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Activation of NRF2 by p62 and proteasome reduction in sphere-forming breast carcinoma cells.

Ryoo IG, Choi BH, Kwak MK - Oncotarget (2015)

Bottom Line: The MCF7 mammospheres expressed significantly higher levels of the NRF2 protein and target gene expression compared to the monolayer.Moreover, unlike the control mammospheres, NRF2-knockdown mammospheres did not develop anticancer drug resistance.Collectively, these results indicated that altered proteasome function and p62 expression caused NRF2 activation in CSC-enriched mammospheres.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, The Catholic University of Korea, Bucheon, Gyeonggi-do, Republic of Korea.

ABSTRACT
Cancer stem cells (CSCs) express high levels of drug efflux transporters and antioxidant genes, and are therefore believed to be responsible for cancer recurrence following chemo/radiotherapy intervention. In this study, we investigated the role of NF-E2-related factor 2 (NRF2), a master regulator of antioxidant gene expression, in the growth and stress resistance of CSC-enriched mammosphere. The MCF7 mammospheres expressed significantly higher levels of the NRF2 protein and target gene expression compared to the monolayer. As underlying mechanisms, we observed that proteolytic activity and expression of the proteasome catalytic subunits were decreased in the mammospheres. Additionally, mammospheres retained a high level of p62 and the silencing of p62 was observed to attenuate NRF2 activation. NRF2 increase was confirmed in sphere-cultures of the colon and ovarian cancer cells. The functional implication of NRF2 was demonstrated in NRF2-knockdown mammospheres. NRF2-silenced mammospheres demonstrated increased cell death and retarded sphere growth as a result of target gene repression. Moreover, unlike the control mammospheres, NRF2-knockdown mammospheres did not develop anticancer drug resistance. Collectively, these results indicated that altered proteasome function and p62 expression caused NRF2 activation in CSC-enriched mammospheres. In addition, NRF2 appeared to play a role in CSC survival and anticancer drug resistance.

No MeSH data available.


Related in: MedlinePlus

Stabilization of NRF2 protein in MCF7 mammospheres(A) NRF2 protein stability was assessed in monolayers and mammospheres following the treatment with protein synthesis inhibitor CHX (10 μg/mL). At each time point, cells were harvested and NRF2 protein levels were monitored by western blot analysis. A bar graph represents the relative NRF2 protein levels at each time point. (B) KEAP1 and CUL3 protein levels were determined in monolayers and mammospheres by western blot analysis. (C–D) For detection of ubiquitinated NRF2, total protein lysates of monolayers and mammospheres were immunoprecipitated with an NRF2 (C) or Ub antibody (D), followed by immunoblot analysis with the Ub or NRF2 antibody.
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Figure 3: Stabilization of NRF2 protein in MCF7 mammospheres(A) NRF2 protein stability was assessed in monolayers and mammospheres following the treatment with protein synthesis inhibitor CHX (10 μg/mL). At each time point, cells were harvested and NRF2 protein levels were monitored by western blot analysis. A bar graph represents the relative NRF2 protein levels at each time point. (B) KEAP1 and CUL3 protein levels were determined in monolayers and mammospheres by western blot analysis. (C–D) For detection of ubiquitinated NRF2, total protein lysates of monolayers and mammospheres were immunoprecipitated with an NRF2 (C) or Ub antibody (D), followed by immunoblot analysis with the Ub or NRF2 antibody.

Mentions: In order to elucidate the mechanism involved in the increase of NRF2 protein in mammospheres, we monitored NRF2 protein stability in the presence of protein synthesis inhibitor cycloheximide (CHX). When cells were incubated with CHX (10 μg/mL) for 0–80 min, NRF2 protein levels rapidly diminished with time in MCF7 monolayers, while NRF2 protein levels did not show any decrease up to 80 min in MCF7 mammospheres (Figure 3A). These results indicate that enhanced NRF2 level in mammospheres is due to protein stabilization. Nonetheless, there was no significant difference in KEAP1 levels between monolayers and mammospheres (Figure 3B). In addition, CUL3 protein level was relatively higher in mammospheres, which does not support NRF2 stabilization.


Activation of NRF2 by p62 and proteasome reduction in sphere-forming breast carcinoma cells.

Ryoo IG, Choi BH, Kwak MK - Oncotarget (2015)

Stabilization of NRF2 protein in MCF7 mammospheres(A) NRF2 protein stability was assessed in monolayers and mammospheres following the treatment with protein synthesis inhibitor CHX (10 μg/mL). At each time point, cells were harvested and NRF2 protein levels were monitored by western blot analysis. A bar graph represents the relative NRF2 protein levels at each time point. (B) KEAP1 and CUL3 protein levels were determined in monolayers and mammospheres by western blot analysis. (C–D) For detection of ubiquitinated NRF2, total protein lysates of monolayers and mammospheres were immunoprecipitated with an NRF2 (C) or Ub antibody (D), followed by immunoblot analysis with the Ub or NRF2 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480743&req=5

Figure 3: Stabilization of NRF2 protein in MCF7 mammospheres(A) NRF2 protein stability was assessed in monolayers and mammospheres following the treatment with protein synthesis inhibitor CHX (10 μg/mL). At each time point, cells were harvested and NRF2 protein levels were monitored by western blot analysis. A bar graph represents the relative NRF2 protein levels at each time point. (B) KEAP1 and CUL3 protein levels were determined in monolayers and mammospheres by western blot analysis. (C–D) For detection of ubiquitinated NRF2, total protein lysates of monolayers and mammospheres were immunoprecipitated with an NRF2 (C) or Ub antibody (D), followed by immunoblot analysis with the Ub or NRF2 antibody.
Mentions: In order to elucidate the mechanism involved in the increase of NRF2 protein in mammospheres, we monitored NRF2 protein stability in the presence of protein synthesis inhibitor cycloheximide (CHX). When cells were incubated with CHX (10 μg/mL) for 0–80 min, NRF2 protein levels rapidly diminished with time in MCF7 monolayers, while NRF2 protein levels did not show any decrease up to 80 min in MCF7 mammospheres (Figure 3A). These results indicate that enhanced NRF2 level in mammospheres is due to protein stabilization. Nonetheless, there was no significant difference in KEAP1 levels between monolayers and mammospheres (Figure 3B). In addition, CUL3 protein level was relatively higher in mammospheres, which does not support NRF2 stabilization.

Bottom Line: The MCF7 mammospheres expressed significantly higher levels of the NRF2 protein and target gene expression compared to the monolayer.Moreover, unlike the control mammospheres, NRF2-knockdown mammospheres did not develop anticancer drug resistance.Collectively, these results indicated that altered proteasome function and p62 expression caused NRF2 activation in CSC-enriched mammospheres.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, The Catholic University of Korea, Bucheon, Gyeonggi-do, Republic of Korea.

ABSTRACT
Cancer stem cells (CSCs) express high levels of drug efflux transporters and antioxidant genes, and are therefore believed to be responsible for cancer recurrence following chemo/radiotherapy intervention. In this study, we investigated the role of NF-E2-related factor 2 (NRF2), a master regulator of antioxidant gene expression, in the growth and stress resistance of CSC-enriched mammosphere. The MCF7 mammospheres expressed significantly higher levels of the NRF2 protein and target gene expression compared to the monolayer. As underlying mechanisms, we observed that proteolytic activity and expression of the proteasome catalytic subunits were decreased in the mammospheres. Additionally, mammospheres retained a high level of p62 and the silencing of p62 was observed to attenuate NRF2 activation. NRF2 increase was confirmed in sphere-cultures of the colon and ovarian cancer cells. The functional implication of NRF2 was demonstrated in NRF2-knockdown mammospheres. NRF2-silenced mammospheres demonstrated increased cell death and retarded sphere growth as a result of target gene repression. Moreover, unlike the control mammospheres, NRF2-knockdown mammospheres did not develop anticancer drug resistance. Collectively, these results indicated that altered proteasome function and p62 expression caused NRF2 activation in CSC-enriched mammospheres. In addition, NRF2 appeared to play a role in CSC survival and anticancer drug resistance.

No MeSH data available.


Related in: MedlinePlus