Non-small cell lung cancer (NSCLC) is the most common form of lung cancer, accounting for approximately 80% of diagnosed cases.1 Epidermal Growth Factor Receptor (EGF R), a receptor tyrosine kinase (RTK), is dysregulated in 40-80% of NSCLC cancers and numerous mutations that activate EGF R have been detected in primary NSCLC tumors.2,3 Therefore, tyrosine kinase inhibitors (TKIs) that target EGF R are commonly used for lung cancer therapy. Erlotinib is a TKI that was approved by the FDA in 2004 for NSCLC patients and has been shown to be effective in those harboring EGF R mutations.4,5 Unfortunately, NSCLC patients develop erlotinib resistance (ER) within 10-14 months of primary treatment.6 In a recent paper, Zhang et al. identified a novel mechanism for ER that could lead to the development of new drug targets for NSCLC patients that have acquired ER.7
To identify candidate genes involved in the development of ER, mRNA expression profiles from cell culture and mouse xenograft models of acquired ER were compared to erlotinib-sensitive controls.7 This analysis revealed that the mRNA levels of AXL, an RTK, were significantly higher in both ER HCC827 human NSCLC cell lines and 15 of 17 ER tumors. Total and phospho-Axl protein levels were also increased in the ER cell lines and tumors. To determine if AXL overexpression had a causative role in ER acquisition, ER cell lines and tumors from the mouse xenograft model of acquired ER were treated with small interfering (si) RNA or short hairpin (sh) RNA, respectively. Sensitivity to erlotinib was restored in both cases. These data suggest that AXL overexpression is necessary for the acquisition of ER in these cell culture and in vivo models.
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Axl Overexpression is a Novel Mechanism of TKI Resistance in EGF R-mutant NSCLC. EGF R is a receptor tyrosine kinase (RTK) that is often mutationally activated in non-small cell lung cancer (NSCLC). Its activity can result in the activation of MAPK (ERK), Akt, and NFkB (RelA) signaling and promote cancer progression. Erlotinib treatment can block the activity of mutant EGF R and its downstream signaling pathways, resulting in cancer regression (left panel). Zhang et al. show that in vitro and in vivo models of NSCLC with acquired erlotinib resistance overexpress Axl, another RTK, which re-activates the MAPK, Akt, and NFkB signaling pathways, and promotes cancer progression in the presence of erlotinib (right panel). Furthermore, Axl overexpression was dependent on the expression of Vimentin, a marker for EMT, suggesting that EMT may play a role in the acquisition of resistance to erlotinib.
To determine if Axl kinase activity was required in this model of ER, Zhang et al. transiently overexpressed either wild-type Axl or a kinase dead Axl mutant in HCC827 cells.7 Only wild-type Axl was able to induce ER, suggesting that its kinase activity was required for the development of ER. Axl has been reported to activate MAPK, Akt, and NFkB signaling in cancer cell lines.8,9 Treatment of ER cells with erlotinib, along with either AXL-specific siRNA or Axl inhibitors, reduced the phosphorylation of ERK, Akt, and RelA, whereas treatment with erlotinib alone did not.7 These results suggested that the MAPK, Akt, and NFkB pathways may mediate the acquisition of ER downstream of Axl kinase activity.
Epithelial to mesenchymal transition (EMT), the process by which cells lose epithelial characteristics and acquire a migratory, mesenchymal phenotype, has been linked to the development of ER.10 Since Axl can be induced during EMT, this process might contribute to Axl-dependent ER acquisition.11 Interestingly, the ER xenograft-derived tumors and ER cell lines expressed several biomarkers of EMT, including Vimentin upregulation.7,12 Additionally, siRNA knockdown experiments showed that Vimentin was required for Axl overexpression and ER acquisition in HCC827 cells.7 These data suggest that EMT may have a role in the development of Axl-dependent ER in this cell culture model.
To determine if Axl-dependent ER might be clinically relevant, the authors asked if Axl overexpression could be detected in NSCLC patients with acquired ER.7 Axl expression was assessed by immunohistochemistry in 35 EGF R-mutant NSCLC specimens from individuals before treatment with erlotinib or gefitinib, another TKI, and then after acquisition of TKI resistance. Axl expression levels were at least two-fold higher in seven out of 35 TKI-resistant specimens compared to pre-treatment specimens. This suggested that overexpression of Axl may be important for ER acquisition in some NSCLC patients.
This new study utilized both cell culture and mouse xenograft models to identify a novel Axl-dependent mechanism by which EGF R-mutant NSCLC can acquire resistance to TKIs. It is likely complex, potentially involving several downstream signaling pathways and an association with EMT. Axl overexpression observed in tumor tissue from patients with TKI-resistant NSCLC supported their model and suggested that this mechanism could have clinical relevance. It is possible that future therapies combining Axl inhibition with TKI treatment could delay or prevent the development of TKI resistance.
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