Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Feb 13;19(1):148.
doi: 10.1186/s12885-019-5360-7.

Hypoxia-induced cancer stemness acquisition is associated with CXCR4 activation by its aberrant promoter demethylation

Nahyeon Kang  1   2 , Su Yeon Choi  1   2 , Bit Na Kim  1   2 , Chang Dong Yeo  1   2 , Chan Kwon Park  1   2 , Young Kyoon Kim  1 , Tae-Jung Kim  3 , Seong-Beom Lee  4 , Sug Hyung Lee  5 , Jong Y Park  6 , Mi Sun Park  7 , Hyeon Woo Yim  7 , Seung Joon Kim  8   9
Affiliations

Hypoxia-induced cancer stemness acquisition is associated with CXCR4 activation by its aberrant promoter demethylation

Nahyeon Kang et al. BMC Cancer. .

Abstract

Background: A hypoxic microenvironment leads to an increase in the invasiveness and the metastatic potential of cancer cells within tumors via the epithelial-mesenchymal transition (EMT) and cancer stemness acquisition. However, hypoxia-induced changes in the expression and function of candidate stem cell markers and their possible molecular mechanism is still not understood.

Methods: Lung cell lines were analyzed in normoxic or hypoxic conditions. For screening among the stem cell markers, a transcriptome analysis using next-generation sequencing was performed. For validation, the EMT and stem cell characteristics were analyzed. To determine whether an epigenetic mechanism was involved, the cell lines were treated with a DNA methyltransferase inhibitor (AZA), and methylation-specific PCR and bisulfite sequencing were performed.

Results: Next-generation sequencing revealed that the CXCR4 expression was significantly higher after the hypoxic condition, which functionally resulted in the EMT and cancer stemness acquisition. The acquisition of the EMT and stemness properties was inhibited by treatment with CXCR4 siRNA. The CXCR4 was activated by either the hypoxic condition or treatment with AZA. The methylation-specific PCR and bisulfite sequencing displayed a decreased CXCR4 promoter methylation in the hypoxic condition.

Conclusions: These results suggest that hypoxia-induced acquisition of cancer stem cell characteristics was associated with CXCR4 activation by its aberrant promoter demethylation.

Keywords: Cancer stem cell; EMT; Hypoxic stimuli; Promoter methylation.

PubMed Disclaimer

Conflict of interest statement

Ethics approval and consent to participate

Animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee of the Catholic University Medical School guidelines (Approval no. CUMC-2015-0080-01).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Transcriptome analysis of the BEAS-2B and A549 cells following hypoxic stimuli for 24 h using next-generation sequencing. a Heat map of the hierarchical clustering shows a distinct separation of mRNA expression patterns of the cells cultured under hypoxic and normoxic conditions. b Levels of mRNA encoding fibronectin, vimentin, α-SMA, Slug, Snail, and ZEB1 were highly induced in cells cultured in hypoxic compared with normoxic conditions; whereas, E-cadherin decreased when the cells were exposed to hypoxic stimuli. c Among the stem cell markers, the expression of CXCR4 increased following hypoxic stimuli in both the BEAS-2B and A549 cells
Fig. 2
Fig. 2
Expression of hypoxia-induced EMT markers and stem cell markers. a E-cadherin expression decreased according to the length of exposure to hypoxia in four lung cell lines (BEAS-2B, A549, H292, and H226). Expression of fibronectin, vimentin, and α-SMA increased; although, the expression levels differed according to the duration of exposure to hypoxic stimuli. b Confocal microscopy images of E-cadherin, α-SMA, and CXCR4 expression. Expression of the epithelial cell marker E-cadherin was lost following hypoxic stimuli; although, the expression of the mesenchymal cell marker α-SMA and the stem cell marker CXCR4 increased following hypoxic stimuli. E-cadherin (gray), α-SMA (red), CXCR4 (green), and DAPI (blue) (scale bar = 50 μm). c The time-dependent mRNA and protein expressions of CXCR4 are shown. Compared with the normoxic condition, the cells exposed to the hypoxic condition displayed increased CXCR4 mRNA and protein expressions. The mRNA expressions of CXCR4 in each cell line increased as early as 2 h; although, the protein expressions were definite in 24 or 48 h according to the cell lines
Fig. 3
Fig. 3
Functional assessment of the EMT with Matrigel invasion assays following hypoxic stimuli. a, b Matrigel invasion assay signifies the hypoxia enhanced transwell invasion in the BEAS-2B, A549, H292, H226, and H460 lung cells, and these increases were reduced after CXCR4 siRNA treatment. Data are presented as mean ± SE (n = 3, *P < 0.05) (scale bar = 1000 μm)
Fig. 4
Fig. 4
Acquisition of stemness in vitro using soft agar colony formation and sphere formation assays. a Colony formation rates of all lung cell lines that were incubated under hypoxic conditions were higher than those of cell lines cultured under normoxic conditions. The increased colony formation under hypoxic conditions was reduced after CXCR4 siRNA treatment (scale bar = 500 μm). b Lung cell lines exposed to hypoxic stimuli exhibited increased sphere formation ability compared with that of the normoxic control (scale bar = 500 μm)
Fig. 5
Fig. 5
Acquisition of stemness and CXCR4 expression in vivo using a mouse tumor model. a Tumor-forming potential of cells cultured under the normoxic or hypoxic conditions was compared following an injection into the BALB/c nude mice. The number of mice that developed tumors was higher in the hypoxic cell group compared with the normoxic control group. In BEAS-2B or H226 cells injected mice, tumor size was significantly higher in the hypoxic cell groups compared with the normoxic groups (P < 0.01). A549 injected mice showed a tendency to increase although not significant. Data are presented as mean ± SE (*P < 0.01). b CXCR4 immunohistochemistry in mouse tumors injected with hypoxic cell groups showed strong CXCR4 expression compared with the normoxic controls (scale bar = 500 μm). Data are presented as mean ± SE (*P < 0.05)
Fig. 6
Fig. 6
Activation of CXCR4 by its promoter DNA demethylation. a Real-time RT-PCR revealed significantly increased CXCR4 expression after treatment with either the DNA methyltransferase inhibitor (AZA) or hypoxic condition for 24 h compared with the controls in the lung cell lines. Data are presented as the mean ± SE (n = 4, *P < 0.05). b Methylation-specific real-time PCR revealed increased promoter demethylation in hypoxic cell groups compared with normoxic controls. Data are presented as mean ± SE (n = 4, *P < 0.05). c Bisulfite sequencing was performed to demonstrate the CpG site methylation status in the promoter region of CXCR4 in lung cell lines. Representative bisulfite sequencing results show 11 CpG sites presented in underlined letters within an 82-bp promoter region of CXCR4. CpG sites which were demethylated following hypoxic condition were demonstrated in red letters. Y indicates heterozygote C/T double peaks
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jung CY, Antonia SJ. Tumor immunology and immune checkpoint inhibitors in non-small cell lung Cancer. Tuberculosis and respiratory diseases. 2018;81(1):29–41. doi: 10.4046/trd.2017.0120. - DOI - PMC - PubMed
    1. Lim SW, Ahn MJ. Current status of immune checkpoint inhibitors in treatment of non-small cell lung cancer. The Korean journal of internal medicine. 2019;34(1):50–59. doi: 10.3904/kjim.2018.179. - DOI - PMC - PubMed
    1. Monteiro J, Fodde R. Cancer stemness and metastasis: therapeutic consequences and perspectives. Eur J Cancer. 2010;46(7):1198–1203. doi: 10.1016/j.ejca.2010.02.030. - DOI - PubMed
    1. Yeo CD, Kang N, Choi SY, Kim BN, Park CK, Kim JW, Kim YK, Kim SJ. The role of hypoxia on the acquisition of epithelial-mesenchymal transition and cancer stemness: a possible link to epigenetic regulation. The Korean journal of internal medicine. 2017;32(4):589–599. doi: 10.3904/kjim.2016.302. - DOI - PMC - PubMed
    1. Wu KJ, Yang MH. Epithelial-mesenchymal transition and cancer stemness: the Twist1-Bmi1 connection. Biosci Rep. 2011;31(6):449–455. doi: 10.1042/BSR20100114. - DOI - PubMed