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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

Involvement of p62 in NRF2 activation in mammospheres(A) LC3 (LI3-I and LC3-II) and p62 protein levels were determined in monolayers and mammospheres. (B)p62 transcript levels were assessed in monolayers and mammospheres by real-time PCR. (C) Transcript levels for p62 transcript levels were monitored in stable MCF7 cell lines expressing the nonspecific scRNA (sc) or p62-specific shRNA (shp62). Values represent the mean ± SE from 3 experiments. (D) p62 protein levels were determined in monolayers and mammospheres of sc and shp62 MCF7. (E) NRF2 protein levels were determined in sc and shp62 mammospheres. Relative NRF2 levels were quantified. (F)GCLC, AKR1c1, and MRP2 transcript levels were assessed in sc and shp62 mammospheres by real-time PCR. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared to sc mammospheres.
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Figure 5: Involvement of p62 in NRF2 activation in mammospheres(A) LC3 (LI3-I and LC3-II) and p62 protein levels were determined in monolayers and mammospheres. (B)p62 transcript levels were assessed in monolayers and mammospheres by real-time PCR. (C) Transcript levels for p62 transcript levels were monitored in stable MCF7 cell lines expressing the nonspecific scRNA (sc) or p62-specific shRNA (shp62). Values represent the mean ± SE from 3 experiments. (D) p62 protein levels were determined in monolayers and mammospheres of sc and shp62 MCF7. (E) NRF2 protein levels were determined in sc and shp62 mammospheres. Relative NRF2 levels were quantified. (F)GCLC, AKR1c1, and MRP2 transcript levels were assessed in sc and shp62 mammospheres by real-time PCR. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared to sc mammospheres.

Mentions: Experimental evidence showed that proteasome inhibition could activate autophagy as a compensatory mechanism [32]. In our mammosphere system, the level of the ubiquitinated total proteins was not higher in MCF7 mammospheres than that in the MCF7 monolayers, although the proteasome function was reduced (Supplementary Figure S3). This may imply the compensatory activation of autophagy in mammospheres. Indeed, LC3-II protein was accumulated in the mammospheres, which reflects an increased autophagy activity (Figure 5A). In addition, both protein and mRNA levels of p62 were increased in the mammospheres (Figure 5A and 5B). Since p62 can regulate NRF2 via a non-canonical pathway [25], these data suggest that altered autophagy activity in mammospheres may be an additional contributing factor to NRF2 stabilization. To investigate a potential correlation between p62 and NRF2, we established the p62-knockdown stable cell line (shp62) and examined NRF2 expression in mammospheres. Our established knockdown cells displayed a 52% reduction in p62 mRNA level when compared to the nonspecific sc control cell line (Figure 5C). After mammosphere formation, the increase in p62 was not observed in shp62 cells, confirming p62 knockdown (Figure 5D). Importantly, we found that p62 knockdown suppressed NRF2 increase in mammospheres. NRF2 protein levels in total cell lysates were not elevated in the p62 knockdown group (Figure 5E). Accordantly, the increase in AKR1c1 and MRP2 transcript levels was significantly repressed by p62 knockdown (Figure 5F). Taken together, these data imply that the increased p62 level in the mammosphere is also associated with NRF2 accumulation and consequent activation.


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

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

Involvement of p62 in NRF2 activation in mammospheres(A) LC3 (LI3-I and LC3-II) and p62 protein levels were determined in monolayers and mammospheres. (B)p62 transcript levels were assessed in monolayers and mammospheres by real-time PCR. (C) Transcript levels for p62 transcript levels were monitored in stable MCF7 cell lines expressing the nonspecific scRNA (sc) or p62-specific shRNA (shp62). Values represent the mean ± SE from 3 experiments. (D) p62 protein levels were determined in monolayers and mammospheres of sc and shp62 MCF7. (E) NRF2 protein levels were determined in sc and shp62 mammospheres. Relative NRF2 levels were quantified. (F)GCLC, AKR1c1, and MRP2 transcript levels were assessed in sc and shp62 mammospheres by real-time PCR. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared to sc mammospheres.
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Related In: Results  -  Collection

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Figure 5: Involvement of p62 in NRF2 activation in mammospheres(A) LC3 (LI3-I and LC3-II) and p62 protein levels were determined in monolayers and mammospheres. (B)p62 transcript levels were assessed in monolayers and mammospheres by real-time PCR. (C) Transcript levels for p62 transcript levels were monitored in stable MCF7 cell lines expressing the nonspecific scRNA (sc) or p62-specific shRNA (shp62). Values represent the mean ± SE from 3 experiments. (D) p62 protein levels were determined in monolayers and mammospheres of sc and shp62 MCF7. (E) NRF2 protein levels were determined in sc and shp62 mammospheres. Relative NRF2 levels were quantified. (F)GCLC, AKR1c1, and MRP2 transcript levels were assessed in sc and shp62 mammospheres by real-time PCR. Values represent the mean ± SE from 3 experiments. aP < 0.05 compared to sc mammospheres.
Mentions: Experimental evidence showed that proteasome inhibition could activate autophagy as a compensatory mechanism [32]. In our mammosphere system, the level of the ubiquitinated total proteins was not higher in MCF7 mammospheres than that in the MCF7 monolayers, although the proteasome function was reduced (Supplementary Figure S3). This may imply the compensatory activation of autophagy in mammospheres. Indeed, LC3-II protein was accumulated in the mammospheres, which reflects an increased autophagy activity (Figure 5A). In addition, both protein and mRNA levels of p62 were increased in the mammospheres (Figure 5A and 5B). Since p62 can regulate NRF2 via a non-canonical pathway [25], these data suggest that altered autophagy activity in mammospheres may be an additional contributing factor to NRF2 stabilization. To investigate a potential correlation between p62 and NRF2, we established the p62-knockdown stable cell line (shp62) and examined NRF2 expression in mammospheres. Our established knockdown cells displayed a 52% reduction in p62 mRNA level when compared to the nonspecific sc control cell line (Figure 5C). After mammosphere formation, the increase in p62 was not observed in shp62 cells, confirming p62 knockdown (Figure 5D). Importantly, we found that p62 knockdown suppressed NRF2 increase in mammospheres. NRF2 protein levels in total cell lysates were not elevated in the p62 knockdown group (Figure 5E). Accordantly, the increase in AKR1c1 and MRP2 transcript levels was significantly repressed by p62 knockdown (Figure 5F). Taken together, these data imply that the increased p62 level in the mammosphere is also associated with NRF2 accumulation and consequent activation.

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