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Integrin α6A splice variant regulates proliferation and the Wnt/β-catenin pathway in human colorectal cancer cells.

Groulx JF, Giroux V, Beauséjour M, Boudjadi S, Basora N, Carrier JC, Beaulieu JF - Carcinogenesis (2014)

Bottom Line: The α6A silencing was also found to be associated with a significant repression of a number of Wnt/β-catenin pathway end points.Moreover, it was accompanied by a reduction in the capacity of these cells to develop tumours in xenografts.Taken together, these results demonstrate that the α6A variant is a pro-proliferative form of the α6 integrin subunit in CRC cells and appears to mediate its effects through the Wnt/β-catenin pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Intestinal Physiopathology, Department of Anatomy and Cell Biology and Department of Medicine, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada.

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Regulation of the Wnt/β-catenin pathway. (A and B) Inhibition of GSK3β rescues Wnt/β-catenin activity. (A) Response of β-catenin/TCF4 promotor activity in α6A knocked down T84 cells and controls ± SB216763. Statistical analysis between shctrl and shα6A: **P ≤ 0.001; statistical analysis between − versus + SB216763: $$P ≤ 0.001; ANOVA, n = 3. (B) qPCR analysis for the expression of LGR5 and CCD2 transcript levels in shctl and shα6A cells. T84 cells were treated for 24h ± SB216763 prior to analysis. Data were normalized to RPLPO as reference gene. Statistical analysis between untreated shctrl and shα6A: **P ≤ 0.001; statistical analysis between SB216763-treated shctrl and shα6A: ##P ≤ 0.001; statistical analysis between − versus + SB216763: $P ≤ 0.05, $$P ≤ 0.001; ANOVA, n = 3. (C and D) Knockdown of α6A reduces DVL2 protein levels in the four cell lines tested. (C) Representative WB and graph of the densiometric analysis of the detection of DVL2 protein levels in shctl and shα6A cells. Statistical analysis between untreated shctrl and shα6A: *P ≤ 0.05, ***P ≤ 0.0001, t-test, n = 3. (D) qPCR analysis for the expression of DVL2 transcript levels in shctl and shα6A cells. Data were normalized to RPLPO levels. (E) Working model for the involvement of the α6Aβ4 integrin in the regulation of the Wnt/β-catenin pathway. (Left) The α6Aβ4 integrin is over-expressed in CRC. When present, α6A regulates positively DVL2 at the protein level. DVL2, which inhibits GSK3β-mediated β-catenin phosphorylation, enhances β-catenin stability and translocation into the nucleus for the activation of the transcription of specific target genes involved in cell proliferation. (Right) Knockdown of the α6A subunit in CRC cells results in a decrease in DVL2 levels, thus allowing β-catenin phosphorylation by GSK3β. β-Catenin being targeted to degradation is no longer translocated to the nucleus resulting in repression of the transcription of Wnt/β-catenin-specific target genes. In this context, pharmacological inhibition of GSK3β with SB216763 restores Wnt/β-catenin pathway activity by bypassing the regulation of DVL2 by the α6A integrin subunit. The ‘?’ box denotes a still unknown mechanism by which α6A could be involved in the repression of key proteins regulating DVL2 degradation. Pathways/molecules activated are in black, whereas those inhibited are in grey.
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Figure 6: Regulation of the Wnt/β-catenin pathway. (A and B) Inhibition of GSK3β rescues Wnt/β-catenin activity. (A) Response of β-catenin/TCF4 promotor activity in α6A knocked down T84 cells and controls ± SB216763. Statistical analysis between shctrl and shα6A: **P ≤ 0.001; statistical analysis between − versus + SB216763: $$P ≤ 0.001; ANOVA, n = 3. (B) qPCR analysis for the expression of LGR5 and CCD2 transcript levels in shctl and shα6A cells. T84 cells were treated for 24h ± SB216763 prior to analysis. Data were normalized to RPLPO as reference gene. Statistical analysis between untreated shctrl and shα6A: **P ≤ 0.001; statistical analysis between SB216763-treated shctrl and shα6A: ##P ≤ 0.001; statistical analysis between − versus + SB216763: $P ≤ 0.05, $$P ≤ 0.001; ANOVA, n = 3. (C and D) Knockdown of α6A reduces DVL2 protein levels in the four cell lines tested. (C) Representative WB and graph of the densiometric analysis of the detection of DVL2 protein levels in shctl and shα6A cells. Statistical analysis between untreated shctrl and shα6A: *P ≤ 0.05, ***P ≤ 0.0001, t-test, n = 3. (D) qPCR analysis for the expression of DVL2 transcript levels in shctl and shα6A cells. Data were normalized to RPLPO levels. (E) Working model for the involvement of the α6Aβ4 integrin in the regulation of the Wnt/β-catenin pathway. (Left) The α6Aβ4 integrin is over-expressed in CRC. When present, α6A regulates positively DVL2 at the protein level. DVL2, which inhibits GSK3β-mediated β-catenin phosphorylation, enhances β-catenin stability and translocation into the nucleus for the activation of the transcription of specific target genes involved in cell proliferation. (Right) Knockdown of the α6A subunit in CRC cells results in a decrease in DVL2 levels, thus allowing β-catenin phosphorylation by GSK3β. β-Catenin being targeted to degradation is no longer translocated to the nucleus resulting in repression of the transcription of Wnt/β-catenin-specific target genes. In this context, pharmacological inhibition of GSK3β with SB216763 restores Wnt/β-catenin pathway activity by bypassing the regulation of DVL2 by the α6A integrin subunit. The ‘?’ box denotes a still unknown mechanism by which α6A could be involved in the repression of key proteins regulating DVL2 degradation. Pathways/molecules activated are in black, whereas those inhibited are in grey.

Mentions: To further investigate the possible contribution of GSK3β to α6A-mediated β-catenin activation, we evaluated the effect of GSK3β inhibition on the rescue of responsive β-catenin/TCF4/LEF reporter plasmid activity and target gene expression in α6A knocked down T84 cells. As shown in Figure 6, we found that the pharmacological inhibition of GSK3β with SB21673 led to a significant stimulation of TOPflash activity in both shctl and shα6A cells (Figure 6A). Moreover, GSK3β inhibition stimulated TOPflash activity to the same level in both shctl and shα6A cells. To further extend these observations, the effect of α6A knockdown and GSK3β inhibition on Wnt/β-catenin target gene expression was analyzed by qPCR. We chose to investigate LGR5, CCD1 and CCD2, three well-documented target genes of the Wnt/β-catenin pathway in the intestine (37,38). First, shα6A cells were found to display a significant reduction of mRNA levels for LGR5 and CCD2 relative to shctl cells (Figure 6B), confirming the inhibition of Wnt/β-catenin transcriptional activity on these two target genes. GSK3β inhibition in shα6A cells resulted in a significant stimulation of LGR5 and CCD2 mRNA expression (Figure 6B). When both shctl and shα6A cells treated with the GSK3β inhibitor were compared, LGR5 mRNA expression was still significantly lower in shα6A cells than in shctl cells, whereas CCD2 mRNA expression was similar (Figure 6B), suggesting at least a partial rescue. No significant change in CCD1 mRNA expression was observed under these conditions (data not shown). These results suggest a role for the α6A integrin subunit in the control of Wnt/β-catenin activity and some of its target genes through GSK3β, which in turn may regulate CRC proliferation.


Integrin α6A splice variant regulates proliferation and the Wnt/β-catenin pathway in human colorectal cancer cells.

Groulx JF, Giroux V, Beauséjour M, Boudjadi S, Basora N, Carrier JC, Beaulieu JF - Carcinogenesis (2014)

Regulation of the Wnt/β-catenin pathway. (A and B) Inhibition of GSK3β rescues Wnt/β-catenin activity. (A) Response of β-catenin/TCF4 promotor activity in α6A knocked down T84 cells and controls ± SB216763. Statistical analysis between shctrl and shα6A: **P ≤ 0.001; statistical analysis between − versus + SB216763: $$P ≤ 0.001; ANOVA, n = 3. (B) qPCR analysis for the expression of LGR5 and CCD2 transcript levels in shctl and shα6A cells. T84 cells were treated for 24h ± SB216763 prior to analysis. Data were normalized to RPLPO as reference gene. Statistical analysis between untreated shctrl and shα6A: **P ≤ 0.001; statistical analysis between SB216763-treated shctrl and shα6A: ##P ≤ 0.001; statistical analysis between − versus + SB216763: $P ≤ 0.05, $$P ≤ 0.001; ANOVA, n = 3. (C and D) Knockdown of α6A reduces DVL2 protein levels in the four cell lines tested. (C) Representative WB and graph of the densiometric analysis of the detection of DVL2 protein levels in shctl and shα6A cells. Statistical analysis between untreated shctrl and shα6A: *P ≤ 0.05, ***P ≤ 0.0001, t-test, n = 3. (D) qPCR analysis for the expression of DVL2 transcript levels in shctl and shα6A cells. Data were normalized to RPLPO levels. (E) Working model for the involvement of the α6Aβ4 integrin in the regulation of the Wnt/β-catenin pathway. (Left) The α6Aβ4 integrin is over-expressed in CRC. When present, α6A regulates positively DVL2 at the protein level. DVL2, which inhibits GSK3β-mediated β-catenin phosphorylation, enhances β-catenin stability and translocation into the nucleus for the activation of the transcription of specific target genes involved in cell proliferation. (Right) Knockdown of the α6A subunit in CRC cells results in a decrease in DVL2 levels, thus allowing β-catenin phosphorylation by GSK3β. β-Catenin being targeted to degradation is no longer translocated to the nucleus resulting in repression of the transcription of Wnt/β-catenin-specific target genes. In this context, pharmacological inhibition of GSK3β with SB216763 restores Wnt/β-catenin pathway activity by bypassing the regulation of DVL2 by the α6A integrin subunit. The ‘?’ box denotes a still unknown mechanism by which α6A could be involved in the repression of key proteins regulating DVL2 degradation. Pathways/molecules activated are in black, whereas those inhibited are in grey.
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Related In: Results  -  Collection

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Figure 6: Regulation of the Wnt/β-catenin pathway. (A and B) Inhibition of GSK3β rescues Wnt/β-catenin activity. (A) Response of β-catenin/TCF4 promotor activity in α6A knocked down T84 cells and controls ± SB216763. Statistical analysis between shctrl and shα6A: **P ≤ 0.001; statistical analysis between − versus + SB216763: $$P ≤ 0.001; ANOVA, n = 3. (B) qPCR analysis for the expression of LGR5 and CCD2 transcript levels in shctl and shα6A cells. T84 cells were treated for 24h ± SB216763 prior to analysis. Data were normalized to RPLPO as reference gene. Statistical analysis between untreated shctrl and shα6A: **P ≤ 0.001; statistical analysis between SB216763-treated shctrl and shα6A: ##P ≤ 0.001; statistical analysis between − versus + SB216763: $P ≤ 0.05, $$P ≤ 0.001; ANOVA, n = 3. (C and D) Knockdown of α6A reduces DVL2 protein levels in the four cell lines tested. (C) Representative WB and graph of the densiometric analysis of the detection of DVL2 protein levels in shctl and shα6A cells. Statistical analysis between untreated shctrl and shα6A: *P ≤ 0.05, ***P ≤ 0.0001, t-test, n = 3. (D) qPCR analysis for the expression of DVL2 transcript levels in shctl and shα6A cells. Data were normalized to RPLPO levels. (E) Working model for the involvement of the α6Aβ4 integrin in the regulation of the Wnt/β-catenin pathway. (Left) The α6Aβ4 integrin is over-expressed in CRC. When present, α6A regulates positively DVL2 at the protein level. DVL2, which inhibits GSK3β-mediated β-catenin phosphorylation, enhances β-catenin stability and translocation into the nucleus for the activation of the transcription of specific target genes involved in cell proliferation. (Right) Knockdown of the α6A subunit in CRC cells results in a decrease in DVL2 levels, thus allowing β-catenin phosphorylation by GSK3β. β-Catenin being targeted to degradation is no longer translocated to the nucleus resulting in repression of the transcription of Wnt/β-catenin-specific target genes. In this context, pharmacological inhibition of GSK3β with SB216763 restores Wnt/β-catenin pathway activity by bypassing the regulation of DVL2 by the α6A integrin subunit. The ‘?’ box denotes a still unknown mechanism by which α6A could be involved in the repression of key proteins regulating DVL2 degradation. Pathways/molecules activated are in black, whereas those inhibited are in grey.
Mentions: To further investigate the possible contribution of GSK3β to α6A-mediated β-catenin activation, we evaluated the effect of GSK3β inhibition on the rescue of responsive β-catenin/TCF4/LEF reporter plasmid activity and target gene expression in α6A knocked down T84 cells. As shown in Figure 6, we found that the pharmacological inhibition of GSK3β with SB21673 led to a significant stimulation of TOPflash activity in both shctl and shα6A cells (Figure 6A). Moreover, GSK3β inhibition stimulated TOPflash activity to the same level in both shctl and shα6A cells. To further extend these observations, the effect of α6A knockdown and GSK3β inhibition on Wnt/β-catenin target gene expression was analyzed by qPCR. We chose to investigate LGR5, CCD1 and CCD2, three well-documented target genes of the Wnt/β-catenin pathway in the intestine (37,38). First, shα6A cells were found to display a significant reduction of mRNA levels for LGR5 and CCD2 relative to shctl cells (Figure 6B), confirming the inhibition of Wnt/β-catenin transcriptional activity on these two target genes. GSK3β inhibition in shα6A cells resulted in a significant stimulation of LGR5 and CCD2 mRNA expression (Figure 6B). When both shctl and shα6A cells treated with the GSK3β inhibitor were compared, LGR5 mRNA expression was still significantly lower in shα6A cells than in shctl cells, whereas CCD2 mRNA expression was similar (Figure 6B), suggesting at least a partial rescue. No significant change in CCD1 mRNA expression was observed under these conditions (data not shown). These results suggest a role for the α6A integrin subunit in the control of Wnt/β-catenin activity and some of its target genes through GSK3β, which in turn may regulate CRC proliferation.

Bottom Line: The α6A silencing was also found to be associated with a significant repression of a number of Wnt/β-catenin pathway end points.Moreover, it was accompanied by a reduction in the capacity of these cells to develop tumours in xenografts.Taken together, these results demonstrate that the α6A variant is a pro-proliferative form of the α6 integrin subunit in CRC cells and appears to mediate its effects through the Wnt/β-catenin pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Intestinal Physiopathology, Department of Anatomy and Cell Biology and Department of Medicine, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada.

Show MeSH
Related in: MedlinePlus