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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

Activation of NRF2 signaling in MCF7 mammospheres(A) Total and nuclear NRF2 protein levels were determined in monolayers and mammospheres by western blot analysis. (B) NRF2 transcription activity was monitored using a NQO-1 ARE-driven luciferase reporter. (C)NRF2 transcript levels were assessed in monolayers and mammospheres by real-time PCR. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared with MCF7 monolayer. (D) NRF2 protein levels were determined in MCF7 monolayers, mammospheres, and re-differentiated MCF7 mammospheres by western blot analysis. (E) MCF7 cells were cultured in a normal adherent plate under normal medium or sphere medium for 3 d and microscopic observation was performed. (F) In these conditions, NRF2 and KLF4 protein levels were assessed by western blot analysis. (G) Levels of NRF2 and KLF4 were determined in sphere-cultured HCT116 and A2780 by western blot analysis.
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Figure 2: Activation of NRF2 signaling in MCF7 mammospheres(A) Total and nuclear NRF2 protein levels were determined in monolayers and mammospheres by western blot analysis. (B) NRF2 transcription activity was monitored using a NQO-1 ARE-driven luciferase reporter. (C)NRF2 transcript levels were assessed in monolayers and mammospheres by real-time PCR. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared with MCF7 monolayer. (D) NRF2 protein levels were determined in MCF7 monolayers, mammospheres, and re-differentiated MCF7 mammospheres by western blot analysis. (E) MCF7 cells were cultured in a normal adherent plate under normal medium or sphere medium for 3 d and microscopic observation was performed. (F) In these conditions, NRF2 and KLF4 protein levels were assessed by western blot analysis. (G) Levels of NRF2 and KLF4 were determined in sphere-cultured HCT116 and A2780 by western blot analysis.

Mentions: Based on the elevated expression of detoxifying/antioxidant genes and drug efflux transporters, we investigated whether the NRF2 signaling pathway was activated in MCF7 mammospheres. First, western blot analysis revealed that the level of NRF2 protein was substantially higher in mammospheres than in the monolayers. NRF2 protein levels in the total cell lysate as well as in the nuclear fraction were higher in the mammospheres (Figure 2A). Accordantly, we observed that ARE-driven luciferase activity was significantly enhanced in mammospheres (Figure 2B), indicating that NRF2 activity increased in the MCF7 mammospheres. In addition, the level of the NRF2 transcript was 1.8 times higher in the mammospheres than in the monolayers (Figure 2C). It is noteworthy that the increase in the level of NRF2 was sphere culture-specific. When MCF7 mammospheres were dissociated and re-differentiated in normal adherent plates for 3 d, the level of NRF2 protein in the total cell lysate returned to the level observed in MCF7 monolayers (Figure 2D). These results indicate the increase in NRF2 signaling is mammosphere-specific. Nonetheless, NRF2 activation could be associated with particular components in the sphere culture medium, such as growth factors. In order to exclude this possibility, we cultured MCF7 cells in a normal adherent plate with the sphere culture medium for 3 d. In these conditions, MCF7 did not form spheres (Figure 2E) and, in contrast to what was observed in the MCF7 mammosphere cultures, NRF2 and KLF4 protein levels were not elevated (Figure 2F). This also verified the mammosphere-specific NRF2 activation. In addition, we could confirm the NRF2 increase in sphere-cultured colon carcinoma HCT116 and ovarian carcinoma A2780: levels of NRF2 protein along with CSC marker KLF4 were significantly higher in spheres when compared to corresponding monolayer cells (Figure 2G). Collectively, these results indicate that NRF2 activation may be involved in the enhanced expression of efflux transporters and detoxifying/antioxidant genes in MCF7 mammospheres.


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

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

Activation of NRF2 signaling in MCF7 mammospheres(A) Total and nuclear NRF2 protein levels were determined in monolayers and mammospheres by western blot analysis. (B) NRF2 transcription activity was monitored using a NQO-1 ARE-driven luciferase reporter. (C)NRF2 transcript levels were assessed in monolayers and mammospheres by real-time PCR. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared with MCF7 monolayer. (D) NRF2 protein levels were determined in MCF7 monolayers, mammospheres, and re-differentiated MCF7 mammospheres by western blot analysis. (E) MCF7 cells were cultured in a normal adherent plate under normal medium or sphere medium for 3 d and microscopic observation was performed. (F) In these conditions, NRF2 and KLF4 protein levels were assessed by western blot analysis. (G) Levels of NRF2 and KLF4 were determined in sphere-cultured HCT116 and A2780 by western blot analysis.
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Figure 2: Activation of NRF2 signaling in MCF7 mammospheres(A) Total and nuclear NRF2 protein levels were determined in monolayers and mammospheres by western blot analysis. (B) NRF2 transcription activity was monitored using a NQO-1 ARE-driven luciferase reporter. (C)NRF2 transcript levels were assessed in monolayers and mammospheres by real-time PCR. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared with MCF7 monolayer. (D) NRF2 protein levels were determined in MCF7 monolayers, mammospheres, and re-differentiated MCF7 mammospheres by western blot analysis. (E) MCF7 cells were cultured in a normal adherent plate under normal medium or sphere medium for 3 d and microscopic observation was performed. (F) In these conditions, NRF2 and KLF4 protein levels were assessed by western blot analysis. (G) Levels of NRF2 and KLF4 were determined in sphere-cultured HCT116 and A2780 by western blot analysis.
Mentions: Based on the elevated expression of detoxifying/antioxidant genes and drug efflux transporters, we investigated whether the NRF2 signaling pathway was activated in MCF7 mammospheres. First, western blot analysis revealed that the level of NRF2 protein was substantially higher in mammospheres than in the monolayers. NRF2 protein levels in the total cell lysate as well as in the nuclear fraction were higher in the mammospheres (Figure 2A). Accordantly, we observed that ARE-driven luciferase activity was significantly enhanced in mammospheres (Figure 2B), indicating that NRF2 activity increased in the MCF7 mammospheres. In addition, the level of the NRF2 transcript was 1.8 times higher in the mammospheres than in the monolayers (Figure 2C). It is noteworthy that the increase in the level of NRF2 was sphere culture-specific. When MCF7 mammospheres were dissociated and re-differentiated in normal adherent plates for 3 d, the level of NRF2 protein in the total cell lysate returned to the level observed in MCF7 monolayers (Figure 2D). These results indicate the increase in NRF2 signaling is mammosphere-specific. Nonetheless, NRF2 activation could be associated with particular components in the sphere culture medium, such as growth factors. In order to exclude this possibility, we cultured MCF7 cells in a normal adherent plate with the sphere culture medium for 3 d. In these conditions, MCF7 did not form spheres (Figure 2E) and, in contrast to what was observed in the MCF7 mammosphere cultures, NRF2 and KLF4 protein levels were not elevated (Figure 2F). This also verified the mammosphere-specific NRF2 activation. In addition, we could confirm the NRF2 increase in sphere-cultured colon carcinoma HCT116 and ovarian carcinoma A2780: levels of NRF2 protein along with CSC marker KLF4 were significantly higher in spheres when compared to corresponding monolayer cells (Figure 2G). Collectively, these results indicate that NRF2 activation may be involved in the enhanced expression of efflux transporters and detoxifying/antioxidant genes in MCF7 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