Quantitative Analysis of Wild Type and the newly generated CRISPR/Cas9 Hpn-/- mouse mammary gland gene expression analysis

Transforming growth factor beta (TGFß) is a multifunctional cytokine with a well-established role in mammary gland development and both oncogenic and tumor-suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFß activity by acting as a storage compartment of latent TGFß, but how TGFß is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFß signaling through the release of latent TGFß from the ECM-storage compartment. Mammary glands in hepsin CRISPR knock-out mice showed reduced TGFß signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent TGFß1, while overexpression of hepsin in mammary tumors increased TGFß signaling. Cell-free and cell-based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent TGFß and, importantly, that the ability of hepsin to activate TGFß signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFß pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin. Overall design: RNA was extracted from 2-6 weeks old wild-type (N=3, refference samples) and Hpn-/- (N=3) virgin female mammary glands and sequenced with Illumina NextSeq500 (Illumina, San Diego, CA, USA) to quantitate transcriptional changes in gene expression. Pathway and statistical enrichment analysis were performed in GSEA 4.0.3 software (Subramanian et al., 2005 PMID: 16199517).