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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

Reduced proteasome function in MCF7 mammospheres(A) To monitor the proteasome function, MCF7 cells in monolayers and mammospheres were transfected with the pLKO.1-ODCpdd-luciferase plasmid. The proteasome sensitive luciferase activity was then assessed. (B) Proteasome catalytic activities were monitored by measuring chymotrypsin-like (Chymo), trypsin-like (Tryp), and caspase-like (Casp) peptidase activities. Values represent the mean ± SE from 4–5 experiments. aP < 0.05 compared with MCF7 monolayer. (C) Protein levels for PSMB5, PSMB6, and PSMB7, catalytic core subunits of the proteasome were determined by western blot analysis.
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Figure 4: Reduced proteasome function in MCF7 mammospheres(A) To monitor the proteasome function, MCF7 cells in monolayers and mammospheres were transfected with the pLKO.1-ODCpdd-luciferase plasmid. The proteasome sensitive luciferase activity was then assessed. (B) Proteasome catalytic activities were monitored by measuring chymotrypsin-like (Chymo), trypsin-like (Tryp), and caspase-like (Casp) peptidase activities. Values represent the mean ± SE from 4–5 experiments. aP < 0.05 compared with MCF7 monolayer. (C) Protein levels for PSMB5, PSMB6, and PSMB7, catalytic core subunits of the proteasome were determined by western blot analysis.

Mentions: In an attempt to elucidate causative mechanisms of NRF2 accumulation, proteasome function was assessed in MCF7 mammospheres. For this, we evaluated the level of proteasome substrate accumulation using the pLKO.1-ODCpdd-luciferase plasmid. In this construct, the proteasome destruction domain (pdd) from ornithine decarboxylase (ODC) was fused to the luciferase-containing pLKO.1 plasmid. The plasmid was transfected in MCF7 to monitor the proteasome function. In mammospheres, the proteasome sensitive luciferase activity was significantly high when compared to MCF7 monolayers. The luciferase activity was similar to that of the MG132-treated monolayer group (Figure 4A), indicating that proteolytic proteasome activity is reduced in the mammospheres. Protease activity of the 26S proteasome is mediated by three catalytic subunits. The catalytic core subunits of the proteasome are PSMB5, PSMB6, and PSMB7, presenting chemotrypsin-like, caspase-like, and trypsin-like peptidase activities, respectively. When these peptidase activities were determined using fluorogenic substrates, the proteasome protease activities were reduced in mammospheres. In particular, trypsin-like and caspase-like activities were significantly low in mammospheres compared to monolayers (Figure 4B). In addition, PSMB5, PSMB6, and PSMB7 protein levels were consistently reduced in mammospheres (Figure 4C). These results indicated that reduced proteasome activity might be responsible for the enhanced ubiquitinated NRF2 accumulation in mammospheres.


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

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

Reduced proteasome function in MCF7 mammospheres(A) To monitor the proteasome function, MCF7 cells in monolayers and mammospheres were transfected with the pLKO.1-ODCpdd-luciferase plasmid. The proteasome sensitive luciferase activity was then assessed. (B) Proteasome catalytic activities were monitored by measuring chymotrypsin-like (Chymo), trypsin-like (Tryp), and caspase-like (Casp) peptidase activities. Values represent the mean ± SE from 4–5 experiments. aP < 0.05 compared with MCF7 monolayer. (C) Protein levels for PSMB5, PSMB6, and PSMB7, catalytic core subunits of the proteasome were determined by western blot analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4480743&req=5

Figure 4: Reduced proteasome function in MCF7 mammospheres(A) To monitor the proteasome function, MCF7 cells in monolayers and mammospheres were transfected with the pLKO.1-ODCpdd-luciferase plasmid. The proteasome sensitive luciferase activity was then assessed. (B) Proteasome catalytic activities were monitored by measuring chymotrypsin-like (Chymo), trypsin-like (Tryp), and caspase-like (Casp) peptidase activities. Values represent the mean ± SE from 4–5 experiments. aP < 0.05 compared with MCF7 monolayer. (C) Protein levels for PSMB5, PSMB6, and PSMB7, catalytic core subunits of the proteasome were determined by western blot analysis.
Mentions: In an attempt to elucidate causative mechanisms of NRF2 accumulation, proteasome function was assessed in MCF7 mammospheres. For this, we evaluated the level of proteasome substrate accumulation using the pLKO.1-ODCpdd-luciferase plasmid. In this construct, the proteasome destruction domain (pdd) from ornithine decarboxylase (ODC) was fused to the luciferase-containing pLKO.1 plasmid. The plasmid was transfected in MCF7 to monitor the proteasome function. In mammospheres, the proteasome sensitive luciferase activity was significantly high when compared to MCF7 monolayers. The luciferase activity was similar to that of the MG132-treated monolayer group (Figure 4A), indicating that proteolytic proteasome activity is reduced in the mammospheres. Protease activity of the 26S proteasome is mediated by three catalytic subunits. The catalytic core subunits of the proteasome are PSMB5, PSMB6, and PSMB7, presenting chemotrypsin-like, caspase-like, and trypsin-like peptidase activities, respectively. When these peptidase activities were determined using fluorogenic substrates, the proteasome protease activities were reduced in mammospheres. In particular, trypsin-like and caspase-like activities were significantly low in mammospheres compared to monolayers (Figure 4B). In addition, PSMB5, PSMB6, and PSMB7 protein levels were consistently reduced in mammospheres (Figure 4C). These results indicated that reduced proteasome activity might be responsible for the enhanced ubiquitinated NRF2 accumulation in mammospheres.

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