The long-term objective is to develop a new treatment for breast cancer based on blockade of the autocrine growth factor activity of procathepsin D. Breast cancer cells secrete procathepsin D, the zymogen from which the aspartic proteinase cathepsin D is generated by removal of an activation peptide (APpCD). Procathepsin D has been identified as an independent prognostic factor in breast cancer. In preliminary experiments, procathepsin D was found to act as a specific autocrine growth factor for breast cancer-derived cells, but not for any other cell type tested. These effects were mediated through a new, previously unknown specific receptor moiety expressed on breast cancer cell lines. The region of procathepsin D responsible for its mitogenic activity was localized in position 36-44 of the APpCD sequence. No growth factor activity could be shown with the mature enzyme cathepsin D. The proposed specific aims are based on the central hypothesis that procathepsin D is involved in breast cancer via a specific receptor that mediates autocrine activation for increased metastatic growth.
For Aim lit is hypothesized that the overproduction of procathepsin D results in an increase in the metastatic potential of breast tumor cells. A low metastatic human breast cancer cell line will be transfected with human procathepsin D cDNA such that the cells will secrete constitutively varying amounts of procathepsin D. The metastatic potential of each transfected cell line will be evaluated both in vitro and in vivo in relationship to the amount of procathepsin D secretion. In addition, the synthesis of pCD will be inhibited using specifically constructed ribozymes. Attempts will be made to determine the exact site in procathepsin D responsible for breast cancer cell growth factor activity. Synthetic peptides representing fragments of APpCD will be prepared. Amino acid substitutions in the most active peptide fragment will be used to map the essential amino acid contact sites for the receptor.
For Aim 2 attempts will be made to identify the membrane receptor for procathepsin D. A synthetic peptide representing the binding site domain of procathepsin D will be used to isolate candidate receptor molecules.
For Aim 3 it is hypothesized that inhibition of the APpCD interaction with its receptor will result in inhibition of cancer cell growth. Peptide analogs or complementary peptides will be prepared with D-amino acids to block the growth and malignancy of cancer cells both in vitro and in vivo. The overall goal is to generate a pharmacological agent for breast cancer based on blockage of the autocrine growth factor activity of pCD.
| Benes, Petr; Vetvicka, Vaclav; Fusek, Martin (2008) Cathepsin D--many functions of one aspartic protease. Crit Rev Oncol Hematol 68:12-28 |
| Ohri, Sujata Saraswat; Vashishta, Aruna; Proctor, Mary et al. (2008) The propeptide of cathepsin D increases proliferation, invasion and metastasis of breast cancer cells. Int J Oncol 32:491-8 |
| Vashishta, Aruna; Saraswat Ohri, Sujata; Vetvickova, Jana et al. (2007) Procathepsin D secreted by HaCaT keratinocyte cells - A novel regulator of keratinocyte growth. Eur J Cell Biol 86:303-13 |
| Vashishta, Aruna; Ohri, Sujata Saraswat; Proctor, Mary et al. (2007) Ribozyme-targeting procathepsin D and its effect on invasion and growth of breast cancer cells: an implication in breast cancer therapy. Int J Oncol 30:1223-30 |
