Maspin: Breast Cancer Suppression through Enzyme Inhibition?

Institution: The Burnham Institute for Medical Research
Investigator(s): Jeffrey Smith, Ph.D. -
Award Cycle: 2004 (Cycle 10) Grant #: 10WB-0083 Award: $196,115
Award Type: STEP Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2004)
In 1994 a class II tumor suppressor gene called maspin was identified from patients with breast cancer. Although a decade of research on maspin has solidified its function as an anti-tumor protein, there are conflicting interpretations of the findings, and differing opinions about maspin's mechanism of action. Opinions differ on maspin's site of action (from the inside of the cell vs. the outside), on its biochemical activity (protease inhibitor vs. cell surface ligand), and even on its effects on tumors (tumor suppressor vs. metastatic gatekeeper). Here we seek to clarify maspin's mechanism of action. Our work on maspin has led us to another protein called the proteasome. The proteasome is found inside cells where it acts as a machine to degrade proteins. This process is a normal function in cells, as they must turnover older proteins. Recently though, the proteasome has been implicated in cancer. Our work connects these two seemingly unrelated regulators of tumor progression.

Our preliminary results show that maspin has very little impact on gene expression, but that is has widespread effects on protein expression. This is an unusual observation, but one that would be expected if maspin regulates the proteasome. Based on these results, we suggest that maspin's anti-metastatic activity results from its ability to regulate protein degradation by the proteasome. To test this hypothesis we propose to: 1) Measure the ability of maspin to directly inhibit protease activity of the proteasome in purified systems and in cells, and 2) Measure the effects of maspin on the compliment of proteins in cells that are marked for proteasome degradation by molecular tags.

One aspect of our study will make use of standard approaches in protein and cellular biochemistry. We will use these techniques to measure the direct effects of purified maspin on proteasome function, and to measure the effects of maspin on the proteasome in breast cancer cells. A separate aspect of the study will involve a highly sophisticated and innovative strategy toward surveying the effects of maspin on the cellular process that tags proteins for degradation by the proteasome. This tagging is called ubiquitination and we plan to study it with a technique called proteomics. Proteomics allows one to measure the effects of a chemical modification (like tagging with ubiquitin) on thousands of proteins at once. This methodology has not been used extensively in breast cancer research.

The proposal is innovate for three reasons: 1) the hypothesis is entirely novel and untested. It connects two seemingly unrelated proteins, maspin and the proteasome, 2) one of the approaches we plan to use to study the effects of maspin is called proteomics. This highly sophisticated and technical strategy allows one to measure small changes to protein structure, like their tagging for degradation. The approach makes use of mass spectrometry to assess changes across the entire complement of cellular proteins in one experiment. The power of proteomic analysis has not been widely applied to gain insight into breast cancer, 3) the results of the study are likely to suggest highly innovative avenues for drug design in breast cancer. If maspin suppresses breast cancer by acting on the proteasome, then this molecular target would become a new and validated target for therapeutic intervention in breast cancer.


Final Report (2005)
In 1994 the breast cancer metastasis suppressor gene called maspin was identified. Although a decade of research on maspin has solidified its function as an anti-metastasis protein, there are conflicting interpretations of the findings, and differing opinions about maspin's mechanism of action. Opinions differ on maspin's site of action (intracellular vs. extracellular), on its biochemical activity (protease inhibitor vs. cell surface ligand), and even on its effects on tumors (tumor suppressor vs. metastatic gatekeeper). The primary objective or our study was to clarify the mechanism by which maspin functions as a metastasis suppressor.

Our proposal was based on the fact that we had uncovered a novel connection between maspin and another protein, called the proteasome. The proteasome is a proteolytic machine that regulates the turnover of the vast majority of proteins expressed in the cell, and therefore controls the complement of proteins expressed in a cell. We discovered that maspin has a major impact on the composition and function of the tumor cell proteasome. Based on this exciting observation, we hypothesized that maspin's primary anti metastatic activity results from its ability to inhibit (and/or regulate) protein degradation by the proteasome.

Our study led to three important results. First, we found that cells expressing maspin also express a distinct form of the proteasome. Unlike the canonical proteasome, the maspin-associated proteasome sediments on sucrose gradients at 23S, as opposed to the 20S and 26S proteasomes described previously. Mass spectrometry has been used to compare the protein composition of the two forms of the proteasome, and major differences in the molecular structure have been defined. These findings indicate that there may be a "metastatic" proteasome" and a "non-metastatic" proteasome (present in maspin-expressing cells, and that there may be ways of selectively targeting proteasomes that drive metastasis.

Second, we have defined regions of maspin that are essential for its anti-metastatic effects. By using mutational analysis we found that residues within a region of the protein called the reactive site loop are necessary for the anti-metastatic function of maspin in vivo. Cells expressing the mutated forms of maspin exhibited high levels of metastasis, but cells expressing wild-type maspin are not metastatic. Therefore, we conclude that the reactive site loop of maspin is necessary for its anti-metastatic effects in vivo. This is the first demonstration that the reactive site loop is involved in the anti-metastatic effects of maspin in vivo.

Third, we have found that cells transfected with mutant forms of maspin express the canonical proteasome, and not the "non-metastatic" proteasome. These findings show provide an additional link between maspin's anti-metastatic effects, and its modulation of proteasome composition.

Altogether then, the Step Award has facilitated the opening of a new area of research linking maspin, the proteasome and breast cancer metastasis. The award has led to one published paper, and our studies funded with the award will lead to two additional papers along with a full grant proposal to study these important connections in metastatic disease.

Maspin alters the carcinoma proteome.
Periodical:FASEB Journal
Index Medicus: FASEB J
Authors: Chen EI, Florens L, Axelrod FT, Monosov E, Barbas CF 3rd, Yates JR 3rd, Felding-Habermann
Yr: 2005 Vol: 19 Nbr: Abs: Pg:1123-4