CD133 Modulate HIF-1α Expression under Hypoxia in EMT Phenotype Pancreatic Cancer Stem-Like Cells

Abstract

Although CD133 is a known representative cancer stem cell marker, its function in tumor aggressiveness under hypoxia is not fully known. The aim of this study is to demonstrate that CD133 regulates hypoxia inducible factor (HIF)-1α expression with tumor migration. The CD133⁺ pancreatic cancer cell line, Capan1M9, was compared with the CD133(-) cell line, shCD133M9, under hypoxia. HIF-1α expression levels were compared by Western blot, HIF-1α nucleus translocation assay and real-time (RT)-PCR. The hypoxia responsive element (HRE) was observed by luciferase assay. The migration ability was analyzed by migration and wound healing assays. Epithelial mesenchymal transition (EMT) related genes were analyzed by real-time RT-PCR. HIF-1α was highly expressed in Capan1M9 compared to shCD133M9 under hypoxia because of the high activation of HRE. Furthermore, the migration ability of Capan1M9 was higher than that of shCD133M9 under hypoxia, suggesting higher expression of EMT related genes in Capan1M9 compared to shCD133M9.

Conclusion: HIF-1α expression under hypoxia in CD133⁺ pancreatic cancer cells correlated with tumor cell migration through EMT gene expression. Understanding the function of CD133 in cancer aggressiveness provides a novel therapeutic approach to eradicate pancreatic cancer stem cells.

Keywords: CD133; EMT; HIF-1α; cancer stem cell; hypoxia; migration; pancreatic cancer.

Figure 1

CD133⁺ cells obtained tolerance to hypoxia. (A) Flow cytometric analysis showed an increase of the rate of CD133⁺ cells under 1% hypoxia and 0.1% hypoxia; (B) Capan1M9 cells showed 84.4% CD133 expression and shCD133M9 cells showed only 20.6% CD133 positive rate by flow cytometric analysis; (C) Fluorescent immunostaining showed that hypoxia inducible factor (HIF)-1α expressed in nuclei of Capan1M9 and shCD133M9 under hypoxia. Scale bars show 50 mm as a red bar; (D) Western blot analysis proved RNAi methods effectively down-regulated CD133 expression and the difference of HIF-1α expression level.

Figure 2

CD133 regulates HIF-1α under hypoxic condition. (A) Western blots confirmed CD133 silencing decreased HIF-1α protein levels in each period, both under 1% hypoxia and normoxia with CoCl₂ administration; (B) In Capan1M9 and shCD133M9 transfected with HIF-1α reporter plasmid, there are significant difference of HIF-1α transactivating activity. CD133 silencing also decreased HIF-1α transactivation activity; (C) The ratio of HIF-1α⁺ cells was counted after 12 h 1% O₂ exposure by HIF-1α nuclear translocation assay. HIF-1α⁺ cells of shCD133M9 were smaller than that of Capan1M9. (D) RT-PCR confirmed CD133 silencing decreased HIF-1α mRNA levels in each hypoxic period. Data represent mean ± SD of three experiments; *** p < 0.001; **** p < 0.0001.

Figure 2

CD133 regulates HIF-1α under hypoxic condition. (A) Western blots confirmed CD133 silencing decreased HIF-1α protein levels in each period, both under 1% hypoxia and normoxia with CoCl₂ administration; (B) In Capan1M9 and shCD133M9 transfected with HIF-1α reporter plasmid, there are significant difference of HIF-1α transactivating activity. CD133 silencing also decreased HIF-1α transactivation activity; (C) The ratio of HIF-1α⁺ cells was counted after 12 h 1% O₂ exposure by HIF-1α nuclear translocation assay. HIF-1α⁺ cells of shCD133M9 were smaller than that of Capan1M9. (D) RT-PCR confirmed CD133 silencing decreased HIF-1α mRNA levels in each hypoxic period. Data represent mean ± SD of three experiments; *** p < 0.001; **** p < 0.0001.

Figure 3

CD133 plays an important role in pancreatic cancer cell migration under hypoxia. (A) Boyden chamber migration assay showed CD133 silencing reduced the migratory ability of Capan1M9 both in normoxia and hypoxia. Capan1M9 only enhanced its migration ability under hypoxia; (B) Wound healing assay also showed CD133 silencing reduced the migratory ability of Capan1M9 in normoxia and hypoxia. Hypoxia discouraged wound healing of Capan1M9 and shCD133M9. Wide error bars for Capan1M9 cell migrations are due to varying cell number between experiments. Data represent mean ± SD of three experiments; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

CD133 contributes to EMT phenomenon under hypoxia. (A) CD133 silencing down-regulated Slug mRNA level significantly. Hypoxia gained the Slug mRNA level only in Capan1M9; (B) Significant difference was not shown in Snail mRNA level; (C) CD133 silencing down-regulated N-cadherin mRNA level. Data represent mean ± SD of three experiments; ** p < 0.01; *** p < 0.001.

Figure 5

Proposed model for CD133-HIF-1α axis in pancreatic cancer stem cells. CD133 positively stimulates HIF-1α expression under hypoxia, and subsequent up-regulation of N-cadherin results in epithelial mesenchymal transition (EMT) phenomenon and tumor migration. Two and three arrows represent N-cadherin up-regulation.