Urinary dipeptidase 1 and trefoil factor 1 are promising biomarkers for early diagnosis of colorectal cancer.

BACKGROUND: Currently utilized serum tumor markers and fecal immunochemical tests do not have sufficient diagnostic power for colorectal cancer (CRC) due to their low sensitivities. To establish non-invasive urinary protein biomarkers for early CRC diagnosis, we performed stepwise analyses employing urine samples from CRCs and healthy controls (HCs). METHODS: Among 474 urine samples, 363 age- and sex-matched participants (188 HCs, 175 stage 0-III CRCs) were randomly divided into discovery (16 HCs, 16 CRCs), training (110 HCs, 110 CRCs), and validation (62 HCs, 49 CRCs) cohorts. RESULTS: Of the 23 urinary protein candidates comprehensively identified from mass spectrometry in the discovery cohort, urinary levels of dipeptidase 1 (uDPEP1) and Trefoil factor1 (uTFF1) were the two most significant diagnostic biomarkers for CRC in both training and validation cohorts using enzyme-linked immunosorbent assays. A urinary biomarker panel comprising uDPEP1 and uTFF1 significantly distinguished CRCs from HCs, showing area under the curves of 0.825-0.956 for stage 0-III CRC and 0.792-0.852 for stage 0/I CRC. uDPEP1 and uTFF1 also significantly distinguished colorectal adenoma (CRA) patients from HCs, with uDPEP1 and uTFF1 increasing significantly in the order of HCs, CRA patients, and CRC patients. Moreover, expression levels of DPEP1 and TFF1 were also significantly higher in the serum and tumor tissues of CRC, compared to HCs and normal tissues, respectively. CONCLUSIONS: This study established a promising and non-invasive urinary protein biomarker panel, which enables the early detection of CRC with high sensitivity.

Phosphotyrosine proteomics in cells synchronized at monopolar cytokinesis reveals EphA2 as functioning in cytokinesis.

Cytokinesis is the final step of the cell division in which cellular components are separated into two daughter cells. This process is regulated through the phosphorylation of different classes of proteins by serine/threonine (Ser/Thr) kinases such as Aurora B and Polo-like kinase 1 (PLK1). Conversely, the role of phosphorylation at tyrosine residues during cytokinesis has not been studied in detail yet. In this study, we performed a phosphotyrosine proteomic analysis of cells undergoing monopolar cytokinesis synchronized by using the Eg5 inhibitor (+)-S-trityl-l-cysteine (STLC) and the CDK1 inhibitor RO-3306. Phosphotyrosine proteomics gave 362 tyrosine-phosphorylated peptides. Western blot analysis of proteins revealed tyrosine phosphorylation in mitogen-activated protein kinase 14 (MAPK14), vimentin, ephrin type-A receptor 2 (EphA2), and myelin protein zero-like protein 1 (MPZL1) during monopolar cytokinesis. Additionally, we demonstrated that EphA2, a protein with unknown function during cytokinesis, is involved in cytokinesis. EphA2 knockdown accelerated epithelial cell transforming 2 (Ect2) knockdown-induced multinucleation, suggesting that EphA2 plays a role in cytokinesis in a particular situation. The list also included many proteins previously reported to play roles during cytokinesis. These results evidence that the identified phosphopeptides facilitate the identification of novel tyrosine phosphorylation signaling involved in regulating cytokinesis.

Integration of pharmacoproteomic and computational approaches reveals the cellular signal transduction pathways affected by apatinib in gastric cancer cell lines.

Apatinib is known to be a highly selective vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor with anti-angiogenic and anti-tumor properties. In a phase III study, the objective response rate to apatinib was low. It remains unclear why the effectivity of apatinib varies among patients and what type of patients are candidates for the treatment. In this study, we investigated the anti-tumor efficacy of apatinib against 13 gastric cancer cell lines and found that it differed depending on the cell line. Using integrated wet and dry approaches, we showed that apatinib was a multi-kinase inhibitor of c-Kit, RAF1, VEGFR1, VEGFR2, and VEGFR3, predominantly inhibiting c-Kit. Notably, KATO-III, which was the most apatinib-sensitive among the gastric cancer cell lines investigated, was the only cell line expressing c-Kit, RAF1, VEGFR1, and VEGFR3 but not VEGFR2. Furthermore, we identified SNW1 as a molecule affected by apatinib that plays an important role in cell survival. Finally, we identified the molecular network related to SNW1 that was affected by treatment with apatinib. These results suggest that the mechanism of action of apatinib in KATO-III cells is independent of VEGFR2 and that the differential efficacy of apatinib was due to differences in expression patterns of receptor tyrosine kinases. Furthermore, our results suggest that the differential efficacy of apatinib in gastric cell lines may be attributed to SNW1 phosphorylation levels at a steady state. These findings contribute to a deeper understanding of the mechanism of action of apatinib in gastric cancer cells.

Alterations in ether lipid metabolism and the consequences for the mouse lipidome.

Alkylglycerol monooxygenase (AGMO) and plasmanylethanolamine desaturase (PEDS1) are enzymes involved in ether lipid metabolism. While AGMO degrades plasmanyl lipids by oxidative cleavage of the ether bond, PEDS1 exclusively synthesizes a specific subclass of ether lipids, the plasmalogens, by introducing a vinyl ether double bond into plasmanylethanolamine phospholipids. Ether lipids are characterized by an ether linkage at the sn-1 position of the glycerol backbone and they are found in membranes of different cell types. Decreased plasmalogen levels have been associated with neurological diseases like Alzheimer's disease. Agmo-deficient mice do not present an obvious phenotype under unchallenged conditions. In contrast, Peds1 knockout mice display a growth phenotype. To investigate the molecular consequences of Agmo and Peds1 deficiency on the mouse lipidome, five tissues from each mouse model were isolated and subjected to high resolution mass spectrometry allowing the characterization of up to 2013 lipid species from 42 lipid subclasses. Agmo knockout mice moderately accumulated plasmanyl and plasmenyl lipid species. Peds1-deficient mice manifested striking changes characterized by a strong reduction of plasmenyl lipids and a concomitant massive accumulation of plasmanyl lipids resulting in increased total ether lipid levels in the analyzed tissues except for the class of phosphatidylethanolamines where total levels remained remarkably constant also in Peds1 knockout mice. The rate-limiting enzyme in ether lipid metabolism, FAR1, was not upregulated in Peds1-deficient mice, indicating that the selective loss of plasmalogens is not sufficient to activate the feedback mechanism observed in total ether lipid deficiency.

Genetic defects in peroxisome morphogenesis (Pex11ß, dynamin-like protein 1, and nucleoside diphosphate kinase 3) affect docosahexaenoic acid-phospholipid metabolism.

Peroxisomes are essential organelles involved in lipid metabolisms including plasmalogen biosynthesis and ß-oxidation of very long-chain fatty acids. Peroxisomes proliferate by the growth and division of pre-existing peroxisomes. The peroxisomal membrane is elongated by Pex11ß and then divided by the dynamin-like GTPase, DLP1 (also known as DRP1 encoded by DNM1L gene), which also functions as a fission factor for mitochondria. Nucleoside diphosphate kinase 3 (NME3) localized in both peroxisomes and mitochondria generates GTP for DLP1 activity. Deficiencies of either of these factors induce abnormal morphology of peroxisomes and/or mitochondria, and are associated with central nervous system dysfunction. To investigate whether the impaired division of peroxisomes affects lipid metabolisms, we assessed the phospholipid composition of cells lacking each of the different division factors. In fibroblasts from the patients deficient in DLP1, NME3, or Pex11ß, docosahexaenoic acid (DHA, C22:6)-containing phospholipids were found to be decreased. Conversely, the levels of several fatty acids such as arachidonic acid (AA, C20:4) and oleic acid (C18:1) were elevated. Mouse embryonic fibroblasts from Drp1- and Pex11ß-knockout mice also showed a decrease in the levels of phospholipids containing DHA and AA. Collectively, these results suggest that the dynamics of organelle morphology exert marked effects on the fatty acid composition of phospholipids.

Longstanding overt ventriculomegaly diagnosed in adolescents, not adults: a pediatric case report.

BACKGROUND: Longstanding overt ventriculomegaly in adults (LOVA) is a new form of progressive hydrocephalus characterized by onset in early childhood and gradual progression into adulthood. Patients with LOVA are usually asymptomatic in childhood. The diagnosis of LOVA in adolescence has not been reported. CASE REPORT: A patient with macrocephaly and mild ventriculomegaly from infancy developed headache exacerbation and cognitive dysfunction at the age of 11 years. Brain magnetic resonance imaging showed mild tri-ventriculomegaly with no radiological aggravation compared to imaging at the age of 8 years. No papilledema was observed. Drainage of 15 ml of spinal fluid via a lumbar puncture relieved the headache and cognitive dysfunction. Based on repeated improvements in cognitive function and headaches after spinal fluid drainage, we diagnosed the patient with LOVA with symptom onset in early adolescence. A ventriculoperitoneal shunt was placed, and the headaches disappeared completely. The full-scale intellectual quotient, verbal comprehension, and working memory improved significantly. CONCLUSIONS: LOVA may manifest as early as adolescence. The clinical presentation, age, clinical, radiological features, and management vary, and a spinal tap exam is useful for diagnosing LOVA, even in children. The spinal tap exam may be indicated in children with longstanding ventriculomegaly and deteriorating neurological symptoms to diagnose this "treatable intellectual disability."

Adeno-associated virus-based malaria booster vaccine following attenuated replication-competent vaccinia virus LC16m8Δ priming.

We previously demonstrated that boosting with adeno-associated virus (AAV) type 1 (AAV1) can induce highly effective and long-lasting protective immune responses against malaria parasites when combined with replication-deficient adenovirus priming in a rodent model. In the present study, we compared the efficacy of two different AAV serotypes, AAV1 and AAV5, as malaria booster vaccines following priming with the attenuated replication-competent vaccinia virus strain LC16m8Δ (m8Δ), which harbors the fusion gene encoding both the pre-erythrocytic stage protein, Plasmodium falciparum circumsporozoite (PfCSP) and the sexual stage protein (Pfs25) in a two-dose heterologous prime-boost immunization regimen. Both regimens, m8Δ/AAV1 and m8Δ/AAV5, induced robust anti-PfCSP and anti-Pfs25 antibodies. To evaluate the protective efficacy, the mice were challenged with sporozoites twice after immunization. At the first sporozoite challenge, m8Δ/AAV5 achieved 100% sterile protection whereas m8Δ/AAV1 achieved 70% protection. However, at the second challenge, 100% of the surviving mice from the first challenge were protected in the m8Δ/AAV1 group whereas only 55.6% of those in the m8Δ/AAV5 group were protected. Regarding the transmission-blocking efficacy, we found that both immunization regimens induced high levels of transmission-reducing activity (>99%) and transmission-blocking activity (>95%). Our data indicate that the AAV5-based multistage malaria vaccine is as effective as the AAV1-based vaccine when administered following an m8Δ-based vaccine. These results suggest that AAV5 could be a viable alternate vaccine vector as a malaria booster vaccine.

Sterile protection and transmission blockade by a multistage anti-malarial vaccine in the pre-clinical study.

The Malaria Vaccine Technology Roadmap 2013 (World Health Organization) aims to develop safe and effective vaccines by 2030 that will offer at least 75% protective efficacy against clinical malaria and reduce parasite transmission. Here, we demonstrate a highly effective multistage vaccine against both the pre-erythrocytic and sexual stages of Plasmodium falciparum that protects and reduces transmission in a murine model. The vaccine is based on a viral-vectored vaccine platform, comprising a highly-attenuated vaccinia virus strain, LC16m8Δ (m8Δ), a genetically stable variant of a licensed and highly effective Japanese smallpox vaccine LC16m8, and an adeno-associated virus (AAV), a viral vector for human gene therapy. The genes encoding P. falciparum circumsporozoite protein (PfCSP) and the ookinete protein P25 (Pfs25) are expressed as a Pfs25-PfCSP fusion protein, and the heterologous m8Δ-prime/AAV-boost immunization regimen in mice provided both 100% protection against PfCSP-transgenic P. berghei sporozoites and up to 100% transmission blocking efficacy, as determined by a direct membrane feeding assay using parasites from P. falciparum-positive, naturally-infected donors from endemic settings. Remarkably, the persistence of vaccine-induced immune responses were over 7 months and additionally provided complete protection against repeated parasite challenge in a murine model. We propose that application of the m8Δ/AAV malaria multistage vaccine platform has the potential to contribute to the landmark goals of the malaria vaccine technology roadmap, to achieve life-long sterile protection and high-level transmission blocking efficacy.

Central-variant posterior reversible encephalopathy syndrome in an infant with mid-aortic syndrome: A rare case of symmetric basal ganglia lesions.

Central-variant posterior reversible encephalopathy syndrome is an atypical subtype of posterior reversible encephalopathy syndrome that occurs during rapid fluctuations in blood pressure, leading to cerebrovascular autoregulatory failure and endothelial dysfunction. Few reports have described posterior reversible encephalopathy syndrome in infants. A 4-month-old girl, who was diagnosed a month before with hypoxic ischemic encephalopathy due to sudden cardiac arrest, showed persistent renovascular hypertension with a systolic blood pressure of 200 mmHg. Computed tomography of the head revealed a new-onset low-attenuation area in the bilateral basal ganglia, and computed tomography of the trunk revealed severe long-segment narrowing of the abdominal aorta encompassing the bilateral renal arteries. She was treated with antihypertensive drugs and peritoneal dialysis. Follow-up imaging after blood pressure stabilization showed resolution of the low-attenuation area in the bilateral basal ganglia. We diagnosed her basal ganglia lesions as central-variant posterior reversible encephalopathy syndrome. She suffered from neurological sequelae attributable to hypoxic ischemic encephalopathy but showed no evidence of basal ganglia dysfunction. Here, we report a case of infantile central-variant posterior reversible encephalopathy syndrome involving bilateral basal ganglia lesions with mid-aortic syndrome. The differential diagnosis of infantile symmetric bilateral basal ganglia lesions is broad and includes genetic, acquired metabolic or toxic, infectious, inflammatory, vascular, and neoplastic pathologies. Among them, central-variant posterior reversible encephalopathy syndrome is rare but important because neurological prognosis may be favorable, and specific treatment, such as administration of antihypertensive drugs or discontinuation of drugs that induce posterior reversible encephalopathy syndrome, is possible.

Src activation in lipid rafts confers epithelial cells with invasive potential to escape from apical extrusion during cell competition.

Abnormal/cancerous cells within healthy epithelial tissues undergo apical extrusion to protect against carcinogenesis, although they acquire invasive capacity once carcinogenesis progresses. However, the molecular mechanisms by which cancer cells escape from apical extrusion and invade surrounding tissues remain elusive. In this study, we demonstrate a molecular mechanism for cell fate switching during epithelial cell competition. We found that during competition within epithelial cell layers, Src transformation promotes maturation of focal adhesions and degradation of extracellular matrix. Src-transformed cells underwent basal delamination by Src activation within sphingolipid/cholesterol-enriched membrane microdomains/lipid rafts, whereas they were apically extruded when Src was outside of lipid rafts. A comparative analysis of contrasting phenotypes revealed that activation of the Src-STAT3-MMP axis through lipid rafts was required for basal delamination. CUB-domain-containing protein 1 (CDCP1) was identified as an Src-activating scaffold and as a Met regulator in lipid rafts, and its overexpression induced basal delamination. In renal cancer models, CDCP1 promoted epithelial-mesenchymal transition-mediated invasive behavior by activating the Src-STAT3-MMP axis through Met activation. Overall, these results suggest that spatial activation of Src signaling in lipid rafts confers resistance to apical extrusion and invasive potential on epithelial cells to promote carcinogenesis.

Fatal X-linked lymphoproliferative disease type 1-associated limbic encephalitis with positive anti-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antibody.

BACKGROUND: X-linked lymphoproliferative disease type 1 (XLP1) is a rare monogenic immune dysregulation disorder caused by a deficiency of a signaling lymphocyte activation molecule-associated protein (SAP). While many patients with XLP1 present with fatal hemophagocytic lymphohistiocytosis upon Epstein Barr virus (EBV) infection, a small fraction present with limbic encephalitis in the absence of EBV infection. It is poorly understood why SAP deficiency may cause limbic encephalitis in XLP1. CASE: A 12-year-old boy presented with seizures, changes in personality, memory loss, and cognitive deficits during treatment for interstitial pneumonia. A diagnosis of limbic encephalitis was made. Despite treatment against CD8+ T cell-mediated autoimmunity with intravenous methylprednisolone, dexamethasone, intravenous immunoglobulin, plasma exchange, cyclosporine, weekly etoposide, mycophenolate mofetil, and adalimumab, encephalitis progressed until the patient died after one month of treatment intitiation. Post-mortem genetic testing revealed a de novo SH2D1A truncating mutation. Tests for EBV infection were negative. Initial spinal fluid revealed markedly elevated protein levels, mild pleocytosis, and elevation of two chemokines (C-X-C motif chemokine ligand [CXCL] 10 and CXCL 13). Moreover, initial spinal fluid was tested positive for anti-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) autoantibody. DISCUSSION: In XLP1-associated limbic encephalitis, anti-AMPAR autoantibody production by the dysregulated immune system due to SAP deficiency might be a pathogenic mechanism of central nervous system manifestations. In addition to the standard treatment for XLP1, targeted treatment against B-cell-mediated immunity might be indicated for patients with XLP1-associated limbic encephalitis.

Systematic identification of ALK substrates by integrated phosphoproteome and interactome analysis.

The sensitivity of phosphorylation site identification by mass spectrometry has improved markedly. However, the lack of kinase-substrate relationship (KSR) data hinders the improvement of the range and accuracy of kinase activity prediction. In this study, we aimed to develop a method for acquiring systematic KSR data on anaplastic lymphoma kinase (ALK) using mass spectrometry and to apply this method to the prediction of kinase activity. Thirty-seven ALK substrate candidates, including 34 phosphorylation sites not annotated in the PhosphoSitePlus database, were identified by integrated analysis of the phosphoproteome and crosslinking interactome of HEK 293 cells with doxycycline-induced ALK overexpression. Furthermore, KSRs of ALK were validated by an in vitro kinase assay. Finally, using phosphoproteomic data from ALK mutant cell lines and patient-derived cells treated with ALK inhibitors, we found that the prediction of ALK activity was improved when the KSRs identified in this study were used instead of the public KSR dataset. Our approach is applicable to other kinases, and future identification of KSRs will facilitate more accurate estimations of kinase activity and elucidation of phosphorylation signals.

Early therapeutic plasma exchange may lead to complete neurological recovery in moderate to severe influenza-associated acute necrotizing encephalopathy.

BACKGROUND: Acute necrotizing encephalopathy (ANE) is a pediatric neurological disease, presumably caused by cytokine storms, with a poor prognosis. Immunomodulatory therapy, including therapeutic plasma exchange (TPE), could be an effective treatment. CASES: Two patients with influenza-associated ANE were treated. The ANE severity scores were 3 and 8 in case 1 (a 3-y-old boy) and case 2 (a 7-y-old boy), respectively. In case 1, intravenous methylprednisolone and TPE were initiated at 8 and 16 h, respectively, after the onset of impaired consciousness. In case 2, multiple organ failure and septic shock persisted even after infusion of fluids and inotropic agents. Intravenous methylprednisolone and TPE were started at 5 and 9 h, respectively, after the onset of impaired consciousness, which improved the inotrope-refractory septic shock. Patient 1 and 2 achieved complete neurological recovery within 4 weeks and after 3 months, respectively. In both patients, cytokine levels were serially measured. There were increased serum interleukin (IL)-6 and IL-10 levels in both patients; patient 1 showed increased IL-6 levels in the initial cerebrospinal fluid sample. There was a post-treatment decrease in serum IL-6 levels in both cases. DISCUSSION: Early intensive immunomodulatory therapy with TPE may improve neurological outcomes in pediatric influenza-associated ANE. Further studies are required to establish the efficacy of TPE for ANE.

Temporal dynamics from phosphoproteomics using endoscopic biopsy specimens provides new therapeutic targets in stage IV gastric cancer.

Phosphoproteomic analysis expands our understanding of cancer biology. However, the feasibility of phosphoproteomic analysis using endoscopically collected tumor samples, especially with regards to dynamic changes upon drug treatment, remains unknown in stage IV gastric cancer. Here, we conducted a phosphoproteomic analysis using paired endoscopic biopsy specimens of pre- and post-treatment tumors (Ts) and non-tumor adjacent tissues (NATs) obtained from 4 HER2-positive gastric cancer patients who received trastuzumab-based treatment and from pre-treatment Ts and NATs of 4 HER2-negative gastric cancer patients. Our analysis identified 14,622 class 1 phosphosites with 12,749 quantified phosphosites and revealed molecular changes by HER2 positivity and treatment. An inhibitory signature of the ErbB signaling was observed in the post-treatment HER2-positive T group compared with the pre-treatment HER2-positive T group. Phosphoproteomic profiles obtained by a case-by-case review using paired pre- and post-treatment HER2-positive T could be utilized to discover predictive or resistant biomarkers. Furthermore, these data nominated therapeutic kinase targets which were exclusively activated in the patient unresponded to the treatment. The present study suggests that a phosphoproteomic analysis of endoscopic biopsy specimens provides information on dynamic molecular changes which can individually characterize biologic features upon drug treatment and identify therapeutic targets in stage IV gastric cancer.

GSK3 inhibition circumvents and overcomes acquired lorlatinib resistance in ALK-rearranged non-small-cell lung cancer.

Anaplastic lymphoma kinase (ALK) fusion is found in ~3%-5% of patients with non-small-cell lung cancers (NSCLCs). Although the third-generation ALK tyrosine kinase inhibitor (TKI) lorlatinib shows high clinical efficacy in ALK-positive NSCLC, most of the patients eventually relapse with acquired resistance. Recently, drug-tolerant persister (DTP) cells have been considered an important seed of acquired resistance cells. In this study, we established lorlatinib intermediate resistant cells from a patient-derived cell model. Glycogen synthase kinase 3 (GSK3) inhibitions significantly suppressed lorlatinib intermediate resistant cell growth. GSK3 inhibition also sensitized acquired resistance cells derived from alectinib-treated patients with or without secondary mutations to lorlatinib. Therefore, GSK3 plays a crucial role in developing acquired resistance against lorlatinib in ALK-positive NSCLC mediated by lorlatinib intermediate resistant cells and could be a potential molecular target to prevent acquired lorlatinib resistance and overcome ALK-TKI resistance.

Intestinal microbe-dependent ω3 lipid metabolite αKetoA prevents inflammatory diseases in mice and cynomolgus macaques.

Dietary ω3 fatty acids have important health benefits and exert their potent bioactivity through conversion to lipid mediators. Here, we demonstrate that microbiota play an essential role in the body's use of dietary lipids for the control of inflammatory diseases. We found that amounts of 10-hydroxy-cis-12-cis-15-octadecadienoic acid (αHYA) and 10-oxo-cis-12-cis-15-octadecadienoic acid (αKetoA) increased in the feces and serum of specific-pathogen-free, but not germ-free, mice when they were maintained on a linseed oil diet, which is high in α-linolenic acid. Intake of αKetoA, but not αHYA, exerted anti-inflammatory properties through a peroxisome proliferator-activated receptor (PPAR)γ-dependent pathway and ameliorated hapten-induced contact hypersensitivity by inhibiting the development of inducible skin-associated lymphoid tissue through suppression of chemokine secretion from macrophages and inhibition of NF-κB activation in mice and cynomolgus macaques. Administering αKetoA also improved diabetic glucose intolerance by inhibiting adipose tissue inflammation and fibrosis through decreased macrophage infiltration in adipose tissues and altering macrophage M1/M2 polarization in mice fed a high-fat diet. These results collectively indicate that αKetoA is a novel postbiotic derived from α-linolenic acid, which controls macrophage-associated inflammatory diseases and may have potential for developing therapeutic drugs as well as probiotic food products.

RoDiCE: robust differential protein co-expression analysis for cancer complexome.

MOTIVATION: The full spectrum of abnormalities in cancer-associated protein complexes remains largely unknown. Comparing the co-expression structure of each protein complex between tumor and healthy cells may provide insights regarding cancer-specific protein dysfunction. However, the technical limitations of mass spectrometry-based proteomics, including contamination with biological protein variants, causes noise that leads to non-negligible over- (or under-) estimating co-expression. RESULTS: We propose a robust algorithm for identifying protein complex aberrations in cancer based on differential protein co-expression testing. Our method based on a copula is sufficient for improving identification accuracy with noisy data compared to conventional linear correlation-based approaches. As an application, we use large-scale proteomic data from renal cancer to show that important protein complexes, regulatory signaling pathways and drug targets can be identified. The proposed approach surpasses traditional linear correlations to provide insights into higher-order differential co-expression structures. AVAILABILITY AND IMPLEMENTATION: https://github.com/ymatts/RoDiCE. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Possible Therapeutic Strategy Involving the Purine Synthesis Pathway Regulated by ITK in Tongue Squamous Cell Carcinoma.

The epidermal growth factor receptor is the only available tyrosine kinase molecular target for treating oral cancer. To improve the prognosis of tongue squamous cell carcinoma (TSCC) patients, a novel molecular target for tyrosine kinases is thus needed. We examined the expression of interleukin-2-inducible T-cell kinase (ITK) using immunohistochemistry, and the biological function of ITK was investigated using biochemical, phosphoproteomic, and metabolomic analyses. We found that ITK is overexpressed in TSCC patients with poor outcomes. The proliferation of oral cancer cell lines expressing ITK via transfection exhibited significant increases in three-dimensional culture assays and murine inoculation models with athymic male nude mice as compared with mock control cells. Suppressing the kinase activity using chemical inhibitors significantly reduced the increase in cell growth induced by ITK expression. Phosphoproteomic analyses revealed that ITK expression triggered phosphorylation of a novel tyrosine residue in trifunctional purine biosynthetic protein adenosine-3, an enzyme in the purine biosynthesis pathway. A significant increase in de novo biosynthesis of purines was observed in cells expressing ITK, which was abolished by the ITK inhibitor. ITK thus represents a potentially useful target for treating TSCC through modulation of purine biosynthesis.

Identification of Blood-Based Biomarkers for the Prediction of the Response to Neoadjuvant Chemoradiation in Rectal Cancer.

The current standard of care for patients with locally advanced rectal cancer (LARC) is neoadjuvant chemoradiation (nCRT) followed by total mesorectal excision surgery. However, the response to nCRT varies among patients and only about 20% of LARC patients achieve a pathologic complete response (pCR) at the time of surgery. Therefore, there is an unmet need for biomarkers that could predict the response to nCRT at an early time point, allowing for the selection of LARC patients who would or would not benefit from nCRT. To identify blood-based biomarkers for prediction of nCRT response, we performed in-depth quantitative proteomic analysis of pretreatment plasma from mice bearing rectal tumors treated with concurrent chemoradiation, resulting in the quantification of 567 proteins. Among the plasma proteins that increased in mice with residual rectal tumor after chemoradiation compared to mice that achieved regression, we selected three proteins (Vascular endothelial growth factor receptor 3 [VEGFR3], Insulin like growth factor binding protein 4 [IGFBP4], and Cathepsin B [CTSB]) for validation in human plasma samples. In addition, we explored whether four tissue protein biomarkers previously shown to predict response to nCRT (Epidermal growth factor receptor [EGFR], Ki-67, E-cadherin, and Prostaglandin G/H synthase 2 [COX2]) also act as potential blood biomarkers. Using immunoassays for these seven biomarker candidates as well as Carcinoembryonic antigen [CEA] levels on plasma collected before nCRT from 34 patients with LARC (6 pCR and 28 non-pCR), we observed that levels of VEGFR3 (p = 0.0451, AUC = 0.720), EGFR (p = 0.0128, AUC = 0.679), and COX2 (p = 0.0397, AUC = 0.679) were significantly increased in the plasma of non-pCR LARC patients compared to those of pCR LARC patients. The performance of the logistic regression model combining VEGFR3, EGFR, and COX2 was significantly improved compared with the performance of each biomarker, yielding an AUC of 0.869 (sensitivity 43% at 95% specificity). Levels of VEGFR3 and EGFR were significantly decreased 5 to 7 months after tumor resection in plasma from 18 surgically resected rectal cancer patients, suggesting that VEGFR3 and EGFR may emanate from tumors. These findings suggest that circulating VEGFR3 can contribute to the prediction of the nCRT response in LARC patients together with circulating EGFR and COX2.