2011;121:2723C2735. with CH12 against EGFRvIII+HER2+ breast cancers, which might be a potential clinical application in the future. resistance), and many trastuzumab-responsive patients develop resistance after continuous treatment (acquired resistance) [9, 10]. Two major categories of SGC-CBP30 trastuzumab resistance mechanisms have been proposed: (I) resistance due to genetic alterations of receptor tyrosine kinases (RTKs) and their downstream signaling targets, such as aberrant activation of the PI3K/AKT pathway due to phosphatase and tensin homolog (PTEN) deficiency or PIK3CA gene activating mutations [11, 12], and the accumulation of truncated HER2 receptors (p95HER2) that lack the trastuzumab-binding domain name ; and (II) acquired resistance primarily due to the acquisition of option RTK or feedback signal activation that compensate for HER2 inhibition after trastuzumab treatment [14C16]. EGFR, an essential RTK, playing an vital role in cell differentiation, proliferation, and survival in a number of human cancers, also contribute to both and acquired trastuzumab resistance [14, 17]. Accumulating reports have exhibited that EGFRvIII, the most common EGFR mutant forms with constitutively activated kinase domain name [18, 19], expresses in various human cancers, including breast malignancy, and it has not been detected in normal adult human tissue [20, 21]. EGFRvIII expression was detected in approximately 5% of primary breast cancer cases and contributes to malignancy stem cell phenotypes in invasive breast carcinomas . Furthermore, approximately 40% of HER2-positive primary breast tumors were found to co-express EGFRvIII, and, even more striking, 75% of HER2-positive metastatic lymph node specimens co-expressed EGFRvIII . EGFRvIII is usually posited to be involved in tumorigenicity, invasiveness, and metastasis in breast cancers [22C24]. Several strategies SGC-CBP30 against EGFRvIII-positive tumors have been explored. For instance, anti-EGFRvIII antibodies, such as mAb 806 and CH12, which selectively bind to a cancer-specific epitope of EGFR or EGFRvIII, have been demonstrated to be capable of efficiently inhibiting the growth of EGFRvIII-positive tumor xenografts [21, 25, 26]. However, it needs to be decided whether these antibodies have efficacy against breast tumors with EGFRvIII overexpression. Considering the co-expression of HER2 and EGFRvIII in breast cancers, we predicted that HER2 and EGFRvIII might cooperate for tumor growth, and EGFRvIII expression might contribute to trastuzumab resistance. However, to date, no treatment NFKBI strategies have been explored on EGFRvIII+HER2+ breast cancers. Therefore, in this study, we examined the combination effect of trastuzumab and CH12 around the EGFRvIII+HER2+ breast cancer cells and the molecular mechanisms underlying their antitumor effects. RESULTS EGFRvIII overexpression decreased the sensitivity of breast cancers to trastuzumab The EGFRvIII encoding sequence was introduced into the HER2-positive breast malignancy cell lines BT474 and SKBR3, and the established EGFRvIII+HER2+ cells (Physique S1) were less sensitive to trastuzumab SGC-CBP30 than their parental cells (Physique ?(Figure1A).1A). Furthermore, the antitumor efficacy of trastuzumab in BT474-EGFRvIII xenografts in nude mice was slightly weaker than that in parental BT474 model (Physique ?(Figure1B).1B). The inhibition rate of trastuzumab at a concentration of 2 mg/kg was 51% in BT474-parental xenograft, while it was 43.7% in BT474-EGFRvIII model ( 0.01). Open in a separate window Physique 1 EGFRvIII overexpression decreased the sensitivity of breast cancers to trastuzumabA. Cell proliferation of parental BT474 and SKBR3 cell lines and their corresponding EGFRvIII overexpression sublines upon treatment with trastuzumab at a concentration of 1 1 g/mL for BT474 and at a concentration of 20 g/mL for SKBR3. Data are expressed as the inhibition rate of cell growth in triplicate experiments (Bars, SD). Statistical signi?cance is indicated parental cells * 0.05, ** 0.01, *** 0.001. B. Growth curve of xenografts derived from either BT474 parental or EGFRvIII overexpression subline upon treatment with vehicle or trastuzumab at a concentration of 2 and 5 mg/kg intraperitoneally, once a week for 2 weeks. The data are expressed as mean tumor volumes SE. C., D. Changes of EGFR or HER2 relevant signaling after trastuzumab treament in BT474 and BT474-EGFRvIII C..