Modulation of cell migration and invasiveness by tumor suppressor TSC2 in lymphangioleiomyomatosis

The loss of TSC2 function is associated with the pathobiology of lymphangioleiomyomatosis (LAM), which is characterized by the abnormal proliferation, migration, and differentiation of smooth muscle-like cells within the lungs. Although the etiology of LAM remains unknown, clinical and genetic evidence provides support for the neoplastic nature of LAM. The goal of this study was to determine the role of tumor suppressor TSC2 in the neoplastic potential of LAM cells. We show that primary cultures of human LAM cells exhibit increased migratory activity and invasiveness, which is abolished by TSC2 re-expression. We found that TSC2 also inhibits cell migration through its N-terminus, independent of its GTPase-activating protein activity. LAM cells show increased stress fiber and focal adhesion formation, which is attenuated by TSC2 re-expression. The small GTPase RhoA is activated in LAM cells compared with normal human mesenchymal cells. Pharmacologic inhibition of Rho activity abrogates LAM cell migration; RhoA activity was also abolished by TSC2 re-expression or TSC1 knockdown with specific siRNA. These data demonstrate that TSC2 controls cell migration through its N-terminus by associating with TSC1 and regulating RhoA activity, suggesting that TSC2 may play a critical role in modulating cell migration and invasiveness, which contributes to the pathobiology of LAM.     

Development of a Lymphangioleiomyomatosis Model by Endonasal Administration of Human TSC2(-/-) Smooth Muscle Cells in Mice

Lymphangioleiomyomatosis (LAM) is an interstitial lung disease characterized by invasion and proliferation of abnormal smooth muscle (ASM) cells in lung parenchyma and axial lymphatics. LAM cells bear mutations in tuberous sclerosis (TSC) genes. TSC2(-/-) ASM cells, derived from a human renal angiomyolipoma, require epidermal growth factor (EGF) for proliferation. Blockade of EGF receptors (EGFR) causes cell death. TSC2(-/-) ASM cells, previously labeled with PKH26-GL dye, were endonasally administered to 5-week-old immunodeficient female nude mice, and 4 or 26 weeks later anti-EGFR antibody or rapamycin was administered twice a week for 4 consecutive weeks. TSC2(-/-) ASM cells infiltrated lymph nodes and alveolar lung walls, causing progressive destruction of parenchyma. Parenchymal destruction was efficiently reversed by anti-EGFR treatment and partially by rapamycin treatment. Following TSC2(-/-) ASM cell administration, lymphangiogenesis increased in lungs as indicated by more diffuse LYVE1 expression and high murine VEGF levels. Anti-EGFR antibody and rapamycin blocked the increase in lymphatic vessels. This study shows that TSC2(-/-) ASM cells can migrate and invade lungs and lymph nodes, and anti-EGFR antibody is more effective than rapamycin in promoting lung repair and reducing lymphangiogenesis. The development of a model to study metastasis by TSC cells will also help to explain how they invade different tissues and metastasize to the lung.     

Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex

Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While energy depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMPK) by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition by hypoxia is not known. Here we show that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex and the hypoxia-inducible gene REDD1/RTP801. Disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets S6K and 4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIF). In contrast to energy depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and REDD1 overexpression is sufficient to down-regulate S6K phosphorylation in a TSC1/TSC2-dependent manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia.     

Targeting the Phosphatidylinositol 3-Kinase Pathway in Airway Smooth Muscle

The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a critical role in regulating cell growth, proliferation, survival, and motility. Structural alterations, e.g. airway remodeling, in asthma and chronic obstructive pulmonary disease (COPD) are associated with increased airway smooth muscle (ASM) cell growth and proliferation due to the frequent stimulation of ASM by inflammatory mediators, contractile agonists, and growth factors. The critical role of the PI3K signaling pathway in regulating ASM cell growth and proliferation is well established. However, recent discovery of the tumor suppressor proteins tuberous sclerosis complex 1 (TSC1) and TSC2, also known as hamartin and tuberin, as downstream effectors of PI3K and upstream regulators of the mammalian target of rapamycin (mTOR) and S6 kinase 1(S6K1) shed a new light on the PI3K signaling cascade in regulating cell growth and proliferation. The activity of TSC1/TSC2 is regulated by growth factors, nutrients, and energy; thus, TSC1/TSC2 serves as a signaling module for protein translational regulation, cell cycle progression, and cell size, which are key events controlling cell growth and proliferation. This article highlights the potential contribution of the PI3K-TSC1/TSC2-mTOR/S6K1 pathway in smooth muscle remodeling. Pharmacologic manipulation of this signaling pathway could have a major impact on treatment of asthma and COPD.     

Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis

Pulmonary lymphangioleiomyomatosis (LAM) is a destructive lung disease characterized by a diffuse hamartomatous proliferation of smooth muscle cells (LAM cells) in the lungs. Pulmonary LAM can occur as an isolated form (sporadic LAM) or in association with tuberous sclerosis complex (TSC) (TSC-LAM), a genetic disorder with autosomal dominant inheritance with various expressivity resulting from mutations of either the TSC1 or TSC2 gene. We examined mutations of both TSC genes in 6 Japanese patients with TSC-LAM and 22 patients with sporadic LAM and identified six unique and novel mutations. TSC2 germline mutations were detected in 2 (33.3%) of 6 patients with TSC-LAM and TSC1 germline mutation in 1 (4.5%) of 22 sporadic LAM patients. In accordance with the tumor-suppressor model, loss of heterozygosity (LOH) was detected in LAM cells from 3 of 4 patients with TSC-LAM and from 4 of 8 patients with sporadic LAM. Furthermore, an identical LOH or two identical somatic mutations were demonstrated in LAM cells microdissected from several tissues, suggesting LAM cells can spread from one lesion to another. Our results from Japanese patients with LAM confirmed the current concept of pathogenesis of LAM: TSC-LAM has a germline mutation but sporadic LAM does not; sporadic LAM is a TSC2 disease with two somatic mutations; and a variety of TSC mutations causes LAM. However, our study indicates that a fraction of sporadic LAM can be a TSC1 disease; therefore, both TSC genes should be examined, even for patients with sporadic LAM. Received: August 30, 2001 / Accepted: November 2, 2001     

Renal angiomyolipomas from patients with sporadic lymphangiomyomatosis contain both neoplastic and non-neoplastic vascular structures

Renal angiomyolipomas are highly vascular tumors that occur sporadically, in women with pulmonary lymphangiomyomatosis (LAM), and in tuberous sclerosis complex (TSC). The goal of this study was to determine whether the distinctive vessels of angiomyolipomas are neoplastic or reactive. We studied angiomyolipomas with loss of heterozygosity (LOH) in the TSC2 region of chromosome 16p13 from patients with LAM. We found that angiomyolipomas contain five morphologically distinct vessel types: cellular, collagenous, hemangiopericytic, glomeruloid, and aneurysmatic. Using laser capture microdissection, we determined that four of the vessel types have TSC2 LOH and are therefore neoplastic. One vessel type, collagenous vessels, did not have LOH, and is presumably reactive. Recently, activation of S6 Kinase and its target S6 ribosomal protein (S6) was demonstrated in cells lacking TSC2 expression. We found that angiomyolipoma vessel types in which LOH were detected were immunoreactive with anti-phospho-S6 antibodies. Angiomyolipoma cells without LOH, including the endothelial component of the vessels, were not immunoreactive. To our knowledge, angiomyolipomas are the first benign vascular tumor in which the vascular cells, rather than the stromal cells, have been found to be neoplastic. Angiomyolipomas appear to reflect novel vascular mechanisms that may be the result of activation of cellular pathways involving S6 Kinase.     

Tuberin Regulates E-Cadherin Localization: Implications in Epithelial-Mesenchymal Transition

The tuberous sclerosis complex 2 (TSC2) gene encodes the protein tuberin, which functions as a key negative regulator of both mammalian target of rapamycin (mTOR) C1-dependent cell growth and proliferation. Loss-of-function mutations of TSC2 result in mTORC1 hyperactivity and predispose individuals to both tuberous sclerosis and lymphangioleiomyomatosis. These overlapping diseases have in common the abnormal proliferation of smooth muscle-like cells. Although the origin of these cells is unknown, accumulating evidence suggests that a metastatic mechanism may be involved, but the means by which the mTOR pathway contributes to this disease process remain poorly understood. In this study, we show that tuberin regulates the localization of E-cadherin via an Akt/mTORC1/CLIP170-dependent, rapamycin-sensitive pathway. Consequently, Tsc2(−/−) epithelial cells display a loss of plasma membrane E-cadherin that leads to reduced cell-cell adhesion. Under confluent conditions, these cells detach, grow in suspension, and undergo epithelial-mesenchymal transition (EMT) that is marked by reduced expression levels of both E-cadherin and occludin and increased expression levels of both Snail and smooth muscle actin. Functionally, the Tsc2(−/−) cells demonstrate anchorage-independent growth, cell scattering, and anoikis resistance. Human renal angiomyolipomas and lymphangioleiomyomatosis also express markers of EMT and exhibit an invasive phenotype that can be interpreted as consistent with EMT. Together, these results suggest a novel relationship between TSC2/mTORC1 and the E-cadherin pathways and implicate EMT in the pathogenesis of tuberous sclerosis complex-related diseases.Mutation of the TSC2 gene gives rise to the autosomal dominant disorder, tuberous sclerosis complex (TSC), that is characterized by “hamartomas” in the brain, kidney, skin, heart, and lung.¹ Genetic and biochemical analyses have highlighted the role of the TSC2 protein, tuberin, in concert with its interacting partner, TSC1 (hamartin), in negatively regulating mammalian target of rapamycin (mTOR) C1 by promoting the hydrolysis of Rheb-GTP.² Multiple factors including growth factors, energy, and oxygen availability converge on the TSC1/TSC2 complex to modulate mTORC1 activity.³ The best characterized function of mTORC1 is the promotion of protein synthesis through its downstream targets, p70S6K and 4E-BP1. In turn, p70S6K mediates phosphorylation of IRS-1 to inhibit phosphatidylinositol 3-kinase/AKT signaling in a negative feedback mechanism.⁴ Consequently, the loss of TSC1 or TSC2 leads to an “overgrowth” phenotype with increased cell size and proliferation, characteristic of the hamartomas seen in tuberous sclerosis. However, many of the clinical and pathological features of TSC remain unexplained by our current understanding of the function of these genes.One such example is the lymphangioleiomyomatosis (LAM) that occurs in ∼40% of females diagnosed with TSC.⁵ The sporadic form of LAM is also associated with mutation of the TSC2 gene.⁶ LAM is a unique disease that affects women of childbearing age and is characterized by the infiltration of smooth muscle-like cells in the lung interstitium, which eventually leads to the progressive loss of pulmonary function and cystic destruction of the lung.⁷ Although LAM is not exclusive to the lungs and can involve the axial lymphatic system and other organs, mortality due to respiratory failure takes place within 8 to 15 years after diagnosis.⁸ LAM and angiomyolipoma (AML) are classified as perivascular epithelioid cell neoplasms), that is, defined as “mesenchymal tumors composed of histologically and immunohistochemically distinctive perivascular epithelioid cells.”⁹ These tumors are characterized histologically by their epithelioid appearance and their physical relationship to blood vessels.⁹ The abnormal cells display a distinct immunophenotype that includes the expression of melanocytic (eg, gp100) and smooth muscle markers (eg, smooth muscle actin) but not epithelial antigens. Because perivascular epithelioid cells have no normal anatomical counterpart, the origin of these tumors remains elusive.One current theory suggests that pulmonary LAM is the result of a metastatic process in which certain precursor cells migrate to the lung and invade the parenchyma.¹⁰ Indeed, primary LAM cells have been shown to be invasive in vitro,¹¹ and these cells have been identified in body fluids, including blood and urine, suggesting that LAM cells are capable of detaching from their primary sites and entering the circulation.¹² Further evidence in support of this hypothesis comes from the observations that pulmonary recurrences of LAM after lung transplant contain cells that originated from the organ recipients,^13,14 and common patterns of TSC2 mutation have been identified in LAM and lymph node disease from the same individual.¹⁵ Finally, the unique immunophenotype in LAM does not reflect an epithelial nor mesenchymal origin but rather a mixture of “epithelioid” and “spindle” cells that is suggestive of a variable differentiation pattern.¹⁶ Nonetheless, both populations of cells are believed to be clonally derived. The spindle cells are reported to be more proliferative and express smooth muscle actin, whereas the epithelioid cells express the melanocytic markers (eg, HMB-45) and are less mitotically active. Collectively, the observed behavior of LAM cells with respect to their infiltrative growth pattern, metastatic potential, and altered cell differentiation is reminiscent of cells undergoing epithelial-mesenchymal transition (EMT).^17,18 Here, we propose that LAM may be a manifestation of EMT and show that human AML and LAM do indeed express markers of EMT.One of the critical steps driving EMT is the repression of E-cadherin, resulting in loss of cell-cell adhesion. E-cadherin is expressed in most epithelial cells in which adherens junctions are formed to create the multicellular organization important for the formation and maintenance of bodily compartments. Structural studies highlight the essential role of calcium in the progressive cis-dimerization of the cadherin ectodomain, leading to the formation of a trans-dimer “zipper” between multiple cis-dimers to form cell adhesion.¹⁹ In this study, we provide evidence that E-cadherin membrane localization is regulated by the Akt/mTORC1 pathway such that the loss of TSC2 leads to significant reduction in membrane E-cadherin. Consequently, cells deficient in Tsc2 are less adhesive and more prone to detach and to undergo EMT. Our findings highlight a novel functional link between tuberin and E-cadherin activity that may contribute to the pathogenesis of TSC and related disorders.     

AMPA type glutamate receptor mediates neurotransmission at turtle vestibular calyx synapse

AMPK is a serine/threonine protein kinase, which serves as an energy sensor in all eukaryotic cell types. Published studies indicate that AMPK activation strongly suppresses cell proliferation in non-malignant cells as well as in tumour cells. These actions of AMPK appear to be mediated through multiple mechanisms including regulation of the cell cycle and inhibition of protein synthesis, de novo fatty acid synthesis, specifically the generation of mevalonate as well as other products downstream of mevalonate in the cholesterol synthesis pathway. Cell cycle regulation by AMPK is mediated by up-regulation of the p53–p21 axis as well as regulation of TSC2–mTOR (mammalian target of rapamycin) pathway. The AMPK signalling network contains a number of tumour suppressor genes including LKB1, p53, TSC1 and TSC2, and overcomes growth factor signalling from a variety of stimuli (via growth factors and by abnormal regulation of cellular proto-oncogenes including PI3K, Akt and ERK). These observations suggest that AMPK activation is a logical therapeutic target for diseases rooted in cellular proliferation, including atherosclerosis and cancer. In this review, we discuss about exciting recent advances indicating that AMPK functions as a suppressor of cell proliferation by controlling a variety of cellular events in normal cells as well as in tumour cells.     

Regulation of T-cell survival and mitochondrial homeostasis by TSC1

The mammalian target of rapamycin (mTOR) is a key regulator of cell growth and metabolism. It associates with multiple proteins and forms two distinct signaling complexes, mTORC1 and mTORC2. Accumulating evidence has revealed critical roles for intact mTOR signaling during T-cell activation and responses to microbial infection. However, the importance of mTOR regulation in T cells has yet to be explored. The TSC1/TSC2 complex has been shown to inhibit mTORC1 signaling in cell line models. We show here that deletion of TSC1 in the murine T-cell lineage results in a dramatic reduction of the peripheral T-cell pool, correlating with increased cell death. While mTORC1 is constitutively activated, mTORC2 signaling, reflected by Akt phosphorylation and activity, is decreased in TSC1-deficient T cells. Furthermore, TSC1-deficient T cells contain elevated reactive oxygen species (ROS) and exhibit decreased mitochondrial content and membrane potential, which is correlated with the activation of the intrinsic death pathway. Overall, our results demonstrate that TSC1 differentially regulates mTORC1 and mTORC2 activity, promotes T-cell survival, and is critical for normal mitochondrial homeostasis in T cells.     

Pathogenesis of multifocal micronodular pneumocyte hyperplasia and lymphangioleiomyomatosis in tuberous sclerosis and association with tuberous sclerosis genes TSC1 and TSC2

Tuberous sclerosis (TSC) is a rare, genetically determined disorder / familial tumor syndrome, currently diagnosed using specific clinical criteria proposed by Gomez, including the presence of multiorgan hamartomas. Pulmonary involvement in TSC is well known as pulmonary lymphangioleiomyomatosis (LAM), which has an incidence of 1-2.3% in TSC patients. LAM has immunohistochemical expression of both smooth-muscle actin and a monoclonal antibody specific for human melanoma, HMB-45. It has recently been reported that multifocal micronodular pneumocyte hyperplasia (MMPH) associated with TSC should be considered as a distinct type of lung lesion, whether it occurs with or without LAM. Two predisposing genes have been found in families affected by TSC; approximately half of the families show linkage to TSC1 at 9q34.3, and the other half show linkage to TSC2 at 16p13.3. TSC genes are considered to be tumor suppressor genes, and mutations in them may lead to abnormal differentiation and proliferation of cells. Tuberin, the TSC2 gene product, has recently been found to be expressed in LAM and MMPH. In this article we discuss the histogenesis and genetic abnormalities of neoplastic lesions associated with TSC, and we review the current understanding of the pathogenesis of pulmonary hamartomatous lesions such as LAM and MMPH in TSC.     

Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling

Tuberous sclerosis complex (TSC) is a genetic disease caused by mutation in either TSC1 or TSC2. The TSC1 and TSC2 gene products form a functional complex and inhibit phosphorylation of S6K and 4EBP1. These functions of TSC1/TSC2 are likely mediated by mTOR. Here we report that TSC2 is a GTPase-activating protein (GAP) toward Rheb, a Ras family GTPase. Rheb stimulates phosphorylation of S6K and 4EBP1. This function of Rheb is blocked by rapamycin and dominant-negative mTOR. Rheb stimulates the phosphorylation of mTOR and plays an essential role in regulation of S6K and 4EBP1 in response to nutrients and cellular energy status. Our data demonstrate that Rheb acts downstream of TSC1/TSC2 and upstream of mTOR to regulate cell growth.     

Frequent [corrected] hyperphosphorylation of ribosomal protein S6 [corrected] in lymphangioleiomyomatosis-associated angiomyolipomas.

Lymphangioleiomyomatosis is a progressive lung disease characterized by a diffuse proliferation of pulmonary smooth muscle cells and cystic degeneration. Lymphangioleiomyomatosis can occur either independently of other disease or in association with tuberous sclerosis complex, a tumor-suppressor gene syndrome caused by mutations that inactivate either TSC1 or TSC2. TSC2 mutations and loss of heterozygosity have been identified in sporadic lymphangioleiomyomatosis-associated angiomyolipomas, thus implicating the TSC/Ras homolog-enriched in brain (Rheb)/mammalian target of Rapamycin (mTOR)/p70 S6 kinase signaling pathway in their pathogenesis. This study was undertaken to determine whether the mTOR/p70 S6 kinase signaling pathway is activated in lymphangioleiomyomatosis-associated angiomyolipomas lacking TSC1/TSC2 loss of heterozygosity. Phospho-ribosomal protein S6 (Ser235/236) immunohistochemistry was performed on five lymphangioleiomyomatosis-associated angiomyolipomas, two matched lymphangioleiomyomatosis pulmonary samples, and three sporadic angiomyolipomas. TSC1/TSC2 loss of heterozygosity was previously excluded in these angiomyolipomas. Moderate or strong phospho-ribosomal protein S6 immunoreactivity was found in all lymphangioleiomyomatosis-associated and sporadic angiomyolipomas, suggesting a high incidence of mTOR/p70 S6 kinase signaling pathway activation despite a lack of TSC1/TSC2 loss of heterozygosity. Focally positive phospho-S6 staining was also evident in both lymphangioleiomyomatosis pulmonary samples. We hypothesized that this S6 hyperphosphorylation could reflect mutational activation of Rheb or Rheb-like protein (RhebL1), Ras family members which directly activate mTOR. Mutational analysis performed on DNA from these eight angiomyolipomas plus five additional sporadic angiomyolipomas did not reveal mutations in exons 3 and 4 (homologous sites of Ras activating mutations) of either Rheb or RhebL1. These data suggest that activation of the Rheb/mTOR/p70 S6 kinase pathway is related to the pathogenesis of lymphangioleiomyomatosis-associated and sporadic angiomyolipomas lacking TSC1/TSC2 loss of heterozygosity. This high incidence of mTOR signaling pathway activation suggests that treatment with mTOR inhibitors, such as Rapamycin, may benefit patients with angiomyolipomas independent of the detection of TSC1/TSC2 loss of heterozygosity.     

Selected case from the Arkadi M. Rywlin International Pathology Slide Seminar: Sporadic lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a systemic, progressive, and fatal condition affecting almost exclusively women in their reproductive years. LAM most often occurs as a sporadic disease, but also occurs in women with tuberous sclerosis complex (TSC) (syndromic LAM). There are no pathologic differences between sporadic and syndromic LAM. Sporadic LAM is a rare disease with prevalence of approximately 1 to 2 cases per million women in the United States and among populations of white descent, and is even rarer among Asian and African individuals. Syndromic LAM affects 4% to 5% of women with TSC. Sporadic LAM is often found also in association with renal angiomyolipoma, the most common sign of TSC, but LAM associated with angiomyolipoma does not define TSC. Although LAM is not diagnostic for TSC either in isolation or in association with angiomyolipoma, still it is considered by some researchers as an incomplete expression (forme fruste) of TSC. LAM may involve the lungs and the axial lymphatics and lymph nodes of the thorax and retroperitoneum. In sporadic LAM, thoracic, intraabdominal, and cervical lymph nodes can be involved with or without lung involvement. The diagnosis of LAM is often delayed. A case of LAM in a young lady, which was complicated with pleural and peritoneal chylous effusions, is presented. The diagnosis was first made on a retroperitoneal lymph node biopsy. The patient had a prolonged prior history of respiratory problems owing to lung involvement, and eventually died 2 years after diagnosis. Focus on the clinicopathologic diagnosis of TSC is also made.     

Lethality of Drosophila lacking TSC tumor suppressor function rescued by reducing dS6K signaling

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in one of two tumor suppressor genes, TSC1 and TSC2. Here, we show that absence of Drosophila Tsc1/2 leads to constitutive dS6K activation and inhibition of dPKB, the latter effect being relieved by loss of dS6K. In contrast, the dPTEN tumor suppressor, a negative effector of PI3K, has little effect on dS6K, but negatively regulates dPKB. More importantly, we demonstrate that reducing dS6K signaling rescues early larval lethality associated with loss of dTsc1/2 function, arguing that the S6K pathway is a promising target for the treatment of TSC.     

Metastasis of benign tumor cells in tuberous sclerosis complex

Lymphangiomyomatosis (LAM) is a life-threatening lung disease affecting almost exclusively young women. Histologically, LAM is characterized by the diffuse, bilateral proliferation of abnormal smooth muscle cells and cystic degeneration of the lung parenchyma. LAM can occur as an isolated disorder (sporadic LAM), or in women with tuberous sclerosis complex (TSC-LAM). Patients with both sporadic LAM and TSC-LAM often have benign renal angiomyolipomas. The smooth muscle cells within the angiomyolipomas are very similar to the smooth muscle cells in pulmonary LAM. Genetic data suggest that pulmonary LAM is the result of a highly unusual disease mechanism: the metastasis of benign cells. If LAM is the result of metastasis, it is remarkable that the metastasis occurs in women, but not in men. In this review, I discuss the genetic data supporting this metastatic model for LAM. The implications of the model for the functions of the TSC1 and TSC2 gene products, hamartin and tuberin, respectively, will also be considered. Hamartin and tuberin may play functional roles in the suppression of cell migration and/or metastasis, possibly through their regulation of the small GTPase Rho.     

一磷酸腺苷激酶在肿瘤细胞增殖及自噬中的研究进展

一磷酸腺苷激酶(AMPK)以异质三联体形式存在于所有真核细胞内,是维持机体能量稳态的感受器。AMPK的激活主要来自avicinD、LKB1等,而AMPK主要激活底物是TSC2、p53和p27kip1,其中TSC2是mTOR的负性调控元件,它的激活可以间接地抑制细胞增殖;p53也可抑制mTOR通路,中断细胞增殖;活化的p27kip1可以诱导自噬,促进细胞死亡,抑制肿瘤生长。这些都表明AMPK在肿瘤细胞增殖和自噬中发挥作用。由于在许多肿瘤细胞中可以发现失活的AMPK,因而研究AMPK与肿瘤发展及细胞自噬的关系,将可能为肿瘤治疗找到新靶点。     

Fetal Brain Lesions in Tuberous Sclerosis Complex: TORC1 Activation and Inflammation

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in either the TSC1 or TSC2 genes and characterized by developmental brain abnormalities. We defined the spectrum of brain abnormalities in fetal TSC brain ranging from 23 to 38 gestational weeks. We hypothesized (i) prenatal activation of the target‐of‐rapamycin complex 1 (TORC1) signaling pathway; and (ii) activation of inflammatory pathways in fetal brain lesions. Immunocytochemical analysis of cortical tubers, as well as subependymal lesions in all cases confirmed the cell‐associated activation of the TORC1 signaling pathway in both the cortical tubers and subependymal lesions (including a congenital subependymal giant cell astrocytoma) with expression of pS6, p4EBP1 and c‐myc proteins, as well as of p70 S6 kinase 1. The lesions contained macrophages and T‐lymphocytes; giant cells within the lesions expressed inflammatory response markers including major histocompatibility complex class I and II, Toll‐like receptors (TLR) 2 and 4 and receptor for advanced glycation end products (RAGE). These observations indicate that brain malformations in TSC are likely a consequence of increased TORC1 activation during embryonic brain development. We also provide evidence supporting the possible immunogenicity of giant cells and the early activation of inflammatory pathways in TSC brain.     

Tissue-specific renin-angiotensin system in pulmonary lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM), a multisystem disease found in middle-aged women, is characterized by cystic lung destruction and abdominal tumors (e.g., angiomyolipomas, lymphangioleimyomas), resulting from proliferation of abnormal-appearing, smooth muscle-like cells (LAM cells). The LAM cells, in combination with other cells, form nodular structures within the lung interstitium and in the walls of the cysts. LAM cells contain mutations in the tuberous sclerosis complex TSC1 and/or TSC2 genes, which lead to dysregulation of the mammalian target of rapamycin, affecting cell growth and proliferation. Proliferation and migration of vascular smooth muscle cells and production of angiogenic factors are regulated, in part, by angiotensin II. To determine whether a LAM-specific renin-angiotensin system might play a role in the pathogenesis of LAM, we investigated the expression of genes and gene products of this system in LAM nodules. mRNA for angiotensinogen was present in RNA isolated by laser-captured microdissection from LAM nodules. Angiotensin I-converting enzyme and chymase-producing mast cells were present within the LAM nodules. We detected renin in LAM cells, as determined by the presence of mRNA and immunohistochemistry. Angiotensin II type 1 and type II receptors were identified in LAM cells by immunohistochemistry and immunoblotting of microdissected LAM nodules. Angiotensin II is localized in cells containing alpha-smooth muscle actin (LAM cells). A LAM-specific renin-angiotensin system appears to function within the LAM nodule as an autocrine system that could promote LAM cell proliferation and migration, and could represent a pharmacologic target.     

Osteoprotegerin Contributes to the Metastatic Potential of Cells with a Dysfunctional TSC2 Tumor-Suppressor Gene

In addition to its effects on bone metabolism, osteoprotegerin (OPG), a soluble member of the tumor necrosis factor family of receptors, promotes smooth muscle cell proliferation and migration and may act as a survival factor for tumor cells. We hypothesized that these cellular mechanisms of OPG may be involved in the growth and proliferation of lymphangioleiomyomatosis (LAM) cells, abnormal smooth muscle-like cells with mutations in one of the tuberous sclerosis complex tumor-suppressor genes (TSC1/TSC2) that cause LAM, a multisystem disease characterized by cystic lung destruction, lymphatic infiltration, and abdominal tumors. Herein, we show that OPG stimulated proliferation of cells cultured from explanted LAM lungs, and selectively induced migration of LAM cells identified by the loss of heterozygosity for TSC2. Consistent with these observations, cells with TSC2 loss of heterozygosity expressed the OPG receptors, receptor activator of NF-κB ligand, syndecan-1, and syndecan-2. LAM lung nodules showed reactivities to antibodies to tumor necrosis factor–related apoptosis-inducing ligand, receptor activator of NF-κB ligand, syndecan-1, and syndecan-2. LAM lung nodules also produced OPG, as shown by expression of OPG mRNA and colocalization of reactivities to anti-OPG and anti-gp100 (HMB45) antibodies in LAM lung nodules. Serum OPG was significantly higher in LAM patients than in normal volunteers. Based on these data, it appears that OPG may have tumor-promoting roles in the pathogenesis of lymphangioleiomyomatosis, perhaps acting as both autocrine and paracrine factors.Osteoprotegerin (OPG; TNFRSF11B), a soluble member of the tumor necrosis factor (TNF) receptor family, is best known as a regulator of bone metabolism that promotes bone formation by inhibiting osteoclast development, thus protecting against osteoporosis.^1,2 OPG, acting as a decoy receptor, binds to receptor activator of NF-κB ligand (RANKL), preventing the interaction of RANKL with its receptor RANK, resulting in the inhibition of osteoclast activation and bone resorption. Polymorphisms in the OPG gene have been linked to development of osteoporosis.^3–6 Patients with juvenile Paget disease, a rare inherited disease affecting children, show increased bone turnover, leading to skeletal deformity. Mutations in the OPG gene determine the severity of the juvenile Paget disease phenotype,⁷ with the loss of the entire gene or mutations leading to the loss of OPG structure resulting in a severe phenotype.More recently, the role of OPG in vascular cell biological characteristics has been studied. OPG knockout mice have both severe osteoporosis and significant arterial calcification,⁸ suggesting that OPG plays a protective role against arterial calcification in mice. OPG serum levels are associated with the severity of cardiovascular disease in humans.^9–11 OPG levels may be higher either directly, through a proatherosclerotic effect, or indirectly, because of an incomplete compensatory mechanism in which increases in serum OPG levels are seen as a response to RANKL activity.^9–11 This compensatory effect may also be invoked to explain high serum levels of OPG, sometimes seen in subjects with osteoporosis.¹²Vascular smooth muscle cells express OPG, and aortic smooth muscle cells proliferate in response to OPG.¹³ OPG induced both the proliferation and migration of pulmonary artery smooth muscle cells¹⁴ and human microvascular endothelial cells.¹⁵ The effects of OPG on human microvascular endothelial cells were mediated through integrins α_Vβ₃ and α_Vβ₅ and the extracellular signal–regulated kinase 1/2. OPG can also stimulate monocyte migration; this effect was shown to involve syndecans and phosphatidylinositol-3-OH kinase/Akt, protein kinase C, and tyrosine kinases.¹⁶OPG also has roles in tumor development and metastasis.^17,18 OPG can bind TNF-related apoptosis-inducing ligand (TRAIL), blocking TRAIL’s apoptotic effects on cancer cells.^19–23 Serum OPG levels may be higher in cancer patients compared with healthy controls, and levels may correlate with cancer stage.^24–27 Tumor growth and metastasis are also supported by OPG’s promotion of endothelial cell survival and angiogenesis.^28,29 Interestingly, some malignant breast cancer tumors show endothelial OPG expression, whereas neighboring normal endothelium does not express high levels of the protein.²⁹Lymphangioleiomyomatosis (LAM) cells are abnormal neoplastic smooth muscle-like cells, with mutations in one of two tuberous sclerosis complex tumor-suppressor genes (TSC1 or TSC2). TSC1 (encoding hamartin) and TSC2 (tuberin) form a complex that regulates the serine/threonine kinase, mammalian target of rapamycin.³⁰ Mutations in TSC1/TSC2 lead to uncontrolled mammalian target of rapamycin activity, resulting in increased cell proliferation and size.³⁰ These LAM cells form nodules covered with type II pneumocytes, with surrounding areas of cystic destruction in the lungs of patients with LAM. In addition to the cystic destruction of lung parenchyma, LAM, a rare multisystem disease affecting women,³¹ is characterized by lymphatic abnormalities and abdominal tumors (eg, angiomyolipomas). LAM cells can metastasize, as LAM cells from lung lesions and angiomyolipomas in the same patient have the same TSC2 mutation.³² Consistent with their migratory behavior, LAM cells have been isolated from blood and other body fluids of patients with LAM.^33,34 LAM cells have characteristics of both smooth muscle cells, such as reactivity with antibodies to smooth muscle actin and desmin, and of melanocytes, with reactivity with HMB45,³⁵ an antibody recognizing gp100, a melanosomal protein.^36–38In this study, we investigated the effect of OPG on the neoplastic smooth muscle cell-like LAM cells. OPG promoted proliferation of cells grown from explanted LAM lungs and specifically induced LAM cell migration. Three OPG receptors, RANKL, syndecan-1, and syndecan-2, were detected on LAM cells and LAM lung nodules. Furthermore, LAM cells produced OPG, and OPG levels were elevated in serum from patients with LAM compared with healthy volunteers, suggesting both autocrine and paracrine effects of OPG in LAM.