Autophagy inhibition enhances vorinostat‐induced apoptosis via ubiquitinated protein accumulation

Abstract

Autophagy is an evolutionarily conserved cell survival pathway that enables cells to recoup ATP and other critical biosynthetic molecules during nutrient deprivation or exposure to hypoxia, which are hallmarks of the tumour microenvironment. Autophagy has been implicated as a potential mechanism of resistance to anticancer agents as it can promote cell survival in the face of stress induced by chemotherapeutic agents by breaking down cellular components to generate alternative sources of energy. Disruption of autophagy with chloroquine (CQ) induces the accumulation of ubiquitin-conjugated proteins in a manner similar to the proteasome inhibitor bortezomib (BZ). However, CQ-induced protein accumulation occurs at a slower rate and is localized to lysosomes in contrast to BZ, which stimulates rapid buildup of ubiquitinated proteins and aggresome formation in the cytosol. The histone deacetylase (HDAC) inhibitor vorinostat (VOR) blocked BZ-induced aggresome formation, but promoted CQ-mediated ubiquitinated protein accumulation. Disruption of autophagy with CQ strongly enhanced VOR-mediated apoptosis in colon cancer cells. Accordingly, knockdown of the essential autophagy gene Atg7 also sensitized cells to VOR-induced apoptosis. Knockdown of HDAC6 greatly enhanced BZ-induced apoptosis, but only marginally sensitized cells to CQ. Subsequent studies determined that the CQ/VOR combination promoted a large increase in superoxide generation that was required for ubiquitinated protein accumulation and cell death. Finally, treatment with the CQ/VOR combination significantly reduced tumour burden and induced apoptosis in a colon cancer xenograft model. Collectively, our results establish that inhibition of autophagy with CQ induces ubiquitinated protein accumulation and VOR potentiates CQ-mediated aggregate formation, superoxide generation and apoptosis.

BAG3 induction is required to mitigate proteotoxicity via selective autophagy following inhibition of constitutive protein degradation pathways

“…21 To this end, proteasome inhibitors such as Bortezomib together with HDAC inhibitors have demonstrated synergistic cytotoxicity in several cancers, for example, ovarian, breast and colon carcinoma. [11][12][13] Although we similarly show in RMS that cotreatment with ST80 and Bortezomib co-operates to trigger cell death, our present study focused on the surviving, but not the dying cell subpopulation, as we aimed at identifying the mechanism of acquired resistance to ST80/Bortezomib-induced proteotoxic stress. Notably, we demonstrate for the first time that BAG3-mediated autophagy is turned on as a compensatory pathway upon concomitant inhibition of the proteasome and HDAC6 that is required for clearance of protein aggregates and cell regrowth during recovery after proteotoxic stress.…”

“…Therefore, proteasome inhibitors have been combined with HDAC inhibitors to concomitantly inhibit the two key constitutive protein degradation pathways in order to increase the antitumor activities of proteasome inhibitors. [11][12][13] In rhabdomyosarcoma (RMS), the most common pediatric soft tissue tumor, initial studies showed that Bortezomib triggers cell death in vitro and in vivo. 14 Therefore, we used RMS cells as a model to study the regulation of constitutive and inducible protein degradation pathways.…”

Reovirus therapy stimulates endoplasmic reticular stress, NOXA induction, and augments bortezomib-mediated apoptosis in multiple myeloma

“…Investigation of the mechanisms of action of bortezomib has determined that it has pleiotropic cellular effects that contribute to its anti-myeloma activity including inhibition of nuclear factor kB, stimulation of ER stress, induction of the pro-apoptotic Bcl-2 homology 3 (BH3)-only family member NOXA, inhibition of tumor angiogenesis and the disruption of survival signaling cascades (Nawrocki et al, 2002;Hideshima et al, 2003;Qin et al, 2005). Bortezomib-mediated proteasomal inhibition results in the accumulation of large quantities of ubiquitinated protein aggregates that induce ER stress, which has been reported to be a significant contributor to bortezomib's anti-cancer activity (Nawrocki et al, 2005a(Nawrocki et al, , b, 2008Fribley et al, 2006;Carew et al, 2010). As such, we posit that agents with the ability to increase ER stress will possess strong antimyeloma activity and augment bortezomib-mediated cell death.…”

Sonic Hedgehog in cancer stem cells: a novel link with autophagy

“…Consistent with our fi ndings, previous reports have suggested that several cytotoxic chemotherapeutic agents induce autophagy, and inhibition of autophagy enhanced their effi ciency in vitro . Combinations of other anticancer drugs with autophagy inhibitors have also shown success in preclinical models (Amaravadi et al, 2007, Carew et al, 2010.…”