Person:

Henske, Elizabeth

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 TGFβ-mediated transcription and chromatin modifications. In aggregate, this work identifies a specific tumor suppressive mechanism for BHD in regulating TGFβ-dependent transcription and apoptosis, which has implications for the development of targeted therapies.

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.

Lymphangioleiomyomatosis (LAM), a multisystem disease of women, is manifest by the proliferation of smooth muscle-like cells in the lung resulting in cystic lung destruction. Women with LAM can also develop renal angiomyolipomas. LAM is caused by mutations in the tuberous sclerosis complex genes (TSC1 or TSC2), resulting in hyperactive mammalian Target of Rapamycin (mTOR) signaling. The mTOR inhibitor, Rapamycin, stabilizes lung function in LAM and decreases the volume of renal angiomyolipomas, but lung function declines and angiomyolipomas regrow when treatment is discontinued, suggesting that factors induced by mTORC1 inhibition may promote the survival of TSC2-deficient cells. Whether microRNA (miRNA, miR) signaling is involved in the response of LAM to mTORC1 inhibition is unknown. We identified Rapamycin-dependent miRNA in LAM patient angiomyolipoma-derived cells using two separate screens. First, we assayed 132 miRNA of known significance to tumor biology. Using a cut-off of >1.5-fold change, 48 microRNA were Rapamycin-induced, while 4 miRs were downregulated. In a second screen encompassing 946 miRNA, 18 miRs were upregulated by Rapamycin, while eight were downregulated. Dysregulation of miRs 29b, 21, 24, 221, 106a and 199a were common to both platforms and were classified as candidate “RapamiRs.” Validation by qRT-PCR confirmed that these microRNA were increased. miR-21, a pro-survival miR, was the most significantly increased by mTOR-inhibition (p<0.01). The regulation of miR-21 by Rapamycin is cell type independent. mTOR inhibition promotes the processing of the miR-21 transcript (pri-miR-21) to a premature form (pre-miR-21). In conclusion, our findings demonstrate that Rapamycin upregulates multiple miRs, including pro-survival miRs, in TSC2-deficient patient-derived cells. The induction of miRs may contribute to the response of LAM and TSC patients to Rapamycin therapy.

Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhdflox/flox mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma.

Background: TSC2-deficient cells can proliferate in the lungs, kidneys, and other organs causing devastating progressive multisystem disorders such as lymphangioleiomyomatosis (LAM) and tuberous sclerosis complex (TSC). Preclinical models utilizing LAM patient-derived cells have been difficult to establish. We developed a novel animal model system to study the molecular mechanisms of TSC/LAM pathogenesis and tumorigenesis and provide a platform for drug testing. Methods and Findings: TSC2-deficient human cells, derived from the angiomyolipoma of a LAM patient, were engineered to co-express both sodium-iodide symporter (NIS) and green fluorescent protein (GFP). Cells were inoculated intraparenchymally, intravenously, or intratracheally into athymic NCr nu/nu mice and cells were tracked and quantified using single photon emission computed tomography (SPECT) and computed tomography (CT). Surprisingly, TSC2-deficient cells administered intratracheally resulted in rapid dissemination to lymph node basins throughout the body, and histopathological changes in the lung consistent with LAM. Estrogen was found to be permissive for tumor growth and dissemination. Rapamycin inhibited tumor growth, but tumors regrew after the drug treatment was withdrawn. Conclusions: We generated homogeneous NIS/GFP co-expressing TSC2-deficient, patient-derived cells that can proliferate and migrate in vivo after intratracheal instillation. Although the animal model we describe has some limitations, we demonstrate that systemic tumors formed from TSC2-deficient cells can be monitored and quantified noninvasively over time using SPECT/CT, thus providing a much needed model system for in vivo drug testing and mechanistic studies of TSC2-deficient cells and their related clinical syndromes.

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).

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.

Tumor suppressor genes and their effector pathways have been identified for many dominantly heritable cancers, enabling efforts to intervene early in the course of disease. Our approach on the subject of early intervention was to investigate gene expression patterns of morphologically normal one-hit cells before they become hemizygous or homozygous for the inherited mutant gene which is usually required for tumor formation. Here, we studied histologically non-transformed renal epithelial cells from patients with inherited disorders that predispose to renal tumors, including von Hippel-Lindau (VHL) disease and Tuberous Sclerosis (TSC). As controls, we studied histologically normal cells from non-cancerous renal epithelium of patients with sporadic clear cell renal cell carcinoma (ccRCC). Gene expression analyses of VHLmut/wt or TSC1/2mut/wt versus wild-type (WT) cells revealed transcriptomic alterations previously implicated in the transition to precancerous renal lesions. For example, the gene expression changes in VHLmut/wt cells were consistent with activation of the hypoxia response, associated, in part, with the Warburg effect. Knockdown of any remaining VHL mRNA using shRNA induced secondary expression changes, such as activation of NF?B and interferon pathways, that are fundamentally important in the development of RCC. We posit that this is a general pattern of hereditary cancer predisposition, wherein haploinsufficiency for VHL or TSC1/2, or potentially other tumor susceptibility genes, is sufficient to promote development of early lesions, while cancer results from inactivation of the remaining normal allele. The gene expression changes identified here are related to the metabolic basis of renal cancer and may constitute suitable targets for early intervention.

Abstract Germline loss‐of‐function BHD mutations cause cystic lung disease and hereditary pneumothorax, yet little is known about the impact of BHD mutations in the lung. Folliculin (FLCN), the product of the Birt–Hogg–Dube (BHD) gene, has been linked to altered cell–cell adhesion and to the AMPK and mTORC1 signaling pathways. We found that downregulation of FLCN in human bronchial epithelial (HBE) cells decreased the phosphorylation of ACC, a marker of AMPK activation, while downregulation of FLCN in small airway epithelial (SAEC) cells increased the activity of phospho‐S6, a marker of mTORC1 activation, highlighting the cell type–dependent functions of FLCN. Cell–cell adhesion forces were significantly increased in FLCN‐deficient HBE cells, consistent with prior findings in FLCN‐deficient human kidney‐derived cells. To determine how these altered cell–cell adhesion forces impact the lung, we exposed mice with heterozygous inactivation of Bhd (similarly to humans with germline inactivation of one BHD allele) to mechanical ventilation at high tidal volumes. Bhd+/− mice exhibited a trend (P = 0.08) toward increased elastance after 6 h of ventilation at 24 cc/kg. Our results indicate that FLCN regulates the AMPK and mTORC1 pathways and cell–cell adhesion in a cell type–dependent manner. FLCN deficiency may impact the physiologic response to inflation‐induced mechanical stress, but further investigation is required. We hypothesize that FLCN‐dependent effects on signaling and cellular adhesion contribute to the pathogenesis of cystic lung disease in BHD patients.

Genetic studies have shown that the tuberous sclerosis complex (TSC) 1–TSC2–mammalian target of Rapamycin (mTOR) and the Hippo–Yes-associated protein 1 (YAP) pathways are master regulators of organ size, which are often involved in tumorigenesis. The crosstalk between these signal transduction pathways in coordinating environmental cues, such as nutritional status and mechanical constraints, is crucial for tissue growth. Whether and how mTOR regulates YAP remains elusive. Here we describe a novel mouse model of TSC which develops renal mesenchymal lesions recapitulating human perivascular epithelioid cell tumors (PEComas) from patients with TSC. We identify that YAP is up-regulated by mTOR in mouse and human PEComas. YAP inhibition blunts abnormal proliferation and induces apoptosis of TSC1–TSC2-deficient cells, both in culture and in mosaic Tsc1 mutant mice. We further delineate that YAP accumulation in TSC1/TSC2-deficient cells is due to impaired degradation of the protein by the autophagosome/lysosome system. Thus, the regulation of YAP by mTOR and autophagy is a novel mechanism of growth control, matching YAP activity with nutrient availability under growth-permissive conditions. YAP may serve as a potential therapeutic target for TSC and other diseases with dysregulated mTOR activity.