TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity.

The transcription factor TRPS1 is a context-dependent oncogene in breast cancer. In the mammary gland, TRPS1 activity is restricted to the luminal population and is critical during puberty and pregnancy. Its function in the resting state remains however unclear. To evaluate whether it could be a target for cancer therapy, we investigated TRPS1 function in the healthy adult mammary gland using a conditional ubiquitous depletion mouse model where long-term depletion does not affect fitness. Using transcriptomic approaches, flow cytometry and functional assays, we show that TRPS1 activity is essential to maintain a functional luminal progenitor compartment. This requires the repression of both YAP/TAZ and SRF/MRTF activities. TRPS1 represses SRF/MRTF activity indirectly by modulating RhoA activity. Our work uncovers a hitherto undisclosed function of TRPS1 in luminal progenitors intrinsically linked to mechanotransduction in the mammary gland. It may also provide new insights into the oncogenic functions of TRPS1 as luminal progenitors are likely the cells of origin of many breast cancers.

Tumor associated macrophages as key contributors and targets in current and future therapies for melanoma.

INTRODUCTION: Despite the success of immunotherapies for melanoma in recent years, there remains a significant proportion of patients who do not yet derive benefit from available treatments. Immunotherapies currently licensed for clinical use target the adaptive immune system, focussing on Tcell interactions and functions. However, the most prevalent immune cells within the tumor microenvironment (TME) of melanoma are macrophages, a diverse immune cell subset displaying high plasticity, to which no current therapies are yet directly targeted. Macrophages have been shown not only to activate the adaptive immune response, and enhance cancer cell killing, but, when influenced by factors within the TME of melanoma, these cells also promote melanoma tumorigenesis and metastasis. AREAS COVERED: We present a review of the most up-to-date literatureavailable on PubMed, focussing on studies from within the last 10 years. We also include data from ongoing and recent clinical trials targeting macrophages in melanoma listed on clinicaltrials.gov. EXPERT OPINION: Understanding the multifaceted role of macrophages in melanoma, including their interactions with immune and cancer cells, the influence of current therapies on macrophage phenotype and functions and how macrophages could be targeted with novel treatment approaches, are all critical for improving outcomes for patients with melanoma.

Organelle adaptations in response to mechanical forces during tumour dissemination.

Cell migration plays a pivotal role in various biological processes including cancer dissemination and successful metastasis, where the role of mechanical signals is increasingly acknowledged. This review focuses on the intricate mechanisms through which cancer cells modulate their migratory strategies via organelle adaptations in response to the extracellular matrix (ECM). Specifically, the nucleus and mitochondria emerge as pivotal mediators in this process. These organelles serve as sensors, translating mechanical stimuli into rapid metabolic alterations that sustain cell migration. Importantly, prolonged exposure to such stimuli can induce transcriptional or epigenetic changes, ultimately enhancing metastatic traits. Deciphering the intricate interplay between ECM properties and organelle adaptations not only advances our understanding of cytoskeletal dynamics but also holds promise for the development of innovative anti-metastatic therapeutic strategies.

LncRNA U90926 is dispensable for the development of obesity-associated phenotypes in vivo.

Obesity is a global health problem characterized by excessive fat accumulation, driven by adipogenesis and lipid accumulation. Long non-coding RNAs (lncRNAs) have recently been implicated in regulating adipogenesis and adipose tissue function. Mouse lncRNA U90926 was previously identified as a repressor of in vitro adipogenesis in 3T3-L1 preadipocytes. Consequently, we hypothesized that, in vivo, U90926 may repress adipogenesis, and hence its deletion would increase weight gain and adiposity. We tested the hypothesis by applying U90926-deficient (U9-KO) mice to a high-throughput phenotyping pipeline. Compared with WT, U9-KO mice showed no major differences across a wide range of behavioral, neurological, and other physiological parameters. In mice fed a standard diet, we have found no differences in obesity-related phenotypes, including weight gain, fat mass, and plasma concentrations of glucose, insulin, triglycerides, and free fatty acids, in U9-KO mice compared to WT. U90926 deficiency lacked a major effect on white adipose tissue morphology and gene expression profile. Furthermore, in mice fed a high-fat diet, we found increased expression of U90926 in adipose tissue stromal vascular cell fraction, yet observed no effect of U90926 deficiency on weight gain, fat mass, adipogenesis marker expression, and immune cell infiltration into the adipose tissue. These data suggest that the U90926 lacks an essential role in obesity-related phenotypes and adipose tissue biology in vivo.

Very-Low-Level Viremia, Inflammatory Biomarkers, and Associated Baseline Variables: Three-Year Results of the Randomized TANGO Study.

Background: We compared proportions of participants with target detected, target not detected (TND), and elevated viral load (VL) and assessed baseline variables associated with week 144 inflammatory biomarker levels between dolutegravir-lamivudine (DTG/3TC) and tenofovir alafenamide-based regimens (TBRs) in the TANGO study (post hoc). Methods: TANGO is an open-label, multicenter, phase 3 study that randomized adults with VL <50 copies/mL to switch to once-daily fixed-dose DTG/3TC or continue TBR. At baseline and each study visit, the VL was measured. Elevated VL event frequencies were assessed, including "blips." Interleukin 6, D-dimer, high-sensitivity C-reactive protein, soluble CD14, and soluble CD163 were measured at baseline and at week 144. Loge-transformed week 144 biomarker levels were compared between treatment groups using an analysis of covariance model adjusting for baseline variables. Results: High, comparable proportions of participants had VL <40 copies/mL and TND at week 144 (DTG/3TC, 279 of 369 [76%]; TBR, 267 of 372 [72%], intention-to-treat exposed Snapshot analysis; adjusted difference, 3.9% [95% confidence interval, -2.5% to 10.2%]), with similar TND proportions at all postbaseline visits (123 of 369 [33%] vs 101 of 372 [27%], respectively). Similar proportions of DTG/3TC participants had ≥1 postbaseline VL ≥50 copies/mL (28 of 369 [8%] vs 42 of 372 [11%] for TBR), primarily blips (18 of 369 [5%] and 26 of 372 [7%], respectively). Week 144 inflammatory biomarker levels were low and comparable between groups and associated with multiple demographic and baseline characteristics, including baseline biomarker levels, indicating a multifactorial inflammatory response. Conclusions: Week 144 biomarker levels were low and generally comparable between treatment groups, reflecting similar, robust, and durable viral suppression observed using the stringent TND end point. Trial registration:  ClinicalTrials.gov, NCT03446573.

Implication of transcription factor FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knockout mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knockout of Foxd2 in ureteric bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift toward a stromal cell identity. Histology of Foxd2 knockout mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.

Evolución de los serotipos causantes de enfermedad neumocócica invasiva aislados durante 2008-2022 en un Hospital Público Madrileño de nivel dos, en relación con su inclusión en diferentes vacunas conjugadas / Evolution of the serotypes causing invasive pneumococcal disease along 2008-2022 in a Level 2 Public Hospital of the Madrid Region, in relation to their inclusion in different conjugate vaccines

Introducción. El uso de vacunas conjugadas frente a Streptococcus pneumoniae ocasiona cambios en la epidemio logía de la Enfermedad Neumocócica Invasiva (ENI). El objetivo de este estudio fue analizar la evolución de los serotipos de S. pneumoniae aislados en el Hospital Universitario de Getafe entre 2008 y 2022. Material y métodos. Se estudiaron 313 cepas de S. pneu moniae. El serotipado se realizó mediante el test de aglutina ción por látex (Pneumotest-latex) y la reacción de Quellung. Además, se determinó la concentración mínima inhibitoria (CMI) frente a penicilina, eritromicina y levofloxacino por el método de gradiente de concentración (E-test) según los cri terios de corte EUCAST. Resultados. Los serotipos más frecuentes en todo el pe riodo de estudio fueron 8, 3, 19A, 1, 11A y 22F correspondien do con el 46,6 % de los aislados. Durante los años 2008-2012, los serotipos 3, 1, 19A, 7F, 6C y 11A supusieron en conjunto el 53,6% de los aislamientos. Entre 2013 y 2017 los serotipos 3, 8, 12F, 19A, 22F y 19F representaron el 51% de los aislados. Entre 2018-2022 los serotipos 8, 3, 11A, 15A, 4 y 6C incluyeron al 55,5% de los casos. En total, 5 cepas (1,6%) se mostraron resistentes a penicilina, 64 (20,4%) resistentes a eritromicina y 11 (3,5%) resistentes a levofloxacino. Los niveles de CMI50 y CMI90 frente a los tres antibióticos se mantuvieron estables a lo largo del tiempo. Conclusiones. El uso de vacunas conjugadas condicionó un descenso de los serotipos cubiertos junto con un aumento de los no vacunales. Los patrones de sensibilidad a eritromicina y levofloxacino se mantuvieron relativamente estables. La re sistencia a penicilina fue muy baja, no encontrándose este tipo de cepas resistentes en el último periodo de estudio (AU)

Introduction. The use of conjugate vaccines against Streptococcus pneumoniae originates changes in the invasive pneumococcal disease (IPD). The aim of this study was to in vestigate the evolution of S. pneumoniae serotypes isolated in the Hospital Universitario de Getafe between 2008 and 2022. Material and Methods. 313 of S. pneumoniae strains were studied. Serotyping was carried out by latex agglutina tion (Pneumotest-latex) and the Quellung reaction. In addi tion, the minimal inhibitory concentration (MIC) was deter mined against penicillin, erythromycin and levofloxacin by the concentration gradient method (E-test) according the EUCAST breakpoints. Results. The most frequent serotypes throughout the study period were 8, 3, 19A, 1, 11A and 22F corresponding to 46.6% of the isolates. Along 2008-2012 the serotypes 3, 1, 19A, 7F, 6C and 11A represented altogether 53.6% of the isolates. Between 2013 and 2017 the serotypes 3, 8, 12F, 19A, 22F and 19F grouped 51% of the isolates. During 2018-2022 the serotypes 8, 3, 11A, 15A, 4 and 6C included the 55.5% of the cases. In total 5 strains (1.6%) were penicillin resistant, 64 (20.4%) erythromycin resistant and 11 (3.5%) levofloxacin re sistant. The MIC50 and MIC90 levels maintained stables along the time. Conclusion. The conjugate vaccines use with different se rotype coverage conditioned a decrease of the vaccine-includ ed and an increase of non-covered. Despite these changes, the global antimicrobial susceptibility patterns to erythromycin and levofloxacin maintained relatively stables. The resistance a penicillin was low, not finding this type of resistant strains in the last study period (AU)

[Evolution of the serotypes causing invasive pneumococcal disease along 2008-2022 in a Level 2 Public Hospital of the Madrid Region, in relation to their inclusion in different conjugate vaccines]. / Evolución de los serotipos causantes de enfermedad neumocócica invasiva aislados durante 2008-2022 en un Hospital Público Madrileño de nivel dos, en relación con su inclusión en diferentes vacunas conjugadas.

OBJECTIVE: The use of conjugate vaccines against Streptococcus pneumoniae originates changes in the invasive pneumococcal disease (IPD). The aim of this study was to in vestigate the evolution of S. pneumoniae serotypes isolated in the Hospital Universitario de Getafe between 2008 and 2022. METHODS: 313 of S. pneumoniae strains were studied. Serotyping was carried out by latex agglutination (Pneumotest-latex) and the Quellung reaction. In addition, the minimal inhibitory concentration (MIC) was determined against penicillin, erythromycin and levofloxacin by the concentration gradient method (E-test) according the EUCAST breakpoints. RESULTS: The most frequent serotypes throughout the study period were 8, 3, 19A, 1, 11A and 22F corresponding to 46.6% of the isolates. Along 2008-2012 the serotypes 3, 1, 19A, 7F, 6C and 11A represented altogether 53.6% of the isolates. Between 2013 and 2017 the serotypes 3, 8, 12F, 19A, 22F and 19F grouped 51% of the isolates. During 2018-2022 the serotypes 8, 3, 11A, 15A, 4 and 6C included the 55.5% of the cases. In total 5 strains (1.6%) were penicillin resistant, 64 (20.4%) erythromycin resistant and 11 (3.5%) levofloxacin resistant. The MIC50 and MIC90 levels maintained stables along the time. CONCLUSIONS: The conjugate vaccines use with different serotype coverage conditioned a decrease of the vaccine-included and an increase of non-covered. Despite these changes, the global antimicrobial susceptibility patterns to erythromycin and levofloxacin maintained relatively stables. The resistance a penicillin was low, not finding this type of resistant strains in the last study period.

CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel of pancreatic cancer cell lines, three-dimensional (3D) invasion systems, microarray gene signatures, microfluidic devices, mouse models, and intravital imaging, we demonstrate that ROCK-Myosin II activity in PDAC cells supports a transcriptional program conferring amoeboid invasive and immunosuppressive traits and in vivo metastatic abilities. Moreover, we find that immune checkpoint CD73 is highly expressed in amoeboid PDAC cells and drives their invasive, metastatic, and immunomodulatory traits. Mechanistically, CD73 activates RhoA-ROCK-Myosin II downstream of PI3K. Tissue microarrays of human PDAC biopsies combined with bioinformatic analysis reveal that rounded-amoeboid invasive cells with high CD73-ROCK-Myosin II activity and their immunosuppressive microenvironment confer poor prognosis to patients. We propose targeting amoeboid PDAC cells as a therapeutic strategy.

AMPK is a mechano-metabolic sensor linking cell adhesion and mitochondrial dynamics to Myosin-dependent cell migration.

Cell migration is crucial for cancer dissemination. We find that AMP-activated protein kinase (AMPK) controls cell migration by acting as an adhesion sensing molecular hub. In 3-dimensional matrices, fast-migrating amoeboid cancer cells exert low adhesion/low traction linked to low ATP/AMP, leading to AMPK activation. In turn, AMPK plays a dual role controlling mitochondrial dynamics and cytoskeletal remodelling. High AMPK activity in low adhering migratory cells, induces mitochondrial fission, resulting in lower oxidative phosphorylation and lower mitochondrial ATP. Concurrently, AMPK inactivates Myosin Phosphatase, increasing Myosin II-dependent amoeboid migration. Reducing adhesion or mitochondrial fusion or activating AMPK induces efficient rounded-amoeboid migration. AMPK inhibition suppresses metastatic potential of amoeboid cancer cells in vivo, while a mitochondrial/AMPK-driven switch is observed in regions of human tumours where amoeboid cells are disseminating. We unveil how mitochondrial dynamics control cell migration and suggest that AMPK is a mechano-metabolic sensor linking energetics and the cytoskeleton.

AOX delays the onset of the lethal phenotype in a mouse model of Uqcrh (complex III) disease.

The alternative oxidase, AOX, provides a by-pass of the cytochrome segment of the mitochondrial respiratory chain when the chain is unavailable. AOX is absent from mammals, but AOX from Ciona intestinalis is benign when expressed in mice. Although non-protonmotive, so does not contribute directly to ATP production, it has been shown to modify and in some cases rescue phenotypes of respiratory-chain disease models. Here we studied the effect of C. intestinalis AOX on mice engineered to express a disease-equivalent mutant of Uqcrh, encoding the hinge subunit of mitochondrial respiratory complex III, which results in a complex metabolic phenotype beginning at 4-5 weeks, rapidly progressing to lethality within a further 6-7 weeks. AOX expression delayed the onset of this phenotype by several weeks, but provided no long-term benefit. We discuss the significance of this finding in light of the known and hypothesized effects of AOX on metabolism, redox homeostasis, oxidative stress and cell signaling. Although not a panacea, the ability of AOX to mitigate disease onset and progression means it could be useful in treatment.

Knockout mouse models as a resource for the study of rare diseases.

Rare diseases (RDs) are a challenge for medicine due to their heterogeneous clinical manifestations and low prevalence. There is a lack of specific treatments and only a few hundred of the approximately 7,000 RDs have an approved regime. Rapid technological development in genome sequencing enables the mass identification of potential candidates that in their mutated form could trigger diseases but are often not confirmed to be causal. Knockout (KO) mouse models are essential to understand the causality of genes by allowing highly standardized research into the pathogenesis of diseases. The German Mouse Clinic (GMC) is one of the pioneers in mouse research and successfully uses (preclinical) data obtained from single-gene KO mutants for research into monogenic RDs. As part of the International Mouse Phenotyping Consortium (IMPC) and INFRAFRONTIER, the pan-European consortium for modeling human diseases, the GMC expands these preclinical data toward global collaborative approaches with researchers, clinicians, and patient groups.Here, we highlight proprietary genes that when deleted mimic clinical phenotypes associated with known RD targets (Nacc1, Bach2, Klotho alpha). We focus on recognized RD genes with no pre-existing KO mouse models (Kansl1l, Acsf3, Pcdhgb2, Rabgap1, Cox7a2) which highlight novel phenotypes capable of optimizing clinical diagnosis. In addition, we present genes with intriguing phenotypic data (Zdhhc5, Wsb2) that are not presently associated with known human RDs.This report provides comprehensive evidence for genes that when deleted cause differences in the KO mouse across multiple organs, providing a huge translational potential for further understanding monogenic RDs and their clinical spectrum. Genetic KO studies in mice are valuable to further explore the underlying physiological mechanisms and their overall therapeutic potential.

RANK is a poor prognosis marker and a therapeutic target in ER-negative postmenopausal breast cancer.

Despite strong preclinical data, the therapeutic benefit of the RANKL inhibitor, denosumab, in breast cancer patients, beyond the bone, is unclear. Aiming to select patients who may benefit from denosumab, we hereby analyzed RANK and RANKL protein expression in more than 2,000 breast tumors (777 estrogen receptor-negative, ER- ) from four independent cohorts. RANK protein expression was more frequent in ER- tumors, where it associated with poor outcome and poor response to chemotherapy. In ER- breast cancer patient-derived orthoxenografts (PDXs), RANKL inhibition reduced tumor cell proliferation and stemness, regulated tumor immunity and metabolism, and improved response to chemotherapy. Intriguingly, tumor RANK protein expression associated with poor prognosis in postmenopausal breast cancer patients, activation of NFKB signaling, and modulation of immune and metabolic pathways, suggesting that RANK signaling increases after menopause. Our results demonstrate that RANK protein expression is an independent biomarker of poor prognosis in postmenopausal and ER- breast cancer patients and support the therapeutic benefit of RANK pathway inhibitors, such as denosumab, in breast cancer patients with RANK+ ER- tumors after menopause.

Implication of FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT).

Background: Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below 30 years of age. Many monogenic forms have been discovered mainly due to comprehensive genetic testing like exome sequencing (ES). However, disease-causing variants in known disease-associated genes still only explain a proportion of cases. Aim of this study was to unravel the underlying molecular mechanism of syndromic CAKUT in two multiplex families with presumed autosomal recessive inheritance. Methods and Results: ES in the index individuals revealed two different rare homozygous variants in FOXD2, a transcription factor not previously implicated in CAKUT in humans: a frameshift in family 1 and a missense variant in family 2 with family segregation patterns consistent with autosomal-recessive inheritance. CRISPR/Cas9-derived Foxd2 knock-out (KO) mice presented with bilateral dilated renal pelvis accompanied by renal papilla atrophy while extrarenal features included mandibular, ophthalmologic, and behavioral anomalies, recapitulating the phenotype of humans with FOXD2 dysfunction. To study the pathomechanism of FOXD2-dysfunction-mediated developmental renal defects, in a complementary approach, we generated CRISPR/Cas9-mediated KO of Foxd2 in ureteric-bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important in renal/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a cell identity shift towards a stromal cell identity. Histology of Foxd2 KO mouse kidneys confirmed increased fibrosis. Further, GWAS data (genome-wide association studies) suggests that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Conclusions: In summary, our data implicate that FOXD2 dysfunction is a very rare cause of autosomal recessive syndromic CAKUT and suggest disturbances of the PAX2-WNT4 cell signaling axis contribute to this phenotype.

Preclinical to clinical utility of ROCK inhibitors in cancer.

ROCK belongs to the AGC family of Ser/Thr protein kinases that are involved in many cellular processes. ROCK-driven actomyosin contractility regulates cytoskeletal dynamics underpinning cell migration, proliferation, and survival in many cancer types. ROCK1/2 play key protumorigenic roles in several subtypes and stages of cancer development. Therefore, successfully targeting ROCK and its downstream effectors presents an interesting avenue for cancer treatment. Because local use of ROCK inhibitors will reduce the side effects of systemic administration, we propose different therapeutic strategies and latest-generation ROCK inhibitors for use in the clinic.

LAP1 supports nuclear adaptability during constrained melanoma cell migration and invasion.

Metastasis involves dissemination of cancer cells away from a primary tumour and colonization at distal sites. During this process, the mechanical properties of the nucleus must be tuned since they pose a challenge to the negotiation of physical constraints imposed by the microenvironment and tissue structure. We discovered increased expression of the inner nuclear membrane protein LAP1 in metastatic melanoma cells, at the invasive front of human primary melanoma tumours and in metastases. Human cells express two LAP1 isoforms (LAP1B and LAP1C), which differ in their amino terminus. Here, using in vitro and in vivo models that recapitulate human melanoma progression, we found that expression of the shorter isoform, LAP1C, supports nuclear envelope blebbing, constrained migration and invasion by allowing a weaker coupling between the nuclear envelope and the nuclear lamina. We propose that LAP1 renders the nucleus highly adaptable and contributes to melanoma aggressiveness.