Vinorelbine Improves the Efficacy of Sorafenib against Hepatocellular Carcinoma: A Promising Therapeutic Approach.

Hepatocellular carcinoma (HCC) is a leading global cause of cancer-related mortality. Despite the widespread adoption of sorafenib as the standard HCC treatment, its efficacy is constrained, frequently encountering resistance. To augment the effectiveness of sorafenib, this study investigated the synergy of sorafenib and vinorelbine using 22 HCC patient-derived xenograft (PDX) models. In this study, mice bearing HCC tumors were treated with the vehicle, sorafenib (15 mg/kg), vinorelbine (3 mg/kg), and sorafenib-vinorelbine combination (Sora/Vino). Rigorous monitoring of the tumor growth and side effects coupled with comprehensive histological and molecular analyses was conducted. The overall survival (OS) of mice bearing HCC orthotopic tumors was also assessed. Our data showed a notable 86.4% response rate to Sora/Vino, surpassing rates of 31.8% for sorafenib and 9.1% for vinorelbine monotherapies. Sora/Vino significantly inhibited tumor growth, prolonged OS of mice bearing HCC orthotopic tumors (p < 0.01), attenuated tumor cell proliferation and angiogenesis, and enhanced necrosis and apoptosis. The combination therapy effectively suppressed the focal adhesion kinase (FAK) pathway, which is a pivotal player in cell proliferation, tumor angiogenesis, survival, and metastasis. The noteworthy antitumor activity in 22 HCC PDX models positions Sora/Vino as a promising candidate for early-phase clinical trials, leveraging the established use of sorafenib and vinorelbine in HCC and other cancers.

Everolimus Acts in Synergy with Vinorelbine to Suppress the Growth of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a challenging cancer to treat, as traditional chemotherapies have shown limited effectiveness. The mammalian target of rapamycin/sirolimus (mTOR) and microtubules are prominent druggable targets for HCC. In this study, we demonstrated that co-targeting mTOR using mTOR inhibitors (everolimus and sirolimus) along with the microtubule inhibitor vinorelbine yielded results superior to those of the monotherapies in HCC PDX models. Our research showed that the vinorelbine arrests cells at the mitotic phase, induces apoptosis, and normalizes tumor blood vessels but upregulates survivin and activates the mTOR/p70S6K/4EBP1 pathway. The addition of the everolimus significantly improved the tumor response to the vinorelbine, leading to improved overall survival (OS) in most tested orthotopic HCC PDX models. The mechanistic investigation revealed that this marked antitumor effect was accompanied by the downregulations of mTOR targets (p-p70S6K, p-4EBP1, and p-S6K); several key cell-cycle regulators; and the antiapoptotic protein survivin. These effects did not compromise the normalization of the blood vessels observed in response to the vinorelbine in the vinorelbine-sensitive PDX models or to the everolimus in the everolimus-sensitive PDX models. The combination of the everolimus and vinorelbine (everolimus/vinorelbine) also promoted apoptosis with minimal toxicity. Given the cost-effectiveness and established effectiveness of everolimus, and especially sirolimus, this strategy warrants further investigation in early-phase clinical trials.

Postoperative Inflammatory Marker Surveillance in Colorectal Peritoneal Carcinomatosis.

BACKGROUND: The prognostic significance of inflammatory markers in solid cancers is well-established, albeit with considerable heterogeneity. This study sought to investigate the postoperative inflammatory marker trend in peritoneal carcinomatosis (PC), with a focus on colorectal PC (CPC), and to propose optimal surveillance periods and cutoffs. METHODS: Data were collected from a prospectively maintained database of PC patients treated at the authors' institution from April 2001 to March 2019. The platelet-lymphocyte ratio (PLR), the neutrophil-lymphocyte ratio (NLR), and the lymphocyte-monocyte ratio (LMR) were collected preoperatively and on postoperative days 0, 1 to 3, 4 to 7, 8 to 21, 22 to 56, and 57 to 90 as averages. Optimal surveillance periods and cutoffs for each marker were determined by maximally selected rank statistics. The Kaplan-Meier method and Cox proportional hazard regression models were used to investigate the association of inflammatory markers with 1-year overall survival (OS) and recurrence-free survival (RFS) using clinicopathologic parameters. RESULTS: The postoperative inflammatory marker trend and levels did not differ between the patients with and those without hyperthermic intraperitoneal chemotherapy (HIPEC). Low postoperative LMR (days 4-7), high postoperative NLR (days 8-21), and high postoperative PLR (days 22-56) were optimal for prognosticating poor 1-year OS, whereas high postoperative PLR and NLR (days 57-90) and low postoperative LMR (days 8-21) were associated with poor 1-year RFS. A composite score of these three markers was prognostic for OS in CPC. CONCLUSIONS: The reported cutoffs should be validated in a larger population of CPC patients. Future studies should account for the inflammatory response profile when selecting appropriate surveillance periods.

Durable engraftment of genetically modified FVIII-secreting autologous bone marrow stromal cells in the intramedullary microenvironment.

Genetically modified FVIII-expressing autologous bone marrow-derived mesenchymal stromal cells (BMSCs) could cure haemophilia A. However, culture-expanded BMSCs engraft poorly in extramedullary sites. Here, we compared the intramedullary cavity, skeletal muscle, subcutaneous tissue and systemic circulation as tissue microenvironments that could support durable engraftment of FVIII-secreting BMSC in vivo. A zinc finger nuclease integrated human FVIII transgene into PPP1R12C (intron 1) of culture-expanded primary canine BMSCs. FVIII-secretory capacity of implanted BMSCs in each dog was expressed as an individualized therapy index (number of viable BMSCs implanted × FVIII activity secreted/million BMSCs/24 hours). Plasma samples before and after implantation were assayed for transgenic FVIII protein using an anti-human FVIII antibody having negligible cross-reactivity with canine FVIII. Plasma transgenic FVIII persisted for at least 48 weeks after implantation in the intramedullary cavity. Transgenic FVIII protein levels were low after intramuscular implantation and undetectable after both intravenous infusion and subcutaneous implantation. All plasma samples were negative for anti-human FVIII antibodies. Plasma concentrations and durability of transgenic FVIII secretion showed no correlation with the therapy index. Thus, the implantation site microenvironment is crucial. The intramedullary microenvironment, but not extramedullary tissues, supported durable engraftment of genetically modified autologous FVIII-secreting BMSCs.

Unique Triphenylphosphonium Derivatives for Enhanced Mitochondrial Uptake and Photodynamic Therapy.

In this study, unique methyl-functionalized derivatives (T*PP+) of the drug carrier triphenylphosphonium (TPP+) that exhibit significant enhancement of the accumulation of both the cation and its conjugated cargo in cell mitochondria are designed. We show that the presence of methyl group(s) at key positions within the phenyl ring results in an increase in the hydrophobicity and solvent accessible surface area of T*PP+. In particular, when the para position of the phenyl ring in T*PP+ is functionalized with a methyl group, the cation is most exposed to the surrounding environment, leading to a large decrease in water entropy and an increase in the level of van der Waals interaction with and partition into a nonpolar solvent. Therefore, stronger binding between the hydrophobic T*PP+ and mitochondrial membrane occurs. This is exemplified in a (hexachloro-fluorescein)-TPP+ conjugate system, where an ∼12 times increase in the rate of mitochondrial uptake and a 2 times increase in photodynamic therapy (PDT) efficacy against HeLa and FU97 cancer cells are achieved when TPP+ is replaced with T*PP+. Importantly, nearly all the FU97 cells treated with the (hexachloro-fluorescein)-T*PP+ conjugate are killed as compared to only half the population of cells in the case of the (hexachloro-fluorescein)-TPP+ conjugate at a similar PDT light dosage. This study thus forms a platform for the healthcare community to explore alternative TPP+ derivatives that can act as optimal drug transporters for enhanced mitochondrially targeted therapies.

Multidimensional Genome-wide Analyses Show Accurate FVIII Integration by ZFN in Primary Human Cells.

Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genome effects is currently lacking. In light of serious adverse effects in clinical trials which employed genome-integrating viral vectors, this study evaluated potential genotoxicity of ZFN-mediated transgenesis using different techniques. We employed deep sequencing of predicted off-target sites, copy number analysis, whole-genome sequencing, and RNA-seq in primary human umbilical cord-lining epithelial cells (CLECs) with AAVS1 ZFN-mediated FVIII transgene integration. We combined molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. We also showed AAVS1 ZFN activity and durable FVIII transgene secretion in primary human dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells. Our study suggests that, with close attention to the molecular design of genome-modifying constructs, AAVS1 ZFN-mediated FVIII integration in several primary human cell types may be safe and efficacious.

Multidimensional identification of tissue biomarkers of gastric cancer.

Gastric cancer remains highly fatal due to a dearth of diagnostic biomarkers for early stage disease and molecular targets for therapy. Plasma membrane proteins, including cluster of differentiation (CD) proteins and receptor tyrosine kinases (RTKs), are a rich reservoir of biomarkers. Recognizing that interrogating plasma membrane proteins individually overlooks extensive interactions among them, we have systematically investigated the membrane proteomes and transcriptomes of six gastric cancer cell lines. Our data revealed aberrantly high expression of proteins whose functions accurately reflect the clinical phenotype of gastric cancer, and prioritized critical RTKs and CD proteins in gastric cancer. Expression of selected surface proteins was confirmed by flow cytometry and immunostaining of clinical gastric cancer tissues. Close to 90% of the gastric cancer tissues in a cohort showed up-regulation of at least one of four proteins, that is, MET, EPHA2, FGFR2, and CD104/ITGB4. All intestinal type gastric cancer tumors in this cohort overexpressed at least one of a panel of three proteins, MET, FGFR2, and EPHA2. This study reports the first quantitative global landscape of the surface proteome of gastric cancer cells and provides a shortlist of gastric cancer biomarkers.

Global molecular dysfunctions in gastric cancer revealed by an integrated analysis of the phosphoproteome and transcriptome.

We integrated LC-MS/MS-based and protein antibody array-based proteomics with genomics approaches to investigate the phosphoproteome and transcriptome of gastric cancer cell lines and endoscopic gastric biopsies from normal subjects and patients with benign gastritis or gastric cancer. More than 3,000 non-redundant phosphorylation sites in over 1,200 proteins were identified in gastric cancer cells. We correlated phosphoproteome data with transcriptome data sets and reported the expression of 41 protein kinases, 5 phosphatases and 65 phosphorylated mitochondrial proteins in gastric cancer cells. Transcriptional expression levels of 190 phosphorylated proteins were >2-fold higher in gastric cancer cells compared to normal stomach tissue. Pathway analysis demonstrated over-presentation of DNA damage response pathway and underscored critical roles of phosphorylated p53 in gastric cancer. This is the first study to comprehensively report the gastric cancer phosphoproteome. Integrative analysis of the phosphoproteome and transcriptome provided an expansive view of molecular signaling pathways in gastric cancer.

Generating patient-derived ascites-dependent xenograft mouse models of peritoneal carcinomatosis.

Clinically relevant animal models are crucial for effective development of therapeutics for peritoneal carcinomatosis (PC). This protocol describes the generation of patient-derived ascites-dependent xenograft (PDADX) models from the cellular component of ascites. The use of routine intraperitoneal injection of the fluid component of ascites is analogous to the biological events occurring intra-abdominally in patients with PC. By serving as a proxy, PDADX models represent a valuable tool for preclinical testing of new therapeutics for PC. For complete details on the use and execution of this protocol, please refer to Hendrikson et al. (2022).

Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis.

Peritoneal carcinomatosis (PC) present a ubiquitous clinical conundrum in all intra-abdominal malignancies. Via functional and transcriptomic experiments of ascites-treated PC cells, we identify STAT3 as a key signaling pathway. Integrative analysis of publicly available databases and correlation with clinical cohorts (n = 7,359) reveal putative clinically significant activating ligands of STAT3 signaling. We further validate a 3-biomarker prognostic panel in ascites independent of clinical covariates in a prospective study (n = 149). Via single-cell sequencing experiments, we uncover that PAI-1, a key component of the prognostic biomarker panel, is largely secreted by fibroblasts and mesothelial cells. Molecular stratification of ascites using PAI-1 levels and STAT3 activation in ascites-treated cells highlight a therapeutic opportunity based on a phenomenon of paracrine addiction. These results are recapitulated in patient-derived ascites-dependent xenografts. Here, we demonstrate therapeutic proof of concept of direct ligand inhibition of a prognostic target within an enclosed biological space.

Biosafety assessment of site-directed transgene integration in human umbilical cord-lining cells.

Biosafety and efficacy considerations that impede clinical application of gene therapy could be addressed by nonviral ex vivo cell therapy, utilizing transgenic cells that have been comprehensively pre-evaluated for genotoxic potential and transgene expression. We evaluated the genotoxic potential of phiC31 bacteriophage integrase-mediated transgene integration in cord-lining epithelial cells (CLECs) readily cultured from the outer membrane of human umbilical cords, by sequencing and mapping integration sites, spectral karyotyping, high-resolution genome copy number, transcriptome, and transgene copy number analyses and in vivo tumorigenicity. Of 44 independent integration events, <5% were exonic and 85% of modified cells had integrated

Gene Expression Changes Associated with Dedifferentiation in Liposarcoma Predict Overall Survival.

Up to 10% of well-differentiated liposarcoma (WDLS) progress to dedifferentiated liposarcoma (DDLS). We aimed to identify gene expression changes associated with dedifferentiation and whether these were informative of tumour biology of DDLS. We analysed datasets from the Gene Expression Omnibus (GEO, ID = GSE30929) database to identify differentially expressed genes between WDLS (n = 52) and DDLS (n = 39). We validated the signature on whole and laser-capture microdissected samples from patients with tumours consisting of mixed WDLS and DDLS components. A subset of this signature was applied to an independent dataset from The Cancer Genome Atlas (TCGA, n = 58 DDLS) database to segregate samples based on gene expression and compared for recurrence and overall survival (OS). A 15-gene signature consisting of genes with increased expression in DDLS compared to WDLS was generated. This signature segregated WDLS and DDLS samples from patients with mixed component tumours and across multiple recurrences. A further subset of this signature, consisting of five genes (AQP7, ACACB, FZD4, GPD1, LEP), segregated DDLS in a TCGA cohort with a significant difference in OS (p = 0.019) and recurrence-free survival (RFS) (p = 0.061). The five-gene model stratified DDLS into prognostic groups and outperformed clinical factors in existing models in retroperitoneal DDLS.

A machine learning approach to identify predictive molecular markers for cisplatin chemosensitivity following surgical resection in ovarian cancer.

Ovarian cancer is associated with poor prognosis. Platinum resistance contributes significantly to the high rate of tumour recurrence. We aimed to identify a set of molecular markers for predicting platinum sensitivity. A signature predicting cisplatin sensitivity was generated using the Genomics of Drug Sensitivity in Cancer and The Cancer Genome Atlas databases. Four potential biomarkers (CYTH3, GALNT3, S100A14, and ERI1) were identified and optimized for immunohistochemistry (IHC). Validation was performed on a cohort of patients (n = 50) treated with surgical resection followed by adjuvant carboplatin. Predictive models were established to predict chemosensitivity. The four biomarkers were also assessed for their ability to prognosticate overall survival in three ovarian cancer microarray expression datasets from The Gene Expression Omnibus. The extreme gradient boosting (XGBoost) algorithm was selected for the final model to validate the accuracy in an independent validation dataset (n = 10). CYTH3 and S100A14, followed by nodal stage, were the features with the greatest importance. The four gene signature had comparable prognostication as clinical information for two-year survival. Assessment of tumour biology by means of gene expression can serve as an adjunct for prediction of chemosensitivity and prognostication. Potentially, the assessment of molecular markers alongside clinical information offers a chance to further optimise therapeutic decision making.

An Optimised Protocol Harnessing Laser Capture Microdissection for Transcriptomic Analysis on Matched Primary and Metastatic Colorectal Tumours.

Generation of large amounts of genomic data is now feasible and cost-effective with improvements in next generation sequencing (NGS) technology. Ribonucleic acid sequencing (RNA-Seq) is becoming the preferred method for comprehensively characterising global transcriptome activity. Unique to cytoreductive surgery (CRS), multiple spatially discrete tumour specimens could be systematically harvested for genomic analysis. To facilitate such downstream analyses, laser capture microdissection (LCM) could be utilized to obtain pure cell populations. The aim of this protocol study was to develop a methodology to obtain high-quality expression data from matched primary tumours and metastases by utilizing LCM to isolate pure cellular populations. We demonstrate an optimized LCM protocol which reproducibly delivered intact RNA used for RNA sequencing and quantitative polymerase chain reaction (qPCR). After pathologic annotation of normal epithelial, tumour and stromal components, LCM coupled with cDNA library generation provided for successful RNA sequencing. To illustrate our framework's potential to identify targets that would otherwise be missed with conventional bulk tumour sequencing, we performed qPCR and immunohistochemical technical validation to show that the genes identified were truly expressed only in certain sub-components. This study suggests that the combination of matched tissue specimens with tissue microdissection and NGS provides a viable platform to unmask hidden biomarkers and provides insight into tumour biology at a higher resolution.

Pairing a prognostic target with potential therapeutic strategy for head and neck cancer.

OBJECTIVES: We have previously identified and validated a panel of molecular prognostic markers (ATP13A3, SSR3, and ANO1) for Head and Neck Squamous Cell Carcinoma (HNSCC). The aim of this study was to investigate the consequence of ATP13A3 dysregulation on signaling pathways, to aid in formulating a therapeutic strategy targeting ATP13A3-overexpressing HNSCC. MATERIALS AND METHODS: Gene Set Enrichment Analysis (GSEA) was performed on HNSCC microarray expression data (Internal local dataset [n = 92], TCGA [n = 232], EMBL [n = 81]) to identify pathways associated with high expression of ATP13A3. Validation was performed using immunohistochemistry (IHC) on tissue microarrays (TMAs) of head and neck cancers (n = 333), staining for ATP13A3 and phosphorylated Aurora kinase A (phospho-T288). Short interfering RNA was used to knockdown ATP13A3 expression in patient derived HNSCC cell lines. Protein expression of ATP13A3 and Aurora kinase A was then assessed by immunoblotting. RESULTS: GSEA identified Aurora kinase pathway to be associated with high expression of ATP13A3 (p = 0.026). The Aurora kinase pathway was also associated with a trend towards poor prognosis and tumor aggressiveness (p = 0.086, 0.094, respectively). Furthermore, the immunohistochemical staining results revealed a significant association between Aurora kinase activity and high ATP13A3 expression (p < 0.001). Knockdown of ATP13A3 in human head and neck cell lines showed decrease in Aurora kinase A levels. CONCLUSION: Tumors with high ATP13A3 are associated with high Aurora kinase activity. This suggests a potential therapeutic role of Aurora kinase inhibitors in a subset of poor prognosis HNSCC patients with overexpression of ATP13A3.

A set of molecular markers predicts chemosensitivity to Mitomycin-C following cytoreductive surgery and hyperthermic intraperitoneal chemotherapy for colorectal peritoneal metastasis.

Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) is associated with significant perioperative morbidity and mortality. We aim to generate and validate a biomarker set predicting sensitivity to Mitomycin-C to refine selection of patients with colorectal peritoneal metastasis (CPM) for this treatment. A signature predicting Mitomycin-C sensitivity was generated using data from Genomics of Drug Sensitivity in Cancer and The Cancer Genome Atlas. Validation was performed on CPM patients who underwent CRS-HIPEC (n = 62) using immunohistochemistry (IHC). We determined predictive significance of our set using overall survival as a surrogate endpoint via a logistic regression model. Three potential biomarkers were identified and optimized for IHC. Patients exhibiting lower expression of PAXIP1 and SSBP2 had poorer survival than those with higher expression (p = 0.045 and 0.140, respectively). No difference was observed in patients with differing DTYMK expression (p = 0.715). Combining PAXIP1 and SSBP2 in a set, patients with two dysregulated protein markers had significantly poorer survival than one or no dysregulated marker (p = 0.016). This set independently predicted survival in a Cox regression model (HR 5.097; 95% CI 1.731-15.007; p = 0.003). We generated and validated an IHC prognostic set which could potentially identify patients who are likely to benefit from HIPEC using Mitomycin-C.

Integrated Multimodal Evaluation of Genotoxicity in ZFN-Modified Primary Human Cells.

Iatrogenic adverse events in clinical trials of retroviral vector-mediated gene-corrected cells have prioritized the urgent need for more comprehensive and stringent assessment of potentially genotoxic off-target alterations and the biosafety of cells intended for therapeutic applications. Genome editing tools such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 nuclease systems are being investigated as safer and efficient alternatives for site-directed genome modification. Using site-specific integration into the AAVS1 locus of primary human cells as an example, we present an integrated approach to multimodal investigation of off-target alterations and an evaluation of potential genotoxicity induced by ZFN-mediated integration of a therapeutic transgene.

Analysis and functional annotation of expressed sequence tags (ESTs) from multiple tissues of oil palm (Elaeis guineensis Jacq.).

BACKGROUND: Oil palm is the second largest source of edible oil which contributes to approximately 20% of the world's production of oils and fats. In order to understand the molecular biology involved in in vitro propagation, flowering, efficient utilization of nitrogen sources and root diseases, we have initiated an expressed sequence tag (EST) analysis on oil palm. RESULTS: In this study, six cDNA libraries from oil palm zygotic embryos, suspension cells, shoot apical meristems, young flowers, mature flowers and roots, were constructed. We have generated a total of 14537 expressed sequence tags (ESTs) from these libraries, from which 6464 tentative unique contigs (TUCs) and 2129 singletons were obtained. Approximately 6008 of these tentative unique genes (TUGs) have significant matches to the non-redundant protein database, from which 2361 were assigned to one or more Gene Ontology categories. Predominant transcripts and differentially expressed genes were identified in multiple oil palm tissues. Homologues of genes involved in many aspects of flower development were also identified among the EST collection, such as CONSTANS-like, AGAMOUS-like (AGL)2, AGL20, LFY-like, SQUAMOSA, SQUAMOSA binding protein (SBP) etc. Majority of them are the first representatives in oil palm, providing opportunities to explore the cause of epigenetic homeotic flowering abnormality in oil palm, given the importance of flowering in fruit production. The transcript levels of two flowering-related genes, EgSBP and EgSEP were analysed in the flower tissues of various developmental stages. Gene homologues for enzymes involved in oil biosynthesis, utilization of nitrogen sources, and scavenging of oxygen radicals, were also uncovered among the oil palm ESTs. CONCLUSION: The EST sequences generated will allow comparative genomic studies between oil palm and other monocotyledonous and dicotyledonous plants, development of gene-targeted markers for the reference genetic map, design and fabrication of DNA array for future studies of oil palm. The outcomes of such studies will contribute to oil palm improvements through the establishment of breeding program using marker-assisted selection, development of diagnostic assays using gene targeted markers, and discovery of candidate genes related to important agronomic traits of oil palm.

Chromosomal breaks at FRA18C: association with reduced DOK6 expression, altered oncogenic signaling and increased gastric cancer survival.

Chromosomal rearrangements are common in cancer. More than 50% occur in common fragile sites and disrupt tumor suppressors. However, such rearrangements are not known in gastric cancer. Here we report recurrent 18q2 breakpoints in 6 of 17 gastric cancer cell lines. The rearranged chromosome 18, t(9;18), in MKN7 cells was flow sorted and identified by reverse chromosome painting. High-resolution tiling array hybridization mapped breakpoints to DOK6 (docking protein 6) intron 4 in FRA18C (18q22.2) and an intergenic region in 9q22.2. The same rearrangement was detected by FISH in 22% of 99 primary gastric cancers. Intron 4 truncation was associated with reduced DOK6 transcription. Analysis of The Cancer Genome Atlas stomach adenocarcinoma cohort showed significant correlation of DOK6 expression with histological and molecular phenotypes. Multiple oncogenic signaling pathways (gastrin-CREB, NGF-neurotrophin, PDGF, EGFR, ERK, ERBB4, FGFR1, RAS, VEGFR2 and RAF/MAP kinase) known to be active in aggressive gastric cancers were strikingly diminished in gastric cancers with low DOK6 expression. Median survival of patients with low DOK6-expressing tumors was 2100 days compared with 533 days in patients with high DOK6-expressing tumors (log-rank P = 0.0027). The level of DOK6 expression in tumors predicted patient survival independent of TNM stage. These findings point to new functions of human DOK6 as an adaptor that interacts with diverse molecular components of signaling pathways. Our data suggest that DOK6 expression is an integrated biomarker of multiple oncogenic signals in gastric cancer and identify FRA18C as a new cancer-associated fragile site.