Prognostic and Predictive Role of SPOP Mutations in Prostate Cancer: A Systematic Review and Meta-analysis.

CONTEXT: Mutations in the speckle-type POZ (SPOP) gene are frequently identified in prostate cancer (PC); yet, prognostic implications for affected patients remain unclear. Limited consensus exists regarding tailored treatments for SPOP-mutant (SPOPmut) PC. OBJECTIVE: To elucidate the prognostic and predictive significance of SPOP mutations across distinct PC stages and treatments. EVIDENCE ACQUISITION: A systematic literature search of PubMed, Embase, and Scopus was conducted up to January 29, 2024. The meta-analysis included studies comparing survival outcomes between SPOPmut and SPOP wild-type (SPOPwt) PC. EVIDENCE SYNTHESIS: From 669 records, 26 studies (including five abstracts) were analyzed. A meta-analysis of metastasis-free survival in localized (hazard ratio [HR]: 0.72, 95% confidence interval [CI]: 0.59-0.88; p < 0.01) and overall survival (OS) in metastatic PC (HR: 0.64, 95% CI: 0.53-0.76; p < 0.01) showed a favorable prognosis for patients with SPOPmut PC. In metastatic settings, SPOP mutations correlated with improved progression-free survival (PFS) and OS in patients undergoing androgen deprivation therapy ± androgen receptor signaling inhibitor (HR: 0.51, 95% CI: 0.35-0.76, p < 0.01, and HR: 0.60, 95% CI:0.46-0.79, p < 0.01, respectively). In metastatic castration-resistant PC, only abiraterone provided improved PFS and OS to patients with SPOP mutations compared with patients with SPOPwt, but data were limited. SPOP mutations did not correlate with improved PFS (p = 0.80) or OS (p = 0.27) for docetaxel. CONCLUSIONS: Patients with SPOPmut PC seem to exhibit superior oncological outcomes compared with patients with SPOPwt. Tailored risk stratification and treatment approaches should be explored in such patients. PATIENT SUMMARY: Speckle-type POZ (SPOP) mutations could be a favorable prognostic factor in patients with prostate cancer (PC) and may also predict better progression-free and overall survival than treatment with hormonal agents. Therefore, less intensified treatments omitting chemotherapy for patients with SPOP-mutant PC should be explored in clinical trials.

Antiandrogens as Therapies for COVID-19: A Systematic Review.

BACKGROUND: In 2019, the breakthrough of the coronavirus 2 disease (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represented one of the major issues of our recent history. Different drugs have been tested to rapidly find effective anti-viral treatments and, among these, antiandrogens have been suggested to play a role in mediating SARS-CoV-2 infection. Considering the high heterogeneity of studies on this topic, we decided to review the current literature. METHODS: We performed a systematic review according to PRISMA guidelines. A search strategy was conducted on PUBMED and Medline. Only original articles published from March 2020 to 31 August 2023 investigating the possible protective role of antiandrogens were included. In vitro or preclinical studies and reports not in the English language were excluded. The main objective was to investigate how antiandrogens may interfere with COVID-19 outcomes. RESULTS: Among 1755 records, we selected 31 studies, the majority of which consisted of retrospective clinical data collections and of randomized clinical trials during the first and second wave of the COVID-19 pandemic. CONCLUSIONS: In conclusion, we can state that antiandrogens do not seem to protect individuals from SARS-CoV-2 infection and COVID-19 severity and, thus, their use should not be encouraged in this field.

The HACE1 E3 ligase mediates RAC1-dependent control of mTOR signaling complexes.

HACE1 is a HECT family E3 ubiquitin-protein ligase with broad but incompletely understood tumor suppressor activity. Here, we report a previously unrecognized link between HACE1 and signaling complexes containing mammalian target of rapamycin (mTOR). HACE1 blocks mTORC1 and mTORC2 activities by reducing mTOR stability in an E3 ligase-dependent manner. Mechanistically, HACE1 binds to and ubiquitylates Ras-related C3 botulinum toxin substrate 1 (RAC1) when RAC1 is associated with mTOR complexes, including at focal adhesions, leading to proteasomal degradation of RAC1. This in turn decreases the stability of mTOR to reduce mTORC1 and mTORC2 activity. HACE1 deficient cells show enhanced mTORC1/2 activity, which is reversed by chemical or genetic RAC1 inactivation but not in cells expressing the HACE1-insensitive mutant, RAC1K147R . In vivo, Rac1 deletion reverses enhanced mTOR expression in KRasG12D -driven lung tumors of Hace1-/- mice. HACE1 co-localizes with mTOR and RAC1, resulting in RAC1-dependent loss of mTOR protein stability. Together, our data demonstrate that HACE1 destabilizes mTOR by targeting RAC1 within mTOR-associated complexes, revealing a unique ubiquitin-dependent process to control the activity of mTOR signaling complexes.

KappaBle fluorescent reporter mice enable low-background single-cell detection of NF-κB transcriptional activity in vivo.

Nuclear factor-κB (NF-κB) is a transcription factor with a key role in a great variety of cellular processes from embryonic development to immunity, the outcome of which depends on the fine-tuning of NF-κB activity. The development of sensitive and faithful reporter systems to accurately monitor the activation status of this transcription factor is therefore desirable. To address this need, over the years a number of different approaches have been used to generate NF-κB reporter mice, which can be broadly subdivided into bioluminescence- and fluorescence-based systems. While the former enables whole-body visualization of the activation status of NF-κB, the latter have the potential to allow the analysis of NF-κB activity at single-cell level. However, fluorescence-based reporters frequently show poor sensitivity and excessive background or are incompatible with high-throughput flow cytometric analysis. In this work we describe the generation and analysis of ROSA26 knock-in NF-κB reporter (KappaBle) mice containing a destabilized EGFP, which showed sensitive, dynamic, and faithful monitoring of NF-κB transcriptional activity at the single-cell level of various cell types during inflammatory and infectious diseases.

Loss of Rnf31 and Vps4b sensitizes pancreatic cancer to T cell-mediated killing.

Pancreatic ductal adenocarcinoma (PDA) is an inherently immune cell deprived tumor, characterized by desmoplastic stroma and suppressive immune cells. Here we systematically dissect PDA intrinsic mechanisms of immune evasion by in vitro and in vivo CRISPR screening, and identify Vps4b and Rnf31 as essential factors required for escaping CD8+ T cell killing. For Vps4b we find that inactivation impairs autophagy, resulting in increased accumulation of CD8+ T cell-derived granzyme B and subsequent tumor cell lysis. For Rnf31 we demonstrate that it protects tumor cells from TNF-mediated caspase 8 cleavage and subsequent apoptosis induction, a mechanism that is conserved in human PDA organoids. Orthotopic transplantation of Vps4b- or Rnf31 deficient pancreatic tumors into immune competent mice, moreover, reveals increased CD8+ T cell infiltration and effector function, and markedly reduced tumor growth. Our work uncovers vulnerabilities in PDA that might be exploited to render these tumors more susceptible to the immune system.

Adult T-cells impair neonatal cardiac regeneration.

AIMS: Newborn mice and humans display transient cardiac regenerative potential that rapidly declines postnatally. Patients who survive a myocardial infarction (MI) often develop chronic heart failure due to the heart's poor regeneration capacity. We hypothesized that the cardiac 'regenerative-to-scarring' transition might be driven by the perinatal shifts observed in the circulating T-cell compartment. METHODS AND RESULTS: Post-MI immune responses were characterized in 1- (P1) vs. 7-day-old (P7) mice subjected to left anterior descending artery ligation. Myocardial infarction induced robust early inflammatory responses (36 h post-MI) in both age groups, but neonatal hearts exhibited rapid resolution of inflammation and full functional recovery. The perinatal loss of myocardial regenerative capacity was paralleled by a baseline increase in αß-T cell (CD4+ and CD8+) numbers. Strikingly, P1-infarcted mice reconstituted with adult T-cells shifted to an adult-like healing phenotype, marked by irreversible cardiac functional impairment and increased fibrosis. Infarcted neonatal mice harbouring adult T-cells also had more monocyte-derived macrophage recruitment, as typically seen in adults. At the transcriptome level, infarcted P1 hearts that received isolated adult T-cells showed enriched gene sets linked to fibrosis, inflammation, and interferon-gamma (IFN-γ) signalling. In contrast, newborn mice that received isolated Ifng-/- adult T-cells prior to MI displayed a regenerative phenotype that resembled that of its age-matched untreated controls. CONCLUSION: Physiological T-cell development or adoptive transfer of adult IFN-γ-producing T-cells into neonates contributed to impaired cardiac regeneration and promoted irreversible structural and functional cardiac damage. These findings reveal a trade-off between myocardial regenerative potential and the development of T-cell competence.

Fibroblast growth factor receptor 3 in hepatocytes protects from toxin-induced liver injury and fibrosis.

The liver's remarkable regenerative capacity is orchestrated by several growth factors and cytokines. Fibroblast growth factor receptor 3 (Fgfr3) is frequently overexpressed in hepatocellular carcinoma and promotes cancer aggressiveness, whereas its role in liver homeostasis, repair and regeneration is unknown. We show here that Fgfr3 is expressed by hepatocytes in the healthy liver. Its major ligand, Fgf9, is mainly expressed by non-parenchymal cells and upregulated upon injury. Mice lacking Fgfr3 in hepatocytes exhibit increased tissue necrosis after acute toxin treatment and more excessive fibrosis after long-term injury. This was not a consequence of immunological alterations in the non-injured liver as revealed by comprehensive flow cytometry analysis. Rather, loss of Fgfr3 altered the expression of metabolic and pro-fibrotic genes in hepatocytes. These results identify a paracrine Fgf9-Fgfr3 signaling pathway that protects from toxin-induced cell death and the resulting liver fibrosis and suggests a potential use of FGFR3 ligands for therapeutic purposes.

Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF.

Intestinal epithelial cell (IEC) NF-κB signaling regulates the balance between mucosal homeostasis and inflammation. It is not fully understood which signals tune this balance and how bacterial exposure elicits the process. Pure LPS induces epithelial NF-κB activation in vivo. However, we found that in mice, IECs do not respond directly to LPS. Instead, tissue-resident lamina propria intercrypt macrophages sense LPS via TLR4 and rapidly secrete TNF to elicit epithelial NF-κB signaling in their immediate neighborhood. This response pattern is relevant also during oral enteropathogen infection. The macrophage-TNF-IEC axis avoids responses to luminal microbiota LPS but enables crypt- or tissue-scale epithelial NF-κB responses in proportion to the microbial threat. Thereby, intercrypt macrophages fulfill important sentinel functions as first responders to Gram-negative microbes breaching the epithelial barrier. The tunability of this crypt response allows the induction of defense mechanisms at an appropriate scale according to the localization and intensity of microbial triggers.

Comparative analysis of the role of mast cells in murine asthma models using Kit-sufficient mast cell-deficient animals.

BACKGROUND: Asthma is a frequent chronic disease that can potentially severely affect the respiratory capacity and well-being of patients. Mast cells (MCs) are regarded as major players in human asthma due to their capacity to release crucial inflammatory mediators following allergen exposure. However, unambiguous characterization of their role in animal models has long been hindered by the unavailability of specific MC-deficient models lacking confounding MC-unrelated effects. This study aims to examine the role of MCs in Kit-sufficient MC-deficient Cpa3Cre/+ mice. METHODS: We used a variety of models of acute and chronic asthma employing distinct routes and regimes of sensitization. These sensitizations were done via the peritoneal cavity, the skin, or the lung. Additionally, different allergens, i.e. ovalbumin and house dust mite extract, were used. RESULTS: Our results show that the absence of MCs had no impact on the severity of allergic airway inflammation in any of the tested mouse models, as measured by leukocyte infiltration in the airways, cytokine expression, antibody production, airway hyper-responsiveness and mucus production. CONCLUSION: This indicates that MCs do not play a major role in murine allergic airway inflammation.

HACE1 blocks HIF1α accumulation under hypoxia in a RAC1 dependent manner.

Uncovering the mechanisms that underpin how tumor cells adapt to microenvironmental stress is essential to better understand cancer progression. The HACE1 (HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase) gene is a tumor suppressor that inhibits the growth, invasive capacity, and metastasis of cancer cells. However, the direct regulatory pathways whereby HACE1 confers this tumor-suppressive effect remain to be fully elucidated. In this report, we establish a link between HACE1 and the major stress factor, hypoxia-inducible factor 1 alpha (HIF1α). We find that HACE1 blocks the accumulation of HIF1α during cellular hypoxia through decreased protein stability. This property is dependent on HACE1 E3 ligase activity and loss of Ras-related C3 botulinum toxin substrate 1 (RAC1), an established target of HACE1 mediated ubiquitinylation and degradation. In vivo, genetic deletion of Rac1 reversed the increased HIF1α expression observed in Hace1-/- mice in murine KRasG12D-driven lung tumors. An inverse relationship was observed between HACE1 and HIF1α levels in tumors compared to patient-matched normal kidney tissues, highlighting the potential pathophysiological significance of our findings. Together, our data uncover a previously unrecognized function for the HACE1 tumor suppressor in blocking HIF1α accumulation under hypoxia in a RAC1-dependent manner.

A crucial role for Jagunal homolog 1 in humoral immunity and antibody glycosylation in mice and humans.

Jagunal homolog 1 (JAGN1) has been identified as a critical regulator of neutrophil biology in mutant mice and rare-disease patients carrying JAGN1 mutations. Here, we report that Jagn1 deficiency results in alterations in the endoplasmic reticulum (ER) of antibody-producing cells as well as decreased antibody production and secretion. Consequently, mice lacking Jagn1 in B cells exhibit reduced serum immunoglobulin (Ig) levels at steady state and fail to mount an efficient humoral immune response upon immunization with specific antigens or when challenged with viral infections. We also demonstrate that Jagn1 deficiency in B cells results in aberrant IgG N-glycosylation leading to enhanced Fc receptor binding. Jagn1 deficiency in particular affects fucosylation of IgG subtypes in mice as well as rare-disease patients with loss-of-function mutations in JAGN1. Moreover, we show that ER stress affects antibody glycosylation. Our data uncover a novel and key role for JAGN1 and ER stress in antibody glycosylation and humoral immunity in mice and humans.

High-Dimensional T Helper Cell Profiling Reveals a Broad Diversity of Stably Committed Effector States and Uncovers Interlineage Relationships.

CD4+ T helper (Th) cells are fundamental players in immunity. Based on the expression of signature cytokines and transcription factors, several Th subsets have been defined. Th cells are thought to be far more heterogeneous and multifunctional than originally believed, but characterization of the full diversity has been hindered by technical limitations. Here, we employ mass cytometry to analyze the diversity of Th cell responses generated in vitro and in animal disease models, revealing a vast heterogeneity of effector states with distinct cytokine footprints. The diversities of cytokine responses established during primary antigen encounters in Th1- and Th2-cell-polarizing conditions are largely maintained after secondary challenge, regardless of the new inflammatory environment, highlighting many of the identified states as stable Th cell sublineages. We also find that Th17 cells tend to upregulate Th2-cell-associated cytokines upon challenge, indicating a closer developmental connection between Th17 and Th2 cells than previously anticipated.

HACE1 Prevents Lung Carcinogenesis via Inhibition of RAC-Family GTPases.

HACE1 is an E3 ubiquitin ligase with important roles in tumor biology and tissue homeostasis. Loss or mutation of HACE1 has been associated with the occurrence of a variety of neoplasms, but the underlying mechanisms have not been defined yet. Here, we report that HACE1 is frequently mutated in human lung cancer. In mice, loss of Hace1 led to enhanced progression of KRasG12D -driven lung tumors. Additional ablation of the oncogenic GTPase Rac1 partially reduced progression of Hace1-/- lung tumors. RAC2, a novel ubiquitylation target of HACE1, could compensate for the absence of its homolog RAC1 in Hace1-deficient, but not in HACE1-sufficient tumors. Accordingly, ablation of both Rac1 and Rac2 fully averted the increased progression of KRasG12D -driven lung tumors in Hace1-/- mice. In patients with lung cancer, increased expression of HACE1 correlated with reduced levels of RAC1 and RAC2 and prolonged survival, whereas elevated expression of RAC1 and RAC2 was associated with poor prognosis. This work defines HACE1 as a crucial regulator of the oncogenic activity of RAC-family GTPases in lung cancer development. SIGNIFICANCE: These findings reveal that mutation of the tumor suppressor HACE1 disrupts its role as a regulator of the oncogenic activity of RAC-family GTPases in human and murine lung cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/14/3009/F1.large.jpg.

Cyclopentenone Prostaglandins and Structurally Related Oxidized Lipid Species Instigate and Share Distinct Pro- and Anti-inflammatory Pathways.

Oxidized lipids play a critical role in a variety of diseases with two faces: pro- and anti-inflammatory. The molecular mechanisms of this Janus-faced activity remain largely unknown. Here, we have identified that cyclopentenone-containing prostaglandins such as 15d-PGJ2 and structurally related oxidized phospholipid species possess a dual and opposing bioactivity in inflammation, depending on their concentration. Exposure of dendritic cells (DCs)/macrophages to low concentrations of such lipids before Toll-like receptor (TLR) stimulation instigates an anti-inflammatory response mediated by nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent inhibition of nuclear factor κB (NF-κB) activation and downstream targets. By contrast, high concentrations of such lipids upon TLR activation of DCs/macrophages result in inflammatory apoptosis characterized by mitochondrial depolarization and caspase-8-mediated interleukin (IL)-1ß maturation independently of Nrf2 and the classical inflammasome pathway. These results uncover unexpected pro- and anti-inflammatory activities of physiologically relevant lipid species generated by enzymatic and non-enzymatic oxidation dependent on their concentration, a phenomenon known as hormesis.

Mice carrying an analogous heterozygous dynamin 2 K562E mutation that causes neuropathy in humans develop predominant characteristics of a primary myopathy.

Some mutations affecting dynamin 2 (DNM2) can cause dominantly inherited Charcot-Marie-Tooth (CMT) neuropathy. Here, we describe the analysis of mice carrying the DNM2 K562E mutation which has been associated with dominant-intermediate CMT type B (CMTDIB). Contrary to our expectations, heterozygous DNM2 K562E mutant mice did not develop definitive signs of an axonal or demyelinating neuropathy. Rather, we found a primary myopathy-like phenotype in these mice. A likely interpretation of these results is that the lack of a neuropathy in this mouse model has allowed the unmasking of a primary myopathy due to the DNM2 K562E mutation which might be overshadowed by the neuropathy in humans. Consequently, we hypothesize that a primary myopathy may also contribute to the disease mechanism in some CMTDIB patients. We propose that these findings should be considered in the evaluation of patients, the determination of the underlying disease processes and the development of tailored potential treatment strategies.

Bhlhe40 and Bhlhe41 transcription factors regulate alveolar macrophage self-renewal and identity.

Tissues in multicellular organisms are populated by resident macrophages, which perform both generic and tissue-specific functions. The latter are induced by signals from the microenvironment and rely on unique tissue-specific molecular programs requiring the combinatorial action of tissue-specific and broadly expressed transcriptional regulators. Here, we identify the transcription factors Bhlhe40 and Bhlhe41 as novel regulators of alveolar macrophages (AMs)-a population that provides the first line of immune defense and executes homeostatic functions in lung alveoli. In the absence of these factors, AMs exhibited decreased proliferation that resulted in a severe disadvantage of knockout AMs in a competitive setting. Gene expression analyses revealed a broad cell-intrinsic footprint of Bhlhe40/Bhlhe41 deficiency manifested by a downregulation of AM signature genes and induction of signature genes of other macrophage lineages. Genome-wide characterization of Bhlhe40 DNA binding suggested that these transcription factors directly repress the expression of lineage-inappropriate genes in AMs. Taken together, these results identify Bhlhe40 and Bhlhe41 as key regulators of AM self-renewal and guardians of their identity.

Publisher Correction: The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy.

Angiogenesis is a hallmark of cancer, promoting growth and metastasis. Anti-angiogenic treatment has limited efficacy due to therapy-induced blood vessel alterations, often followed by local hypoxia, tumor adaptation, progression, and metastasis. It is therefore paramount to overcome therapy-induced resistance. We show that Apelin inhibition potently remodels the tumor microenvironment, reducing angiogenesis, and effectively blunting tumor growth. Functionally, targeting Apelin improves vessel function and reduces polymorphonuclear myeloid-derived suppressor cell infiltration. Importantly, in mammary and lung cancer, Apelin prevents resistance to anti-angiogenic receptor tyrosine kinase (RTK) inhibitor therapy, reducing growth and angiogenesis in lung and breast cancer models without increased hypoxia in the tumor microenvironment. Apelin blockage also prevents RTK inhibitor-induced metastases, and high Apelin levels correlate with poor prognosis of anti-angiogenic therapy patients. These data identify a druggable anti-angiogenic drug target that reduces tumor blood vessel densities and normalizes the tumor vasculature to decrease metastases.

IL-21 promotes allergic airway inflammation by driving apoptosis of FoxP3+ regulatory T cells.

BACKGROUND: IL-21 is a key player of adaptive immunity, with well-established roles in B-cell and cytotoxic T-cell responses. IL-21 has been implicated in promotion of effector CD4+ T cells and inhibition of forkhead box P3-positive regulatory T (Treg) cells, but the mechanism and functional relevance of these findings remain controversial. OBJECTIVE: We sought to understand the mechanisms by which IL-21 controls effector CD4+ cell responses and Treg cell homeostasis. METHODS: We used IL-21 receptor-deficient mice to study the effect of IL-21 on T-cell responses in models of asthma and colitis. We used mixed bone marrow chimeras and adoptive transfer of naive CD4+ T cells and Treg cells into lymphopenic mice to assess the cell-intrinsic effects of IL-21. Using various in vitro T-cell assays, we characterized the mechanism of IL-21-mediated inhibition of Treg cells. RESULTS: We show that IL-21 production by TH2 and follicular helper T/ex-follicular helper T cells promotes asthma by inhibiting Treg cells. Il21r-/- mice displayed reduced generation of TH2 cells and increased generation of Treg cells. In mixed chimeras we demonstrate that IL-21 promotes TH2 responses indirectly through inhibition of Treg cells. Depleting Treg cells in Il21r-/- mice restored TH2 generation and eosinophilia. Furthermore, IL-21 inhibited Treg cell generation in mice with colitis. Using competitive transfer of Il21r+/+ and Il21r-/- CD4+ cells, we show that IL-21 directly inhibited expansion of differentiated Treg cells but was dispensable for TH1/TH17 effectors. We show that IL-21 sensitizes Treg cells to apoptosis by interfering with the expression of Bcl-2 family genes. CONCLUSION: IL-21 directly promotes apoptosis of Treg cells and therefore indirectly sustains generation of inflammatory TH cells and related effector responses.

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.