Molecular Context of theEGFRMutations: Evidence for the Activation of mTOR/S6K Signaling

Abstract

Purpose: Activating somatic mutations in the epidermal growth factor receptor (EGFR) gene are present in a small subset of lung adenocarcinomas.These mutations cluster in specific regions and confer sensitivity to inhibitors of the tyrosine kinase activity of EGFR. To further determine the genetic and molecular characteristics of tumors carrying EGFR gene mutations, we investigated the EGFR gene status in lung adenocarcinomas and evaluated its association with specific characteristics of the patients and tumors, such as mutations at KRAS and p53, EGFR and ErbB2 gene amplification, levels of EGFR and HER2 proteins, and levels of downstream effectors of EGFR, such as phospho^extracellular signal-regulated kinase and phospho-S6 proteins. Experimental Design: The mutational status of EGFR was determined by direct sequencing in 86 primary lung adenocarcinomas and12 lung cancer cell lines, and was correlated with a number of variables relating to the tumor and patient. A tissue microarray containing 37 lung tumors was constructed to determine, by fluorescence in situ hybridization analysis, the number of copies of EGFR and ErbB2 genes and, by immunohistochemistry, the levels of EGFR, HER2, phospho-ERK, and phospho-S6 proteins. Results: EGFR gene mutations were identified in 13% of the primary tumors. The type and clustering of the mutations were identical to those previously reported. Amplification of the EGFR occurred in14% of the tumors and could arise in tumors with EGFR mutations. Interestingly, mTOR activation, as measured indirectly by augmented levels of phospho-S6 protein, was more frequent in tumors with gene alterations in either EGFR or KRAS (P = 0.00005; Fisher's exact test) than in their wild-type counterparts. Conclusions: Our data agree with the accumulation of EGFR mutations in a subset of patients with lung cancer. Moreover, we report EGFR gene amplification in EGFR-mutant tumors and a positive correlation between EGFR or KRAS alterations and activation of mTOR signaling.

Dysfunctional AMPK activity, signalling through mTOR and survival in response to energetic stress in LKB1-deficient lung cancer

“…The cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA), tested negative for mycoplasma infection and grown under recommended conditions. Thirtyseven lung primary tumours from formalin-fixed, paraffinembedded tissue blocks were used for the tissue microarray (TMA), as described previously (Conde et al, 2006).…”

“…Technical details of the Western blots are included as Supplementary methods. Immunohistochemical staining of LKB1 and phospho-acetyl CoA carboxylase (ACC) proteins was performed on 3-mm-thick sections from the TMA as described previously (Conde et al, 2006). Immunostaining was evaluated by a pathologist (EC).…”

C5 Extract Induces Apoptosis in B16F10 Murine Melanoma Cells through Extrinsic and Intrinsic Apoptotic Pathways and Sub-G1 Phase Arrest

“…Importantly, mTOR activation was more frequent in tumors with gene alterations such as EGFR mutations or PI3K/Akt over-expression [37]. Phosphorylation of mTOR was decreased by C5E and a combined effect was caused by 0.4 mg/ml C5E plus vinblastine ( Figure 6C).…”

A comparison of four methods for detecting KRAS mutations in formalin-fixed specimens from metastatic colorectal cancer patients

“…The four detection assays were conducted at the same institution under the same conditions. Direct sequencing for exon 2 of the KRAS gene was carried out using PCR and 2X bidirectional direct sequencing following previously described protocols (11,12). Tumor DNA for exon 3 was amplified using the following primers: forward, 5'-CAC TGT AAT AAT CCA GAC TGTG-3' and reverse, 5'-CCC ACC TAT AAT GGT GAA TATC-3'.…”