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1.  Autophagy 
Autophagy  2011;7(11):1400-1401.
Mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is activated in tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), is a master regulator of cell growth, cellular metabolism and autophagy. Treatment of TSC and LAM patients with mTORC1 inhibitors partially decreases the size of brain and kidney tumors, and stabilizes pulmonary function. However, the tumors regrow and lung function continues to decline when treatment is discontinued. We hypothesized that dysregulation of autophagy plays a critical role in the pathogenesis of tumors with mTORC1 hyperactivation and in their response to mTORC1-targeted therapy. We found that cells lacking TSC2 have low levels of autophagy under basal and cellular stress conditions. Using genetic and pharmacological approaches, we discovered that the survival of Tsc2-deficient tumor cells is dependent on autophagy induction. Thus, autophagy inhibitors may have therapeutic potential in TSC and LAM, either as single agent therapy or in combination with mTORC1 inhibitors.
PMCID: PMC3242802  PMID: 21997371
autophagy; p62/sequestosome 1; tuberin; sirolimus; mTOR; chloroquine; tuberous sclerosis complex; lymphangioleiomyomatosis; metabolism
2.  mTOR Activation, Lymphangiogenesis, and Estrogen-Mediated Cell Survival: The “Perfect Storm” of Pro-Metastatic Factors in LAM Pathogenesis 
Research interest in lymphangioleiomyomatosis (LAM) has grown dramatically in the past decade, particularly among cancer biologists. There are at least two reasons for this: first, the discovery in the year 2000 that LAM cells carry TSC2 gene mutations, linking LAM with cellular pathways including the PI3K/Akt/mTOR axis, and allowing the Tuberous Sclerosis Complex (TSC)-regulated pathways that are believed to underlie LAM pathogenesis to be studied in cells, yeast, Drosophila, and mice. A second reason for the rising interest in LAM is the discovery that LAM cells can travel to the lung, including repopulating a donor lung after lung transplantation, despite the fact that LAM cells are histologically benign. This “benign metastasis” underpinning suggests that elucidating LAM pathogenesis will unlock a set of fundamental mechanisms that underlie metastatic potential in the context of a cell that has not yet undergone malignant transformation. Here, we will outline the data supporting the metastatic model of LAM, consider the biochemical and cellular mechanisms that may enable LAM cells to metastasize, including both cell autonomous and non-cell autonomous factors, and highlight a mouse model in which estrogen promotes the metastasis and survival of TSC2-deficient cells in a MEK-dependent manner. We propose a multistep model of LAM cell metastasis that highlights multiple opportunities for therapeutic intervention. Taken together, the metastatic behavior of LAM cells and the involvement of tumor-related signaling pathways lead to optimism that cancer-related paradigms for diagnosis, staging, and therapy will lead to therapeutic breakthroughs for women living with LAM.
PMCID: PMC2883473  PMID: 20235886
3.  Mammalian Target of Rapamycin Signaling and Autophagy 
The pace of progress in lymphangioleiomyomatosis (LAM) is remarkable. In the year 2000, TSC2 gene mutations were found in LAM cells; in 2001 the tuberous sclerosis complex (TSC) genes were discovered to regulate cell size in Drosophila via the kinase TOR (target of rapamycin); and in 2008 the results were published of a clinical trial of rapamycin, a specific inhibitor of TOR, in patients with TSC and LAM with renal angiomyolipomas. This interval of just 8 years between a genetic discovery for which the relevant signaling pathway was as yet unknown, to the initiation, completion, and publication of a clinical trial, is an almost unparalleled accomplishment in modern biomedical research. This robust foundation of basic, translational, and clinical research in TOR, TSC, and LAM is now poised to optimize and validate effective therapeutic strategies for LAM. An immediate challenge is to deduce the mechanisms underlying the partial response of renal angiomyolipomas to rapamycin, and thereby guide the design of combinatorial approaches. TOR complex 1 (TORC1), which is known to be active in LAM cells, is a key inhibitor of autophagy. One hypothesis, which will be explored here, is that low levels of autophagy in TSC2-null LAM cells limits their survival under conditions of bioenergetic stress. A corollary of this hypothesis is that rapamycin, by inducing autophagy, promotes the survival of LAM cells, while simultaneously arresting their growth. If this hypothesis proves to be correct, then combining TORC1 inhibition with autophagy inhibition may represent an effective clinical strategy for LAM.
PMCID: PMC3137149  PMID: 20160148
tuberin; rapamycin; chloroquine; Rheb; tuberous sclerosis
4.  The evolutionarily conserved TSC/Rheb pathway activates Notch in tuberous sclerosis complex and Drosophila external sensory organ development  
Mutations in either of the genes encoding the tuberous sclerosis complex (TSC), TSC1 and TSC2, result in a multisystem tumor disorder characterized by lesions with unusual lineage expression patterns. How these unusual cell-fate determination patterns are generated is unclear. We therefore investigated the role of the TSC in the Drosophila external sensory organ (ESO), a classic model of asymmetric cell division. In normal development, the sensory organ precursor cell divides asymmetrically through differential regulation of Notch signaling to produce a pIIa and a pIIb cell. We report here that inactivation of Tsc1 and overexpression of the Ras homolog Rheb each resulted in duplication of the bristle and socket cells, progeny of the pIIa cell, and loss of the neuronal cell, a product of pIIb cell division. Live imaging of ESO development revealed this cell-fate switch occurred at the pIIa-pIIb 2-cell stage. In human angiomyolipomas, benign renal neoplasms often found in tuberous sclerosis patients, we found evidence of Notch receptor cleavage and Notch target gene activation. Further, an angiomyolipoma-derived cell line carrying biallelic TSC2 mutations exhibited TSC2- and Rheb-dependent Notch activation. Finally, inhibition of Notch signaling using a γ-secretase inhibitor suppressed proliferation of Tsc2-null rat cells in a xenograft model. Together, these data indicate that the TSC and Rheb regulate Notch-dependent cell-fate decision in Drosophila and Notch activity in mammalian cells and that Notch dysregulation may underlie some of the distinctive clinical and pathologic features of TSC.
PMCID: PMC2798691  PMID: 20038815
5.  Estradiol and mTORC2 cooperate to enhance prostaglandin biosynthesis and tumorigenesis in TSC2-deficient LAM cells 
Estradiol enhances COX-2 expression and prostaglandin biosynthesis in TSC2-deficient cells via a rapamycin-insensitive, mTORC2-dependent mechanism.
Lymphangioleiomyomatosis (LAM) is a progressive neoplastic disorder that leads to lung destruction and respiratory failure primarily in women. LAM is typically caused by tuberous sclerosis complex 2 (TSC2) mutations resulting in mTORC1 activation in proliferative smooth muscle–like cells in the lung. The female predominance of LAM suggests that estradiol contributes to disease development. Metabolomic profiling identified an estradiol-enhanced prostaglandin biosynthesis signature in Tsc2-deficient (TSC−) cells, both in vitro and in vivo. Estradiol increased the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin biosynthesis, which was also increased at baseline in TSC-deficient cells and was not affected by rapamycin treatment. However, both Torin 1 treatment and Rictor knockdown led to reduced COX-2 expression and phospho-Akt-S473. Prostaglandin production was also increased in TSC-deficient cells. In preclinical models, both Celecoxib and aspirin reduced tumor development. LAM patients had significantly higher serum prostaglandin levels than healthy women. 15-epi-lipoxin-A4 was identified in exhaled breath condensate from LAM subjects and was increased by aspirin treatment, indicative of functional COX-2 expression in the LAM airway. In vitro, 15-epi-lipoxin-A4 reduced the proliferation of LAM patient–derived cells in a dose-dependent manner. Targeting COX-2 and prostaglandin pathways may have therapeutic value in LAM and TSC-related diseases, and possibly in other conditions associated with mTOR hyperactivation.
PMCID: PMC3892971  PMID: 24395886
6.  Non-canonical functions of the tuberous sclerosis complex-Rheb signalling axis 
EMBO Molecular Medicine  2011;3(4):189-200.
The protein products of the tuberous sclerosis complex (TSC) genes, TSC1 and TSC2, form a complex, which inhibits the small G-protein, Ras homolog enriched in brain (Rheb). The vast majority of research regarding these proteins has focused on mammalian Target of Rapamycin (mTOR), a target of Rheb. Here, we propose that there are clinically relevant functions and targets of TSC1, TSC2 and Rheb, which are independent of mTOR. We present evidence that such non-canonical functions of the TSC-Rheb signalling network exist, propose a standard of evidence for these non-canonical functions, and discuss their potential clinical and therapeutic implications for patients with TSC and lymphangioleiomyomatosis (LAM).
PMCID: PMC3377068  PMID: 21412983
lymphangioleiomyomatosis; mTOR; non-canonical; Rheb; tuberous sclerosis complex
7.  Differential Requirement of CAAX-mediated Posttranslational Processing for Rheb Localization and Signaling 
Oncogene  2009;29(3):380-391.
The Rheb1 and Rheb2 small GTPases and their effector mTOR are aberrantly activated in human cancer and are attractive targets for anti-cancer drug discovery. Rheb is targeted to endomembranes via its C-terminal CAAX (C = cysteine, A = aliphatic, X = terminal amino acid) motif, a substrate for posttranslational modification by a farnesyl isoprenoid. Following farnesylation, Rheb undergoes two additional CAAX-signaled processing steps, Rce1-catalyzed cleavage of the AAX residues and Icmt-mediated carboxylmethylation of the farnesylated cysteine. However, whether these post-prenylation processing steps are required for Rheb signaling through mTOR is not known. We found that Rheb1 and Rheb2 localize primarily to the endoplasmic reticulum and Golgi apparatus. We determined that Icmt and Rce1 processing is required for Rheb localization, but is dispensable for Rheb-induced activation of the mTOR substrate p70 S6 kinase (S6K). Finally, we evaluated whether farnesylthiosalicylic acid (FTS) blocks Rheb localization and function. Surprisingly, FTS prevented S6K activation induced by a constitutively active mTOR mutant, indicating that FTS inhibits mTOR at a level downstream of Rheb. We conclude that inhibitors of Icmt and Rce1 will not block Rheb function, but FTS could be a promising treatment for Rheb- and mTOR-dependent cancers.
PMCID: PMC2809798  PMID: 19838215
Rheb; mTOR; Ras converting enzyme 1 (Rce1); Isoprenylcysteine carboxyl methyltransferase (Icmt); S-trans, trans-farnesylthiosalicylic acid (FTS)

Results 1-7 (7)