The online version of this article (doi:10.1186/1476-4598-11-73) contains supplementary material, which is available to authorized users.
We do not have any conflict of interest.
SS conducted the experiments and wrote the initial version of the manuscript; JT and NBS conducted certain experiments; DC did pathological analysis of the samples; EH provided intellectual input; SA provided human tumor xenografts and input; SC directed the project and finalized the manuscript. All authors read and approved the final manuscript.
Cancer stem cells are thought to be responsible for the initiation and progression of cancers. In non-small cell lung cancers (NSCLCs), Hoechst 33342 dye effluxing side population (SP) cells are shown to have stem cell like properties. The oncogenic capacity of cancer stem-like cells is in part due to their ability to self-renew; however the mechanistic correlation between oncogenic pathways and self-renewal of cancer stem-like cells has remained elusive. Here we characterized the SP cells at the molecular level and evaluated its ability to generate tumors at the orthotopic site in the lung microenvironment. Further, we investigated if the self-renewal of SP cells is dependent on EGFR mediated signaling.
SP cells were detected and isolated from multiple NSCLC cell lines (H1650, H1975, A549), as well as primary human tumor explants grown in nude mice. SP cells demonstrated stem-like properties including ability to self-renew and grow as spheres; they were able to generate primary and metastatic tumors upon orthotopic implantation into the lung of SCID mice. In vitro study revealed elevated expression of stem cell associated markers like Oct4, Sox2 and Nanog as well as demonstrated intrinsic epithelial to mesenchymal transition features in SP cells. Further, we show that abrogation of EGFR, Src and Akt signaling through pharmacological or genetic inhibitors suppresses the self-renewal growth and expansion of SP-cells and resulted in specific downregulation of Sox2 protein expression. siRNA mediated depletion of Sox2 significantly blocked the SP phenotype as well as its self-renewal capacity; whereas other transcription factors like Oct4 and Nanog played a relatively lesser role in regulating self-renewal. Interestingly, Sox2 was elevated in metastatic foci of human NSCLC samples.
Our findings suggest that Sox2 is a novel target of EGFR-Src-Akt signaling in NSCLCs that modulates self-renewal and expansion of stem-like cells from NSCLC. Therefore, the outcome of the EGFR-Src-Akt targeted therapy may rely upon the expression and function of Sox2 within the NSCLC-CSCs.
Additional file 1: Figure S1. BIBW2992 inhibits EGFR phophorylation. H1975 cells were treated with 500 nM gefitinib or 200 nM BIBW2992 for 5 days. EGFR phosphorylation and total EGFR expression was detected in presence or absence of drug treatment. Figure S2. Downregulation of Sox2 expression after EGFR and Src inhibition. H1650-SPAdh cells were treated plated over PDL-Laminin coated glass surface and treated with indicated drugs for 4 days. (A) Expression of Sox2 was monitored by immunofluorescence confocal imaging. Isotype antibody was used to show the specific staining of Sox2. (B) Number of Sox2 positive cells for each treatment condition were converted into percentage and plotted. P values were calculated from three different experiments and suggested a significant decrease in Sox2 positive cells after EGFR and Src inhibition. (C) Under similar treatm,ent conditions cells were stained with Nanog specific antibodies. Drug treatment did not alter the expression of Nanog in H1650-SPAdh cells. Figure S3. Depletion of Sox2 expression suppresses SP frequency. (A) A549, H1650 and H1975 cells were transiently transfected with second set siRNA (purchased from Origene). 48 h after transfection, cells were analyzed for SP frequency. Similar to first set of siRNA (purchased from SantaCruz), depletion of Sox2 resulted in significant decrease in SP frequency in NSCLCs. (B) NSCLC cells were transfected with Sox2 SIRNA and ABCG2 expression was detected by western blotting. β-Actin was used as internal control for equal loading. * p<0.05. (DOCX 1 MB)12943_2012_1040_MOESM1_ESM.docx
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- EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer
Srikumar P Chellappan
- BioMed Central
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