Clinical characteristics and comorbidities associated with testosterone prescribing in men.

OBJECTIVE: Testosterone replacement therapy (TRT) is recommended for the treatment of symptomatic hypogonadism in men. Data on prescription behaviours are, however, limited and conflicting. The objective of this study was to investigate clinical characteristics associated with the likelihood of being prescribed TRT by general practitioners (GP) in North-West London (NWL). DESIGN: Retrospective cohort study using Discover database of GP-registered patients in NWL between 2015 and 2019. PATIENTS: We identified 20,299 men aged ≥18 years with serum total testosterone measurement (TT) and without prior TRT prescription records. MEASUREMENTS: We determined whether TRT was subsequently commenced, while analysing clinical characteristics related to hypogonadism. RESULTS: Of all men having TT measurement, 19,583 (96.4%) were not commenced on TRT (Group A) and 716 (3.5%) men were commenced on TRT (Group B). Men prescribed TRT (Group B) had higher mean age, body mass index (BMI) and higher risks of hypertension, depression type 2 diabetes and ischaemic heart disease; conversely, men in Group B had lower mean pretreatment TT and were less likely to have prostate cancer. Four-hundred and thirty-six men (24.3%) with TT < 8 nmol/L and symptoms of low libido were not prescribed TRT. CONCLUSIONS: Our study highlights several factors which may influence the decisions made by clinicians when initiating TRT in primary care. Clearer guidance for clinicians may help to improve the consistency of treatment of men with hypogonadism.

Transient over-expression of estrogen receptor-α in breast cancer cells promotes cell survival and estrogen-independent growth.

Estrogen receptor-α (ERα) positive breast cancer frequently responds to inhibitors of ERα activity, such as tamoxifen, and/or to aromatase inhibitors that block estrogen biosynthesis. However, many patients become resistant to these agents through mechanisms that remain unclear. Previous studies have shown that expression of ERα in ERα-negative breast cancer cell lines frequently inhibits their growth. In order to determine the consequence of ERα over-expression in ERα-positive breast cancer cells, we over-expressed ERα in the MCF-7 breast cancer cell line using adenovirus gene transduction. ERα over-expression led to ligand-independent expression of the estrogen-regulated genes pS2 and PR and growth in the absence of estrogen. Interestingly, prolonged culturing of these cells in estrogen-free conditions led to the outgrowth of cells capable of growth in cultures from ERα transduced, but not in control cultures. From these cultures a line, MLET5, was established which remained ERα-positive, but grew in an estrogen-independent manner. Moreover, MLET5 cells were inhibited by anti-estrogens showing that ERα remains important for their growth. Gene expression microarray analysis comparing MCF-7 cells with MLET5 highlighted apoptosis as a major functional grouping that is altered in MLET5 cells, such that cell survival would be favoured. This conclusion was further substantiated by the demonstration that MLET5 show resistance to etoposide-induced apoptosis. As the gene expression microarray analysis also shows that the apoptosis gene set differentially expressed in MLET5 is enriched for estrogen-regulated genes, our findings suggest that transient over-expression of ERα could lead to increased cell survival and the development of estrogen-independent growth, thereby contributing to resistance to endocrine therapies in breast cancer patients.

ZNF366 is an estrogen receptor corepressor that acts through CtBP and histone deacetylases.

The regulation of gene expression by estrogen receptor-alpha (ERalpha) requires the coordinated and temporal recruitment of diverse sets of transcriptional co-regulator complexes, which mediate nucleosome remodelling and histone modification. Using ERalpha as bait in a yeast two-hybrid screen, we have identified a novel ERalpha-interacting protein, ZNF366, which is a potent corepressor of ERalpha activity. The interaction between ZNF366 and ERalpha has been confirmed in vitro and in vivo, and is mediated by the zinc finger domains of the two proteins. Further, we show that ZNF366 acts as a corepressor by interacting with other known ERalpha corepressors, namely RIP140 and CtBP, to inhibit expression of estrogen-responsive genes in vivo. Together, our results indicate that ZNF366 may play an important role in regulating the expression of genes in response to estrogen.

A novel HSV-1 virus, JS1/34.5-/47-, purges contaminating breast cancer cells from bone marrow.

PURPOSE: Oncolytic herpes simplex virus type 1 (HSV-1) vectors show considerable promise as agents for cancer therapy. We have developed a novel recombinant HSV-1 virus (JS1/34.5-/47-) for purging of occult breast cancer cells from bone marrow of patients. Here, we evaluate the therapeutic efficacy of this oncolytic virus. EXPERIMENTAL DESIGN: Electron microscopy was used to determine whether human breast cancer and bone marrow cells are permissive for JS1/34.5-/47- infection. Subsequently, the biological effects of JS1/34.5-/47- infection on human breast cancer cells and bone marrow were established using cell proliferation and colony formation assays, and the efficiency of cell kill was evaluated. Finally, the efficiency of JS1/34.5-/47- purging of breast cancer cells was examined in cocultures of breast cancer cells with bone marrow as well as bone marrow samples from high-risk breast cancer patients. RESULTS: We show effective killing of human breast cancer cell lines with the JS1/34.5-/47- virus. Furthermore, we show that treatment with JS1/34.5-/47- can significantly inhibit the growth of breast cancer cell lines without affecting cocultured mononuclear hematopoietic cells. Finally, we have found that the virus is effective in destroying disseminated tumors cells in bone marrow taken from breast cancer patients, without affecting the hematopoietic contents in these samples. CONCLUSION: Collectively, our data show that the JS1/34.5-/47- virus can selectively target breast cancer cells while sparing hematopoietic cells, suggesting that JS1/34.5-/47- can be used to purge contaminating breast cancer cells from human bone marrow in the setting of autologous hematopoietic cell transplantation.

Silencing of androgen-regulated genes using a fusion of AR with the PLZF transcriptional repressor.

The androgen receptor (AR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors and plays a key role in the development and progression of prostate cancer. Current therapies include the use of antiandrogens aimed at inhibiting the transcriptional activation of AR-regulated genes by AR. Here, we explore a strategy aimed at obtaining silencing of AR-regulated genes, based on the properties of the transcriptional repressor promyelocytic leukamia zinc-finger protein (PLZF). In order to do this, we have made a fusion protein between PLZF and AR, named PLZF-AR, and show that PLZF-AR is able to bring about silencing of genomically encoded AR-regulated genes and inhibit the androgen-regulated growth of LNCaP prostate cancer cells. Together, our results show that this strategy is able to bring about potent repression of AR-regulated responses and, therefore, could be of value in the development of new therapies for prostate cancer.

In vitro deregulation of markers characteristic of human prostate epithelial cells.

We have screened primary cultures of human prostate for the expression of markers reported to be characteristic of specific cell lineages in vivo, in order to ascertain whether human prostate cells in vitro maintain and reflect their in vivo differentiated phenotypes and to evaluate the homogeneity of the populations of cells that can be derived from this tissue. Using single and dual stain immunofluorescent microscopy to analyse very early organoid and subsequently derived monolayer stage cultures, we have observed that expression of markers characteristic of human prostate epithelial cells in vivo is deregulated within 48h, indicating that dissociation of human prostate tissue and cultivation of prostate epithelial cells in culture can result in promiscuous expression of cell type specific markers of prostate epithelial cells. These observations have important implications for studies of cell lineage and differentiation of prostate cells in vitro.