2009 Scientific Report

VARI | 2009
Project 2: Integrin and AR relationships in prostate cancer
All primary and metastatic prostate cancers express the androgen receptor (AR), and in late-stage disease it is often amplified
or mutated. In the normal gland, the AR-expressing epithelial cells do not interact with the ECM in the basement membrane;
however, all AR-expressing tumor cells do have such interactions. In normal cells, AR expression suppresses growth and
promotes differentiation, but in tumor cells AR expression promotes cell growth and is required for cell survival. The mechanisms
that lead to the change from growth inhibition and differentiation to growth promotion and survival are unknown. Our
hypothesis is that adhesion to the ECM by the tumor cells is responsible for driving the change in AR function by initiating
crosstalk between AR and integrins.
AR and integrin-mediated survival signaling in prostate tumor cells
Adhesion of PC3 metastatic prostate cancer cells to laminin and treatment with PI-3K inhibitors induces cell death. However,
we found that reexpression of AR prevented that cell death in an androgen-independent manner. We have determined that
AR expression results in increased expression of a6b1 integrin, the receptor for laminin. In addition, there is an increase in
Bcl-xL levels. The increase in Bcl-xL is dependent on a6 integrin, and both integrin and Bcl-xL are dependent on AR. Thus,
AR-expressing tumor cells are likely to survive better when they remain adherent to the laminin-rich ECM that is present in the
prostate gland. Survival under these conditions appears to depend on the ability of AR to enhance expression of the laminin
receptor, a6b1 integrin, which in turn stimulates Bcl-xL expression. These findings have broad implications for therapies
specifically targeting the PI-3K pathway, in that AR-expressing cells may harbor an alternative survival pathway via integrins.
We are currently determining how AR regulates the expression of a6 integrin and whether the transcriptional function of AR is
required for the survival phenotype.
AR-expressing cells also have elevated Src activity. Loss of Src did not impact cell survival, but these cells display increased
cell adhesion, spreading, and migration. Future studies will be aimed at determining if these cells are also more aggressive in
our in vivo metastasis models and if AR is responsible for controlling this. The survival signaling pathways observed in vitro will
also be tested in our metastasis animal models.
AR and integrin crosstalk in primary prostate epithelial cells
Our ability to understand AR function in tumor cells relative to normal cells is hampered by the lack of a cell culture model in
which normal cells naturally express AR. We sought to solve this problem by identifying the conditions necessary to induce
the differentiation of normal human prostate basal epithelial cells (which do not express AR) into secretory AR-expressing
cells. Combined treatment of confluent monolayers of human basal prostate epithelial cells with KGF and DHT stimulates the
production of a second layer of cells, analogous to a stratified epithelium. The upper-layer cells express epithelial differentiation
markers, AR, and AR-regulated genes, but no longer express integrins or basal cell markers. The upper secretory cell layer can
easily be dissociated from the bottom basal cell layer and analyzed biochemically. Because integrins are no longer expressed
in the secretory cells (as seen in vivo), we sought to determine how these cells survive. We found a dramatic increase in
E-cadherin expression in the differentiated cells. The secretory AR-positive cells no longer rely on integrin or integrin-activated
signaling pathways such as EGFR/c-Met/Erk; they now depend on E-cadherin and PI-3K signaling for their survival. Also, as
has been demonstrated in in vivo models, these cells do not need androgen or AR for survival.
Now that we have established a working differentiation model, we are poised to manipulate the cells by systematic introduction
of oncogenic mutations known to be associated with the development of prostate cancer. As proof of concept, we are also
capable of inducing the differentiation response in our immortalized PECs. Our hypothesis is that activation of oncogenes
during differentiation will cause a dependence on AR for survival, which will elevate a6b1 integrin.
Thus we have established two different models for studying AR in prostate cells. In both models the expression of AR has a
major impact on integrin expression and function, indicating there is significant “crosstalk” between integrins and AR.
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