Paradoxical induction of growth arrest and apoptosis by EGF via the up-regulation of PTEN by activating Redox factor-1/Egr-1 in human lung cancer cells

Epidermal growth factor (EGF) signaling promotes cell proliferation and survival in several types of cancer. Here, however, we showed that EGF inhibits proliferation and promotes programmed cell death in non-small cell lung cancer (NSCLC) cells. In A549 cells, EGF increased redox factor-1 (Ref-1) expression and the association of Ref-1 with zinc finger-containing transcriptional regulator (EGR1) via activation of p22phox, RAC1, and an NADPH oxidase subunit. EGF increased p22phox and RAC1 expression through activation of purinergic receptors (P2Y). Elevated Ref-1/EGR1 levels increased phosphatase and tensin homolog (PTEN) levels, leading to inhibition of the Akt pathway. EGF-induced PTEN upregulation increased apoptosis and autophagy-induced damage in A549 cells, whereas Ref-1 knockdown blocked EGF-induced PTEN upregulation in an NADPH oxidase p22phox subunit-independent manner. In addition, p22phox knockdown restored EGF-induced effects, implying that changes in P2Y activity caused by EGF, which activates NADPH oxidase via RAC1, influenced Ref-1-mediated redox regulation. Finally, EGF similarly attenuated cell proliferation and promoted autophagy and apoptosis in vivo in a xenograft model using A549 cells. These findings reveal that EGF-induced redox signaling is linked to Ref-1-induced death in NSCLC cells.


Supplementary Figure S8: Quantification of mitochondria and phagosomes of tumor tissue in electron microscopic
images. EGF-treated cells exhibited some structural changes in control tumor tissue and tumor tissues from mice injected with 1 mg/kg EGF, including swelling and differences in mitochondrial morphology, as revealed by electron microscopy (EM; original magnification, 12000×). Normal mitochondria, dysfunctional mitochondria, and phagosomes were enumerated in 10 EM images. Data were analyzed by unpaired t-tests. **p < 0.01, ***p < 0.001.

Supplementary Figure S9: Body weight and tumor size measurement in the lung cancer animal model. (A) Body
weights of tumor-bearing control mice (black circles) and EGF-injected tumor-bearing mice (white circles). The x-axis values represent the number of days after EGF injection. (B) Tumor size was calculated by multiplying the shortest and longest diameters using digital calipers. Treatments with EGF were administered after a couple of weeks when tumors reached an average volume of 50 mm3. Tumor tissues were derived from 6-week-old immunodeficient mice (nu/nu) that had been treated daily with EGF (0 or 1 mg/kg/PBS) for 21 days. Differences between PBS-injected tumor-bearing control mice (n = 6, whit circles) and EGF-injected tumor-bearing mice (n = 8, black circles) were analyzed using unpaired t-tests. ***p < 0.001. Data are means and standard deviations. Figure S7: Cell cycle of A549 cells. A549 cells were incubated with 100 ng/mL hrEGF for 0, 24, 48, 72, or 96 h.

Supplementary
The changes in cell cycle viabilities after treatment with EGF were then measured and analyzed using PI staining with flow cytometry. Representative results of three independent experiments are shown.

Authentication of NSCLCs and knockdown of specific genes in A549 cell lines by analysis of short tandem repeats (STRs)
Authentication of the cells (A549, EKVX, NCI-H23, NCI-H266, NCI-H322M, and NCI-H522; Supplementary Table S1) was performed by analysis of STRs and comparison with the original cell line (Supplementary  Table S2). To demonstrate the derivation of control KD, EGFR1 KD, p22 phox KD, and Ref-1 KD cell lines from the parental A549 cell line, STRs of all five cell lines were performed and compared with the original parental A549 cells. The cells exhibited consistency with parental A549 cells (Supplementary Table S3). All cell-specific tetranucleotide repeat loci and the amelogenin genderdetermining marker were amplified using AmpFLSTR Identifiler Plus PCR Amplification Kit (Applied Biosystems, CA, USA) and analyzed using a 3730 DNA analyzer and peak scanner (Applied Biosystems).

Colony formation assay
Untreated-control and pretreated-EGF cells were plated at a low density (300 ~ 600 cells/well) in RPMI-1640 media. Cells were fed fresh medium in the presence of EGF (100 ng/ml) once every three days. After two weeks of culture, cells were fixed with 4% formaldehyde for 10 min and stained with crystal violet (0.05%, 2 h).

Measurement of mRNA expression levels
Total RNA was extracted from A549 cells using TRIzol reagent (Invitrogen), and 1 μg total RNA from each sample was reverse transcribed. The RT-PCR protocol was as follows: 27-30 cycles at 94°C for 30 s, 58°C for 50 s, and 72°C for 1 min. The primer sequences for human EGFR1, human EGFR2, and human Ref-1 are shown in Supplementary Table S2). As an internal standard, human GAPDH mRNA was amplified and analyzed under identical conditions using a pair of specific primers.

Detection of intracellular ROS generation
For the flow cytometric analysis of ROS production, EGF-treated cells were monitored by 2′,7′-dichlorofluorescin diacetate (H2DCFDA; CellRox Deep Red; cat. #C10422; Molecular Probes Inc., OR, USA). Briefly, control and treated cells were loaded with 5 μM H2DCFDA in RPMI 1640 medium for 30 min at 37°C. The cells were then washed twice with fresh DPBS. Data were obtained using a flow cytometer (FACSCalibur; BD Bioscience, CA, USA) with an FL-4 filter and analyzed using Cell Quest Pro software (BD Bioscience).

ATP measurement
Intracellular ATP was measured using an ENLITEN ATP assay system bioluminescence detection kit (#TB267; Promega Corp., Madison, WI, USA), according to the manufacturer's instructions. Briefly, 5 × 10 4 cells/well were incubated with EGF for different times. Supernatants were then removed, and cells were washed with PBS. The cells of each well were lysed, and intracellular ATP was quantified using a fluorometer (VICTOR3 Multilabel Reader; PerkinElmer).

Cell cycle analysis
A549, control KD, p22 phox KD, or Ref-1KD was treated with 100 ng/ml EGF. The cell cycle was analyzed using a flow cytometer.