MicroRNA-21 is Induced by Rapamycin in a Model of Tuberous Sclerosis (TSC) and Lymphangioleiomyomatosis (LAM)

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

Folliculin, the Product of the Birt-Hogg-Dube Tumor Suppressor Gene, Interacts with the Adherens Junction Protein p0071 to Regulate Cell-Cell Adhesion

Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhdflox/flox mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma

Folliculin regulates cell–cell adhesion, AMPK, and mTORC1 in a cell‐type‐specific manner in lung‐derived cells

Abstract Germline loss‐of‐function BHD mutations cause cystic lung disease and hereditary pneumothorax, yet little is known about the impact of BHD mutations in the lung. Folliculin (FLCN), the product of the Birt–Hogg–Dube (BHD) gene, has been linked to altered cell–cell adhesion and to the AMPK and mTORC1 signaling pathways. We found that downregulation of FLCN in human bronchial epithelial (HBE) cells decreased the phosphorylation of ACC, a marker of AMPK activation, while downregulation of FLCN in small airway epithelial (SAEC) cells increased the activity of phospho‐S6, a marker of mTORC1 activation, highlighting the cell type–dependent functions of FLCN. Cell–cell adhesion forces were significantly increased in FLCN‐deficient HBE cells, consistent with prior findings in FLCN‐deficient human kidney‐derived cells. To determine how these altered cell–cell adhesion forces impact the lung, we exposed mice with heterozygous inactivation of Bhd (similarly to humans with germline inactivation of one BHD allele) to mechanical ventilation at high tidal volumes. Bhd+/− mice exhibited a trend (P = 0.08) toward increased elastance after 6 h of ventilation at 24 cc/kg. Our results indicate that FLCN regulates the AMPK and mTORC1 pathways and cell–cell adhesion in a cell type–dependent manner. FLCN deficiency may impact the physiologic response to inflation‐induced mechanical stress, but further investigation is required. We hypothesize that FLCN‐dependent effects on signaling and cellular adhesion contribute to the pathogenesis of cystic lung disease in BHD patients

Rapamycin induces miR-21 expression via an AKT-independent mechanism.

<p><b>A</b>) Western blot analysis of 621-101 cells treated with DMSO (lane 1), Rapamycin (20 nM, 24 h - lane 2), the AKT inhibitor MK2206 (10 nM, 24 h - lane 3), and Rapamycin and MK2206 (lane 4). Rapamycin treatment induces AKT phosphorylation at S473 and MK2206 abrogates Rapamycin's effect on phosphor-Akt. <b>B</b>) Expression of miR-21, 24, 29b, and 221 in 621-101 cells treated as in A). miR-21 levels are induced by Rapamycin, however the addition of MK2206 has no effect suggesting an AKT-independent mechanism.</p

Exiqon miRNA microarray confirms 8 Rapamycin-dependent miRNA.

<p>621-101 cells were treated with Rapamycin 20 nM or DMSO for 24 hours. Total RNA was isolated and applied to the Exiqon platform, which assays 946 human miRNA. <b>A</b>) Heat map of miRNA dysregulated by Rapamycin >1.5-fold, log₂ scale. RNA from three biologic replicates per condition was pooled; each miRNA was assayed in quadruplet on the array. <b>B</b>) miRNA dysregulated by Rapamycin >1.5-fold (normalized to RNU44). Highlighted miRNA (except miR-31 and 210) are common to both the Exiqon and Signosis platforms. miR-21 is circled.</p

qRT-PCR confirmation of Rapamycin-dependent miRNA in TSC2-deficient cells.

<p>TSC2−/− cells were treated with Rapamycin 20 nM or DMSO for 24 hr and miRNA expression was assessed by qRT-PCR. <b>A</b>) miRNA expression is similar in 621-101 cells using RNU44 (left panel) or RNU48 (right panel) for normalization. <b>B</b>) miRNA expression in 621-101 cells normalized to RNU44. Highlighted results are significant using a Bonferroni correction.</p

Rapamycin-regulated miRNA in LAM patient-derived cells identified by the Exiqon Array (Fold Change >1.5, normalized to RNU44).

<p>Rapamycin-regulated miRNA in LAM patient-derived cells identified by the Exiqon Array (Fold Change >1.5, normalized to RNU44).</p

miR-21 is mTOR-dependent and may be TSC2-independent.

<p><b>A</b>) Stable downregulation of tuberin in C3H-10T1/2 pre-pericytes results in increased phosphorylation of ribosomal protein S6, as expected. Treatment with Rapamycin (20 nM, 24 h) inhibits phosphorylation of S6. <b>B</b>) Downregulation of TSC2 in C3H-10T1/2 cells does not affect miR-21 expression. Inhibition of mTORC1 with Rapamycin induces ∼2-fold increase in miR-21 expression in both control shRNA and TSC2 shRNA cells. Bars represent the mean of two biologic replicates +/− SD. * p<0.05. <b>C</b>) LAM patient-derived cells (621-101), TSC2-null rat uterine leiomyoma-derived cells (ELT3), TSC2-null mouse embryonic fibroblasts (MEFs), HEK293 and lung adenocarcinoma (A549) cells were treated with Rapamycin 20 nM vs Control for 24 h. Relative MiR-21 expression was determined by qRT-PCR. Human cells were normalized to RNU44, mouse cells to snora202 and rat cells to U87, which are all small nucleolar RNA molecules. For all charts, bars represent the mean of three biologic replicates +/− standard error. * p<0.05. ** p<0.01.</p