Global Protease Activity Profiling Identifies HER2-Driven Proteolysis in Breast Cancer

Differential
expression of extracellular proteases and endogenous
protease inhibitors has been associated with distinct molecular subtypes
of breast cancer. However, due to the tight post-translational regulation
of protease activity, protease expression-level data alone are not
sufficient to understand the role of proteases in malignant transformation.
Therefore, we hypothesized that global profiles of extracellular protease
activity could more completely reflect differences observed at the
transcriptional level in breast cancer and that subtype-associated
protease activity may be leveraged to identify specific proteases
that play a functional role in cancer signaling. Here, we used a global
peptide library-based approach to profile the activities of proteases
within distinct breast cancer subtypes. Analysis of 3651 total peptide
cleavages from a panel of well-characterized breast cancer cell lines
demonstrated differences in proteolytic signatures between cell lines.
Cell line clustering based on protease cleavages within the peptide
library expanded upon the expected classification derived from transcriptional
profiling. An isogenic cell line model developed to further interrogate
proteolysis in the HER2 subtype revealed a proteolytic signature consistent
with activation of TGF-β signaling. Specifically, we determined
that a metalloprotease involved in TGF-β signaling, BMP1, was
upregulated at both the protein (2-fold, P = 0.001)
and activity (P = 0.0599) levels. Inhibition of BMP1
and HER2 suppressed invasion of HER2-expressing cells by 35% (P < 0.0001), compared to 15% (P = 0.0086)
observed in cells where only HER2 was inhibited. In summary, through
global identification of extracellular proteolysis in breast cancer
cell lines, we demonstrate subtype-specific differences in protease
activity and elucidate proteolysis associated with HER2-mediated signaling.