In the current study, we used the SP phenotype to identify and enrich a subpopulation of NSCLCs with the properties ascribed to CSCs. The studies presented here demonstrates a specific and significant role for EGFR signaling cascade in facilitating the self-renewal growth and expansion of the side population cells from NSCLCs.
Our study, in accordance with earlier studies , , confirmed the presence of SP cells in established human NSCLC cell lines and in human tumor xenografts with the properties of CSCs. Comparing the self-renewal ability of SP and MP cells isolated from human tumor xenografts, we found that approximately 0.2% SP cells were able to self-renew and form spheres, whereas MP cells were unable to self-renew. Comparing the percentage of sphere forming cells in SP cells, we estimate that approximately 1-2% of SP cells from established cell lines may have stem-like properties; therefore, SP phenotype may not be the exclusive marker for CSCs, but can be used to enrich stem-like cells from NSCLCs.
SP cells were found to be more tumorigenic in vivo, confirming the enrichment of tumor initiating cells in SP compartment. These cells were able to produce highly invasive disease upon implantation into the lungs. Also, the direct association of stem-like cells with generation of metastatic disease may be supported by our observation where a significant correlation was observed between high Sox2 expressions in the metastatic tumors of lung adenocarcinoma patients. Recent reports indicate that the normal epithelial cells acquire the CSCs properties upon induction of EMT governed by various cytokines and growth factors from stromal cells [10, 25]. Our results demonstrate that SP-cells intrinsically exhibit loss of epithelial markers and/or the gain of mesenchymal markers as compared to MP cells and could be due to the higher expression of transcription factors Twist, Slug and Snail, which are known to be involved in maintaining the mesenchymal phenotype. Together with the expression of embryonic stem cell transcription factors like Oct4, Sox2, and Nanog along with the exhibition of EMT like features and orthotopic tumor-forming ability, collectively suggest that SP cells isolated from NSCLC cell lines and tumors have stem-like properties. The observation that EGFR signaling affects stem-like functions of SP cells is intriguing, given that several EGFR tyrosine-kinase inhibitors have efficacy against NSCLCs [34, 35]. Interestingly, EGFR appears to regulate Sox2 levels, through the Src-Akt pathway; Sox2 has been shown to be regulated by Akt in ES cells, through the inhibition of proteasomal degradation . Consistent with these results, our observation suggest that inhibition of EGFR-Src-Akt signaling downregulates Sox2 levels along with a reduction in ABCG2 levels. This decrease in ABCG2 expression upon EGFR inhibition is probably a causal effect of Sox2 depletion-mediated differentiation of SP into MP cells.
The fact that EGFR-pathway inhibition resulted in specific depletion of Sox2 without any significant effect on Oct4 or Nanog expression suggests that their expression may be regulated through independent mechanisms in NSCLC SP cells. Our results as well as an earlier report  suggest that Sox2 is expressed in both low as well as high stage adenocarcinomas irrespective of their grades. However, Oct4 or Nanog expression was found to be associated only with the high grade lung adenocarcinoma and not expressed in low grade tumors [17, 37]. Therefore, we predict that the EGFR pathway inhibition may exert its favorable effects only for those tumors where Sox2 is the major determinant in controlling the self-renewal of CSCs. Interestingly, a recent study showed that the ectopic overexpression of Oct4 and Nanog increases the tumor initiating property of A549 cells . In agreement with these reports, we find that specific and independent depletion of Oct4 or Nanog also resulted in decrease in SP phenotype but in a cell type dependent fashion (Data not shown). Two recent reports demonstrate that ectopic expression of Sox2 increased the frequency of side population cells and tumor formation in mouse and human NSCLC cell lines [33, 38]. These reports strongly suggest that Sox2 expressing cells harbor the stem cell-like properties. Our observation further strengthens this postulation where we demonstrate that Sox2 depletion was sufficient to inhibit the self-renewing property SP cells in all the three NSCLC cell lines.
In addition to the mutation in EGFR signaling, perturbation of p53 activity is another important event occurs in initiation and progression of NSCLCs . Recently, p53 is shown to have certain roles in promoting the differentiation of human embryonic stem cell through repression of factors like Oct4, Klf4, Lin28A, and Sox2 . However, there is not much information available on the direct role of p53 transcriptional activities in regulating Sox2 expression in stem-like cells in cancer, and would be interesting to explore in future.