Access Type

Open Access Dissertation

Date of Award

December 2011

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Cancer Biology

First Advisor

Bonnie F. Sloane

Abstract

FUNCTIONAL IN VITRO ANALYSES OF LIPID RAFT-ASSOCIATED CATHEPSIN B: IMPLICATION FOR THE INVASIVE PHENOTYPE

OF INFLAMMATORY BREAST CANCER

by

BERNADETTE C. VICTOR

December 2011

Advisor: Dr. Bonnie F. Sloane

Major: Cancer Biology

Degree: Doctor of Philosophy

Inflammatory breast cancer (IBC) is an aggressive, metastatic and

highly angiogenic form of locally advanced breast cancer. Breast

cancer invasion has been linked to proteolytic activity at the tumor

cell surface. We observed that uPA, uPAR and enzymatically active

cathepsin B were all present in caveolae fractions isolated from

SUM149 cells. Using a live-cell proteolysis assay, we demonstrated

that both IBC cell lines degrade type IV collagen. The SUM149 cells

exhibit predominantly pericellular proteolysis, consistent with

localization of constituents of a proteolytic pathway to membrane

microdomains. A functional role for cathepsin B was confirmed by

the ability of CA074, a cell impermeable and highly selective

cathepsin B inhibitor, to significantly reduce pericellular proteolysis

and invasion by SUM149 cells. A statistically significant co-

expression of cathepsin B and caveolin-1 was found in IBC patient

biopsies, thus validating our in vitro data. To determine whether the

uPA and cathepsin B present at the cell surface is localized

specifically to caveolae and investigate if caveolae are a functional

component of the IBC phenotype, we knocked down the expression

of caveolin-1 in SUM149 cells. The subcellular distribution of

cathepsin B was unchanged in caveolin-1 knockdown SUM149 cells.

In contrast, knockdown decreased the association of both uPA and

EGFR with caveolae. Knockdown of caveolin-1 also decreased

degradation of type IV collagen and invasion by IBC cells. We also

determined that targeted disruption of lipid microdomains by use of

lovastatin diminished the aggressiveness of IBC cells. Cathepsin B

activity and association with lipid microdomains was reduced in

lovastatin treated IBC cells. Lovastatin treatment also reduced type

IV collagen degradation and invasion by IBC cells. In conclusion, we

have shown that cathepsin B and caveolin-1 both contribute to the

aggressiveness of IBC, albeit by different mechanisms.

Share

COinS