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     

Mutational analysis of the tuberous sclerosis gene TSC2 in patients with pulmonary lymphangioleiomyomatosis

Pulmonary lymphangioleiomyomatosis (LAM) is a rare disorder limited almost exclusively to women of reproductive age. LAM affects about 5% of women with tuberous sclerosis complex (TSC). LAM also occurs in women who do not have TSC (sporadic LAM). TSC is a tumour suppressor gene syndrome characterised by seizures, mental retardation, and tumours in the brain, heart, and kidney. Angiomyolipomas, which are benign tumours with smooth muscle, fat, and dysplastic vascular components, are the most common renal tumour in TSC. Renal angiomyolipomas also occur in 63% of sporadic LAM patients. We recently found that 54% of these angiomyolipomas have TSC2 loss of heterozygosity, leading to the hypothesis that sporadic LAM is genetically related to TSC. In this study, we screened DNA from 21 women with sporadic LAM for mutations in all 41 exons of TSC2. Twelve of the patients had known renal angiomyolipomas. No TSC2 mutations were detected. We did find three silent TSC2 polymorphisms. We conclude that patients with sporadic LAM, including those with renal angiomyolipomas, do not have a high frequency of germline mutations in the coding region of TSC2.     

The TSC-2 product tuberin is expressed in lymphangioleiomyomatosis and angiomyolipoma

AIMS: Lymphangioleiomyomatosis is categorized by proliferation of abnormal smooth muscle cells (LAM cells) in the lungs and lymphatics and the presence of angiomyolipomas. Recently mutations in the tuberous sclerosis complex-2 gene (TSC-2) have been described in LAM cells and angiomyolipomas. The TSC-2 protein tuberin is a tumour suppressor and its loss may result in cellular proliferation. We used immunohistochemistry to test the hypothesis that uncontrolled cellular proliferation in lymphangioleiomyomatosis is the result of reduced tuberin protein expression. METHODS AND RESULTS: Tissue from normal lung, normal kidney, lymphangioleiomyomatosis and angiomyolipomas was immunostained with three separate anti-tuberin antibodies. Tuberin staining in normal tissues was similar to that previously described. Surprisingly, tuberin was strongly expressed in the LAM cells of all cases of lymphangioleiomyomatosis and angiomyolipoma at a greater level than in normal smooth muscle cells. The perivascular cells of angiomyolipomas, however, did not stain for tuberin. CONCLUSIONS: Our results suggest that a loss of tuberin protein in LAM cells is not the cause of the cellular proliferation seen in lymphangioleiomyomatosis. Lymphangioleiomyomatosis may result either from the expression of a mutant tuberin with abnormal function, as a result of mutations in functionally related proteins, or from more than one mechanism.     

Loss of tuberin in both subependymal giant cell astrocytomas and angiomyolipomas supports a two-hit model for the pathogenesis of tuberous sclerosis tumors.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation, and tumors of skin, brain, heart, and kidney. In this study, we focused on two of the most frequent tumors in TSC patients, renal angiomyolipomas and subependymal giant cell astrocytomas (SEGAs). Two questions were addressed. First, is loss of tuberin, the product of the TSC2 gene, seen in both renal and central nervous system tumors from TSC patients? Second, when loss of tuberin occurs, does it affect each of the cell types seen in these tumors? We used a loss of heterozygosity approach to identify tumors from TSC2 patients. We found loss of tuberin immunostaining in the spindle and epithelioid cells but not in the giant cells of six TSC2 SEGAs. We also found loss of tuberin immunostaining in all three cell types (smooth muscle, fat, and vessels) of six TSC2 angiomyolipomas. Chromosome 16p13 loss of heterozygosity occurred in both spindle and epithelioid cells of a SEGA and in smooth muscle and fat but not the vessels of two angiomyolipomas. These results support a two-hit tumor suppressor model for the pathogenesis of SEGAs and angiomyolipomas. The vascular elements of angiomyolipomas and the giant cells of SEGAs may be reactive rather than neoplastic.     

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.     

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.     

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.     

The expression of hamartin, the product of the TSC1 gene, in normal human tissues and in TSC1- and TSC2-linked angiomyolipomas.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation, and hamartomatous tumors in multiple organs, including subependymal giant cell astrocytomas, cardiac rhabdomyomas, and renal angiomyolipomas. Mutations in two genes are associated with TSC: TSC1, which was cloned in 1997, and TSC2, which was cloned in 1993. We report here the expression of hamartin, the product of the TSC1 gene, in normal human tissues and in renal angiomyolipomas from TSC1- and TSC2-linked patients. By Western blot analysis, hamartin is strongly expressed in brain, kidney, and heart, all of which are frequently affected in TSC. By immunohistochemical analysis, the expression pattern of hamartin in normal human tissues was almost identical to that of tuberin, the product of the TSC2 gene. This is consistent with the recent finding that tuberin and hamartin interact and with the clinical similarity between TSC1- and TSC2-linked disease. Strong hamartin expression was seen in cortical neurons, renal tubular epithelial cells, pancreatic islet cells, bronchial epithelial cells, and pulmonary macrophages. Hamartin was also expressed in endocrine tissues, including islet cells of the pancreas, follicular cells of the thyroid, and the zona reticularis of the adrenal cortex. In eight angiomyolipomas from a TSC1-linked patient, no hamartin expression was detected, whereas tuberin, the product of the TSC2 gene, was expressed. In 19 angiomyolipomas from a TSC2-linked patient, in whose angiomyolipomas loss of tuberin expression had previously been shown, hamartin expression was present. These data suggest that tuberin and hamartin immunoreactivity can distinguish tumors with underlying TSC1 mutations from those with TSC2 mutations. This differentiation might have diagnostic implications.     

β-Catenin is a useful adjunct immunohistochemical marker for the diagnosis of pulmonary lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare multisystem disease leading to cystic destruction of the lung parenchyma and is associated with abnormal smooth muscle proliferation affecting airways, lymphatics, and blood vessels. LAM occurs sporadically or in association with the tuberous sclerosis complex (TSC). Recent evidence demonstrates the role of aberrant β-catenin signaling in TSC. To further understand the pathogenesis of LAM and to examine the diagnostic usefulness of β-catenin, we examined protein expression in 28 pulmonary LAM cases and 10 cases of renal angiomyolipoma resected from patients with sporadic LAM. Immunohistochemical analysis was performed for established markers of LAM cells (HMB45, estrogen receptor [ER]-α, and progesterone receptor [PR]) and β-catenin. All LAM cases were positive for β-catenin and demonstrated high specificity with overall immunoreactivity superior to HMB45, ER-α, and PR. Similar expression was demonstrated in renal angiomyolipoma. Our results indicate that β-catenin is a useful marker of LAM and may be clinically useful in the diagnostic setting.     

Matrix proteoglycans and remodelling of interstitial lung tissue in lymphangioleiomyomatosis

The interstitial lung disease lymphangioleiomyomatosis (LAM) is characterized by diffuse proliferation of smooth muscle cells (SMCs), which in many patients show TSC2 (tuberin) gene mutations, in addition to thickening of interstitial tissues, loss of alveoli, and the development of cystic spaces. While SMC proliferation is the defining feature of LAM, a significant proportion of LAM lung tissue consists of expanded interstitial connective tissue that is negative for smooth muscle actin and TSC2 mutations. The importance of this actin-negative interstitial tissue to the pathophysiology of LAM is not clear. The present study has determined the contribution of this interstitial tissue to LAM lung volume by morphometric analysis and has examined its cell and matrix proteoglycan composition by immunohistochemistry. Lung tissue from nine LAM patients and four control subjects was examined. LAM lung contained twice as much interstitial tissue as control lung (27% versus 13% of total lung volume), with SMCs accounting for less than 25% of the interstitial volume. Areas of interstitial tissue stained strongly for the matrix proteoglycans versican and biglycan. Decorin was prominent in association with collagen bundles. SMCs did not stain, or stained lightly, for proteoglycans. Versican and biglycan deposits were closely associated with actin-negative interstitial fibroblasts identified by prolyl 4-hydroxylase immuno-staining. Comparatively normal alveolar walls in LAM lung also stained strongly for versican and had a reduced elastin content. Thickened interstitial regions contained significant amounts of elastin (approximately 13% of interstitial volume) but with fibres in disorganized patterns. Elastic fibres were absent from areas that stained strongly for versican and biglycan. These areas also showed weak staining for elastin binding protein (EBP), consistent with proteoglycan-induced shedding of EBP and inhibition of elastic fibre formation. These findings point to a significant contribution from matrix proteoglycans to the expanded and remodelled interstitial lung tissue of LAM patients.     

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.     

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.     

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.     

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.     

Multifocal micronodular pneumocyte hyperplasia and lymphangioleiomyomatosis in tuberous sclerosis with a TSC2 gene.

A 45-year-old woman with a long-standing diagnosis of tuberous sclerosis (TSC) is presented. She has multifocal micronodular pneumocyte hyperplasia (MMPH) and lymphangioleiomyomatosis (LAM) of the lung, together with the detection of TSC2 gene mutation. During surgery for spontaneous pneumothorax, an open-lung biopsy was performed. Micronodules were well defined, measuring approximately 4 mm in diameter. These MMPHs were histologically composed of papillary proliferation of Type II pneumocytes, with positive immunoreactivity of keratin and surfactant apoprotein. The cystlike spaces, with dilatation and destruction of air spaces, were diffusely formed, and the walls were composed of the spindle cells. Such LAM showed positive immunoreactivity for HMB-45 (a monoclonal antibody specific for human melanoma) and tuberin (the gene product of TSC2). On germline mutation analysis using leukocytes of the present patient, a TSC2 gene mutation was confirmed as a deletion of G (or g) on Exon 9 by polymerase chain reaction-single-strand conformational polymorphism. However, no mutation was detected in her son. With microdissection analysis using paraffin-embedding lung tissues, LOH of the TSC2 gene preliminarily was detected in a LAM lesion but not in MMPH. It is suggested that MMPH, in addition to LAM, could be another pulmonary lesion in TSC patients and that the detection of TSC2 and/or TSC1 gene could essentially be useful for the pathogenesis of MMPH and LAM in TSC patients.     

Glutamine synthetase interpretation in hepatocellular adenoma

Lymphangioleiomyomatosis (LAM) of the lung is a rare low-grade malignancy affecting primarily women of childbearing age. LAM is characterized by the proliferation of SMA and HMB-45 positive spindle-shaped and epithelioid cells throughout the lung in the form of discrete lesions causing cystic destruction and ultimately respiratory insufficiency. LAM occurs sporadically or in patients with tuberous sclerosis complex (TSC) and is etiologically linked to mutations in the TSC1 and TSC2 genes. Although LAM cells are known to express estrogen and progesterone receptors (ER and PR, respectively), their respective expression level was never determined. Therefore, here we measured the immunohistochemical expression of ERs and PRs in a large series of pulmonary LAM cases using the Aperio Spectrum Analysis Platform. Our case series comprised open lung biopsy specimens from 20 LAM patients and lungs explanted during the course of lung transplant from 24 patients. All cases were positive for ER and PR. PR expression was statistically significantly higher than ER in 80 % of the biopsies while ER predominated only in one case. Specimens from explanted cases of LAM had relatively fewer PR-positive nuclei. As a result, PR expression was significantly higher than ER in 38 % of the cases, whereas ER predominated in 33 %. Overall, PR expression predominated in 57 % of cases and ER in 21 %. These data indicate that PR frequently prevails over ER in pulmonary LAM. LAM is unusual in its high PR/ER ratio; other female neoplasms show a definite prevalence of ER. Our findings therefore warrant further study of PR function in LAM.     

Molecular pathogenesis of lymphangioleiomyomatosis: lessons learned from orphans

Lymphangioleiomyomatosis (LAM) is a rare progressive cystic lung disease affecting young women. The pivotal observation that LAM occurs both spontaneously and as part of the tuberous sclerosis complex (TSC) led to the hypothesis that these disorders share common genetic and pathogenetic mechanisms. In this review we describe the evolution of our understanding of the molecular and cellular basis of LAM and TSC, beginning with the discovery of the TSC1 and TSC2 genes and the demonstration of their involvement in sporadic (non-TSC) LAM. This was followed by rapid delineation of the signaling pathways in Drosophila melanogaster with confirmation in mice and humans. This knowledge served as the foundation for novel therapeutic approaches that are currently being used in human clinical trials.     

Vascular Endothelial Growth Factors C and D Induces Proliferation of Lymphangioleiomyomatosis Cells through Autocrine Crosstalk with Endothelium

Lymphangioleiomyomatosis (LAM) is a potentially fatal lung disease characterized by nodules of proliferative smooth muscle-like cells. The exact nature of these LAM cells and their proliferative stimuli are poorly characterized. Herein we report the novel findings that the lymphangiogenic vascular endothelial growth factors (VEGF) C and D induce LAM cell proliferation through activation of their cognate receptor VEGF-R3 and activation of the signaling intermediates Akt/mTOR/S6. Furthermore, we identify expression of the proteoglycan NG2, a marker of immature smooth muscle cells, as a characteristic of LAM cells both in vitro and in human lung tissue. VEGF-C-induced LAM cell proliferation was in part a result of autocrine stimulation that resulted from cross talk with lymphatic endothelial cells. Ultimately, these findings identify the lymphangiogenic VEGF proteins as pathogenic growth factors in LAM disease and at the same time provide a novel pharmacotherapeutic target for a lung disease that to date has no known effective treatment.The understanding of lymphatic biology has expanded rapidly with the discovery of the lymphangiogenic proteins vascular endothelial growth factors (VEGF) C and D and their cognate receptor VEGF-R3.^1,2 They are members of the VEGF family of growth factors, having 40% homology with VEGF-A, and their importance in lymph vasculature biology is well demonstrated by the finding that VEGF-C null mice die as a result of abnormal lymphatic development.³ Increasingly it has become clear that the biological role of these VEGF proteins have expanded beyond effects on the lymphatic endothelium alone to include the peri-lymphatic milieu and the investing immature vascular smooth muscle or pericytes.⁴ Furthermore, they have now been implicated in both pathogenic and non-pathogenic human processes including cancer growth and metastasis, wound healing, and immune regulation.The lymphatic endothelial cell (LEC) is distinct from blood vessel endothelium with respect to their biological function, structure, and protein repertoire.⁵ As such they represent a unique cell that may play a distinctive role in the pathogenesis of human disease. Like blood vessel endothelium they share their basement membrane with surrounding immature smooth muscle cells called pericytes. It is clear that along with VEGF, pericyte-endothelial cell interactions play an important role in modulating vascular biology.⁴Until recently the lymphatic vasculature has been considered an innocent bystander in the pathogenesis of pulmonary disease. Historically, one such disease has been pulmonary lymphangioleiomyomatosis (LAM), a progressive and fatal lung disease that almost exclusively affects women in their reproductive years.^6,7 Pathologically, LAM is characterized by proliferation of abnormal smooth muscle-like cells that form lung nodules distributed in a peri-lymphatic manner. The etiology of LAM has been linked to mutations in the TSC2 gene; however, the nature and site-of-origin of LAM cells remains speculative.⁸ Based on the LAM cells proximity to lymphatic vessels, we hypothesized that the LEC and lymphangiogenic proteins VEGF-C and/or -D might play a role in their proliferation; and that these smooth muscle- like LAM cells represented perivascular mural cells that were proliferating through a mechanism involving LEC cross talk.Using both in vitro primary LAM-derived cells (LDC)⁹ and immunohistochemistry of human lung LAM tissues, we report that VEGF-C and -D induce LDC proliferation through activation of their cognate receptor VEGF-R3 and subsequently phosphatidylinositol 3-kinase (PI3K)/mTOR/S6 signaling. Of note, this VEGF-R3 proliferative signaling pathway did not appear to be inhibited by functional tuberin. Furthermore, LDC proliferation was induced by cross talk between LEC and LDC and mediated through LDC production of VEGF-C. Taken together, these results identify the lymphangiogenic VEGF proteins as novel pathogenic growth factors in LAM disease and the lymphatic endothelium as a modifying factor in LAM cell proliferation.