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

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

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