Development and characterization of sequential prostate cancer in vitro model

Abstract

Prostate cancer (PCa) is the second most common type of cancer in men globally, and the fourth most common type of cancer overall. PCa is mostly diagnosed in older men, to whom it arises as a result of sporadic mutations collected over time. Most newly discovered PCa cases are indolent, and do not require immediate medical intervention, but as the cancer progresses, its mortality rate dramatically increases. PCa is characterized by its dependence on androgens, the male sex hormones that normally direct the development and growth of the prostate. Androgen deprivation therapy (ADT) is the most efficient treatment option for advanced PCa, but the cancer can adapt to lose its androgen dependency. This adaptation arises from genetic alterations of androgen receptor (AR), which becomes activated when androgen, such as dihydrotestosterone (DHT), binds to the receptor. Alterations that increase AR expression or activation drive progression of PCa, and alterations that make AR activate without androgen ligand drive disease progression. Other PCa driving genetic alteration commonly found is the genetic fusion of promoter region of AR regulated gene, such as TMPRSS2, and oncogene ERG (TMPRSS2-ERG). This fusion is typical for early-stage premalignant lesions, that precede PCa. Another important aberration is the loss of PTEN tumor suppressor gene, often caused by gene deletion, leading to increased PCa cell proliferation.

Due to its prevalence, PCa has been the subject of much research. Majority of the re search has focused on disease progression, and thus there is a lack of knowledge about the earliest steps of PCa establishment. This lack of information is partly caused by the lack of good models to study PCa carcinogenesis, as most of the used cell lines are derived from progressed disease. To address this limitation, a cell line model using non-cancerous RWPE-1 prostate epi thelial cells has been established. This cell model has been modified with three genetic alterations of interest, so that the model can serve as a basis for studying prostate carcinogenesis. In this work, the model was further characterized by studying the produced sub cell lines: AR expressing RWPE-1-AR, AR and ERG expressing RWPE-1-AR-T2E, one with heterozygous PTEN deletion RWPE-1-AR-T2E-PTEN+/-, one with heterozygous PTEN but no ERG expression RWPE-1-AR PTEN+/-, and one with homozygous PTEN deletion but no ERG expression RWPE-1-AR-PTEN-/-. The model is missing a cell line with AR and ERG expression with homozygous PTEN deletion, thus this cell line was tried to be produced by using CRISPR/Cas9 technique on the heterozygous PTEN deletion line. Ultimately this was unsuccessful, as all created clones still expressed PTEN.

Previous studies have shown that DHT has a negative effect on the proliferation of this cell model, and this effect was further studied by inspecting how DHT influences the cell cycle. It was shown that DHT arrests cell cycle in the G1-phase, and the effect is apparent even before 24h timepoint. DHT does not seem to make cells apoptotic or senescent. The effect is likely me diated through AR signaling, which drives the cells to differentiate, explaining why cells cease to grow and divide. ERG expression is strongly induced by DHT, although the exogenous TMPRSS2-ERG fusion gene in the model is not under direct AR regulation. Understanding the DHT-responsiveness of ERG expression requires additional studies. While the development of the model has had unexpected challenges, the RWPE-1 model has shown its value for modeling early PCa carcinogenesis. The model still needs more optimization and characterization, but when completed, it can prove to be a excellent tool for PCa research.