Cathepsin d projects
We established that procathepsin D as an enzymatically inactive zymogen acts as an autocrine growth factor specific for breast, lung and prostate cancer-derived cells. Furthermore, the growth of cancer cells in vivo could be inhibited by anti-procathepsin D antibodies. This function was mediated through a specific receptor expressed on all human cancer cell lines examined. This receptor was found to be distinct from the well known mannose-6-phosphate receptor, which has been shown to function as the cathepsin D receptor in most other systems. The parts of the procathepsin D molecule responsible for its mitogenic activity were localized near to amino acids 27-44 of the activation peptide sequence. In all our experiments, the mature enzyme cathepsin D had no role in metastasis. Accordingly, the current research is focused entirely on the biological activity of procathepsin D. When we inhibited secretion of procathepsin D by genetic manipulation (antisense, ribozyme or RNA interference), we significantly inhibited the breast and lung cancer development. Based on above mentioned data, we proposed a model of mechanism of procathepsin D action. The over-expressed procathepsin D escapes normal targeting pathways and is secreted out of the cancer cells. Subsequently, procathepsin D interacts with surrounding proteins and is recognized via its activation peptide part by a yet unidentified cell surface receptor. This interaction release a signal (e.g. activation of MAPK pathway) that results in differential expression of cancer promoting genes including various cytokines that in turn stimulate tumor growth.
One of the promising tumor markers in breast cancer is procathepsin D. Many clinical studies revealed an association between its level and prognosis, incidence of metastasis, and aggressiveness in a variety of solid tumor types, with most research done on breast cancer. Major studies and one meta-analysis found that procathepsin D levels in tumor homogenates represent an independent prognostic factor. Fully mature enzyme cathepsin D, originally suggested as a prognostic/diagnostic marker was later found to have questionable value, as several several forms of cathepsin D – inactive precursor procathepsin D, enzymatically active intermediate (single chain) cathepsin D and mature (two chains) cathepsin D are simultaneously present in and around cancer tissue, with detecting antibodies not distinguishing among them. In addition, several forms are also present in stromal cells resulting in questionable quantification in tumor tissues and consequently its prognostic significance.
The current situation in biomarkers in breast cancer is not satisfactory. The only validated serum biomarkers including carcinoembryonic antigen, CA15.3 and CA27.29, do not have sufficient sensitivity for detection in early diagnoses. Validation of several suggested markers such as BC1, BC2, BC3 or inter-alpha trypsin inhibitor H4 or M/z 4292 gave contradictory results. In addition, the use of SELDI does not really represent an easy and inexpensive assay.
The existence of autoantibodies in cancer patient serum is well established. These autoantibodies produced by the patient's immune system upon exposure to or tumor-related molecules are emerging as promising biomarkers for the early detection of cancer. These antibodies are specific, secreted in adequate quantities despite the presence of a relatively small amount of the corresponding antigen and most of all, are present even before first clinical signs. In addition, these antibodies have persistent concentrations and long half-lives. However, attempts to use them as diagnostic markers were mostly unsuccessful most possibly due to the fact that the sensitivities to these autoantibodies to individual tumor-specific antigens are not sufficient to establish a reliable diagnostic test.
Recently, a new possibility to use the current knowledge of association of the procathepsin D with various types of cancer emerged. The major advantage is the presence of procathepsin D in the plasma of breast cancer patients). As procathepsin D is under normal physiological conditions found only inside the cells, parts of this molecule are considered non-self and able to induce antibody response. Research conducted in our laboratory revealed the formation of anti-procathepsin D autoantibodies, which was confirmed by other laboratories. These autoantibodies are specific to procathepsin D and do not interact with the mature enzyme cathepsin D, making it easier to distinguish between these two molecules. In the current project, we focused on the hypothesis that anti-procathepsin D autoantibodies correlate with the stage of breast cancer, thus offering a possibility to develop a non-invasive screening test.
summary of the role
- Procathepsin D and its activation peptide act as an autocrine mitogen in human lung and breast cancer-derived cell lines
- The growth factor activity of procathepsin D can be localized in position 36-44 of the activation peptide
- In vivo experiments demonstrated that anti-procathepsin D (or anti-36-44 AA fragment) antibodies can suppress the growth of lung and breast cancer in mice
- Secretion of procathepsin D from the cancer cells correlates with level of invasiveness in vitro
- The mitogenic activity of procathepsin D is not dependent on proteolytic activity or glycosylation
- Both ribozymes and siRNA blocking pCD secretion also inhibits lung and breast cancer
- Transfection of control cells with pCD cDNA will switch their mutagenicity
- Various cytokines are involved in pCD-stimulated proliferation
- Numerous genes (including NFkB2 and Cdc25) are involved in effects of pCD
- The activation peptide of procathepsin D is a new potential target for inhibition of cancer growth
- Release of activation peptide and subsequent formation of specific antibodies can be used for a simple cancer screening