Repurposing Nitazoxanide for Potential Treatment of Rare Disease Lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare genetic lung disease. Unfortunately, treatment with the mTORC1 inhibitor Rapamycin only slows disease progression, and incomplete responses are common. Thus, there remains an urgent need to identify new targets for the development of curative LAM treatments. Nitazoxanide (NTZ) is an orally bioavailable antiprotozoal small molecule drug approved for the treatment of diarrhea caused by Giardia lamblia or Cryptosporidium parvum in children and adults, with a demonstrated mTORC1 inhibitory effect in several human cell lines. NTZ's excellent safety profile characterized by its more than 20 years of clinical use makes it a promising candidate for repurposing. Our rationale for this study was to further investigate NTZ's effect using in vitro and in vivo LAM models and to elucidate the underlying molecular mechanism beyond mTORC1 inhibition. For this purpose, we investigated cell proliferation, cell viability, and changes in protein phosphorylation and expression in primary human cell cultures derived from LAM lung samples before translating our results into a syngeneic mouse model utilizing Tsc2-null cells. NTZ reduced cell growth for all tested cell lines at a dose of about 30 µM. Lower doses than that had no effect on cell viability, but doses above 45 µM lowered the viability by about 10 to 15% compared to control. Interestingly, our western blot revealed no inhibition of mTORC1 and only a mild effect on active ß-Catenin. Instead, NTZ had a pronounced effect on reducing pAkt. In the mouse model, prophylactic NTZ treatment via the intraperitoneal and oral routes had some effects on reducing lung lesions and improving body weight retention, but the results remain inconclusive.

Hyperactive mTORC1 in lung mesenchyme induces endothelial cell dysfunction and pulmonary vascular remodeling.

Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease caused by tuberous sclerosis complex 1/2 (TSC1/2) gene mutations in pulmonary mesenchymal cells, resulting in activation of the mechanistic target of rapamycin complex 1 (mTORC1). A subset of patients with LAM develop pulmonary vascular remodeling and pulmonary hypertension. Little, however, is known regarding how LAM cells communicate with endothelial cells (ECs) to trigger vascular remodeling. In end-stage LAM lung explants, we identified EC dysfunction characterized by increased EC proliferation and migration, defective angiogenesis, and dysmorphic endothelial tube network formation. To model LAM disease, we used an mTORC1 gain-of-function mouse model with a Tsc2 KO (Tsc2KO) specific to lung mesenchyme (Tbx4LME-Cre Tsc2fl/fl), similar to the mesenchyme-specific genetic alterations seen in human disease. As early as 8 weeks of age, ECs from mice exhibited marked transcriptomic changes despite an absence of morphological changes to the distal lung microvasculature. In contrast, 1-year-old Tbx4LME-Cre Tsc2fl/fl mice spontaneously developed pulmonary vascular remodeling with increased medial thickness. Single-cell RNA-Seq of 1-year-old mouse lung cells identified paracrine ligands originating from Tsc2KO mesenchyme, which can signal through receptors in arterial ECs. These ECs had transcriptionally altered genes including those in pathways associated with blood vessel remodeling. The proposed pathophysiologic mesenchymal ligand-EC receptor crosstalk highlights the importance of an altered mesenchymal cell/EC axis in LAM and other hyperactive mTORC1-driven diseases. Since ECs in patients with LAM and in Tbx4LME-Cre Tsc2fl/fl mice did not harbor TSC2 mutations, our study demonstrates that constitutively active mTORC1 lung mesenchymal cells orchestrated dysfunctional EC responses that contributed to pulmonary vascular remodeling.

Lost in translation: a neglected mTOR target for lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a cystic lung disease of women resulting from mutations in tuberous sclerosis complex (TSC) genes that suppress the mammalian target of rapamycin complex 1 (mTORC1) pathway. mTORC1 activation enhances a plethora of anabolic cellular functions, mainly via the activation of mRNA translation through stimulation of ribosomal protein S6 kinase (S6K1)/ribosomal protein S6 (S6) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)/eukaryotic translation initiation factor 4E (eIF4E). Rapamycin (sirolimus), an allosteric inhibitor of mTORC1, stabilises lung function in many but not all LAM patients and, upon cessation of the drug, disease progression resumes. At clinically tolerable concentrations, rapamycin potently inhibits the ribosomal S6K1/S6 translation ribosome biogenesis and elongation axis, but not the translation 4E-BP1/eIF4E initiation axis. In this mini-review, we propose that inhibition of mTORC1-driven translation initiation is an obvious but underappreciated therapeutic strategy in LAM, TSC and other mTORC1-driven diseases.

Sorafenib inhibits invasion of multicellular organoids that mimic Lymphangioleiomyomatosis nodules.

Lymphangioleiomyomatosis (LAM) is a debilitating, progressive lung disease with few therapeutic options, largely due to a paucity of mechanistic knowledge of disease pathogenesis. Lymphatic endothelial cells (LECs) are known to envelope and invade clusters of LAM-cells, comprising of smooth muscle α-actin and/or HMB-45 positive "smooth muscle-like cells" however the role of LECs in LAM pathogenesis is still unknown. To address this critical knowledge gap, we investigated wether LECs interact with LAM-cells to augment their metastatic behaviour of LAM-cells. We performed in situ spatialomics and identified a core of transcriptomically related cells within the LAM nodules. Pathway analysis highlights wound and pulmonary healing, VEGF signaling, extracellular matrix/actin cytoskeletal regulating and the HOTAIR regulatory pathway enriched in the LAM Core cells. We developed an organoid co-culture model combining primary LAM-cells with LECs and applied this to evaluate invasion, migration, and the impact of Sorafenib, a multi-kinase inhibitor. LAM-LEC organoids had significantly higher extracellular matrix invasion, decreased solidity and a greater perimeter, reflecting increased invasion compared to non-LAM control smooth muscle cells. Sorafenib significantly inhibited this invasion in both LAM spheroids and LAM-LEC organoids compared to their respective controls. We identified TGFß1ι1, a molecular adapter coordinating protein-protein interactions at the focal adhesion complex and known to regulate VEGF, TGFß and Wnt signalling, as a Sorafenib-regulated kinase in LAM-cells. In conclusion we have developed a novel 3D co-culture LAM model and have demonstrated the effectiveness of Sorafenib to inhibit LAM-cell invasion, identifying new avenues for therapeutic intervention.

Normalization of Enzyme Expression and Activity Regulating Vitamin A Metabolism Increases RAR-Beta Expression and Reduces Cellular Migration and Proliferation in Diseases Caused by Tuberous Sclerosis Gene Mutations.

BACKGROUND: Mutation in a tuberous sclerosis gene (TSC1 or 2) leads to continuous activation of the mammalian target of rapamycin (mTOR). mTOR activation alters cellular including vitamin A metabolism and retinoic acid receptor beta (RARß) expression. The goal of the present study was to investigate the molecular connection between vitamin A metabolism and TSC mutation. We also aimed to investigate the effect of the FDA approved drug rapamycin and the vitamin A metabolite retinoic acid (RA) in cell lines with TSC mutation. METHODS: Expression and activity of vitamin A associated metabolic enzymes and RARß were assessed in human kidney angiomyolipoma derived cell lines, primary lymphangioleiomyomatosis (LAM) tissue derived LAM cell lines. RARß protein levels were also tested in primary LAM lung tissue sections. TaqMan arrays, enzyme activities, qRT-PCRs, immunohistochemistry, immunofluorescent staining, and western blotting were performed and analysed. The functional effects of retinoic acid (RA) and rapamycin were tested in a scratch and a BrDU assay to assess cell migration and proliferation. RESULTS: Metabolic enzyme arrays revealed a general deregulation of many enzymes involved in vitamin A metabolism including aldehyde dehydrogenases (ALDHs), alcohol dehydrogenases (ADHs) and Cytochrome P450 2E1 (CYP2E1). Furthermore, RARß downregulation was a characteristic feature of all TSC-deficient cell lines and primary tissues. Combination of the two FDA approved drugs -RA for acute myeloid leukaemia and rapamycin for TSC mutation- normalised ALDH and ADH expression and activity, restored RARß expression and reduced cellular proliferation and migration. CONCLUSION: Deregulation of vitamin A metabolizing enzymes is a feature of TSC mutation. RA can normalize RARß levels and limit cell migration but does not have a significant effect on proliferation. Based on our data, translational studies could confirm whether combination of RA with reduced dosage of rapamycin would have more beneficial effects to higher dosage of rapamycin monotherapy meanwhile reducing adverse effects of rapamycin for patients with TSC mutation.

Activated p53 in the anti-apoptotic milieu of tuberous sclerosis gene mutation induced diseases leads to cell death if thioredoxin reductase is inhibited.

Tuberous sclerosis, angiomyolipoma and lymphangioleiomyomatosis are a group of diseases characterized by mutation in tuberous sclerosis genes (TSC 1-2). TSC mutation leads to continuous activation of the mTOR pathway that requires adaptation to increased ATP requirement. With limited treatment options, there is an increasing demand to identify novel therapeutic targets and to understand the correlations between mTOR pathway activation and the lack of cell death in the presence of TSC mutation. In the current study, we demonstrate deregulation of p53 controlled and mitochondria associated cell death processes. The study also reveals that treatment of TSC mutant cells with the drug candidate Proxison combined with reduced concentration of rapamycin can increase production of reactive oxygen species (ROS), can modify miRNA expression pattern associated with p53 regulation and can reduce cell viability.

CrossTORC and WNTegration in Disease: Focus on Lymphangioleiomyomatosis.

The mechanistic target of rapamycin (mTOR) and wingless-related integration site (Wnt) signal transduction networks are evolutionarily conserved mammalian growth and cellular development networks. Most cells express many of the proteins in both pathways, and this review will briefly describe only the key proteins and their intra- and extracellular crosstalk. These complex interactions will be discussed in relation to cancer development, drug resistance, and stem cell exhaustion. This review will also highlight the tumor-suppressive tuberous sclerosis complex (TSC) mutated, mTOR-hyperactive lung disease of women, lymphangioleiomyomatosis (LAM). We will summarize recent advances in the targeting of these pathways by monotherapy or combination therapy, as well as future potential treatments.

Therapeutic Targeting of DGKA-Mediated Macropinocytosis Leads to Phospholipid Reprogramming in Tuberous Sclerosis Complex.

Lymphangioleiomyomatosis is a rare destructive lung disease affecting primarily women and is the primary lung manifestation of tuberous sclerosis complex (TSC). In lymphangioleiomyomatosis, biallelic loss of TSC1/2 leads to hyperactivation of mTORC1 and inhibition of autophagy. To determine how the metabolic vulnerabilities of TSC2-deficient cells can be targeted, we performed a high-throughput screen utilizing the "Repurposing" library at the Broad Institute of MIT and Harvard (Cambridge, MA), with or without the autophagy inhibitor chloroquine. Ritanserin, an inhibitor of diacylglycerol kinase alpha (DGKA), was identified as a selective inhibitor of proliferation of Tsc2-/- mouse embryonic fibroblasts (MEF), with no impact on Tsc2+/+ MEFs. DGKA is a lipid kinase that metabolizes diacylglycerol to phosphatidic acid, a key component of plasma membranes. Phosphatidic acid levels were increased 5-fold in Tsc2-/- MEFs compared with Tsc2+/+ MEFs, and treatment of Tsc2-/- MEFs with ritanserin led to depletion of phosphatidic acid as well as rewiring of phospholipid metabolism. Macropinocytosis is known to be upregulated in TSC2-deficient cells. Ritanserin decreased macropinocytic uptake of albumin, limited the number of lysosomes, and reduced lysosomal activity in Tsc2-/- MEFs. In a mouse model of TSC, ritanserin treatment decreased cyst frequency and volume, and in a mouse model of lymphangioleiomyomatosis, genetic downregulation of DGKA prevented alveolar destruction and airspace enlargement. Collectively, these data indicate that DGKA supports macropinocytosis in TSC2-deficient cells to maintain phospholipid homeostasis and promote proliferation. Targeting macropinocytosis with ritanserin may represent a novel therapeutic approach for the treatment of TSC and lymphangioleiomyomatosis. SIGNIFICANCE: This study identifies macropinocytosis and phospholipid metabolism as novel mechanisms of metabolic homeostasis in mTORC1-hyperactive cells and suggest ritanserin as a novel therapeutic strategy for use in mTORC1-hyperactive tumors, including pancreatic cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2086/F1.large.jpg.

mTORC1 activation in lung mesenchyme drives sex- and age-dependent pulmonary structure and function decline.

Lymphangioleiomyomatosis (LAM) is a rare fatal cystic lung disease due to bi-allelic inactivating mutations in tuberous sclerosis complex (TSC1/TSC2) genes coding for suppressors of the mechanistic target of rapamycin complex 1 (mTORC1). The origin of LAM cells is still unknown. Here, we profile a LAM lung compared to an age- and sex-matched healthy control lung as a hypothesis-generating approach to identify cell subtypes that are specific to LAM. Our single-cell RNA sequencing (scRNA-seq) analysis reveals novel mesenchymal and transitional alveolar epithelial states unique to LAM lung. This analysis identifies a mesenchymal cell hub coordinating the LAM disease phenotype. Mesenchymal-restricted deletion of Tsc2 in the mouse lung produces a mTORC1-driven pulmonary phenotype, with a progressive disruption of alveolar structure, a decline in pulmonary function, increase of rapamycin-sensitive expression of WNT ligands, and profound female-specific changes in mesenchymal and epithelial lung cell gene expression. Genetic inactivation of WNT signaling reverses age-dependent changes of mTORC1-driven lung phenotype, but WNT activation alone in lung mesenchyme is not sufficient for the development of mouse LAM-like phenotype. The alterations in gene expression are driven by distinctive crosstalk between mesenchymal and epithelial subsets of cells observed in mesenchymal Tsc2-deficient lungs. This study identifies sex- and age-specific gene changes in the mTORC1-activated lung mesenchyme and establishes the importance of the WNT signaling pathway in the mTORC1-driven lung phenotype.

A phase II clinical trial of the Safety Of Simvastatin (SOS) in patients with pulmonary lymphangioleiomyomatosis and with tuberous sclerosis complex.

INRODUCTION: The mechanistic target of rapamycin inhibitors (mTORi) sirolimus and everolimus stabilize lung function in patients with pulmonary lymphangioleiomyomatosis (LAM) but do not induce remission. Pre-clinical studies suggest that simvastatin in combination with sirolimus induces LAM cell death. The objective of this study was to assess the safety of simvastatin with either sirolimus or everolimus in LAM patients. METHODS: This was a phase II single arm trial evaluating the safety of escalating daily simvastatin (20-40 mg) in LAM patients already treated with sirolimus or everolimus. Adverse events and changes in lipid panel profile, pulmonary function tests, and VEGF-D were assessed. RESULTS: Ten LAM patients on a stable dose of mTORi for >3 months were treated with 20 mg simvastatin for two months followed by 40 mg for two months. The most common adverse events were peripheral edema (30%), cough (30%), and diarrhea (30%). No patients withdrew or had a reduction in simvastatin dose because of adverse events. Two patients required sirolumus dose reduction for supratherapeutic trough levels following simvastatin initiation. Total cholesterol and low density lipoproteins declined over the study period (-46.0 mg/dL±20.8, p = 0.008; -41.9 mg/dL±22.0, p = 0.01, respectively). There was also a decline in FEV1 (-82.0 mL±86.4, p = 0.02) but no significant change in FVC, DLCO, or VEGF-D. CONCLUSIONS: The combination of simvastatin with mTORi in LAM patients is safe and well-tolerated from an adverse events perspective. The addition of simvastatin, however, was associated with decline in FEV1 and the efficacy of this combination should be explored in larger trials.

Adoptive T-Cell Transfer to Treat Lymphangioleiomyomatosis.

Patients with lymphangioleiomyomatosis (LAM) develop pulmonary cysts associated with neoplastic, smooth muscle-like cells that feature neuroendocrine cell markers. The disease preferentially affects premenopausal women. Existing therapeutics do not cure LAM. As gp100 is a diagnostic marker expressed by LAM lesions, we proposed to target this immunogenic glycoprotein using TCR transgenic T cells. To reproduce the genetic mutations underlying LAM, we cultured Tsc2-/- kidney tumor cells from aged Tsc2 heterozygous mice and generated a stable gp100-expressing cell line by lentiviral transduction. T cells were isolated from major histocompatibility complex-matched TCR transgenic pmel-1 mice to measure cytotoxicity in vitro, and 80% cytotoxicity was observed within 48 hours. Antigen-specific cytotoxicity was likewise observed using pmel-1 TCR-transduced mouse T cells, suggesting that transgenic T cells may likewise be useful to treat LAM in vivo. On intravenous injection, slow-growing gp100+ LAM-like cells formed lung nodules that were readily detectable in severe combined immunodeficient/beige mice. Adoptive transfer of gp100-reactive but not wild-type T cells into mice significantly shrunk established lung tumors, even in the absence of anti-PD-1 therapy. These results demonstrate the treatment potential of adoptively transferred T cells to eliminate pulmonary lesions in LAM.

Inhibition of Growth of TSC2-Null Cells by a PI3K/mTOR Inhibitor but Not by a Selective MNK1/2 Inhibitor.

Lymphangioleiomyomatosis (LAM) is a rare metastatic cystic lung disease due to a mutation in a TSC tumor suppressor, resulting in hyperactive mTOR growth pathways. Sirolimus (rapamycin), an allosteric mTORC1 inhibitor, is a therapeutic option for women with LAM but it only maintains lung volume during treatment and does not provide benefit for all LAM patients. The two major mTORC1 protein synthesis pathways are via S6K/S6 or 4E-BP/eIF4E activation. We aimed to investigate rapamycin in combination with compounds that target associated growth pathways, with the potential to be additive to rapamycin. In this study we demonstrated that rapamycin, at a clinically tolerable concentration (10 nM), inhibited the phosphorylation of S6, but not the critical eIF4E releasing Thr 37/46 phosphorylation sites of 4E-BP1 in TSC2-deficient LAM-derived cells. We also characterized the abundant protein expression of peIF4E within LAM lesions. A selective MNK1/2 inhibitor eFT508 inhibited the phosphorylation of eIF4E but did not reduce TSC2-null cell growth. In contrast, a PI3K/mTOR inhibitor omipalisib blocked the phosphorylation of Akt and both S6K/S6 and 4E-BP/eIF4E branches, and additively decreased the growth of TSC2-null cells with rapamycin. Omipalisib, or another inhibitor of both major mTORC1 growth pathways and pAkt, might provide therapeutic options for TSC2-deficient cancers including, but not limited to, LAM.

Immunotherapy for Lymphangioleiomyomatosis and Tuberous Sclerosis: Progress and Future Directions.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare genetic multisystem disease characterized by the nodular proliferation of smooth muscle-like LAM cells, progressive cystic changes of the lung, lymphatic abnormalities, and renal angiomyolipomas (AMLs). LAM can arise sporadically or in women with the autosomal dominant disorder, tuberous sclerosis complex (TSC), in which hamartomatous tumors of brain, heart, skin, kidney, and lung are found. LAM and TSC are caused by mutations in the TSC1 or TSC2 tumor suppressor genes leading to elevated mechanistic/mammalian target of rapamycin complex activity. Recent data indicate that T cells within LAM nodules and renal AMLs exhibit features of T-cell exhaustion, with coinhibitory receptor programmed cell death protein 1 (PD-1) expression on tumor-infiltrating T cells. Treatment of animal models of TSC and LAM with anti-PD-1 antibodies or with the combination of anti-PD-1 and anti-CTLA4 antibodies has led to remarkable results, suppressing TSC2-null tumor growth and inducing tumor rejection. Here we review our current knowledge about the potential for immunotherapy for the treatment of LAM and TSC and highlight critical unknowns and key next steps.

Immune Checkpoint Ligand PD-L1 Is Upregulated in Pulmonary Lymphangioleiomyomatosis.

Pulmonary lymphangioleiomyomatosis (LAM) is a slow-progressing metastatic disease that is driven by mutations in the tumor suppressor tuberous sclerosis complex 1/2 (TSC1/2). Rapamycin inhibits LAM cell proliferation and is the only approved treatment, but it cannot cause the regression of existing lesions and can only stabilize the disease. However, in other cancers, immunotherapies such as checkpoint blockade against PD-1 and its ligand PD-L1 have shown promise in causing tumor regression and even curing some patients. Thus, we asked whether PD-L1 has a role in LAM progression. In vitro, PD-L1 expression in murine Tsc2-null cells is unaffected by mTOR inhibition with torin but can be upregulated by IFN-γ. Using immunohistochemistry and single-cell flow cytometry, we found increased PD-L1 expression both in human lung tissue from patients with LAM and in Tsc2-null lesions in a murine model of LAM. In this model, PD-L1 is highly expressed in the lung by antigen-presenting and stromal cells, and activated T cells expressing PD-1 infiltrate the affected lung. In vivo treatment with anti-PD-1 antibody significantly prolongs mouse survival in the model of LAM. Together, these data demonstrate that PD-1/PD-L1-mediated immunosuppression may occur in LAM, and suggest new opportunities for therapeutic targeting that may provide benefits beyond those of rapamycin.

Rapamycin-independent IGF2 expression in Tsc2-null mouse embryo fibroblasts and human lymphangioleiomyomatosis cells.

Lymphangioleiomyomatosis (LAM) is a rare, almost exclusively female lung disease linked to inactivating mutations in tuberous sclerosis complex 2 (TSC2), a tumor suppressor gene that controls cell metabolic state and growth via regulation of the mechanistic target of rapamycin (mTORC1) signaling. mTORC1 is frequently activated in human cancers and, although the mTORC1 inhibitor rapamycin has a cytostatic effect, it is, in general, unable to elicit a robust curative effect or tumor regression. Using RNA-Seq, we identified (1) Insulin-like Growth Factor (IGF2) as one of the genes with the highest fold-change difference between human TSC2-null and TSC2-expressing angiomyolipoma cells from a patient with LAM, and (2) the mouse IGF2 homolog Igf2, as a top-ranking gene according to fold change between Tsc2-/- and Tsc2+/+ mouse embryo fibroblasts (MEFs). We extended transcript-level findings to protein level, observing increased Igf2 protein expression and Igf2 secretion by Tsc2-/- MEFs. Increased Igf2 expression was not due to epigenetic imprinting, but was partially mediated through the Stat3 pathway and was completely insensitive to rapamycin treatment. An siRNA-mediated decrease of Igf2 resulted in decreased Stat3 phosphorylation, suggesting presence of an autocrine Igf2/Stat3 amplification cycle in Tsc2-/- MEFs. In human pulmonary LAM lesions and metastatic cell clusters, high levels of IGF2 were associated with mTORC1 activation. In addition, treatment of three primary IGF2-expressing LAM lung cell lines with rapamycin did not result in IGF2 level changes. Thus, targeting of IGF2 signaling may be of therapeutic value to LAM patients, particularly those who are unresponsive to rapamycin.

Urokinase-type plasminogen activator (uPA) is critical for progression of tuberous sclerosis complex 2 (TSC2)-deficient tumors.

Lymphangioleiomyomatosis (LAM) is a fatal lung disease associated with germline or somatic inactivating mutations in tuberous sclerosis complex genes (TSC1 or TSC2). LAM is characterized by neoplastic growth of smooth muscle-α-actin-positive cells that destroy lung parenchyma and by the formation of benign renal neoplasms called angiolipomas. The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin slows progression of these diseases but is not curative and associated with notable toxicity at clinically effective doses, highlighting the need for better understanding LAM's molecular etiology. We report here that LAM lesions and angiomyolipomas overexpress urokinase-type plasminogen activator (uPA). Tsc1-/- and Tsc2-/- mouse embryonic fibroblasts expressed higher uPA levels than their WT counterparts, resulting from the TSC inactivation. Inhibition of uPA expression in Tsc2-null cells reduced the growth and invasiveness and increased susceptibility to apoptosis. However, rapamycin further increased uPA expression in TSC2-null tumor cells and immortalized TSC2-null angiomyolipoma cells, but not in cells with intact TSC. Induction of glucocorticoid receptor signaling or forkhead box (FOXO) 1/3 inhibition abolished the rapamycin-induced uPA expression in TSC-compromised cells. Moreover, rapamycin-enhanced migration of TSC2-null cells was inhibited by the uPA inhibitor UK122, dexamethasone, and a FOXO inhibitor. uPA-knock-out mice developed fewer and smaller TSC2-null lung tumors, and introduction of uPA shRNA in tumor cells or amiloride-induced uPA inhibition reduced tumorigenesis in vivo These findings suggest that interference with the uPA-dependent pathway, when used along with rapamycin, might attenuate LAM progression and potentially other TSC-related disorders.

Lymphangioleiomyomatosis: A Monogenic Model of Malignancy.

Lymphangioleiomyomatosis (LAM) is a rare, low-grade, metastasizing neoplasm that arises from an unknown source, spreads via the lymphatics, and targets the lungs. All pulmonary structures become infiltrated with benign-appearing spindle and epithelioid cells (LAM cells) that express smooth-muscle and melanocyte-lineage markers, harbor mTOR-activating mutations in tuberous sclerosis complex (TSC) genes, and recruit abundant stromal cells. Elaboration of lymphangiogenic growth factors and matrix remodeling enzymes by LAM cells enables their access to lymphatic channels and likely drives the cystic lung remodeling that often culminates in respiratory failure. Dysregulated cellular signaling results in a shift from oxidative phosphorylation to glycolysis as the preferred mode of energy generation, to allow for the accumulation of biomass required for cell growth and tolerance of nutrient-poor, anaerobic environments. Symptomatic LAM occurs almost exclusively in females after menarche, highlighting the central but as yet poorly understood role for sex-restricted anatomical structures and/or hormones in disease pathogenesis. LAM is an elegant model of malignancy because biallelic mutations at a single genetic locus confer all features that define cancer upon the LAM cell-metabolic reprogramming and proliferative signals that drive uncontrolled growth and inappropriate migration and invasion, the capacity to exploit the lymphatic circulation as a vehicle for metastasis and access to the lungs, and destruction of remote tissues. The direct benefit of the study of this rare disease has been the rapid identification of an effective FDA-approved therapy, and the collateral benefits have included elucidation of the pivotal roles of mTOR signaling in the regulation of cellular metabolism and the pathogenesis of cancer.

Idiopathic multicentric Castleman's disease: a systematic literature review.

BACKGROUND: Multicentric Castleman's disease describes a group of poorly understood lymphoproliferative disorders driven by proinflammatory hypercytokinaemia. Patients have heterogeneous clinical features, characteristic lymph node histopathology, and often deadly multiple organ dysfunction. Human herpesvirus 8 (HHV8) causes multicentric Castleman's disease in immunosuppressed patients. The cause of HHV8-negative multicentric Castleman's disease is idiopathic; such cases are called idiopathic multicentric Castleman's disease. An absence of centralised information about idiopathic multicentric Castleman's disease represents a major challenge for clinicians and researchers. We aimed to characterise clinical features of, treatments for, and outcomes of idiopathic multicentric Castleman's disease. METHODS: We did a systematic literature review and searched PubMed, the Cochrane database, and ClinicalTrials.gov from January, 1995, with keywords including "Castleman's disease" and "giant lymph node hyperplasia". Inclusion criteria were pathology-confirmed Castleman's disease in multiple nodes and minimum clinical and treatment information on individual patients. Patients with HHV8 or HIV infection or diseases known to cause Castleman-like histopathology were excluded. FINDINGS: Our search identified 626 (33%) patients with HHV8-negative multicentric Castleman's disease from 1923 cases of multicentric Castleman's disease. 128 patients with idiopathic multicentric Castleman's disease met all inclusion criteria for the systematic review. Furthermore, aggregated data for 127 patients with idiopathic multicentric Castleman's disease were presented from clinical trials, which were excluded from primary analyses because patient-level data were not available. Clinical features of idiopathic multicentric Castleman's disease included multicentric lymphadenopathy (128/128), anaemia (79/91), elevated C-reactive protein (65/79), hypergammaglobulinaemia (63/82), hypoalbuminaemia (57/63), elevated interleukin 6 (57/63), hepatomegaly or splenomegaly (52/67), fever (33/64), oedema, ascites, anasarca, or a combination (29/37), elevated soluble interleukin 2 receptor (20/21), and elevated VEGF (16/20). First-line treatments for idiopathic multicentric Castleman's disease included corticosteroids (47/128 [37%]), cytotoxic chemotherapy (47/128 [37%]), and anti-interleukin 6 therapy (11/128 [9%]). 49 (42%) of 116 patients failed first-line therapy, 2-year survival was 88% (95% CI 81-95; 114 total patients, 12 events, 36 censored), and 27 (22%) of 121 patients died by the end of their observed follow-up (median 29 months [IQR 12-50]). 24 (19%) of 128 patients with idiopathic multicentric Castleman's disease had a diagnosis of a separate malignant disease, significantly higher than the frequency expected in age-matched controls (6%). INTERPRETATION: Our systematic review provides comprehensive information about clinical features, treatment, and outcomes of idiopathic multicentric Castleman's disease, which accounts for at least 33% of all cases of multicentric Castleman's disease. Our findings will assist with prompt recognition, diagnostic criteria development, and effective management of the disease. FUNDING: None.