Neuregulin Signaling Is a Mechanism of Therapeutic Resistance in Head and Neck Squamous Cell Carcinoma.

EGFR signaling confers resistance to radiotherapy and is a validated target in head and neck squamous cell carcinoma (HNSCC). The inhibition of EGFR in combination with radiotherapy improves local control and overall survival in these patients; however, therapeutic resistance limits the efficacy of this approach. We therefore sought to identify cellular mechanisms that cause resistance to EGFR inhibition and radiotherapy in HNSCC. Though clonal isolation of carcinoma cells exposed to increasing concentrations of cetuximab, we found that resistant cells upregulate prosurvival ErbB3 and AKT signaling. Using EFM-19 cells and confirmatory analysis of protein levels, we demonstrate that cetuximab resistance is characterized by enhanced neuregulin expression identifying a novel adaptive mechanism of therapeutic resistance. Inhibition of this autocrine loop with CDX-3379 (an ErbB3 specific antibody) was sufficient to block ErbB3/AKT signaling in cetuximab resistant cells. The combination of CDX-3379 and cetuximab reduced proliferation and survival after radiotherapy in several HNSCC cell lines. These in vitro findings were confirmed in xenograft tumor growth experiments including an approach using growth factor-supplemented Matrigel. In vivo, the delivery of EGFR and ErbB3 antibodies significantly reduced tumor growth in cetuximab-resistant FaDu and CAL27 xenografts. In summary, this work demonstrates that autocrine NRG ligand secretion is a mechanism for therapeutic resistance to cetuximab and radiotherapy. This cross-resistance to both therapeutic modalities identifies NRG as an actionable therapeutic target for improving treatment regimens in HNSCC.

Oligosaccharyltransferase Inhibition Reduces Receptor Tyrosine Kinase Activation and Enhances Glioma Radiosensitivity.

PURPOSE: Parallel signaling reduces the effects of receptor tyrosine kinase (RTK)-targeted therapies in glioma. We hypothesized that inhibition of protein N-linked glycosylation, an endoplasmic reticulum co- and posttranslational modification crucial for RTK maturation and activation, could provide a new therapeutic approach for glioma radiosensitization.Experimental Design: We investigated the effects of a small-molecule inhibitor of the oligosaccharyltransferase (NGI-1) on EGFR family receptors, MET, PDGFR, and FGFR1. The influence of glycosylation state on tumor cell radiosensitivity, chemotherapy-induced cell toxicity, DNA damage, and cell-cycle arrest were determined and correlated with glioma cell receptor expression profiles. The effects of NGI-1 on xenograft tumor growth were tested using a nanoparticle formulation validated by in vivo molecular imaging. A mechanistic role for RTK signaling was evaluated through the expression of a glycosylation-independent CD8-EGFR chimera. RESULTS: NGI-1 reduced glycosylation, protein levels, and activation of most RTKs. NGI-1 also enhanced the radiosensitivity and cytotoxic effects of chemotherapy in those glioma cells with elevated ErbB family activation, but not in cells without high levels of RTK activation. NGI-1 radiosensitization was associated with increases in both DNA damage and G1 cell-cycle arrest. Combined treatment of glioma xenografts with fractionated radiotherapy and NGI-1 significantly reduced tumor growth compared with controls. Expression of the CD8-EGFR eliminated the effects of NGI-1 on G1 arrest, DNA damage, and cellular radiosensitivity, identifying RTK inhibition as the principal mechanism for the NGI-1 effect. CONCLUSIONS: This study suggests that oligosaccharyltransferase inhibition with NGI-1 is a novel approach to radiosensitize malignant gliomas with enhanced RTK signaling.See related commentary by Wahl and Lawrence, p. 455.

Oligosaccharyltransferase Inhibition Overcomes Therapeutic Resistance to EGFR Tyrosine Kinase Inhibitors.

Asparagine (N)-linked glycosylation is a posttranslational modification essential for the function of complex transmembrane proteins. However, targeting glycosylation for cancer therapy has not been feasible due to generalized effects on all glycoproteins. Here, we perform sensitivity screening of 94 lung cancer cell lines using NGI-1, a small-molecule inhibitor of the oligosaccharyltransferase (OST) that partially disrupts N-linked glycosylation, and demonstrate a selective loss of tumor cell viability. This screen revealed NGI-1 sensitivity in just 11 of 94 (12%) cell lines, with a significant correlation between OST and EGFR inhibitors. In EGFR-mutant non-small cell lung cancer with EGFR tyrosine kinase inhibitor (TKI) resistance (PC9-GR, HCC827-GR, and H1975-OR), OST inhibition maintained its ability to induce cell-cycle arrest and a proliferative block. Addition of NGI-1 to EGFR TKI treatment was synthetic lethal in cells resistant to gefitinib, erlotinib, or osimertinib. OST inhibition invariably disrupted EGFR N-linked glycosylation and reduced activation of receptors either with or without the T790M TKI resistance mutation. OST inhibition also dissociated EGFR signaling from other coexpressed receptors like MET via altered receptor compartmentalization. Translation of this approach to preclinical models was accomplished through synthesis and delivery of NGI-1 nanoparticles, confirmation of in vivo activity through molecular imaging, and demonstration of significant tumor growth delay in TKI-resistant HCC827 and H1975 xenografts. This therapeutic strategy breaks from kinase-targeted approaches and validates N-linked glycosylation as an effective target in tumors driven by glycoprotein signaling.Significance:EGFR-mutant NSCLC is incurable despite the marked sensitivity of these tumors to EGFR TKIs. These findings identify N-linked glycosylation, a posttranslational modification common to EGFR and other oncogenic signaling proteins, as an effective therapeutic target that enhances tumor responses for EGFR-mutant NSCLC. Cancer Res; 78(17); 5094-106. ©2018 AACR.

Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells.

Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high-throughput screen and lead-compound-optimization campaign that delivered a cell-permeable inhibitor, NGI-1. NGI-1 targets oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.

Calreticulin-dimerization induced by post-translational arginylation is critical for stress granules scaffolding.

Protein arginylation mediated by arginyl-tRNA protein transferase is a post-translational modification that occurs widely in biology, it has been shown to regulate protein and properties and functions. Post-translational arginylation is critical for embryogenesis, cardiovascular development and angiogenesis but the molecular effects of proteins arginylated in vivo are largely unknown. In the present study, we demonstrate that arginylation reduces CRT (calreticulin) thermostability and induces a greater degree of dimerization and oligomerization. R-CRT (arginylated calreticulin) forms disulfide-bridged dimers that are increased in low Ca(2+) conditions at physiological temperatures, a similar condition to the cellular environment that it required for arginylation of CRT. Moreover, R-CRT self-oligomerizes through non-covalent interactions that are enhanced at temperatures above 40 °C, condition that mimics the heat shock treatment where R-CRT is the only isoespecies of CRT that associates in cells to SGs (stress granules). We show that in cells lacking CRT the scaffolding of larger SGs is impaired; the transfection with CRT (hence R-CRT expression) restores SGs assembly whereas the transfection with CRT mutated in Cys146 does not. Thus, R-CRT disulfide-bridged dimers (through Cys146) are essential for the scaffolding of larger SGs under heat shock, although these dimers are not required for R-CRT association to SGs. The alteration in SGs assembly is critical for the normal cellular recover of cells after heat induced stress. We conclude that R-CRT is emerging as a novel protein that has an impact on the regulation of SGs scaffolding and cell survival.

Arginylated calreticulin at plasma membrane increases susceptibility of cells to apoptosis.

Post-translational modifications of proteins are important for the regulation of cell fate and functions; one of these post-translational modifications is arginylation. We have previously established that calreticulin (CRT), an endoplasmic reticulum resident, is also one of the arginylated substrates found in the cytoplasm. In the present study, we describe that arginylated CRT (R-CRT) binds to the cell membrane and identified its role as a preapoptotic signal. We also show that cells lacking arginyl-tRNA protein transferase are less susceptible to apoptosis than wild type cells. Under these conditions R-CRT is present on the cell membrane but at early stages is differently localized in stress granules. Moreover, cells induced to undergo apoptosis by arsenite show increased R-CRT on their cell surface. Exogenously applied R-CRT binds to the cell membrane and is able to both increase the number of cells undergoing apoptosis in wild type cells and overcome apoptosis resistance in cells lacking arginyl-tRNA protein transferase that express R-CRT on the cell surface. Thus, these results demonstrate the importance of surface R-CRT in the apoptotic response of cells, implying that post-translational arginylation of CRT can regulate its intracellular localization, cell function, and survival.

The arginylation-dependent association of calreticulin with stress granules is regulated by calcium.

Post-translational modifications of proteins are important for the regulation of cell functions; one of these modifications is post-translational arginylation. In the present study, we show that cytoplasmic CRT (calreticulin) is arginylated by ATE1 (arginyl-tRNA protein transferase). We also show that a pool of CRT undergoes retrotranslocation from the ER (endoplasmic reticulum) to the cytosol, because in CRT-knockout cells transfected with full-length CRT (that has the signal peptide), cytoplasmic CRT appears as a consequence of its expression and processing in the ER. After the cleavage of the signal peptide, an N-terminal arginylatable residue is revealed prior to retrotranslocation to the cytoplasm where arginylation takes place. SGs (stress granules) from ATE1-knockout cells do not contain CRT, indicating that CRT arginylation is required for its association to SGs. Furthermore, R-CRT (arginylated CRT) in the cytoplasm associates with SGs in cells treated with several stressors that lead to a reduction of intracellular Ca2+ levels. However, in the presence of stressors that do not affect Ca2+ levels, R-CRT is not recruited to these loci despite the fact that SGs are formed, demonstrating Ca2+-dependent R-CRT association to SGs. We conclude that post-translational arginylation of retrotranslocated CRT, together with the decrease in intracellular Ca2+, promotes the association of CRT to SGs.