Truncated p110 ERBB2 induces mammary epithelial cell migration, invasion and orthotopic xenograft formation, and is associated with loss of phosphorylated STAT5

Abstract

Truncated-ERBB2 isoforms (t-ERBB2s), resulting from receptor proteolysis or alternative translation of the ERBB2 mRNA, exist in a subset of human breast tumors. t-ERBB2s lack the receptor extracellular domain targeted by therapeutic anti-ERBB2 antibodies and antibody-drug conjugates, including trastuzumab, trastuzumab-DM1 and pertuzumab. In clinical studies, expression of t-ERBB2 in breast tumors correlates with metastasis as well as trastuzumab resistance. By using a novel immuno-microarray method, we detect a significant t-ERBB2 fraction in 18 of 31 (58%) of immunohistochemistry (IHC)3+ ERBB2+ human tumor specimens, and further show that t-ERBB2 isoforms are phosphorylated in a subset of IHC3+ samples (10 of 31, 32%). We investigated t-ERBB2 biological activity via engineered expression of full-length and truncated ERBB2 isoforms in human mammary epithelial cells (HMECs), including HMEC and MCF10A cells. Expression of p110 t-ERBB2, but not p95m (m=membrane, also 648CTF) or intracellular ERBB2s, significantly enhanced cell migration and invasion in multiple cell types. In addition, only expression of the p110 isoform led to human breast epithelial cell (HMLE) xenograft formation in vivo. Expression of t-ERBB2s did not result in hyperactivation of the phosphoinositide kinase-3/AKT or mitogen-activated protein kinase signaling pathways in these cells; rather, phosphoproteomic array profiling revealed attenuation of phosphorylated signal transducer and activator of transcription 5 (STAT5) in p110-t-ERBB2-expressing cells compared to controls. Short hairpin-mediated silencing of STAT5 phenocopied p110-t-ERBB2-driven cell migration and invasion, while expression of constitutively active STAT5 reversed these effects. Thus, we provide novel evidence that (1) expression of p110 t-ERBB2 is sufficient for full transformation of HMEC, yielding in vivo xenograft formation, and (2) truncated p110 t-ERBB2 expression is associated with decreased phosphorylation of STAT5.

Figure 1

Detection of ERBB2 isoforms in human breast cancer cell lines. (a) Schematic representation of full-length and truncated ERBB2 isoforms. p185, p110 and p95m isoforms contain transmembrane domain, whereas p95cyto lacks this domain. p95n is targeted to the nucleus by the addition of two tandem nuclear localization sequence motifs on the C terminus. ECD, extracellular domain; TKD, tyrosine kinase domain; NLS, nuclear localization sequence. (b) Western blots of SKBR3 and BT474 lysates reveal detectable expression of truncated ERBB2 isoforms in human ERBB2+ breast cancer cell lines. (c) Schematic of the CEER detection method. Target substrate is immobilized with a capture antibody (green). The first detector antibody (red) conjugated to GO binds to the captured target substrate using a different epitope than the capture antibody. The second detector antibody (blue) conjugated to HRP binds to a third epitope and completes the formation of the immuno-complex necessary for signal generation. GO, glucose oxidase; HRP, horseradish peroxidase; P, phosphorylated residue. (d) Detection of t-ERBB2 isoforms in BT474 cells using the CEER assay. Total protein lysate from 25 BT474 cells (left panel) or protein lysate from 250 BT474 cells from which p185 ERBB2 was removed with an ECD targeting antibody (right panel) were tested for the expression of ERBB2 using antibodies targeting both the ERBB2-ECD and ICD. Following removal of p185 ERBB2 (right panel), primarily t-ERBB2 isoforms are present, which are detected with the ERBB2-ICD targeting antibody (red signal is near maximal signal), but not with an ECD targeting antibody (light blue is close to the background signal). The image for post-p185 ERBB2 removed BT474 profile is shown at a higher photomultiplier tube (Photo Multiplier Tube, hence higher background signal) gain set as the signal was almost non-detectable with the ERBB2-ECD antibody. Utilizing BT474 lysates with and without p185ERBB2 removal and ICD capture configuration, the number of t-ERBB2 per cell were determined to be ∼5.3 × 10⁴ receptors per cell, compared to 1.2 × 10⁶ total ERBB2 receptors per cell. Therefore, the percentage of t-ERBB2 in BT474 cells is determined to be approximately 4.3% (13/300).

Figure 2

Detection of ERBB2 isoforms in primary human breast tumor samples. (a) CEER analysis of human tumor samples reveals the presence and phosphorylation of t-ERBB2s. Lysates of tumors from three patients with high (no. 26811) or intermediate (nos. 24913 and 25882) ERBB2 expression as scored by IHC were tested. Assay configuration is illustrated below the CEER panels. Antibodies were arrayed in triplicate at three concentrations. Left panel antibodies for total p185 ERBB2 assay: control IgG (pink arrows), ECD-ERBB2 (yellow arrows) and cytokeratin (CK, green arrows). Center and right panel antibodies for total and phosphorylated t-ERBB2 assay: control IgG (pink arrows), ECD-ERBB2 (yellow arrows), ICD-ERBB2 (dark and light blue arrows) and phosphorylated ERBB2 (red arrows). Before t-ERBB2 CEER assay (center and right panels), full-length p185 ERBB2 was removed with an N-terminal ECD-directed antibody. (b) Western blot analysis of ERBB2 isoforms in human breast tumor samples with varying levels of ERBB2 (by IHC) assayed in part (a). Patient specimen lysates 26811, 24913 and 25882 were probed with antibody against ICD-ERBB2. Patient 26811 with positive western analysis also showed high level of ERBB2 as well as t-ERBB2 expression by CEER, with the use of much less cell lysate. Using CEER configuration, a quantitative detection of full-length ERBB2 and t-ERBB2 was achieved in samples (24913 and 25882) with IHC2+ status. Of note, t-ERBB2 was not detected by western blot analysis in these samples. Furthermore, a robust phosphorylation of t-ERBB2 was observed in patient 26811, whereas lower level was detected in patient 24913.

Figure 3

Expression and localization of ERBB2 receptor isoforms in HMLE cells. (a) Lysates from parental or recombinant ERBB2-transduced HMLE cells were probed with anti-ERBB2 antibody in a western blot assay. Actin was blotted as loading control. (b) Recombinant HMLE cells were fractionated into cytoplasmic ‘C', membrane ‘M' and nuclear ‘N' fractions, followed by western blotting for ERBB2. Control antibodies identify proteins restricted to the cytoplasm (GAPDH, actin), the plasma membrane (NaK-ATPase) and the nucleus (Lamin A/C). p110 HMLE also express the p95cyto isoform due to translation from the AUG codon corresponding to methionine 687. (c) Confocal microscopy of recombinant HMLE cells. Plasma membranes were stained with concanavalin A (green), followed by a mouse anti-ERBB2 antibody and anti-mouse Cy5 (red) for receptor labeling. p185, p110 and p95m reside primarily in the plasma membrane, whereas p95cyto and p95n reside in the cytoplasm and nucleus, respectively. Images are representative of the population at large and were taken at × 80 magnification on a Zeiss confocal microscope. Cells expressing p95n were analyzed at higher magnification to confirm nuclear localization.

Figure 4

p110 t-ERBB2 increases migration and invasion of HMLE and HME cells in vitro. (a) Migration of parental and recombinant HMLE cells. Numbers of cells in nine random fields on the lower sides of triplicate 8 μm filters were counted after 24 h. *P=0.04 (Student's t-test). Shown are representative images of HMLE p185 and HMLE p110 migration transwells after staining with crystal violet. (b) Invasion through Matrigel of parental and recombinant HMLE cells. Numbers of cells on the lower sides of triplicate 8 μm filters were counted after 36 h. *P=0.03 (Student's t-test). Shown are representative images of HMLE p185 and HMLE p110 invasion transwells after staining with crystal violet. (c) HME cells expressing p185 and p110 isoforms of t-ERBB2 were used in migration assays, and representative images of transwell membranes are shown. (d) HME cells expressing p185 and p110 isoforms of t-ERBB2 were used in invasion assays, and representative images of transwell membranes are shown.

Figure 5

p110 t-ERBB2 promotes HMLE orthotopic xenograft formation. (a) Growth curves of parental and recombinant HMLE xenografts over 150 days. (b) Photographs and hematoxylin and eosin staining of a representative p110-HMLE tumor. (c) Polymerase chain reaction using vector and ERBB2 targeted primers and genomic DNA from p110-HMLE tumors as template. Polymerase chain reaction lacking template was used for the negative control reaction.

Figure 6

STAT5 phosphorylation is attenuated in p110 t-ERBB2-expressing cells, and elicits migration and invasion of HMLE cells. (a) Western blot of phospho-STAT5 in lysates from recombinant HMLE cells expressing ERBB2 isoforms. (b) Western blot of phospho-STAT5 in lysates from recombinant HME cells expressing ERBB2 isoforms. (c) Western blot of p21 in lysates from recombinant HMLE cells expressing ERBB2 isoforms. (d) Western blot of total STAT5 protein in lysates from HMLE transduced with non-silencing control (NSC) shRNA or STAT5-targeted shRNA. (e) Migration (left panels) and invasion (right panels) of control HMLE (top rows) and HMLE with silenced STAT5 expression (bottom rows) from (d).

Figure 7

Constitutively active STAT5 reverses p110-driven migration and invasion of HMLE cells. HMLE cells stably expressing p110 t-ERBB2 were transduced with empty vector or constitutively active STAT5b vector. (a) Migration of p110-HMLE cells expressing empty vector (top panels) or caSTAT5b (lower panels). Representative photographs of transwell membranes are shown. (b) Invasion through Matrigel by p110-HMLE cells expressing empty vector (top panels) or caSTAT5b (lower panels). Representative photographs of transwell membranes are shown.