PHRF1 Promotes Cell Invasion by Modulating SOX4 Expression in Colorectal Cancer HCT116-p53-/- Cells.

BACKGROUND/AIM: PHD and RING finger domain-containing protein 1 (PHRF1) ubiquitinates TGIP (TG-interacting protein) and redistributes cPML (cytoplasmic variant of PML) to the cytoplasm to enhance TGF-ß signaling by. It is unclear whether PHRF1 affects invasion and survival when both mutations of the activated oncogene Kras and inactivation of the tumor suppressor p53 are present. MATERIALS AND METHODS: We knockout PHRF1 expression using Crispr-Cas9 editing in HCT116-p53-/- (KrasG13D/p53-/-) cells and analyzed the expression profile in HCT116-p53-/-PHRF1-/- cells. RESULTS: In contrast to lung cancer A549 (KrasG12S/p53wt) cells, the expression of Zeb1, a transcription factor for epidermal-mesenchymal transition (EMT), was not affected in PHRF1-knockout HCT116 p53-/- cells. Instead, SOX4 displayed a significant contribution to the impaired invasion in HCT116-p53-/-PHRF1-/- cells. Mechanistically, the C-terminal SRI domain of PHRF1 was required for both transwell invasion and SOX4 expression. The reintroduction of SOX4 into HCT116-p53-/- PHRF1-/- cells partially restored their invasive capability. CONCLUSION: This study sheds light on the role of PHRF1 in the invasion of colorectal cancer HCT116-p53-/- cells, which harbor the oncogenic KrasG13D mutation and lack p53. These findings provide novel insights regarding the role of PHRF1 in invasion by modulating SOX4 expression in colorectal cancer HCT116-p53-/- cells.

HDGF stimulates liver tumorigenesis by enhancing reactive oxygen species generation in mitochondria.

Hepatoma-derived growth factor (HDGF) overexpression and uncontrolled reactive oxygen species (ROS) accumulation are involved in malignant transformation and poor prognosis in various types of cancer. However, the interplay between HDGF and ROS generation has not been elucidated in hepatocellular carcinoma. Here, we first analyzed the profile of HDGF expression and ROS production in newly generated orthotopic hepatomas by ultrasound-guided implantation. In situ superoxide detection showed that HDGF-overexpressing hepatomas had significantly elevated ROS levels compared with adjacent nontumor tissues. Consistently, liver tissues from HDGF-deficient mice exhibited lower ROS fluorescence than those from age- and sex-matched WT mice. ROS-detecting fluorescent dyes and flow cytometry revealed that recombinant HDGF (rHDGF) stimulated the production of superoxide anion, hydrogen peroxide, and mitochondrial ROS generation in cultured hepatoma cells in a dose-dependent manner. In contrast, the inactive Ser103Ala rHDGF mutant failed to promote ROS generation or oncogenic behaviors. Seahorse metabolic flux assays revealed that rHDGF dose dependently upregulated bioenergetics through enhanced basal and total oxygen consumption rate, extracellular acidification rate, and oxidative phosphorylation in hepatoma cells. Moreover, antioxidants of N-acetyl cysteine and MitoQ treatment significantly inhibited HDGF-mediated cell proliferation and invasive capacity. Genetic silencing of superoxide dismutase 2 augmented the HDGF-induced ROS generation and oncogenic behaviors of hepatoma cells. Finally, genetic knockdown nucleolin (NCL) and antibody neutralization of surface NCL, the HDGF receptor, abolished the HDGF-induced increase in ROS and mitochondrial energetics. In conclusion, this study has demonstrated for the first time that the HDGF/NCL signaling axis induces ROS generation by elevating ROS generation in mitochondria, thereby stimulating liver carcinogenesis.

Phosphorylation of PUF-A/PUM3 on Y259 modulates PUF-A stability and cell proliferation.

Human PUF-A/PUM3 is a RNA and DNA binding protein participating in the nucleolar processing of 7S to 5.8S rRNA. The nucleolar localization of PUF-A redistributes to the nucleoplasm upon the exposure to genotoxic agents in cells. However, little is known regarding the roles of PUF-A in tumor progression. Phosphoprotein database analysis revealed that Y259 phosphorylation of PUF-A is the most prevalent residue modified. Here, we reported the importance of PUF-A's phosphorylation on Y259 in tumorigenesis. PUF-A gene was knocked out by the Crispr/Cas9 method in human cervix epithelial HeLa cells. Loss of PUF-A in HeLa cells resulted in reduced clonogenic and lower transwell invasion capacity. Introduction of PUF-AY259F to PUF-A deficient HeLa cells was unable to restore colony formation. In addition, the unphosphorylated mutant of PUF-A, PUF-AY259F, attenuated PUF-A protein stability. Our results suggest the important role of Y259 phosphorylation of PUF-A in cell proliferation.

An important role of PHRF1 in dendritic architecture and memory formation by modulating TGF-ß signaling.

PHRF1 is involved in transforming growth factor ß (TGF-ß) signaling to constrain the formation of acute promyelocytic leukemia (APL) in mouse APL models. PHRF1 also participates in modulating non-homologous end-joining. However, the role of PHRF1 in mammalian dendrite architecture and synaptic plasticity is unclear. Here, we investigated the role of PHRF1 in dendritic formation in the murine hippocampus using Camk2a promoter driven-iCre recombinase to conduct a PHRF1 conditional knockout, namely PHRF1Δ/Δ, in the forebrain region. PHRF1Δ/Δ mice developed normally, but exhibited anxiety-like behaviors and displayed defective spatial memory. Alterations of dendritic complexity in apical and basal dendrites of pyramidal neurons were noticed in PHRF1Δ/Δ mutants. Furthermore, electrical stimulation in the hippocampal CA1 region after the TGF-ß1 treatment showed a reduced synaptic plasticity in PHRF1Δ/Δ mice. Immunoblotting analysis indicated that PHRF1 ablation affected the TGF-ß signaling. Collectively, our results demonstrate that PHRF1 is important for the dendritic architecture and required for spatial memory formation in the hippocampus.