Proteomic Profiling of Adhesive Structures in Breast Cancer
| Institution: | The Burnham Institute for Medical Research | ||
| Investigator(s): |
Jason Bush , Ph.D. -
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| Award Cycle: | 2004 (Cycle 10) | Grant #: 10FB-0115 | Award: $90,000 |
| Award Type: | Postdoctoral Fellowship | ||
| Research Priorities | |||
| Pathogenesis>Unraveling the path to breast cancer: tumor progression | |||
Initial Award Abstract (2004)
When normal breast cells in the breast undergo a change called transformation, they acquire a different appearance and become more mobile. Despite these characteristic changes, the factors controlling this process are still poorly understood. We know that the loss of adhesion both between cells and to the cell matrix plays a critical role. Cell adhesion is modulated by several multi-protein complexes that transmit signals from the outside of the cell to its interior. In breast cancer, one of these complexes is associated with normal cell behavior and forms a stable structure while the other is associated with a pre-cancerous state and forms a transient structure. Many of the proteins that exist in these complexes are known and common to both, but new factors are constantly recruited to these sites to accommodate changes in the environment. Our goal is to better define the components of these structures using cutting-edge approaches. We believe that a more comprehensive list of components will not only open up new avenues for therapeutic advances in breast cancer but also provide basic biological ideas for other cancer types. I hypothesize that the factors that drive the formation of stable adhesive structures are distinct from the factors that promote transient adhesion sites. I am asking the simple question: what determines the formation of one structure over another? Furthermore, I suggest that defining the composition of proteins found in each type of adhesive structure will provide a fundamental basis for their assembly, and will provide a menu of potential drug targets. I am using well-defined breast cancer cells and placing them on substrates that mimic the signals from the normal physiological environment. By stimulating the cells with a specific growth factor, I can force them to change the type of adhesion structure--we then have a 2-state system for analysis. Following this change, I am using a combination of detergents and mechanical disruption to remove the majority of the cell and leave a footprint of the adhesion structure which I can pool. Once collected, the samples are labeled with a reagent called ICAT that can be used for downstream analysis by mass spectrometry. The ICAT reagents will provide me with information about the expression levels of proteins in the form of a ratio. Ratios that differ significantly represent potential protein targets for further biochemical examination. Those targets that are increased in the transient adhesive structures would be considered pro-tumorigenic and so, we will use strategies to reduce the expression of these targets, thus converting the adhesive sites to a more stable structure associated with normal appearance. Our goal is to identify unique molecules causally involved in cell transformation. The proposed study has two innovative elements: (1) We are using state-of-the-art proteomic tools and software to label and analyze the samples, and (2) The new isolation procedures refined in this lab to investigate the biological process will provide more significant samples not previously obtained.
Final Report (2006)
Breast cancer metastasis can occur when tumor cells acquire the ability to move and migrate from the original tumor site to other organs. By necessity, these cancer cells have changed the way they interact with other cells and their immediate environment. A class of proteins called, integrins, help establish and regulate these cellular interactions. The overall goal of this proposal was to find new proteins that may interact with integrins in breast cancer model systems. The original cell system proposed was severely limited and did not allow for sufficient purification or enrichment of the integrin fraction. This technical hurdle was noted as a pitfall by the review panel and so, an alternative model system was developed and utilized. It became clear that a model system where integrin was either present or absent represented the best way of determining any differences in the kinds of proteins to be isolated. Using such a system combined with large-scale protein profiling technologies, we were able to generate a substantial dataset and then define and validate a new biochemical relationship between an integrin and a specific protease, a molecule often increased in breast cancer. We were able to extrapolate these findings to human cells that mimic the system and are now confirming the observations in certain breast cancer cell lines. The new observations created by this proposed project have directly led to a submitted manuscript and formed the basis for grant applications applying the cell biology that was learned. Understanding the biochemical relationship between these two molecules could help define some essential cell biology involved in cancer spread and may lead to new ways of intervening in breast cancer.
Symposium Abstract (2005)
Understanding the molecular events that occur at the cell membrane in cancer cells has received a tremendous amount of investigation. Cathepsin B (cathB) is a secreted protein that is often elevated in many kinds of carcinoma (breast, prostate, and colon) where it can be associated with the invasive front of a tumor cell or within adjacent supporting fibroblasts. An increase in another membrane protein called beta3 integrin is also often associated with aggressive tumor status and bone metastases in several cancer types. To explore the biological differences associated with beta3 integrin near the membrane, we first utilized cells from a mouse model which completely lacks all beta3 integrin. This was followed by various strategies to simplify the protein mixtures through multiple purification steps that included tagging techniques and proteomic analyses for identification of proteins that were present at different levels in mice with completely normal beta3 integrin (beta3+/+) and mice with no beta3 integrin (beta3-/-). By these stringent methods, we were able to identify 113 unique proteins from a partial membrane fraction, of which 16% of the proteins differed by a significant 2-fold change. Positive hits were clustered according to common biological functions, with proteases (protein-degrading enzymes) predominating. Specifically, we found that cathepsin B, which is both intracellular and secreted, was increased by over 3.5-fold in the beta3-/- cells. Additional studies further confirmed both the increased presence and activity of cathB in beta3-/- cells. Overabundance of a functional cathB chimeric protein in beta3-/- cells compared to beta3+/+ cells did not localize to the normal intracellular structures (the lysosomes) as expected, but presented a more diffuse staining that we associated with the internal protein sorting system of the cell (the Golgi apparatus). Supporting these results, secreted levels of cathepsin B were reduced in beta3-/- cells. Thus, our data suggests that beta3 integrin may be involved in the localization of cathB to the typical secretion structures or possibly trafficking to the membrane through the normal protein maturation process. This unexpected link between two markers of advanced breast cancer may provide insight for alternative therapeutic intervention.
