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2009 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Research Interests Our laboratory is interested in understanding the mechanisms by which integrin receptors, interacting with the extracellular matrix (ECM), regulate cell processes involved in the development and progression of cancer. Using tissue culture models, biochemistry, molecular genetics, and mouse models, we are defining the cellular and molecular events involved in integrindependent adhesion and downstream signaling that are important for prostate tumorigenesis and metastasis. Project 1: Integrin crosstalk in normal and tumor prostate epithelial cells In the human prostate gland, a3b1 and a6b4 integrins on epithelial cells bind to the ECM protein laminin 5 in the basement membrane. In tumor cells, however, the a3 and b4 integrin subunits disappear—as does laminin 5—and the tumor cells express primarily a6b1 and adhere to a basement membrane containing laminin 10. There is also an increase in the expression of the receptor tyrosine kinases EGFR and c-Met in tumor cells, and our laboratory has demonstrated that integrins cooperate with these receptors. Two fundamental questions in our lab are whether the changes in integrin and matrix interactions that occur in tumor cells are required for or help to drive the survival of tumor cells, and whether integrin cooperation with EGFR or c-Met is important for that cell survival. Integrins and RTKs in prostate epithelial cell survival By interacting with the ECM, integrins stimulate intracellular signaling transduction pathways that regulate cell shape, proliferation, migration, survival, gene expression, and differentiation. Integrins do not act autonomously, but “crosstalk” or cooperate with receptor tyrosine kinases (RTKs) to regulate many of these cellular processes. Published studies from our lab indicate that integrin-mediated adhesion to ECM proteins activates the epidermal growth factor receptors EGFR/ErbB2 and the HGF/ SF receptor c-Met. We have shown that integrin-mediated activation of these receptors is ligand-independent and is required for integrin-mediated cell survival of prostate epithelial cells. However, the mechanisms by which the RTKs cooperate with integrins to regulate survival are different. The ability of EGFR to support integrin-mediated cell survival of normal primary prostate epithelial cells (PECs) on their endogenous matrix, laminin 5, is mediated through a3b1 integrin and requires signaling downstream to Erk. Disruption of this pathway leads to a caspase-independent mechanism of cell death resembling senescence/differentiation. On the other hand, loss of c-Met results in classic apoptotic cell death. Surprisingly, we found that c-Met regulates integrin-mediated survival by stabilizing a3b1 integrin expression and that regulation of integrin expression by c-Met occurs independently of its kinase activity. We are mapping the domains on c-Met that are required to rescue a3b1 integrin expression. The hypothesis being tested involves a potential scaffolding function of c-Met in suppressing the function of a cell surface death receptor called Fas and preventing the loss of a3b1 integrin and induction of death. Integrin control of the autophagy survival pathway During these studies, we also discovered that growth factor–deprived PECs adherent to laminin 5 robustly activate the autophagy survival pathway. Disruption of this pathway leads to apoptotic cell death, and a3b1 integrin is required for efficient autophagy induction. Because loss of c-Met reduces a3b1 integrin expression, autophagy induction is blocked in c-Met-inhibited cells. Preliminary data suggest that a3b1 integrin regulates the assembly of autophagosomes. Future work will be focused on identifying which molecules in the autophagy pathway are controlled by integrins. Our hypothesis is that under starvation conditions, integrins regulate the assembly of a FAK/FIP200 complex that controls autophagy. It is quite controversial as to whether inhibition or augmentation of autophagy is required for tumorigenesis and metastasis. Interestingly, immortalization of PECs or fibroblasts completely blocks the ability of autophagy-inducing stimuli to induce autophagy. In PECs immortalized by HPV E6/E7, we discovered a dramatic increase in PI-3K activity, which is known to inhibit autophagy via activation of mTor. However, blocking this pathway failed to restore the autophagic response. E7 is known to inhibit PP2A, and PP2A is required for autophagy induction in yeast downstream of mTor, but its role in mammalian cells has not been investigated. We will be following this line of investigation in both the virally immortalized PECs and in spontaneously immortalized cells. Ultimately, the effect of introducing prostate-specific oncogenes into PECs on the autophagy response will be analyzed. 36

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. 37

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