RESUMO
The poly(rC) binding protein 1 gene (PCBP1) encodes the heterogeneous nuclear ribonucleoprotein E1 (hnRNPE1), a nucleic acid-binding protein that plays a tumor-suppressive role in the mammary epithelium by regulating phenotypic plasticity and cell fate. Following the loss of PCBP1 function, the FAM3C gene (encoding the Interleukin-like EMT inducer, or "ILEI" protein) and the leukemia inhibitory factor receptor (LIFR) gene are upregulated. Interaction between FAM3C and LIFR in the extracellular space induces phosphorylation of signal transducer and activator of transcription 3 (pSTAT3). Overexpression and/or hyperactivity of STAT3 has been detected in 40% of breast cancer cases and is associated with a poor prognosis. Herein, we characterize feed-forward regulation of LIFR expression in response to FAM3C/LIFR/STAT3 signaling in mammary epithelial cells. We show that PCBP1 upregulates LIFR transcription through activity at the LIFR promoter, and that FAM3C participates in transcriptional regulation of LIFR. Additionally, our bioinformatic analysis reveals a signature of transcriptional regulation associated with FAM3C/LIFR interaction and identifies the TWIST1 transcription factor as a downstream effector that participates in the maintenance of LIFR expression. Finally, we characterize the effect of LIFR expression in cell-based experiments that demonstrate the promotion of invasion, migration, and self-renewal of breast cancer stem cells (BCSCs), consistent with previous studies linking LIFR expression to tumor initiation and metastasis in mammary epithelial cells.
Assuntos
Neoplasias da Mama , Proteínas de Ligação a DNA , Proteínas de Ligação a RNA , Feminino , Humanos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Autorrenovação Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Proteínas de Neoplasias/genética , Receptores de OSM-LIF/genética , Receptores de OSM-LIF/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Invasividade NeoplásicaRESUMO
Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) is a tumor suppressor protein that binds site- and structure-specifically to RNA sequences to regulate mRNA stability, facilitate alternative splicing, and suppress protein translation on several metastasis-associated mRNAs. Here, we show that hnRNP E1 binds polycytosine-rich DNA tracts present throughout the genome, including those at promoters of several oncogenes and telomeres and monitors genome integrity. It binds DNA in a site- and structure-specific manner. hnRNP E1-knockdown cells displayed increased DNA damage signals including γ-H2AX at its binding sites and also showed increased mutations. UV and hydroxyurea treatment of hnRNP E1-knockdown cells exacerbated the basal DNA damage signals with increased cell cycle arrest, activation of checkpoint proteins, and monoubiquitination of proliferating cell nuclear antigen despite no changes in deubiquitinating enzymes. DNA damage caused by genotoxin treatment localized to hnRNP E1 binding sites. Our work suggests that hnRNP E1 facilitates functions of DNA integrity proteins at polycytosine tracts and monitors DNA integrity at these sites.
Assuntos
Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Instabilidade Genômica , Poli C , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , DNA/química , DNA/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Humanos , Camundongos , Modelos Biológicos , Mutação , Taxa de Mutação , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Poli C/química , Ligação Proteica , Transdução de SinaisRESUMO
The cardiac transcription factor Nkx2-5 is essential for normal outflow tract (OFT) and right ventricle (RV) development. Nkx2-5-/- null mouse embryos display severe OFT and RV hypoplasia and a single ventricle phenotype due to decreased proliferation of Second Heart Field (SHF) cells, a pool of cardiac progenitors present in anterior pharyngeal arch mesoderm at mid-gestation. However, definition of the precise role of Nkx2-5 in facilitating SHF expansion is incomplete. We have found that Nkx2-5 positively and directly regulates a novel target gene, Ccdc117, in cells of the SHF at these stages. The nuclear/mitotic spindle associated protein Ccdc117 interacts with the MIP18/MMS19 cytoplasmic iron-sulfur (FeS) cluster assembly (CIA) complex, which transfers critical FeS clusters to several key enzymes with functions in DNA repair and replication. Loss of cellular Ccdc117 expression results in reduced proliferation rates associated with a delay at the G1-S transition, decreased rates of DNA synthesis, and unresolved DNA damage. These results implicate a novel role for Nkx2-5 in the regulation of cell cycle events in the developing heart, through Ccdc117's interaction with elements of the CIA pathway and the facilitation of DNA replication during SHF expansion.