Coexpression of multiple ErbB family members is more predictive of shortened survival than expression of EGFR alone (12), and coactivation of EGFR with HER2 has been implicated in resistance to trastuzumab, a HER2-targeting agent, in breast cancer models (13). EGFR is also shown to be upregulated after long-term exposure to GDC-0941 trastuzumab (14), further reinforcing the critical nature of these redundant pathways to cellular growth in malignancies. Trastuzumab has been shown to resensitize lung cancer cells to cetuximab in vitro (15), likely because HER2 signaling occurs through many of the same downstream effectors as EGFR including mitogen- activated protein kinase (MAPK) and phosphoinositide 3-kinase Although cetuximab produces strong antitumor effects on human cancer cells in vivo (17, 18), it has suboptimal antiproliferative effects in vitro (19, 20) and is best modeled in vitro using invasion assays (21). In the present study, we generated an in vivo model of cetuximab resistance. This in vivo generated model of cetuximab resistance provides a means to biochemically examine relevant mechanisms of resistance.

Furthermore, this model may be used to test the targeting of such resistance EGFR(HER) inhibition mechanisms in vivo to overcome resistance to cetuximab. Here, for the first time in the context of resistance to an EGFR-targeting agent, we describe increased phosphorylation of 611-CTF, a truncated fragment of HER2, in our cetuximab-resistant model. We also show in vivo that combined inhibition of EGFR and HER2 with a dual kinase-targeting agent can overcome resistance to cetuximab. maintained in Dulbecco’s Modified Eagle’s Media (DMEM) with 10% FBS and 0.4 mg/mL hydrocortisone (15). OSC-19 cells were maintained in Minimum Essential Medium with 10% FBS and 1% nonessential amino acids. CAL33, T24, and A431 cells were maintained in DMEM þ 10% FBS. All cell lines were validated by genotyping within 6 months of their use with the AmpFlSTR Identifiler System (Applied Biosystems). Cetuximab-resistant clones were maintained in media with 100 nmol/L cetuximab. Cetuximab (Erbitux; ImClone Systems and Bristol-Myers Squibb) was purchased from the School of Pharmacy, University of Pittsburgh. Afatinib was obtained from Boehringer Ingelheim as a powder and resuspended in dimethyl sulfoxide for in vitro studies or 0.5% methylcellulose with 0.4% Tween 80 in saline for animal studies.

Trastuzumab (Herceptin; Genentech) was purchased from the School of EGFR(HER) inhibitor in clinical trials Pharmacy, University of Pittsburgh, and diluted as recommended in the package insert. Erlotinib was purchased from ChemieTek. Subcutaneous xenografts were generated from 6 different epithelial cancer cell lines (T24, CAL33, A431, OSC-19, SCC1, and SCC1c8; n¼6 for all cell lines except T24, where n ¼ 12) in athymic nude mice, using 1  106 cells with Matrigel (BD Biosciences). After tumor formation (7–10 days), mice received 0.8 mg of cetuximab by intraperitoneal (i.p.) injection twice weekly. Tumors were measured twice weekly. If tumors progressed after 14 days of treatment, dosing was increased to 1.0 mg of cetuximab twice weekly and then 0.8 mg of cetuximab 3 times per week after 28 days. If no tumors were present, the animal was sacrificed after 90 days of treatment. If tumors were present, the animal was sacrificed at 90 days or when the tumor diameter exceeded 20 mm. Tumors were removed, digested, and suspended as single cells, which were propagated in culture and reinoculated as 2 subcutaneous xenografts. These tumors were treated with 0.8 mg of cetuximab 3 times per week immediately following tumor formation. For the differential sensitivity study, 1  106 parental and resistant cells were blindly injected on opposite flanks of the same mouse (n¼7) with Matrigel. Treatment began following tumor formation. Animals were treated with 2.0 mg of cetuximab 3 times weekly by i.p. injection. For the combination EGFR(HER) inhibitor drug study, 2  106 parental and resistant cells were injected on opposite flanks of the same mouse (n ¼ 40) with Matrigel, and animals were stratified by tumor volume (22) into 4 groups and then randomly distributed from each group into 4 treatment groups with 10 animals per group. Animals were treated with cetuximab, afatinib, or both. The treatments and measurements were conducted by an individual blinded to the treatment. One milligram of cetuximab or vehicle control was given by i.p.