Key Points Abnormal signatures in TGF-β1 signaling gene expression were identified in spleen and marrow from the Gata1low model of MF. These signatures include abnormalities in individual gene (Id2, Stat1, mTOR) in spleen and of gene pathways (Smads and BMPs) in marrow.
The fragile X mental retardation protein (FMRP) is lacking or mutated in patients with the fragile X syndrome (FXS), the most frequent form of inherited intellectual disability. FMRP affects metastasis formation in a mouse model for breast cancer. Here we show that FMRP is overexpressed in human melanoma with high Breslow thickness and high Clark level. Furthermore, meta-analysis of the TCGA melanoma data revealed that high levels of FMRP expression correlate significantly with metastatic tumor tissues, risk of relapsing and disease-free survival. Reduction of FMRP in metastatic melanoma cell lines impinges on cell migration, invasion and adhesion. Next-generation sequencing in human melanoma cells revealed that FMRP regulates a large number of mRNAs involved in relevant processes of melanoma progression. Our findings suggest an association between FMRP levels and the invasive phenotype in melanoma and might open new avenues towards the discovery of novel therapeutic targets.
Splenomegaly is a major manifestation of primary myelofibrosis (PMF) contributing to clinical symptoms and hematologic abnormalities. The spleen from PMF patients contains increased numbers of hematopoietic stem cells (HSC) and megakaryocytes (MK). These MK express high levels of P-selectin (P-sel) that, by triggering neutrophil emperipolesis, may cause TGF-b release and disease progression. This hypothesis was tested by deleting the P-sel gene in the myelofibrosis mouse model carrying the hypomorphic Gata1 low mutation that induces megakaryocyte abnormalities that recapitulate those observed in PMF. P-sel null Gata1 low mice survived splenectomy and lived 3 months longer than P-sel WT Gata1low littermates and expressed limited fibrosis and osteosclerosis in the marrow or splenomegaly. Furthermore, deletion of P-sel disrupted megakaryocyte/neutrophil interactions in spleen, reduced TGF-b content, and corrected the HSC distribution that in Gata1 low mice, as in PMF patients, is abnormally expanded in spleen. Conversely, pharmacological inhibition of TGF-b reduced P-sel expression in MK and corrected HSC distribution. Spleens, but not marrow, of Gata1 low mice contained numerous cKIT pos activated fibrocytes, probably of dendritic cell origin, whose membrane protrusions interacted with MK establishing niches hosting immature cKIT pos hematopoietic cells. These activated fibrocytes were not detected in spleens from P-sel null Gata1 low or TGF-b-inhibited Gata1 low littermates and were observed in spleen, but not in marrow, from PMF patients. Therefore, in Gata1 low mice, and possibly in PMF, abnormal P-sel expression in MK may mediate the pathological cell interactions that increase TGF-b content in MK and favor establishment of a microenvironment that supports myelofibrosis-related HSC in spleen. STEM CELLS 2016;34:67-82 SIGNIFICANCE STATEMENTIn this manuscript, we describe experiments aimed at defining mechanisms by which splenic hematopoiesis dominates in a mouse model of primary myelofibrosis (PMF). We show that a unique stem cell niche is formed by progeny of hematopoietic stem cells carrying an enhancer deletion in the gene encoding Gata1. The mutant niche is shown to be central to the myelofibrosis phenotype, and is dependent upon TGF-b signaling and requires P-selectin to mediate cell-cell interactions. These results are presented in the context of cancer-supporting environments (niches) with the novel observation that in this model, the mutant hematopoietic stem cell generates progeny that participate in the formation of the niche. Analysis of human PMF corroborates our mouse studies.
The fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the absence of FMRP, a protein regulating RNA metabolism. Recently, an unexpected function of FMRP in modulating the activity of Adenosine Deaminase Acting on RNA (ADAR) enzymes has been reported both in Drosophila and Zebrafish. ADARs are RNA-binding proteins that increase transcriptional complexity through a post-transcriptional mechanism called RNA editing.To evaluate the ADAR2-FMRP interaction in mammals we analyzed several RNA editing re-coding sites in the fmr1 knockout (KO) mice. Ex vivo and in vitro analysis revealed that absence of FMRP leads to an increase in the editing levels of brain specific mRNAs, indicating that FMRP might act as an inhibitor of editing activity. Proximity Ligation Assay (PLA) in mouse primary cortical neurons and in non-neuronal cells revealed that ADAR2 and FMRP co-localize in the nucleus. The ADAR2-FMRP co-localization was further observed by double-immunogold Electron Microscopy (EM) in the hippocampus. Moreover, ADAR2-FMRP interaction appeared to be RNA independent.Because changes in the editing pattern are associated with neuropsychiatric and neurodevelopmental disorders, we propose that the increased editing observed in the fmr1-KO mice might contribute to the FXS molecular phenotypes.
Myelofibrosis (MF) is characterized by hyperactivation of thrombopoietin (TPO) signaling, which induces a RPS14 deficiency that de-regulates GATA1 in megakaryocytes by hampering its mRNA translation. As mice carrying the hypomorphic Gata1low mutation, which reduces the levels of Gata1 mRNA in megakaryocytes, develop MF, we investigated whether the TPO axis is hyperactive in this model. Gata1low mice contained two times more Tpo mRNA in liver and TPO in plasma than wild-type littermates. Furthermore, Gata1low LSKs expressed levels of Mpl mRNA (five times greater than normal) and protein (two times lower than normal) similar to those expressed by LSKs from TPO-treated wild-type mice. Gata1low marrow and spleen contained more JAK2/STAT5 than wild-type tissues, an indication that these organs were reach of TPO-responsive cells. Moreover, treatment of Gata1low mice with the JAK inhibitor ruxolitinib reduced their splenomegaly. Also in Gata1low mice activation of the TPO/MPL axis was associated with a RSP14 deficiency and a discordant microarray ribosome signature (reduced RPS24, RPS26 and SBDS expression). Finally, electron microscopy revealed that Gata1low megakaryocytes contained poorly developed endoplasmic reticulum with rare polysomes. In summary, Gata1low mice are a bona fide model of MF, which recapitulates the hyperactivation of the TPO/MPL/JAK2 axis observed in megakaryocytes from myelofibrotic patients.
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