Growth and development of the prostate are regulated by androgens and mesenchymalepithelial interactions. Prostate growth starts in the embryo, continues throughout
neonatal life, and is completed at puberty. The male and female embryonic urogenital
sinus (UGS) both contain a prostate precursor, but due to the action of testosterone only
the male will develop a prostate. Testosterone acts in concert with paracrine signalling
from inductive mesenchyme to regulate epithelial growth and branching of the prostate.
The ventral mesenchymal pad (VMP) is an area of inductive mesenchyme that is rich in
regulatory growth factors of which few have been identified at present. Recently, the
role of mesenchyme/stroma in cancer has been established and developmental factors
may be involved in mediating the effects of cancer stroma. The aim of this thesis was to
identify mesenchymal factors and characterise their expression and function in the
developing prostate, and to investigate their expression in prostate cancer stroma.
The LongSAGE technique was used to generate a comprehensive transcriptional profile
of the neonatal rat prostate rudiment. LongSAGE is an open-ended and unbiased geneprofiling method. Two libraries were made from tissues in the female UGS; one library
was prepared from the whole female prostate rudiment and the other library was
prepared from the inductive mesenchyme (VMP) isolated from within the rudiment.
Several essential factors known to be involved in prostate development were identified
in the libraries, including low abundance cDNAs such as AR and FgflO. This suggested
that the approach had sufficient sensitivity to identify key mesenchymal factors. The
two libraries were compared, and the comparison was statistically analysed,
highlighting genes that were VMP-enriched (P<0.05). Candidate mesenchymal
transcripts were selected from the VSU and VMP Libraries by two different approaches,
either because of their status as VMP -enriched (P<0.05) transcripts, or by an 'intuitive'
approach, because the transcripts were associated with genes that are known members
of developmental pathways and/or have been associated with prostate cancer. The
expression and abundance of candidate transcripts were quantified by qRT-PCR in the
male and female neonatal UGS. Subsequently, candidate transcripts were verified as
VMP-enriched and were quantified during prostate development by Northern blot
analysis. The protein distribution of selected candidates were localised within the
neonatal rat UGS by immunocytochemistry, and the effect of testosterone treatment on
the protein distribution was studied. To test the function of one of the candidates on
prostate growth and development, recombinant protein was added to prostate rudiments
grown in vitro. Finally, candidate transcripts were investigated in human prostate
cancer associated fibroblast cells (CAFs) and normal prostate fibroblast cells (NPFs) by
RT-PCR and Northern blot analysis.
SAGE analysis, qRT-PCR and Northen blot analysis identified six candidate transcripts
as VMP-enriched; Dlkl, Notch2, Ptn Nell2, MMP2 and MMP14. The transcript
expression of each candidate was most abundant in the developing rat prostate during
the perinatal period. Proteins for Dlkl, Ptn and Notch2 were localised to mesenchymal
cells of the neonatal VMP and ventral prostate (VP). Ptn expression was also associated
with the basement membrane and cell-surface of the epithelial duct cells of the VP.
Treatment of VP organs with recombinant DLK1 in vitro increased the organ size and
epithelial branching. Also, PTN, NOTCH2, MMP2 and MMP14 transcript expression
was observed in CAFs and NPFs. PTN and NOTCH2 showed a decrease in CAFs
compared to NPFs suggesting a tumour-suppressive role.
In summary, a comprehensive gene profiling technique was used to identify
mesenchyme specific/enriched transcripts in the developing prostate. The expression,
distribution and function of candidate transcripts and proteins were investigated in the
developing prostate and in prostate cancer stroma.