Conditional Inactivation of Pten with EGFR Overexpression in Schwann Cells Models Sporadic MPNST

The genetic mechanisms involved in the transformation from a benign neurofibroma to a malignant sarcoma in patients with neurofibromatosis-type-1- (NF1-)associated or sporadic malignant peripheral nerve sheath tumors (MPNSTs) remain unclear. It is hypothesized that many genetic changes are involved in transformation. Recently, it has been shown that both phosphatase and tensin homolog (PTEN) and epidermal growth factor receptor (EGFR) play important roles in the initiation of peripheral nerve sheath tumors (PNSTs). In human MPNSTs, PTEN expression is often reduced, while EGFR expression is often induced. We tested if these two genes cooperate in the evolution of PNSTs. Transgenic mice were generated carrying conditional floxed alleles of Pten, and EGFR was expressed under the control of the 2′,3′-cyclic nucleotide 3′phosphodiesterase (Cnp) promoter and a desert hedgehog (Dhh) regulatory element driving Cre recombinase transgenic mice (Dhh-Cre). Complete loss of Pten and EGFR overexpression in Schwann cells led to the development of high-grade PNSTs. In vitro experiments using immortalized human Schwann cells demonstrated that loss of PTEN and overexpression of EGFR cooperate to increase cellular proliferation and anchorage-independent colony formation. This mouse model can rapidly recapitulate PNST onset and progression to high-grade PNSTs, as seen in sporadic MPNST patients

Overexpression of HGF/MET axis along with p53 inhibition induces de novo glioma formation in mice

BACKGROUND: Aberrant MET receptor tyrosine kinase (RTK) activation leads to invasive tumor growth in different types of cancer. Overexpression of MET and its ligand hepatocyte growth factor (HGF) occurs more frequently in glioblastoma (GBM) than in low-grade gliomas. Although we have shown previously that HGF-autocrine activation predicts sensitivity to MET tyrosine kinase inhibitors (TKIs) in GBM, whether it initiates tumorigenesis remains elusive. METHODS: Using a well-established Sleeping Beauty (SB) transposon strategy, we injected human and cDNA together with a short hairpin siRNA against (SB-hHgf.Met.ShP53) into the lateral ventricle of neonatal mice to induce spontaneous glioma initiation and characterized the tumors with H&E and immunohistochemistry analysis. Glioma sphere cells also were isolated for measuring the sensitivity to specific MET TKIs. RESULTS: Mixed injection of SB-hHgf.Met.ShP53 plasmids induced de novo glioma formation with invasive tumor growth accompanied by HGF and MET overexpression. While glioma stem cells (GSCs) are considered as the tumor-initiating cells in GBM, both SB-hHgf.Met.ShP53 tumor sections and glioma spheres harvested from these tumors expressed GSC markers nestin, GFAP, and Sox 2. Moreover, specific MET TKIs significantly inhibited tumor spheres\u27 proliferation and MET/MAPK/AKT signaling. CONCLUSIONS: Overexpression of the HGF/MET axis along with p53 attenuation may transform neural stem cells into GSCs, resulting in GBM formation in mice. These tumors are primarily driven by the MET RTK pathway activation and are sensitive to MET TKIs. The SB-hHgf.Met.ShP53 spontaneous mouse glioma model provides a useful tool for studying GBM tumor biology and MET-targeting therapeutics

Identification of <i>Rtl1</i>, a Retrotransposon-Derived Imprinted Gene, as a Novel Driver of Hepatocarcinogenesis

<div><p>We previously utilized a Sleeping Beauty (SB) transposon mutagenesis screen to discover novel drivers of HCC. This approach identified recurrent mutations within the <i>Dlk1-Dio3</i> imprinted domain, indicating that alteration of one or more elements within the domain provides a selective advantage to cells during the process of hepatocarcinogenesis. For the current study, we performed transcriptome and small RNA sequencing to profile gene expression in SB–induced HCCs in an attempt to clarify the genetic element(s) contributing to tumorigenesis. We identified strong induction of <i>Retrotransposon-like 1</i> (<i>Rtl1</i>) expression as the only consistent alteration detected in all SB–induced tumors with <i>Dlk1-Dio3</i> integrations, suggesting that <i>Rtl1</i> activation serves as a driver of HCC. While previous studies have identified correlations between disrupted expression of multiple <i>Dlk1-Dio3</i> domain members and HCC, we show here that direct modulation of a single domain member, <i>Rtl1</i>, can promote hepatocarcinogenesis <i>in vivo</i>. Overexpression of <i>Rtl1</i> in the livers of adult mice using a hydrodynamic gene delivery technique resulted in highly penetrant (86%) tumor formation. Additionally, we detected overexpression of <i>RTL1</i> in 30% of analyzed human HCC samples, indicating the potential relevance of this locus as a therapeutic target for patients. The <i>Rtl1</i> locus is evolutionarily derived from the domestication of a retrotransposon. In addition to identifying <i>Rtl1</i> as a novel driver of HCC, our study represents one of the first direct <i>in vivo</i> demonstrations of a role for such a co-opted genetic element in promoting carcinogenesis.</p> </div

Integrated transposons drive overexpression of <i>Rtl1</i>.

<p>(A) Strand-specific RT-PCR detected activation of <i>Rtl1</i> expression in SB-induced HCCs, with minimal activation of <i>Rtl1as</i> observed. No expression of either transcript was detected in normal liver. (B) Transposons integrated upstream of <i>Rtl1</i> drive its expression by generating fusion transcripts. Transcription initiated from the CAG promoter within the transposon splices into <i>Rtl1</i>, either directly or via inclusion of an upstream cryptic exon. PCR to detect fusion transcripts was performed on cDNA from SB-induced HCCs using the indicated primers. Transposon-driven expression of <i>Rtl1</i> was detected for all of the tumor samples harboring <i>Dlk1-Dio3</i> domain integrations. sd, splice donor.</p

Rtl1 promotes growth of cultured hepatocytes in extracellular matrix.

<p>Two weeks after plating cultured hepatocytes in a matrix of Matrigel, cells transfected with an empty vector construct (A) failed to grow significantly. Cells transfected with an <i>Rtl1</i> expression construct (B) grew to form several large colonies. (C–D) Rtl1 promotes growth of large cyst-like structures. Increased magnification reveals that cells lacking Rtl1 (C) form small, dense colonies, while those expressing Rtl1 (D) form large cyst-like colonies composed of several cells. (E) Quantification of colonies per well formed by each cell line in a 24-well plate. The results depicted are based on three experimental replicates per condition and are representative of experiments conducted on three separate days. Scale bars = 0.5 cm (A–B) and 100 µm (C–D).</p

<i>Dlk1-Dio3</i> domain transposon integration sites in SB–induced HCC and effects on domain expression.

<p>(A) The <i>Dlk1-Dio3</i> imprinted domain spans ∼800 kilobases at the distal end of mouse chromosome 12 (human chr14q32). Three protein-coding genes are expressed from the paternal allele (<i>Dlk1</i>, <i>Rtl1</i>, and <i>Dio3</i>). The maternal allele encodes four lncRNAs (<i>Meg3</i>, <i>Rtl1as</i>, <i>Rian</i>, and <i>Mirg</i>), as well as several miRNAs and snoRNAs. SB transposon and AAV integration sites found to be associated with HCC development in mice are depicted. (B) Intensity plot showing normalized expression levels of long transcripts within and surrounding the <i>Dlk1-Dio3</i> domain in SB-induced HCCs and normal livers. (C) Intensity plot showing normalized expression of <i>Dlk1-Dio3</i> domain miRNAs. miRNAs contained within lncRNAs are indicated. miRNAs with no detected expression across all samples were omitted.</p

Expression of <i>RTL1</i> in human HCC.

<p>(A) <i>RTL1</i> expression in human HCC and matched benign liver samples was analyzed by RT-PCR. Plotted values represent normalized band intensities from imaged gels. The threshold above which a sample was scored as positive for significant <i>RTL1</i> expression (dashed line) was set at three standard deviations above the average intensity value in benign samples lacking detectable expression. For the one patient with significant <i>RTL1</i> expression detected in benign tissue, the matched HCC sample also displayed expression (indicated with arrows). (B) Plot of <i>RTL1</i> expression in human HCC and normal liver samples based on RNASeq data available through TCGA. The threshold above which a sample was scored as positive for significant <i>RTL1</i> expression (dashed line) was set at one standard deviation above the average expression level in tumor-free liver. RSEM, RNASeq by Expectation Maximization.</p

<i>Rtl1</i>-expressing mouse HCCs resemble human S1 subclass.

<p>Expression levels for the gene sets defining human HCC subclasses S1, S2, and S3 were analyzed in SB-induced HCCs and normal livers. Gene Set Enrichment Analysis (GSEA) was conducted for each subclass independently to assess the significance of differential expression between tumor and normal samples. Heat maps generated by GSEA are shown. This analysis revealed a significant (p = 0.039) overexpression of the genes defining human subclass S1 in SB-induced HCCs, as compared to normal liver.</p

<i>In vivo</i> hepatic overexpression of <i>Rtl1</i> promotes tumorigenesis.

<p>(A–C) Macroscopic images of tumor-containing whole livers from mice injected with <i>Rtl1</i> overexpression constructs via hydrodynamic tail vein injection. Mice were euthanized and livers collected nine months post-injection. (D) A normal liver from a hydrodynamically injected mouse is shown for comparison. Scale bars = 1 cm.</p

Insertional Mutagenesis Identifies a STAT3/Arid1b/β-catenin Pathway Driving Neurofibroma Initiation

To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1) neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/β-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs) and Schwann cells (SCs) prevents neurofibroma formation, decreasing SCP self-renewal and β-catenin activity. β-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and β-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3β and the SWI/SNF gene Arid1b to increase β-catenin. Knockdown of Arid1b or Gsk3β in Stat3fl/fl;Nf1fl/fl;DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/β-catenin pathway inhibitors in neurofibroma therapeutic trials