HER2YVMA drives rapid development of adenosquamous lung tumors in mice that are sensitive to BIBW2992 and rapamycin combination therapy

Abstract

Mutations in the HER2 kinase domain have been identified in human clinical lung cancer specimens. Here we demonstrate that inducible expression of the most common HER2 mutant (HER2(YVMA)) in mouse lung epithelium causes invasive adenosquamous carcinomas restricted to proximal and distal bronchioles. Continuous expression of HER2(YVMA) is essential for tumor maintenance, suggesting a key role for HER2 in lung adenosquamous tumorigenesis. Preclinical studies assessing the in vivo effect of erlotinib, trastuzumab, BIBW2992, and/or rapamycin on HER2(YVMA) transgenic mice or H1781 xenografts with documented tumor burden revealed that the combination of BIBW2992 and rapamycin is the most effective treatment paradigm causing significant tumor shrinkage. Immunohistochemical analysis of lung tumors treated with BIBW2992 and rapamycin combination revealed decreased phosphorylation levels for proteins in both upstream and downstream arms of MAPK and Akt/mTOR signaling axes, indicating inhibition of these pathways. Based on these findings, clinical testing of the BIBW2992/rapamycin combination in non-small cell lung cancer patients with tumors expressing HER2 mutations is warranted.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Expression of hHER2YVMA rapidly induces carcinomas in the bronchioles at 2 different latencies. (A) Time course analysis of lung tumorigenesis after doxycycline administration using both MRI and histology. MRI photographs demonstrate rapid increase in tumor volume over a 2-week time span for founder 26 whereas 5B shows only a small lesion at 2 weeks (arrow). H&E-stained high-power and low-power (Insets) magnifications show histopathologically similar tumors for 2 founders. Histology was done on moribund mice killed at 4 (founder line 26) or 15 (founder line 5B) weeks. (B) Kaplan–Meier survival curves for 2 different founders. 26 has median latency of 5.4 weeks, and 5B has a median latency of 14 weeks. (C) Western blots for HER2, phospho-HER2, and β-actin proteins from lungs of 26 and 5B founder line mice that were fed a normal diet (−), 1 week of doxycycline diet (+), and 5 weeks of doxycycline diet followed by 3 days of normal diet (+/−).

Fig. 2.

Tumors depend on hHER2YVMA expression for proliferation but apoptose on hHER2YVMA deinduction. (A) Representative photomicrographs of cross-sectional tumors stained with the indicated antibodies from hHER2YVMA founder line 26 on doxycycline diet for 6 weeks (On doxy) and another put on a normal diet for 3 days after 7 weeks of doxycyline diet (Off doxy). At least 3 mice from each founder were analyzed for histology. (B) Representative MRI and histology of 1 of 3 mice taken given a normal diet for 1, 2, or 12 weeks after at least 4 weeks of doxycycline. The tumor around a bronchiolar branch identifiable by MRI (arrow) at 4 weeks on doxycycline is not visible after 1, 2, or 12 weeks off diet (H, heart area). By histology, residual tumor (arrow) is seen up to 2 weeks on normal diet but is completely cleared on prolonged administration of normal diet.

Fig. 3.

Differential response of adenosquamous tumors driven by hHER2YVMA expression to 2-week treatment with erlotinib, trastuzumab, BIBW2992, rapamycin, or BIBW2992/rapamycin combination. (A) MRI, histology, and relative tumor volume of bitransgenic tet-op-hHER2YVMA/CCSP-rtTA mice treated with empty vehicle, erlotinib at 50 mg/kg, trastuzumab at 100 mg/kg twice per week, BIBW2992 at 20 mg/kg, rapamycin at 2 mg/kg, or both BIBW2992 at 20 mg/kg orally and rapamycin at 2 mg/kg every other day by i.p. injection. The mice were imaged with MRI before and after 2 weeks of treatment. Representative imaging is shown for MRI (H, heart area). Histology is from the same mice shown by MRI. The bar in inset is 100 μm. (B) Dot plot diagram expressed with mean ± SEM illustrates the relative tumor regression measured by MRI before and after 2 weeks of treatment for 4–5 different mice. Statistical analyses were performed by using Student's 2-tailed t test. (C) Response of established H1781 NSCLC tumors to BIBW2992, rapamycin, or a combination thereof—BIBW2992 was administered daily p.o. at 20 mg/kg (■) and rapamycin i.p. at 2 mg/kg (▴). In the combination arm (•) both drugs were given concomitantly at the same doses and regimen as the single agents. (♦), growth of untreated tumors. Each group comprised 7 animals; data points are averages, and error bars represent standard deviations. Apoptosis and proliferation assessed by cCK18 staining and Ki-67, respectively, were quantified from 4 tumor fields of 2 different tumors and graphed as a percentage of the total area (Lower). P, placebo; U, untreated; E, erlotinib; T, trastuzumab; B, BIBW2992; R, rapamycin.

Fig. 4.

Immunohistochemical assessment reveals that phosphorylation of HER2 and downstream signaling molecules are most decreased with combination of BIBW2992 and rapamycin. Bitransgenic tet-op-hHER2YVMA/CCSP-rtTA mice were treated with trastuzumab, BIBW2992, rapamycin, or the combination of BIBW2992 and rapamycin therapy for 72 h, and lung sections were then stained with antibodies recognizing phospho-HER2, phospho-EGFR, phospho-Akt, phospho-MAPK, and phospho-S6. IHC photographs are representative fields from duplicate animals in each group. The bar in each photomicrograph is 20 μm. Quantitative analyses of 6 different high-power tumor images from 2 mice are presented as percent positive cells in a tumor area in the bar graphs.