60 Chapter 3 be the use of a variety and potentially unreliable antibodies. Therefore, we investigated the specificity and sensitivity of single batches of five commercial antibodies directed against the C-terminus of MET under reducing, native and FFPE conditions before establishing the receptor’s influence on the survival of patients with oral and oropharyngeal SCC. Two out of five antibodies, D1C2 and CVD13, specifically detected MET across all examined conditions, confirming the need for biomarker validation and testing as discussed by Hayes et al. (41). At this point, it should be stressed that the observed specificity of D1C2 and CVD13 should not be taken for granted, since the reliability of antibodies is known to vary between manufacturing lots. Consequently, one must always verify the specificity of D1C2 and CVD13 with changing lot numbers before using them in a research and/or clinical environment (33). Besides the precursor (p170MET) and the b-chain (p145MET), D1C2 and CVD13 detected five additional MET C-terminal protein fragments (p40MET, p55MET, p50MET, p60MET and p70MET) under reducing conditions. This finding is not unexpected as MET is subject to proteolysis (42). Under conditions of stress, MET is cleaved by caspases leading to the generation of a proapoptotic intracellular 40 kDa C-terminal fragment, referred to as p40MET (43, 44). Also, independently of ligand stimulation, MET can be cleaved within its extracellular juxtamembrane domain by membrane metalloproteases. This leads to the generation of a soluble MET N-terminal fragment which is shed into the extracellular space (42) – a process referred to as ectodomain shedding (45) – and a membrane-anchored C-terminal fragment (MET-CTF) of 55 kDa, referred to as p55MET (46). These specific MET-CTFs (55 kDa) can be processed through direct lysosomal degradation (46) or are cleaved at the membrane by the g-secretase complex, a process known as presenilin-regulated intramembrane proteolysis. The generated 50 kDa intracellular domain of MET (MET-ICD), referred to as p50MET, is subsequently released into the cytosol and degraded by the proteasome (36). Finally, the observation that the rate of MET ectodomain shedding – in terms of the number of N-terminal fragments – increases with increasing malignant behavior of breast cancer cells (45), provides an explanation for the p70MET and p60MET C-terminal fragments observed in HeLa, HT-29 and PC3 using D1C2 under reducing conditions. More specifically – among the observed shed N-terminal fragments – two fragments were observed of 75 and 85 kDa. These specific N-terminal fragments – theoretically – give rise to MET-CTFs of 70 and 60 kDa.
RkJQdWJsaXNoZXIy MTk4NDMw