Cancer stem-like cells can be induced through dedifferentiation under hypoxic conditions in glioma, hepatoma and lung cancer

Abstract

Traditional studies have shown that transcription factors, including SOX-2, OCT-4, KLF-4, Nanog and Lin-28A, contribute to the dedifferentiation and reprogramming process in normal tissues. Hypoxia is a physiological phenomenon that exists in tumors and promotes the expression of SOX-2, OCT-4, KLF-4, Nanog and Lin-28A. Therefore, an interesting question is whether hypoxia as a stimulating factor promotes the process of dedifferentiation and induces the formation of cancer stem-like cells. Studies have shown that OCT-4 and Nanog overexpression induced the formation of cancer stem cell-like cells through dedifferentiation and enhanced malignancy in lung adenocarcinoma, and reprogramming SOX-2 in pancreatic cancer cells also promoted the dedifferentiation process. Therefore, we investigated this phenomenon in glioma, lung cancer and hepatoma cells and found that the transcription factors mentioned above were highly expressed under hypoxic conditions and induced the formation of spheres, which exhibited asymmetric division and cell cycle arrest. The dedifferentiation process induced by hypoxia highlights a new pattern of cancer development and recurrence, demonstrating that all kinds of cancer cells and the hypoxic microenvironment should be taken into consideration when developing tumor therapies.

Figure 1

Hypoxia promoted an increase in the expression of SOX-2, OCT-4, KLF-4, Nanog, Lin-28A and CD133. (a) The RT-qPCR analysis showed an upregulation in the expression of SOX-2, OCT-4, KLF-4, Nanog, Lin-28A and CD133 in a time-dependent manner in sorted A549 cells under hypoxic conditions (*P<0.05). For SOX-2, the peak expression was after 12 h of hypoxia, and the expression then decreased slightly but remained statistically higher than that of the control cells. The expression of OCT-4, KLF-4, Nanog Lin-28A and CD133 was highest after 9 h of hypoxia. (b, c) Western blot analysis showed that the transcription factors and stem cell markers were not expressed under normoxia. However, there was an increase in the expression of SOX-2, OCT-4, KLF-4, Nanog, Lin-28A and CD133 in a time-dependent manner after hypoxia treatment in sorted A549 cells (*P<0.05).

Figure 2

(a–f) Immunofluorescence staining showed that A549 CD133-negative cells highly expressed SOX-2, OCT-4, KLF-4, Nanog, Lin-28A and CD133 after 48 h of 1% O₂ exposure. (g–l) Immunofluorescence staining showed that HepG2 CD133-negative cells highly expressed SOX-2, OCT-4, KLF-4, Nanog, Lin-28A and CD133 after 48 h of 1% O₂ exposure.

Figure 3

Hypoxia increased the expression of putative CSC markers. (a) In sorted A549 cells, the proportion of CD133-positive cells was only 5.59%±2.272 in the control cells, and this proportion increased in a time-dependent manner under hypoxic conditions. After 15 days of hypoxia, the proportion of CD133-positive cells reached 49.2%±3.125. The CD133 expression in sorted HepG2 cells also increased from 2.04%±1.253 to 40.6%±2.871 after 15 days of hypoxia. For GL261 cells, the proportion of CD133-positive cells was 7.03%±3.425 in the normoxia control cells, and this rate increased remarkably from 9 days to 15 days of hypoxia exposure from 20.3%±2.547 to 97.6%±3.791. In addition, the results also showed that the expression of two other stem cell markers, CD15 and NESTIN, was upregulated, increasing from 0.56%±0.251 to 60.2%±3.472 and 3.96%±5.231 to 60.3%±5.284, respectively, after 15 days of hypoxia. (b) The proportion of stem cell markers in sorted cells increased significantly under hypoxic conditions (*P<0.05).

Figure 4

The spheres formed by single sorted cancer cells under hypoxia. (a) Single cancer cell seeding model; non-cancer stem cells were sorted using magnetic cell sorting, counted and diluted to 1500 cells/1 ml DMEM/F12+10% FBS, and then 1 μl of the suspension was seeded into each well of 96-well plates containing 170 μl of DMEM/F12 without serum. (b, c) Single sorted (CD133⁻ cells for A549 and HepG2, CD133⁻CD15⁻NESTIN⁻ cells for GL261) cancer cells formed a sphere after 21 days of hypoxia exposure; however, the cells under normoxia were dead, and there was no sphere formation (*P<0.05).

Figure 5

Newly formed spheres highly expressed transcription factors and showed asymmetric division. (a) Immunofluorescence staining showed that the newly formed GL261 spheres highly expressed SOX-2, OCT-4, KLF-4, Nanog, Lin-28A, CD133, CD15 and NESTIN. (b) These newly formed GL261 spheres kept growing in a suspension and proliferated extensively in stem cell culture medium (DMEM/F12+EGF+FGF2+B27) but presented an adherent phenotype in differentiated medium (DMEM/F12+10% FBS).

Figure 6

Cells under hypoxia showed cell cycle arrest and lower cell apoptosis. (a) A representative graph of the cell cycle arrest of sorted GL261 cells after hypoxia treatment. (b) Sorted GL261, A549 and HepG2 cells in hypoxia exhibited an increase in cells in the G₀/G₁ phase and a decrease in the proportion of cells in the G₁/G₂ and S phase (*P<0.05, ^#P<0.05). (c) Flow cytometry detected the apoptosis of sorted GL261 cells under hypoxia conditions. (d) The cell apoptosis rate was much higher in cells treated with normoxia than those treated with hypoxia (*P<0.05).