Identification of ADAM10 as a major source of HER2 ectodomain sheddase activity in HER2 overexpressing breast cancer cells

Abstract

Overexpression and activating mutations of ErbB family members have been implicated in the development and progression of a variety of tumor types. Cleavage of the HER2 receptor by an as yet unidentified ectodomain sheddase has been shown to liberate the HER2 extracellular domain (ECD) leaving a fragment with constitutive kinase activity that can provide ligand-independent growth and survival signals to the cell. This process is clinically relevant since HER2 ECD serum levels in metastatic breast cancer patients are associated with a poorer prognosis. Thus, inhibition of the HER2 sheddase may provide a novel therapeutic approach for breast cancer. We describe the use of transcriptional profiling, pharmacological and in vitro approaches to identify the major source of HER2 sheddase activity. Real-time PCR was used to identify those ADAM family members which were expressed in HER2 shedding cell lines. siRNAs that selectively inhibited ADAM10 expression reduced HER2 shedding. In addition, we profiled over 1000 small molecules for in vitro inhibition of a panel of ADAM and MMP proteins; a positive correlation was observed only between ADAM10 inhibition and reduction of HER2 ECD shedding in a cell based assay. Finally, in vitro studies demonstrate that in combination with low doses of Herceptin, selective ADAM10 inhibitors decrease proliferation in HER2 overexpressing cell lines while inhibitors, that do not inhibit ADAM10, have no impact. These results are consistent with ADAM10 being a major determinant of HER2 shedding, the inhibition of which, may provide a novel therapeutic approach for treating a variety of cancers with active HER2 signaling.

A Naturally Occurring HER2 Carboxy-Terminal Fragment Promotes Mammary Tumor Growth and Metastasis

“…Probably because of the generality of this type of endoproteolysis, it has been widely assumed that HER2 CTFs occurring in breast cancer patients arise exclusively through this mechanism. In accordance with this assumption, and despite the fact that they constitute a group of proteins ranging from ϳ90 to 120 kDa, HER2 CTFs are frequently referred to as P95 (6,23,33), the predicted molecular weight of the transmembrane product of HER2 ectodomain shedding. In a previous publication (1), we showed the existence of an additional mechanism of CTF generation, the alternative initiation of translation.…”

Targeting the function of the HER2 oncogene in human cancer therapeutics

“…Trastuzumab binding inhibits the proteolytic cleavage and shedding of HER2 by ADAM proteases (Molina et al, 2001;Liu et al, 2006). This may, in part, inhibit the invasive properties of HER2-transformed cells, since truncated HER2 induces a more invasive morphology and is associated with increased kinase activity, increased transforming efficiency, and is increased in patients with metastatic disease (Segatto et al, 1988;Christianson et al, 1998;Egeblad et al, 2001;Molina et al, 2002).…”

Improved decision making for prioritizing tumor targeting antibodies in human xenografts: Utility of fluorescence imaging to verify tumor target expression, antibody binding and optimization of dosage and application schedule

“…In addition to these fixation and antigen retrieval problems, the fact that TAAs can be modulated during the growth of tumor cells must be taken into consideration. 51,52 In this context, it was shown that extracellular receptor domains can be shed 53,54 and found in serum [55][56][57][58] or internalized into tumor cells. 59 Furthermore, different disintegrin and metalloproteinases (ADAMs), which are considered to be responsible for shedding, 60,61 can cleave antibodies, thereby compromising antitumoral efficacy.…”