Downregulation of transcription factor SOX2 in cancer stem cells suppresses growth and metastasis of lung cancer

Abstract

Background: The cancer stem cell hypothesis suggests that neoplastic clones are maintained exclusively by a small subpopulation of cells, which have indefinite proliferation and differentiation potentials and give rise to phenotypically diverse cancer cells. Cancer stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the 'side population' (SP).

Methods and results: The Hoechst efflux assay was used to isolate and characterize the SP from murine D121 lung carcinoma cells. Here, we demonstrated that D121-SP cells contain cancer stem cell characteristics, that is, upregulation of the transcription factors SOX2 and Oct 4 in D121-SP cells. In addition, the migration of D121-SP was decreased, and apoptosis of D121-SP was upregulated following knocking down of SOX2 in D121 cells. Importantly, downregulation of SOX2 in D121 cells markedly suppressed their metastatic potential in syngeneic mice.

Conclusions: These results suggest that the SP is an enriched source of lung tumour cells with stem cell properties and that SOX2 has an important role in maintaining stem cell properties and functions that may be a potential target for effective lung cancer therapy.

Figure 1

D121 non-small cell lung carcinoma cells contain a SP with cancer stem cell properties. (A) D121 lung cancer cells and mouse bone marrow cells were stained with Hoechst 33342 to determine a dye effluxing SP. (B) The MHC-I antigen expression on D121-SP cells was upregulated by incubation with INF-γ. (C) The SP of D121 cells was sorted by flow cytometry after being stained with Hoechst 33342 dye, and a group of C57/BL mice were implanted in the cleared fat pad with either the SP or non-SP fraction of D121 cancer cells at 1 × 10⁵ per mouse. (D) All mice were killed on day 25 after tumour cell implantation and analysed for the metastasis score on lungs.

Figure 2

Upregulation of stem cell markers on D121-SP population. (A) Expressions of ABCG2 and Sca-1 stem cell markers were upregulated in the SP cells derived from D121 lung tumour cells as detected by flow cytometry, immunofluorescence histology staining (B) and RT–PCR (C).

Figure 3

Upregulation of stem cell-associated transcription factors SOX2 and Oct 4 on D121-SP cells. (A) The expression of SOX2 and Oct 4, but not Wnt-1 and 10 were found to be upregulated in the SP cells derived from D121 lung cancer cells as detected by RT–PCR and immunoflorence histology staining (B).

Figure 4

Inhibition of migration and increased apoptosis of D121 lung carcinoma cells by downregulation of SOX2 gene expression. (A) The expression of SOX2 in D121 lung cancer cells was downregulated by siRNA as verified by western blotting and RT–PCR. (B) Downregulation of TGF-β and Oct 4 while SOX2 expression of SOX2 was knocked down by siRNA. (C) Inhibition of D121 cells migration after downregulation of SOX2. (D–F) Increased apoptosis of D121-SP cells, which was indicated by increasing Caspase-3 and Annexin V after SOX2 knockdown in D121 cells.

Figure 5

SOX2 knockdown in D121 lung carcinoma cells suppressed experimental pulmonary metastases in syngeneic C57/BL mice. (A) The data indicate the tumour foci on the lung surface of mice. (B) The lung weights of mice is shown by bar graphs.