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. 2024 Jan 24;13(3):216.
doi: 10.3390/cells13030216.

SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

Seung-Hyun Bae  1   2 , Kyung Yong Lee  2   3 , Suji Han  1 , Chul Won Yun  1 , ChanHyeok Park  1   2 , Hyonchol Jang  1   2
Affiliations

SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

Seung-Hyun Bae et al. Cells. .

Abstract

Effectively targeting cancer stemness is essential for successful cancer therapy. Recent studies have revealed that SOX2, a pluripotent stem cell factor, significantly contributes to cancer stem cell (CSC)-like characteristics closely associated with cancer malignancy. However, its contradictory impact on patient survival in specific cancer types, including lung adenocarcinoma (LUAD), underscores the need for more comprehensive research to clarify its functional effect on cancer stemness. In this study, we demonstrate that SOX2 is not universally required for the regulation of CSC-like properties in LUAD. We generated SOX2 knockouts in A549, H358, and HCC827 LUAD cells using the CRISPR/Cas9 system. Our results reveal unchanged CSC characteristics, including sustained proliferation, tumor sphere formation, invasion, migration, and therapy resistance, compared to normal cells. Conversely, SOX2 knockdown using conditional shRNA targeting SOX2, significantly reduced CSC traits. However, these loss-of-function effects were not rescued by SOX2 resistant to shRNA, underscoring the potential for SOX2 protein level-independent results in prior siRNA- or shRNA-based research. Ultimately, our findings demonstrate that SOX2 is not absolutely essential in LUAD cancer cells. This emphasizes the necessity of considering cancer subtype-dependent and context-dependent factors when targeting SOX2 overexpression as a potential therapeutic vulnerability in diverse cancers.

Keywords: CRISPR/Cas9; SOX2; cancer stem cell-like properties; lung adenocarcinoma; shRNA.

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Conflict of interest statement

The authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1
Inter- and intra-cellular heterogeneity in Lung adenocarcinoma (LUAD) cell lines regarding SOX2 expression. (A) SOX2 mRNA expression in LUAD cells. Semi-quantitative RT-PCR was conducted to analyze the mRNA expression of SOX2 and ACTB (β-actin) using total RNA extracted from LUAD cells A549, H23, and H358. NCCIT, an embryonic carcinoma cell line, was used as a positive control. (B) SOX2 protein expression in LUAD cells. The antibodies mentioned were employed in Western blotting, with β-actin serving as a loading control. (C) Flow cytometry (FACSVerse) was utilized to assess SOX2 protein expression in individual LUAD cells, employing an anti-SOX2 antibody and secondary goat anti-rabbit Alexa 488. A Rabbit IgG was used as a negative control. (D) Representative images of immunofluorescence staining with anti-SOX2 antibody in A549 and H358 cells. Nuclei stained with DAPI. Scale bar: 20 μm. (E) Quantification of SOX2-positive cells using immunostaining. Cells labeled with SOX2, exhibiting fluorescence intensity surpassing that of the highest controls, were considered as SOX2-positive cells. n = 50 for each sample.
Figure 2
Figure 2
SOX2 knockout does not perturb the cell proliferation of LUAD. (A) A schematic representation illustrating the gSOX2 target site on SOX2 Exon1 for the generation of SOX2 knockout LUAD cell lines using the CRISPR/Cas9 system. (B) Immunoblots showing the SOX2 protein level in A549 and H358 cells stably expressing gMock or gSOX2. (C) Immunocytochemical analysis using anti-SOX2 antibody in Mock and SOX2 knockout LUAD cells. Scale bar: 20 μm. (D) No impact of SOX2 knockout on the cell proliferation of LUAD cells. Cellular proliferation was measured using sulforhodamine B (SRB) assays, and each value was presented as optical density at 515 nm (OD515). ns, not significant. (E) No difference in the ability of a single cell to grow into a colony in LUAD SOX2 knockout cells. Representative images (left) and the quantification (right) of relative colony number in each sample are shown. The crystal violet staining was performed 7 days after seeding of A549 cells and 14 days for H358 cells. Values represent means ± SD from three independent experiments. ns, not significant.
Figure 3
Figure 3
SOX2 is not required for tumor sphere formation, invasion, and migration of certain LUAD cells. (A) No impact of SOX2 knockout on tumor sphere (TS) formation in LUAD cells. TS formation assays were conducted 14 days post-seeding using A549 and H358 cells stably expressing gMock or gSOX2. Representative images (top) and the quantification of TS number (bottom) are shown. Results from each independent experiment (Experiment 1, 2 and 3) are presented as means ± SD (n = 3). ns, not significant. Scale bar: 100 μm. (B,C) No impact of SOX2 knockout on the migration and invasion of LUAD cells. Migration (B) and invasion (C) assays were carried out using Mock and SOX2 knockout A549 and H358 cells. Representative images (left) are shown, and the total number of migrated and invaded cells was normalized to that of gMock. Mean ± SD from three independent experiments are presented. Scale bar: 100 μm. ns, not significant.
Figure 4
Figure 4
shRNA−resistant SOX2 is unable to rescue impaired proliferation by shSOX2 in LUAD. (A) A schematic showing the target site for SOX2 knockdown, with DNA sequence substitutions for shRNA-resistant SOX2. The target site (red line) was used to generate Tet-inducible SOX2 knockdown (shSOX2) in A549 and H358 cells. Red letters indicate three synonymous substitutions, reconstituting shRNA-resistant SOX2 in shSOX2 cell lines (shSOX2/+SOX2). (B) SOX2 knockdown and its reconstitution in shSOX2 LUAD cell lines were assessed. Cells were treated with 1 μg/mL of doxycycline (Dox) for 4 days, and SOX2 protein level was accessed by Western blotting. shMock and shMock + Mock were used as negative controls. (C,D) Reduced cell survival caused by shSOX2 is not restored by SOX2 reconstitution in LUAD cells. Clonogenic assays were conducted using indicated A549 and H358 cell lines. Representative images (C) and the quantification (D) are shown. The colony number of each sample was normalized to that of shMock without Dox. Mean ± SD from three independent experiments. ***, p < 0.001; ns, not significant. p values were compared with shMock without Dox.
Figure 5
Figure 5
shRNA−resistant SOX2 is unable to rescue suppression of sphere formation, invasion, and migration observed in shSOX2 LUAD cells. (A,B) The impaired sphere formation caused by shSOX2 is not restored by the reconstitution of SOX2 in LUAD cells. ‘+’ Dox indicates that 1 μg/mL Dox was treated. Representative images (top) and the quantification of TS number (bottom) are shown. TS formation assays were conducted using shMock, shSOX2, shSOX2/+Mock, and shSOX2/+SOX2 in A549 (A) and H358 (B) cells. Results from each independent experiment (Experiment 1,2 and 3) are presented as means ± SD (n = 3). (C,D) Reconstitution of SOX2 is unable to restore the reduced migration and invasion observed in shSOX2 LUAD cells. Migration (C) and invasion (D) assays using indicated A549 with or without Dox treatment. Means ± SD from three independent experiments are presented. ***, p < 0.001; **, p < 0.01 when compared with the shMock without Dox; ns, not significant; TS, tumor sphere. Scale bar: 100 μm.
Figure 6
Figure 6
SOX2 expression does not significantly affect cancer stem cell-like properties in certain LUAD cell lines. (A,B) Cellular response to chemotherapeutic drugs is not altered by SOX2 knockout in LUAD cells. Indicated concentrations of cisplatin (A) or paclitaxel (B) were treated for 72 h in A549 and H358 cells stably expressing gMock and gSOX2. Cell viability was measured by SRB assay, and the values are presented as the mean ± SD from three independent experiments. ns, not significant.
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