Elizabeth P. Henske

HEF1-dependent Aurora A activation induces disassembly of the primary cilium

The mammalian cilium protrudes from the apical/lumenal surface of polarized cells and acts as a sensor of environmental cues. Numerous developmental disorders and pathological conditions have been shown to arise from defects in cilia-associated signaling proteins. Despite mounting evidence that cilia are essential sites for coordination of cell signaling, little is known about the cellular mechanisms controlling their formation and disassembly. Here, we show that interactions between the prometastatic scaffolding protein HEF1/Cas-L/NEDD9 and the oncogenic Aurora A (AurA) kinase at the basal body of cilia causes phosphorylation and activation of HDAC6, a tubulin deacetylase, promoting ciliary disassembly. We show that this pathway is both necessary and sufficient for ciliary resorption and that it constitutes an unexpected nonmitotic activity of AurA in vertebrates. Moreover, we demonstrate that small molecule inhibitors of AurA and HDAC6 selectively stabilize cilia from regulated resorption cues, suggesting a novel mode of action for these clinical agents.

The mTORC1 Pathway Stimulates Glutamine Metabolism and Cell Proliferation by Repressing SIRT4

Proliferating mammalian cells use glutamine as a source of nitrogen and as a key anaplerotic source to provide metabolites to the tricarboxylic acid cycle (TCA) for biosynthesis. Recently, mammalian target of rapamycin complex 1 (mTORC1) activation has been correlated with increased nutrient uptake and metabolism, but no molecular connection to glutaminolysis has been reported. Here, we show that mTORC1 promotes glutamine anaplerosis by activating glutamate dehydrogenase (GDH). This regulation requires transcriptional repression of SIRT4, the mitochondrial-localized sirtuin that inhibits GDH. Mechanistically, mTORC1 represses SIRT4 by promoting the proteasome-mediated destabilization of cAMP-responsive element binding 2 (CREB2). Thus, a relationship between mTORC1, SIRT4, and cancer is suggested by our findings. Indeed, SIRT4 expression is reduced in human cancer, and its overexpression reduces cell proliferation, transformation, and tumor development. Finally, our data indicate that targeting nutrient metabolism in energy-addicted cancers with high mTORC1 signaling may be an effective therapeutic approach.

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.

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.

Tuberous sclerosis complex: Linking growth and energy signaling pathways with human disease

The most exciting advances in the tuberous sclerosis complex (TSC) field occurred in 1993 and 1997 with the cloning of the TSC2 and TSC1 genes, respectively, and in 2003 with the identification of Rheb as the target of tuberins (TSC2) GTPase activating protein (GAP) domain. Rheb has a dual role: it activates mTOR and inactivates B-Raf. Activation of mTOR leads to increased protein synthesis through phosphorylation of p70S6K and 4E-BP1. Upon insulin or growth factor stimulation, tuberin is phosphorylated by several kinases, including AKT/PKB, thereby suppressing its GAP activity and activating mTOR. Phosphorylation of hamartin (TSC1) by CDK1 also negatively regulates the activity of the hamartin/tuberin complex. Despite these biochemical advances, exactly how mutations in TSC1 or TSC2 lead to the clinical manifestations of TSC is far from being understood. Two of the most unusual phenotypes in TSC are the apparent metastasis of benign cells carrying TSC1 and TSC2 mutations, resulting in pulmonary lymphangiomyomatosis, and the ability of cells with TSC1 or TSC2 mutations to differentiate into the separate components of renal angiomyolipomas (vessels, smooth muscle and fat). We will discuss how the TSC signaling pathways are affected by mutations in TSC1 or TSC2, focusing on how these mutations may lead to the renal and pulmonary manifestations of TSC.

Mutation in TSC2 and activation of mammalian target of rapamycin signalling pathway in renal angiomyolipoma

Mutations that inactivate either TSC1 or TSC2 cause tuberous sclerosis. We have used immunoblotting and immunohistochemical analysis to see whether there is phosphorylation of p70 S6 kinase, and the ribosomal S6 protein in angiomyolipomas occurring in tuberous scierosis. Hamartin (encoded by TSC1) and S6K was expressed in all samples. Tuberin (TSC2) was weak or absent in angiomyolipomas, but present in healthy kidney, whereas, phosphorylated p70 S6 kinase and p56 were present only in angiomyolipomas. Our results indicate activation of a mammalian target of rapamycin metabolic pathway in tuberous sclerosis lesions, which contributes to their growth. We suggest that treatment with rapamycin and its analogues could benefit such patients.

Lymphangioleiomyomatosis — a wolf in sheep's clothing

Lymphangioleiomyomatosis (LAM) is a rare progressive lung disease of women. LAM is caused by mutations in the tuberous sclerosis genes, resulting in activation of the mTOR complex 1 signaling network. Over the past 11 years, there has been remarkable progress in the understanding of LAM and rapid translation of this knowledge to an effective therapy. LAM pathogenic mechanisms mirror those of many forms of human cancer, including mutation, metabolic reprogramming, inappropriate growth and survival, metastasis via blood and lymphatic circulation, infiltration/invasion, sex steroid sensitivity, and local and remote tissue destruction. However, the smooth muscle cell that metastasizes, infiltrates, and destroys the lung in LAM arises from an unknown source and has an innocent histological appearance, with little evidence of proliferation. Thus, LAM is as an elegant, monogenic model of neoplasia, defying categorization as either benign or malignant.

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.

Selective alterations in glutamate and GABA receptor subunit mRNA expression in dysplastic neurons and giant cells of cortical tubers

The molecular pharmacologic basis of epileptogenesis in cortical tubers in the tuberous sclerosis complex is unknown. Altered transcription of genes encoding glutamatergic and γ‐aminobutyric acid (GABA)‐ergic receptors and uptake sites may contribute to seizure initiation and may occur selectively in dysplastic neurons and giant cells. Arrays containing GABA A (GABAAR), GluR, NMDA receptor (NR) subunits, GAD65, the vesicular GABA transporter (VGAT), and the neuronal glutamate transporter (EAAC1) cDNAs were probed with amplified poly (A) mRNA from tubers or normal neocortex to identify changes in gene expression. Increased levels of EAAC1, and NR2B and 2D subunit mRNAs and diminished levels of GAD65, VGAT, GluR1, and GABAAR α1 and α2 were observed in tubers. Ligand‐binding experiments in frozen tuber homogenates demonstrated an increase in functional NR2B‐containing receptors. Arrays were then probed with poly (A) mRNA from single, microdissected dysplastic neurons, giant cells, or normal neurons (n = 30 each). Enhanced expression of GluR 3, 4, and 6 and NR2B and 2C subunit mRNAs was noted in the dysplastic neurons, whereas only the NR2D mRNA was upregulated in giant cells. GABAAR α1 and α2 mRNA levels were reduced in both dysplastic neurons and giant cells compared to control neurons. Differential expression of GluR, NR, and GABAAR mRNAs in tubers reflects cell‐specific changes in gene transcription that argue for a distinct molecular phenotype of dysplastic neurons and giant cells and suggests that dysplastic neurons and giant cells make differential contributions to epileptogenesis in the tuberous sclerosis complex. Ann Neurol 2001;49:67–78

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.