Delineating a Functional Role for the Urinary Biomarker Lipocalin 2 in Prostate Cancer

PhDProstate cancer (PCa) is the most commonly diagnosed cancer amongst Western males. PCa progression is strongly linked to steroid receptor signalling, however the modulation of steroid receptor expression in PCa is incompletely understood. Lipocalin 2 (LCN2) is a secreted protein which binds to Fe3+-containing siderophores and was originally identified as part of the innate immune response. LCN2 has been proposed as a potential biomarker for a range of cancers. However, LCN2 effects appear to be tissue specific. LCN2 expression is associated with poor prognosis in breast cancer, but with good prognosis in pancreatic cancer where it has been used therapeutically. The role of LCN2 in prostate cancer is poorly understood, in particular its effects on steroid receptor regulation. To elucidate the role of LCN2 in prostate cancer, the LCN2 gene was ectopically expressed in LNCaP cells to generate the LNCaP-LCN2 cell line. LNCaP-LCN2 cells had elevated androgen receptor expression which was linked to increased levels of KLK3 (PSA). LNCaP-LCN2 cells also had reduced levels of Estrogen receptor α (ERα), but increased expression of ERβ. This was combined with higher levels of E-cadherin, but not to changes in other EMT markers. Reciprocally, LCN2 was suppressed using RNAi in the PC3 cell line to generate PC3-shLCN2 cells. PC3-shLCN2 displayed a distinct change in morphology, with increased cell size and a sub-population of multi-nucleated and highly enlarged cells. PC3-shLCN2 cells had reduced proliferation, and lost the ability to form colonies in a 3D substrate. With regards to steroid receptors, PC3-shLCN2 cells had increased ERα expression, but reduced ERβ expression. This was also combined with a loss of E-cadherin and EGFR. Microarray analysis of PC3-shLCN2 cells identified changes to expression of a wide range of genes including VEGF-R, SPARC and KLK6. Functional grouping of differentially expressed genes suggests that LCN2 in involved in a range of cellular processes including hormone receptor response, Wnt signalling and cell cytoskeletal integrity. Many, but not all genes identified by microarray were responsive to recombinant LCN2 protein indicating a paracrine function for the protein. Treatment of PC3 cells with the iron chelator Deferoxamine resulted in phenotypic changes similar to those found in PC3-shLCN2 cells which suggest that LCN2 functions in part due to intracellular iron regulation. In summary, the data presented in this thesis suggests that LCN2 has both pro- and anti- tumourigenic properties in prostate cancer and that the protein is involved in a much wider range of functions than previously described

GLI1 Confers Profound Phenotypic Changes upon LNCaP Prostate Cancer Cells That Include the Acquisition of a Hormone Independent State

The GLI (GLI1/GLI2) transcription factors have been implicated in the development and progression of prostate cancer although our understanding of how they actually contribute to the biology of these common tumours is limited. We observed that GLI reporter activity was higher in normal (PNT-2) and tumourigenic (DU145 and PC-3) androgen-independent cells compared to androgen-dependent LNCaP prostate cancer cells and, accordingly, GLI mRNA levels were also elevated. Ectopic expression of GLI1 or the constitutively active ΔNGLI2 mutant induced a distinct cobblestone-like morphology in LNCaP cells that, regarding the former, correlated with increased GLI2 as well as expression of the basal/stem-like markers CD44, β1-integrin, ΔNp63 and BMI1, and decreased expression of the luminal marker AR (androgen receptor). LNCaP-GLI1 cells were viable in the presence of the AR inhibitor bicalutamide and gene expression profiling revealed that the transcriptome of LNCaP-GLI1 cells was significantly closer to DU145 and PC-3 cells than to control LNCaP-pBP (empty vector) cells, as well as identifying LCN2/NGAL as a highly induced transcript which is associated with hormone independence in breast and prostate cancer. Functionally, LNCaP-GLI1 cells displayed greater clonal growth and were more invasive than control cells but they did not form colonies in soft agar or prostaspheres in suspension suggesting that they do not possess inherent stem cell properties. Moreover, targeted suppression of GLI1 or GLI2 with siRNA did not reverse the transformed phenotype of LNCaP-GLI1 cells nor did double GLI1/GLI2 knockdowns activate AR expression in DU145 or PC-3 cells. As such, early targeting of the GLI oncoproteins may hinder progression to a hormone independent state but a more detailed understanding of the mechanisms that maintain this phenotype is required to determine if their inhibition will enhance the efficacy of anti-hormonal therapy through the induction of a luminal phenotype and increased dependency upon AR function

Refractory Pseudomonas aeruginosa infections treated with phage PASA16: A compassionate use case series

BACKGROUND: A growing number of compassionate phage therapy cases were reported in the last decade, with a limited number of clinical trials conducted and few unsuccessful clinical trials reported. There is only a little evidence on the role of phages in refractory infections. Our objective here was to present the largest compassionate-use single-organism/phage case series in 16 patients with non-resolving Pseudomonas aeruginosa infections. METHODS: We summarized clinical phage microbiology susceptibility data, administration protocol, clinical data, and outcomes of all cases treated with PASA16 phage. In all intravenous phage administrations, PASA16 phage was manufactured and provided pro bono by Adaptive Phage Therapeutics. PASA16 was administered intravenously, locally to infection site, or by topical use to 16 patients, with data available for 15 patients, mainly with osteoarticular and foreign-device-associated infections. FINDINGS: A few minor side effects were noted, including elevated liver function enzymes and a transient reduction in white blood cell count. Good clinical outcome was documented in 13 out of 15 patients (86.6%). Two clinical failures were reported. The minimum therapy duration was 8 days with a once- to twice-daily regimen. CONCLUSIONS: PASA16 with antibiotics was found to be relatively successful in patients for whom traditional treatment approaches have failed previously. Such pre-phase-1 cohorts can outline potential clinical protocols and facilitate the design of future trials. FUNDING: The study was funded in part by The Israeli Science Foundation IPMP (ISF_1349/20), Rosetrees Trust (A2232), United States-Israel Binational Science Foundation (2017123), and the Milgrom Family Support Program