Ectodomain shedding of the cell adhesion molecule Nectin-4 in ovarian cancer is mediated by ADAM10 and ADAM17

Petra C Buchanan,Kristin L.M Boylan,Bruce Walcheck,Rachel Heinze,Melissa A Geller,Peter A Argenta,Amy P.N Skubitz

doi:10.1074/jbc.m116.746859

Petra C Buchanan, Kristin L.M Boylan + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m116.746859

Copy DOI

Abstract

We previously showed that the cell adhesion molecule Nectin-4 is overexpressed in ovarian cancer tumors, and its cleaved extracellular domain can be detected in the serum of ovarian cancer patients. The ADAM (adisintegrin and metalloproteinase) proteases are involved in ectodomain cleavage of transmembrane proteins, and ADAM17 is known to cleave Nectin-4 in breast cancer. However, the mechanism of Nectin-4 cleavage in ovarian cancer has not yet been determined. Analysis of ovarian cancer gene microarray data showed that higher expression of Nectin-4, ADAM10, and ADAM17 is associated with significantly decreased progression-free survival. We quantified Nectin-4 shedding from the surface of ovarian cancer cells after stimulation with lysophosphatidic acid. We report that ADAM17 and ADAM10 cleave Nectin-4 and release soluble Nectin-4 (sN4). Small molecule inhibitors and siRNA knockdown of both ADAM proteases confirmed these results. In matched samples from 11 high-grade serous ovarian cancer patients, we detected 2-20-fold more sN4 in ascites fluid than serum. Co-incubation of ovarian cancer cells with ascites fluid significantly increased sN4 shedding, which could be blocked using a dual inhibitor of ADAM10 and ADAM17. Furthermore, we detected RNA for Nectin-4, ADAM10, and ADAM17 in primary ovarian carcinoma tumors, secondary omental metastases, and ascites cells isolated from serous ovarian cancer patients. In a signaling pathway screen, lysophosphatidic acid increased phosphorylation of AKT, EGF receptor, ERK1/2, JNK1/2/3, and c-Jun. Understanding the function of Nectin-4 shedding in ovarian cancer progression is critical to facilitate its development as both a serum biomarker and a therapeutic target for ovarian cancer.

Highlights

We analyzed progression-free survival (PFS) data of grade 1 and 2 serous ovarian cancer patients using the Kaplan-Meier plotter for ovarian cancer, which incorporates data from Affymetrix microarrays from the European Genome-phenome Archive (EGA), as well as The Cancer Genome Atlas (TCGA) [29]
PFS data were plotted for Nectin-4, ADAM17, and ADAM10, respectively (Fig. 1, A–C)
Our findings are the first to demonstrate that both ADAM10 and ADAM17 are major sheddases of Nectin-4 from the surface of ovarian cancer cells

Summary

To whom correspondence should be addressed

Deaths annually in the United States [1]. Currently, early detection tests are lacking, and as a result most women are diagnosed at late stages, portending a poor prognosis. Nectin-4 promotes anchorage independence in breast cancer cells [16], and the extracellular domain of some Nectins can bind to growth factor receptors, which may play a role in the regulation of cell proliferation, migration, and apoptosis [17]. Ectodomain shedding of membrane proteins by ADAMs regulates cell proliferation, adhesion, and migration and is involved in cell signaling. They play important roles in cancer formation, progression, and resistance to therapy [20]. We investigated the role of ADAM10 and ADAM17 in ectodomain shedding of the cell adhesion molecule Nectin-4 in the NIH: OVCAR5 ovarian cancer cell line, patients’ serum, ascites fluid, and solid tumor samples

Results

Discussion

Experimental procedures