Impact of p53 Status on Radiosensitization of Tumor Cells by MET Inhibition-Associated Checkpoint Abrogation

Signaling via the MET receptor tyrosine kinase has been implicated in crosstalk with cellular responses to DNA damage. Our group previously demonstrated that MET inhibition in tumor cells with deregulated MET activity results in radiosensitization via downregulation of the ATR-CHK1-CDC25 pathway, a major signaling cascade responsible for intra-S and G2/M cell cycle arrest following DNA damage. Here we aimed at studying the potential therapeutic application of ionizing radiation in combination with a MET inhibitor, EMD-1214063, in p53-deficient cancer cells that harbor impaired G1/S checkpoint regulation upon DNA damage. We hypothesized that upon MET inhibition, p53-deficient cells would bypass both G1/S and G2/M checkpoints, promoting premature mitotic entry with substantial DNA lesions and cell death in a greater extent than p53-proficient cells. Our data suggest that p53-deficient cells are more susceptible to EMD-1214063 and combined treatment with irradiation than wildtype p53 lines as inferred from elevated γH2AX expression and increased cytotoxicity. Furthermore, cell cycle distribution profiling indicates constantly lower G1 and higher G2/M population as well as higher expression of a mitotic marker p-histone H3 following the dual treatment in p53 knockdown isogenic variant, compared to the parental counterpart. IMPLICATIONS The concept of MET inhibition-mediated radiosensitization enhanced by p53 deficiency is of high clinical relevance, since p53 is frequently mutated in numerous types of human cancer. The current data point for a therapeutic advantage for an approach combining MET targeting along with DNA damaging agents for MET positive/p53 negative tumors

MicroRNAs interfere with DNA methylation in rheumatoid arthritis synovial fibroblasts

BACKGROUND The DNA of rheumatoid arthritis synovial fibroblasts (RASF) is globally hypomethylated; this contributes to an aggressive behaviour. In an attempt to remethylate these cells, we supplemented with methyl donors. We investigated the possible interference of microRNAs (miRs). MATERIAL AND METHODS RASF were treated with L-methionine or betaine. Transcripts of de novo methyltransferases (DNMTs) and miRs were measured by real-time PCR, and a transcription PCR array was performed. Levels of homocysteine, matrix metalloproteinase-1 (MMP-1) and global DNA methylation were determined. Transfection with lipofectamine was performed with specific pre-miRs and anti-miRs, such as miR29 and let7f. RESULTS L-methionine was more efficient to increase DNA methylation than betaine. This was associated with a reduced expression of DNMT3A mRNA in betaine-treated RASF. Betaine increases the expression of miR29 in RASF which targets DNMT3A, thereby limiting the remethylation process. Nevertheless, betaine inhibited the expression of multiple transcription factors, decreased the release of MMP-1, biosynthesis of homocysteine and cell migration. CONCLUSION Alterations in cellular miRs profiles, in particular the upregulation of miR29, which targets DNMT3A, may limit the efficiency of betaine if it is used as DNA remethylating agent. However, L-methionine also has similar impact on miR29 expression. On the other hand, betaine has multiple other beneficial effects on the activated phenotype of RASF; it is not excluded that the effect of betaine on DNMT3A is, at least in part, indirect. Clinical trials with betaine could be promising

Deciphering MET-dependent modulation of global cellular responses to DNA damage by quantitative phosphoproteomics.

Increasing evidence suggests that interference with growth factor receptor tyrosine kinases (RTKs) signaling can affect DNA damage response (DDR) networks, with a consequent impact on cellular responses to DNA-damaging agents widely used in cancer treatment. In that respect, the MET RTK is deregulated in abundance and/or activity in a variety of human tumors. Using two proteomic techniques, we explored how disrupting MET signaling modulates global cellular phosphorylation response to ionizing radiation (IR). Following an immunoaffinity-based phosphoproteomic discovery survey we selected candidate phosphorylation sites for extensive characterization by targeted proteomics focusing on phosphorylation sites in both signaling networks. Several substrates of the DDR were confirmed to be modulated by sequential MET inhibition and IR, or MET inhibition alone. Upon combined treatment, for two substrates, NUMA1 S395 and CHEK1 S345, the gain and loss of phosphorylation, respectively, were recapitulated using in vivo tumor models by immunohistochemistry, with possible utility in future translational research. Overall, we have corroborated phosphorylation sites at the intersection between MET and the DDR signaling networks, and suggest that these represent a class of proteins at the interface between oncogene-driven proliferation and genomic stability

Identification of a MET-eIF4G1 translational regulation axis that controls HIF-1α levels under hypoxia.

Poor oxygenation is a common hallmark of solid cancers that strongly associates with aggressive tumor progression and treatment resistance. While a hypoxia-inducible factor 1α (HIF-1α)-associated transcriptional overexpression of the hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK) MET has been previously documented, any regulation of the HIF-1α system through MET downstream signaling in hypoxic tumors has not been yet described. By using MET-driven in vitro as well as ex vivo tumor organotypic fresh tissue models we report that MET targeting results in depletion of HIF-1α and its various downstream targets. Mechanistically, we provide evidence that MET regulates HIF-1α levels through a protein translation mechanism that relies on phosphorylation modulation of the eukaryotic initiation factor 4G1 (eIF4G1) on serine 1232 (Ser-1232). Targeted phosphoproteomics data demonstrate a significant drop in eIF4G1 Ser-1232 phosphorylation following MET targeting, which is linked to an increased affinity between eIF4G1 and eIF4E. Since phosphorylation of eIF4G1 on Ser-1232 is largely mediated through mitogen-activated protein kinase (MAPK), we show that expression of a constitutively active K-RAS variant is sufficient to abrogate the inhibitory effect of MET targeting on the HIF-1α pathway with subsequent resistance of tumor cells to MET targeting under hypoxic conditions. Analysis of The Cancer Genome Atlas data demonstrates frequent co-expression of MET, HIF-1α and eIF4G1 in various solid tumors and its impact on disease-free survival of non-small cell lung cancer patients. Clinical relevance of the MET-eIF4G1-HIF-1α pathway is further supported by a co-occurrence of their expression in common tumor regions of individual lung cancer patients