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 integrin-dependent 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, α3β1 and α6β4 integrins on epithelial cells bind to the ECM protein laminin 5 in the basement membrane. In tumor cells, however, the α3 and β4 integrin subunits disappear—as does laminin 5—and the tumor cells express primarily α6β1 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 α3β1 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 α3β1 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 α3β1 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 α3β1 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 α3β1 integrin is required for efficient autophagy induction. Because loss of c-Met reduces α3β1 integrin expression, autophagy induction is blocked in c-Met-inhibited cells. Preliminary data suggest that α3β1 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.
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 AR-negative 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 transcriptional activation and expression of α6β1 integrin, the receptor for laminin. Engagement of laminin by α6β1 integrin increases NF-κB activity and Bcl-xL levels, all of which are required for cell survival. 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.. 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 and it will require that both pathways be targeted therapeutically. We are currently determining how AR regulates the expression of α6 integrin.
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, which is dependent on both AR and Src. 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 have begun manipulating the cells by systematically introducing oncogenic mutations known to be associated with the development of prostate cancer. Our hypothesis is that activation of oncogenes during differentiation will cause a dependence on AR for survival, which will be mediated in part by elevated expression of α6β1 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.
Project 3: Role of CD82 in prostate cancer metastasis
Death from prostate cancer is due to the development of metastatic disease, which is difficult to control. CD82/KAI1 is a metastasis suppressor gene whose expression is specifically lost in metastatic cancer, but not in primary tumors. Interestingly, CD82/KAI1 (a member of the tetraspanin family) is known to associate with both integrins and receptor tyrosine kinases. Our goal has been to determine how loss of CD82/KAI1 expression promotes metastasis by studying the role of CD82/KAI1 in integrin and receptor tyrosine kinase crosstalk.
Mechanism of CD82 suppression of c-Met
We have found that reexpression of CD82/KAI1 in metastatic tumor cells suppresses laminin-specific migration and invasion via suppression of both integrin- and ligand-induced activation of c-Met. Thus, CD82/KAI1 normally acts to regulate signaling through c-Met; upon CD82 loss in tumor cells, signaling through c-Met is increased, leading to increased invasion. We are currently determining the mechanism by which CD82/KAI1 down-regulates c-Met signaling. So far our studies indicate that c-Met and CD82 do not directly interact, and CD82 may act to suppress c-Met signaling indirectly by dispersing the c-Met aggregates on metastatic tumor cells into monomers, thus blocking signaling. We have generated mutants of CD82 to determine which part of the CD82 molecule is required for suppression of c-Met activity. In addition, we have determined that reexpression of CD82 in tumor cells induces a physical association between CD82 and a related family member, CD151. Loss of CD151 prevents CD82 from suppressing c-Met. We are currently determining whether CD82/CD151 association with integrins is required to suppress c-Met.
CD82 control of metastasis and c-Met activation in vivo
We have initiated several mouse studies to determine the mechanism by which loss of CD82 promotes metastasis in vivo. The ability of DU145 prostate cancer cells to metastasize depends on activation of c-Met. Using transgenic SCID mice that express the human version of HGF (only human HGF will activate human c-Met), we have been able to demonstrate that DU145 tumor cells will metastasize only in the HGF/SCID mice, but not in regular SCID mice. Under these conditions, reexpression of CD82 completely suppresses metastasis and there is a dramatic reduction in c-Met activity in the tumors. Mutants that no longer suppress c-Met activity in vitro will be used to demonstrate that they are also no longer capable of suppressing metastasis in the HGF/SCID mice.
In addition, we have generated mice with conditional loss of CD82 expression in the prostate, as well as mice with complete CD82 loss in all tissues. These mice have been crossed with mice that develop only primary tumors (Pten conditional) in order to determine if the loss of CD82 is sufficient to induce prostate cancer metastasis. Thus, far none of the mice have developed metastases, suggesting that additional factors are required. Future studies will be aimed at determining what other genetic events are required to convert primary tumors to metastatic tumors. In addition, we are currently assessing other non-tumor-related phenotypes observed in mice in which CD82 is lost in all tissues.
