A DNA-PK phosphorylation site on MET regulates its signaling interface with the DNA damage response.

The DNA damage response (DDR) is intertwined with signaling pathways downstream of oncogenic receptor tyrosine kinases (RTKs). To drive research into the application of targeted therapies as radiosensitizers, a better understanding of this molecular crosstalk is necessary. We present here the characterization of a previously unreported MET RTK phosphosite, Serine 1016 (S1016) that represents a potential DDR-MET interface. MET S1016 phosphorylation increases in response to irradiation and is mainly targeted by DNA-dependent protein kinase (DNA-PK). Phosphoproteomics unveils an impact of the S1016A substitution on the overall long-term cell cycle regulation following DNA damage. Accordingly, the abrogation of this phosphosite strongly perturbs the phosphorylation of proteins involved in the cell cycle and formation of the mitotic spindle, enabling cells to bypass a G2 arrest upon irradiation and leading to the entry into mitosis despite compromised genome integrity. This results in the formation of abnormal mitotic spindles and a lower proliferation rate. Altogether, the current data uncover a novel signaling mechanism through which the DDR uses a growth factor receptor system for regulating and maintaining genome stability.

The human vault RNA enhances tumorigenesis and chemoresistance through the lysosome in hepatocellular carcinoma.

The small non-coding VTRNA1-1 (vault RNA 1-1) is known to confer resistance to apoptosis in several malignant cell lines and to also modulate the macroautophagic/autophagic flux in hepatocytes, thus highlighting its pro-survival role. Here we describe a new function of VTRNA1-1 in regulating in vitro and in vivo tumor cell proliferation, tumorigenesis and chemoresistance. Knockout (KO) of VTRNA1-1 in human hepatocellular carcinoma cells reduced nuclear localization of TFEB (transcription factor EB), leading to a downregulation of the coordinated lysosomal expression and regulation (CLEAR) network genes and lysosomal compartment dysfunction. We demonstrate further that impaired lysosome function due to loss of VTRNA1-1 potentiates the anticancer effect of conventional chemotherapeutic drugs. Finally, loss of VTRNA1-1 reduced drug lysosomotropism allowing higher intracellular compound availability and thereby significantly reducing tumor cell proliferation in vitro and in vivo. These findings reveal a so far unknown role of VTRNA1-1 in the intracellular catabolic compartment and describe its contribution to lysosome-mediated chemotherapy resistance.Abbreviations: ATP6V0D2: ATPase H+ transporting V0 subunit d2; BafA: bafilomycin A1; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; DMSO: dimethyl sulfoxide; GST-BHMT: glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase; HCC: hepatocellular carcinoma; LAMP1: lysosomal associated membrane protein 1; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAPK: mitogen-activated protein kinase; MITF: melanocyte inducing transcription factor; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ncRNA: non-coding RNA; RNP: ribonucleoprotein; SF: sorafenib; SQSTM1/p62: sequestosome 1; STS: staurosporine; tdRs: tRNA-derived RNAs; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; vtRNA: vault RNA transcript.

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.

CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre-loxP-controlled lentiviral vectors expressing CRIPTO in cell line-derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft-derived ex vivo tumour slices. CRIPTO-overexpressing patient-derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial-to-mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2-CRIPTO cells formed tumours when injected into immune-compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High-level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO-expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Sinomenine blocks tissue remodeling in a rat model of chronic cardiac allograft rejection.

BACKGROUND: Chronic rejection (CR) with graft vasculopathy is recognized as a major cause of graft loss over time. Sinomenine (SN) has anti-inflammatory, antirheumatic, and immunomodulatory effects. Previously, we demonstrated antimacrophage and anti-T cell effects of SN in acute rejection. In the current study, we investigated the effect of SN in a rat cardiac allograft model of CR. MATERIALS AND METHODS: After a brief course of cyclosporine A (CsA), Lewis recipients of F344 hearts were treated with SN alone, CsA alone, or a combination of both drugs. Grafts were analyzed morphometrically and by immunohistochemistry. Expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and endothelin 1 was assessed by reverse transcription-polymerase chain reaction. Antidonor IgM formation was investigated by FACS. RESULTS: Cardiac grafts from SN-treated rats showed less pronounced vasculopathy in comparison with untreated rats or CsA-treated recipients. After treatment with a combination of both drugs, rats had significantly less graft vasculopathy than rats receiving either drug alone. Treatment with CsA alone led to a decrease in bFGF expression, whereas SN alone did not affect gene expression. SN in combination with CsA, however, markedly reduced expression of bFGF, vascular endothelial growth factor, and endothelin 1. SN alone did not inhibit antidonor antibody formation. CONCLUSION: These studies demonstrate for the first time the therapeutic value of SN in a model of chronic cardiac allograft rejection. SN in combination with low-dose T cell-targeted immunosuppression is effective in controlling tissue remodeling in the context of CR and is associated with inhibition of intragraft expression of mediators involved in angiogenesis, vascular tone, and tissue remodeling.

Apoptosis in hypoxic human pancreatic islets correlates with HIF-1alpha expression.

To become insulin independent, patients with type 1 diabetes mellitus require transplantation of at least two donor pancreata because of massive beta-cell loss in the early post-transplantation period. Many studies describing the introduction of new immunosuppressive protocols have shown that this loss is due to not only immunological events but also nonimmunological factors. To test to what extent hypoxia may contribute to early graft loss, we analyzed the occurrence of apoptotic events and the expression of hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-dependent alpha subunit and a constitutive beta subunit. Histological analysis of human and rat islets revealed nuclear pyknosis as early as 6 h after hypoxic exposure (1% O2). Moreover, immunoreactivity to activated caspase-3 was observed in the core region of isolated human islets. Of note, both of these markers of apoptosis topographically overlap with HIF-1alpha immunoreactivity. HIF-1alpha mRNA was detected in islets from human and rat as well as in several murine beta-cell lines. When exposed to hypoxia, mouse insulinoma cells (MIN6) had an increased HIF-1alpha protein level, whereas its mRNA level did not alter. In conclusion, our data provide convincing evidence that reduced oxygenation is an important cause of beta-cell loss and suggest that HIF-1alpha protein level is an indicator for hypoxic regions undergoing apoptotic cell death. These observations suggest that gene expression under the control of HIF-1 represents a potential therapeutic tool for improving engraftment of transplanted islets.