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Hypoxia-inducible factors modulate the stemness and malignancy of colon cancer cells by playing opposite roles in canonical Wnt signaling.

Santoyo-Ramos P, Likhatcheva M, García-Zepeda EA, Castañeda-Patlán MC, Robles-Flores M - PLoS ONE (2014)

Bottom Line: This study examined the role played by hypoxia-inducible factors (HIFs) in malignant phenotype maintenance and canonical Wnt signaling.The stable knockdown of HIF-1α or HIF-2α expression induced negative effects on the malignant phenotype of colon cancer cells, with lactate production, the rate of apoptosis, migration, CXCR4-mediated chemotaxis, and tumorigenic activity all being significantly affected by HIF knockdown and with HIF-1α depletion exerting greater effects.In SW480 cells, HIF-2α knockdown did not affect β-catenin levels, increasing the transcriptional activity of β-catenin by inducing its nuclear accumulation, whereas HIF-1α silencing negatively affected the stability and transcriptional activity of β-catenin, inducing its exit from the nuclei and its recruitment to the cell membrane by E-cadherin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Medicine, and Biomedical Research Institute, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico.

ABSTRACT
This study examined the role played by hypoxia-inducible factors (HIFs) in malignant phenotype maintenance and canonical Wnt signaling. Under normoxia, we determined that both HIF-1α and HIF-2α are expressed in human colon cancer cells but not in their non-malignant counterparts. The stable knockdown of HIF-1α or HIF-2α expression induced negative effects on the malignant phenotype of colon cancer cells, with lactate production, the rate of apoptosis, migration, CXCR4-mediated chemotaxis, and tumorigenic activity all being significantly affected by HIF knockdown and with HIF-1α depletion exerting greater effects. Knockdown of these two HIF transcripts induced different and even opposite effects on β-catenin transcriptional activity in colon cancer cells with different genetic Wnt signaling pathways. In SW480 cells, HIF-2α knockdown did not affect β-catenin levels, increasing the transcriptional activity of β-catenin by inducing its nuclear accumulation, whereas HIF-1α silencing negatively affected the stability and transcriptional activity of β-catenin, inducing its exit from the nuclei and its recruitment to the cell membrane by E-cadherin. In addition, although HIF-1α depletion induced a reversal of the epithelial-to-mesenchymal transition (EMT), HIF-2α silencing altered the expression of the stem cell markers CD44, Oct4, and CD24 and of the differentiation marker CK20 in the opposite direction as HIF-1α silencing. Remarkably, HIF-2α knockdown also enhanced β-catenin transcriptional activity under hypoxia in cells that displayed normal Wnt signaling, suggesting that the gene negatively modulates canonical Wnt signaling in colon cancer cells. Taken together, our results indicate that HIFs play opposing roles in canonical Wnt signaling and are essential for the stemness and malignancy maintenance of colon cancer cells.

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HIF-1α and HIF-2α knockdown exerted opposing effects on the expression of the stem cell markers CD44 and Oct4, but only HIF-1α depletion increased the expression of the differentiation marker CK20.Stable control (scrambled shRNA plasmid) or HIF-1α- or HIF-2α- silenced SW480 cells were incubated in the presence of EDTA, washed, and incubated with mouse anti-CD44, rabbit anti-Oct4, mouse anti-CD24 or mouse anti-CK20 antibodies. The cells were washed, stained with Alexa 647-conjugated goat anti-mouse/rabbit secondary antibody, and examined by flow cytometry. The figure shows the overlapping histograms of labeled SW480 control cells (blue line and Ctr in each bar graph), HIF-1α-knockdown cells (green line), and HIF-2α-knockdown cells (orange line). A representative histogram of the data is shown, and the bar graphs represent the means of the median fluorescence intensity ± SEM from at least four independent experiments. *: p<0.05; **: p<0.01.
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pone-0112580-g009: HIF-1α and HIF-2α knockdown exerted opposing effects on the expression of the stem cell markers CD44 and Oct4, but only HIF-1α depletion increased the expression of the differentiation marker CK20.Stable control (scrambled shRNA plasmid) or HIF-1α- or HIF-2α- silenced SW480 cells were incubated in the presence of EDTA, washed, and incubated with mouse anti-CD44, rabbit anti-Oct4, mouse anti-CD24 or mouse anti-CK20 antibodies. The cells were washed, stained with Alexa 647-conjugated goat anti-mouse/rabbit secondary antibody, and examined by flow cytometry. The figure shows the overlapping histograms of labeled SW480 control cells (blue line and Ctr in each bar graph), HIF-1α-knockdown cells (green line), and HIF-2α-knockdown cells (orange line). A representative histogram of the data is shown, and the bar graphs represent the means of the median fluorescence intensity ± SEM from at least four independent experiments. *: p<0.05; **: p<0.01.

Mentions: We then examined the effect of HIF-1α or HIF-2α depletion on the expression profiles of stem cell markers by flow cytometry. SW480 cells have been reported to express CD44, with a subpopulation of these cells also expressing CD24 [24], reported to be a stem cell marker and adhesion molecule. In addition, HIFs have been reported to induce the gene expression signatures characteristic of human embryonic stem cells in aggressive tumors [25], including Oct4 and Sox2. The presence of the CD44 and Oct4 stem cell markers and the possible presence of CD24 and CK20, which has been widely used as an epithelial differentiation marker, were then analyzed by FACS. The results showed that the depletion of HIF-1α decreased the expression of CD44 and Oct4 and increased the expression of CD24 and CK20 with respect to the controls (Figure 9). In marked contrast to HIF-1α, HIF-2α knockdown did not affect the expression of CD24 and CK20 but did increase the expression of the stem cell markers CD44 and Oct4 compared with the controls. These findings are consistent with the hypotheses that HIF-1α participates in the induction of an EMT genetic program that allows cancer cells to acquire features of mesenchymal-like cells, and importantly, that HIF-1α and HIF-2α do not activate the same pathways to regulate stem cell maintenance or differentiation, arguing that these proteins play complementary and non-redundant roles in tumor biology.


Hypoxia-inducible factors modulate the stemness and malignancy of colon cancer cells by playing opposite roles in canonical Wnt signaling.

Santoyo-Ramos P, Likhatcheva M, García-Zepeda EA, Castañeda-Patlán MC, Robles-Flores M - PLoS ONE (2014)

HIF-1α and HIF-2α knockdown exerted opposing effects on the expression of the stem cell markers CD44 and Oct4, but only HIF-1α depletion increased the expression of the differentiation marker CK20.Stable control (scrambled shRNA plasmid) or HIF-1α- or HIF-2α- silenced SW480 cells were incubated in the presence of EDTA, washed, and incubated with mouse anti-CD44, rabbit anti-Oct4, mouse anti-CD24 or mouse anti-CK20 antibodies. The cells were washed, stained with Alexa 647-conjugated goat anti-mouse/rabbit secondary antibody, and examined by flow cytometry. The figure shows the overlapping histograms of labeled SW480 control cells (blue line and Ctr in each bar graph), HIF-1α-knockdown cells (green line), and HIF-2α-knockdown cells (orange line). A representative histogram of the data is shown, and the bar graphs represent the means of the median fluorescence intensity ± SEM from at least four independent experiments. *: p<0.05; **: p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4232394&req=5

pone-0112580-g009: HIF-1α and HIF-2α knockdown exerted opposing effects on the expression of the stem cell markers CD44 and Oct4, but only HIF-1α depletion increased the expression of the differentiation marker CK20.Stable control (scrambled shRNA plasmid) or HIF-1α- or HIF-2α- silenced SW480 cells were incubated in the presence of EDTA, washed, and incubated with mouse anti-CD44, rabbit anti-Oct4, mouse anti-CD24 or mouse anti-CK20 antibodies. The cells were washed, stained with Alexa 647-conjugated goat anti-mouse/rabbit secondary antibody, and examined by flow cytometry. The figure shows the overlapping histograms of labeled SW480 control cells (blue line and Ctr in each bar graph), HIF-1α-knockdown cells (green line), and HIF-2α-knockdown cells (orange line). A representative histogram of the data is shown, and the bar graphs represent the means of the median fluorescence intensity ± SEM from at least four independent experiments. *: p<0.05; **: p<0.01.
Mentions: We then examined the effect of HIF-1α or HIF-2α depletion on the expression profiles of stem cell markers by flow cytometry. SW480 cells have been reported to express CD44, with a subpopulation of these cells also expressing CD24 [24], reported to be a stem cell marker and adhesion molecule. In addition, HIFs have been reported to induce the gene expression signatures characteristic of human embryonic stem cells in aggressive tumors [25], including Oct4 and Sox2. The presence of the CD44 and Oct4 stem cell markers and the possible presence of CD24 and CK20, which has been widely used as an epithelial differentiation marker, were then analyzed by FACS. The results showed that the depletion of HIF-1α decreased the expression of CD44 and Oct4 and increased the expression of CD24 and CK20 with respect to the controls (Figure 9). In marked contrast to HIF-1α, HIF-2α knockdown did not affect the expression of CD24 and CK20 but did increase the expression of the stem cell markers CD44 and Oct4 compared with the controls. These findings are consistent with the hypotheses that HIF-1α participates in the induction of an EMT genetic program that allows cancer cells to acquire features of mesenchymal-like cells, and importantly, that HIF-1α and HIF-2α do not activate the same pathways to regulate stem cell maintenance or differentiation, arguing that these proteins play complementary and non-redundant roles in tumor biology.

Bottom Line: This study examined the role played by hypoxia-inducible factors (HIFs) in malignant phenotype maintenance and canonical Wnt signaling.The stable knockdown of HIF-1α or HIF-2α expression induced negative effects on the malignant phenotype of colon cancer cells, with lactate production, the rate of apoptosis, migration, CXCR4-mediated chemotaxis, and tumorigenic activity all being significantly affected by HIF knockdown and with HIF-1α depletion exerting greater effects.In SW480 cells, HIF-2α knockdown did not affect β-catenin levels, increasing the transcriptional activity of β-catenin by inducing its nuclear accumulation, whereas HIF-1α silencing negatively affected the stability and transcriptional activity of β-catenin, inducing its exit from the nuclei and its recruitment to the cell membrane by E-cadherin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Medicine, and Biomedical Research Institute, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico.

ABSTRACT
This study examined the role played by hypoxia-inducible factors (HIFs) in malignant phenotype maintenance and canonical Wnt signaling. Under normoxia, we determined that both HIF-1α and HIF-2α are expressed in human colon cancer cells but not in their non-malignant counterparts. The stable knockdown of HIF-1α or HIF-2α expression induced negative effects on the malignant phenotype of colon cancer cells, with lactate production, the rate of apoptosis, migration, CXCR4-mediated chemotaxis, and tumorigenic activity all being significantly affected by HIF knockdown and with HIF-1α depletion exerting greater effects. Knockdown of these two HIF transcripts induced different and even opposite effects on β-catenin transcriptional activity in colon cancer cells with different genetic Wnt signaling pathways. In SW480 cells, HIF-2α knockdown did not affect β-catenin levels, increasing the transcriptional activity of β-catenin by inducing its nuclear accumulation, whereas HIF-1α silencing negatively affected the stability and transcriptional activity of β-catenin, inducing its exit from the nuclei and its recruitment to the cell membrane by E-cadherin. In addition, although HIF-1α depletion induced a reversal of the epithelial-to-mesenchymal transition (EMT), HIF-2α silencing altered the expression of the stem cell markers CD44, Oct4, and CD24 and of the differentiation marker CK20 in the opposite direction as HIF-1α silencing. Remarkably, HIF-2α knockdown also enhanced β-catenin transcriptional activity under hypoxia in cells that displayed normal Wnt signaling, suggesting that the gene negatively modulates canonical Wnt signaling in colon cancer cells. Taken together, our results indicate that HIFs play opposing roles in canonical Wnt signaling and are essential for the stemness and malignancy maintenance of colon cancer cells.

Show MeSH
Related in: MedlinePlus