ErbB2, EphrinB1, Src Kinase and PTPN13 Signaling Complex Regulates MAP Kinase Signaling in Human Cancers

In non-cancerous cells, phosphorylated proteins exist transiently, becoming de-phosphorylated by specific phosphatases that terminate propagation of signaling pathways. In cancers, compromised phosphatase activity and/or expression occur and contribute to tumor phenotype. The non-receptor phosphatase, PTPN13, has recently been dubbed a putative tumor suppressor. It decreased expression in breast cancer correlates with decreased overall survival. Here we show that PTPN13 regulates a new signaling complex in breast cancer consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation and localization studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further enhances signaling. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in breast cancers harboring ErbB2-activating mutations and decreased PTPN13 expression

mNeuNT is required for EphrinB1 activation and initiation of signaling.

<p>(<b>A</b>) Western blot analysis of a human keratinocyte cell line, HaCaT cells (control as well as cells knocked-down for PTPN13), and breast cancer cell lines (MCF7, BT474, HCC1953) immunoprecipitated (IP) for ErbB2 and immunoblotted (IB) for EphrinB1. Membrane was re-probed for ErbB2. GAPDH was used as a loading control. (<b>B</b>) <i>En face</i> confocal images of HaCaT cells immunolocalizing surface EphrinB (green) and total ErbB2 (red). Nuclei are counterstained with DaPi (blue). Scale bar 20 µm. (<b>C</b>) Western blot analysis of breast cancer cell lines: T47D, BT474 and MCF7. (<b>D</b>) HEK293 cells transiently transfected with either wildtype PTPN13 or the C/S PTPN13 mutant analyzed by western blot. (<b>E</b>) HEK293 cells transiently transfected with either eGFP alone, or a combination of EphrinB1, ErbB2 (wildtype or mNeuNT), and PTPN13 (wildtype or C/S mutant) and analyzed by western blot. (<b>F</b>) <i>En face</i> confocal images of cells transfected in <b>E</b> processed for immunolocalization of phosphorylated EphrinB (green) and ErbB2 (red). Nuclei counterstained with DaPi (blue). Scale bar 20 µm.</p

ErbB2 and EphrinB1 associate in a manner that likely requires the transmembrane domains.

<p>(<b>A</b>) Schematic representation of ErbB2 and EphrinB1 mutants. LBD, ligand binding domain. * , PDZ binding domain (PDZBD). Numbers refers to amino acid number. (<b>B</b>) HEK293 cells were transiently transfected with HA-tagged wildtype or mutant ErbB2 constructs and analyzed by western blot. (<b>C</b>) HEK293 cells were transiently transfected with FLAG-tagged wildtype or mutant EphrinB1 constructs and analyzed by western blot. (<b>D</b>) HEK293 cells transfected in <b>B</b> and <b>C</b> were further analyzed by immunofluorescence and confocal microscopy. <i>En face</i> confocal images of immuno-localized HA-tagged ErbB2 (red, top panels) and endogenous EphrinB1 (endog. EphrinB1,green, top panels). Yellow color signifies co-localization. Immunostaining of FLAG-tagged EphrinB1 (green, bottom panels) and wild-type ErbB2 (red, bottom panels). Yellow color signifies co-localization. Nuclei were counterstained with DaPi (blue). Scale bar 20 µm.</p

mNeuNT associates with activated Src which phosphorylates EphrinB1.

<p>(<b>A</b>) HEK293 cells transiently transfected with either wildtype ErbB2 or mNeuNT and analyzed by western blot. (<b>B</b>) HEK293 cells transiently transfected with a combination of EphrinB1, mNeuNT, and PTPN13 (wildtype or C/S mutant) were treated with or without PP2 and analyzed by western blot. (<b>C</b>) Untransfected HEK293 cells treated with increasing doses of saracatinib and analyzed by western blot for expression of endogenous activated Src, total Src, phosphorylated EphrinB1 and immunoprecipitated EphrinB1.</p

Decreased PTPN13 expression occurs in BL tumors and correlates with increased EphrinB1 and Erk1/2 signaling.

<p>(<b>A</b>) Relative PTPN13 mRNA expression of PTPN13 in molecularly characterized breast tumors. Basal-like (BL) breast cancer PTPN13 expression is decreased relative to normal breast (p = 0.00044 for basal vs. normal). (<b>B</b>) Western blot analysis of breast cancer cell lines. MDA-MB231, MDA-MB468, HCC1143, HCC1954 are breast cancer cell lines with BL breast cancer characteristics. The BT474 cell line has Her2/ErbB2 over-expressing breast cancer characteristics. MCF7 and T47D are breast cancer cell lines with luminal characteristics. HEK293 cells over-expressing PTPN13 served as a positive control. (<b>C</b>) BL breast cancer cell lines, MDA-MB231 and MDA-MB468, expressing low or high PTPN13, respectively, were analyzed by western blot. (<b>D</b>) HEK293 cells stably knocked-down for EphrinB1 (sh EphrinB1) or control were analyzed by western blot. (<b>E</b>) MDA-MB468 cells were transiently transfected with an shRNA plasmid targeting PTPN13 (shPTPN13) or a non-silencing shRNA construct (Non-silencing) and analyzed by western blot for the indicated proteins. (<b>F</b>) HaCaT cells, a human keratinocyte cell line, and UM-SCC84 cells, an HPV-negative head and neck squamous cell carcinoma cell line, stably knocked-down for PTPN13 (sh PTPN13) or control lines were analyzed by western blot. (<b>G</b>) HaCaT cells stably knocked-down for PTPN13 (sh PTPN13) or over-expressing HPV16 E6 protein (PHV16 E6) or control were analyzed by western blot for phosphorylated EphrinB1, phosphorylated Erk1/2, total Erk1/2, and GAPDH.</p