2014;5:503C517. 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 Rivaroxaban (Xarelto) combination with lysosomal inhibition, as a therapeutic approach for Rivaroxaban (Xarelto) the treatment of TNBC. quadrants. (B) quantification of the increase in Annexin-V and 7-AAD double-positive cell populations (shaded area) in MDA-MB-231 (quantification of the ER stress-associated proteins/amido black ratio. 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 M 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 ?Figure5A,5A, both amino acid starved and RA-9 treated ovarian cancer cells displayed punctate LC3 localization characteristic of autophagosome formation. Figure ?Figure5B5B show the percent of cells that contained visible puncta per each condition. Consistent Mapkap1 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 ?(Figure5C).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 ?Figure5D,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]. Open in a separate window 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 M 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 Rivaroxaban (Xarelto) of the LC3II/-actin ratio. -actin was used as loading control. (D) autophagy flux measured in ES-2 ovarian cancer cells either mock treated or treated with 5 M b-AP15 alone, combination of 5 M b-AP15 and 50 M of the autophagy inhibitor Chloroquine or 50 M of the autophagy inhibitor Chloroquine alone over a period of 18 hours. Synergistic effect of DUB and autophagy inhibitors in inducing apoptosis in TNBC cells Recent data suggest that, in cancer, proteasome- and lysosome-assisted protein degradation are functionally coupled. [20, 28] Our data indicate that following proteasome-associated DUB inhibition, TNBC cells activate autophagy as a protective mechanism to decrease levels of proteotoxic stress. Therefore, we hypothesized that concomitant inhibition of DUBs and autophagy would synergistically trigger cell death. Specifically, we tested the effect of exposing TNBC cell lines, including MDA-MB-435, MDA-MB-231 and MDA-MB-468, to the combination of the proteasome-associated inhibitor b-AP15 and Vorinostat or Chloriquine over a period of 48 hours. As shown in Figure ?Figure6A,6A, analyses of cell death indicated synergistic activity for the b-AP15 and Vorinostat combination in all of cell lines tested, with the CI of 0.80, 0.51 and 0.55 for MDA-MB-435, MDA-MB-231 and MDA-MB-468, respectively. Likewise, the combination of b-AP15 and the clinically approved lysosome inhibitor Chloroquine shows synergistic cell killing with CIs of 0.84, 0.83 and 0.78 for MDA-MB-435, MDA-MB-231 and MDA-MB-468, respectively, though to a lesser extent. These data indicate that rather than a simple additive killing, the combination of a DUB and lysosome inhibitor is Rivaroxaban (Xarelto) highly to moderately synergistic. Open in a separate window Figure 6 DUB and autophagy inhibitors synergistically kill TNBC cells(A) Cultures of MDA-MB-435 (< 0.01. (C) GFP-LC3 expressing ES-2 ovarian cancer cells were either mock treated or incubated with 5 M b-AP15, 20 M of Vorinostat or the combination of both for 18 h before fixation and immuno-fluorescent staining of DNA (blue) and LC3 (green). To further confirm that inhibition of autophagy is responsible for the increased cell killing observed following DUB inhibition and.