Activation of HER Family Signaling as a Mechanism of Acquired Resistance to ALK Inhibitors in EML4-ALK–Positive Non–Small Cell Lung Cancer

Abstract

Purpose: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI) such as crizotinib show marked efficacy in patients with non-small cell lung cancer positive for the echinoderm microtubuleassociated protein-like 4 (EML4)-ALK fusion protein. However, acquired resistance to these agents has already been described in treated patients, and the mechanisms of such resistance remain largely unknown.Experimental Design: We established lines of EML4-ALK-positive H3122 lung cancer cells that are resistant to the ALK inhibitor TAE684 (H3122/TR cells) and investigated their resistance mechanism with the use of immunoblot analysis, ELISA, reverse transcription and real-time PCR analysis, and an annexin V binding assay. We isolated EML4-ALK-positive lung cancer cells (K-3) from a patient who developed resistance to crizotinib and investigated their characteristics.Results: The expression of EML4-ALK was reduced at the transcriptional level, whereas phosphorylation of epidermal growth factor receptor (EGFR), HER2, and HER3 was upregulated, in H3122/TR cells compared with those in H3122 cells. This activation of HER family proteins was accompanied by increased secretion of EGF. Treatment with an EGFR-TKI induced apoptosis in H3122/TR cells, but not in H3122 cells. The TAE684-induced inhibition of extracellular signal-regulated kinase (ERK) and STAT3 phosphorylation observed in parental cells was prevented by exposure of these cells to exogenous EGF, resulting in a reduced sensitivity of cell growth to TAE684. K-3 cells also manifested HER family activation accompanied by increased EGF secretion.Conclusions: EGF-mediated activation of HER family signaling is associated with ALK-TKI resistance in lung cancer positive for EML4-ALK.

Target therapy in NSCLC patients: Relevant clinical agents and tumour molecular characterisation

“…These mutations either involve the 'gatekeeper' residue (L1196) or sites at a distance from crizotinib binding (F1174L and C1156Y) (56,57). Other mechanisms, such as the activation of HER family signalling, have been demonstrated as ALK-TKI-resistant (58). The cytotoxic activity of crizotinib has also been demonstrated against tumours carrying the ROS1 rearrangement (4,59) and MET amplification (5).…”

Systemic treatment in EGFR-ALK NSCLC patients: second line therapy and beyond

“…Bypass signaling, by v-Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) or EGFR, has also been described as a mechanism of resistance to crizotinib in ALK positive NSCLC 51 . EGF-induced activation of EGFR, HER2, and HER3, as well as a reduced level of ALK activation, was associated with sustained downstream signaling in the presence of ALK inhibitors, indicative of a shift in survival dependency from the ALK signaling pathway to HER family pathways in ALK-TKI-resistant cells 56 . .…”

Emerging uses of biomarkers in lung cancer management: molecular mechanisms of resistance

“…Numerous examples of bypass signaling activation have been discovered. For example, gene expression profiling of crizotinib-resistant versus crizotinibnaive NSCLC tumor samples identified EGFR and HER2 signatures as two of the most enriched gene expression signatures in resistant tumors (51,56,57). In one study, increased EGFR activation was identified in 4 of 9 cases (44.4%) when biopsy samples prior to crizotinib initiation and after development of resistance were compared (51).…”