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Deciphering the molecular mechanism of enhanced tumor activity of the EGFR variant T790M/L858R using melanoma cell
Hanshuang Shao1,2, Alan Wells1,2
1Department of Pathology, University of Pittsburgh, Pittsburgh, United States.
Introduction:
The abnormal expression and mutagenesis of EGFR drives both the development and progression of a multitude of human cancers. Further mutations within the tyrosine kinase region of the EGFR subsequently contribute to resistance to targeted drugs. What is not known is how these mutations affect progression-related behaviors of cancer cells.
Methods:
The mutagenesis of EGFR T790M, L858R, and T790M/L858R was performed via oligo primer-guided polymerase chain reaction (PCR). GFP-tagged mammalian expression vectors were constructed and confirmed. Stable melanoma cell lines WM983A and WM983B expressing WT or mutant EGFRs were generated for determining the functions of WT and mutant EGFRs in migration, invasion, and resistance to doxorubicin. Immunoblotting and immunofluorescence were performed to detect the transphosphorylation and autophosphorylation of WT and mutant EGFRs and other molecules.
Results:
The EGFR mutant T790M/L858R showed significantly higher basal autophosphorylation in melanoma cell lines WM983A and WM983B. Overexpression of WT EGFR significantly enhanced the protein level of E-cadherin (E-cad) via upregulating its mRNA. In contrast, L858R significantly downregulated E-cad. Biological activity assays show that T790M/L858R presented significant enhancement in vitro in invasion and migration, while WT and T790M moderately inhibited invasion and migration. In WM983A cells, enhanced invasion and migration by T790M/L858R required the downstream signaling pathways through Akt and p38. T790M/L858R dramatically triggers phosphorylation of actin cross-linking protein alpha-actinin-4 in the absence of EGF. This double mutant also conferred resistance to a general chemotherapy doxorubicin through Akt but not the p38 signaling pathway.
Conclusion:
These findings suggest that T790M/L858R not only confers enhanced therapeutic resistance in cancer cell lines but also may promote tumor metastasis via its boosted downstream signaling pathways and/or direct phosphorylation of other key proteins.
Insights
The EGFR T790M/L858R mutation enhances cancer cell invasion, migration, and doxorubicin resistance by activating Akt and p38 signaling pathways. This double mutant may promote tumor metastasis and therapeutic resistance.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Genetics
Background:
- Epidermal Growth Factor Receptor (EGFR) mutations drive cancer development and progression.
- Mutations in the EGFR tyrosine kinase region confer resistance to targeted therapies.
- The impact of specific EGFR mutations on cancer cell progression remains largely unknown.
Purpose of the Study:
- To investigate the functional consequences of EGFR mutations, specifically T790M, L858R, and the double mutant T790M/L858R, on melanoma cell behavior.
- To determine the role of these EGFR mutants in cell migration, invasion, and resistance to doxorubicin.
- To elucidate the downstream signaling pathways involved in the phenotypic changes induced by mutant EGFRs.
Main Methods:
- Generated stable melanoma cell lines (WM983A and WM983B) expressing wild-type (WT) or mutant EGFRs (T790M, L858R, T790M/L858R) using GFP-tagged mammalian expression vectors.
- Assessed cell migration and invasion in vitro using biological activity assays.
- Determined doxorubicin resistance in engineered cell lines.
- Utilized immunoblotting and immunofluorescence to detect protein phosphorylation, including EGFR autophosphorylation and downstream signaling molecules like Akt and p38.
- Investigated the phosphorylation of alpha-actinin-4.
Main Results:
- The EGFR T790M/L858R mutant exhibited significantly higher basal autophosphorylation in melanoma cells.
- WT EGFR overexpression increased E-cadherin (E-cad) levels, while L858R downregulated E-cad.
- T790M/L858R significantly enhanced in vitro invasion and migration, whereas WT and T790M moderately inhibited these processes.
- Enhanced invasion and migration by T790M/L858R were dependent on Akt and p38 signaling pathways.
- T790M/L858R triggered alpha-actinin-4 phosphorylation independently of EGF and conferred doxorubicin resistance via the Akt pathway.
Conclusions:
- The EGFR T790M/L858R mutation confers enhanced therapeutic resistance and potentially promotes tumor metastasis.
- Boosted downstream signaling pathways, including Akt and p38, and direct phosphorylation of key proteins like alpha-actinin-4 contribute to the pro-metastatic phenotype.
- Understanding these mechanisms is crucial for developing effective cancer therapies targeting EGFR mutations.
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