Article Figures & Data
Figures
- Figure 1.
Expression of HER2 and HER2Δ16 in human tissues and breast tumors. A, schematic of mRNA from wild-type HER2 and the exon 16 deletion isoform HER2Δ16 with relative positions of RT-PCR primers and predicted size of each PCR-amplified product indicated. B, RT-PCR detection of HER2 and HER2Δ16 expression in normal human tissue RNA. C, RT-PCR detection of HER2 and HER2Δ16 expression in RNA isolated from normal and malignant breast tissue. Representative examples of tumors with HER2 overexpression (1–3), tumors lacking HER2 overexpression (4–6), and tumors with HER2 and HER2Δ16 overexpression (7–9).
- Figure 2.
HER2Δ16 forms stable dimers and activates multiple signaling pathways in breast tumor cells. A, Western blot analysis of total and site-specific HER2 and HER2Δ16 phosphorylated activation in stable MCF-7 cell lines. B, assay of constitutive HER2 or HER2Δ16 receptor dimerization from indicated stable MCF-7 cell line lysates analyzed by native nonreducing PAGE and Western blot. C, expression and phosphorylated activation of HER/ErbB family members in response to ectopic HER2 and HER2Δ16 expression in stable MCF-7 cell lines. Each HER receptor was immunoprecipitated (IP) from the indicated MCF-7 cell line and analyzed for phosphorylated activation by anti-P-Tyr Western immunoblot (IB) analysis. D, protein lysates from the indicated MCF-7 stable cell lines were analyzed by Western blot for activation of multiple oncogenic signaling pathways.
- Figure 3.
HER2Δ16 promotes proliferation and invasion of tumor cells. A, cell growth assay of NIH3T3 cells expressing HER2 or HER2Δ16. Each cell line was plated in duplicate at 0.5 × 105 cells per plate and cell numbers were counted. Inset, Western blot analysis of each stable cell line. B, indicated MCF-7 stable cell lines were plated in triplicate in a 96-well plate at 2 × 103 cells per well and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay was done after 7 d using standard procedures. C, colony formation assay with each cell line seeded in duplicate into a 6-well plate at 1 × 103 cells per well and incubated for 7 to 10 d. Colony number and size were calculated using a ColCount colony counter (Oxford Optronix). Data are normalized to the MCF-7/pcDNA vector control. Representative colony formation results. D, invasion and migration of each stable MCF-7 cell line was determined using a BD BioCoat Invasion Chamber with (invasion) or without (migration) Matrigel insert. Invading/migrating cells were counted after 22 h. B to D, mean ± SE. Asterisk, HER2Δ16 cells significantly different from both vector control and HER2-expressing cells.
- Figure 4.
HER2Δ16 promotes trastuzumab resistance. A, schematic of HER2 protein with positions of putative signal peptide (SP), two cysteine-rich regions (cys1 and cys2), transmembrane region (TM), and intracellular tyrosine kinase domain (TK). The exon 16 deletion in HER2Δ16 (light gray) is positioned in a region carboxyl to the binding site of trastuzumab. B, trastuzumab binds to the cell surface of HER2Δ16-expressing MCF-7 cells. Immunofluorescence assay of the indicated stable MCF-7 cell lines showing overlap between cell surface binding of a HER2 polyclonal antibody (red) and the humanized HER2 antibody trastuzumab (green) in both HER2- and HER2Δ16-expressing cells. Binding of HER2 antibody or trastuzumab was not observed in the MCF-7/vector control cells. C, indicated stable MCF-7 cell lines were treated with 10 μg/mL trastuzumab for 48 h and protein lysates were analyzed by Western blot. Asterisk, trastuzumab dephosphorylated activation of PTEN in HER2-expressing cells. D, HER2Δ16-expressing MCF-7 cells are trastuzumab resistant. Each stable MCF-7 cell line was treated with 10 μg/mL trastuzumab in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay or colony formation assay. Only the invasive HER2Δ16 cell line was treated with 10 μg/mL trastuzumab in an invasion assay using a BD BioCoat Invasion Chamber. Each entire experiment was repeated at least three times. Mean ± SE of percent inhibition of treated versus untreated cells.
- Figure 5.
HER2Δ16 directly couples to Src kinase in breast tumor cells. A, immunofluorescent detection of HER2 or HER2Δ16 (green) and Src kinase (red) in HER2Δ16- and HER2-expressing MCF-7 cells and analyzed by deconvolution microscopy. B, Src kinase coimmunoprecipitates with HER2Δ16 but not HER2. HEK293T cells were cotransfected with chicken Src kinase and HER2-Flag or HER2Δ16-Flag. Src and Flag immunoprecipitates were probed by Western blot for Src kinase or Flag-tagged HER2 and HER2Δ16. C, suppression of Src kinase destabilizes HER2Δ16 and inhibits cell invasion. Invasion assay of MCF-7/HER2Δ16 cells using a BD BioCoat Invasion Chamber following treatment with nonspecific (NS) or Src targeting RNAi. Control Western blot showing suppression of Src expression and loss of HER2Δ16 protein following treatment with Src RNAi. Asterisks, nonspecific band observed in Src kinase Western blot. D, activated Src kinase is coexpressed with HER2Δ16 in invasive breast tumors. HER2Δ16(+) and HER2Δ16(−) primary invasive breast tumors (Supplementary Tables S1 and S2) were stained by immunohistochemistry for activated Src kinase P-Src Y416. Membrane associated and activated Src kinase was observed in 44% (4 of 9) of HER2Δ16(+) tumors and 25% (1 of 4) of HER2Δ16(−) tumors.
- Figure 6.
Trastuzumab-resistant HER2Δ16 cells are sensitive to the tyrosine kinase inhibitor dasatinib. A, each stable MCF-7 cell line was treated with increasing amounts of dasatinib (0, 10, 100, or 1,000 nmol/L), and after 24 h, protein lysates were analyzed by Western blot. Asterisks, dasatinib-induced suppression of Src kinase and loss of HER2Δ16. B, dasatinib dose-response. Each stable MCF-7 cell line was treated with the indicated concentration of dasatinib and viability was determined using a colony formation assay. C, trastuzumab-resistant and trastuzumab-sensitive tumor cell lines are sensitive to dasatinib. Each indicated stable MCF-7 cell line was treated with 10 μg/mL trastuzumab and/or 100 nmol/L of the tyrosine kinase inhibitor dasatinib in a colony formation assay and the invasive MCF-7/HER2Δ16 was similarly treated in an invasion assay using a BD BioCoat Invasion Chamber. B and C, each experiment was repeated at least three times. Mean and ± SE of percent viability of treated versus untreated cell lines.
Additional Files
Supplementary Data
Files in this Data Supplement:
Supplementary Data
- Supplementary Tables S1-S2 - Supplementary Table 1. Clinical characteristics of 85 primary breast tumors used for statistical analysis; Supplementary Table 2. HER2 and HER2�16 coexpression compared clinicopathological parameters.
Volume 8, Issue 8
