HER2 Carboxyl-terminal Fragments Regulate Cell Migration and Cortactin Phosphorylation

A group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF.Deregulation of the epidermal growth factor receptor signaling network contributes to initiate and/or maintain malignant growth (1). One of these alterations, aberrant cellular motility, is necessary for invasive growth, which eventually culminates with the establishment of distant metastases, the leading cause of death in patients with cancer.The epidermal growth factor receptor is the prototype of a family that also includes HER2 (ErbB2, Neu), HER3, and HER4 (ErbB3 and ErbB4). The analysis of cells expressing various HER receptors indicated that HER2 plays a critical role in the regulation of motility (2, 3). Upon activation through homo- or heterodimerization with other HER receptors, several tyrosines in the cytoplasmic tail of HER2 are phosphorylated and initiate intracellular signaling pathways, including the phospholipase C-γ1 and phosphatidylinositol 3-kinase pathways (4), which, in turn, promote cell migration through partially understood cascades. These cascades are largely regulated by phosphorylation/dephosphorylation of cellular components.A subgroup of HER2-positive patients expresses a series of carboxyl-terminal fragments (CTFs)⁵ of HER2. HER2 CTFs can be generated by two independent mechanisms: proteolytic processing and alternative initiation of translation. Metalloproteases with the so-called α-secretase activity shed the extracellular domain of HER2, leaving behind a fragment, known as P95, that starts around alanine 648 (5) (see also Fig. 1A). Alternative initiation of translation of the mRNA encoding HER2 from the methionine codons 611 and 687 generates two fragments: 611- and 687-CTF. These differ by a stretch of 76 amino acids, which includes the transmembrane domain and a cysteine-rich short extracellular domain (6) (see also Fig. 1A).Open in a separate windowFIGURE 1.Effect of HER2 CTFs on cell migration. A, schematic showing the different constructs used in these studies. The primary sequence of the extracellular and intracellular juxtamembrane domains of HER2 is shown. The transmembrane domain (TM) is represented by a hatched box. The positions of amino acids 611, 648, and 687 are indicated. The N at the beginning of the rectangle representing HER2 identifies the amino terminus. The vertical bold double line represents the plasma membrane, and the extracellular (out) and intracellular (in) regions are marked. B, MCF7 Tet-Off cells stably transfected with the empty vector (Vector) or with the vector containing the cDNAs encoding HER2 or 611-, 648-, or 687-CTFs under the control of a Tet/Dox-responsive element were kept with or without doxycycline for 24 h, lysed, and analyzed by Western blot with CB11, an antibody against the cytoplasmic domain of HER2. C, MCF7 Tet-Off cells transfected with empty vector or with 611-CTF were seeded in the absence of doxycycline, and motility was monitored by time lapse video microscopy. Representative fields of migrated cells at the indicated times are shown. Results indistinguishable from those corresponding to MCF7 Tet-Off cells transfected with vector and treated without doxycycline were observed when analyzing MCF7/611-CTF Tet-Off cells in the presence of doxycycline (data not shown). D, representative examples of the migratory behavior of MCF7 Tet-Off cells treated as in C. Digital images were taken every 30 min for 24 h (see supplemental videos), and the tracks were manually drawn. Results indistinguishable from those corresponding to MCF7 Tet-Off cells transfected with vector and treated without doxycycline were observed when analyzing MCF7/611-CTF Tet-Off cells in the presence of doxycycline (data not shown). E, tracks from cells analyzed as in D were measured in mm. Bars, average length ± S.D. of the tracks of five cells, each one on a different culture plate. F, MCF7 Tet-Off cells transfected with 611-CTF were seeded in the absence or the presence of doxycycline on transwell plates, as described under “Experimental Procedures.” After 24 h, cells were fixed and stained with DAPI. Representative fields are shown. G, MCF7 Tet-Off cells stably transfected with the empty vector (Vector) or with the vector containing the cDNAs encoding HER2 or 611-, 648-, or 687-CTFs were seeded on transwell plates with or without doxycycline as indicated. After 24 h, cells were fixed, stained with DAPI, and counted. Bars, average of the cells counted in three independent experiments, each one performed in triplicate, ± S.D. H, representative examples of the images obtained by time lapse microscopy of the closure of wounds made in a monolayer of control MCF7 Tet-Off cells stably transfected with 611-CTF and treated with or without doxycycline as indicated. The lines drawn in the images represent reference lines marking the width of the scratch when it was made. I, average values of migration distances in scratch wound assays as in H. Bars, means from three independent experiments, each one derived from evaluation of 10 fields ± S.D.We have recently shown that 687-CTF seems to be inactive (7). In contrast, the two CTFs containing the transmembrane domain, 648- and 611-CTFs, expressed at levels similar to those found in human breast tumors, can activate different intracellular signal transduction pathways (7). The level of activation of these pathways by HER2 CTFs is quite different. 611-CTF activates the mitogen-activated protein kinase and the Akt pathways to a greater extent because it constitutively forms homodimers maintained through disulfide bonds (7). In contrast, 648-CTF does not seem to form homodimers, and its activity is comparable with that of full-length HER2 (7). Therefore, cells expressing transmembrane CTFs, particularly 611-CTF, constitute a relevant model to study the consequences of the overactivation of HER2 signaling in tumors. Supporting this conclusion, it has been shown that breast cancer patients expressing CTFs have worse prognosis and are more likely to develop nodal metastasis compared with patients expressing predominantly full-length HER2 (8).Here we show that expression of 611-CTF enhances the migration of breast cancer cells as judged by monitoring single-cell migration, transwell migration, and wound healing assays. Since cell migration is frequently regulated by phosphorylation/dephosphorylation, we searched for phosphoproteins regulated by 611-CTF and probably contributing to cell migration using two independent proteomic approaches. The results of these analyses showed that difference gel electrophoresis (DIGE) is a particularly convenient methodology to analyze the regulation of multiphosphorylated proteins.Cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, was identified by DIGE as a phosphoprotein likely to be regulated by 611-CTF. Several assays showed that expression of 611-CTF leads to an increase in the phosphorylation of cortactin and to the generation of cell protrusions resembling lamellipodia or invadopodia. Confirming a role of cortactin on the increased cell migration induced by 611-CTF, down-modulation of the former with short hairpin RNAs leads to an impairment of the cell migration induced by the HER2 fragment. These results unveil a role of cortactin in the increased cell migration induced by hyperactive HER2 and strongly suggest that cortactin-dependent increased cell migration contributes to the tendency of breast tumors expressing CTFs to metastasize.