1. Gokhan Gorgisen1, Derya OZES1, Suray Pehlivanoglu1, Abdullah ERDOGAN2, Levent DERTSIZ2, Gulay OZBILIM3, Irem Hicran OZBUDAK3 ,
Osman Nidai OZES1
1Department of Medical Biology, Faculty of Medicine, Akdeniz University, Antalya, Turkey.
2Department of Chest Surgery, Faculty of Medicine, Akdeniz University, Antalya, Turkey.
3Department of Medical Pathology, Faculty of Medicine, Akdeniz University, Antalya, Turkey.
16 NSCLC
and control
samples
Preparation
protein lysates
Determine the expression
levels and or
phosphorylation of EGFR,
STAT3, ERK, TWIST1,
GAPDH, and AKT.
DNA isolation
DNA sequencing of EGFR
exon 18, 19, 21 to see the
presence or absence of
mutations
Members of Human Epidermal Growth Factor Receptor family (EGFR1-4) have been shown to be over-expressed
in 88% of NSCLC. Tyrosine kinase (TK) inhibitors, Erlotinib and Gefitinib have been used to treat lung cancer but
they are almost completely ineffective in patients who do not show EGFR over-expression or mutations. Therefore,
before initiating therapy with these TK inhibitors, EGFR expression or mutation profiles must be determined.
On the basis of the results obtained from samples of 16 tumor samples , we did not detect any activating
mutations of EGFR1and K-RAS. Nonetheless, Western Blot analysis of the mentioned oncogenes indicate highly
elevated levels of phosphorylation or expression in some samples. Among the patients different levels of induced
phosphorylation have been found; these levels are, 2 to 23 fold induced phosphorylation of ERK, 1.5 to 20 in AKT, 2
to 10 in EGFR, and 2 to 24 in TWIST1 and 2-450 in GAPDH. Not only induced phosphorylation levels are elevated
but also regular protein levels are increased in tumor samples. These levels are 2 fold expression of ERK, 1,5 to 278
fold in STAT3, 2,5 to 18 fold in AKT. These results indicate that even in the absence of any activating mutations of
EGFR or K-Ras, the EGFR pathway is still very active in some NSCLC patients.
Thus, in NSCLC patients, the tumor micro-environment created by the tumor or surrounding cells can be as
important as the activation mutations of EGFR1, promoting the survival and metastasis of the cell. More
importantly, if activating mutations of EGFR1 is considered to be the main reason for TK therapy, then TK therapy
may not be the optimal decision to make.
STAT3
pSTAT3
β actin
ERK
pERK
β actin
TWIST1
β actin
AKT
pAKT
β actin
GAPDH
β actin
AKT
pAKT
β actin
GAPDH
β actin
STAT3
pSTAT3
β actin
ERK
pERK
β actin
TWIST1
β actin
GAPDH
β actin
AKT
pAKT
β actin
STAT3
pSTAT3
β actin
ERK
pERK
β actin
TWIST1
β actin
STAT3
pSTAT3
β actin
ERK
pERK
β actin
TWIST1
β actin
GAPDH
β actin
AKT
pAKT
β actin
IP:EGFR
IB:pY
EGFR
pY
IP:EGFR
IB:pY
EGFR
pY
IP:EGFR
IB:pY
EGFR
pY
N1 T1 N2 T2 N3 T3 N4 T4 N1 T1 N2 T2 N3 T3 N4 T4
N1 T1 N2 T2 N3 T3 N4 T4 N1 T1 N2 T2 N3 T3 N4 T4
N1 T1 N2 T2 N3 T3 N4 T4 N1 T1 N2 T2 N3 T3 N4 T4 N5 T5 N6 T6 N7 T7 N8 T8 N5 T5 N6 T6 N7 T7 N8 T8
N5 T5 N6 T6 N7 T7 N8 T8N5 T5 N6 T6 N7 T7 N8 T8
N5 T5 N6 T6 N7 T7 N8 T8 N5 T5 N6 T6 N7 T7 N8 T8 N9 T9 N10 T10 N11 T11 N12 T12 N9 T9 N10 T10 N11 T11 N12 T12
N9 T9 N10 T10 N11 T11 N12 T12N9 T9 N10 T10 N11 T11 N12 T12
N9 T9 N10 T10 N11 T11 N12 T12 N9 T9 N10 T10 N11 T11 N12 T12 N13 T13 N14 T14 N15 T15N16 T16 N13 T13 N14 T14 N15 T15N16 T16
N13 T13 N14 T14 N15 T15 N16 T16N13 T13 N14 T14 N15 T15 N16 T16
N13 T13 N14 T14 N15 T15 N16 T16
Abbr: N: Normal Tissue
T:Tumor Tissue