Exploring anti-androgen therapies in hormone dependent prostate cancer and new therapeutic routes for castration resistant prostate cancer.

Androgen deprivation therapies (ADTs) are important treatments which inhibit androgen-induced prostate cancer (PCa) progression by either preventing androgen biosynthesis (e.g. abiraterone) or by antagonizing androgen receptor (AR) function (e.g. bicalutamide, enzalutamide, darolutamide). A major limitation of current ADTs is they often remain effective for limited durations after which patients commonly progress to a lethal and incurable form of PCa, called castration-resistant prostate cancer (CRPC) where the AR continues to orchestrate pro-oncogenic signalling. Indeed, the increasing numbers of ADT-related treatment-emergent neuroendocrine-like prostate cancers (NePC), which lack AR and are thus insensitive to ADT, represents a major therapeutic challenge. There is therefore an urgent need to better understand the mechanisms of AR action in hormone dependent disease and the progression to CRPC, to enable the development of new approaches to prevent, reverse or delay ADT-resistance. Interestingly the AR regulates distinct transcriptional networks in hormone dependent and CRPC, and this appears to be related to the aberrant function of key AR-epigenetic coregulator enzymes including the lysine demethylase 1 (LSD1/KDM1A). In this review we summarize the current best status of anti-androgen clinical trials, the potential for novel combination therapies and we explore recent advances in the development of novel epigenetic targeted therapies that may be relevant to prevent or reverse disease progression in patients with advanced CRPC.

Leucine induced dephosphorylation of Sestrin2 promotes mTORC1 activation.

The studies described herein were designed to explore the role of Sestrin2 in mediating the selective action of leucine to activate mTORC1. The results demonstrate that Sestrin2 is a phosphoprotein and that its phosphorylation state is responsive to the availability of leucine, but not other essential amino acids. Moreover, leucine availability-induced alterations in Sestrin2 phosphorylation correlated temporally and dose dependently with the activation state of mTORC1, there being a reciprocal relationship between the degree of phosphorylation of Sestrin2 and the extent of repression of mTORC1. With leucine deprivation, Sestrin2 became more highly phosphorylated and interacted more strongly with proteins of the GATOR2 complex. Notably, in cells lacking the protein kinase ULK1, the activation state of mTORC1 was elevated in leucine-deficient medium, such that the effect of re-addition of the amino acid was blunted. In contrast, overexpression of ULK1 led to hyperphosphorylation of Sestrin2 and enhanced its interaction with GATOR2. Neither rapamycin nor Torin2 had any effect on Sestrin2 phosphorylation, suggesting that leucine deprivation-induced repression of mTORC1 was not responsible for the action of ULK1 on Sestrin2. Mass spectrometry analysis of Sestrin2 revealed three phosphorylation sites that are conserved across mammalian species. Mutation of the three sites to phospho-mimetic amino acids in exogenously expressed Sestrin2 promoted its interaction with GATOR2 and dramatically repressed mTORC1 even in the presence of leucine. Overall, the results support a model in which leucine selectively promotes dephosphorylation of Sestrin2, causing it to dissociate from and thereby activate GATOR2, leading to activation of mTORC1.

Validation of normal human bronchial epithelial cells as a model for influenza A infections in human distal trachea.

Primary normal human bronchial/tracheal epithelial (NHBE) cells, derived from the distal-most aspect of the trachea at the bifurcation, have been used for a number of studies in respiratory disease research. Differences between the source tissue and the differentiated primary cells may impact infection studies based on this model. Therefore, we examined how well-differentiated NHBE cells compared with their source tissue, the human distal trachea, as well as the ramifications of these differences on influenza A viral pathogenesis research using this model. We employed a histological analysis including morphological measurements, electron microscopy, multi-label immunofluorescence confocal microscopy, lectin histochemistry, and microarray expression analysis to compare differentiated NHBEs to human distal tracheal epithelium. Pseudostratified epithelial height, cell type variety and distribution varied significantly. Electron microscopy confirmed differences in cellular attachment and paracellular junctions. Influenza receptor lectin histochemistry revealed that α2,3 sialic acids were rarely present on the apical aspect of the differentiated NHBE cells, but were present in low numbers in the distal trachea. We bound fluorochrome bioconjugated virus to respiratory tissue and NHBE cells and infected NHBE cells with human influenza A viruses. Both indicated that the pattern of infection progression in these cells correlated with autopsy studies of fatal cases from the 2009 pandemic.

Characterizing and Diminishing Autofluorescence in Formalin-fixed Paraffin-embedded Human Respiratory Tissue.

Tissue autofluorescence frequently hampers visualization of immunofluorescent markers in formalin-fixed paraffin-embedded respiratory tissues. We assessed nine treatments reported to have efficacy in reducing autofluorescence in other tissue types. The three most efficacious were Eriochrome black T, Sudan black B and sodium borohydride, as measured using white light laser confocal Λ2 (multi-lambda) analysis. We also assessed the impact of steam antigen retrieval and serum application on human tracheal tissue autofluorescence. Functionally fitting this Λ2 data to 2-dimensional Gaussian surfaces revealed that steam antigen retrieval and serum application contribute minimally to autofluorescence and that the three treatments are disparately efficacious. Together, these studies provide a set of guidelines for diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue. Additionally, these characterization techniques are transferable to similar questions in other tissue types, as demonstrated on frozen human liver tissue and paraffin-embedded mouse lung tissue fixed in different fixatives.