Autophagy inhibition enhances vorinostat-induced apoptosis via ubiquitinated protein accumulation.
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.
Carew
Jennifer S
JS
Institute for Drug Development, University of Texas Health Science Center, San Antonio, TX 78245, USA.
Medina
Ernest C
EC
Esquivel
Juan A
JA
Mahalingam
Devalingam
D
Swords
Ronan
R
Kelly
Kevin
K
Zhang
Hui
H
Huang
Peng
P
Mita
Alain C
AC
Mita
Monica M
MM
Giles
Francis J
FJ
Nawrocki
Steffan T
ST
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis; D001343:Autophagy; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D002277:Carcinoma / Q000188:drug therapy / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival; D002738:Chloroquine / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism; D005260:Female; D019073:HT29 Cells; D056572:Histone Deacetylase Inhibitors / Q000378:metabolism; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D057149:Ubiquitinated Proteins / Q000378:metabolism*; D000077337:Vorinostat; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D001897:Boronic Acids; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D011719:Pyrazines; D057149:Ubiquitinated Proteins; D013481:Superoxides; D000077337:Vorinostat; D000069286:Bortezomib; D002738:Chloroquine; D046988:Proteasome Endopeptidase Complex; D006655:Histone Deacetylases