ErbB3 ablation impairs PI3K/Akt-dependent mammary tumorigenesis - PubMed (original) (raw)

. 2011 Jun 1;71(11):3941-51.

doi: 10.1158/0008-5472.CAN-10-3775. Epub 2011 Apr 11.

Joan T Garrett, Violeta Sánchez, Jamie C Stanford, Christian Young, Anindita Chakrabarty, Cammie Rinehart, Yixian Zhang, Yaming Wu, Lee Greenberger, Ivan D Horak, Carlos L Arteaga

Affiliations

ErbB3 ablation impairs PI3K/Akt-dependent mammary tumorigenesis

Rebecca S Cook et al. Cancer Res. 2011.

Abstract

The ErbB receptor family member ErbB3 has been implicated in breast cancer growth, but it has yet to be determined whether its disruption is therapeutically valuable. In a mouse model of mammary carcinoma driven by the polyomavirus middle T (PyVmT) oncogene, the ErbB2 tyrosine kinase inhibitor lapatinib reduced the activation of ErbB3 and Akt as well as tumor cell growth. In this phosphatidylinositol-3 kinase (PI3K)-dependent tumor model, ErbB2 is part of a complex containing PyVmT, p85 (PI3K), and ErbB3, that is disrupted by treatment with lapatinib. Thus, full engagement of PI3K/Akt by ErbB2 in this oncogene-induced mouse tumor model may involve its ability to dimerize with and phosphorylate ErbB3, which itself directly binds PI3K. In this article, we report that ErbB3 is critical for PI3K/Akt-driven tumor formation triggered by the PyVmT oncogene. Tissue-specific, Cre-mediated deletion of ErbB3 reduced Akt phosphorylation, primary tumor growth, and pulmonary metastasis. Furthermore, EZN-3920, a chemically stabilized antisense oligonucleotide that targets the ErbB3 mRNA in vivo, produced similar effects while causing no toxicity in the mouse model. Our findings offer further preclinical evidence that ErbB3 ablation may be therapeutically effective in tumors where ErbB3 engages PI3K/Akt signaling.

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Figures

Figure 1

Figure 1. Inhibition of ErbB2 impairs growth of MMTV-PyVmT tumors

A Whole cell extracts prepared from HC11 mouse mammary cells or MMTV-Neu (Neu) and MMTV-PyVmT (mT) primary tumor cells, were analyzed by immunoblot for the proteins indicated at the right of each panel. B–C. Whole cell extracts from MMTV-PyVmT primary tumor cells cultured in serum-free media for 6 h ± lapatinib (1 μM) and an additional 5 min ± heregulin (HRG; 2 ng/ml; in B) were used for western analysis or for IP followed by western analysis to detect the proteins indicated at the right of each panel. D. MMTV-PyVmT cells were embedded in growth factor-reduced Matrigel with increasing concentrations of lapatinib. Medium and lapatinib were replenished every two days. Digital images were analyzed with Olympus DP2 software to measure colony area in pixels. At least 50 colonies per well x3 wells per condition were measured and used to calculate the average colony size per well. Values represent the average total colony area per well ± S.D. E. MMTV-PyVmT primary tumors cells (1×106) were injected into the inguinal mammary fat pad of 5-week old WT FVB female mice. Tumor- bearing mice (tumor volume ≥200 mm3) were treated ± lapatinib (100 mg/kg/day x28). Tumor volume was measured weekly as indicated in Methods. Each data point represents the mean tumor volume in mm3 ± SD (_n_=10; _p_=0.0021, Mann-Whitney test). F–H. Whole cell extracts from MMTV-PyVmT primary tumor cells cultured in serum-free media with lapatinib (1 μM) for 24 h were used for immunoprecipitation (IP) with antibodies against the following: ErbB3 and p85 (F), ErbB3 (G), and PyVmT (H). Immune complexes were separated by SDS-PAGE and next subjected to immunoblot (IB) analysis using the indicated antibodies as described in Methods.

Figure 2

Figure 2. Absence of ErbB3 impairs the formation of MMTV-PyVmT multi-focal tumors

A Whole mount hematoxylin stained inguinal mammary glands of 8-week old virgin female mice. B. Tumor-free curve was generated by documenting the time at which tumors were originally palpated. The average tumor latency (T50) was calculated using the Kaplan-Meier test (_n_=20 for each genotype; p<0.0001, Log-rank test). C. Lung metastases were identified in histological sections and enumerated. The midlines indicate the average number of lung metastases for each genotype ± S.D. D. Ten mammary glands per mouse were harvested and weighed together. The values show the average total mammary/tumor wet weight ± S.E. (n=6; _p_=0.015). E. H&E-stained tumor sections (left panels), and TUNEL-stained tumor sections (right panels) from 11-week old virgin female mice. F. Whole tumor lysates were prepared as described in Methods and used for western analysis using the antibodies indicated at the right of the panels. Genotype of each tumor (with respect to the targeted Erbb3 and MMTV-Cre alleles) is indicated at top (n.s.= non-specific). G. Whole tumor extracts harvested from 3 mice per genotype were precipitated with a p85 antibody. Immune complexes were separated by SDS-PAGE followed by western analysis for p85 and PyVmT as indicated in Methods.

Figure 3

Figure 3. EZN-3920 inhibits tumor formation and P-Akt in mammary glands of MMTV-PyVmT mice

A Primary MMTV-PyVmT tumor cells were treated in culture with 2.5 μM EZN-3920 or EZN-4455 for 72 h. Whole cell extracts were used directly for western analysis to detect expression of ErbB3, ErbB2, and EGFR, or used for p85 IP followed by western analysis for ErbB3 and p85. B–F. Treatment with EZN-4455 or EZN-3920 (25 mg/kg, twice weekly) began when mice were 3 weeks of age and continued for 5 weeks (total of 10 doses). Tissues were harvested 24 h after the final dose. B–C. Low-power magnification of inguinal mammary glands of mice treated with EZN-4455 (B) or EZN-3920 (C) illustrate profound inhibition of tumor progression in mice treated with EZN-3920. CLN: central lymph node; Ad: adipose tissue; duct: normal ductal epithelium; vess: blood vessel; hyperpl: hyperplastic nodule. D. High-power magnification of mammary epithelium from mouse treated with EZN-3920. Note single layer of epithelium surrounding a lumen**. E.** Immunohistochemical detection of S473 P-Akt. F. Quantitation of the rate of P-Akt positivity in mammary epithelium of mice treated with EZN-4455 and EZN-3920. Values represent the average number ± S.D. of P-Akt+ epithelial cells per total number of epithelial cells in five 400x fields per sample x 5 samples per condition (_p_=0.00001; Student’s unpaired T-test).

Figure 4

Figure 4. Genetic ablation of ErbB3 inhibits growth of PyVmT tumors

A ErbB3fl/fl.MTB-TCre X PyVmT primary tumor cells were treated with tetracycline for 9 days; whole cell lysates were prepared and analyzed by western blot to detect ErbB3, P-Akt, and actin (control). B. ErbB3fl/fl.MTB-TCre X PyVmT primary tumor cells were injected into the inguinal mammary fatpad of wild-type female FVB mice. When tumors reached ~50 mm3, mice were randomized into treatment groups receiving DOX for an 8-week period or receiving DOX for one-week followed by DOX removal for the remaining 7 weeks of the study. The third group did not receive DOX. Tumor volume was calculated at the end of the 8-week treatment period. Values shown represent the average tumor volume relative to the volume of the tumor with the smallest volume (144 mm3). C. Western analysis of whole tumor lysates harvested from mice at the end of the 8-week period of treatment with DOX. Antibodies used for western analysis are shown at right of each panel. D. Immunohistochemical detection of ErbB3 in tumors from DOX-treated mice. Tumors shown are from mice maintained in DOX for 8 weeks or not.

Figure 5

Figure 5. Systemic delivery of ErbB3 antisense inhibits growth of established MMTV-PyVmT mammary tumors

A MMTV-PyVmT primary tumor cells were injected into the inguinal mammary fat pad of 5-week old wild-type FVB mice. When tumors reached a volume ≥200 mm3, mice were randomized to receiving 25 mg/kg EZN-3920 or EZN-4455 twice weekly via tail vein injection. Tumor volumes were monitored twice weekly and tumor volumes calculated as indicated in Methods. Each data point represents the mean tumor volume ± S.D. (_n_=7; _p_=0.006, Student’s T-test). + indicates identification of dead mouse in cage. @ indicates mouse removed from study due to excessive tumor volume. B–E. Tumors were harvested 24 h after the final of 8 doses and examined histologically. B. Immunohistochemical detection of ErbB3 as described in Methods. C. H&E-stained sections of tumors. Lower magnification is shown in left panels. Boxed areas are viewed in higher power magnification in the right panels. Two boxed areas are indicated in the EZN-3920 samples to demonstrate the acellular compartment (AcC) and the area of actively growing tumor (AGT). D. Immunohistochemical detection of S473 P-Akt. E. The index of cells staining positive for P-Akt and Ki67 was calculated as the number of positive cells divided by the total number of cells. Five randomly chosen fields from each of 5 samples were counted. Values represent the average ± S.D.

Figure 6

Figure 6. ErbB3 ablation with EZN-3920 cooperates with lapatinib to induce tumor cell death

A Cells were treated with EZN-4455 or EZN-3920 for 6 days prior to harvesting whole cell extracts, which were used directly for western analysis or for immunoprecipitation of p85 followed by western analysis. B. Cells were transfected with EZN-3920 or EZN-4455 (5 μM) and then embedded in growth factor-reduced Matrigel. Cells were imaged at day 14. C. Cells cultured in monolayer were transfected with EZN-3920 or EZN-4455 (5 μM) for 72 h. Cells were treated for the final 16 h with trastuzumab (20 μg/ml), lapatinib (1 μM) or DMSO. Cells were fixed and stained for evidence of apoptosis using the Apo-BrdU assay, and measured using flow cytometry. Values shown represent the average percentage of the cell population that was TUNEL-positive, ± S.E.M. (n=3; lapatinib vs. EZN3920 plus lapatinib, p<0.01 for BT-474, SKBR3, and MDA-MB-453 cells). D. Cells were transfected with EZN-3920 or EZN-4455 (5 μM) for 72 h. Cells were treated for the final 1, 4, 24, or 48 h of culture with lapatinib (1 μM) in the presence of absence of trastuzumab (20 μg/ml) or with DMSO. Whole cell extracts were analyzed by western blot with Y1197 P-ErbB3 and total ErbB3 antibodies.

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