Chronic intermittent hypoxia promoted lung cancer stem cell-like properties via enhancing Bach1 expression

Abstract

Background: An adverse role for obstructive sleep apnea (OSA) in cancer aggressiveness and mortality has recently emerged from clinical and animal studies, and the reasons have not been fully determined. Cancer stem cells (CSCs) are regarded as the main cause of carcinoma metastasis. So far, the relationship between OSA and lung CSCs has not been explored.

Method: In the present study, we established an orthotopic mouse model of primary lung cancer and utilized chronic intermittent hypoxia (CIH) exposure to mimic OSA status.

Results: We observed that CIH endows lung cancer with greater metastatic potential, evidenced by increased tumor growth, tumor seeding, and upregulated CSC-related gene expression in the lungs. Notably, the transcription factor BTB and CNC homology 1 (Bach1), a key factor in responding to conditions of oxidative stress, is increased in lung cancer after CIH exposure in vitro and in vivo. Meanwhile, exposing lung cancer cells to CIH promoted cell proliferation, clonal diversity, induced stem-like cell marker expression, and gave rise to CSCs at a relatively higher frequency. Furthermore, the increase of mitochondrial ROS (mtROS) and CSC-marker expression induced by CIH exposure was abolished in Bach1 shRNA-treated lung cancer cells.

Conclusions: Our results indicated that CIH promoted lung CSC-like properties by activating mtROS, which was partially mediated by Bach1.

Keywords: Bach1; Cancer stem cells; Chronic intermittent hypoxia; Lung cancer; Obstructive sleep apnea.

Fig. 1

Lung cancer in CIH-treated mice has greater CSC-like potential. a The timeline of this experiment. Briefly, after injected with urethane, mice were cultured for 6 months, then exposed to CIH for 1 month. All mice were harvest at the end of the experiment. b Survival rates of C57Bl/6 wild-type male mice. Tumor + Nor, n = 12, Tumor + CIH, n = 12. c The representative images of cancer loci in lungs from Tumor + Nor and Tumor + CIH groups. d The number of cancer loci observed in the lungs of mice. Data are shown as mean ± SEM. e Images of lung cancer nodules in H&E-stained lungs of mice. (f and g) The protein levels of HIF-1α, CD44, ABCG2, Nanog, SOX2, and Oct4 in the lung cancer nodules were detected by western blot analysis. GADPH expression served as a loading control. Data are shown as mean ± SEM. Error bars represent the mean. *P < 0.05, **P < 0.01 and ***P < 0.001. CIH chronic intermittent hypoxia, Nor normoxia, CSC cancer stem cell

Fig. 2

CIH promoted lung cancer cell proliferation. Cell validations of A549 (a) and SPCA1 (b) were detected by CCK-8 assay after exposure to Nor or CIH for 24, 48, 72, and 96 h. Cell proliferation of A549 (c) or SPCA1 (d) after 48-h Nor or CIH exposure was assessed using Edu assay. e The percentage of Edu positive cells in A549 or SPCA1 was analyzed and illustrated. f, g Assessment of cancer genericity was performed by colony formation analysis after CIH treatment. h The number of colonies was measured and illustrated. Error bars represent the mean ± SEM of at least triplicate experiments. *P < 0.05, **P < 0.01. CIH chronic intermittent hypoxia

Fig. 3

CIH promoted CSC-like properties in NSCLC cells. a Microscopic observation of NSCLC cells. A549 and SPCA1 spheroids were obtained and then cultured under Nor or CIH conditions for 24 h. b qRCP analyses in triplicate of CD44, Sox2, Nanog, Oct4, and CD133 in NSCLC cells. Adherent A549 and SPCA1 were treated with 24 h-Nor or CIH exposure. GADPH expression served as an internal control. c Flow cytometry analyses of the percentage of CD44⁺CD133⁺ cells in A549 or SPCA1 populations. Error bars represent the mean ± SEM of at least triplicate experiments. *P < 0.05, **P < 0.01. CIH chronic intermittent hypoxia, CSC cancer stem cell

Fig. 4

CIH induces mitochondrial ROS accumulation in NSCLC cells. Flow cytometric analysis of A549 (a) or SPCA1 (b) treated with MitoSOX after CIH exposure for 48hrs is shown. c Quantitative analysis of MitoSOX-positive cells is shown. All data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control group. Every experiment was repeated at least three times. CIH chronic intermittent hypoxia

Fig. 5

CIH increased Bach1 expression and CSC-like property. a The protein expression of Bach1 in lung cancer nodules from Tumor + Nor or Tumor + CIH mice by Western blot analysis. b Quantification of relative protein expression was performed by densitometric analysis, and GADPH acted as an internal control. The protein expression of Bach1, Nanog, SOX2, and OCT4 in A549 (c) or SPCA1 (d) after CIH exposure for 48 h by Western blot analysis. e Quantification of relative protein expression was performed by densitometric analysis, and GADPH acted as an internal control. All data are presented as the mean ± SEM. *P < 0.05 compared with the control group. Every experiment was repeated at least three times. CIH chronic intermittent hypoxia, CSC cancer stem cell

Fig. 6

Knockdown of Bach1 decreased the stemness and mitochondrial ROS accumulation in CIH-treated NSCLCs. Bach1 shRNA or parental negative control (NC) was transfected into A549 or SPCA1. Then the cells were cultured under CIH conditions for 48hrs. Flow cytometry analyses of CD44⁺ CD133⁺ cells in Nor or CIH-treated A549 (a) or SPCA1 (b). c The percentage of CD44⁺ CD133⁺ cells in Nor or CIH-treated cells was measured and illustrated. Fluorescence microscopy analysis for localization of mtROS production in A549 (d) and SPCA1 (e). Nuclei was stained with Hoechst (blue), mitochondria ROS were stained with MitoSOX-red. The merged panels showed the MitoSOX-red positive cells in total cells. f The percentage of MitoSOX positive cells was measured and illustrated. All experiments were performed in triplicate, and data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared with the control group