1A and S1A) [11]. Apoptosis induction has been implicated as a critical mechanism of targeted agents including anti-EGFR antibodies [3, 4]. Stress-induced apoptosis in mammalian cells is mediated by the Bcl-2 family proteins, which regulate cell death through 3CAI a series of events, including permeabilization of the outer mitochondrial membrane, cytosolic release of cytochrome or encodes a small GTPase that mediates signals from growth factor receptors to downstream PI3K/AKT and MEK/ERK effector pathways [8]. mutations, mostly at codons 12, 13, and 61, lock KRAS into a constitutively active GTP-bound form, resulting in hyperactive PI3K/AKT and RAF/MEK/ERK signaling [9], as well as resistance to anti-EGFR antibody therapy [10]. It has been shown that continuous exposure of sensitive CRC cells to anti-EGFR antibodies enriches a small fraction of cells with a mutation or genomic amplification, which underlies acquired resistance to anti-EGFR antibodies [11, 12]. However, the exact mechanisms by which mutant KRAS confers resistance to anti-EGFR therapy remains unclear. A variety of approaches have been explored to target mutant KRAS, such as directly inhibiting KRAS [13], and targeting KRAS downstream effector pathways [4, 14]. Despite these efforts, mutant KRAS has remained one of the most challenging oncology targets. Novel mechanistic insight and targeting approaches for KRAS-mediated resistance are urgently needed. In this study, we identified p53-upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 family member [15], as a critical mediator of apoptotic response to anti-EGFR antibodies in CRC cells. PUMA induction by anti-EGFR antibodies is mediated by the p53 homologue p73, and consistently abrogated in CRC cells with acquired resistance. We also found that inhibitors of aurora kinases can overcome the resistance 3CAI to anti-EGFR antibodies by restoring PUMA induction, providing a rationale to improve the efficacy of EGFR-targeted therapy. Results PUMA induction mediates apoptotic response to anti-EGFR antibodies in CRC cells To determine the response mechanisms, we analyzed several CRC cell lines that are exquisitely sensitive to anti-EGFR antibodies [16]. Treating DiFi and CCK-81 CRC cells with cetuximab or panitumumab suppressed cell growth in a dose-dependent manner (Fig. 1A and S1A) [11]. Cetuximab or panitumumab at 5 or 10 nM markedly induced cell death with characteristics of apoptosis, including nuclear condensation and fragmentation (Fig. 1B), Annexin V staining of plasma membrane (Fig. 1C), and activation of caspase-9 and caspases-3/7 (Fig. 1D, 1E, S1B, and S1C). We 3CAI also detected permeabilization of mitochondrial outer membrane by MitoTracker staining (Fig. 1F), as well as cytosolic release of mitochondrial cytochrome (Fig. 2G), following cetuximab or panitumumab treatment. The growth suppressive and apoptotic effects of the anti-EGFR antibodies were abolished by the pan-caspase inhibitor z-VAD-fmk (Fig. 1, A and G), indicating a critical role of apoptotic cell death. Open in a separate window Figure 1 Anti-EGFR antibodies induce mitochondria-dependent apoptosis in sensitive CRC cells(A) MTS analysis of DiFi colon cancer cells treated with cetuximab (Cmab) or panitumumab (Pmab) at the indicated doses for 72 hr. For comparison, DiFi cells pre-treated with Influenza B virus Nucleoprotein antibody 10 M z-VAD-fmk (z-VAD) for 1 hr were also analyzed. (B) Apoptosis in DiFi cells treated Cmab or Pmab at the indicated doses for 72 hr was analyzed by counting condensed and fragmented nuclei after nuclear staining with Hoechst 33258. (C) Apoptosis in DiFi cells treated 10 nM Cmab or Pmab for 72 hr was analyzed 3CAI by Annexin V/PI staining followed by flow cytometry. (D) Caspase-3/7 activity in DiFi cells treated with 10 nM Cmab or Pmab for 24 hr was measured using the Caspase-3/-7 Activation kit. (E) 3CAI Western blotting of cleaved (C) caspase-3 and caspase-9 in DiFi cells treated as in (D). (F) Mitochondrial membrane integrity in DiFi cells treated with 10 nM of Cmab or Pmab for 72 hr was analyzed by MitoTracker Red CMXRos staining followed by flow cytometry. (G) DiFi cells with or without pre-treatment with 10 M z-VAD-fmk (z-VAD) for 1 hr were treated with 10 nM Cmab for 72 hr. Apoptosis was analyzed as in (B). Results in (A), (B), (D) and (G) were expressed as means s.e.m. of triplicates in two independent experiments. *, <0.05; **, <0.01; ***, <0.001 Open in a separate window Figure 2 PUMA is a critical mediator of the apoptotic response to anti-EGFR antibodies(A) DiFi cells were treated with 10.