First, EGFR and HER3 expression were knocked down with siRNAs. 
siRNAs
Compared to the Ctx(S) parental cell line (HP), all Ctx(R) clones exhibited robust decreases in cell proliferation upon dual knockdown.  Analysis of Ctx(R) clones indicated that neuregulin-1 was highly overexpressed compared to HP cells.  Incubation of HP cells with neuregulin-1 rendered them resistant to cetuximab. 
HP
Next, dual treatment of Ctx(R) clones with cetuximab and the HER3 neutralizing monoclonal antibody (mAb) U3-1287 led to potent anti-proliferative effects.  Blockade of EGFR with cetuximab resulted in inactivation of MAPK, while blockade of HER3 with U3-1287 resulted in the inactivation of AKT.  Tumors that had acquired resistance to cetuximab were significantly growth delayed upon dual treatment of U3-1287 and cetuximab compared to those continued on cetuximab only. 
U3-1287
Overcoming acquired resistance to cetuximab by dual targeting HER family receptors with antibody-based therapy.BACKGROUND
 Cetuximab, an anti-EGFR monoclonal antibody, is used to treat several cancers.  However, many patients who initially respond to cetuximab acquire resistance.  To examine mechanisms of acquired resistance, we developed a series of cetuximab-resistant (Ctx(R)) clones derived from the cetuximab sensitive (CtxS) non-small cell lung cancer (NSCLC) cell line H226.  Incubation of HP cells with neuregulin-1 rendered them resistant to cetuximab.  Next, dual treatment of Ctx(R) clones with cetuximab and the HER3 neutralizing monoclonal antibody (mAb) U3-1287 led to potent anti-proliferative effects.  Blockade of EGFR with cetuximab resulted in inactivation of MAPK, while blockade of HER3 with U3-1287 resulted in the inactivation of AKT.  De novo CtxR H226 mouse xenografts were established to determine if dual therapy could overcome acquired resistance to cetuximab in vivo.  Tumors that had acquired resistance to cetuximab were significantly growth delayed upon dual treatment of U3-1287 and cetuximab compared to those continued on cetuximab only.  These studies demonstrate that dually targeting HER family receptors with antibody-based therapies can overcome acquired resistance to cetuximab.
cetuximab
Next, dual treatment of Ctx(R) clones with cetuximab and the HER3 neutralizing monoclonal antibody (mAb) U3-1287 led to potent anti-proliferative effects.  Blockade of EGFR with cetuximab resulted in inactivation of MAPK, while blockade of HER3 with U3-1287 resulted in the inactivation of AKT.  Tumors that had acquired resistance to cetuximab were significantly growth delayed upon dual treatment of U3-1287 and cetuximab compared to those continued on cetuximab only. 
U3-1287
Overcoming acquired resistance to cetuximab by dual targeting HER family receptors with antibody-based therapy.BACKGROUND
 Cetuximab, an anti-EGFR monoclonal antibody, is used to treat several cancers.  However, many patients who initially respond to cetuximab acquire resistance.  To examine mechanisms of acquired resistance, we developed a series of cetuximab-resistant (Ctx(R)) clones derived from the cetuximab sensitive (CtxS) non-small cell lung cancer (NSCLC) cell line H226.  Incubation of HP cells with neuregulin-1 rendered them resistant to cetuximab.  Next, dual treatment of Ctx(R) clones with cetuximab and the HER3 neutralizing monoclonal antibody (mAb) U3-1287 led to potent anti-proliferative effects.  Blockade of EGFR with cetuximab resulted in inactivation of MAPK, while blockade of HER3 with U3-1287 resulted in the inactivation of AKT.  De novo CtxR H226 mouse xenografts were established to determine if dual therapy could overcome acquired resistance to cetuximab in vivo.  Tumors that had acquired resistance to cetuximab were significantly growth delayed upon dual treatment of U3-1287 and cetuximab compared to those continued on cetuximab only.  These studies demonstrate that dually targeting HER family receptors with antibody-based therapies can overcome acquired resistance to cetuximab.
cetuximab