BackgroundProtein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy.Methods and FindingsEncouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors.ConclusionsOur results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.
Anterior gradient 2 (AGR2), a protein belonging to the protein disulfide isomerase (PDI) family, is overexpressed in multiple cancers and promotes angiogenesis to drive cancer progression. The mechanisms controlling AGR2 abundance in cancer remain largely unknown. Here, we observed that AGR2 expression is significantly suppressed by proteasome inhibitor MG132/bortezomib at mRNA and protein levels in lung cancer cells. MG132-mediated repression of AGR2 transcription was independent of ROS generation and ER stress induction, but partially resulted from the downregulated E2F1. Further investigation revealed that MG132 facilitated polyubiquitinated AGR2 degradation through activation of autophagy, as evidenced by predominant restoration of AGR2 level in cells genetic depletion of Atg5 and Atg7, or by autophagy inhibitors. Activation of autophagy by rapamycin noticeably reduced the AGR2 protein in cells and in the mouse tissue samples administrated with bortezomib. We also provided evidence identifying the K48-linked polyubiquitin chains conjugating onto K89 of AGR2 by an E3 ligase UBR5. In addition, an autophagy receptor NBR1 was demonstrated to be important in polyubiquitinated AGR2 clearance in response to MG132 or bortezomib. Importantly, downregulation of AGR2 by proteasome inhibition significantly enhanced antitumor activity of bevacizumab, highlighting the importance of AGR2 as a predictive marker for selection of subgroup patients in chemotherapy.
The role of RNA methylation on the sixth N atom of adenylate (m 6 A) in acute kidney injury (AKI) is unknown. FTO (fat mass and obesity-associated protein) reverses the m 6 A modification in cisplatin-induced AKI. Here, we aimed to determine FTO's role in AKI. We induced AKI in c57BL/6 mice by intraperitoneal cisplatin injection and treated the animal with vehicle or an FTO inhibitor meclofenamic acid (MA) for 3 days. Moreover, as an in vitro model, human kidney proximal tubular cells (HK2 cells) were treated with cisplatin. We found that the cisplatin treatment reduces FTO expression and increases m 6 A levels in vivo and in vitro. MA aggravated renal damage and increased apoptosis in cisplatin-treated kidneys, phenotypes that were correlated with reduced FTO expression and increased m 6 A levels. Moreover, MA promoted apoptosis in cisplatin-treated HK2 cells, which was correlated with the reduced FTO expression and increased m 6 A in HK2 cells. FTO protein overexpression reduced m 6 A levels and inhibited apoptosis in cisplatin-treated HK2 cells and also blocked the MA-induced increase in m 6 A levels and apoptosis rates. In agreement, overexpression of the m 6 A-generating methyltransferase-like 3 and 14 (METTL3 and METTL14) or siRNA-mediated FTO knockdown promoted apoptosis and enhanced m 6 A levels in cisplatin-treated HK2 cells. MA increased p53 mRNA and protein levels in AKI both in vitro and in vivo, and FTO overexpression reduced p53 expression and reversed the MA-induced p53 increase in AKI. In conclusion, reduced renal FTO expression in cisplatin-induced AKI increases RNA m 6 A levels and aggravates renal damages.
Cisplatin is an effective chemotherapeutic agent and widely used in treatment of various solid organ malignancies, including head and neck, ovarian, and testicular cancers. However, the induction of acute kidney injury (AKI) is one of its main side effects. Leukotriene B receptor 1 (BLT1) mediates the majority of physiological effects of leukotriene B (LTB), a potent lipid chemoattractant generated at inflammation sites, but the role of the LTB-BLT1 axis in cisplatin-induced AKI remains unknown. Here we found upregulated LTB synthesis and BLT1 expression in the kidney after cisplatin administration. Cisplatin was found to directly upregulate gene expression of leukotriene A hydrolase and stimulate LTB production in renal tubular epithelial cells. Reduced kidney structural/functional damage, inflammation, and apoptosis were observed in BLT1 mice, as well as in wild-type mice treated with the LTA4H inhibitor SC-57461A and the BLT1 antagonist U-75302. Neutrophils were likely the target of this pathway, as BLT1 absence induced a significant decrease in infiltrating neutrophils in the kidney. Adoptive transfer of neutrophils from wild-type mice restored kidney injury in BLT1 mice following cisplatin challenge. Thus, the LTB-BLT1 axis contributes to cisplatin-induced AKI by mediating kidney recruitment of neutrophils, which induce inflammation and apoptosis in the kidney. Hence, the LTB-BLT1 axis could be a potential therapeutic target in cisplatin-induced AKI.
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