A Molecular Strategy to Inhibit Breast Cancer Metastasis

Institution: University of California, San Francisco
Investigator(s): Frances Brodsky, D.Phil. -
Award Cycle: 2009 (Cycle 15) Grant #: 15IB-0035 Award: $150,000
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2009)

A novel cellular pathway recently discovered in the principal investigator’s laboratory is likely to contribute to breast cancer metastasis. Research in the laboratory has focused for many years on a protein, called clathrin, and its role in membrane traffic. Clathrin facilitates the formation of small vesicles within cells. Clathrin-coated vesicles selectively sort materials to be transported within cells at the cell membrane, the internal Golgi network, and other internal cell compartments for “traffic control.” In the context of breast cancer, clathrin controls hormone uptake into the cell. Recent work in the laboratory has revealed that clathrin through its interaction with a second protein called Hip1 (Huntingtin Interacting Protein-1), also controls how cells move and can spread through the body. It is already known that the Hip1 protein is present in excess in certain types of breast cancer, and correlates with poor survival in patients with prostate cancer. We believe that excess Hip1 in breast cancer cells might be linked to increased spreading behavior. Our studies of clathrin and Hip1 also allow us to search for potential drugs that might block their interaction providing the basis for designing new breast cancer therapeutics.

First, our studies will quantify Hip1 expression at both the mRNA and protein levels in a panel of 51 breast cancer cell lines established by the Breast Cancer Special Program for Research Excellence (SPORE) under the auspices of Dr. Joe W. Gray (Lawrence Berkeley Labs and UCSF). Hip expression will be evaluated and correlated with other characterized properties of the cell lines (already published), including migratory behavior. Cell lines with reduced Hip expression will be tested for increased migratory behavior upon induced over-expression of Hip proteins. To validate whether elevated levels of Hip expression correlate with increased metastatic potential, experimental metastasis assays in mice will be performed with Dr. Andrei Goga (UCSF), using breast cancer cell lines engineered to express different levels of Hip proteins. In addition, the ability of these cell lines to form tumors in vivo will be assessed after subcutaneous injection. Other work in our lab will employ recombinant protein expression and peptide binding assays to develop an automated, high-throughput screen to identify small molecule inhibitors of Hip-clathrin interactions. Identification of effective small molecule inhibitors provides a starting point for further drug discovery.

This research project will investigate a novel pathway that has not yet been clearly established as playing a role in breast cancer, although research elsewhere has shown strong indications that clathrin and Hip proteins may play a substantial role in tumor biology processes related to cell movement.

Final Report (2011)

This project elaborates on our recent discovery of a role for the clathrin protein in cell migration. Cell migration plays a key role in breast cancer spread (metastasis). Clathrin has been studied in our laboratory for many years, but appreciating its role in migration is novel. Because of our knowledge about clathrin, we hypothesized that its function in migration involves interaction with a protein called Hip. The Hip protein has been documented to be present in excess in metastatic breast cancer cells. Our previous research demonstrated that interaction between Hip and clathrin might easily be prevented with small molecules and this proposal aimed to screen for such inhibitory molecules that could potentially be developed into therapeutic agents to block the breast cancer metastasis pathway involving clathrin-Hip protein interactions. Current treatments for breast cancer are particularly effective against metastatic tumors, so this project aims to redress that problem.

The aims of this project were first to validate our hypothesis that clathrin-Hip interaction plays a role in metastasis of breast cancer cells using cell culture and models of cancer metastasis in mice. The second aim was to identify small molecules that can inhibit the clathrin-Hip interaction. We have achieved about half of the first aim by validating a role for clathrin-Hip interaction in migration of breast cancer cells in cell culture. For these studies we we utilized siRNA knockdown of Hip1 and associated clathrin subunit proteins and analyzed the effect son cell migration (matrix interactions) and cell-cell interactions using fluorescent microscopy techniques. Essentially, these “wound healing” type assays duplicate the mechanistic properties for breast cancer cell metastatic migration. Next, we have also started to generate the tools needed to confirm these findings in an animal model (xenografts) of human breast cancer. We are about halfway towards setting up the screen for a small molecule inhibitor, as we have generated one of two components needed.

Barriers that were overcome included: 1) establishing a culture system to study breast cancer cell migration, and 2) improving methods for producing protein components for the small molecule screen. A barrier that was not foreseen is finding the right approach to manipulating Hip protein levels in cells to be used for the animal model work. While this needs more development work, it is certainly feasible. Future work in the lab will continue to develop the animal model for testing the effects of inhibiting clathrin-Hip interactions. We will also proceed with the small molecule screen, provided future support can be obtained.

Symposium Abstract (2010)

This study examines a novel cellular pathway as a potential target for breast cancer therapeutics. Clathrin is a major coat protein which sorts and transports materials at the cell membrane, the internal Golgi network and other internal cell compartments. Our recent work demonstrated that clathrin through its interaction with a second protein called Hip1 (Huntingtin interaction protein 1) also controls how cells move and spread. We found that cells depleted of clathrin or Hip1 are defective in movement. It is known that the Hip1 is present in excess in many breast cancer cells. Excessive Hip1 in breast cancer cells might be linked to their increased spreading behavior. To validate whether elevated levels of Hip1 expression correlate with increased metastatic potential, we will perform metastasis assay in mice using breast cancer cell lines expressing different levels of Hip1 and assess the tumorigenic ability. Small molecule inhibitors will be screened for competing with Hip1 and clathrin interaction using recombinant protein expression and peptide binding assays. Identification of effective small molecule inhibitors provides a starting point for further drug discovery.

Clathrin phosphorylation is required for actin recruitment at sites of bacterial adhesion and internalization.
Periodical:Journal of Cell Biology
Index Medicus: J Cell Biol
Authors: Bonazzi M, Vasudevan L, et al, Brodsky FM, Cossart P.
Yr: 2011 Vol: 195 Nbr: 3 Abs: Pg:525-36