Kinase mTOR: regulation and role in maintenance of cellular homeostasis, tumor development, and aging.

Serine/threonine protein kinase mTOR regulates the maintenance of cellular homeostasis by coordinating transcription, translation, metabolism, and autophagy with availability of amino acids, growth factors, ATP, and oxygen. The mTOR kinase is a component of two protein complexes, mTORC1 and mTORC2, which are different in their composition and regulate different cellular processes. An uncontrolled activation of the mTOR kinase is observed in cells of the majority of tumors, as well as in diabetes and neurodegenerative and some other diseases. At present, inhibitors of the kinase complex mTORC1 are undergoing clinical trials. This review focuses on different aspects of the regulation of the mTORC1 and mTORC2 complexes, on their role in the regulation of protein synthesis, metabolism, and autophagy, as well as on using mTOR inhibitors for treatment of tumors and slowing of aging.

Autophagy: mechanisms, regulation, and its role in tumorigenesis.

Autophagy (from Greek "auto" - self, "phagos" - to eat) is the major catabolic process involved in the delivery and lysosomal degradation of long-lived intracellular components: proteins, lipids, nucleic acids, and organelles. Since the discovery of genes involved in regulation of autophagy in the 1990s, there has been a significant increase in studies of autophagy as a process involved in maintaining cellular homeostasis, as well as its role in the development of different pathologies. This review focuses on the basics of autophagy and its regulatory mechanisms. The role of autophagy in the maintenance of cellular homeostasis and tumorigenesis is also discussed.

A tissue-bioengineering strategy for modeling rare human kidney diseases in vivo.

The lack of animal models for some human diseases precludes our understanding of disease mechanisms and our ability to test prospective therapies in vivo. Generation of kidney organoids from Tuberous Sclerosis Complex (TSC) patient-derived-hiPSCs allows us to recapitulate a rare kidney tumor called angiomyolipoma (AML). Organoids derived from TSC2-/- hiPSCs but not from isogenic TSC2+/- or TSC2+/+ hiPSCs share a common transcriptional signature and a myomelanocytic cell phenotype with kidney AMLs, and develop epithelial cysts, replicating two major TSC-associated kidney lesions driven by genetic mechanisms that cannot be consistently recapitulated with transgenic mice. Transplantation of multiple TSC2-/- renal organoids into the kidneys of immunodeficient rats allows us to model AML in vivo for the study of tumor mechanisms, and to test the efficacy of rapamycin-loaded nanoparticles as an approach to rapidly ablate AMLs. Collectively, our experimental approaches represent an innovative and scalable tissue-bioengineering strategy for modeling rare kidney disease in vivo.

Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the widespread development of distinctive tumors termed hamartomas. TSC-determining loci have been mapped to chromosomes 9q34 (TSC1) and 16p13 (TSC2). The TSC1 gene was identified from a 900-kilobase region containing at least 30 genes. The 8.6-kilobase TSC1 transcript is widely expressed and encodes a protein of 130 kilodaltons (hamartin) that has homology to a putative yeast protein of unknown function. Thirty-two distinct mutations were identified in TSC1, 30 of which were truncating, and a single mutation (2105delAAAG) was seen in six apparently unrelated patients. In one of these six, a somatic mutation in the wild-type allele was found in a TSC-associated renal carcinoma, which suggests that hamartin acts as a tumor suppressor.

Loss of the polycystic kidney disease (PKD1) region of chromosome 16p13 in renal cyst cells supports a loss-of-function model for cyst pathogenesis.

It is not known whether mutations in the PKD1 gene cause autosomal dominant polycystic kidney disease (PKD) by an activating (gain-of-function) or an inactivating (loss-of-function) model. We analyzed DNA from cyst epithelial cells for loss of heterozygosity (LOH) in the PKD1 region of chromosome 16p13 using microsatellite markers. 29 cysts from four patients were studied. Five cysts from three patients had chromosome 16p13 LOH. Four of the cysts had loss of two chromosome 16p13 markers that flank the PKD1 gene. In two patients, microsatellite analysis of family members was consistent with loss of the wild-type copy of PKD1 in the cysts. In the third patient, 16p13 LOH was detected in three separate cysts, all of which showed loss of the same alleles. Chromosome 3p21 LOH was detected in one cyst. No LOH was detected in four other genomic regions. These results demonstrate that some renal cyst epithelial cells exhibit clonal chromosomal abnormalities with loss of the wild-type copy of PKD1. This supports a loss-of-function model for autosomal dominant PKD, with a germline mutation inactivating one copy of PKD1 and somatic mutation or deletion inactivating the remaining wild-type copy.

Hamartin, the product of the tuberous sclerosis 1 (TSC1) gene, interacts with tuberin and appears to be localized to cytoplasmic vesicles.

Tuberous sclerosis is an inherited syndrome associated with mutations in two tumor suppressor genes: TSC1 and TSC2. Tuberin, the product of TSC2, appears to be localized to the Golgi apparatus and may have a function in vesicular transport. The function of hamartin, the product of TSC1, is not known. In this report, we demonstrate an interaction between hamartin and tuberin, which is detectable at endogenous protein levels. Hamartin is present in a cell line derived from the Eker rat that lacks functional tuberin, indicating that the stability of hamartin is not dependent on its interaction with tuberin. Hamartin is localized to the membrane/particulate (P100) fraction of cultured cells. The P100 localization is unchanged in the Eker cells. Finally, we show that at endogenous expression levels, hamartin has a punctate pattern of immunofluorescence in the cytoplasm. Taken together, the presence of hamartin in the membrane/particulate fraction and its pattern of cytoplasmic staining suggest that it is localized to cytoplasmic vesicles. If altered vesicular trafficking leads to tumorigenesis in tuberous sclerosis, TSC1 and TSC2 may have a novel mechanism of tumor suppression.

Cosmid contigs spanning 9q34 including the candidate region for TSC1.

The tuberous sclerosis disease gene TSC1 has been mapped to 9q34. However, its precise localisation has proved problematic because of conflicting recombination data. Therefore, we have attempted to clone the entire target area into cosmid contigs prior to gene isolation studies. We have used Alu-PCR from irradiation hybrids to produce complex probes from the target region which have identified 1,400 cosmids from a chromosome-specific library. These, along with cosmids obtained by other methods, have been assembled into contigs by a fingerprinting technique. We estimate that we have obtained most of the region in cosmid contigs. These cosmids are a resource for the isolation of expressed genes within the TSC1 interval. In addition, the cosmid contig assembly has demonstrated a number of previously unknown physical connections between genes and markers in 9q34.

New developments in the neurobiology of the tuberous sclerosis complex.

OBJECTIVE: To outline recent developments in the neurobiology of the tuberous sclerosis complex (TSC). BACKGROUND: TSC may be associated with neuropsychiatric disorders including epilepsy, mental retardation, and autism. The uncontrolled growth of subependymal giant cell astrocytomas may lead to hydrocephalus and death. The recent identification of mutations in two genes (TSC1 and TSC2) that cause TSC has led to rapid progress in understanding the molecular and cellular pathogenesis of this disorder. How distinct mutations lead to the varied clinical phenotype of TSC is under intense investigation. RESULTS: We report the recent diagnostic criteria for TSC and provide an overview of the molecular genetics, molecular pathophysiology, and neuropathology of TSC. Important diagnostic criteria for TSC include facial angiofibromas, ungual fibromas, retinal hamartomas, and cortical tubers. Both familial and sporadic TSC cases occur. Approximately 50% of TSC families show genetic linkage to TSC1 and 50% to TSC2. Among sporadic TSC cases, mutations in TSC2 are more frequent and often accompanied by more severe neurologic deficits. Multiple mutational subtypes have been identified in the TSC1 and TSC2 genes. The TSC1 (chromosome 9) and TSC2 (chromosome 16) genes encode distinct proteins, hamartin and tuberin, respectively, which are widely expressed in the brain and may interact as part of a cascade pathway that modulates cellular differentiation, tumor suppression, and intracellular signaling. Tuberin has a GTPase activating protein-related domain that may contribute to a role in cell cycle passage and intracellular vesicular trafficking. CONCLUSION: Identification of tuberous sclerosis complex (TSC) gene mutations has fostered understanding of how brain lesions in TSC are formed. Further characterization of the roles of hamartin and tuberin will provide potential therapeutic avenues to treat seizures, mental retardation, and tumor growth in TSC.

Apparent renal cell carcinomas in tuberous sclerosis are heterogeneous: the identification of malignant epithelioid angiomyolipoma.

Renal epithelial tumors (carcinoma and oncocytoma) have been reported with higher a frequency than expected in patients with the tuberous sclerosis complex. However, the recent identification of a monotypic, epithelioid variant of angiomyolipoma, closely simulating renal cell carcinoma, has cast doubt on the real frequency of carcinoma. Immunohistochemical analysis with a panel of antibodies, including melanogenesis marker HMB45, can discriminate between carcinoma and carcinoma-like angiomyolipoma. We studied five tumors previously reported as carcinoma and found that only one of them showed an immunohistochemical phenotype indicative of an epithelial tumor (Ker+, HMB45-). Three tumors exhibited a phenotype compatible with the monotypic epithelioid variant of angiomyolipoma (HMB45+, Ker-), and two of the three patients died of metastatic disease. The last patient had unusual clinical features, and the tumor was positive both for HMB45 and keratin. It is concluded that (1) renal cell carcinoma is less common in tuberous sclerosis complex than previously believed, (2) some cases called renal cell carcinoma probably represent a monotypic, epithelioid variant of angiomyolipoma, and (3) epithelioid angiomyolipoma is a potentially malignant tumor with invasion and metastases. These findings indicate that all reported renal carcinomas in tuberous sclerosis complex, therefore, must be reevaluated.

Frequent estrogen and progesterone receptor immunoreactivity in renal angiomyolipomas from women with pulmonary lymphangioleiomyomatosis.

OBJECTIVE: To determine whether renal angiomyolipomas from women with pulmonary lymphangioleiomyomatosis (LAM) express estrogen receptor (ER) and progesterone receptor (PR). DESIGN: Retrospective study of archival tissue. PATIENTS: Twelve women with LAM and angiomyolipomas. SETTING: Fox Chase Cancer Center. INTERVENTIONS: ER and PR expression was studied using immunohistochemistry. The hormonal status of the patients at the time of resection of the angiomyolipoma was determined. RESULTS: Ten of the angiomyolipomas had ER immunoreactivity (83%), and all 12 had PR immunoreactivity (100%). The ER and PR positivity was in the smooth muscle component of the angiomyolipomas only. For five women, pulmonary LAM specimens were also available; two were ER positive (40%), and all five were PR positive (100%). All four angiomyolipomas from women receiving progesterone therapy were ER and PR positive. One tumor from a woman receiving tamoxifen was ER negative and strongly PR positive. One woman was pregnant; her tumor was ER and PR positive. CONCLUSIONS: ER and PR expression is frequent in renal angiomyolipoma cells from women with LAM. PR was more consistently present than ER in angiomyolipomas and in LAM. Our data suggest that angiomyolipoma growth could be affected by hormonal factors. If the growth of LAM-associated angiomyolipomas slows during hormonal therapy, there are two potential implications for LAM patients: first, angiomyolipoma size could serve as a measurable indication of response to hormonal therapy; and second, surgical removal of angiomyolipomas might be avoided in some cases.

Renal angiomyolipomas, cysts, and cancer in tuberous sclerosis complex.

Tuberous sclerosis is a multisystem syndrome characterized by neurological symptoms and tumors in multiple organs, including kidney, brain, skin, eyes, heart, and lung. The kidney and brain are the two most frequently affected organs in TSC, and renal disease is a leading cause of death in TSC patients. Three types of tumors occur in TSC kidneys: (1) angiomyolipomas, which are benign tumors composed of smooth muscle, fat, and vessels; (2) epithelial cysts; and (3) malignant tumors. This review focuses on the clinical, pathological, and molecular features of these tumors.

Estradiol promotes pentose phosphate pathway addiction and cell survival via reactivation of Akt in mTORC1 hyperactive cells.

Lymphangioleiomyomatosis (LAM) is a female-predominant interstitial lung disease that can lead to respiratory failure. LAM cells typically have inactivating TSC2 mutations, leading to mTORC1 activation. The gender specificity of LAM suggests that estradiol contributes to disease development, yet the underlying pathogenic mechanisms are not completely understood. Using metabolomic profiling, we identified an estradiol-enhanced pentose phosphate pathway signature in Tsc2-deficient cells. Estradiol increased levels of cellular NADPH, decreased levels of reactive oxygen species, and enhanced cell survival under oxidative stress. Mechanistically, estradiol reactivated Akt in TSC2-deficient cells in vitro and in vivo, induced membrane translocation of glucose transporters (GLUT1 or GLUT4), and increased glucose uptake in an Akt-dependent manner. (18)F-FDG-PET imaging demonstrated enhanced glucose uptake in xenograft tumors of Tsc2-deficient cells from estradiol-treated mice. Expression array study identified estradiol-enhanced transcript levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway. Consistent with this, G6PD was abundant in xenograft tumors and lung metastatic lesions of Tsc2-deficient cells from estradiol-treated mice. Molecular depletion of G6PD attenuated estradiol-enhanced survival in vitro, and treatment with 6-aminonicotinamide, a competitive inhibitor of G6PD, reduced lung colonization of Tsc2-deficient cells. Collectively, these data indicate that estradiol promotes glucose metabolism in mTORC1 hyperactive cells through the pentose phosphate pathway via Akt reactivation and G6PD upregulation, thereby enhancing cell survival under oxidative stress. Interestingly, a strong correlation between estrogen exposure and G6PD was also found in breast cancer cells. Targeting the pentose phosphate pathway may have therapeutic benefit for LAM and possibly other hormonally dependent neoplasms.

Rabin8 Protein Interacts with GTPase Rheb and Inhibits Phosphorylation of Ser235/Ser236 in Small Ribosomal Subunit Protein S6.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that in association with Raptor, mLST8, PRAS40 and Deptor forms a complex (mTORC1) playing the key role in the regulation of protein biosynthesis, transcription, cellular metabolism, apoptosis and autophagy; mainly via direct phosphorylation of S6 kinases. mTORC1 is activated by growth factors and amino acids via the activation of Rheb GTPase. In the current study, we demonstrate for the first time that the over-expression of Rabin8, which functions as a guanine nucleotide exchange factor for Rab8 GTPase, suppresses phosphorylation of Ser235/Ser236 in ribosomal protein S6. Downregulation of Rabin8 using small interfering RNA (siRNA) increases the phosphorylation of Ser235/Ser236 in ribosomal protein S6. Furthermore, Rabin8 can be immunoprecipitated with Rheb GTPase. These results suggest the existence of a novel mechanism of mTORС1 regulation and its downstream processes. Since Rabin8 is a known regulator of ciliogenesis, a potential link can exist between regulation of Rheb/mTORC1 and ciliogenesis.

Loss of the Birt-Hogg-Dubé tumor suppressor results in apoptotic resistance due to aberrant TGFß-mediated transcription.

Birt-Hogg-Dubé (BHD) syndrome is an inherited cancer susceptibility disease characterized by skin and kidney tumors, as well as cystic lung disease, which results from loss-of-function mutations in the BHD gene. BHD is also inactivated in a significant fraction of patients with sporadic renal cancers and idiopathic cystic lung disease, and little is known about its mode of action. To investigate the molecular and cellular basis of BHD tumor suppressor activity, we generated mutant Bhd mice and embryonic stem cell lines. BHD-deficient cells exhibited defects in cell-intrinsic apoptosis that correlated with reduced expression of the BH3-only protein Bim, which was similarly observed in all human and murine BHD-related tumors examined. We further demonstrate that Bim deficiency in Bhd(-/-) cells is not a consequence of elevated mTOR or ERK activity, but results instead from reduced Bim transcription associated with a general loss of TGFß-mediated transcription and chromatin modifications. In aggregate, this work identifies a specific tumor suppressive mechanism for BHD in regulating TGFß-dependent transcription and apoptosis, which has implications for the development of targeted therapies.

Differential requirement of CAAX-mediated posttranslational processing for Rheb localization and signaling.

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. After farnesylation, Rheb undergoes two additional CAAX-signaled processing steps, Ras converting enzyme 1 (Rce1)-catalyzed cleavage of the AAX residues and isoprenylcysteine carboxyl methyltransferase (Icmt)-mediated carboxylmethylation of the farnesylated cysteine. However, whether these postprenylation 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.

The role of the Birt-Hogg-Dubé protein in mTOR activation and renal tumorigenesis.

Birt-Hogg-Dubé (BHD) syndrome is a tumor-suppressor gene disorder characterized by skin tumors, cystic lung disease and renal cell carcinoma. Very little is known about the molecular pathogenesis of BHD. Clinical similarities between BHD and tuberous sclerosis complex (TSC) suggest that the BHD and TSC proteins may function within a common pathway. The TSC proteins inhibit the activity of the mammalian target of rapamycin complex 1 (TORC1), and in Schizosaccharomyces pombe, Bhd and Tsc1/Tsc2 have opposing roles in the regulation of amino-acid homeostasis. We report here that in mammalian cells, downregulation of BHD reduces the phosphorylation of ribosomal protein S6, an indicator of TORC1 activity. To determine whether folliculin, the product of the BHD gene, regulates mammalian target of rapamycin activity in vivo, we generated a mouse with targeted inactivation of the Bhd gene. The mice developed spontaneous oncocytic cysts and tumors composed of cells that resemble the renal cell carcinomas in BHD patients. The cysts and tumors had low levels of phospho-S6. Taken together, these data indicate that folliculin regulates the activity of TORC1, and suggest a new paradigm in which both inappropriately high and inappropriately low levels of TORC1 activity can be associated with renal tumorigenesis.

A 5.4-Mb continuous pulsed-field gel electrophoresis map of human 9q34.1 between ABL and D9S114, including the tuberous sclerosis (TSC1) region.

We constructed a long-range restriction map of the tuberous sclerosis (TSC1) region of human chromosome 9q34, extending from ABL (T39-2-2) to D9S114. The physical map includes five genes and seven anonymous markers. The maximum distance between ABL and D9S114 is 5.4 Mb. The TSC1 critical region, between D9S149 and D9S114, has a maximum distance of 2.7 Mb. The ratio of genetic and physical distance in the region is 1 cM:600 kb. We also used Southern blot analysis and a radiation hybrid cell line, E6B, to exclude 3 genes--PBX3A, RXR alpha, and TAN1--from the AK1 to D9S114 interval. This excludes them as disease genes for tuberous sclerosis.

Chromosome 16 loss of heterozygosity in tuberous sclerosis and sporadic lymphangiomyomatosis.

In previous work we found loss of heterozygosity (LOH) of the wild-type TSC2 allele in the abnormal pulmonary smooth muscle cells and renal angiomyolipoma cells from patients with sporadic pulmonary lymphangiomyomatosis (LAM). Here we report TSC2 LOH in microdissected pulmonary LAM cells from a patient with tuberous sclerosis complex (TSC), demonstrating for the first time that the two-hit tumor suppressor gene model applies to the TSC-associated, as well as sporadic LAM. We also compared the chromosome 16 LOH region between angiomyolipoma and pulmonary LAM from two patients with sporadic LAM. Previously we found that these patients had TSC2 mutations and TSC2 LOH in their angiomyolipomas and pulmonary LAM cells but not in normal lung or kidney cells. This suggests that pulmonary LAM may result from the migration of smooth muscle cells from renal angiomyolipomas to the lung. In this case, one would predict that the angiomyolipoma and LAM cells would have identical LOH patterns. We found that at each chromosome 16 marker, the results were concordant between angiomyolipoma and LAM. This is consistent with a model in which pulmonary LAM cells and angiomyolipoma cells have a common genetic origin.

Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a progressive and often fatal interstitial lung disease characterized by a diffuse proliferation of abnormal smooth muscle cells in the lungs. LAM is of unusual interest biologically because it affects almost exclusively young women. LAM can occur as an isolated disorder (sporadic LAM) or in association with tuberous sclerosis complex. Renal angiomyolipomas, which are found in most tuberous sclerosis patients, also occur in 60% of sporadic LAM patients. We previously found TSC2 loss of heterozygosity in 7 of 13 (54%) of angiomyolipomas from sporadic LAM patients, suggesting that LAM and TSC could have a common genetic basis. In this study, we report the identification of somatic TSC2 mutations in five of seven angiomyolipomas from sporadic LAM patients. In all four patients from whom lung tissue was available, the same mutation found in the angiomyolipoma was present in the abnormal pulmonary smooth muscle cells. In no case was the mutation present in normal kidney, morphologically normal lung, or lymphoblastoid cells. Our data demonstrate that somatic mutations in the TSC2 gene occur in the angiomyolipomas and pulmonary LAM cells of women with sporadic LAM, strongly supporting a direct role of TSC2 in the pathogenesis of this disease.