Breast cancer that lacks expression of the estrogen receptor (ER), progesterone receptor(PR), and the epidermal growth factor receptor 2 (Her2/Neu/ErbB2) is known as the triple negative subset of basal-like breast cancers. There are currently no known targeted therapies for this disease. These cancers are characterized by a high propensity to metastasize due to the dysregulation of factors contributing to cell migration, invasion and survival. In addition, triple-negative breast cancers commonly harbor mutations of p53. It has been shown recently that mutant p53 contributes to the metastasis of cancer cells by multiple mechanisms, all of which require further study in the triple negative setting. Triple-negative breast cancer cell lines show characteristically low expression of the p53 inhibitors HDM2 and HDMX. Because p53 binds to these proteins via its transactivation domain and not the commonly mutated DNA binding domain, we are currently using a stapled alpha helical peptide against the transactivation domain (SAH-p53) to study the regulation of mutant p53 in the triple-negative breast cancer cell line MDA-MB-231. The MCF7 cell line that expresses wild-type p53 and high levels of the inhibitory proteins is used as a positive control for peptide activity. Preliminary experiments have showed an inhibition of MDA-MB-231 cell migration and invasion through Matrigel with 5-10 uM of the SAH-p53 peptide in the presence or absence of epidermal growth factor (EGF) used as a chemoattractant. In addition, MTT and trypan blue assays have shown a dose-dependent decrease in mitochondrial metabolism and viability, respectively, suggesting that the effects of SAH-p53 in triple-negative cells are independent of the transcriptional activity of p53. Interestingly, the MTT and trypan blue data also held true for a p53-null triple-negative breast cancer cell line MDA-MB-157. The effects of SAH-p53 on the migration and invasion properties on the p53-null line are currently underway and have the potential to confirm a p53-independent effect of the peptide on the metastatic potential of triple-negative cells. The observed effects warrant investigations into the signaling pathways affected by SAH-p53 in triple-negative lines. We have established that the canonical p53 transcriptional pathway that is induced in MCF7 cells is not induced by SAH-p53 in MDA-MB-231 cells. We are currently attempting to pinpoint the pathways that are altered in the mutant cells. We are investigating the effect of SAH-p53 on events resulting from alterations in p63/p63 activity and modulation of sterol biosynthesis, both of which are known to have roles at the mitochondria and in cell migration, though this has not been well characterized. The fact that effects of SAH-p53 are as strong in cells that overexpress HDM2 or HDMX as in cells with very low levels suggests that SAH-p53 may have a secondary target or these proteins have other binding partners that elicit this response. To this end, we have designed biotinylated photoactivatable SAH-p53 peptides that are used to cross-link the peptide to its target in the presence of UV light. These peptides can be used in conjugation with strepavidin beads to elute target proteins. We have established that these peptides successfully bind to and can be cross-linked to HDM2, and the work on HDMX is underway. We are currently exposing whole protein lysates from MDA-MB-231 and MCF7 cells to these peptides and have preliminary results suggesting that they do cross-link to multiple proteins. Experiments in the near future will determine the identity of these proteins. In addition, a stable HDMX shRNA MDA-MB-231 isogenic cell line is being created to determine the necessity of HDMX in the response to the SAH-p53 peptides.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011376-03
Application #
8763478
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2013
Total Cost
$289,804
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code