TGF-β1 exposure induces epithelial to mesenchymal transition both in CSCs and non-CSCs of the A549 cell line, leading to an increase of migration ability in the CD133+ A549 cell fraction

Abstract

Metastasis is the leading cause of death by cancer. Non-small-cell lung cancer (NSCLC) represents nearly 85% of primary malignant lung tumours. Recent researches have demonstrated that epithelial-to-mesenchymal transition (EMT) plays a key role in the early process of metastasis of cancer cells. Transforming growth factor-β1 (TGF-β1) is the major inductor of EMT. The aim of this study is to investigate TGF-β1's effect on cancer stem cells (CSCs) identified as cells positive for CD133, side population (SP) and non-cancer stem cells (non-CSCs) identified as cells negative for CD133, and SP in the A549 cell line. We demonstrate that TGF-β1 induces EMT in both CSC and non-CSC A549 sublines, upregulating the expression of mesenchymal markers such as vimentin and Slug, and downregulating levels of epithelial markers such as e-cadherin and cytokeratins. CSC and non-CSC A549 sublines undergoing EMT show a strong migration and strong levels of MMP9 except for the CD133(-) cell fraction. OCT4 levels are strongly upregulated in all cell fractions except CD133(-) cells. On the contrary, wound size reveals that TGF-β1 enhances motility in wild-type A549 as well as CD133(+) and SP(+) cells. For CD133(-) and SP(-) cells, TGF-β1 exposure does not change the motility. Finally, assessment of growth kinetics reveals major colony-forming efficiency in CD133(+) A549 cells. In particular, SP(+) and SP(-) A549 cells show more efficiency to form colonies than untreated corresponding cells, while for CD133(-) cells no change in colony number was observable after TGF-β1 exposure. We conclude that it is possible to highlight different cell subpopulations with different grades of stemness. Each population seems to be involved in different biological mechanisms such as stemness maintenance, tumorigenicity, invasion and migration.

Figure 1

Cytometric analyses for CD133 expression and side population profile. (a) Expression levels of CD133 were ∼3.9%. The purity of sorted populations for CD133 was routinely 90%. (b) SP cells were detected in A549 cells, with a percentage of about 12.3% of the total cell population, and the SP cell fraction was abolished in the presence of verapamil. The purity of sorted populations for side population was routinely 90%

Figure 2

Light microscope analyses. Both WT and sorted A549 showed a morphological change with elongated, fibroblast-like cells after TGF-β1 treatment. Original magnification: × 100

Figure 3

Immunofluorescence analyses of EMT-related proteins. Both WT and sorted A549 showed an increase of vimentin and, in parallel, a decrease of cytokeratins after TGF-β1 treatment. Original magnification: × 100

Figure 4

RT-PCR analyses and densitometry evaluation of EMT-related proteins. Both WT and sorted A549 showed an increase of vimentin and Slug and, in parallel, a decrease of e-cadherin after TGF-β1 treatment. *P<0.001, **P<0.0005, ***P<0.0001 compared to the parental cell line (day 0 of treatment)

Figure 5

RT-PCR, densitometry and western blot analyses of OCT4. (a) RT-PCR and densitometry evaluation showed an increase of OCT4 in all sorted and unsorted A549 cells except in CD133⁻ A549 cells, after TGF-β1 treatment. (b) Western blot analyses showed an increase of OCT4 in all sorted and unsorted A549 cells except in CD133⁻ A549 cells, after TGF-β1 treatment. β-Actin is used as loading control. *P<0.001, **P<0.0005, compared to the parental cell line (day 0 of treatment)

Figure 6

Migration potential analyses. (a) Migration assay showed that TGF-β1 significantly enhanced the migration of all samples compared with their controls except for CD133⁻ fraction. (b) RT-PCR and densitometry analyses showed that MMP-9 gene expression levels were increased in all sorted fractions except for the CD133⁻ fraction. The CD133⁺ fraction showed higher levels of MMP-9 gene expression than other fractions. (c) Western blot analyses for MMP-9 confirmed RT-PCR results. β-Actin is used as loading control. **P<0.0005, ***P<0.0001 compared to the parental cell line (day 0 of treatment)

Figure 7

Wound healing analyses. Wound size analyses performed at 24, 48, 72 and 96 h, after TGF-β1 treatment, showed that TGF-β1 enhanced motility in WT A549 as well as CD133⁺ and SP⁺ cells compared to untreated controls. CD133⁺ cells showed higher motility than other cell fractions. For CD133⁻ and SP⁻ cells, TGF-β1 exposure did not change the motility

Figure 8

Soft agar analyses. Colony growth kinetics revealed major colony-forming efficiency in CD133⁺ A549 cells compared to both untreated cells and all cell fractions sorted. SP⁺ and SP⁻ A549 cells showed more efficiency to form colonies than untreated cells. For CD133⁻ cells, no change in colony number was observable after TGF-β1 exposure