milk cells. Using the techniques and findings we've developed in this study; future research may focus on further characterising HMCs and the functional secretory mammary epithelium during lactation.

Advances in stem cells and regenerative medicine: single-cell dynamics, new models and translational perspectives.

An international cohort of over 300 stem cell biologists came together in Heidelberg, Germany in May 2017 as delegates of the 'Advances in Stem Cells and Regenerative Medicine' conference run through the European Molecular Biology Organization. This Meeting Review highlights the novel insights into stem cell regulation, new technologies aiding in discovery and exciting breakthroughs in the field of regenerative medicine that emerged from the meeting.

ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition.

Ferroptosis is a form of regulated necrotic cell death controlled by glutathione peroxidase 4 (GPX4). At present, mechanisms that could predict sensitivity and/or resistance and that may be exploited to modulate ferroptosis are needed. We applied two independent approaches-a genome-wide CRISPR-based genetic screen and microarray analysis of ferroptosis-resistant cell lines-to uncover acyl-CoA synthetase long-chain family member 4 (ACSL4) as an essential component for ferroptosis execution. Specifically, Gpx4-Acsl4 double-knockout cells showed marked resistance to ferroptosis. Mechanistically, ACSL4 enriched cellular membranes with long polyunsaturated ω6 fatty acids. Moreover, ACSL4 was preferentially expressed in a panel of basal-like breast cancer cell lines and predicted their sensitivity to ferroptosis. Pharmacological targeting of ACSL4 with thiazolidinediones, a class of antidiabetic compound, ameliorated tissue demise in a mouse model of ferroptosis, suggesting that ACSL4 inhibition is a viable therapeutic approach to preventing ferroptosis-related diseases.

BRD4 promotes p63 and GRHL3 expression downstream of FOXO in mammary epithelial cells.

Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression.

Quantification of regenerative potential in primary human mammary epithelial cells.

We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49f(hi)/EpCAM(-) population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis.

Antimicrobial Resistance Among Nontyphoidal Salmonella Isolated From Blood in the United States, 2003-2013.

BACKGROUND: Salmonella causes an estimated 100 000 antimicrobial-resistant infections annually in the United States. Salmonella antimicrobial resistance may result in bacteremia and poor outcomes. We describe antimicrobial resistance among nontyphoidal Salmonella blood isolates, using data from the National Antimicrobial Resistance Monitoring System. METHODS: Human nontyphoidal Salmonella isolates from 2003 to 2013 were classified as fully susceptible, resistant to ≥1 antimicrobial agent, or resistant to a first-line agent. Logistic regression was used to compare resistance patterns, serotypes, and patient characteristics for Salmonella isolated from blood versus stool and to determine resistance trends over time. RESULTS: Approximately 20% of blood isolates had antimicrobial resistance to a first-line treatment agent. Bacteremia was associated with male sex, age ≥65 years, and specific serotypes. Blood isolates were more likely to be resistant to ≥1 agent for serotypes Enteritidis, Javiana, Panama, and Typhimurium. Blood isolates were most commonly resistant to tetracycline (19%), and more likely resistant to a first-line agent (odds ratio, 1.81; 95% confidence interval, 1.56-2.11) than stool isolates. Ceftriaxone resistance increased in blood isolates from 2003 to 2013 (odd ratio, 1.12; 95% confidence interval, 1.02-1.22). CONCLUSIONS: Resistance to first-line treatment agents in patients with Salmonella bacteremia is a concern for public health and for informing clinical decisions. Judicious antimicrobial use is crucial to limit resistance.