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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 ER stress responses and onset of autophagy in TNBC cells(A) MDA-MB-231 breast cancer cells stably expressing the tandem-tagged mCherry-GFP-LC3 were either mock treated or exposed to 5 μM of b-AP15 over a period of 18 hours and LC3 puncta were visualized by fluorescence microscopy (objective, 60X). (B)left panel, dose-dependent accumulation of LC3-II isoforms in MDA-MB-231 TNBC cells exposed to the indicated dose of b-AP15 over 18 hours and quantification of the LC3II/β-actin ratio. Middle panel, dose-dependent accumulation of LC3-II isoforms in MDA-MB-468 TNBC cells exposed to the indicated dose of b-AP15 over 18 hours and quantification of the LC3II/β-actin ratio. Right panel, time-dependent accumulation of LC3-II isoforms in MDA-MB-231 breast cancer cells exposed to 5 μM RA-9 for the indicated time and quantification of the LC3II/β-actin ratio. β-actin was used as loading control. (C)Left panel, MDA-MB-231 breast cancer cells exposed to 5 μM of b-AP15 over a period of 24 h following Western blot analysis with specific antibody against the ER stress-associated proteins GRP-78, amido black was used as loading control. Right panel, quantification of the ER stress-associated proteins/amido black ratio. (D)Left panel, MDA-MB-468 breast cancer cells exposed to 5 μM of b-AP15 over a period of 24 h following Western blot analysis with specific antibody against the ER stress-associated proteins GRP-78, amido black was used as loading control. Right panel, quantification of the ER stress-associated proteins/amido black ratio.
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Figure 4: Inhibition of proteasome-associated DUBs induces ER stress responses and onset of autophagy in TNBC cells(A) MDA-MB-231 breast cancer cells stably expressing the tandem-tagged mCherry-GFP-LC3 were either mock treated or exposed to 5 μM of b-AP15 over a period of 18 hours and LC3 puncta were visualized by fluorescence microscopy (objective, 60X). (B)left panel, dose-dependent accumulation of LC3-II isoforms in MDA-MB-231 TNBC cells exposed to the indicated dose of b-AP15 over 18 hours and quantification of the LC3II/β-actin ratio. Middle panel, dose-dependent accumulation of LC3-II isoforms in MDA-MB-468 TNBC cells exposed to the indicated dose of b-AP15 over 18 hours and quantification of the LC3II/β-actin ratio. Right panel, time-dependent accumulation of LC3-II isoforms in MDA-MB-231 breast cancer cells exposed to 5 μM RA-9 for the indicated time and quantification of the LC3II/β-actin ratio. β-actin was used as loading control. (C)Left panel, MDA-MB-231 breast cancer cells exposed to 5 μM of b-AP15 over a period of 24 h following Western blot analysis with specific antibody against the ER stress-associated proteins GRP-78, amido black was used as loading control. Right panel, quantification of the ER stress-associated proteins/amido black ratio. (D)Left panel, MDA-MB-468 breast cancer cells exposed to 5 μM of b-AP15 over a period of 24 h following Western blot analysis with specific antibody against the ER stress-associated proteins GRP-78, amido black was used as loading control. Right panel, quantification of the ER stress-associated proteins/amido black ratio.

Mentions: We have previously shown that inhibition of proteasome-associated DUBs via small-molecule inhibitors induce endoplasmic reticulum (ER) stress response as a mechanism to compensate for unsustainable proteotoxic stress [23]. Here, we evaluated whether inhibition of proteasome-associated DUBs with the specific inhibitors b-AP15 or RA-9 would trigger autophagy as a cytoprotective response in TNBC cells. MDA-MB-231 cells were transiently transfected with the mCherry-GFP-LC3 reporter and exposed to mock (control) or 5 μM b-AP15 treatment for 18 hours. Onset of autophagy was visualized by immunofluorescence microscopy. As shown in Figure 4A, TNBC cells treated with b-AP15 displayed a punctate LC3 localization characteristic of autophagosome formation. Autophagy activation following inhibition of proteasome-associated DUBs was further confirmed by measuring protein expression levels of the LC3-I and LC3-II isoforms by Western blot analysis. During autophagy, the cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes. This causes accumulation of the LC3-II isoform over the LC3-I isoform [24]. Here, MDA-MB-231 and MDA-MB-468 TNBC cells were exposed to increasing doses of b-AP15 over 18 hours and analyzed by Western blot with an anti-LC3 antibody to assess levels of both LC3-I and its lipidated, autophagic vescicle-associated LC3-II isoform [25, 26]. Consistent with our initial hypothesis of autophagy activation, b-AP15 treatment caused dose-dependent accumulation of LC3-II isoforms in each cell line (Figure 4B, left and middle panels) and RA-9 treatment caused time-dependent accumulation of LC3-II isoforms in MDA-MB-231 (Figure 4B, right panel). We have recently shown that inhibition of ubiquitin-dependent protein degradation upstream of proteasome causes activation of ER stress responses in ovarian cancer cells [27]. To test whether a similar event occurs in TNBC, MDA-MB-231 and MDA-MB-468 cells were exposed to 5 μM b-AP15 over a period of 24 h and the cell lysates subjected to Western blot analysis for the ER-stress marker GRP-78. We found that b-AP15 exposure caused a time-dependent increase in the steady levels of GRP-78 in both MDA-MB-231 (Figure 4C) and MDA-MB-468 cells (Figure 4D). Quantification of the changes in the steady state levels of GRP-78 in MDA-MB-231 and MDA-MB-468 cells are provided in the right panels of Figures 4C and 4D, respectively.


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 ER stress responses and onset of autophagy in TNBC cells(A) MDA-MB-231 breast cancer cells stably expressing the tandem-tagged mCherry-GFP-LC3 were either mock treated or exposed to 5 μM of b-AP15 over a period of 18 hours and LC3 puncta were visualized by fluorescence microscopy (objective, 60X). (B)left panel, dose-dependent accumulation of LC3-II isoforms in MDA-MB-231 TNBC cells exposed to the indicated dose of b-AP15 over 18 hours and quantification of the LC3II/β-actin ratio. Middle panel, dose-dependent accumulation of LC3-II isoforms in MDA-MB-468 TNBC cells exposed to the indicated dose of b-AP15 over 18 hours and quantification of the LC3II/β-actin ratio. Right panel, time-dependent accumulation of LC3-II isoforms in MDA-MB-231 breast cancer cells exposed to 5 μM RA-9 for the indicated time and quantification of the LC3II/β-actin ratio. β-actin was used as loading control. (C)Left panel, MDA-MB-231 breast cancer cells exposed to 5 μM of b-AP15 over a period of 24 h following Western blot analysis with specific antibody against the ER stress-associated proteins GRP-78, amido black was used as loading control. Right panel, quantification of the ER stress-associated proteins/amido black ratio. (D)Left panel, MDA-MB-468 breast cancer cells exposed to 5 μM of b-AP15 over a period of 24 h following Western blot analysis with specific antibody against the ER stress-associated proteins GRP-78, amido black was used as loading control. Right panel, quantification of the ER stress-associated proteins/amido black ratio.
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Figure 4: Inhibition of proteasome-associated DUBs induces ER stress responses and onset of autophagy in TNBC cells(A) MDA-MB-231 breast cancer cells stably expressing the tandem-tagged mCherry-GFP-LC3 were either mock treated or exposed to 5 μM of b-AP15 over a period of 18 hours and LC3 puncta were visualized by fluorescence microscopy (objective, 60X). (B)left panel, dose-dependent accumulation of LC3-II isoforms in MDA-MB-231 TNBC cells exposed to the indicated dose of b-AP15 over 18 hours and quantification of the LC3II/β-actin ratio. Middle panel, dose-dependent accumulation of LC3-II isoforms in MDA-MB-468 TNBC cells exposed to the indicated dose of b-AP15 over 18 hours and quantification of the LC3II/β-actin ratio. Right panel, time-dependent accumulation of LC3-II isoforms in MDA-MB-231 breast cancer cells exposed to 5 μM RA-9 for the indicated time and quantification of the LC3II/β-actin ratio. β-actin was used as loading control. (C)Left panel, MDA-MB-231 breast cancer cells exposed to 5 μM of b-AP15 over a period of 24 h following Western blot analysis with specific antibody against the ER stress-associated proteins GRP-78, amido black was used as loading control. Right panel, quantification of the ER stress-associated proteins/amido black ratio. (D)Left panel, MDA-MB-468 breast cancer cells exposed to 5 μM of b-AP15 over a period of 24 h following Western blot analysis with specific antibody against the ER stress-associated proteins GRP-78, amido black was used as loading control. Right panel, quantification of the ER stress-associated proteins/amido black ratio.
Mentions: We have previously shown that inhibition of proteasome-associated DUBs via small-molecule inhibitors induce endoplasmic reticulum (ER) stress response as a mechanism to compensate for unsustainable proteotoxic stress [23]. Here, we evaluated whether inhibition of proteasome-associated DUBs with the specific inhibitors b-AP15 or RA-9 would trigger autophagy as a cytoprotective response in TNBC cells. MDA-MB-231 cells were transiently transfected with the mCherry-GFP-LC3 reporter and exposed to mock (control) or 5 μM b-AP15 treatment for 18 hours. Onset of autophagy was visualized by immunofluorescence microscopy. As shown in Figure 4A, TNBC cells treated with b-AP15 displayed a punctate LC3 localization characteristic of autophagosome formation. Autophagy activation following inhibition of proteasome-associated DUBs was further confirmed by measuring protein expression levels of the LC3-I and LC3-II isoforms by Western blot analysis. During autophagy, the cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes. This causes accumulation of the LC3-II isoform over the LC3-I isoform [24]. Here, MDA-MB-231 and MDA-MB-468 TNBC cells were exposed to increasing doses of b-AP15 over 18 hours and analyzed by Western blot with an anti-LC3 antibody to assess levels of both LC3-I and its lipidated, autophagic vescicle-associated LC3-II isoform [25, 26]. Consistent with our initial hypothesis of autophagy activation, b-AP15 treatment caused dose-dependent accumulation of LC3-II isoforms in each cell line (Figure 4B, left and middle panels) and RA-9 treatment caused time-dependent accumulation of LC3-II isoforms in MDA-MB-231 (Figure 4B, right panel). We have recently shown that inhibition of ubiquitin-dependent protein degradation upstream of proteasome causes activation of ER stress responses in ovarian cancer cells [27]. To test whether a similar event occurs in TNBC, MDA-MB-231 and MDA-MB-468 cells were exposed to 5 μM b-AP15 over a period of 24 h and the cell lysates subjected to Western blot analysis for the ER-stress marker GRP-78. We found that b-AP15 exposure caused a time-dependent increase in the steady levels of GRP-78 in both MDA-MB-231 (Figure 4C) and MDA-MB-468 cells (Figure 4D). Quantification of the changes in the steady state levels of GRP-78 in MDA-MB-231 and MDA-MB-468 cells are provided in the right panels of Figures 4C and 4D, respectively.

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