Towards improving lymphangioleiomyomatosis care: a study of biomarkers and therapies

Lymphangioleiomyomatosis (LAM) is a rare lung disease primarily affecting women of childbearing age and characterized by cystic lung destruction. The disease is caused by loss-of-function mutations in tuberous sclerosis complex 1 and 2 (TSC1/2) genes (1,2). In LAM, the dysfunction of the products of TSC1 and TSC2 results in the loss of regulation and the constitutive activation of the mammalian target of rapamycin complex 1 (mTORC1) (3). The increase in the activity of this pathway leads to the uncontrolled growth of LAM cells and their acquisition of migration capacities to the lung. LAM is caused by the proliferation of stem cell-like cells with lung metastatic potential whose origin is currently unknown. The identification of the origin may help to isolate circulating LAM cells, and their molecular characterization would be extremely valuable for further understanding of disease development, lung tropism, and neoplastic progression. In breast cancer, lung metastasis events have been associated with low expression of TSC1/2. Moreover, the expression of these genes correlates with the expression of a set of genes described as lung metastasis mediators (4). In addition, lung metastasis mediators and breast stem cell markers are relatively overexpressed in LAM tissues and cell models (5). For these reasons, we aimed the following goal: - To assess the level of tumorigenesis and metastatic potential to the lung of Tsc2-deficient mammary epithelial cells We generated a cohort of female mice with conditional deletion of the Tsc2 gene in a specific stem/progenitor cell population of the mammary epithelium. The Tsc2 deletion was directed by the Cre recombinase, and Cre expression was under the control of the promoter of the beta-lactoglobulin (BLG). The BLG was only expressed in the mammary epithelium cells responsible for milk production. We expected that some of the Tsc2 knock-out cells were tumorigenic and had metastatic potential to migrate to the lung. However, any histopathological changes were observed in breast or lung tissues. CONCLUSION 1: Breast cell origin has not been demonstrated. However, these results do not rule out the possibility of mammary cells being a source of LAM disease, but our study had some limitations that could have influenced the results. LAM diagnosis and monitoring can be challenging due to the heterogeneity of symptoms and the insufficiency of non-invasive tests (6,7). There are some cases in which non-invasive techniques such as high-resolution computed tomography or the measurement of vascular endothelial growth factor D (VEGF-D) (8) in plasma are not definitive for diagnosis. Therefore, we considered important to improve the non-invasive differential diagnostic and monitoring protocols, and for that reason, we purpose the following goal: - To identify LAM plasma biomarkers that are complementary to VEGF-D Following on the hypothesis that the transcriptional programs of the breast cancer cells that metastasize to lung can reveal mechanistic similarities to those of LAM cells, 30 enzymes were predicted to be over-expressed in LAM samples. Thus, a targeted metabolomic analysis in a set of serum samples from LAM patients, controls and patients with other rare lung diseases was performed. We were able to asses by LC-MS/MS that the levels of four metabolites produced by some of the selected enzymes may be over-expressed in the serum of LAM patients relative to controls. Methylimidazoleacetic acid (MIAA) is the most promising metabolite due to its overexpression in the serum of LAM patients relative not only to controls but also to the other rare lung disease patients. The two monoamine oxidases (MAOA and MAOB) are some of the enzymes responsible for the production of MIAA. MAOs were overexpressed in LAM cell lines compared with their controls. In addition, the activity of these enzymes was increased in LAM cells and human LAM lesions expressed these proteins. Furthermore, MIAA is the major histamine-derived metabolite. The histamine receptor 1 (HRH1) was overexpressed in LAM cell lines and it was expressed in human LAM lesions. Finally, the histamine signaling pathway was more activated in LAM cells. CONCLUSION 2: MIAA is more abundant in LAM plasma compared with control groups and it may be complementary to VEGF-D determination. In addition, the expression and function of MAOs and HRH1 may be relevant to LAM biology. Rapamycin, a mTORC1 inhibitor, is the standard of care for LAM and significantly stabilizes pulmonary function and decreases angiomyolipomas size in most cases (9). However, this treatment does not fully eradicate LAM cells and a substantial proportion of patients treated with this compound show a continued decline in lung function. Consequently, to improve LAM care, we aimed the following goal: - To Discover of novel therapeutic strategies Based on the high levels of expression and activity of enzymes participating in monoamine catabolism, the consequences of pharmacological inhibition of the corresponding proteins were assessed in vitro and in vivo. We were interested in testing the effect of MAO inhibitors (clorgyline and rasagiline) and an HRH1 antagonist (loratadine) alone and in combination with rapamycin to determine whether LAM care can be improved. We performed several phenotypic assays in vitro using these combinations, but none of them were conclusive. Then, we analyzed the effect of immunodeficient and immunocompetent mouse models. In the immunodeficient model, the combinations of rapamycin with clorgyline and rasagiline showed higher tumor volume reduction than rapamycin alone. In the immunocompetent model, the three monotherapies reduced the tumor growth more than the control and the combinations of rapamycin with clorgyline and loratadine also showed higher tumor volume reduction than rapamycin alone. CONCLUSION 3: Combinations of mTOR and MAO/HRH1 inhibitors appear to be more efficient than rapamycin alone in reducing LAM tumorigenesis. In addition, monotherapies based on MAO inhibition and HRH1 antagonism may also be beneficial in some cases. (1) Darling, T. N. et al. 2010. Lymphangioleiomyomatosis and TSC2-/- cells. Lymphat. Res. Biol. 8, 59–69. (2) Hammes, S. R. & Krymskaya, V. P. 2013. Targeted Approaches toward Understanding and Treating Pulmonary Lymphangioleiomyomatosis (LAM). Horm. Cancer 4, 70–77. 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(9) McCormack, F.X. et al. 2011. Efficacy and safety of sirolimus in lymphangioleiomyomatosis. N. Engl. J. Med. 364, 1595–1606.