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Down regulation of Wnt signaling mitigates hypoxia-induced chemoresistance in human osteosarcoma cells.

Scholten DJ, Timmer CM, Peacock JD, Pelle DW, Williams BO, Steensma MR - PLoS ONE (2014)

Bottom Line: Hypoxia is an element intrinsic to most solid-tumor microenvironments, including that of OS, and is associated with resistance to therapy, poor survival, and a malignant phenotype.Finally, we show that hypoxia results in resistance of human OS cells to doxorubicin-mediated toxicity (6-13 fold increase, p<0.01).These data support the conclusion that Wnt/β-catenin signaling is down-regulated in human OS cells under hypoxia and that this signaling alteration may represent a viable target to combat chemoresistant OS subpopulations in a hypoxic niche.

View Article: PubMed Central - PubMed

Affiliation: Michigan State University College of Human Medicine, Grand Rapids, Michigan, United States of America; Van Andel Research Institute, Grand Rapids, Michigan, United States of America.

ABSTRACT
Osteosarcoma (OS) is the most common type of solid bone cancer and remains the second leading cause of cancer-related death for children and young adults. Hypoxia is an element intrinsic to most solid-tumor microenvironments, including that of OS, and is associated with resistance to therapy, poor survival, and a malignant phenotype. Cells respond to hypoxia through alterations in gene expression, mediated most notably through the hypoxia-inducible factor (HIF) class of transcription factors. Here we investigate hypoxia-induced changes in the Wnt/β-catenin signaling pathway, a key signaling cascade involved in OS pathogenesis. We show that hypoxia results in increased expression and signaling activation of HIF proteins in human osteosarcoma cells. Wnt/β-catenin signaling is down-regulated by hypoxia in human OS cells, as demonstrated by decreased active β-catenin protein levels and axin2 mRNA expression (p<0.05). This down-regulation appears to rely on both HIF-independent and HIF-dependent mechanisms, with HIF-1α standing out as an important regulator. Finally, we show that hypoxia results in resistance of human OS cells to doxorubicin-mediated toxicity (6-13 fold increase, p<0.01). These hypoxic OS cells can be sensitized to doxorubicin treatment by further inhibition of the Wnt/β-catenin signaling pathway (p<0.05). These data support the conclusion that Wnt/β-catenin signaling is down-regulated in human OS cells under hypoxia and that this signaling alteration may represent a viable target to combat chemoresistant OS subpopulations in a hypoxic niche.

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Wnt/β-catenin signaling down-regulation is both dependent and independent of HIF expression.A, Levels of active β-catenin were determined by western blot at different hypoxia time points for each of the shRNA in MNNG/HOS cells. Overall levels of active β-catenin decreased regardless of HIF expression, although the magnitude of decrease was not equal across all lines. Active and total β-catenin protein levels were decreased at the 72 hour time point. Actin was the loading control. B, Decreased Wnt/β-catenin signaling activity under hypoxia was confirmed by measuring axin2 mRNA levels via qrt-PCR in MNNG/HOS cells. Hypoxia (72 hour, 0.5% O2) resulted in decreased axin2 mRNA expression relative to normoxia. C, When analyzed relative to the shnon hypoxia mRNA, axin2 mRNA was increased in the shHIF-1α MNNG/HOS cell lines. Asterisks indicate statistical significance (*p<0.05, **p<0.01, ***p<0.001). shNON: non-targeting shRNA; HIF shRNAs used are indicated in parentheses.
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pone-0111431-g003: Wnt/β-catenin signaling down-regulation is both dependent and independent of HIF expression.A, Levels of active β-catenin were determined by western blot at different hypoxia time points for each of the shRNA in MNNG/HOS cells. Overall levels of active β-catenin decreased regardless of HIF expression, although the magnitude of decrease was not equal across all lines. Active and total β-catenin protein levels were decreased at the 72 hour time point. Actin was the loading control. B, Decreased Wnt/β-catenin signaling activity under hypoxia was confirmed by measuring axin2 mRNA levels via qrt-PCR in MNNG/HOS cells. Hypoxia (72 hour, 0.5% O2) resulted in decreased axin2 mRNA expression relative to normoxia. C, When analyzed relative to the shnon hypoxia mRNA, axin2 mRNA was increased in the shHIF-1α MNNG/HOS cell lines. Asterisks indicate statistical significance (*p<0.05, **p<0.01, ***p<0.001). shNON: non-targeting shRNA; HIF shRNAs used are indicated in parentheses.

Mentions: The observed down-regulation in Wnt/β-catenin signaling raises mechanistic questions, especially regarding the potential involvement of HIF proteins. To determine whether the expression of either HIF protein is needed for the decrease in Wnt/β-catenin signaling, we stably transduced MNNG/HOS cells with shRNA targeting either HIF-1α or HIF-2α. We confirmed knockdown at the mRNA (80–90% knockdown) and protein level specific to each HIF protein relative to a non-targeting shRNA control for two independent shRNA's targeting each HIFα subunit (data not shown). Next we examined Wnt/β-catenin signaling activity under hypoxic conditions between the shHIF and non-targeting shRNA MNNG/HOS cells. Active β-catenin protein levels were decreased under hypoxic conditions in both the non-targeting shRNA and the shHIFα cell lines (Figure 3A), as was seen before in the MG-63 cell line (see Fig. 2A). There was also a decrease in total β-catenin at the 72 hour time point (Figure 3A). We assessed axin2 mRNA expression across the shHIF-modified cell lines, comparing normoxic to hypoxic conditions to determine whether β-catenin transcriptional activity was truly altered, or whether the decrease in active β-catenin protein only reflected a decrease in total β-catenin. There was a statistically significant decrease in axin2 mRNA expression under hypoxic conditions in the non-targeting shRNA MNNG/HOS cell line (Figure 3B, p<0.01), just as noted earlier for the MG-63 and 143B lines (see Figure 2B). That decrease was also consistent in the shHIF-1α- and shHIF-2α-treated MNNG/HOS cell lines, indicating that HIF-independent mechanisms contribute to Wnt/β-catenin signaling down-regulation under hypoxia (Figure 3B, p<0.05). However, when the data were normalized to the hypoxia shnon control, axin2 mRNA expression in both hypoxia shHIF-1α cell lines was increased, implying that HIF-1α contributes to Wnt/β-catenin signaling down-regulation (Figure 3C, p<0.05). Thus, this data suggests that both HIF-dependent and HIF-independent mechanisms contribute to the hypoxia-mediated down-regulation of Wnt/β-catenin signaling in human OS cells.


Down regulation of Wnt signaling mitigates hypoxia-induced chemoresistance in human osteosarcoma cells.

Scholten DJ, Timmer CM, Peacock JD, Pelle DW, Williams BO, Steensma MR - PLoS ONE (2014)

Wnt/β-catenin signaling down-regulation is both dependent and independent of HIF expression.A, Levels of active β-catenin were determined by western blot at different hypoxia time points for each of the shRNA in MNNG/HOS cells. Overall levels of active β-catenin decreased regardless of HIF expression, although the magnitude of decrease was not equal across all lines. Active and total β-catenin protein levels were decreased at the 72 hour time point. Actin was the loading control. B, Decreased Wnt/β-catenin signaling activity under hypoxia was confirmed by measuring axin2 mRNA levels via qrt-PCR in MNNG/HOS cells. Hypoxia (72 hour, 0.5% O2) resulted in decreased axin2 mRNA expression relative to normoxia. C, When analyzed relative to the shnon hypoxia mRNA, axin2 mRNA was increased in the shHIF-1α MNNG/HOS cell lines. Asterisks indicate statistical significance (*p<0.05, **p<0.01, ***p<0.001). shNON: non-targeting shRNA; HIF shRNAs used are indicated in parentheses.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4210185&req=5

pone-0111431-g003: Wnt/β-catenin signaling down-regulation is both dependent and independent of HIF expression.A, Levels of active β-catenin were determined by western blot at different hypoxia time points for each of the shRNA in MNNG/HOS cells. Overall levels of active β-catenin decreased regardless of HIF expression, although the magnitude of decrease was not equal across all lines. Active and total β-catenin protein levels were decreased at the 72 hour time point. Actin was the loading control. B, Decreased Wnt/β-catenin signaling activity under hypoxia was confirmed by measuring axin2 mRNA levels via qrt-PCR in MNNG/HOS cells. Hypoxia (72 hour, 0.5% O2) resulted in decreased axin2 mRNA expression relative to normoxia. C, When analyzed relative to the shnon hypoxia mRNA, axin2 mRNA was increased in the shHIF-1α MNNG/HOS cell lines. Asterisks indicate statistical significance (*p<0.05, **p<0.01, ***p<0.001). shNON: non-targeting shRNA; HIF shRNAs used are indicated in parentheses.
Mentions: The observed down-regulation in Wnt/β-catenin signaling raises mechanistic questions, especially regarding the potential involvement of HIF proteins. To determine whether the expression of either HIF protein is needed for the decrease in Wnt/β-catenin signaling, we stably transduced MNNG/HOS cells with shRNA targeting either HIF-1α or HIF-2α. We confirmed knockdown at the mRNA (80–90% knockdown) and protein level specific to each HIF protein relative to a non-targeting shRNA control for two independent shRNA's targeting each HIFα subunit (data not shown). Next we examined Wnt/β-catenin signaling activity under hypoxic conditions between the shHIF and non-targeting shRNA MNNG/HOS cells. Active β-catenin protein levels were decreased under hypoxic conditions in both the non-targeting shRNA and the shHIFα cell lines (Figure 3A), as was seen before in the MG-63 cell line (see Fig. 2A). There was also a decrease in total β-catenin at the 72 hour time point (Figure 3A). We assessed axin2 mRNA expression across the shHIF-modified cell lines, comparing normoxic to hypoxic conditions to determine whether β-catenin transcriptional activity was truly altered, or whether the decrease in active β-catenin protein only reflected a decrease in total β-catenin. There was a statistically significant decrease in axin2 mRNA expression under hypoxic conditions in the non-targeting shRNA MNNG/HOS cell line (Figure 3B, p<0.01), just as noted earlier for the MG-63 and 143B lines (see Figure 2B). That decrease was also consistent in the shHIF-1α- and shHIF-2α-treated MNNG/HOS cell lines, indicating that HIF-independent mechanisms contribute to Wnt/β-catenin signaling down-regulation under hypoxia (Figure 3B, p<0.05). However, when the data were normalized to the hypoxia shnon control, axin2 mRNA expression in both hypoxia shHIF-1α cell lines was increased, implying that HIF-1α contributes to Wnt/β-catenin signaling down-regulation (Figure 3C, p<0.05). Thus, this data suggests that both HIF-dependent and HIF-independent mechanisms contribute to the hypoxia-mediated down-regulation of Wnt/β-catenin signaling in human OS cells.

Bottom Line: Hypoxia is an element intrinsic to most solid-tumor microenvironments, including that of OS, and is associated with resistance to therapy, poor survival, and a malignant phenotype.Finally, we show that hypoxia results in resistance of human OS cells to doxorubicin-mediated toxicity (6-13 fold increase, p<0.01).These data support the conclusion that Wnt/β-catenin signaling is down-regulated in human OS cells under hypoxia and that this signaling alteration may represent a viable target to combat chemoresistant OS subpopulations in a hypoxic niche.

View Article: PubMed Central - PubMed

Affiliation: Michigan State University College of Human Medicine, Grand Rapids, Michigan, United States of America; Van Andel Research Institute, Grand Rapids, Michigan, United States of America.

ABSTRACT
Osteosarcoma (OS) is the most common type of solid bone cancer and remains the second leading cause of cancer-related death for children and young adults. Hypoxia is an element intrinsic to most solid-tumor microenvironments, including that of OS, and is associated with resistance to therapy, poor survival, and a malignant phenotype. Cells respond to hypoxia through alterations in gene expression, mediated most notably through the hypoxia-inducible factor (HIF) class of transcription factors. Here we investigate hypoxia-induced changes in the Wnt/β-catenin signaling pathway, a key signaling cascade involved in OS pathogenesis. We show that hypoxia results in increased expression and signaling activation of HIF proteins in human osteosarcoma cells. Wnt/β-catenin signaling is down-regulated by hypoxia in human OS cells, as demonstrated by decreased active β-catenin protein levels and axin2 mRNA expression (p<0.05). This down-regulation appears to rely on both HIF-independent and HIF-dependent mechanisms, with HIF-1α standing out as an important regulator. Finally, we show that hypoxia results in resistance of human OS cells to doxorubicin-mediated toxicity (6-13 fold increase, p<0.01). These hypoxic OS cells can be sensitized to doxorubicin treatment by further inhibition of the Wnt/β-catenin signaling pathway (p<0.05). These data support the conclusion that Wnt/β-catenin signaling is down-regulated in human OS cells under hypoxia and that this signaling alteration may represent a viable target to combat chemoresistant OS subpopulations in a hypoxic niche.

Show MeSH
Related in: MedlinePlus