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Simultaneous inhibition of deubiquitinating enzymes (DUBs) and autophagy synergistically kills breast cancer cells.

Vogel RI, Coughlin K, Scotti A, Iizuka Y, Anchoori R, Roden RB, Marastoni M, Bazzaro M - Oncotarget (2015)

Bottom Line: We evaluated the effect of the DUB inhibitors b-AP15 and RA-9 alone and in combination with early- and late-stage lysosomal inhibitors on cell viability in a panel of triple negative breast cancer (TNBC) cell lines.Our results indicate small-molecule DUB inhibitors have a profound effect on TNBC viability and lead to activation of autophagy as a cellular mechanism to compensate for ubiquitin-proteasome-system stress.This supports the evaluation of DUB inhibition, in combination with lysosomal inhibition, as a therapeutic approach for the treatment of TNBC.

View Article: PubMed Central - PubMed

Affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA.

ABSTRACT
Breast cancer is one of the leading causes of cancer death among women in the United States. Patients expressing the estrogen and progesterone receptor (ER and PR) and human epidermal growth factor 2 (HER-2) tumor markers have favorable prognosis and efficacious therapeutic options. In contrast, tumors that are negative for these markers (triple-negative) have a disproportionate share of morbidity and mortality due to lack of a validated molecular target. Deubiquitinating enzymes (DUBs) are a critical component of ubiquitin-proteasome-system degradation and have been shown to be differentially expressed and activated in a number of cancers, including breast, with their aberrant activity linked to cancer prognosis and clinical outcome. We evaluated the effect of the DUB inhibitors b-AP15 and RA-9 alone and in combination with early- and late-stage lysosomal inhibitors on cell viability in a panel of triple negative breast cancer (TNBC) cell lines. Our results indicate small-molecule DUB inhibitors have a profound effect on TNBC viability and lead to activation of autophagy as a cellular mechanism to compensate for ubiquitin-proteasome-system stress. Treatment with sub-optimal doses of DUB and lysosome inhibitors synergistically kills TNBC cells. This supports the evaluation of DUB inhibition, in combination with lysosomal inhibition, as a therapeutic approach for the treatment of TNBC.

No MeSH data available.


Related in: MedlinePlus

Inhibition of proteasome-associated DUBs induces autophagic flux in cancer cells(A) ES-2 ovarian cancer cells stably expressing the tandem-tagged mCherry-GFP-LC3 were either mock treated or exposed to 5 μM of RA-9 over a period of 18 hours and LC3 puncta were visualized by fluorescence microscopy. PBS was used as positive control autophagy inducer (objective, 60X). (B) quantification of the number of cells with visible puncta in treated versus controls. (C) dose-dependent accumulation of LC3-II isoforms in ES-2 cells exposed to the indicated dose of RA-9 over 24 hours and quantification of the LC3II/β-actin ratio. β-actin was used as loading control. (D) autophagy flux measured in ES-2 ovarian cancer cells either mock treated or treated with 5 μM b-AP15 alone, combination of 5 μM b-AP15 and 50 μM of the autophagy inhibitor Chloroquine or 50 μM of the autophagy inhibitor Chloroquine alone over a period of 18 hours.
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Figure 5: Inhibition of proteasome-associated DUBs induces autophagic flux in cancer cells(A) ES-2 ovarian cancer cells stably expressing the tandem-tagged mCherry-GFP-LC3 were either mock treated or exposed to 5 μM of RA-9 over a period of 18 hours and LC3 puncta were visualized by fluorescence microscopy. PBS was used as positive control autophagy inducer (objective, 60X). (B) quantification of the number of cells with visible puncta in treated versus controls. (C) dose-dependent accumulation of LC3-II isoforms in ES-2 cells exposed to the indicated dose of RA-9 over 24 hours and quantification of the LC3II/β-actin ratio. β-actin was used as loading control. (D) autophagy flux measured in ES-2 ovarian cancer cells either mock treated or treated with 5 μM b-AP15 alone, combination of 5 μM b-AP15 and 50 μM of the autophagy inhibitor Chloroquine or 50 μM of the autophagy inhibitor Chloroquine alone over a period of 18 hours.

Mentions: Next, we wanted to test whether tested whether activation of autophagy following DUB inhibition is a unique event in breast cancer cells or a common feature in other cancer types. To this end, ES-2 ovarian cancer cells were exposed to mock, 5 μM RA-9 treatment for 18 h, or, as a positive control, autophagy was induced by amino acid starvation in presence of PBS for a period of 3 h. As shown in Figure 5A, both amino acid starved and RA-9 treated ovarian cancer cells displayed punctate LC3 localization characteristic of autophagosome formation. Figure 5B show the percent of cells that contained visible puncta per each condition. Consistent with the observation of increased LC3-II levels in TNBC cells following DUB inhibition, RA-9 treatment also resulted in increased levels of the LC3-II lipidated form in the ovarian cancer cell. (Figure 5C). However, because LC3-II degradation occurs via autophagy, stabilization of its lipidated isoform could be the result of autophagy inhibition rather than its activation. To rule out this possibility, we measured the autophagic flux cancer cells treated with either b-AP15 and the autophagy inhibitor Chloroquine alone or in combination. As shown in Figure 5D, blocking the last step of autophagic flux with Chloroquine prevented the lysosomal degradation of LC3-II in autophagosomes, resulting in further LC3-II isoform accumulation in the ovarian cancer cells. Taken together this strongly suggests that inhibition of protein-associated DUBs causes onset autophagy flux as an alternative pathway to proteasomal degradation [28, 29].


Simultaneous inhibition of deubiquitinating enzymes (DUBs) and autophagy synergistically kills breast cancer cells.

Vogel RI, Coughlin K, Scotti A, Iizuka Y, Anchoori R, Roden RB, Marastoni M, Bazzaro M - Oncotarget (2015)

Inhibition of proteasome-associated DUBs induces autophagic flux in cancer cells(A) ES-2 ovarian cancer cells stably expressing the tandem-tagged mCherry-GFP-LC3 were either mock treated or exposed to 5 μM of RA-9 over a period of 18 hours and LC3 puncta were visualized by fluorescence microscopy. PBS was used as positive control autophagy inducer (objective, 60X). (B) quantification of the number of cells with visible puncta in treated versus controls. (C) dose-dependent accumulation of LC3-II isoforms in ES-2 cells exposed to the indicated dose of RA-9 over 24 hours and quantification of the LC3II/β-actin ratio. β-actin was used as loading control. (D) autophagy flux measured in ES-2 ovarian cancer cells either mock treated or treated with 5 μM b-AP15 alone, combination of 5 μM b-AP15 and 50 μM of the autophagy inhibitor Chloroquine or 50 μM of the autophagy inhibitor Chloroquine alone over a period of 18 hours.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Inhibition of proteasome-associated DUBs induces autophagic flux in cancer cells(A) ES-2 ovarian cancer cells stably expressing the tandem-tagged mCherry-GFP-LC3 were either mock treated or exposed to 5 μM of RA-9 over a period of 18 hours and LC3 puncta were visualized by fluorescence microscopy. PBS was used as positive control autophagy inducer (objective, 60X). (B) quantification of the number of cells with visible puncta in treated versus controls. (C) dose-dependent accumulation of LC3-II isoforms in ES-2 cells exposed to the indicated dose of RA-9 over 24 hours and quantification of the LC3II/β-actin ratio. β-actin was used as loading control. (D) autophagy flux measured in ES-2 ovarian cancer cells either mock treated or treated with 5 μM b-AP15 alone, combination of 5 μM b-AP15 and 50 μM of the autophagy inhibitor Chloroquine or 50 μM of the autophagy inhibitor Chloroquine alone over a period of 18 hours.
Mentions: Next, we wanted to test whether tested whether activation of autophagy following DUB inhibition is a unique event in breast cancer cells or a common feature in other cancer types. To this end, ES-2 ovarian cancer cells were exposed to mock, 5 μM RA-9 treatment for 18 h, or, as a positive control, autophagy was induced by amino acid starvation in presence of PBS for a period of 3 h. As shown in Figure 5A, both amino acid starved and RA-9 treated ovarian cancer cells displayed punctate LC3 localization characteristic of autophagosome formation. Figure 5B show the percent of cells that contained visible puncta per each condition. Consistent with the observation of increased LC3-II levels in TNBC cells following DUB inhibition, RA-9 treatment also resulted in increased levels of the LC3-II lipidated form in the ovarian cancer cell. (Figure 5C). However, because LC3-II degradation occurs via autophagy, stabilization of its lipidated isoform could be the result of autophagy inhibition rather than its activation. To rule out this possibility, we measured the autophagic flux cancer cells treated with either b-AP15 and the autophagy inhibitor Chloroquine alone or in combination. As shown in Figure 5D, blocking the last step of autophagic flux with Chloroquine prevented the lysosomal degradation of LC3-II in autophagosomes, resulting in further LC3-II isoform accumulation in the ovarian cancer cells. Taken together this strongly suggests that inhibition of protein-associated DUBs causes onset autophagy flux as an alternative pathway to proteasomal degradation [28, 29].

Bottom Line: We evaluated the effect of the DUB inhibitors b-AP15 and RA-9 alone and in combination with early- and late-stage lysosomal inhibitors on cell viability in a panel of triple negative breast cancer (TNBC) cell lines.Our results indicate small-molecule DUB inhibitors have a profound effect on TNBC viability and lead to activation of autophagy as a cellular mechanism to compensate for ubiquitin-proteasome-system stress.This supports the evaluation of DUB inhibition, in combination with lysosomal inhibition, as a therapeutic approach for the treatment of TNBC.

View Article: PubMed Central - PubMed

Affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA.

ABSTRACT
Breast cancer is one of the leading causes of cancer death among women in the United States. Patients expressing the estrogen and progesterone receptor (ER and PR) and human epidermal growth factor 2 (HER-2) tumor markers have favorable prognosis and efficacious therapeutic options. In contrast, tumors that are negative for these markers (triple-negative) have a disproportionate share of morbidity and mortality due to lack of a validated molecular target. Deubiquitinating enzymes (DUBs) are a critical component of ubiquitin-proteasome-system degradation and have been shown to be differentially expressed and activated in a number of cancers, including breast, with their aberrant activity linked to cancer prognosis and clinical outcome. We evaluated the effect of the DUB inhibitors b-AP15 and RA-9 alone and in combination with early- and late-stage lysosomal inhibitors on cell viability in a panel of triple negative breast cancer (TNBC) cell lines. Our results indicate small-molecule DUB inhibitors have a profound effect on TNBC viability and lead to activation of autophagy as a cellular mechanism to compensate for ubiquitin-proteasome-system stress. Treatment with sub-optimal doses of DUB and lysosome inhibitors synergistically kills TNBC cells. This supports the evaluation of DUB inhibition, in combination with lysosomal inhibition, as a therapeutic approach for the treatment of TNBC.

No MeSH data available.


Related in: MedlinePlus