Abstract

A primary role of the IGF-II/M6P receptor is to target lysosomal enzymes from the golgi to the lysosomes. This receptor has distinct binding sites for IGF-II and M6P, however, reciprocal interactions between these ligands have been observed (Kiess et al. 1989, 1990). Since IGF-II modulates the routing of cathepsin D in MCF-7 cells by blocking the intracellular binding of cathepsin D to the IGF-II/M6P receptor (De León et al. 1996), we hypothesized that expressing a mutant form of IGF-II that does not bind the IGF-II/M6P receptor will not interfere with lysosomal enzyme trafficking.

In our present study, we report the effect in cathepsin D trafficking when Arg54 Arg55 IGF-II is expressed in MCF-7 breast cancer cells. Arg54 Arg55 IGF-II has no affinity for the IGF-II/M6P receptor but maintains a high affinity for the IGF-I and insulin receptors (Sakano et al. 1991).

Western and radioimmunoassay analyses demonstrated that transfected MCF-7 cells secrete high levels of mutant IGF-II. Receptor binding assays using CM from Arg54 Arg55 IGF-II secreting cells also confirmed that indeed, the mutant IGF-II has a significant decreased binding to the IGF-II/M6P receptor. Utilizing this model and testing our hypothesis by Western blotting and metabolic labeling analyses, we demonstrated that Arg54 Arg55 IGF-II expression does not affect cathepsin D routing. This data, thus, confirms our hypothesis that IGF-II interference with cathepsin D routing is receptor mediated.

Additional studies with this model also showed that morphological changes, indicative of a more aggressive and invasive phenotype, were associated with IGF-II expression. Specifically, changes in the expression of extracellular matrix (ECM) proteins such as vitronectin, Cadherin-5, and E-Cadherin were observed in cells expressing IGF-II. In particular, free-floating Arg54 Arg55 IGF-II cells displayed a significant reduction in an unknown 140 kD complex observed in adhering Arg54 Arg55 IGF-II cells. Thus, changes in IGF-II alter extracellular matrix proteins and facilitate the expression of a phenotype characteristic of more aggressive cancer cells.

In summary, our studies demonstrate that lysosomal enzyme trafficking of cathepsin D is not affected when Arg54 Arg55 IGF-II is overexpressed, thus, providing direct evidence that the IGF-II effect is receptor mediated. The morphological changes observed in the transfected cells provide further evidence of the importance of IGF-II interactions with the IGF-II/M6P receptor in breast cancer.

LLU Discipline

Physiology

Department

Physiology

School

Graduate School

First Advisor

Daisy D. De León

Second Advisor

Daila S. Gridley

Third Advisor

Mark S. Johnson

Fourth Advisor

Thomas A. Linkhart

Fifth Advisor

Subburaman Mohan

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2000

Date (Title Page)

9-2000

Language

English

Library of Congress/MESH Subject Headings

Breast Neoplams; Insulin-Like Growth Factor II -- biosynthesis; Mink Cell Focus-Inducing Viruses -- metabolism; Cathepsin D -- biosynthesis; Neoplasm Metastasis; Cell Division

Type

Thesis

Page Count

xiv; 87

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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