Role of FBXW7 in the quiescence of gefitinib-resistant lung cancer stem cells in EGFR-mutant non-small cell lung cancer

Several recent studies suggest that cancer stem cells (CSCs) are involved in intrinsic resistance to cancer treatment. Maintenance of quiescence is crucial for establishing resistance of CSCs to cancer therapeutics. F-box/WD repeat-containing protein 7 (FBXW7) is a ubiquitin ligase that regulates quiescence by targeting the c-MYC protein for ubiquitination. We previously reported that gefitinib-resistant persisters (GRPs) in EGFR-mutant non-small cell lung cancer (NSCLC) cells highly expressed octamer-binding transcription factor 4 (Oct-4) as well as the lung CSC marker CD133, and they exhibited distinctive features of the CSC phenotype. However, the role of FBXW7 in lung CSCs and their resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors in NSCLC is not fully understood. In this study, we developed GRPs from the two NSCLC cell lines PC9 and HCC827, which express an EGFR exon 19 deletion mutation, by treatment with a high concentration of gefitinib. The GRPs from both PC9 and HCC827 cells expressed high levels of CD133 and FBXW7, but low levels of c-MYC. Cell cycle analysis demonstrated that the majority of GRPs existed in the G0/G1 phase. Knockdown of the FBXW7 gene significantly reduced the cell number of CD133-positive GRPs and reversed the cell population in the G0/G1-phase. We also found that FBXW7 expression in CD133-positive cells was increased and c-MYC expression was decreased in gefitinib-resistant tumors of PC9 cells in mice and in 9 out of 14 tumor specimens from EGFR-mutant NSCLC patients with acquired resistance to gefitinib. These findings suggest that FBXW7 plays a pivotal role in the maintenance of quiescence in gefitinib-resistant lung CSCs in EGFR mutation-positive NSCLC

Deficiency of the eIF4E isoform nCBP limits the cell-to-cell movement of a plant virus encoding triple-gene-block proteins in Arabidopsis thaliana

One of the important antiviral genetic strategies used in crop breeding is recessive resistance. Two eukaryotic translation initiation factor 4E family genes, eIF4E and eIFiso4E, are the most common recessive resistance genes whose absence inhibits infection by plant viruses in Potyviridae, Carmovirus, and Cucumovirus. Here, we show that another eIF4E family gene, nCBP, acts as a novel recessive resistance gene in Arabidopsis thaliana toward plant viruses in Alpha- and Betaflexiviridae. We found that infection by Plantago asiatica mosaic virus (PlAMV), a potexvirus, was delayed in ncbp mutants of A. thaliana. Virus replication efficiency did not differ between an ncbp mutant and a wild type plant in single cells, but viral cell-to-cell movement was significantly delayed in the ncbp mutant. Furthermore, the accumulation of triple-gene-block protein 2 (TGB2) and TGB3, the movement proteins of potexviruses, decreased in the ncbp mutant. Inoculation experiments with several viruses showed that the accumulation of viruses encoding TGBs in their genomes decreased in the ncbp mutant. These results indicate that nCBP is a novel member of the eIF4E family recessive resistance genes whose loss impairs viral cell-to-cell movement by inhibiting the efficient accumulation of TGB2 and TGB3

EXA1, a GYF domain protein, is responsible for loss-of-susceptibility to plantago asiatica mosaic virus in Arabidopsis thaliana

One of the plant host resistance machineries to viruses is attributed to recessive alleles of genes encoding critical host factors for virus infection. This type of resistance, also referred to as recessive resistance, is useful for revealing plant–virus interactions and for breeding antivirus resistance in crop plants. Therefore, it is important to identify a novel host factor responsible for robust recessive resistance to plant viruses. Here, we identified a mutant from an ethylmethane sulfonate (EMS)-mutagenized Arabidopsis population which confers resistance to plantago asiatica mosaic virus (PlAMV, genus Potexvirus). Based on map-based cloning and single nucleotide polymorphism analysis, we identified a premature termination codon in a functionally unknown gene containing a GYF domain, which binds to proline-rich sequences in eukaryotes. Complementation analyses and robust resistance to PlAMV in a T-DNA mutant demonstrated that this gene, named Essential for poteXvirus Accumulation 1 (EXA1), is indispensable for PlAMV infection. EXA1 contains a GYF domain and a conserved motif for interaction with eukaryotic translation initiation factor 4E (eIF4E), and is highly conserved among monocot and dicot species. Analysis using qRT-PCR and immunoblotting revealed that EXA1 was expressed in all tissues, and was not transcriptionally responsive to PlAMV infection in Arabidopsis plants. Moreover, accumulation of PlAMV and a PlAMV-derived replicon was drastically diminished in the initially infected cells by the EXA1 deficiency. Accumulation of two other potexviruses also decreased in exa1-1 mutant plants. Our results provided a functional annotation to GYF domain-containing proteins by revealing the function of the highly conserved EXA1 gene in plant–virus interactions

Dual targeting of a virus movement protein to ER and plasma membrane subdomains is essential for plasmodesmata localization

<div><p>Plant virus movement proteins (MPs) localize to plasmodesmata (PD) to facilitate virus cell-to-cell movement. Numerous studies have suggested that MPs use a pathway either through the ER or through the plasma membrane (PM). Furthermore, recent studies reported that ER-PM contact sites and PM microdomains, which are subdomains found in the ER and PM, are involved in virus cell-to-cell movement. However, functional relationship of these subdomains in MP traffic to PD has not been described previously. We demonstrate here the intracellular trafficking of fig mosaic virus MP (MP_FMV) using live cell imaging, focusing on its ER-directing signal peptide (SP_FMV). Transiently expressed MP_FMV was distributed predominantly in PD and patchy microdomains of the PM. Investigation of ER translocation efficiency revealed that SP_FMV has quite low efficiency compared with SPs of well-characterized plant proteins, calreticulin and CLAVATA3. An MP_FMV mutant lacking SP_FMV localized exclusively to the PM microdomains, whereas SP chimeras, in which the SP of MP_FMV was replaced by an SP of calreticulin or CLAVATA3, localized exclusively to the nodes of the ER, which was labeled with <i>Arabidopsis</i> synaptotagmin 1, a major component of ER-PM contact sites. From these results, we speculated that the low translocation efficiency of SP_FMV contributes to the generation of ER-translocated and the microdomain-localized populations, both of which are necessary for PD localization. Consistent with this hypothesis, SP-deficient MP_FMV became localized to PD when co-expressed with an SP chimera. Here we propose a new model for the intracellular trafficking of a viral MP. A substantial portion of MP_FMV that fails to be translocated is transferred to the microdomains, whereas the remainder of MP_FMV that is successfully translocated into the ER subsequently localizes to ER-PM contact sites and plays an important role in the entry of the microdomain-localized MP_FMV into PD.</p></div

A model for the intracellular trafficking of MP_FMV.

<p>A model for the intracellular trafficking of MP_FMV.</p

Investigation of the subcellular distribution of MP_FMV:YFP.

<p>(A–C) Confocal imaging of MP_FMV:YFP-expressing epidermal cells at 36 hours post-infiltration (hpi). YFP fluorescence was pseudocolored by magenta. (A) (i) Cells expressing MP_FMV:YFP were treated with aniline blue. Arrowheads indicate plasmodesma. The mean ± SD of Pearson correlation coefficient (PCC [–1:1]) is given in the image. Bars = 5 μm. (ii) Fluorescence intensity along the arrow across plasmodesma. The dotted line in the confocal image indicates the cell wall. Bar = 1 μm. (B) Plasmolyzed cells expressing (i) MP_FMV:YFP or (ii) 30K:GFP. Arrows indicate Hechtian strands extended from the PM. Arrowheads indicate plasmodesmata. The dotted line indicates the cell wall. cw, cell wall; cyt, cytoplasm. Bars = 10 μm. (C) Surface views of cells (i) expressing MP_FMV:YFP or (ii) treated with FM4-64. (iii) Co-expression of MP_FMV:CFP and YFP:REM1.3. To obtain higher resolution images, images were processed by a deconvolution algorithm. Bars = 5 μm. (D) 1% TritonX-100 treatment of membranes. Membrane-enriched fractions prepared from FMV-infected fig leaves and <i>N</i>. <i>benthamiana</i> leaves expressing MP_FMV:FLAG or YFP:REM1.3 at 36 hpi were treated with 1% TritonX-100. Anti-FLAG, anti-MP_FMV, anti-GFP, anti-H⁺ATPase and anti-BIP antibodies were used for the detection of MP_FMV:FLAG, MP_FMV, YFP:REM1.3, a PM marker H⁺ATPase and an ER marker BIP. S; soluble fraction. P; insoluble fraction.</p