Research Grants Awarded
The Role Of Giv/Girdin And G Proteins In Metastatic Progression Of Breast Cancer
Tumor metastases relies heavily on efficient cell migration. In fact, migration is a prerequisite for metastasis and there is a correlation between the metastatic and the migratory potentials of a tumor cell. In this context, Akt (aka PKB) has been shown to be not only a prosurvival protein, but also a positive regulator of cell migration. In fact, Akt1 is an oncoprotein frequently hyperactivated in human cancers; it has been recently reported that Akt1 activation is upregulated in breast cancer cell lines representing advanced stages of metastasis and that it is required for breast cancer progression in vivo. Akt is already a target for chemotherapy drug development. In 2005, GIV (aka Girdin) was identified simultaneously by 4 different groups, which claimed interactions of GIV with different cellular components including Akt. GIV was shown as an Akt enhancer with promigratory functions. We showed that GIV binds the trimeric G protein Gai3, and serves as master switch in the control of cell migration. Additionally, we have observed that this interaction also controls migration of highly metastatic breast cancer cell lines (i.e. MDA-MD-231). Depletion of GIV from these cells dramatically impaired Akt activation and migration. We also tested 3 breast cancer cell lines with variable in-vivo metastatic potential for GIV expression, and we found that GIV is expressed exclusively in tumor cells that had high in-vivo metastatic potential. An identical pattern (i.e. expression of GIV correlated with higher metastatic potential) was recognized among 7 other tumor cell lines from colon and skin. Thus we have discovered two new regulators of the Akt pathway that control breast cancer metastases, providing a new attractive target in breast cancer research.
We hypothesize that expression of GIV provides the Gai3-GIV molecular switch that endows breast cancer tumor cells with increased metastatic potential via enhancement of Akt signaling and cell motility. We postulate that manipulations directed towards the disruption of the Gai3-GIV interaction will lead to a reduction of the breast cancer invasiveness.
Validation of GIV as a biomarker of risk for breast cancer metastasis and understanding of the molecular basis by which GIV and Gai3 interact to control cell migration and breast cancer metastasis.
SA#1. To Investigate Whether GIV Expression Correlates with the Stage of Breast Cancer.
Our results already indicate that GIV is expressed exclusively in those breast cancer cell lines with high in-vivo metastatic potential. Now we plan to investigate if GIV expression is upregulated in the 21T series of breast mammary epithelial cells, which represent different stages of breast cancer (normal to highly metastatic) from the same patient. We will also use a combination of immunohistochemistry and FISH mRNA on human tumor samples to investigate if GIV expression correlates with the clinical staging of breast cancer and the GIV intratumoral pattern of expression. Finally, we will also use PCR techniques on poorly metastatic breast cancer cell lines in order to look for the mechanism of silencing of GIV in the tumor cells that migrate/metastasize poorly.
SA#2. To Elucidate if GIV is Necessary and Sufficient to Promote Breast Cancer Metastases.
MDA-MB-231 breast tumor cells will be stably depleted of GIV and MCF7 cells will be stably transfected with GIV. These cell lines will be compared with their untransfected counterparts in two different scenarios: 1) in tissue culture, where Akt activation and invasive migration will be studied using TransWell chambers, and 2) in athymic mouse models, where the metastatic colonization of distal tissues after mammary fat pad inoculation will be evaluated.
SA#3. To Investigate how GIV and Gai3 Cooperate and Co-regulate each other During Breast Tumor Cell Migration and Metastasis.
We will search for the structural motifs responsible for the interaction between GIV and Gai3. After identifying these motifs we will generate GIV mutants unable to interact with Gai3 and will 1) directly observe the effect of expressing these mutants on cell migration and Gai3 localization by live cell imaging and 2) assess their ability to enhance Akt activation, migration in tissue culture and metastatic colonization of distal mouse tissues after mammary fat pad inoculation.
Research outcomes and clinical impact:
The impact of this research on the reduction of breast cancer mortality caused by cancer metastasis can be summarized in the two following points:
1-GIV as a Biomarker: If expression of GIV in a tumor cell positively impacts its ability to metastasize, GIV is a candidate oncoprotein that can serve as a biomarker for identifying patients at risk for breast cancer metastases. In the clinical setting, this may help in decision making whether a patient with seemingly ?limited disease? but GIV positive tumor should receive adjuvant therapy to treat micrometastases before they clinically manifest as metachronous metastases.
2-GIV as a Therapeutic Target: The PI3K-Akt pathway is critical in tumor initiation and progression, being already a molecular target for drug development. GIV is an enhancer of Akt and is essential for cell migration and our results indicate that its functions are regulated by the G protein Gai3. Although the proposed work is in an early stage regarding the development of a pharmacological tool, the generation of a novel anti-metastatic drug based on the outcome of this research should be plausible by the end of the funding period.
The American Cancer Society estimates that ~170,000 new cases of breast cancer will be reported in 2007 in the United States and ~40,000 patients will die. The majority of deaths are due to development of tumor metastases; for localized tumors, the 5-year survival rate exceeds 95%, whereas for metastatic breast cancer, it is less than 30%. Cancer metastases signify systemic, progressive, and essentially incurable disease where palliation becomes the theme of care. Metastasis is a multistep process that requires tumor cells to migrate into the lymphatic or blood vasculature, become blood borne and lodge in another organ where they invade and grow. A prerequisite to metastasis is efficient cell migration and it has been shown that cancer cells of highly metastatic tumors exhibit high-velocity migration as a result of chemotaxis (i.e., movement in the direction of stimuli). During chemotaxis, signals from outside the cell are perceived by direction-sensing receptors and relayed via exceedingly complex molecular assemblies to the interior to initiate events necessary for efficient directional motility.
Among the molecular components required in migration, it was described in 2005 that a protein called Girdin was necessary for cell migration. We discovered the same protein that year in a different context and gave it the name, GIV. We defined GIV/ Girdin based on its binding to the heterotrimeric G alpha-subunit, G(alpha)i3 and later demonstrated that the interaction between these to proteins work as master switch in cell migration: cells migrate or not depending on whether these two molecules interac or not. This ?switch? is conserved across many cell types, including human cervical, colon and breast cancer cells. When we eliminated GIV from highly metastatic breast tumor cells, these cells failed to migrate in tissue culture. we believe that GIV is a key regulatory element in all migration and not ?just another protein implicated in cell migration?. We examined the levels of expression of GIV in three different breast cancer cell lines and observed that only those with high metastatic potential expressed this protein, whereas in those with poorly metastatic potential GIV was virtually absent. Noteworthy, we found this same correlation after analyzing another seven cancer cell lines from colon and skin, i.e. GIV expression was always high in cells with known high metastatic potential. Our data so far indicates that we are dealing with a novel target in breast cancer metastasis research.
We hypothesize that when a breast tumor cell express GIV they acquire the ability to ?activate? the switch formed with G(alpha)i3 and increase the potential of these cells to migrate and metastasize. Therefore, GIV could serve as a biomarker to identify breast cancer patients with high risk of developing metastasis, which would provide valuable information in the clinical decision for a more effective treatment in early stages of the disease. In addition, understanding the basis of how the ?switch? of GIV with G(alpha)i3 is formed will establish the first step in the generation of a novel anti-metastatic drug. Novel chemical approaches are available based on how the molecular interaction of two proteins is governed that allow the rapid generation of synthetic drugs. Our objectives are to validate GIV as a biomarker of risk for breast cancer metastasis and understand the molecular basis by which GIV and G(alpha)i3 interact in order to better control cell migration and breast cancer metastasis.
The way we plan to achieve these goals is briefly described next. We will refine our observation that GIV expression correlates with metastatic potential in different cell lines by analyzing the expression in breast tumor cells of different stages (normal to highly metastatic) from the same patient. The next step is to extend this study to a larger number of histological human samples of breast cancer in different stages. Another critical point we need to address is whether GIV is necessary and sufficient to promote breast cancer metastasis. To this end we will remove or incorporate this protein into breast cancer tumor cells and study how these modifications affect the ability of these cells to metastasize in mouse models. We have already obtained preliminary information that cells without GIV are less metastatic and cells containing GIV are likely to develop a more aggressive breast cancer. Finally, we will use ?in vitro? techniques to discover how GIV and G(alpha)i3 interact at the molecular level. This will allow us to design modifications on GIV to disrupt the interaction with G(alpha)i3 and test if under these conditions breast tumor cells fail to metastasize.
The research proposed would set up a novel approach in the treatment of breast cancer metastasis through the new target we have discovered. The outcomes and impact on the reduction of breast cancer mortality can be summarized in the next two main points:
1-GIV as a Biomarker: If expression of GIV in a tumor cell positively impacts its ability to metastasize, GIV is a candidate oncoprotein that can serve as a biomarker for identifying patients at risk for breast cancer metastases. In the clinical setting, this may help in decision making whether a patient with seemingly ?limited disease? but GIV positive tumor should receive adjuvant therapy to treat metastases in early stages.
2-GIV as a Therapeutic Target: Although the proposed work does not propose the development of a specific pharmacological tool, the generation of a novel anti-metastatic drug based on the outcome of this research should be followed rapidly.