Human prostate organoid generation and the identification of prostate development drivers using inductive rodent tissues.

The reactivation of developmental genes and pathways during adulthood may contribute to pathogenesis of diseases such as prostate cancer. Analysis of the mechanistic links between development and disease could be exploited to identify signalling pathways leading to disease in the prostate. However, the mechanisms underpinning prostate development require further characterisation to interrogate fully the link between development and disease. Previously, our group developed methods to produce prostate organoids using induced pluripotent stem cells (iPSCs). Here, we show that human iPSCs can be differentiated into prostate organoids using neonatal rat seminal vesicle mesenchyme in vitro. The organoids can be used to study prostate development or modified to study prostate cancer. We also elucidated molecular drivers of prostate induction through RNA-sequencing analyses of the rat urogenital sinus and neonatal seminal vesicles. We identified candidate drivers of prostate development evident in the inductive mesenchyme and epithelium involved with prostate specification. Our top candidates included Spx, Trib3, Snai1, Snai2, Nrg2 and Lrp4. This work lays the foundations for further interrogation of the reactivation of developmental genes in adulthood, leading to prostate disease.

Defining splicing factor requirements for androgen receptor variant synthesis in advanced prostate cancer.

Resistance to androgen receptor (AR)-targeted therapies represent a major challenge in prostate cancer (PC). A key mechanism of treatment resistance in patients who progress to castrate-resistant PC (CRPC) is the generation of alternatively spliced androgen receptor variants (AR-Vs). Unlike full-length AR (FL-AR) isoforms, AR-Vs are constitutively active and refractory to current receptor-targeting agents hence drive tumour progression. Identifying regulators of AR-V synthesis may therefore provide new therapeutic opportunities in combination with conventional AR-targeting agents. Our understanding of AR transcript splicing, and the factors that control the synthesis of AR-Vs, remains limited. While candidate-based approaches have identified a small number of AR-V splicing regulators, an unbiased analysis of splicing factors important for AR-V generation is required to fill an important knowledge gap and furnish the field with novel and tractable targets for PC treatment. To that end, we conducted a bespoke CRISPR screen to profile splicing factor requirements for AR-V synthesis. MFAP1 and CWC22 were shown to be required for the generation of AR-V mRNA transcripts and their depletion resulted in reduced AR-V protein abundance and cell proliferation in several CRPC models. Global transcriptomic analysis of MFAP1-depleted cells revealed both AR-dependent and -independent transcriptional impact, including genes associated with DDR. As such, MFAP1 downregulation sensitised PC cells to ionising radiation suggesting therapeutically targeting AR-V splicing could provide novel cellular vulnerabilities which can be exploited in CRPC. Implications: We have utilised a CRISPR screening approach to identify key regulators of pathogenic AR splicing in prostate cancer.

Suppressed basal mitophagy drives cellular aging phenotypes that can be reversed by a p62-targeting small molecule.

Selective degradation of damaged mitochondria by autophagy (mitophagy) is proposed to play an important role in cellular homeostasis. However, the molecular mechanisms and the requirement of mitochondrial quality control by mitophagy for cellular physiology are poorly understood. Here, we demonstrated that primary human cells maintain highly active basal mitophagy initiated by mitochondrial superoxide signaling. Mitophagy was found to be mediated by PINK1/Parkin-dependent pathway involving p62 as a selective autophagy receptor (SAR). Importantly, this pathway was suppressed upon the induction of cellular senescence and in naturally aged cells, leading to a robust shutdown of mitophagy. Inhibition of mitophagy in proliferating cells was sufficient to trigger the senescence program, while reactivation of mitophagy was necessary for the anti-senescence effects of NAD precursors or rapamycin. Furthermore, reactivation of mitophagy by a p62-targeting small molecule rescued markers of cellular aging, which establishes mitochondrial quality control as a promising target for anti-aging interventions.

ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3Gal1) synthesis of Siglec ligands mediates anti-tumour immunity in prostate cancer.

Immune checkpoint blockade has yet to produce robust anti-cancer responses for prostate cancer. Sialyltransferases have been shown across several solid tumours, including breast, melanoma, colorectal and prostate to promote immune suppression by synthesising sialoglycans, which act as ligands for Siglec receptors. We report that ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3Gal1) levels negatively correlate with androgen signalling in prostate tumours. We demonstrate that ST3Gal1 plays an important role in modulating tumour immune evasion through the synthesises of sialoglycans with the capacity to engage the Siglec-7 and Siglec-9 immunoreceptors preventing immune clearance of cancer cells. Here, we provide evidence of the expression of Siglec-7/9 ligands and their respective immunoreceptors in prostate tumours. These interactions can be modulated by enzalutamide and may maintain immune suppression in enzalutamide treated tumours. We conclude that the activity of ST3Gal1 is critical to prostate cancer anti-tumour immunity and provide rationale for the use of glyco-immune checkpoint targeting therapies in advanced prostate cancer.

Mechanisms involved in the enhancement of mammalian target of rapamycin signalling and hypertrophy in skeletal muscle of myostatin-deficient mice.

Myostatin deficiency leads to both an increased rate of protein synthesis and skeletal muscle hypertrophy. However, the mechanisms involved in mediating these effects are not yet fully understood. Here, we demonstrate that genetic loss of myostatin leads to enhanced muscle expression of both protein kinase B and mammalian target of rapamycin/S6K signalling components, consistent with their elevated activity. This is associated with a reduction in the expression of PGC1alpha and COX IV, proteins which play important roles in maintaining mitochondrial function. Furthermore, we show that these changes in signalling and protein expression are largely independent of alterations in intramuscular amino acid content. Our findings, therefore, reveal potential new mechanisms and further contribute to our understanding of myostatin-regulated skeletal muscle growth and function.

8-Methoxyquinoline-5-carboxamides as PDE4 inhibitors: a potential treatment for asthma.

A series of bicyclic heteroaryl ring systems was considered as a replacement for the 3-cyclopentyloxy-4-methoxyphenyl moiety in rolipram resulting in the discovery of 8-methoxyquinoline-5-carboxamides as potent inhibitors of phosphodiesterase type 4 (PDE4).

8-Methoxyquinolines as PDE4 inhibitors.

The synthesis and pharmacological profile of a novel series of 2-substituted 8-methoxyquinolines is described. The 2-trifluoromethyl compound was found to be a potent inhibitor of phosphodiesterase type 4 (PDE4).

7-Methoxyfuro[2,3-c]pyridine-4-carboxamides as PDE4 inhibitors: a potential treatment for asthma.

The synthesis and pharmacological profile of a novel series of 7-methoxy-furo[2,3-c]pyridine-4-carboxamides is described. Some of these compounds were found to be potent inhibitors of phosphodiesterase type 4 (PDE4).

Synthesis and profile of SCH351591, a novel PDE4 inhibitor.

The syntheses and pharmacological profiles of some 2-trifluoromethyl-8-methoxyquinoline-5-carboxamides are described. SCH351591 is a potent selective inhibitor of phosphodiesterase type 4 (PDE4).