Involvement of X-box binding protein 1 and reactive oxygen species pathways in the pathogenesis of tumour necrosis factor receptor-associated periodic syndrome.

OBJECTIVES: To investigate convergence of endoplasmic reticulum stress pathways and enhanced reactive oxygen species (ROS) production, due to intracellular retention of mutant tumour necrosis factor receptor 1 (TNFR1), as a disease mechanism in TNFR-associated periodic syndrome (TRAPS). METHODS: Peripheral blood mononuclear cells from patients with TRAPS (n=16) and healthy controls (HC) (n=22) were studied alongside HEK293T cells expressing wild type-TNFR1 or TRAPS-associated mutations. Unfolded protein response (UPR)-associated proteins (protein kinase-like endoplasmic reticulum kinase, PERK), phosphorylated-PERK (p-PERK), phosphorylated inositol-requiring enzyme 1α (p-IRE1α) and spliced X-box binding protein 1 (sXBP1)) were measured by flow cytometry. XBP1 splicing and UPR-associated transcript expression were assessed by reverse transcription PCR/quantitative real-time PCR. ROS levels were measured using CM-H(2)DCFDA and MitoSOX Red in patients' monocytes or HEK293T cells by flow cytometry. RESULTS: Mutant TNFR1-expressing HEK293T cells had increased TNFR1 expression associated with intracellular aggregation. TRAPS patients had increased sXBP1 transcripts (p<0.01) compared with HC. Raised p-PERK protein was seen, indicative of an UPR, but other UPR-associated transcripts were normal. Increased ROS levels were observed in TRAPS monocytes compared with HCs (p<0.02); these increased further upon IL-6 stimulation (p<0.01). Lipopolysaccharide-stimulated peripheral blood mononuclear cells of patients with TRAPS, but not HCs, demonstrated increased sXBP1 levels (p<0.01), which were reduced by antioxidant treatment (p<0.05). CONCLUSIONS: Patients with TRAPS have evidence of increased sXBP1 and PERK expression but without other signs of classical UPR, and also with high ROS generation that may contribute to the pro-inflammatory state associated with TRAPS. The authors propose a non-traditional XBP1 pathway with enhanced sXBP1 as a novel disease-contributing mechanism in TRAPS.

Different CFTR modulator combinations downregulate inflammation differently in cystic fibrosis.

Previously, we showed that serum and monocytes from patients with CF exhibit an enhanced NLRP3-inflammasome signature with increased IL-18, IL-1ß, caspase-1 activity and ASC speck release (Scambler et al. eLife 2019). Here we show that CFTR modulators down regulate this exaggerated proinflammatory response following LPS/ATP stimulation. In vitro application of ivacaftor/lumacaftor or ivacaftor/tezacaftor to CF monocytes showed a significant reduction in IL-18, whereas IL-1ß was only reduced with ivacaftor/tezacaftor. Thirteen adults starting ivacaftor/lumacaftor and eight starting ivacaftor/tezacaftor were assessed over three months. Serum IL-18 and TNF decreased significantly with treatments, but IL-1ß only declined following ivacaftor/tezacaftor. In (LPS/ATP-stimulated) PBMCs, IL-18/TNF/caspase-1 were all significantly decreased and IL-10 was increased with both combinations. Ivacaftor/tezacaftor alone showed a significant reduction in IL-1ß and pro-IL-1ß mRNA. This study demonstrates that these CFTR modulator combinations have potent anti-inflammatory properties, in addition to their ability to stimulate CFTR function, which could contribute to improved clinical outcomes.

Efficacy, Safety, and Pharmacokinetics of Axitinib in Nasopharyngeal Carcinoma: A Preclinical and Phase II Correlative Study.

Purpose: We hypothesized that axitinib is active with an improved safety profile in nasopharyngeal carcinoma (NPC).Experimental Design: We evaluated axitinib in preclinical models of NPC and studied its efficacy in a phase II clinical trial in recurrent or metastatic NPC patients who progressed after at least one line of prior platinum-based chemotherapy. We excluded patients with local recurrence or vascular invasion. Axitinib was started at 5 mg twice daily in continuous 4-week cycles. Primary endpoint was clinical benefit rate (CBR), defined as the percentage of patients achieving complete response, partial response, or stable disease by RECIST criteria for more than 3 months.Results: We recruited 40 patients, who received a median of 3 lines of prior chemotherapy. Axitinib was administered for a mean of 5.6 cycles, with 16 patients (40%) receiving ≥6 cycles. Of 37 patients evaluable for response, CBR was 78.4% (95% CI, 65.6%-91.2%) at 3 months and 43.2% (30.4%-56.1%) at 6 months. Grade 3/4 toxicities were uncommon, including hypertension (8%), diarrhea (5%), weight loss (5%), and pain (5%). All hemorrhagic events were grade 1 (15%) or grade 2 (3%). Elevated diastolic blood pressure during the first 3 months of axitinib treatment was significantly associated with improved overall survival (HR, 0.29; 95% CI, 0.13-0.64, P = 0.0012). Patient-reported fatigue symptom was associated with hypothyroidism (P = 0.039). Axitinib PK parameters (Cmax and AUC(0-t)) were significantly correlated with tumor response, toxicity, and serum thyroid-stimulating hormone changes.Conclusions: Axitinib achieved durable disease control with a favorable safety profile in heavily pretreated NPC patients. Clin Cancer Res; 24(5); 1030-7. ©2018 AACR.

Genomic analysis of liver cancer unveils novel driver genes and distinct prognostic features.

Objective: Hepatocellular carcinoma (HCC) is a highly heterogeneous disease with a dismal prognosis. However, driver genes and prognostic markers in HCC remain to be identified. It is hoped that in-depth analysis of HCC genomes in relation to available clinicopathological information will give rise to novel molecular prognostic markers. Methods: We collected genomic data of 1,061 HCC patients from previous studies, and performed integrative analysis to identify significantly mutated genes and molecular prognosticators. We employed three MutSig algorithms (MutSigCV, MutSigCL and MutSigFN) to identify significantly mutated genes. The GISTIC2 algorithm was used to delineate focally amplified and deleted genomic regions. Nonnegative matrix factorization (NMF) was utilized to decipher mutational signatures. Kaplan-Meier survival and Cox regression analyses were used to associate gene mutation and copy number alteration with survival outcome. Logistic regression model was applied to test association between gene mutation and mutational signatures. Results: We discovered 11 novel driver genes, including RNF213, VAV3 and TNRC6B, with mutational prevalence ranging from 1% to 3%. Seven mutational signatures were also identified in HCC, some of which were associated with mutations of classical driver genes (e.g., TP53, TERT) as well as alcohol consumption. Focal amplifications of TERT and other druggable targets, including AURKA, were also revealed. Targeting AURKA by a small-molecule inhibitor potently induced apoptosis in HCC cells. We further demonstrated that HCC patients with TERT amplification displayed shortened overall survival independent of other clinicopathological parameters. In conclusion, our study identified novel cancer driver genes and prognostic markers in HCC, reiterating the translational importance of omics data in the precision medicine era.

Preclinical evaluation of the mTOR-PI3K inhibitor BEZ235 in nasopharyngeal cancer models.

The dual PI3K-mTOR inhibitor BEZ235 was evaluated in preclinical models of nasopharyngeal carcinoma (NPC). The IC50 value of BEZ235 for growth was in the nanomolar range in vitro, induce G1 cycle arrest and apoptosis, and inhibited AKT and mTOR signaling in most NPC cell lines. No synergistic effect was observed when BEZ235 was combined with chemotherapy. BEZ235 increased MAPK activation in vitro but not in vivo. A daily schedule was more effective than a weekly schedule on tumor growth and inhibition of downstream mTOR signaling in vivo. The activity of BEZ235 maybe independent of the PIK3CA amplification and mutation status.

Proteomic analyses of intermediate filaments reveals cytokeratin8 is highly acetylated--implications for colorectal epithelial homeostasis.

Butyrate is a critical cancer-preventive element in the colon milieu whose mechanism of action is unclear, but appears to be mediated through inhibition of histone deacetylases (HDACs) and consequent alterations in global protein acetylation. Cytokeratins (CKs) have roles in cytoskeletal function as components of the intermediate filaments (IFs) and this involves CKs in the regulation of tissue homeostasis of high-turnover epithelia such as the colon. We used a 2-D gel/MS analysis to characterise the proteome of IFs, and a novel monitoring-initiated detection and sequencing (MIDAS) approach to identify acetylation sites on principal proteins. We report that CKs are highly acetylated in a colon cancer cell line, with five acetylation sites characterised on CK8 and a further one on CK18. Acetylation of CK8 is responsive to butyrate. HDAC5 is the deacetylase associated with IFs. These data indicate a novel action of butyrate as a cancer preventive agent. Acetylation of CK8 may be associated with IFs stabilisation and thereby provide a candidate mechanism for the appropriate retention or loss of epithelial cells from the flat mucosa.

Coordination of chromosome alignment and mitotic progression by the chromosome-based Ran signal.

Mitotic spindle assembly and chromosome segregation are controlled by the cell cycle machinery and by the guanosine triphosphatase Ran (RanGTPase). We developed a spatial model that allowed us to simulate RanGTP production with different degrees of chromosome alignment in mitosis. Aided by this model, we defined three factors that modulate mitotic RanGTP gradients and mitotic progression in somatic cells. First, the completion of chromosome alignment at the metaphase plate could generate highest local RanGTP concentrations on chromosomes that could lead to spindle checkpoint silencing and metaphase-anaphase transition. Second, the concentration of RanGTP-transport-receptor (represented by RanGTP-importin beta) and its spatial distribution are very sensitive to the level of RanBP1. Reduction of RanBP1 leads to an elevated RanGTP-transport-receptor concentration throughout the cell, which disrupts spindle assembly and weakens spindle checkpoint control. Finally, chromosomal RanGTP production could be dampened by a reduction of RCC1 phosphorylation by Cdc2 in mitosis. Our spatial simulation of RanGTP production using individual chromosomes should provide means to further understand how the Ran system and the cell cycle machinery coordinately regulate mitosis.

Global profiling of surface plasma membrane proteome of oviductal epithelial cells.

In mammalian reproduction, many important events occur within the female reproductive tract, especially within the oviduct. These include transport and final maturation of the female and male gametes, fertilization, embryonic development, and transport of the embryo to the uterus. The plasma membrane molecules of oviductal epithelia that are in direct contact with gametes and embryo(s) and potentially mediate these processes are poorly characterized, and their function is poorly understood. Defining the oviductal cell surface proteome could provide a better understanding of the basis of reproductive processes taking place within the oviduct. We aimed to provide a detailed profile of the surface plasma membrane proteome of the oviductal epithelium by biotinylation of proteins at the cell surface, followed by highly specific purification of these proteins using avidin. This approach for enrichment of oviductal cell surface proteome was validated by immunohistochemistry, gel electrophoresis, and western blot analysis experiments. The enriched molecules were identified using two different technologies: (i) the combination of 2D gel electrophoresis with mass spectrometry and (ii) 1D gel electrophoresis with mass spectrometry (a modified multidimensional protein identification technology (MudPIT) technique). The number of proteins identified using the MudPIT approach was approximately 7 times the number of proteins identified by 2D gel electrophoresis using the same samples (40 versus 276, respectively). Some of the proteins found at the surface of oviductal cells had previously been reported as present in the oviduct and to have known functions in relation to reproductive processes. The other category of proteins that were highly represented in the oviductal surface proteome were various members of the family of heat-shock proteins. To the best of our knowledge, this is the first comprehensive study to identify and characterize proteins at the surface of the epithelium of the mammalian oviduct.

Gametes alter the oviductal secretory proteome.

The mammalian oviduct provides an optimal environment for the maturation of gametes, fertilization, and early embryonic development. Secretory cells lining the lumen of the mammalian oviduct synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. We hypothesized that the presence of gametes in the oviduct alters the oviductal secretory proteomic profile. We used a combination of two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry to identify oviductal protein secretions that were altered in response to the presence of gametes in the oviduct. The oviductal response to spermatozoa was different from its response to oocytes as verified by Western blotting. The presence of spermatozoa or oocytes in the oviduct altered the secretion of specific proteins. Most of these proteins are known to have an influence on gamete maturation, viability, and function, and there is evidence to suggest these proteins may prepare the oviductal environment for arrival of the zygote. Our findings suggest the presence of a gamete recognition system within the oviduct capable of distinguishing between spermatozoa and oocytes.