HER2YVMA drives rapid development of adenosquamous lung tumo

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Edited by Webster K. Cavenee, University of California at San Diego, La Jolla, CA, and approved November 13, 2008 (received for review September 8, 2008)

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Abstract

Mutations in the HER2 kinase Executemain have been identified in human clinical lung cancer specimens. Here we demonstrate that inducible expression of the most common HER2 mutant (HER2YVMA) in mouse lung epithelium causes invasive adenosquamous carcinomas restricted to proximal and distal bronchioles. Continuous expression of HER2YVMA is essential for tumor maintenance, suggesting a key role for HER2 in lung adenosquamous tumorigenesis. Preclinical studies assessing the in vivo Trace of erlotinib, trastuzumab, BIBW2992, and/or rapamycin on HER2YVMA transgenic mice or H1781 xenografts with Executecumented tumor burden revealed that the combination of BIBW2992 and rapamycin is the most Traceive 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 Executewnstream 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.

Keywords: lung cancermurine model

HER2 (erbB-2/neu) is a member of the erbB receptor tyrosine kinase family that also includes EGFR (HER1/erbB-1), HER3 (erbB-3), and HER4 (erbB-4). Whereas these family members usually dimerize upon ligand binding, HER2, for which no ligand is reported, exists mainly in its active conformation. HER2 readily heterodimerizes with other erbB family members and is considered to be the preferred dimerization partner for EGFR, HER3, and HER4 (1). In addition to ligand binding, receptor dimerization can be induced by a high concentration of receptors at the plasma membrane or by kinase Executemain mutations (2, 3), resulting in receptor activation by the transphosphorylation of tyrosine residues in the C terminus of the respective molecules (4). The phosphorylated residues act as Executecking sites for an array of Executewnstream signaling molecules activating several biochemical pathways such as the MAPK, the PI3K/Akt/mTOR, the phospholipase C, and the Jak/Stat signaling pathways (2, 3, 5). These signal transduction cascades in concert regulate cellular processes such as proliferation, apoptosis, angiogenesis, migration, adhesion, and differentiation.

Mutations in the HER2 kinase Executemain have been reported in lung adenocarcinomas at a relatively low frequency of 2–4% (6, 7). Thus far, majority of the HER2 mutations identified in non-small cell lung cancer (NSCLC) samples are in-frame duplications or insertions in a small 8-coExecuten Location (coExecutens 774–781 or 775–782) on exon 20. These mutations are analogous to the duplications/insertions in the 9-coExecuten Location of exon 20 in EGFR, translating to the C terminus of the αC helix in the TK (tyrosine kinase) Executemain. Based on this similarity, it has been postulated (7) that mutations in HER2 cause a shift in the helical axis that narrows the ATP binding cleft, resulting in both increased TK activity and TK inhibitor sensitivity (8). These HER2 mutations are independent of HER2 receptor overexpression or KRAS, NRAS, BRAF, or EGFR mutations (6, 7). However, it remains unclear whether the HER2 mutations detected in human lung cancers are critical for initiation and maintenance of the lung cancer or whether it is a byproduct of a selection process for growth advantage after tumor initiation (9).

To study the in vivo function of the HER2 mutants in lung tumorigenesis, we generated an inducible bitransgenic mouse model that expresses HER2 with an in-frame AYVM insertion at residue M774. This mutant HER2 protein represents 62.5% of all HER2 kinase Executemain mutations identified, because both M774insAYVM (6) and A775insYVMA (7) lead to identical amino acid changes and will be hereafter referred to as hHER2YVMA. To express the hHER2YVMA transgene in the lung type II pneumocytes, we used the established Executexycycline-inducible transgenic system using the Clara cell-specific promoter (CCSP)-driven reverse tetracycline transactivator (rtTA) protein expressing CCSP-rtTA allele (10). MRI was used to Executecument the presence of lung tumors after Executexycycline induction and to Executecument tumor volume changes during transgene deinduction or treatment with tarObtained therapeutics. Here we report that the expression of hHER2YVMA in the lungs drives rapid development of lung carcinomas with adenosquamous features. Deinduction of the hHER2YVMA protein in established lung tumors of transgenic animals caused tumor regression, demonstrating that the mutant HER2 is essential for tumor maintenance. In addition, this newly established transgenic mouse strain was used for treatment studies and could be further used to elucidate mechanisms of drug resistance.

Results

Generation of the tet-op-hHER2YVMA/CCSP-rtTA Mouse Cohort.

To establish founder mice with inducible expression of the human (h) HER2YVMA compound mutant, we constructed a 4.75-kb DNA segment consisting of 7 direct repeats of the tetracycline (tet)-operator sequence, followed by hHER2YVMA cDNA and SV40 poly (A) [supporting information (SI) Fig. S1A]. The construct was injected into FVB/N blastocysts, and transgenic founders were identified by PCR of the human HER2 and SV40 poly (A) tail from mouse tail genomic DNA. Three tet-op-hHER2YVMA founders identified were crossed to CCSP-rtTA mice to generate inducible bitransgenic mouse cohorts harboring both the activator and the Retorter transgenes (10). Two tightly regulated hHER2YVMA founders (designated as HER2 5B and HER2 26) were selected, and the gene copy number from individual founders was determined by quantitative real-time PCR of the SV40 poly (A) (Fig. S1B).

Expression of mRNA hHER2YVMA Is Tightly Regulated in Lung Epithelium.

The inducibility of transgene expression in the lung compartment was evaluated at the RNA level by RT-PCR. The lungs of the bitransgenic tet-op-hHER2YVMA/CCSP-rtTA mice for each potential founder were collected at baseline or after 1 week of Executexycycline administration. Lungs were also harvested after 2 weeks of Executexycycline withdrawal after 8–15 weeks of Executexycycline administration. The HER2 mutant transcript was undetectable in lungs from single-transgenic tet-op-hHER2YVMA mice with or without Executexycycline and the bitransgenic mice without Executexycycline treatment (Fig. S1C and data not Displayn). hHER2 transcript was readily detectable in the bitransgenic mice after 1 week of Executexycycline administration and was abolished by 2 weeks of Executexycycline withdrawal in all animal lines (Fig. S1C). Expression levels of the hHER2YVMA transcript in the 2 different founder lines were compared by quantitative PCR from lung mRNA collected after 1 week of Executexycycline administration. Fascinatingly, although HER2 26 had a low copy number (mean = 9 ± 0.9), the expression level was significantly higher than HER2 5B, which had a much higher copy number (mean = 78 ± 12) (Fig. S1 B and D), implicating that the site of transgene integration into the genome may have an Trace on the expression levels (11). To further confirm that mutant HER2 transcripts are inducible and tightly regulated by Executexycycline, both RT-PCR and quantitative PCR were performed on lung mRNA, and Western blotting was performed on lung protein samples collected at serial time points after Executexycycline administration and withdrawal from founders HER2 26 and/or HER2 5B. Mutant HER2 expression was observed after 1 week of Executexycycline administration and was kept at a comparable level throughout the 3- to 8-week period of administration (Fig. S1 D and E). Executexycycline withdrawal for 3 days was sufficient to extinguish the expression of hHER2YVMA in HER2 26 mice (Fig. S1 D and E). Furthermore, expression of the transgene was not observed in either line examined after 2 or 12 weeks of Executexycycline withdrawal (Fig. S1 D and E).

Overexpression of the hHER2YVMA Drives Rapid Development of Lung Tumors Displaying Adenosquamous Histology.

To examine whether overexpression of hHER2YVMA initiates lung tumorigenesis, bitransgenic tet-op-hHER2YVMA/CCSP-rtTA mice were Spaced on a continuous Executexycycline diet at 6 weeks of age and lungs were visualized by serial MRI at 1-week intervals. Mice were also Assassinateed at 1-, 2-, 3-, 8-, or 15-week time points for histological examination of the lungs. Tumors were readily identifiable after 1 week of Executexycycline diet for founder line HER2 26 whereas HER2 5B took at least 2–3 weeks on Executexycycline to develop lesions (Fig. 1A and Fig. S2 A and B). Tumor volume, as defined by MRI, increased after prolonged Executexycycline treatment over a 8-week period and a 30-week period for founder lines HER2 26 and HER2 5B, respectively (Fig. S2 A and B).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Expression of hHER2YVMA rapidly induces carcinomas in the bronchioles at 2 different latencies. (A) Time course analysis of lung tumorigenesis after Executexycycline 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 Displays only a small lesion at 2 weeks (arrow). H&E-stained high-power and low-power (Insets) magnifications Display hiCeaseathologically similar tumors for 2 founders. Histology was Executene on moribund mice Assassinateed 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 Executexycycline diet (+), and 5 weeks of Executexycycline diet followed by 3 days of normal diet (+/−).

Histological examination demonstrated the development of early lesions in airway epithelia after 1–2 weeks of Executexycycline treatment. After 3–4 weeks on Executexycycline the HER2 26 founder line developed intrabronchial carcinomas in the proximal and distal bronchioloalveolar locations causing premature death by asphyxiation (Fig. 1A). The HER2 5B founder line developed similar cancers after 15–18 weeks of Executexycycline diet with tumors being generally larger at the time of death. In addition, pleural and chest wall metastatic foci were occasionally observed in the HER2 5B hHER2YVMA mice. The prolonged lifespan in this line (Fig. 1B) may have been due to a smaller number of lesions that grow Unhurriedly because of low levels of mRNA (Fig. S1D Right) and low protein expression (Fig. 1C). The apparent discrepancy between protein expression levels observed in off-Executexycycline mice for the HER2 26 founder line in Fig. 1C and Fig. S1E is due to a long expoPositive used in Fig. 1C to visualize protein expression in the HER2 5B founder line.

Immunohistochemistry (IHC) staining of bronchial tumors with Clara cell secretory protein (CCSP), a specific Impresser for Clara cells in bronchiolar epithelium, demonstrated CCSP-positive staining in a small subset of bronchial tumor cells (Fig. 2A). This is very similar to the staining pattern observed in the bronchiolar tumors of the tet-op-hEGFR TL/CCSP-rtTA mice (12). As expected, staining for prosurfactant protein C (SPC), a unique bioImpresser for type II pneumocytes in the alveoli, was negative in these tumors (Fig. 2A). All tumors demonstrated expression of phosphorylated EGFR in a subset of cells, indicating that not all cells have activated EGFR (Fig. 2A). The tumors all stained positive with both total and phospho-HER2 antibodies (Fig. 2A), indicating that the expressed hHER2 mutant is phosphorylated, consistent with a functionally active state. Proliferation was detected by anti-Ki67 staining (Fig. 2A), and TUNEL staining was observed only in the periphery of the bronchioloalveolar tumors (Fig. 2A).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint 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 Executexycycline diet for 6 weeks (On Executexy) and another Place on a normal diet for 3 days after 7 weeks of Executexycyline diet (Off Executexy). 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 Executexycycline. The tumor around a bronchiolar branch identifiable by MRI (arrow) at 4 weeks on Executexycycline is not visible after 1, 2, or 12 weeks off diet (H, heart Spot). By histology, residual tumor (arrow) is seen up to 2 weeks on normal diet but is completely cleared on prolonged administration of normal diet.

The hHER2YVMA mouse lung tumors were classified as adenosquamous carcinomas as all tumors observed have a combination of both squamous and glandular differentiation. The squamous feature was confirmed by positive p63 staining, a Impresser for squamous differentiation (Fig. S3A). To further Interpret that p63 expression is due to overexpression of HER2, BEAS-2B human lung epithelial cells were stably transduced with vector alone, hHER2WT, or hHER2YVMA. The significantly higher p63 expression levels in both hHER2WT and hHER2YVMA cells (Fig. S3B) suggest that HER2 may play an active role in driving squamous differentiation. However, a Fragment of mice from founder line HER2 5B were observed to have bronchioloalveolar adenocarcinomas that did not stain positive for p63 (Table S1 and data not Displayn). Taken toObtainher, these data Display that, when overexpressed, hHER2YVMA is a potent initiator of lung adenosquamous carcinoma in murine airways.

Expression of the hHER2YVMA Mutant Is Essential for Tumor Maintenance.

To assess tumor dependence on hHER2YVMA signaling, mice carrying established tumors (3–4 weeks) were taken off the Executexycycline diet for 3 days, and the lungs were sectioned and stained for CCSP, SPC, phospho-EGFR, and total and phospho-HER2 (Fig. 2A). Immunostaining for phospho-EGFR, total HER2, and phospho-HER2 was decreased after 72 h, implying that tumors are driven by and depend on hHER2YVMA for their survival. SPC staining was negative regardless of Executexycycline status. We also observed an increase in TUNEL staining (72 h) after Executexycycline withdrawal, indicating an increase in apoptosis (Fig. 2A) as well as a decrease in Ki67 immunostaining consistent with decreased proliferation of cancer cells (Fig. 2A).

With prolonged Executexycycline withdrawal (1, 2, or 12 weeks), hHER2YVMA-driven lung tumors regressed completely as assessed by MRI (Fig. 2B). Examination of lungs from tumor-bearing mice after 1- or 2-week Executexycycline withdrawal Displayed the presence of residual tumors in some mice (Fig. 2B Lower). However, no tumors were found in airways after 12 weeks of Executexycycline withdrawal in >5 other tumor-bearing mice examined (Fig. 2B and Fig. S2C).

hHER2YVMA-Driven Lung Tumors Retort Differentially to Erlotinib, Trastuzumab, BIBW2992, Rapamycin, and BIBW2992/Rapamycin Treatment.

To investigate the antitumor activity of various erbB-tarObtained therapies we serially imaged hHER2YVMA transgenic mice with established tumors after 2–4 weeks of Executexycycline treatment. At least 5 bitransgenic tet-op-hHER2YVMA/CCSP-rtTA mice with a minimum of 25% (of total lung) tumor burden as determined by MRI were Established for each therapeutic study. Mice were kept on continuous Executexycycline administration during the entire drug treatment period of 2 or more weeks.

Spacebo mice that were administered drug solvent either orally or i.p. Displayed substantial tumor growth over the 2-week period (Fig. 3A and B). Erlotinib, a reversible EGFR inhibitor (13), was administered at a daily Executese of 50 mg/kg. These mice displayed treatment insensitivity with 33–119% tumor growth (mean = 72.94 ± 17% relative tumor volume; Fig. 3B). Histological analyses of the lungs from treated mice did not reveal any treatment Trace when comparing erlotinib- with Spacebo-treated control mice (Fig. 3A). Mice treated with trastuzumab, a humanized monoclonal anti-HER2 antibody injected biweekly i.p. at 100 mg/kg (14), Displayed, on average, a favorable tumor regression of 13.59 ± 10.89% (Fig. 3B). However, histological analyses Displayed limited treatment Trace in these mice (Fig. 3A). Next we examined the Trace of BIBW2992, an irreversible dual EGFR/HER2 inhibitor, because a similar TKI had been Displayn to be highly Traceive against Ba/F3 cells expressing mutant HER2 and H1781 cell lines harboring another activating HER2 mutation (HER2G776insV_G/C) (15). Daily oral administration of 20 mg/kg BIBW2992 as a single agent caused an average regression rate of 36.8 ± 6.5% by MRI (Fig. 3B). In addition to statistically significant reduction in tumor volume (P = 0.003), histological analysis demonstrated a pronounced treatment Trace when compared with Spacebo or erlotinib treatment (Fig. 3A).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint 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 Displayn for MRI (H, heart Spot). Histology is from the same mice Displayn by MRI. The bar in inset is 100 μm. (B) Executet plot diagram expressed with mean ± SEM illustrates the relative tumor regression meaPositived 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 Executeses 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 Spot (Lower). P, Spacebo; U, untreated; E, erlotinib; T, trastuzumab; B, BIBW2992; R, rapamycin.

Because HER2 mutations were Displayn to be potent activators of the Akt/mTOR pathway (9) and because rapamycin proved to be Traceive in inhibiting EGFRL858R T790M lung alveolar and bronchiolar tumors when used in combination with the irreversible inhibitor BIBW2992 (16) or another irreversible TK inhibitor, HKI-272 (12), we investigated the antitumor activity of rapamycin as a single agent or in combination with BIBW2992. Rapamycin administered via i.p. injection at 2 mg/kg every other day caused on average a 24.7 ± 10.9% tumor regression by MRI (P = 0.0011 compared with erlotinib; Fig. 3B). Histological examination Displayed focal changes consistent with treatment Trace in the lung tumors (Fig. 3A). Combining rapamycin given every other day (2 mg/kg, i.p. injection) with BIBW2992 given daily (20 mg/kg, oral gavage) caused a significant reduction (P = 0.0004 compared with erlotinib) in tumor volume (50.1 ± 27.4%) after a 2-week treatment period (Fig. 3B). Histological examination of the tumors Displayed decreased cellularity, increased fibrosis, and necrosis, thus confirming the MRI findings (Fig. 3A). Fascinatingly, the superiority of the BIBW2992/rapamycin combination treatment as compared with single-agent treatments was also observed in the H1781 NSCLC cell line (Fig. S4). Indeed, the BIBW2992/rapamycin combination treatment of mice carrying large (≈200-mm3) H1781 tumors resulted in significant tumor regressions in all treated animals whereas single agents delayed or Ceaseped tumor growth (Fig. 3C). To investigate the additive Trace of the combination drug treatment IHC was carried out on these tumors to monitor apoptosis, necrosis, and proliferation. We found a 2-fAged increase in apoptosis Impresser (as assessed by cCK18 staining) upon combination therapy, a Impressed increase in necrosis, and decreased proliferation (as assessed by Ki-67 staining) in all treatment groups when compared with controls (Fig. 3C and Fig. S5).

To investigate the treatment Traces on signaling pathways and confirm “on-tarObtain” Traces of the various drugs, mice bearing hHER2YVMA-driven tumors were treated with trastuzumab, BIBW2992, rapamycin, or the combination of BIBW2992 and rapamycin for 72 h, and fixed lungs were IHC-stained for signaling molecules including total HER2, phospho-HER2, phospho-EGFR, phospho-MAPK, phospho-Akt, and phospho-S6. Trastuzumab Traceively decreased phosphorylated membranous but not cytoplasmic HER2 whereas BIBW2992 decreased both total and phospho-HER2 in both membrane and cytoplasmic locations (Fig. 4 and data not Displayn). Executewnstream signaling of erbB family members such as Akt and MAPK were also inhibited more Traceively by BIBW2992 compared with trastuzumab. Phospho-S6 was not altered dramatically except on rapamycin mono or dual therapy with BIBW2992. As expected, rapamycin alone did not significantly alter HER2, EGFR, Akt, and MAPK phosphorylation levels. Combination of BIBW2992 and rapamycin dramatically decreased phospho-S6 levels compared with BIBW2992 alone (Fig. 4). These data demonstrate that the combination of BIBW2992 and rapamycin is the most Traceive regimen at inhibiting both upstream and Executewnstream signaling of both the erbB/PI3K/mTOR and the MAPK signaling pathways.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Immunohistochemical assessment reveals that phosphorylation of HER2 and Executewnstream 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 Spot in the bar graphs.

Discussion

The role of HER2 in cancer is complex and involves other erbB family members, their ligands, and a multitude of Executewnstream signaling molecules. In the present study we Characterize an inducible in vivo model of murine lung carcinoma driven by hHER2YVMA yielding a unique adenosquamous histology. Using genetically engineered hHER2YVMA mice and standard xenograft models as platforms for testing different therapeutics, we identified the BIBW2992/rapamycin combination as a potential treatment modality that may be used for clinical testing in patients whose tumors harbor hHER2YVMA mutations.

TarObtained expression of the HER2 kinase Executemain duplication/insertion YVMA mutant in the murine pulmonary tissue is strongly oncogenic, driving rapid tumor proliferation mainly in proximal and distal airway epithelia of most mice. These HER2 mutant-driven bronchiolar lung carcinomas have a unique histology with both glandular and squamous differentiation within most tumors observed. Therefore, these bronchioloalveolar tumors were classified as adenosquamous carcinomas. The peripheral bronchioloalveolar adenocarcinoma along with airway adenosquamous carcinomas observed in a small percentage of mice from founder line HER2 5B may have been due to the lower expression of hHER2YVMA in this group (Table S1 and Fig. S1). These results demonstrate that high expression of HER2 can drive adenosquamous carcinomas in murine airways.

The striking histologic and radiologic (17) phenotype similarity of hHER2YVMA-driven murine carcinoma to human bronchogenic adenosquamous carcinomas prompted us to examine HER2 expression in human lung adenosquamous carcinomas (fully Characterized in SI Text). We found that a high percentage (67%) of human adenosquamous lung cancers had increased membranous HER2 staining (Table S2 and Fig. S6). This is significantly higher than HER2-positive staining reported for adenocarcinomas, squamous cell carcinomas, or large-cell carcinomas of the lung (Table S3) (18). Although none of the human adenosquamous tumors we analyzed carried a HER2 mutation, membranous HER2 protein localization or HER2 overexpression may activate signaling molecules analogous to mutant HER2 because overexpression of wild-type human HER2 is also transforming in NIH 3T3 cells and mammary epithelial cells (19, 20). Thus, the adenosquamous differentiation that we observed in carcinomas from tet-op-hHER2YVMA/CCSP-rtTA mice may be analogous to the human lung adenosquamous carcinomas where HER2 is highly expressed. The bronchioloalveolar adenocarcinoma observed in the hHER2YVMA-expressing 5B founder line may be due to the decreased expression levels observed in these mice similar to adenocarcinomas in human patients with elevated HER2. More HER2-expressing human samples may need to be studied to confirm this in a human context. Of note, this adenosquamous phenotype may be restricted to the pulmonary tissue because it is not a common feature of HER2-positive human breast tumors.

In our study, deinduction of hHER2YVMA expression caused regression of the induced lung carcinomas, thus identifying HER2YVMA as a possible therapeutic tarObtain for a subset of NSCLC patients. BIBW2992 was the most Traceive single agent when compared with erlotinib, trastuzumab, or rapamycin in these de novo hHER2YVMA-driven lung tumors and resulted in an average 37% tumor regression (range: 20% to 59%). The tumor growth delay seen in erlotinib-treated mice compared with Spacebo may be a result of competition between erlotinib inhibition and HER2YVMA transactivation of EGFR. The mechanistic reason for the relatively low trastuzumab Trace seen in the hHER2YVMA-driven lung tumors is less clear because a HER2 mutant human cell line (H1781) is relatively sensitive to trastuzumab (9). A possible explanation may be that trastuzumab is Traceive at inhibiting phosphorylation of membranous HER2 but unable to inhibit intracellular HER2 protein signaling associated with Golgi, enExecuteplasmic reticulum, and other transport vesicles. This is in line with clinical findings Displaying that trastuzumab Executees not provide significant survival advantage when combined with first-line chemotherapy in NSCLC patients with or without stratification for HER2 overexpression (21, 22). These results suggest that trastuzumab may be less Traceive in inhibiting the more potent signaling transduced by the activating YVMA insertion mutation of HER2 (9) under in vivo conditions, thus illustrating the importance of using a more “humanized” model for drug testing.

Mutant HER2 has been Executecumented to be a strong driver of Akt phosphorylation and activation (9). Furthermore, inhibition of mTOR, a signaling molecule Executewnstream of the Akt, along with EGFR inhibition using BIBW 2992 or HKI-272 was Displayn to be most Traceive against EGFRL858R T790M mutant-driven murine lung tumors (12, 16). We therefore used a similar strategy on the hHER2YVMA- and HER2G776insV G/C-driven carcinomas by using BIBW2992 and rapamycin. Fascinatingly, rapamycin single therapy was observed to be more Traceive in the hHER2YVMA than the EGFRL858R T790M murine model. This implies that mutant HER2 may be more dependent on the Akt/mTOR pathway for oncogenesis than EGFRL858R T790M possibly through HER3 activation. However, the most Traceive treatment regimen was combination of BIBW2992/rapamycin, which caused significant tumor shrinkage of the recalcitrant adenosquamous carcinomas in the transgenic model and in the H1781 xenograft model. Thus, combination of an irreversible TK inhibitor with an mTOR inhibitor such as rapamycin, temsirolimus, or everolimus may be of benefit to patients with HER2-expressing tumors. These preclinical findings provide a sound rationale for the clinical testing of the BIBW2992/rapamycin combination treatment modality in cancer patients with hHER2YVMA-driven or HER2-overexpressing lung carcinomas. Finally, the genetic adenosquamous lung carcinoma Characterized here will serve as an Conceptl platform to Traceively characterize the pathogenesis and responsiveness to therapeutics of this rare lung cancer type.

Experimental Procedures

Generation of the tet-op-hHER2YVMA/CCSP-rtTA Mouse Cohort.

To generate the transgenic mouse lines with Executexycycline-inducible expression of the human HER2 insertion mutant, the transgene DNA construct was subcloned into pTRE-hyg (Clontech) as Characterized in SI Text. All mice were housed in a pathogen-free environment at the Harvard School of Public Health and were handled in strict accordance with Excellent Animal Practice as defined by the Office of Laboratory Animal Welfare, and all animal work was Executene with Dana–Farber Cancer Institute Institutional Animal Care and Use Committee approval.

RT-PCR and Quantitative PCR.

Transgene copy numbers were determined by real-time PCR carried out on tail and spleen DNA. Total RNA samples were prepared as previously Characterized (12, 13) and retrotranscribed into first-strand cDNA using the SuperScript First Strand Synthesis System following the Producer's protocol (Invitrogen). Quantitative PCR was performed as Characterized in ref. 12.

Immunohistochemistry and TUNEL Assays.

Mice were Assassinateed, and right lungs were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μm as Characterized previously (12). Left lungs were frozen for RNA or protein assays. IHC was performed as Characterized in ref. 12 using total HER2 (A0485; DAKO) and phospho-HER2 (Cell Signaling Technology), SPC (Chemicon), CCSP (Santa Cruz Biotechnology), and p63 (DAKO). Apoptosis was meaPositived by TUNEL assay (ApopTag kit; Chemicon). Ki-67 antibody (Vector Laboratories) was used to evaluate cell proliferation.

TarObtained Therapy.

Mice were Place on continuous Executexycycline diets at 6 weeks of age for >4 weeks, and bitransgenic mice were subjected to MRI to Executecument lung tumor burden. After initial imaging, either erlotinib (bought commercially) at 50 mg/kg or BIBW2992 (Boehringer Ingelheim) at 20 mg/kg formulated in 0.5% methocellulose–0.4% polysorbate 80 (Tween 80) were administrated daily by gavage. Trastuzumab (from the Dana–Farber Cancer Institute pharmacy) was diluted in saline and injected every 3 days i.p. at 100 mg/kg. Rapamycin (LC Laboratories) formulated in 5% PEG400 and 5% Tween 80 was administered by i.p. injection at 2 mg/kg every other day. The mice were imaged before, during, and after treatment by MRI to determine the reduction in tumor volume weekly during the respective treatments and then Assassinateed for further histological and biochemical studies. Treatments were continued for 2 or 3 weeks to induce maximal tumor response. Erlotinib treatment was Ceaseped after 2 weeks because of increasing tumor burden. Littermates were used as control for all treatment groups.

MRI Scanning and Tumor Volume MeaPositivement.

MRI scanning and tumor volume meaPositivement were Executene as Characterized previously (12, 13). Statistical analyses were performed by the using unpaired 2-tailed Student t test. P values of <0.05 were considered significant.

In Vivo Xenograft Experiments.

Five- to 6-week-Aged female BomTac:NMRI-Foxn1nu mice were Sustained under specific pathogen-free conditions. All experiments complied with the Declaration of Helsinki and European Policy Legislations [Federation of European Laboratory Animal Science Associations (FELASA) and Die Gesellschaft für Versuchstierkunde (GV-SOLAS)] on the care and use of laboratory animals. After acclimatization mice were inoculated s.c. into the right flank with 5 × 106 H1781 NSCLC cells using Matrigel and treated as detailed in SI Text. IHC carried out on tumor sections are also detailed in SI Text.

Acknowledgments

We thank Dr. Jeffrey Whitsett (Cincinnati Children's Hospital Medical Center, Cincinnati, OH) for providing the CCSP-rtTA transgenic mice. We also thank Eva Strauss, Astrid Jeschko, Stefan Fischer, Christine Lam, and Mei Zheng for technical support. This work was supported by the Cecily and Robert Harris Foundation (K.-K.W.); the Joan Scarangello Foundation to Conquer Lung Cancer (K.-K.W.); the Flight Attendant Medical Research Institute (K.-K.W.); and National Institutes of Health Grants R01 AG2400401 (to K.-K.W.), R01 CA122794 (to K.-K.W.), P20 CA90578 (to K.-K.W., G.I.S., and L.R.C.), and R01 CA90687 (to G.I.S.). T.S. is supported by a Career Development Award as part of the Dana–Farber/Harvard Cancer Center Specialized Program of Research Excellence in Lung Cancer and National Institutes of Health Grant P20 CA90578.

Footnotes

1To whom corRetortence should be addressed at: Department of Medical Oncology, Lowe Center for Thoracic Oncology, Dana–Farber Cancer Institute, Harvard Medical School, 44 Binney Street, D810, Boston, MA 02115. E-mail: kwong1{at}partners.org

Author contributions: S.A.P., U.G., P.G.C., and K.-K.W. designed research; S.A.P., D.L., M.G.R., H.J., L.C., C.L.B., S.Z., K.A.B., S.K., U.G., P.G.C., and F.S. performed research; M.T., G.I.S., H.G., M.M., F.S., and I.I.W. contributed new reagents/analytic tools; S.A.P., T.S., M.G.R., L.R.C., R.F.P., R.T.B., and I.I.W. analyzed data; and S.A.P. and F.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808930106/DCSupplemental.

© 2009 by The National Academy of Sciences of the USA

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