Cetuximabsensitive tumors showed a 64.8% reduction in tumor volume on day 10 of cetuximab treatment compared with a 3.9-fold increase in cetuximab-resistant tumor volumes on day 10 of cetuximab treatment (Fig. 2A; P ¼ 0.002). Frozen tumors were fixed, cryosectioned, and TUNEL-stained to detect apoptotic cells. A total of 61.7% of cells from cetuximab-sensitive tumors (T24) were apoptotic compared with only 26.3% of the cells from tumors derived from cetuximab-resistant cells (T24PR3, Fig. 2A; P ¼ 0.03). These results show that by gradually increasing the dose of cetuximab in vivo over the Ispinesib course of 28 days, cetuximab-resistant tumors can be generated. To show the differential cetuximab sensitivity of this model in vitro, we conducted invasion assays, as cetuximab does not inhibit proliferation in vitro (20).

Cetuximab has been previously reported by us and others to successfully decrease cell invasion through a Matrigel-coated Transwell migration chamber (23, 24). In this model, cetuximab decreased the invasion of parental T24 cells by 55.5% after 24 hours. In contrast, cetuximab only inhibited the invasion of T24PR3 and T24PR4 cells by 1.7% (P¼0.0009) and 8.7% (P ¼ 0.0001), respectively (Fig. 2B). We used a candidate-based approach to explore differences in the cetuximab-sensitive and cetuximab-resistant cells, focusing primarily on the expression and phosphorylation of ErbB family members. Consistent with other in vitro studies of cetuximab resistance (25), EGFR was Entinostat MS-275 downregulated in cetuximab-resistant T24PR3 and T24PR4 cells compared with the isogenic parental T24 cells and the other cetuximab-sensitive cell lines used in this study (Fig. 3A). HER3 was expressed at low levels in T24, T24PR3, and T24PR4 clones, and we observed no significant difference in expression of total or phosphorylated levels of HER3 across these cell lines (data not shown). Furthermore, although there was no significant change in the expression or phosphorylation status of full-length HER2 among cetuximab-sensitive and cetuximab-resistant cells, we observed a marked increase in phosphorylation of 611- CTF, a C-terminal fragment of HER2 containing the transmembrane domain, in only the cetuximab-resistant cells (Fig. 3A).

Despite the abundance of total 611-CTF protein in T24, T24PR3, T24PR4, and other cells, 611-CTF seems to be phosphorylated at Tyr1248, the site responsible for MAPK activation, in only the cetuximab-resistant clones T24PR3 and T24PR4. Densitometry buy Entinostat confirms T24PR3 and T24PR4 cells to significantly express phosphorylated 611- CTF at levels 5.6-fold (P ¼ 0.0223) and 5.9-fold (P ¼ 0.0309) higher, respectively, than T24 cells (Fig. 3A). Although no significant changes were observed in expression of basal or phosphorylated MAPK or AKT between the cetuximab-sensitive and cetuximab-resistant clones (data not shown), we did observe increased phosphorylation of cortactin, a known downstream target of 611-CTF (Fig. 3B; P ¼ 0.039; ref. 26). To determine the functional role of phosphorylated 611- CTF in mitigating resistance to cetuximab, we treated T24PR3 cells with cetuximab and HER2 shRNA or various HER2-targeting agents. First, we used lentiviral shRNA transduction to knockdown full-length HER2 and 611- CTF in 4 separate clones of T24PR3 (Fig. 4A). HER2 knockdown in clones 2 and 4 reduced full-length HER2 by 70% and 78%, respectively, compared with nontargeting scrambled shRNA–transduced control cells. Likewise, HER2 knockdown in clones 2 and 4 reduced 611-CTF expression by 46% and 56%, respectively, compared with scrambled shRNA–transduced cells. This HER2 knockdown of full-length HER2 and 611-CTF could restore the effect of cetuximab on T24PR3 cells in culture. Cetuximab decreased invasion of the

HER2 shRNA–transduced cells by 54.9% (P ¼ 0.047) and 49.5% (P ¼ 0.034), respectively, after 24 hours. To determine whether the effects of HER2 knockdown were due to knockdown of the full-length HER2 or the 611- CTF fragment, we used purchase Entinostat HER2-targeting agents to selectively and functionally inhibit HER2 activity. Trastuzumab is a monoclonal antibody targeting exclusively full-length HER2 and should not interact directly with 611-CTF, which lacks the extracellular region containing the trastuzumab epitope (27).