Analysis of Nkx3.1:Cre-driven Erk5 deletion reveals a profound spinal deformity which is linked to increased osteoclast activity

Extracellular signal-regulated protein kinase 5 (ERK5) has been implicated during development and carcinogenesis. Nkx3.1-mediated Cre expression is a useful strategy to genetically manipulate the mouse prostate. While grossly normal at birth, we observed an unexpected phenotype of spinal protrusion in Nkx3.1:Cre;Erk5fl/fl (Erk5fl/fl) mice by ~6–8 weeks of age. X-ray, histological and micro CT (µCT) analyses showed that 100% of male and female Erk5fl/fl mice had a severely deformed curved thoracic spine, with an associated loss of trabecular bone volume. Although sex-specific differences were observed, histomorphometry measurements revealed that both bone resorption and bone formation parameters were increased in male Erk5fl/fl mice compared to wild type (WT) littermates. Osteopenia occurs where the rate of bone resorption exceeds that of bone formation, so we investigated the role of the osteoclast compartment. We found that treatment of RANKL-stimulated primary bone marrow-derived macrophage (BMDM) cultures with small molecule ERK5 pathway inhibitors increased osteoclast numbers. Furthermore, osteoclast numbers and expression of osteoclast marker genes were increased in parallel with reduced Erk5 expression in cultures generated from Erk5fl/fl mice compared to WT mice. Collectively, these results reveal a novel role for Erk5 during bone maturation and homeostasis in vivo

Sleeping Beauty screen reveals Pparg activation in metastatic prostate cancer

Prostate cancer (CaP) is the most common adult male cancer in the developed world. The paucity of biomarkers to predict prostate tumor biology makes it important to identify key pathways that confer poor prognosis and guide potential targeted therapy. Using a murine forward mutagenesis screen in a Pten-null background, we identified peroxisome proliferator-activated receptor gamma (Pparg), encoding a ligand-activated transcription factor, as a promoter of metastatic CaP through activation of lipid signaling pathways, including up-regulation of lipid synthesis enzymes [fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACLY)]. Importantly, inhibition of PPARG suppressed tumor growth in vivo, with down-regulation of the lipid synthesis program. We show that elevated levels of PPARG strongly correlate with elevation of FASN in human CaP and that high levels of PPARG/FASN and PI3K/pAKT pathway activation confer a poor prognosis. These data suggest that CaP patients could be stratified in terms of PPARG/FASN and PTEN levels to identify patients with aggressive CaP who may respond favorably to PPARG/FASN inhibition

Sprouty2 loss‐induced IL6 drives castration‐resistant prostate cancer through scavenger receptor B1

Metastatic castration‐resistant prostate cancer (mCRPC) is a lethal form of treatment‐resistant prostate cancer and poses significant therapeutic challenges. Deregulated receptor tyrosine kinase (RTK) signalling mediated by loss of tumour suppressor Sprouty2 (SPRY2) is associated with treatment resistance. Using pre‐clinical human and murine mCRPC models, we show that SPRY2 deficiency leads to an androgen self‐sufficient form of CRPC. Mechanistically, HER2‐IL6 signalling axis enhances the expression of androgen biosynthetic enzyme HSD3B1 and increases SRB1‐mediated cholesterol uptake in SPRY2‐deficient tumours. Systemically, IL6 elevated the levels of circulating cholesterol by inducing host adipose lipolysis and hepatic cholesterol biosynthesis. SPRY2‐deficient CRPC is dependent on cholesterol bioavailability and SRB1‐mediated tumoral cholesterol uptake for androgen biosynthesis. Importantly, treatment with ITX5061, a clinically safe SRB1 antagonist, decreased treatment resistance. Our results indicate that cholesterol transport blockade may be effective against SPRY2‐deficient CRPC

BRF1 accelerates prostate tumourigenesis and perturbs immune infiltration

BRF1 is a rate-limiting factor for RNA Polymerase III-mediated transcription and is elevated in numerous cancers. Here, we report that elevated levels of BRF1 associate with poor prognosis in human prostate cancer. In vitro studies in human prostate cancer cell lines demonstrated that transient overexpression of BRF1 increased cell proliferation whereas the transient downregulation of BRF1 reduced proliferation and mediated cell cycle arrest. Consistent with our clinical observations, BRF1 overexpression in a Pten-deficient mouse (Pten BRF1 ) prostate cancer model accelerated prostate carcinogenesis and shortened survival. In Pten BRF1 tumours, immune and inflammatory processes were altered, with reduced tumoral infiltration of neutrophils and CD4 positive T cells, which can be explained by decreased levels of complement factor D (CFD) and C7 components of the complement cascade, an innate immune pathway that influences the adaptive immune response. We tested if the secretome was involved in BRF1-driven tumorigenesis. Unbiased proteomic analysis on BRF1-overexpresing PC3 cells confirmed reduced levels of CFD in the secretome, implicating the complement system in prostate carcinogenesis. We further identify that expression of C7 significantly correlates with expression of CD4 and has the potential to alter clinical outcome in human prostate cancer, where low levels of C7 associate with poorer prognosis

New aspects of sphingosine 1-phosphate signaling in mammalian cells

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is formed by the phosphorylation of sphingosine by the enzyme, sphingosine kinase. S1P regulates cell function via a family of five closely related G-protein-coupled receptors (GPCR) termed S1P1-5 which bind S1P with high affinity (Chun et al., 2002). The S1P receptors relay the effects of S1P through signaling networks that regulate cell proliferation, survival, migration etc. A number of growth factors also activate sphingosine kinase to produce intracellular S1P which binds to putative intracellular proteins to produce cellular responses. There are two isoforms of sphingosine kinase termed SK1 and SK2. Sphingosine kinase 1 (SK1) was purified as a 49 kDa protein from rat kidney (Olivera et al., 1998) and subsequently two variants termed SK1a and SK1b were cloned from mouse (Kohama et al., 1998). The identification of SK2 was by BLAST searches of EST data using the mouse SK1 sequence. SK2 has regions of high homology with SK1, but contains an additional 240 amino acids located at the N-terminus and in the centre of the protein (Liu et al., 2000). Multiple spliced variant forms of SK1 and SK2 have subsequently been cloned (reviewed by Alemany et al., 2007). SK1 and SK2 are partially redundant since SK1−/− or SK2−/− mice are normal whereas elimination of both genes is embryonic lethal ([Allende et al., 2004] and [Mizugishi et al., 2005]). However, these enzymes may also have different sub-cellular localisations and antagonistic roles. For instance, SK1 enhances growth and survival whereas SK2 has been shown to be involved in the regulation of apoptosis (Spiegel and Milstien, 2003

p300-mediated acetylation of COMMD1 regulates its stability, and the ubiquitylation and nucleolar translocation of the RelA NF-κB subunit

Nucleolar sequestration of the RelA subunit of nuclear factor (NF)-κB is an important mechanism for regulating NF-κB transcriptional activity. Ubiquitylation, facilitated by COMMD1 (also known as MURR1), acts as a crucial nucleolar-targeting signal for RelA, but how this ubiquitylation is regulated, and how it differs from cytokine-mediated ubiquitylation, which causes proteasomal degradation of RelA, is poorly understood. Here, we report a new role for p300 (also known as EP300) in controlling stimulus-specific ubiquitylation of RelA, through modulation of COMMD1. We show that p300 is required for stress-mediated ubiquitylation and nucleolar translocation of RelA, but that this effect is indirect. We also demonstrate that COMMD1 is acetylated by p300 and that acetylation protects COMMD1 from XIAP-mediated proteosomal degradation. Furthermore, we demonstrate that COMMD1 acetylation is enhanced by aspirin-mediated stress, and that this acetylation is absolutely required for the protein to bind RelA under these conditions. In contrast, tumour necrosis factor (TNF) has no effect on COMMD1 acetylation. Finally, we demonstrate these findings have relevance in a whole tissue setting. These data offer a new paradigm for the regulation of NF-κB transcriptional activity, and the multiple other pathways controlled by COMMD1

What brought us together to form a community for scholarship

We are a group of teaching-focused academics who share a passion for learning, teaching and the Scholarship of Teaching and Learning (SoTL) in Higher Education (HE). In order to understand how practitioners from a diversity of backgrounds and disciplines came to be in their present LTS (Learning, Teaching & Scholarship) academic roles, we embarked on a Collaborative Autoethnography (CAE). This approach allowed us to use our personal narratives to explore what it means to be a SoTL practitioner in HE, and to analyse these narratives by using textual analysis. This paper unpacks these narratives, focusing on three themes: our rich and diverse backgrounds, influences on our routes to our current academic roles, and how we are loud and proud to be on a teaching-focused career path. It will be of interest to academics who are on, or contemplating, a teaching focused contract. It will also be of relevance to senior staff in HE who wish to understand the nature of these roles, and who wish to consider how to provide appropriate institutional structures to support and nurture these staff

FTY720 and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 and promote its proteasomal degradation in human pulmonary artery smooth muscle, breast cancer and androgen-independent prostate cancer cells

Sphingosine kinase 1 (SK1) is an enzyme that catalyses the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that FTY720 (Fingolimod™) and (S)-FTY720 vinylphosphonate are novel inhibitors of SK1 catalytic activity and induce the proteasomal degradation of this enzyme in human pulmonary artery smooth muscle cells, MCF-7 breast cancer cells and androgen-independent LNCaP-AI prostate cancer cells. Proteasomal degradation of SK1 in response to FTY720 and (S)-FTY720 vinylphosphonate is associated with the down-regulation of the androgen receptor in LNCaP-AI cells. (S)-FTY720 vinylphosphonate also induces the apoptosis of these cells. These findings indicate that SK1 is involved in protecting LNCaP-AI from apoptosis. This protection might be mediated by so-called ‘inside-out’ signalling by S1P, as LNCaP-AI cells exhibit increased expression of S1P2/3 receptors and reduced lipid phosphate phosphatase expression (compared with androgen-sensitive LNCaP cells) thereby potentially increasing the bioavailability of S1P at S1P2/3 receptors

Increased T cell infiltration elicited by Erk5 deletion in a Pten-deficient mouse model of prostate carcinogenesis

Prostate cancer (PCa) does not appear to respond to immune checkpoint therapies where T cell infiltration may be a key limiting factor. Here we report evidence that ablating the growth regulatory kinase Erk5 can increase T cell infiltration in an established Pten-deficient mouse model of human PCa. Mice that were doubly mutant in prostate tissue for Pten and Erk5 (prostate DKO) exhibited a markedly increased median survival with reduced tumor size and proliferation compared to control Pten-mutant mice, the latter of which exhibited increased Erk5 mRNA expression. A comparative transcriptomic analysis revealed upregulation in prostate DKO mice of the chemokines Ccl5 and Cxcl10, two potent chemoattractants for T lymphocytes. Consistent with this effect, we observed a relative increase in a predominantly CD4+ T cell infiltrate in the prostate epithelial and stroma of tumors from DKO mice. Collectively, our results offer a preclinical proof of concept for ERK5 as a target to enhance T cell infiltrates in prostate cancer, with possible implications for leveraging immune therapy in this disease

Activation of β-catenin cooperates with loss of Pten to drive AR-independent castration-resistant prostate cancer

Inhibition of the androgen receptor (AR) is the main strategy to treat advanced prostate cancers. AR-independent treatment-resistant prostate cancer is a major unresolved clinical problem. Prostate cancer patients with alterations in canonical WNT pathway genes, which lead to β-catenin activation, are refractory to AR-targeted therapies. Here using clinically relevant murine prostate cancer models, we investigated the significance of β-catenin activation in prostate cancer progression and treatment resistance. β-catenin activation, independent of the cell-of-origin, cooperated with Pten loss to drive AR-independent castration-resistant prostate cancer. Prostate tumours with β-catenin activation relied on the non-canonical WNT ligand WNT5a for sustained growth. WNT5a repressed AR expression and maintained the expression of c-Myc, an oncogenic effector of β-catenin activation, by mediating nuclear localization of NFƙBp65 and β-catenin. Overall, WNT/β-catenin and AR signalling are reciprocally inhibited. Therefore, inhibiting WNT/β-catenin signalling by limiting WNT secretion in concert with AR inhibition may be useful for treating prostate cancers with alterations in WNT pathway genes