Regulation of breast cancer growth by activation peptide

描述（由申请人提供）：长期目标是开发一种基于阻断原组织蛋白酶D自分泌生长因子活性的乳腺癌治疗方法。乳腺癌细胞分泌原组织蛋白酶D，该酶原通过去除激活肽（APpCD）产生天冬氨酸蛋白酶D。血凝素原D已被确定为乳腺癌的独立预后因素。在初步实验中，我们发现血凝素D原是乳腺癌细胞的一种特殊的自分泌生长因子，但对其他类型的细胞没有作用。这些作用是通过一种新的，以前未知的特异性受体片段在乳腺癌细胞系上表达介导的。APpCD序列的第36-44位定位有丝分裂活性的原athepsin D区。成熟的组织蛋白酶D没有显示出生长因子活性。提出的具体目标是基于组织蛋白酶D原通过一种特定受体参与乳腺癌，该受体介导自分泌激活以增加转移性生长。对于Aim来说，有一种假设是，蛋白酶原D的过量产生导致乳腺肿瘤细胞转移潜力的增加。用人血凝素D原cDNA转染低转移性人乳腺癌细胞系，使细胞分泌不同量的血凝素D原。每个转染细胞系的转移潜力将根据血凝素D原分泌量在体内和体外进行评估。此外，pCD的合成将被特异性构建的核酶所抑制。我们将尝试确定血凝素原D中与乳腺癌细胞生长因子活性有关的确切位置。将制备代表APpCD片段的合成肽。在最活跃的肽片段中的氨基酸替换将用于绘制受体的必需氨基酸接触位点。在Aim 2中，将尝试鉴定原athepsin D的膜受体。一个代表原athepsin D结合位点区域的合成肽将被用来分离候选受体分子。对于Aim 3，假设抑制APpCD与其受体的相互作用将导致抑制癌细胞生长。利用d -氨基酸制备肽类似物或互补肽，可在体内和体外阻断癌细胞的生长和恶性。总体目标是产生一种基于阻断pCD自分泌生长因子活性的乳腺癌药理学药物。

DESCRIPTION (provided by applicant): 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.