Oncologia

Dimerization of the extracellular domains leads to interaction between the intracellular kinases of the HER receptors and subsequent transphosphorylation of certain tyrosine residues in the C-terminal tail. These phosphotyrosines act as docking sites for a group of intracellular phosphotyrosinebinding proteins that transduce signals from the plasma membrane to the nucleus via different signalling pathways, including the mitogen activated protein kinases (MAPKs), PI(3)K-activated Akt, Src and phospholipase C gamma (PLCgamma) pathways. These signalling circuits control the expression of target genes that act in coordination to modify key aspects of cellular biology, including proliferation, migration, survival and differentiation.

In addition to the canonical mode, HER receptors, or fragments of them, seem to be endowed with direct signalling abilities. HER2 is a substrate of metalloproteases collectively known as alpha-secretases, which release the extracellular domain, leaving behind the transmembrane-cytoplasmic fragment, known as P95. By analogy with other transmembrane proteins also cleaved by alpha-secretases, it has been suggested that the cleavage of P95 can also be achieved by gamma-secretases, which release the intracellular domain in a process known as RIP (regulated intramembrane

proteolysis).

Although P95 has been poorly characterized, partly because it is produced at very low levels in cultured cell lines, it has been suggested that it is active. However, since P95 lacks the extracellular domain, it is not predicted to form hetero- or homodimers. Thus, the mechanism of activation of P95 remains unexplained. We have recently identifi ed alternative initiation of translation as an additional mechanism that generates CTFs of HER2 similar, but not identical, to P95. Initiation of translation from methionine codons, located upstream or downstream of the transmembrane domain, leads to the generation of two diff erent CTFs.

Although preliminary evidence suggests that CTFs generated by translation are active, as in the case of P95, the mechanism of activation is unknown. In summary, at least four diff erent HER2 CTFs are generated by two independent mechanisms: proteolytic processing and alternative initiation of translation. Two HER2 CTFs contain the transmembrane and cytoplasmic domains while two are predicted to be soluble intracellular proteins encompassing most of the cytoplasmic domain.

Breast cancer patients expressing CTFs of HER2 are more likely to develop nodal metastasis and have a worse prognosis than those expressing predominantly the fulllength receptor. Furthermore, the presence of CTFs seems to be relevant for tumor treatment. Currently, two types of drugs targeting HER2 are used in clinical practice: monoclonal antibodies against the extracellular domain and smallmolecule inhibitors that block the kinase activity of the receptor. We have recently shown that approximately 90% of breast cancer patients expressing CTFs are resistant to treatment with the anti-HER2 antibody Herceptin (trastuzumab).

However, the CTFs expressed in tumors have not been characterized in detail and it is not known if these fragments arise in tumors by proteolysis and/or alternative initiation of translation. Furthermore, since the activity of the diff erent CTFs has not been analyzed individually, their relative contribution to the malignant phenotype has not been determined

In 2010 we fi nished a comprehensive analysis of the diff erent CTFs of HER2 expressed in breast cancers. In addition we were able to generate and characterize monoclonal antibodies that recognize epitopes exposed in the fragments but masked in full-length HER2. These antibodies constitute a useful tool to identify p95HER2-positive tumors and to fi nd a better treatment for this subtype of patients.