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. Previous studies characterizing this model revealed that: 1) EGFR was robustly overexpressed in Ctx(R) clones due to decreased EGFR ubiquitination and degradation and 2) Ctx(R) clones expressed increased HER2 and HER3 activation resulting in constitutive activation of the PI3K/AKT signaling axis. These findings suggest that dual targeting HER family receptors would be highly beneficial in the Ctx(R) setting. RESULTS: Since HER3 has been implicated in resistance to EGFR inhibitors, the efficacy of dually targeting both EGFR and HER3 in Ctx(R) models was evaluated. First, EGFR and HER3 expression were knocked down with 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. 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. Treatment with both mAbs resulted in knockdown of both signaling pathways simultaneously. HER2 was also strongly inactivated upon dual mAb therapy, suggesting that this treatment regimen can diminish signaling from three HER family receptors. 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. Combinatorial-treated xenograft tumors expressed decreased Ki67 and increased cleaved caspase-3 levels compared to tumors treated with either monotherapy. CONCLUSIONS: These studies demonstrate that dually targeting HER family receptors with antibody-based therapies can overcome acquired resistance to cetuximab.

Sym004, a novel EGFR antibody mixture, can overcome acquired resistance to cetuximab.

The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in a variety of human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for head and neck and colorectal cancer treatment, but many patients treated with cetuximab don't respond or eventually acquire resistance. To determine how tumor cells acquire resistance to cetuximab, we previously developed a model of acquired resistance using the non-small cell lung cancer line NCI-H226. These cetuximab-resistant (Ctx(R)) cells exhibit increased steady-state EGFR expression secondary to alterations in EGFR trafficking and degradation and, further, retained dependence on EGFR signaling for enhanced growth potential. Here, we examined Sym004, a novel mixture of antibodies directed against distinct epitopes on the extracellular domain of EGFR, as an alternative therapy for Ctx(R) tumor cells. Sym004 treatment of Ctx(R) clones resulted in rapid EGFR degradation, followed by robust inhibition of cell proliferation and down-regulation of several mitogen-activated protein kinase pathways. To determine whether Sym004 could have therapeutic benefit in vivo, we established de novo Ctx(R) NCI-H226 mouse xenografts and subsequently treated Ctx(R) tumors with Sym004. Sym004 treatment of mice harboring Ctx(R) tumors resulted in growth delay compared to mice continued on cetuximab. Levels of total and phospho-EGFR were robustly decreased in Ctx(R) tumors treated with Sym004. Immunohistochemical analysis of these Sym004-treated xenograft tumors further demonstrated decreased expression of Ki67, and phospho-rpS6, as well as a modest increase in cleaved caspase-3. These results indicate that Sym004 may be an effective targeted therapy for Ctx(R) tumors.

Nuclear EGFR as a molecular target in cancer.

The epidermal growth factor receptor (EGFR) has been one of the most targeted receptors in the field of oncology. While anti-EGFR inhibitors have demonstrated clinical success in specific cancers, most patients demonstrate either intrinsic or acquired resistance within one year of treatment. Many mechanisms of resistance to EGFR inhibitors have been identified, one of these being attributed to alternatively localized EGFR from the cell membrane into the cell's nucleus. Inside the nucleus, EGFR functions as a co-transcription factor for several genes involved in cell proliferation and angiogenesis, and as a tyrosine kinase to activate and stabilize proliferating cell nuclear antigen and DNA dependent protein kinase. Nuclear localized EGFR is highly associated with disease progression, worse overall survival in numerous cancers, and enhanced resistance to radiation, chemotherapy, and the anti-EGFR therapies gefitinib and cetuximab. In this review the current knowledge of how nuclear EGFR enhances resistance to cancer therapeutics is discussed, in addition to highlighting ways to target nuclear EGFR as an anti-cancer strategy in the future.