Human TSC-associated renal angiomyolipoma cells are hypersensitive to ER stress
ABSTRACT 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.
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ABSTRACT: Nearly all patients with tuberous sclerosis complex (TSC) develop renal angiomyolipomas, although the tumor cell of origin is unknown. We observed decreased renal angiomyolipoma development in patients with the TSC2-PKD1 deletion syndrome and hypertension that were treated from an early age with angiotensin converting enzyme inhibitors or angiotensin receptor blockers. TSC-associated renal angiomyolipomas expressed angiotensin II type 1 receptor, platelet derived growth factor receptor β and VEGFR2, but did not express the adipocyte marker S100 or the endothelial marker CD31. Sera of TSC patients exhibited increased vascular mural cell secreted peptides such as vascular endothelial growth factor (VEGF) D, sVEGFR2, and collagen IV. These findings suggest that angiomyolipomas may arise from renal pericytes. Angiotensin II treatment of angiomyolipoma cells in vitro resulted in an exaggerated intracellular calcium response and increased proliferation that were blocked by the angiotensin type II receptor 1 antagonist valsartan. Blockade of angiotensin II signaling may have preventative therapeutic potential for angiomyolipomas.American journal of physiology. Renal physiology 06/2014; 307(5). DOI:10.1152/ajprenal.00569.2013 · 3.30 Impact Factor
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ABSTRACT: Mutations in tuberous sclerosis (TSC) genes cause the genetic disorder TSC, as well as other neoplasms, including lymphangioleiomyomatosis (LAM) and angiomyolipomas (AMLs). AMLs are benign renal tumors occur both in sporadic LAM and in TSC. As they carry the same mutations, AML cell lines serve as a model for TSC and LAM. Rheb/mammalian target of rapamycin complex 1 (mTORC1) pathway is chronically activated in TSC-deficient cells, and this activation can be diminished using the appropriate inhibitors. Rapamycin (sirolimus) is a known specific inhibitor of mTORC1, whereas S-trans,trans-farnesylthiosalicylic acid (FTS; salirasib) has been shown to inhibit Rheb. To examine the effect of the Rheb/mTOR inhibition pathway, we used human TSC2-deficient AML cells, derived from a LAM patient. FTS indeed inhibited Rheb in these cells and attenuated their proliferation. After comparative treatments with FTS or rapamycin or by re-expression of TSC2, we carried out a gene array analysis. This yielded a substantial number of commonly altered genes, many of which we identified as downstream targets of the interferon (IFN) regulatory factor 7 (IRF7) transcription factor, a central activator of the IFN type 1 immune response. Furthermore, nuclear localization of IRF7 was impaired by each of the three treatments. Interestingly, the phenomena seen on FTS or rapamycin treatment were selective for TSC2-deficient cells. Moreover, knockdown of IRF7 by siRNA mimicked the decrease in number of the abovementioned genes and also inhibited AML cell proliferation. Altogether, these findings support FTS as a potential treatment for TSC and its related pathologies and IRF7 as a novel target for treatment.Cell Death & Disease 12/2014; 5(12):e1557. DOI:10.1038/cddis.2014.502 · 5.18 Impact Factor
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ABSTRACT: The mammalian target of rapamycin (mTOR) nucleates two complexes, namely mTOR complex 1 and 2 (mTORC1 and mTORC2), which are implicated in cell growth, survival, metabolism and cancer. Phosphorylation of the alpha-subunit of translation initiation factor eIF2 at serine 51 (eIF2lalphaS51P) is a key event of mRNA translation initiation and a master regulator of cell fate during cellular stress. Recent studies have implicated mTOR signaling in the stress response but its connection to eIF2alphaS51P has remained unclear. Herein, we report that genetic as well as catalytic inhibition of mTORC2 induces eIF2alphaS51P. On the other hand, the allosteric inhibitor rapamycin induces eIF2alphaS51P through pathways that are independent of mTORC1 inactivation. Increased eIF2alphaS51P by impaired mTORC2 depends on the inactivation of Akt, which primes the activation of the endoplasmic reticulum (ER)-resident kinase PERK/PEK. The biological function of eIF2alphaS51P was characterized in tuberous sclerosis complex (TSC)-mutant cells, which are defective in mTORC2 and Akt activity. TSC-mutant cells exhibit increased PERK activity, which is down regulated by the reconstitution of the cells with an activated form of Akt1. Also, TSC-mutant cells are increasingly susceptible to ER stress, which is reversed by Akt1 reconstitution. The susceptibility of TSC-mutant cells to ER stress is further enhanced by the pharmacological inhibition of PERK or genetic inactivation of eIF2alphaS51P. Thus, the PERK/eIF2alphaS51P arm is an important compensatory pro-survival mechanism, which substitutes for the loss of Akt under ER stress. A novel mechanistic link between mTOR function and protein synthesis is identified in TSC-null tumor cells under stress and reveals potential for the development of anti-tumor treatments with stress-inducing chemotherapeutics. Copyright © 2015, American Association for Cancer Research.Molecular Cancer Research 06/2015; DOI:10.1158/1541-7786.MCR-15-0184-T · 4.50 Impact Factor