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The Role of the Vacuolar H+‐ATPase Subunit a Isoforms in Secretion and Activation of Cathepsin L in Human Breast Cancer Cells
Author(s) -
Folkers Kelly McBride,
Hinton Ayana Makeda
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.956.5
Subject(s) - cathepsin , secretion , cathepsin l , microbiology and biotechnology , cathepsin b , gene isoform , cancer cell , protein subunit , small interfering rna , cell culture , intracellular , biology , chemistry , cancer , biochemistry , transfection , enzyme , genetics , gene
V‐ATPases are multi‐subunit H + pumps that acidify intracellular compartments and are essential for bone degradation and synaptic signaling. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231, and may contribute to the metastatic phenotype. We hypothesize that V‐ATPases create a low pH environment that promotes tumor cell invasion by activating secreted cathepsins. Cathepsins cleave extracellular matrix proteins and activate other proteases, facilitating local tumor invasion. We are investigating the role of subunit a isoforms in secretion and activation of cathepsin L. The 4 subunit a isoforms are responsible for cellular localization of the V‐ATPase. siRNAs were used to reduce mRNA expression of each isoform. RT PCR showed that each isoform was successfully and specifically knocked down. Immunoblotting and activity assays are being used to analyze cathepsin L expression and activity in conditioned media of MB231 cells. Knockdown of the a4 isoform decreased the level of processed cathepsin L in conditioned media, but not the unprocessed form. Activity assays will be used to confirm that reduction of a4 affects the activity of secreted cathepsin L. Our previous findings indicate a4 targets V‐ATPases to the cell surface, thus our data support their role in activation of secreted cathepsin L in the tumor microenvironment of MB231 cells. Funded by DU Anderson Fellowship.