Human TSC-associated renal angiomyolipoma cells are hypersensitive to ER stress.
Tuberous sclerosis complex (TSC), an inherited tumor predisposition syndrome associated with mutations in TSC1 or TSC2, affects ∼1 in 6,000 individuals. Eighty percent of TSC patients develop renal angiomyolipomas, and renal involvement is a major contributor to patient morbidity and mortality. Recent work has shown that mammalian target of rapamycin complex 1 (mTORC1) inhibition caused angiomyolipoma shrinkage but that this treatment may cause cytostatic not a cytotoxic effect. Endoplasmic reticulum (ER) stress can develop in TSC-associated cells due to mTORC1-driven protein translation. We hypothesized that renal angiomyolipoma cells experience ER stress that can be leveraged to result in targeted cytotoxicity. We used immortalized human angiomyolipoma cells stably transfected with empty vector or TSC2 (encoding tuberin). Using cell number quantification and cell death assays, we found that mTORC1 inhibition with RAD001 suppressed angiomyolipoma cell proliferation in a cytostatic manner. Angiomyolipoma cells exhibited enhanced sensitivity to proteasome inhibitor-induced ER stress compared with TSC2-rescued cells. After proteasome inhibition with MG-132, Western blot analyses showed greater induction of C/EBP-homologous protein (CHOP) and more poly (ADP-ribose) polymerase (PARP) and caspase-3 cleavage, supporting ER stress-induced apoptosis. Live cell numbers also were decreased and cell death increased by MG-132 in angiomyolipoma cells compared with TSC2 rescued. Intriguingly, while pretreatment of angiomyolipoma cells with RAD001 attenuated CHOP and BiP induction, apoptotic markers cleaved PARP and caspase-3 and eukaryotic translation initiation factor 2α phosphorylation were increased, along with evidence of increased autophagy. These results suggest that human angiomyolipoma cells are uniquely susceptible to agents that exacerbate ER stress and that additional synergy may be achievable with targeted combination therapy.
Division of Nephrology and Hypertension, Cincinnati Children's Hospital Medical Center, OH 45229-3039, USA. john.bissler@cchmc.org
Brian J
BJ
Siroky
Hong
H
Yin
Justin T
JT
Babcock
Lu
L
Lu
Anna R
AR
Hellmann
Bradley P
BP
Dixon
Lawrence A
LA
Quilliam
John J
JJ
Bissler
D018207:Angiomyolipoma / Q000209:etiology / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D059865:Endoplasmic Reticulum Stress; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D000068338:Everolimus; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007680:Kidney Neoplasms / Q000209:etiology / Q000235:genetics / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D020123:Sirolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D051742:Transcription Factor CHOP / Q000378:metabolism; D014162:Transfection; D014402:Tuberous Sclerosis / Q000150:complications* / Q000235:genetics / Q000378:metabolism; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D000970:Antineoplastic Agents; D015852:Eukaryotic Initiation Factor-2; D007166:Immunosuppressive Agents; D007976:Leupeptins; D046912:Multiprotein Complexes; D061988:Proteasome Inhibitors; D011506:Proteins; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D051742:Transcription Factor CHOP; D000068338:Everolimus; D011065:Poly(ADP-ribose) Polymerases; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D053148:Caspase 3; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D020123:Sirolimus