Tubulin polyglutamylation is a reversible post-translational modification, serving important roles in microtubule (MT)-related processes. Polyglutamylases of the tubulin tyrosine ligaselike (TTLL) family add glutamate moieties to specific tubulin glutamate residues, whereas as yet unknown deglutamylases shorten polyglutamate chains. First we investigated regulatory machinery of tubulin glutamylation in MT-based sensory cilia of the roundworm Caenorhabditis elegans. We found that ciliary MTs were polyglutamylated by a process requiring ttll-4. Conversely, loss of ccpp-6 gene function, which encodes one of two cytosolic carboxypeptidases (CCPs), resulted in elevated levels of ciliary MT polyglutamylation. Consistent with a deglutamylase function for ccpp-6, overexpression of this gene in ciliated cells decreased polyglutamylation signals. Similarly, we confirmed that overexpression of murine CCP5, one of two sequence orthologs of nematode ccpp-6, caused a dramatic loss of MT polyglutamylation in cultured mammalian cells. Finally, using an in vitro assay for tubulin glutamylation, we found that recombinantly expressed Myc-tagged CCP5 exhibited deglutamylase biochemical activities. Together, these data from two evolutionarily divergent systems identify C. elegans CCPP-6 and its mammalian ortholog CCP5 as a tubulin deglutamylase. The microtubule (MT)2 cytoskeleton plays critical roles in multiple cellular processes such as chromosome segregation, intracellular transport, cell morphogenesis, and polarity. Tubulin, which is the major protein subunit of MT fibers, exhibits polymorphic protein variation and undergoes a variety of unique post-translational modifications at its C-terminal tail, such as detyrosination/tyrosination (1, 2), polyglycylation (3), and polyglutamylation (4). These modifications regulate tubulin and MT function. Polyglutamylation enzymes add multiple glutamate moieties to specific glutamate residues within substrate proteins. Physiological roles of tubulin polyglutamylation for MT-related processes were reported recently (5, 6) and reviewed by Ikegami and Setou (7).Polyglutamylase enzymes have recently been identified as belonging to the tubulin tyrosine ligase-like (TTLL) protein family (8 -10) (supplemental Fig. 1). In contrast, the enzyme(s) underlying deglutamylation have not been discovered yet, although such proteins are suggested to be present in mouse brain neurons and in cultured cells (11,12).In this study, we employed the genetically tractable nematode, Caenorhabditis elegans, to identify genes that regulate tubulin polyglutamylation/deglutamylation. Specifically, we investigated tubulin post-translational modification in C. elegans sensory cilia, which are MT-based organelles that extend from the distal dendrite tips of 60 sensory neurons found in sensory organs named amphid (head) and phasmid (tail) sensilla (see Fig. 1A). In this system, we identified a cytosolic carboxypeptidase (ccpp) gene, ccpp-6, as a candidate tubulin deglutamylase gene, whose functional properties are opposite ...

The 14-3-3 proteins form a highly conserved family of dimeric proteins that interact with various signal transduction proteins and regulate cell cycle, apoptosis, stress response, and malignant transformation. We previously demonstrated that the ␤ isoform of 14-3-3 proteins promotes tumorigenicity and angiogenesis of rat hepatoma K2 cells. In this study, to analyze the mechanism of 14-3-3␤-induced malignant transformation, yeast two-hybrid screening was performed, and a novel 14-3-3␤-binding factor, FBI1 (fourteen-three-three beta interactant 1), was identified. In vitro binding and co-immunoprecipitation analyses verified specific interaction of 14-3-3␤ with FBI1. The strong expression of FBI1 was observed in several tumor cell lines but not in non-tumor cell lines. Forced expression of antisense FBI1 in K2 cells inhibited anchorage-independent growth but had no significant effect on cell proliferation in monolayer culture. Down-regulation of FBI1 also inhibited tumorigenicity and metastasis accompanying a decrease in MMP-9 (matrix metalloproteinase-9) expression. In addition, the duration of ERK1/2 activation was curtailed in antisense FBI1-expressing K2 cells. A luciferase reporter assay revealed that the FBI1-14-3-3␤ complex could act as a transcriptional silencer, and MKP-1 (MAPK phosphatase-1) was one of the target genes of the FBI1-14-3-3␤ complex. Moreover, chromatin immunoprecipitation analysis demonstrated that FBI1 and 14-3-3␤ were presented on the MKP-1 promoter. These results indicate that FBI1 promotes sustained ERK1/2 activation through repression of MKP-1 transcription, resulting in promotion of tumorigenicity and metastasis.

In immune cells, CD73 dephosphorylates and converts extracellular AMP into adenosine, which binds the A2A adenosine receptor (A2AR). Blockade of this interaction, which induces an immunosuppressed niche in the tumor microenvironment, represents a potential novel treatment strategy. The clinical significance of CD73 and A2AR expression in non-small-cell lung cancer (NSCLC), however, has yet to be thoroughly investigated. Here we evaluated CD73 and A2AR protein expression levels using immunohistochemistry in tissue microarrays containing 642 resected NSCLC specimens. Furthermore, we compared the expression profiles of 133 paired primary tumors and lymph node metastases. CD73 and A2AR expression levels were significantly higher in females than in males, in never smokers than in ever smokers, and in adenocarcinomas than in squamous cell carcinomas. Among adenocarcinomas, significantly higher CD73 and A2AR expression was observed in TTF-1-positive and mutant EGFR-positive tumors than in their counterparts. Compared with CD73, A2AR expression was more inconsistent between primary tumors and lymph node metastases. Among NSCLC patients, high CD73 expression was an independent indicator of poor prognosis in multivariate Cox regression analyses for overall survival [hazard ratio (HR), 2.18; 95% confidence interval (CI), 1.38–3.46] and recurrence-free survival (HR, 2.05; 95% CI, 1.42–2.95). In contrast, high A2AR expression was an independent predictor of favorable prognosis for overall survival (HR, 0.70; 95% CI, 0.50–0.98) and recurrence-free survival (HR, 0.74; 95% CI, 0.56–0.97). Together, these findings indicate that CD73 and A2AR have opposing prognostic effects, although cases involving CD73 or A2AR expression share some clinicopathological features.

New reliable biomarkers are needed to predict the response to immune checkpoint inhibitors against programmed death-1 (PD-1) and its ligand (PD-L1), because PD-L1 expression on tumor cells has limited power for selecting patients who may benefit from such therapy. Here we investigated the significance of PD-L1 and PD-L2 gene copy number gains using fluorescence in situ hybridization as well as PD-L1 and PD-L2 expression in 654 patients with resected non-small-cell lung cancer. The prevalence of PD-L1 amplification and polysomy was 3.1% and 13.2%, respectively. The PD-L1 gene copy number status was in agreement with both the PD-L2 and Janus kinase 2 gene copy number statuses. PD-L1 and PD-L2 expression was observed in 30.7% and 13.1%, respectively. Both PD-L1 copy number gains and expression were associated with smoking-related tumors. Tumor cells with PD-L1 genomic gains exhibited significantly higher levels of PD-L1 expression than those without, but PD-L2 copy number gains were not related to PD-L2 augmentation. PD-L1 gene amplification and polysomy were independently associated with PD-L1 expression, with high immune infiltrates and EGFR expression in a multivariate logistic regression model. Comparative analysis between primary tumors and synchronous regional lymph node metastases revealed that the PD-L1 gene copy number alterations were highly consistent and reproducible compared with the PD-L1 expression. Both PD-L1 amplification and level of protein expression were predictors of poor survival using Cox univariate analyses. Therefore, we conclude that an increase in PD-L1 gene copy number can be a feasible alternative biomarker for predicting response to anti-PD-1/PD-L1 therapy.

The identification of cancer biomarkers is critical for target-linked cancer therapy. The overall level of phosphatidylcholine (PC) is elevated in colorectal cancer (CRC). To investigate which species of PC is overexpressed in colorectal cancer, an imaging mass spectrometry was performed using a panel of non-neoplastic mucosal and CRC tissues. In the present study, we identified a novel biomarker, PC(16:0 ⁄ 16:1), in CRC using imaging mass spectrometry. Specifically, elevated levels of PC(16:0 ⁄ 16:1) expression were observed in the more advanced stage of CRC. Our data further showed that PC(16:0 ⁄ 16:1) was specifically localized in the cancer region when examined using imaging mass spectrometry. Notably, because the ratio of PC(16:0 ⁄ 16:1) to lyso-PC(16:0) was higher in CRC, we postulated that lyso-PC acyltransferase (LPCAT) activity is elevated in CRC. In an in vitro analysis, we showed that LPCAT4 is involved in the deregulation of PC (16: (1) Previous studies have reported that CRC contains increased amounts of phospholipids as well as an altered phospholipid composition of the CRC cell membrane.(2,3) These changes in membrane phospholipid levels can affect cell proliferation, viability and tumor development.(2,4) Moreover, although phosphatidylcholine (PC) is the most dominant phospholipid in both non-neoplastic and cancer tissues,the amount of PC is highly increased in CRC cells.(2) In addition, the changes in membrane potential and the increased PC ⁄ phosphatidylethanolamine (PE) composition rate are related to the grade of CRC malignancy.(3) In higher eukaryotes, PC is synthesized via two pathways: (i) the triple methylation of PE; and (ii) the cytidine diphosphate (CDP)-choline pathway. (6) However, the steady-state composition of PC species is maintained by the remodeling cycle (Lands' cycle).(7) The precise and concerted deacylation by phospholipase A 2 (PLA 2 ) and reacylation by lyso-PC acyltransferase (LPCAT) are required for normal cell functioning. Notably, the elevated expression of LPCAT1 was associated with colon cancer growth. (8) LPCAT1 is a member of the LPCAT family (LPCAT1-4) and shows LPCAT activity, preferentially incorporating palmitate into PC.(8) However, the relationship between PC remodeling and the progression of cancer, as well as the class of LPCAT involved in this process, remains unclear.Direct mass spectrometry (MS) of biological tissue sections using matrix-assisted laser desorption ⁄ ionization (MALDI) can profile many molecules including phospholipid subtypes. (9) Furthermore, this approach can be extended to imaging MS, which can visualize the distribution of biomolecules in the tissue section.(10-13) Because specific antibodies against lipids and macromolecules are often difficult to obtain, MALDI imaging is a suitable option for detecting distinct species of these molecules directly in a tissue section. This technique has already been applied to various human cancers including prostate and gastric cancer. (14,15) Although this emerging analytical technique was initial...