Activation of ß-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. Patients with prostate cancer with alterations in canonical WNT pathway genes, which lead to ß-catenin activation, are refractory to AR-targeted therapies. Here, using clinically relevant murine prostate cancer models, we investigated the significance of ß-catenin activation in prostate cancer progression and treatment resistance. ß-Catenin activation, independent of the cell of origin, cooperated with Pten loss to drive AR-independent castration-resistant prostate cancer. Prostate tumors with ß-catenin activation relied on the noncanonical WNT ligand WNT5a for sustained growth. WNT5a repressed AR expression and maintained the expression of c-Myc, an oncogenic effector of ß-catenin activation, by mediating nuclear localization of NFκBp65 and ß-catenin. Overall, WNT/ß-catenin and AR signaling are reciprocally inhibited. Therefore, inhibiting WNT/ß-catenin signaling by limiting WNT secretion in concert with AR inhibition may be useful for treating prostate cancers with alterations in WNT pathway genes. SIGNIFICANCE: Targeting of both AR and WNT/ß-catenin signaling may be required to treat prostate cancers that exhibit alterations of the WNT pathway.

Correction: BRF1 accelerates prostate tumourigenesis and perturbs immune infiltration.

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

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 (PtenΔ/Δ BRF1Tg) prostate cancer model accelerated prostate carcinogenesis and shortened survival. In PtenΔ/Δ BRF1Tg 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.

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;Erk5 fl/fl (Erk5 fl/fl) mice by ~6-8 weeks of age. X-ray, histological and micro CT (µCT) analyses showed that 100% of male and female Erk5 fl/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 Erk5 fl/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 Erk5 fl/fl mice compared to WT mice. Collectively, these results reveal a novel role for Erk5 during bone maturation and homeostasis in vivo.

Increased T-cell Infiltration Elicited by Erk5 Deletion in a Pten-Deficient Mouse Model of Prostate Carcinogenesis.

Prostate cancer 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 prostate cancer. 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 with 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. Cancer Res; 77(12); 3158-68. ©2017 AACR.

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.

c-Src dependency of NSAID-induced effects on NF-κB-mediated apoptosis in colorectal cancer cells.

Long-term aspirin or related non-steroidal anti-inflammatory drugs (NSAIDs) ingestion can protect against colorectal cancer (CRC). NSAIDs have a pro-apoptotic activity and we have shown that stimulation of the nuclear factor-kappaB (NF-κB) pathway is a key component of this pro-apoptotic effect. However, the upstream pathways have yet to be fully elucidated. Here, we demonstrate that aspirin activates the c-Src tyrosine kinase pathway in CRC cells. We show that c-Src activation occurs in a time- and dose-dependent manner, preceding aspirin-mediated degradation of IκBα, nuclear/nucleolar translocation of NF-κB/RelA and induction of apoptosis. Furthermore, inhibition of c-Src activity, by chemical inhibition or expression of a kinase dead form of the protein abrogates aspirin-mediated degradation of IκBα, nuclear translocation of RelA and apoptosis, suggesting a causal link. Expression of constitutively active c-Src mimics aspirin-induced stimulation of the NF-κB pathway. The NSAIDs sulindac, sulindac sulphone and indomethacin all similarly activate a c-Src-dependent NF-κB and apoptotic response. These data provide compelling evidence that c-Src is an upstream mediator of aspirin/NSAID effects on NF-κB signalling and apoptosis in CRC cells and have relevance to the development of future chemotherapeutic/chemopreventative agents.

Nucleolar targeting of RelA(p65) is regulated by COMMD1-dependent ubiquitination.

Stimulation of the NF-kappaB pathway can have proapoptotic or antiapoptotic consequences, and one mechanism that determines the outcome is the nuclear distribution of RelA. Certain stress stimuli induce nucleolar accumulation of RelA thereby mediating apoptosis, whereas others induce nucleoplasmic accumulation and inhibition of apoptosis. Here we investigated the mechanisms that regulate the nuclear distribution of RelA, specifically, the role of the ubiquitin/proteasome system. We found that stress-induced nucleolar translocation of RelA is preceded by ubiquitination of the protein. We also found that chemical proteasome inhibitors induce the ubiquitination and nucleolar translocation of RelA and that this is required for the apoptotic response to these agents. We show that the RelA nucleolar localization signal (amino acids 27-30) is a critical domain for ubiquitination of the protein but that the lysine residue within this motif is not a direct target. We show that RelA binds COMMD1, the rate-limiting component of the RelA ubiquitin ligase complex, in response to stress. Furthermore, we show that overexpression of COMMD1 promotes stress-mediated nucleolar targeting of RelA, whereas knockdown of COMMD1 blocks this effect, causing RelA to remain in the nucleoplasm. These data identify a new role for COMMD1 in regulating the nuclear/nucleolar distribution of RelA and suggest that ubiquitination acts as a signal for transport of RelA to the nucleolus. These findings have relevance to the design of chemopreventative/anticancer agents that act by targeting RelA to the nucleolar compartment.