Scientific Overview: Integrin Signaling and Tumorigenesis

Research Interests

Our laboratory is interested in 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.

Integrins are transmembrane proteins that serve as receptors for ECM proteins. By interacting with the ECM, integrins stimulate intracellular signaling transduction pathways to regulate cell shape, proliferation, migration, survival, gene expression, and differentiation. Integrins do not act autonomously, but “crosstalk” with receptor tyrosine kinases (RTKs) to regulate many of these cellular processes. Studies in our lab indicate that integrin-mediated adhesion to ECM proteins activates epidermal growth factor receptors EGFR/ErbB2 and the HGF/SF receptor c-Met. Integrin-mediated activation of these RTKs is ligand-independent and required for the activation of a subset of intracellular signaling molecules in response to cell adhesion.

The prostate gland and cancer

Tumors that develop from cells of epithelial origin, i.e., carcinomas, represent the largest tumor burden in the United States. Prostate cancer is the most frequently diagnosed cancer in American men and the second leading cause of cancer death in men. Patients who at the time of diagnosis have androgen-dependent and organ-confined prostate cancer are relatively easy to cure through radical prostatectomy or localized radiotherapy, but patients with aggressive and metastatic disease have fewer options. Androgen ablation can significantly reduce the tumor burden in the latter patients, but the potential for relapse and the development of androgen-independent cancer is high. Currently there are no effective treatments for patients who reach this stage of disease.

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 expression of the RTKs EGFR and c-Met in the tumor cells. Two fundamental questions 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 crosstalk with RTKs is important.

Integrins and RTKs in prostate epithelial cell survival

How integrin engagement of various ECMs regulates survival pathways in normal and tumor cells is poorly understood. We recently initiated studies to determine how adhesion to matrix regulates cell survival in normal epithelial cells. We have shown that integrin-induced activation of EGFR in normal primary prostate epithelial cells is required for survival on their endogenous matrix, laminin 5. The ability of EGFR to support integrin-mediated cell survival on laminin 5 is mediated through α3β1 integrin and requires signaling downstream to Erk. Surprisingly, we found that the death induced by inhibition of EGFR in normal primary prostate cells is not mediated through or dependent on classical caspase-mediated apoptosis. The presence of an autophagic survival pathway (Fig. 1), regulated by adhesion to matrix, prevents the induction of caspases when EGFR is inhibited. Suppression of autophagy is sufficient to induce caspase activation and apoptosis in laminin 5–adherent primary prostate epithelial cells. Thus, adhesion of normal cells to matrix regulates survival through at least two mechanisms, crosstalk with EGFR and Erk and the maintenance of an autophagic survival pathway (Fig. 2).

Figure 1. Induction of autophagy in primary prostate epithelial cells as shown by punctate staining of the autophagic LC3 protein using fluorescence microscopy.	Figure 2. Laminin-mediated survival pathways in primary prostate epithelial cells.

Interestingly, both of these pathways are absent in at least one metastatic prostate cancer cell line, PC3. Accordingly, integrin-mediated survival of PC3 cells does not depend on EGFR or Erk, but is instead dependent on PI-3K. The PI-3K pathway is inhibitory to autophagy. We are currently testing additional prostate tumor cells lines to determine if this switch in matrix-mediated survival pathways is found in all prostate cancers.

Our next step is to determine how integrins regulate survival through autophagy. Since loss of autophagy results in activation of caspases and classical apoptosis, we have been searching for signaling pathways whose inhibition also results in caspase activation. We have tentatively identified two important molecules, the RTK c-Met and the anti-apoptotic protein Bcl-XL. Inhibition of either molecule leads to caspase-induced cell death, indicating that they may be involved in regulating integrin-mediated autophagy. Future studies in our lab will be aimed at deciphering this pathway.

The androgen receptor in integrin-mediated survival

All primary and metastatic prostate cancers express the intracellular steroid receptor for androgen, AR. In the normal gland, the AR-expressing cells do not interact with the ECM in the basement membrane; however, all AR-expressing tumor cells do adhere to the ECM in the basement membrane. 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 switch 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 switch in AR function.

When prostate tumor cells are placed in culture, they lose expression of AR. The reason for this is not clear, but it may have to do with loss of the appropriate ECM-containing basement membrane. When we introduce AR into prostate tumor cells, it actually suppresses their growth and induces cell death. However, if we place the AR-expressing tumor cells on laminin (the ECM found in tumors), these cells no longer die. The mechanisms responsible for this change in survival are unknown. Preliminary studies indicate that there are changes in integrin expression upon expression of AR that may enhance specific signaling pathways when those integrins bind to matrix. We are currently determining which cell survival pathways are activated by AR upon integrin engagement.

CD82 and integrin signaling in prostate cancer metastasis

Death from prostate cancer is due to the development of metastatic disease, which is difficult to control and occurs by mechanisms that are not understood. One approach we are taking is to characterize genes that are specifically associated with metastatic prostate cancer. CD82/KAI1 is a metastasis suppressor gene whose expression is specifically lost in metastatic cancer but not in primary tumors. Interestingly, CD82/KAI1 is known to associate with both integrins and RTKs. Our goal has been to determine how loss of CD82/KAI1 expression promotes metastasis.

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 the RTK c-Met. Interestingly, c-Met is often overexpressed in metastatic prostate cancer. Thus, CD82/KAI1 normally acts to regulate signaling through c-Met such that 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. Our studies indicate that c-Met and CD82 do not directly interact, and CD82 may act to suppress c-Met signaling indirectly by dispersing c-Met aggregates present on metastatic tumor cells. We have developed mutants of CD82 in order to determine which part of the CD82 molecule is required for suppression of c-Met activity. Also, we have determined that reexpression of CD82 in tumor cells induces a physical association between CD82 and a related family member, CD9. We are determining whether this association is important for suppressing c-Met activity.

We have also initiated mouse studies to demonstrate the importance of CD82 in regulating metastasis in vivo. Using orthotopic injection of wild-type or CD82-expressing metastatic prostate tumor cells directly into the prostate, we found that CD82 also suppresses metastasis in vivo. We are continuing these studies to determine if CD82’s ability to specifically affect c-Met is responsible for metastasis suppression. In addition, we are generating mice in which CD82 expression is specifically lost in the epithelial cells of the prostate gland. This approach will allow us to determine if CD82 is important for the normal biology of prostate epithelial cells in vivo. Furthermore, we will be able to determine if loss of CD82 in the mouse prostate gland will lead to an increased ability to produce metastatic prostate cancer.

External Collaborators

• Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington
• Senthil Muthuswamy, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
• Ilan Tsarfaty, Tel Aviv University, Israel
• Valera Vasioukin, Fred Hutchinson Cancer Research Center, Seattle, Washington
• Xin Zhang, University of Tennessee, Memphis