High-Throughput Screening for Novel Prostate Cancer Drug Targets –Getting Personal

Prostate cancers form a heterogeneous group of diseases and there is a need for novel biomarkers, and for more efficient and targeted methods of treatment. In this thesis, the potential of microarray data, RNA interference (RNAi) and compound screens were utilized in order to identify novel biomarkers, drug targets and drugs for future personalized prostate cancer therapeutics. First, a bioinformatic mRNA expression analysis covering 9873 human tissue and cell samples, including 349 prostate cancer and 147 normal prostate samples, was used to distinguish in silico prevalidated putative prostate cancer biomarkers and drug targets. Second, RNAi based high-throughput (HT) functional profiling of 295 prostate and prostate cancer tissue specific genes was performed in cultured prostate cancer cells. Third, a HT compound screen approach using a library of 4910 drugs and drug-like molecules was exploited to identify potential drugs inhibiting prostate cancer cell growth. Nine candidate drug targets, with biomarker potential, and one cancer selective compound were validated in vitro and in vivo. In addition to androgen receptor (AR) signaling, endoplasmic reticulum (ER) function, arachidonic acid (AA) pathway, redox homeostasis and mitosis were identified as vital processes in prostate cancer cells. ERG oncogene positive cancer cells exhibited sensitivity to induction of oxidative and ER stress, whereas advanced and castrate-resistant prostate cancer (CRPC) could be potentially targeted through AR signaling and mitosis. In conclusion, this thesis illustrates the power of systems biological data analysis in the discovery of potential vulnerabilities present in prostate cancer cells, as well as novel options for personalized cancer management.

Characterisation of Human Neutral and Lysosomal alpha-Mannosidases

Neutral alpha-mannosidase and lysosomal MAN2B1 alpha-mannosidase belong to glycoside hydrolase family 38, which contains essential enzymes required for the modification and catabolism of asparagine-linked glycans on proteins. MAN2B1 catalyses lysosomal glycan degradation, while neutral α-mannosidase is most likely involved in the catabolism of cytosolic free oligosaccharides. These mannose containing saccharides are generated during glycosylation or released from misfolded glycoproteins, which are detected by quality control in the endoplasmic reticulum. To characterise the biological function of human neutral α-mannosidase, I cloned the alpha-mannosidase cDNA and recombinantly expressed the enzyme. The purified enzyme trimmed the putative natural substrate Man9GlcNAc to Man5GlcNAc, whereas the reducing end GlcNAc2 limited trimming to Man8GlcNAc2. Neutral α-mannosidase showed highest enzyme activity at neutral pH and was activated by the cations Fe₂+, Co2+ and Mn₂+, Cu2+ in turn had a strong inhibitory effect on alpha-mannosidase activity. Analysis of its intracellular localisation revealed that neutral alpha-mannosidase is cytosolic and colocalises with proteasomes. Further work showed that the overexpression of neutral alpha-mannosidase affected the cytosolic free oligosaccharide content and led to enhanced endoplasmic reticulum associated degradation and underglycosylation of secreted proteins. The second part of the study focused on MAN2B1 and the inherited lysosomal storage disorder α-mannosidosis. In this disorder, deficient MAN2B1 activity is associated with mutations in the MAN2B1 gene. The thesis reports the molecular consequences of 35 alpha-mannosidosis associated mutations, including 29 novel missense mutations. According to experimental analyses, the mutations fall into four groups: Mutations, which prevent transport to lysosomes are accompanied with a lack of proteolytic processing of the enzyme (groups 1 and 3). Although the rest of the mutations (groups 2 and 4) allow transport to lysosomes, the mutated proteins are less efficiently processed to their mature form than is wild type MAN2B1. Analysis of the effect of the mutations on the model structure of human lysosomal alpha-mannosidase provides insights on their structural consequences. Mutations, which affect amino acids important for folding (prolines, glycines, cysteines) or domain interface interactions (arginines), arrest the enzyme in the endoplasmic reticulum. Surface mutations and changes, which do not drastically alter residue volume, are tolerated better. Descriptions of the mutations and clinical data are compiled in an α-mannosidosis database, which will be available for the scientific community. This thesis provides a detailed insight into two ubiquitous human alpha-mannosidases. It demonstrates that neutral alpha-mannosidase is involved in the degradation of cytosolic oligosaccharides and suggests that the regulation of this α-mannosidase is important for maintaining the cellular homeostasis of N-glycosylation and glycan degradation. The study on alpha-mannosidosis associated mutations identifies multiple mechanisms for how these mutations are detrimental for MAN2B1 activity. The α-mannosidosis database will benefit both clinicians and scientific research on lysosomal alpha‑mannosidosis.

Regulation of epidermal tight junctions by calcium ATPases and p38

The epidermis is the upper layer of the skin and keratinocytes are its most abundant cells. Tight junctions are cell junctions located in the granular layer of the epidermis. They maintain the polarity of the cells and regulate the movement of water-soluble molecules. Epidermal tight junctions may lose their integrity when there are defects in intercellular calcium regulation. Hailey-Hailey and Darier´s disease are dominantly inherited, blistering skin diseases. Hailey-Hailey disease is caused by mutations in the ATP2C1 gene encoding a calcium/manganese ATPase SPCA1 of the Golgi apparatus. Darier´s disease is caused by mutations in the ATP2A2 gene encoding a calcium ATPase SERCA2 of the endoplasmic reticulum. p38 regulates the differentiation of keratinocytes. The overall regulation of epidermal tight junctions is not well understood. The present study examined the regulation of tight junctions in the human epidermis with a focus on calcium ATPases and p38. Skin from Hailey-Hailey and Darier´s disease patients was studied by using immunofluorescence labeling which targeted intercellular junction proteins. Transepidermal water loss was also measured. ATP2C1 gene expression was silenced in cultured keratinocytes, by siRNA, which modeled Hailey-Hailey disease. Expression of intercellular junction proteins was studied at the mRNA and protein levels. Squamous cell carcinoma and normal human keratinocytes were used as a model for impaired and normal keratinocyte differentiation, and the role of p38 isoforms alpha and delta in the regulation of intercellular junction proteins was studied. Both p38 isoforms were silenced by adenovirus cell transduction, chemical inhibitors or siRNA and keratinocyte differentiation was assessed. The results of this thesis revealed that: i.) intercellular junction proteins are expressed normally in acantholytic skin areas of patients with Hailey-Hailey or Darier´s disease but the localization of ZO-1 expanded to the stratum spinosum; ii.) tight junction proteins, claudin-1 and -4, are regulated by ATP2C1 in non-differentiating keratinocytes; and iii.) p38 delta regulates the expression of tight junction protein ZO-1 in proliferating keratinocytes and in squamous cell carcinoma derived cells. ZO-1 silencing, however, did not affect the expression of other tight junction proteins, suggesting that they are differently regulated. This thesis introduces new mechanisms involved in the regulation of tight junctions revealing new interactions. It provides novel evidence linking intracellular calcium regulation and tight junctions.