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

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

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

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

Generation and characterization of <i>in vivo</i> trackable TSC2-deficient cells.

<p>A. Expression cassettes of adenoviral (Ad-NIS/GFP) and retroviral (Retro-NIS/GFP) vectors. CMV  =  cytomegalovirus promoter, NIS  =  sodium-iodide symporter, LITR  =  left-handed inverted terminal repeat, RITR  =  right-handed inverted terminal repeat, GFP  =  green fluorescent protein, ΔE1, ΔE3 =  E1 and E3 deletions of adenovirus type 5 (Ad5) backbone sequence, 5′ LTR = 5′ long terminal repeat, φ signal  =  virus packing signal, IRES  =  internal ribosome entry site, 3′ LTR  = 3′ long terminal repeat. B. <i>In vitro</i> uptake of ^99mTc-pertechnetate and GFP fluorescence in stable Retro-NIS−/GFP-expressing 621-327 cells (top row) and control 621-101 cells (bottom row). Scale bar = 6 mm. C. Immunofluorescent detection of GFP, NIS, and tuberin in TSC2-deficient 621-327 cells (top row), control 621-101 cells (middle row), and HeLa cells (bottom row). GFP fluorescence (2nd column), staining with primary anti-NIS specific antibody (3rd column) or anti-tuberin antibody (right column) with secondary Cy3 antibody conjugates is shown along with phase contrast (left column). Scale bar = 50 µm. D. Western blot analysis of TSC2-expressing HEK293T control cells, and TSC2-deficient 621-101 cells and 621-327 cells, using antibodies to key signaling proteins, NIS, and GFP. A beta actin loading control is also shown, as are long exposure times (long exp.) for tuberin and hamartin.</p

Quantitation of LAM/TSC tumor response to drug treatment.

<p>A. Typical SPECT/CT images of mice treated with rapamycin or vehicle for 4 weeks (i.e., 6 weeks after intratracheal administration of 621-327 cells). T  =  thyroid; S  =  stomach; B  =  bladder. Arrows indicate tumors. Scale bars = 1 cm. B.^99mTc-pertechnetate uptake in LAM/TSC tumors before, during, and after treatment with rapamycin or vehicle control. 621-327 cells were administered intratracheally at time = 0. Rapamycin treatment was during weeks 2 to 6. Mice were followed an additional 2 weeks off drug.</p

Proliferation and pathogenesis of TSC2-deficient cells: pulmonary LAM like-nodules and cysts.

<p>A. <i>Ex vivo</i> SPECT/CT imaging and antibody and H&E staining of lung. Resected whole lungs (left column) from LAM/TSC-bearing mice (top row) or control mice (bottom row) at 2 weeks postadministration of 621-327 cells. Scale bar = 1 mm. Also shown are H&E staining (middle column) and anti-GFP antibody staining (right column) of frozen sections from lungs at 4 weeks postadministration of 621-327 cells. Scale bars = 50 µm. B. H&E staining of paraffin-embedded lung tissue 15 weeks postadministration of 621-327 cells (top row) at low (top left) and high (top right and bottom row of dotted rectangles) magnification. Scale bars = 50 µm. C. Same as (B) except frozen sections at 26 weeks postadministration of 621-327 cells (left) with anti-human NIS antibody staining (right). Scale bars = 50 µm.</p

Lymph node metastasis and invasion by TSC2-deficient cells.

<p>A. Normal lymph node (LN; top row) and lymph node identified by GFP and NIS expression (bottom row) 4 weeks after intratracheal administration of 621-327 cells. Left column shows <i>in vivo</i> color video image. Right columns show same nodes <i>ex vivo</i> after resection, placement on black paper, and imaging using color video, GFP fluorescence, and SPECT/CT, respectively. Scale bars = 1 mm. B. Hematoxylin and eosin (H&E) staining of frozen sections from normal (top row) and tumor-infiltrated (bottom row) lymph nodes 2 weeks after intratracheal administration of 621-327 cells. Dotted rectangle inset  =  higher magnification. Consecutive tissue sections were also stained with anti-GFP antibody. Scale bars = 50 µm. C. H&E staining of paraffin-embedded, tumor-infiltrated lymph nodes at 15 weeks post-administration of 621-327 cells. Dotted rectangle inset  =  higher magnification. Scale bars = 50 µm.</p

The effect of sex and exogenous estrogen on TSC2-deficient tumor growth.

<p>Female (left) or male (right) mice were implanted with 0.18 mg 17ß-estradiol pellets or control placebo pellets subcutaneously. Ten days later, 621-327 cells were administered intratracheally and tumors quantified every other week by SPECT/CT. Shown are the results after 4 weeks of hormone treatment. T  =  thyroid; S  =  stomach; B  =  bladder. Arrows indicate tumors. Scale bars  = 1 cm. +/−  = 0.3–1.4; +  = 1.5 - 2.5; ++  = 2.6–3.7; +++  =  ≥3.8%ID/mm³ (×10⁻⁵).</p