Circulating Lymphangioleiomyomatosis Tumor Cells With Loss of Heterozygosity in the TSC2 Gene Show Increased Aldehyde Dehydrogenase Activity.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a destructive metastasizing neoplasm of the lung characterized by proliferation of LAM cells in specialized lung nodules. LAM cells are characterized by expression of the prometastatic and cancer-initiating hyaluronan receptor CD44v6, and loss of heterozygosity (LOH) of TSC1 and TSC2. The circulating neoplastic LAM cells are thought to be involved in metastasis. Because LAM cells display properties of neoplastic, metastatic, and stem cell-like cancer cells, we hypothesized that elevated aldehyde dehydrogenase (ALDH) activity, characteristic of cancer and stem cells, is a property of LAM cells. METHODS: We performed an in silico search of ALDH genes in microdissected LAM lung nodules. To identify circulating LAM cells, we osmotically removed red blood cells from whole blood to obtain peripheral blood mononuclear cells, which were then sorted by fluorescence-activated cell sorting based on their level of ALDH activity. RESULTS: Microdissected LAM lung nodules possess a distinctive ALDH gene profile. The cell subpopulation with high ALDH activity, isolated from circulating cells, possessed TSC2 LOH in 8 of 14 patients with LAM. Approximately 60% of the circulating cells with high ALDH activity expressed CD44v6. Cells with TSC2 LOH from patients with LAM and LAM/TSC exhibited different properties in different body locations, but all cell types showed high ALDH activity. CONCLUSIONS: This new procedure allows for isolation of circulating LAM cells from cultured cells, blood, and chylous effusions and shows that circulating LAM cells are heterogeneous with neoplastic, metastatic, and cancer-stem cell-like properties.

Somatic activating mutations in PIK3CA cause generalized lymphatic anomaly.

Generalized lymphatic anomaly (GLA) is a vascular disorder characterized by diffuse or multifocal lymphatic malformations (LMs). The etiology of GLA is poorly understood. We identified four distinct somatic PIK3CA variants (Glu542Lys, Gln546Lys, His1047Arg, and His1047Leu) in tissue samples from five out of nine patients with GLA. These same PIK3CA variants occur in PIK3CA-related overgrowth spectrum and cause hyperactivation of the PI3K-AKT-mTOR pathway. We found that the mTOR inhibitor, rapamycin, prevented lymphatic hyperplasia and dysfunction in mice that expressed an active form of PIK3CA (His1047Arg) in their lymphatics. We also found that rapamycin reduced pain in patients with GLA. In conclusion, we report that somatic activating PIK3CA mutations can cause GLA, and we provide preclinical and clinical evidence to support the use of rapamycin for the treatment of this disabling and deadly disease.

The effect of mTOR inhibitors on respiratory infections in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a destructive cystic lung disease. Mammalian target of rapamycin (mTOR) inhibitors are the primary treatment for LAM but it is unknown whether these immunosuppressing medications increase the risk for or the severity of respiratory infections in LAM patients.We searched multiple databases for original articles that reported the rate of respiratory infections in LAM patients treated with mTOR inhibitors or placebo. We calculated incidence rates for respiratory infections in these groups and incidence rate ratios for respiratory infections and severe respiratory infections in mTOR inhibitors treated versus placebo treated patients.11 studies were included. There were 294 patients in the treatment groups and 93 patients in the placebo groups. Among subjects in placebo arms, the incidence rate of respiratory infections was 58.8 per 100 patient-years (95% CI 35.3-82.3 per 100 patient-years). The incidence-rate ratio (IRR) for respiratory infection among treated subjects was 0.71 (95% CI 0.50-1.02; p=0.06 compared to placebo subjects). The IRR for severe respiratory infections among treated subjects was 1.56 (95% CI 0.43-8.55; p=0.52).We found that respiratory infections are common in patients with LAM. Importantly, treatment with mTOR inhibitors does not increase the incidence of these infections and may be protective.

mTOR treatment in lymphangioleiomyomatosis: the role of everolimus.

The orphan lung disease lymphangioleiomyomatosis (LAM) has until recently been untreatable other than by lung transplantation. However, improved understanding of underlying disease mechanisms has revealed the central role of constitutive up-regulation of the mammalian target of rapamycin (mTOR) pathway in this disease. Although other pathways exist and are under investigation for treatment, several mTOR inhibitors are currently available and emerging information suggests that these may have some efficacy in preventing loss of lung function in LAM. This paper summarizes current understanding of treatment with mTOR inhibitors in LAM, and everolimus in particular. It outlines pharmacokinetics and pharmacodynamics relevant to the clinician, recent clinical studies, and issues with potential side effects. mTOR treatment is not yet available in most countries for LAM, but current data for treatment efficacy are impressive, and it is hoped that mTOR inhibition will soon be recognised as an important treatment of this disease.

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

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

Rapamycin-resistant poly (ADP-ribose) polymerase-1 overexpression is a potential therapeutic target in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a female-predominant cystic lung disease that can lead to respiratory failure. LAM cells typically have inactivating tuberous sclerosis complex 2 (TSC2) mutations and mammalian target of rapamycin (mTOR) complex (mTORC) 1 activation. Clinical response to the mTORC1 inhibitors has been limited, prompting a search for additional therapy for LAM. In this study, we investigated the impact of TSC2 on the expression of poly (ADP-ribose) polymerase (PARP)-1 that initiates the DNA repair pathway, and tested the efficacy of PARP1 inhibitors in the survival of TSC2-deficient (TSC2(-)) cells. We analyzed publicly available expression arrays of TSC2(-) cells and validated the findings using real-time RT-PCR, immunoblotting, and immunohistochemistry. We examined the impact of rapamycin and Torin 1 on PARP1 expression. We also tested the effect of PARP1 inhibitors, 8-hydroxy-2-methylquinazoline-4-one and 3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-isoquinoline, on the survival of TSC2(-) cells. We identified the up-regulation of PARP1 in TSC2(-) cells relative to cells in which wild-type TSC2 has been reintroduced (TSC2-addback [TSC2(+)] cells). The transcript levels of PARP1 in TSC2(-) cells were not affected by rapamycin. PARP1 levels were increased in TSC2(-) cells, xenograft tumors of rat-derived TSC2(-) cells, renal cystadenomas from Tsc2(+/-) mice, and human LAM nodules. RNA interference of mTOR failed to reduce PARP1 levels. Proliferation and survival of TSC2(-) cells was reduced in response to PARP1 inhibitor treatment, more so than TSC2(+) cells. TSC2(-) cells exhibit higher levels of PARP1 relative to TSC2(+) cells in an mTOR-insensitive manner. PARP1 inhibitors selectively suppress the growth and induce apoptosis of TSC2(-) cells from patients with LAM. Targeting PARP1 may be beneficial in the treatment of LAM and other neoplasm with mTORC1 activation.

SRC kinase is a novel therapeutic target in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease affecting some women with tuberous sclerosis complex (TSC). Sporadic LAM can develop in women without TSC, owing to somatic mutations in the TSC2 gene. Accumulating evidence supports the view of LAM as a low-grade, destructive, metastasizing neoplasm. The mechanisms underlying the metastatic capability of LAM cells remain poorly understood. The observed behavior of LAM cells with respect to their infiltrative growth pattern, metastatic potential, and altered cell differentiation bears similarity to cells undergoing epithelial-mesenchymal transition. Here, we report increased levels of active Src kinase in LAM lungs and in TSC2(-/-) cells, caused by a reduction of autophagy. Furthermore, increased Src kinase activation promoted migration, invasion, and inhibition of E-cadherin expression in TSC2(-/-) cells by upregulating the transcription factor Snail. Notably, Src kinase inhibitors reduced migration and invasion properties of TSC2(-/-) cells and attenuated lung colonization of intravenously injected TSC2(-/-) cells in vivo to a greater extent than control TSC2(+/+) cells. Our results reveal mechanistic basis for the pathogenicity of LAM cells and they rationalize Src kinase as a novel therapeutic target for treatment of LAM and TSC.

Cathepsin K expression in the spectrum of perivascular epithelioid cell (PEC) lesions of the kidney.

The perivascular epithelioid cell (PEC) is a unique cell type coexpressing contractile proteins (mainly α-smooth muscle actin), melanocytic markers, including microphthalmia-associated transcription factor (MITF), and estrogen and progesterone receptors. It is constantly present in a group of tumors called PEComas. Renal PEComas include the common angiomyolipoma as well as less common lesions such as microscopic angiomyolipoma, intraglomerular lesions, angiomyolipoma with epithelial cysts, epithelioid angiomyolipoma, oncocytoma-like angiomyolipoma and lymphangioleiomyomatosis of the renal sinus. It has been demonstrated that most of these lesions are determined by mutations affecting genes of the tuberous sclerosis complex, tuberous sclerosis 1 (TSC1) and tuberous sclerosis 2 (TSC2), with eventual deregulation of the RHEB/MTOR/RPS6KB2 pathway, and it has been observed that some PEComas regressed during sirolimus therapy, an MTOR inhibitor. Recently, overexpression of MITF has been related to the expression of the papain-like cysteine protease cathepsin K in osteoclasts where it has inhibited MTOR. The aim of this study is to evaluate cathepsin K immunohistochemically in the entire spectrum of PEComa lesions in the kidney. The study population consisted of 84 renal PEComa lesions, including 5 composed predominantly of fat (lipoma-like angiomyolipoma), 15 almost exclusively composed of spindle-shaped smooth muscle cells (leiomyoma-like angiomyolipoma) and 31 common angiomyolipomas composed of a mixture of fat, spindle and epithelioid smooth muscle cells, and abnormal thick-walled blood vessels, 15 microscopic angiomyolipomas, 5 intraglomerular lesions, 2 oncocytoma-like angiomyolipomas, 8 epithelioid angiomyolipomas, 2 angiomyolipomas with epithelial cysts and 1 example of lymphangioleiomyomatosis of the renal sinus. In all of the renal PEComas, cathepsin K was found to be constantly and strongly expressed and seems to be a more powerful marker than other commonly used markers for their identification, especially to confirm the diagnosis on needle biopsies.

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.

Cathepsin-k expression in pulmonary lymphangioleiomyomatosis.

Lymphangioleiomyomatosis is a rare and progressive lung cystic disease, caused by the infiltration of lung parenchyma by mesenchymal cells characterized by co-expression of contractile proteins and melanocytic markers. The pathogenesis of lymphangioleiomyomatosis is determined by mutations affecting tuberous sclerosis complex (TSC) genes, with eventual deregulation of the Rheb/mTOR/p70S6K pathway, and the potential therapeutic activity of mTOR inhibitors is currently under investigation. To better understand the molecular mechanisms involved in the pathogenesis of lymphangioleiomyomatosis, we investigated the expression of cathepsin-k (a papain-like cysteine protease with high matrix-degrading activity). The rationale of this choice was based on the recent demonstration that mTOR inhibitors can regulate major functional activities of osteoclasts, including the expression of cathepsin-k. The immunohistochemical study included 12 cases of lymphangioleiomyomatosis. Twelve angiomyolipomas and several lung diseases (sarcoidosis, organizing pneumonia, usual interstitial pneumonia, emphysema) were investigated as controls. In all lymphangioleiomyomatosis cases, strong cathepsin-k immunoreactivity was demonstrated, restricted to lymphangioleiomyomatosis cells. Similar expression levels were observed in renal angiomyolipomas. These observations extend the knowledge regarding the immunophenotypic profile of lymphangioleiomyomatosis cells, and provide a useful new marker for diagnosis in difficult cases (eg, in small transbronchial biopsies). The strong expression of such a potent papain-like cysteine protease in lymphangioleiomyomatosis cells can significantly contribute to the progressive remodelling of lung parenchyma observed in this deadly disease, with eventual formation of lung cysts. It is possible to speculate that mTOR inhibitors may exert part of their action by limiting the destructive remodelling of lung structure.

Activation of the estrogen receptor contributes to the progression of pulmonary lymphangioleiomyomatosis via matrix metalloproteinase-induced cell invasiveness.

CONTEXT: The role of estrogens in the pathogenesis of lymphangioleiomyomatosis (LAM), an aggressive and destructive, eventually fatal lung disease of women, is poorly understood. OBJECTIVE: The study was conducted to test the hypothesis that the lung disease in LAM is estrogen mediated and to determine whether estrogens contribute to the invasiveness of LAM. DESIGN: In vitro cell culture of spindle-shaped LAM cells (LAMD-SM) were isolated and propagated from affected lungs. Estrogen receptor (ER)-alpha and ERbeta analyses were conducted by RT-PCR. ERalpha and ERbeta, tissue inhibitor of metalloproteinase-2, and matrix metalloproteinases (MMP)-2 had Western blot analysis for protein assessment. Activity assays were performed for MT1-MMP, MMP-2, and tissue inhibitor of metalloproteinase-2. Assessment of MMP-2 promoter function was done via transfection assays. Cell invasion chambers were used to determine and quantitate cell invasiveness. SETTING: The study was conducted at an academic medical center. PATIENTS: Tissue and cells were obtained from patients as outlined in approved institution review board protocol (97/007). INTERVENTION: LAMD-SM cells were treated with a specific MMP-2 antibody or a nonspecific inhibitor, doxycycline. MAIN OUTCOME MEASURES: Activity of MMP-2 and invasiveness of LAMD-SM cells were measured. RESULTS: LAMD-SM cells express functional ERs (ERalpha and ERbeta), which undergo rapid intracellular turnover in their unbound state. 17beta-estradiol (E(2)) enhances the transcriptional ER activity. E(2)-induced ER activation increases synthesis and activity of MMP-2 through posttranscriptional mechanisms in LAMD-SM. The E(2)/ER-mediated increase of MMP-2 promotes LAMD-SM invasiveness, in assays in vitro, which can be inhibited by specific antibodies against MMP-2 or doxycycline, an inhibitor of MMPs. CONCLUSION: The invasion and destruction of lung parenchyma in LAM is, at least partially, an estrogen-MMP-driven process, which has major implications for therapeutic interventions.

Expression of human telomerase reverse transcriptase in lymphangioleiomyomatosis.

Telomerase synthesizes nucleotide hexameric repeats (telomeres) at the ends of chromosomes, replacing base sequences that are lost from these sites during each mitotic cycle and protecting these ends against the action of exonucleases and ligases. Therefore, telomerase is essential for maintaining cellular replication. To evaluate the role of telomerase in the proliferation of abnormal smooth muscle cells (lymphangioleiomyomatosis [LAM] cells) in LAM, we performed immunostaining and in situ hybridization studies to identify telomerase protein and messenger RNA (mRNA), respectively, in pulmonary (n = 18) and extrapulmonary (n = 4) lesions from 22 women with LAM (14 untreated and 8 treated with progesterone or tamoxifen). Immunoreactivity and hybridization signals for telomerase were observed in 5 to 20% of LAM cells, mostly of the spindle-shaped type, in 21 of the 22 patients, and were less intense in the treated group. Other types of cells were unreactive in both groups. Telomerase colocalized in the same cells with alpha-smooth muscle actin, but only rarely with HMB-45 antibody (a marker for epithelioid LAM cells); colocalization with proliferating cell nuclear antigen was incomplete. The telomerase-positive LAM cells may constitute the sources of renewal of LAM cells. Modulation of telomerase may be involved in the control of LAM cell proliferation.

Role for activation of matrix metalloproteinases in the pathogenesis of pulmonary lymphangioleiomyomatosis.

BACKGROUND: Matrix metalloproteinases (MMPs) have been shown to be involved in the pathogenesis of the destructive pulmonary lesions in patients with lymphangioleiomyomatosis (LAM); in the present report, the activation of these enzymes is examined. OBJECTIVE: To evaluate the role of MMPs and their activating enzymes, immunohistochemical and confocalmicroscopic techniques were used to localize alpha-smooth muscle actin (alpha-SMA), HMB-45, proliferating cell nuclear antigen (PCNA), MMP-2, membrane-type 1 MMP (MT1-MMP), MT2-MMP, and MT3-MMP in lung tissues from 10 women with LAM. Tissue samples were obtained from 5 patients before treatment and in 5 patients after hormone treatment (progesterone and/or tamoxifen citrate). RESULTS: Staining for alpha-SMA and MMP-2 was present in all the abnormal smooth muscle cells (LAM cells) in both groups. The percentages of PCNA-, MMP-2-, or MT1-MMP-positive LAM cells were much higher in the untreated group than in the treated group, whereas the percentages of HMB-45-reactive LAM cells were similar in both groups. The reactions for MT1-MMP and PCNA were preferentially localized in small spindle-shaped LAM cells; the reaction for HMB-45 was found in large epithelioid LAM cells. Many of the PCNA-positive cells were also positive for MT1-MMP. Staining for MT2-MMP and MT3-MMP was negative. CONCLUSIONS: This study demonstrates an association between cellular proliferation and the presence of MT1-MMP in LAM cells. The activation of MMP-2 by MT1-MMP may play an important role in the destruction of lung tissue in this disorder.