Rapamycin-independent <i>IGF2</i> expression in <i>Tsc2</i>-null mouse embryo fibroblasts and human lymphangioleiomyomatosis cells

<div><p>Lymphangioleiomyomatosis (LAM) is a rare, almost exclusively female lung disease linked to inactivating mutations in <i>tuberous sclerosis complex 2</i> (<i>TSC2)</i>, 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) <i>Insulin-like Growth Factor</i> (<i>IGF2</i>) as one of the genes with the highest fold-change difference between human <i>TSC</i>2-null and <i>TSC</i>2-expressing angiomyolipoma cells from a patient with LAM, and (2) the mouse <i>IGF2</i> homolog <i>Igf2</i>, as a top-ranking gene according to fold change between <i>Tsc</i>2^-/- and <i>Tsc</i>2^+/+ mouse embryo fibroblasts (MEFs). We extended transcript-level findings to protein level, observing increased Igf2 protein expression and Igf2 secretion by <i>Tsc</i>2^-/- 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 <i>Tsc2</i>^<i>-/-</i> 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.</p></div

Increased expression of <i>IGF2</i> transcripts in TSC2— human LAM cells and <i>Tsc2</i>^<i>-/-</i> MEFs.

<p>(A) TSC2 levels in human TSC2-null LAM 621–102 cells (TSC2—) cells and TSC2 re-expressing 621–103 LAM (TSC2++) cells. (B) RNA-Seq results show increased <i>IGF2</i> transcripts per kilobase million (TPM) in TSC2— cells. (C) Corresponding plot of mapped reads along the hg38 reference genome corresponding to <i>IGF2</i>. (D) Verification that <i>Tsc2</i> is not expressed in <i>Tsc2</i>^<i>-/-</i> MEFs. (E) RNA-Seq results show increased <i>Igf2</i> TPMs in <i>Tsc2</i>^<i>-/-</i> vs. <i>Tsc2</i>^<i>+/+</i> MEFs. (F) Corresponding plot of mapped reads along the mm10 reference genome corresponding to <i>Igf2</i>.</p

STAT3-dependent upregulation of IGF2 in TSC2-null cells.

<p>(A) Re-expression of TSC2 (TSC2++) in TSC2-null LAM 102 (TSC2—) cells decreased STAT3 expression and activation. (B) siRNA-induced knockdown of STAT3 decreased STAT3 levels in TSC2— cells. (C) RNA-Seq results show upregulated <i>IGF2</i> transcripts per kilobase million (TPM) in TSC2— cells transfected with either NT siRNA (Control) or STAT3 siRNA (siSTAT3). (D) STAT3 binding sites in human IGF2 and mouse Igf2 promoter regions. STAT3 enrichment in specific promoter regions of (E) human <i>IGF2</i> and (F) mouse <i>Igf2</i> genes was detected by ChIP-qPCR. Treatment of <i>Tsc2</i>^<i>-/-</i> MEFs with Stat3 inhibitor S3I-201 (100 nM for 18 hr) decreased Igf2 protein (G) expression as measured via Western blot and (H) secretion as measured via ELISA. (I) siRNA-mediated Stat3 knockdown also decreased Igf2 protein expression in <i>Tsc2</i>^<i>-/-</i> MEFs. (J) siRNA-mediated Igf2 knockdown decreased Stat3 phosphorylation but not total Stat3.</p

IGF2 expression in LAM lungs and <i>Tsc2</i>^<i>-/-</i> MEFs.

<p>Representative images of IHC analysis show IGF2 expression in (A) LAM lesion and (B) LAM cluster detected with specific antibodies. Non-immune IgG was used as a control (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197105#pone.0197105.s001" target="_blank">S1 Fig</a>). Igf2 expression in <i>Tsc2</i>^<i>-/-</i> MEFs was detected by (C) qPCR (D) Western blot and (E) ELISA. (F) <i>Tsc2</i>^<i>-/-</i> MEFs were transfected with 50nM <i>Igf2</i> siRNA (siIGF2) or NT siRNA (siNT) for 48 hrs. Decreased levels of Igf2 protein expression were confirmed via western blot with β-actin as an internal loading control. (G) Decreased Igf2 protein secretion was confirmed via ELISA. Igf2 knockdown resulted in (H) increased cleaved caspase-3 levels as measured via immunocytostaining and flow for Alexa Fluor® 488 -Cleaved Caspase 3 where the population of positively stained MEFs was normalized to the control population, and (J) decreased cell viability as assessed by 0.4% Trypan Blue staining normalized to the control cell viability. Student's t-tests were used to determine the statistical significance of the differences, and <i>p</i>-values reflect a sample size of 3 replicates.</p

IGF2 expression is rapamycin-insensitive in <i>Tsc2</i>^<i>-/-</i> MEFs and human LAM cells.

<p><i>Tsc2</i>^<i>-/-</i> and <i>Tsc2</i>^<i>+/+</i> MEFs were grown to near confluence, serum deprived for 2 hr and treated with indicated concentrations of rapamycin for 24 hr, followed by western blot analysis with indicated antibodies. (A) Treatment with 10nM rapamycin for 24 hr did not decrease Igf2 protein expression, although this dose completely suppressed pS6. (B) Igf2, Stat3, and pStat3 protein expression levels were unaffected by rapamycin treatment over a range of concentrations, while it completely inhibited pS6 at 2nM and 20nM concentrations. (C) IGF2 protein levels did not change in TSC2— and TSC2++ cells that were serum deprived for 2 hr and treated with 20nM rapamycin for 24 hr, as measured by western blot analysis. (D) IGF2 protein levels did not change in primary human LAM cells (LAM 111, LAM 105, LAM116) that were serum deprived for 2 hr and treated with 10nM rapamycin for 16 hr, as measured by western blot analysis. Images are representative of western blot analysis performed at least in three separate experiments.</p

RhoA is essential for two stages of platelet production.

<p>RhoA coordinates cytokinesis of promegakaryocytes and endomitosis of megakaryocytes by regulating effectors that control the actin contractile ring. The contractile ring underlies and constricts the cleavage furrow, which facilitates cell division. Another potential site of regulation is the ROCK-myosin pathway during thrombopoiesis. Actomyosin forces limit proplatelet formation, which ultimately controls platelet size.</p

Mice with targeted deletion of RhoA in megakaryocytes and in platelets exhibit macrothrombocytopenia and impaired MLC phosphorylation.

<p>(A) The RhoA transgene construct contains loxP sites flanking exon three. These mice were crossed with mice expressing PF4 promoter-driven CRE recombinase. (B) Western blotting confirmed that platelets from RhoA^fl/fl PF4CRE⁺ positive mice did not express detectable RhoA protein, but had normal amounts of Rac1 and CDC42. (C) Cell blood counts were normal, except that the RhoA^fl/fl PF4CRE⁺ mice were macrothrombocytopenic. * indicates p<0.005, ** indicates p<0.05. The immunoblot of thrombin-treated platelets were probed with antibodies against the phospho-MLC2 Thr¹⁸ (D) or Ser¹⁹ (E). Phosphorylation of the MLC2 Ser¹⁹ residue was normal (D), but phosphorylation of the MLC2 Thr¹⁸ residue was undetectable in RhoA-null platelets for all stimulation times (E).</p

Platelet production in RhoA-null megakaryocytes is impaired.

<p>(A) Mice were injected with GPIbα antibodies, and platelet levels were counted manually from blood smears. While the control mice began to recover from the depletion after two days, RhoA^fl/fl PF4CRE⁺ mice did not begin to recover until after four days. Mean ±SE is shown; * indicates P<0.05; ** indicates P<0.005. N = 5. (B) The platelet survival of infusing either RhoA-null megakaryocytes or controls into the mαIIb^−/−/hαIIb^+/+ mice was measured. Platelet production of the infused RhoA-null megakaryocytes diminished over a 24-hour period of time as compared to the controls (* indicates P<0.005). The mean ±SE is shown. N = 5. The red line shows the “best fit” trajectory of platelet depletion during the first eight hours, while the blue line approximates the platelet clearance rate from eight hours onward.</p

Large RhoA-null megakaryocytes are less compliant than normal sized megakaryocytes.

<p>(A) Pictured are representative cells being aspirated with micropipettes (ΔP 0.3–5.7 kPa) causing the cell membrane protrusion (arrows) to extend in length over time. Compliance (J) was computed from the length of the protrusion, and plotted as a function of time. This data exhibited power law behavior: J(t) = A*(x/x₀)∧B, where A determines elasticity and B determines rheostatic properties. (B) When the data was not normalized by size, RhoA^fl/fl PF4CRE⁺ derived megakaryocytes were less compliant than the control RhoA^fl/fl PF4CRE⁻ derived megakaryocytes at all times tested. Paired t-test analysis demonstrated that P<0.05 for time points indicated by a "*". (C) When only similarly sized megakaryocytes were compared (large controls vs. KO), RhoA-null megakaryocytes had similar compliance curves to that of their controls. Control RhoA^fl/fl PF4CRE⁻: n = 25. RhoA^fl/fl PF4CRE⁺: n = 22. Shown is the mean ± standard deviation for three independent experiments. Paired t-test analysis demonstrated that P>0.05 at all analyzed time points. (D) The basis for normalizing elasticity with size was established as control megakaryocyte elasticity was bimodally distributed, segregating into two groups, WT1 and WT2. Based upon the WT threshold criterion for elasticity, RhoA-null megakaryocytes (KO) were segregated into two groups, KO1 and KO2. The differences in membrane compliance can be entirely accountable by the differences in the sizes of wild type and mutant megakaryocytes.</p

RhoA deletion disrupts megakaryocyte development.

<p>(A) The differences in spleen weights were not statistically significant. (B) When <i>adult</i> RhoA-null platelets were infused into mαIIb^−/−/hαIIb^+/+ mice, the time course of platelet survival was similar to that of their controls. Differences were not statistically significant. Shown is the mean ±SE (N = 3). Values were normalized to platelet levels at the initial draw for each experimental group (30 minutes after infusion). (C) Flow cytometry revealed that the megakaryocyte population, as a percentage of the total bone marrow cells, was diminished in the RhoA^fl/fl PF4CRE⁺mice. Megakaryocytes were discriminated by size and by the use of a FITC-labeled anti-CD41 antibody. Mean ± SE is shown. N = 3. (D) On the top is a representative image of a normal megakaryocyte, and on the bottom is a megakaryocyte (arrow) undergoing apoptosis that is exhibiting dark, pyknotic nuclei and scant cytoplasm. In the bone marrow and in the spleen of RhoA^fl/fl PF4CRE⁺ mice, megakaryocytes are more likely to exhibit this phenotype. Magnification is 40×. (E) The RhoA-null megakaryocyte population has a higher ploidy than normal. Ploidy was calculated by DNA staining with propidium iodide, and we measured staining intensity with flow cytometry. Mean ± SE is shown; * indicates P<0.05. N = 3. (F) Megakaryocyte size (forward scatter) was measured as a function of ploidy. The results show that RhoA-null megakaryocytes were larger than the control cells at all ploidy. Mean ± SE is shown; * indicates P<0.05; ** indicates P<0.01. N = 4.</p