Functional in vitro analyses of lipid raft-associated cathepsin b: implication for the invasive phenotype of inflammatory breast cancer

<p>FUNCTIONAL IN VITRO ANALYSES OF LIPID RAFT-ASSOCIATED CATHEPSIN B: IMPLICATION FOR THE INVASIVE PHENOTYPE </p> <p>OF INFLAMMATORY BREAST CANCER</p> <p>by</p> <p>BERNADETTE C. VICTOR</p> <p>December 2011</p> <p>Advisor: Dr. Bonnie F. Sloane</p> <p>Major: Cancer Biology </p> <p>Degree: Doctor of Philosophy</p> <p>Inflammatory breast cancer (IBC) is an aggressive, metastatic and </p> <p>highly angiogenic form of locally advanced breast cancer. Breast </p> <p>cancer invasion has been linked to proteolytic activity at the tumor </p> <p>cell surface. We observed that uPA, uPAR and enzymatically active </p> <p>cathepsin B were all present in caveolae fractions isolated from </p> <p>SUM149 cells. Using a live-cell proteolysis assay, we demonstrated </p> <p>that both IBC cell lines degrade type IV collagen. The SUM149 cells </p> <p>exhibit predominantly pericellular proteolysis, consistent with </p> <p>localization of constituents of a proteolytic pathway to membrane </p> <p>microdomains. A functional role for cathepsin B was confirmed by </p> <p>the ability of CA074, a cell impermeable and highly selective </p> <p>cathepsin B inhibitor, to significantly reduce pericellular proteolysis </p> <p>and invasion by SUM149 cells. A statistically significant co-</p> <p>expression of cathepsin B and caveolin-1 was found in IBC patient </p> <p>biopsies, thus validating our in vitro data. To determine whether the </p> <p>uPA and cathepsin B present at the cell surface is localized </p> <p>specifically to caveolae and investigate if caveolae are a functional </p> <p>component of the IBC phenotype, we knocked down the expression </p> <p>of caveolin-1 in SUM149 cells. The subcellular distribution of </p> <p>cathepsin B was unchanged in caveolin-1 knockdown SUM149 cells. </p> <p>In contrast, knockdown decreased the association of both uPA and </p> <p>EGFR with caveolae. Knockdown of caveolin-1 also decreased </p> <p>degradation of type IV collagen and invasion by IBC cells. We also </p> <p>determined that targeted disruption of lipid microdomains by use of </p> <p>lovastatin diminished the aggressiveness of IBC cells. Cathepsin B </p> <p>activity and association with lipid microdomains was reduced in </p> <p>lovastatin treated IBC cells. Lovastatin treatment also reduced type </p> <p>IV collagen degradation and invasion by IBC cells. In conclusion, we </p> <p>have shown that cathepsin B and caveolin-1 both contribute to the </p> <p>aggressiveness of IBC, albeit by different mechanisms.</p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Open Access Dissertation

Discipline thesis:degree_discipline

Cancer Biology

Year dc:date.available

2011

Author dc:creator

• Victor, Bernadette Caroline

Contributors dc:contributor

• Bonnie F. Sloane

• cathepsin B, caveolae, inflammatory breast cancer, invasion, proteolysis
• Cell Biology

Repository record dc:identifier

https://digitalcommons.wayne.edu/oa_dissertations/400

OAI identifier oai:identifier

oai:digitalcommons.wayne.edu:oa_dissertations-1399