Person:

Priolo, Carmen

Lymphangioleiomyomatosis (LAM), a multisystem disease of women, is manifest by the proliferation of smooth muscle-like cells in the lung resulting in cystic lung destruction. Women with LAM can also develop renal angiomyolipomas. LAM is caused by mutations in the tuberous sclerosis complex genes (TSC1 or TSC2), resulting in hyperactive mammalian Target of Rapamycin (mTOR) signaling. The mTOR inhibitor, Rapamycin, stabilizes lung function in LAM and decreases the volume of renal angiomyolipomas, but lung function declines and angiomyolipomas regrow when treatment is discontinued, suggesting that factors induced by mTORC1 inhibition may promote the survival of TSC2-deficient cells. Whether microRNA (miRNA, miR) signaling is involved in the response of LAM to mTORC1 inhibition is unknown. We identified Rapamycin-dependent miRNA in LAM patient angiomyolipoma-derived cells using two separate screens. First, we assayed 132 miRNA of known significance to tumor biology. Using a cut-off of >1.5-fold change, 48 microRNA were Rapamycin-induced, while 4 miRs were downregulated. In a second screen encompassing 946 miRNA, 18 miRs were upregulated by Rapamycin, while eight were downregulated. Dysregulation of miRs 29b, 21, 24, 221, 106a and 199a were common to both platforms and were classified as candidate “RapamiRs.” Validation by qRT-PCR confirmed that these microRNA were increased. miR-21, a pro-survival miR, was the most significantly increased by mTOR-inhibition (p<0.01). The regulation of miR-21 by Rapamycin is cell type independent. mTOR inhibition promotes the processing of the miR-21 transcript (pri-miR-21) to a premature form (pre-miR-21). In conclusion, our findings demonstrate that Rapamycin upregulates multiple miRs, including pro-survival miRs, in TSC2-deficient patient-derived cells. The induction of miRs may contribute to the response of LAM and TSC patients to Rapamycin therapy.

Lymphangioleiomyomatosis (LAM) is a progressive neoplastic disorder that leads to lung destruction and respiratory failure primarily in women. LAM is typically caused by tuberous sclerosis complex 2 (TSC2) mutations resulting in mTORC1 activation in proliferative smooth muscle–like cells in the lung. The female predominance of LAM suggests that estradiol contributes to disease development. Metabolomic profiling identified an estradiol-enhanced prostaglandin biosynthesis signature in Tsc2-deficient (TSC−) cells, both in vitro and in vivo. Estradiol increased the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin biosynthesis, which was also increased at baseline in TSC-deficient cells and was not affected by rapamycin treatment. However, both Torin 1 treatment and Rictor knockdown led to reduced COX-2 expression and phospho-Akt-S473. Prostaglandin production was also increased in TSC-deficient cells. In preclinical models, both Celecoxib and aspirin reduced tumor development. LAM patients had significantly higher serum prostaglandin levels than healthy women. 15-epi-lipoxin-A4 was identified in exhaled breath condensate from LAM subjects and was increased by aspirin treatment, indicative of functional COX-2 expression in the LAM airway. In vitro, 15-epi-lipoxin-A4 reduced the proliferation of LAM patient–derived cells in a dose-dependent manner. Targeting COX-2 and prostaglandin pathways may have therapeutic value in LAM and TSC-related diseases, and possibly in other conditions associated with mTOR hyperactivation.