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Robert J Coffey

Publications and source records attributed to Robert J Coffey.

At least 19 recordsLinked to original sources
PubMed2025

A comprehensive analysis of supermere, exomere, and extracellular vesicle isolation and cargo in colorectal cancer.

Biofluids contain a heterogeneous mixture of extracellular vesicles and non-vesicular nanoparticles (including exomeres and supermeres) that transport a diverse array of proteins, RNA, and lipids. Our previous efforts to characterize the contents of these carriers in colorectal cancer relied on 2D culture systems requiring large-scale setups and time-consuming ultracentrifugation-based isolation. To streamline this process, we have combined 3D hollow-fiber bioreactor production and fast-protein liquid chromatography-based size-exclusion chromatography. Here, we compare the impact of culture methods and purification strategies on small extracellular vesicle, exomere, and supermere cargo. Proteomic analyses show consistently distinct profiles for extracellular vesicles, exomeres, and supermeres regardless of culture conditions or isolation method. In contrast, these two variables influence small RNAs, their base modifications, and lipidomic profiles. We present an online tool to query these and future secretome datasets (https://superomics.shinyapps.io/browse).

Humans↗
PubMed2025

Cysteinyl leukotrienes stimulate gut absorption of food allergens to promote anaphylaxis in mice.

Food-specific immunoglobulin E (IgE) triggers life-threatening anaphylaxis; however, for unclear reasons, some people with food-specific IgE are asymptomatic upon allergen consumption. We studied strains of mice with different sensitivities to anaphylaxis when orally challenged with allergen to identify possible causes. In resistant C57BL/6 mice, intestinal goblet cells transported less food allergen than did anaphylaxis-susceptible strains, even before allergic sensitization. In a forward genetic screen, resistance was correlated with dipeptidase 1 (Dpep1) variants. DPEP1 is expressed in intestinal epithelium and catabolizes leukotriene D4 (LTD4). Blocking DPEP1 with cilastatin, deleting Dpep1, or administering LTD4 orally enhanced allergen transport in resistant mice. Conversely, pretreatment of susceptible mice with a synthesis inhibitor, zileuton, abrogated allergen absorption and oral anaphylaxis, indicating that this could be an approach to treating food allergy.

Animals↗
PubMed2007

Oncogenic KRAS provides a uniquely powerful and variable oncogenic contribution among RAS family members in the colonic epithelium.

Activating mutations of the RAS family of small GTPases are among the most common genetic events in human tumorigenesis. Constitutive activation of the three canonical family members, KRAS, NRAS, and HRAS segregate strongly by tissue type. Of these, KRAS mutations predominate in human tumors, including those arising from the colon and lung. We sought to compare the oncogenic contributions of different RAS isoforms in a comparable genetic setting and to explore downstream molecular changes that may explain the apparent differential oncogenic effects of the various RAS family members. We utilized colorectal cancer cell lines characterized by oncogenic KRAS in parallel with isogenically derived lines in which the mutant allele has been disrupted. We additionally attempted to reconstitute the isogenic derivatives with oncogenic forms of other RAS family members and analyze them in parallel. Pairwise analysis of HCT 116 and DLD-1 cell lines as well as their isogenic derivatives reveals distinct K-RAS(G13D) signatures despite the genetic similarities of these cell lines. In DLD-1, for example, oncogenic K-RAS enhances the motility of these cells by downregulation of Rap1 activity, yet is not associated with increased ERK1/2 phosphorylation. In HCT 116, however, ERK1/2 phosphorylation is elevated relative to the isogenic derivative, but Rap1 activity is unchanged. K-RAS is uniquely oncogenic in the colonic epithelium, though the molecular aspects of its oncogenic contribution are not necessarily conserved across cell lines. We therefore conclude that the oncogenic contribution of K-RAS is a function of its multifaceted functionality and is highly context-dependent.

Cell Line, Tumor↗
PubMed2007

Ménétrier disease and gastrointestinal stromal tumors: hyperproliferative disorders of the stomach.

Ménétrier disease and gastrointestinal stromal tumors (GISTs) are hyperproliferative disorders of the stomach caused by dysregulated receptor tyrosine kinases (RTKs). In Ménétrier disease, overexpression of TGF-alpha, a ligand for the RTK EGFR, results in selective expansion of surface mucous cells in the body and fundus of the stomach. In GISTs, somatic mutations of the genes encoding the RTK KIT (or PDGFRA in a minority of cases) result in constitutive kinase activity and neoplastic transformation of gut pacemaker cells (interstitial cells of Cajal). On the basis of the involvement of these RTKs in the pathogenesis of these disorders, Ménétrier disease patients have been effectively treated with a blocking monoclonal antibody specific for EGFR and GIST patients with KIT and PDGFRA tyrosine kinase inhibitors.

Cell Division↗
PubMed2006

Structural studies of human Naked2: a biologically active intrinsically unstructured protein.

Naked1 and 2 are two mammalian orthologs of Naked Cuticle, a canonical Wnt signaling antagonist in Drosophila. Naked2, but not Naked1, interacts with transforming growth factor-alpha (TGFalpha) and escorts TGFalpha-containing vesicles to the basolateral membrane of polarized epithelial cells. Full-length Naked2 is poorly soluble. Since most functional domains, including the Dishevelled binding region, EF-hand, vesicle recognition, and membrane targeting motifs, reside in the N-terminal half of the protein, we expressed and purified the first 217 residues of human Naked2 and performed a functional analysis of this fragment. Its circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra showed no evidence of secondary and/or tertiary structure. The fragment did not bind calcium or zinc. These results indicate that the N-terminal half of Naked2 behaves as an intrinsically unstructured protein.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Urine PGE-M: A metabolite of prostaglandin E2 as a potential biomarker of advanced colorectal neoplasia.

BACKGROUND & AIMS: The enzyme cyclooxygenase-2 is expressed in a majority of colorectal carcinomas (CRCs) and is important in prostaglandin production. We have developed an accurate method to measure the urinary metabolite of prostaglandin E(2) (PGE-M) using recently developed mass spectrometric techniques. The purpose of this pre-validation study was to determine if urinary PGE-M levels can be used as a biomarker to discriminate between healthy patients and those with colorectal disease. METHODS: Urine PGE-M was assessed in a total of 228 patients with CRC, colonic adenomatous polyps, Crohn's disease, and in subjects with no endoscopically detectable disease. Thirteen rectal carcinoma patients were treated with celecoxib and urinary PGE-M was measured before and after treatment. RESULTS: Urine PGE-M levels were increased among healthy men compared with healthy women (median, 8.59 [interquartile range (IQR), 5.67-22.3] vs 4.25 [IQR, 2.35-6.03], P = .0027). Urine PGE-M levels among patients with Crohn's disease (median, 19.85 [IQR, 6.89-90.2]), CRC (median, 14.65 [IQR, 5.94-92.1]), or large adenomas greater than 1 cm in size (median, 18.85 [IQR, 11.9-25.6]) were significantly increased when compared with patients who had either small polyps less than 1 cm in size (median, 9.69 [IQR, 6.41-22.2]), or no polyps (median, 7.05 [IQR, 2.35-24.7]) (P = .0001). PGE-M levels decreased significantly after celecoxib treatment in patients with rectal cancer (median, 21.7 [IQR, 16.2-29.9] vs 9.14 [IQR, 7.14-13.2], P = .009). CONCLUSIONS: The increase in urinary PGE-M in patients with colorectal cancers and large adenomas suggests that urinary PGE-M is a potentially useful biomarker for the detection of advanced colorectal neoplasia.

Adenoma↗
PubMed2006

Randomized controlled trial of intraputamenal glial cell line-derived neurotrophic factor infusion in Parkinson disease.

OBJECTIVE: Glial cell line-derived neurotrophic factor (GDNF) exerts potent trophic influence on midbrain dopaminergic neurons. This randomized controlled clinical trial was designed to confirm initial clinical benefits observed in a small, open-label trial using intraputamenal (Ipu) infusion of recombinant human GDNF (liatermin). METHODS: Thirty-four PD patients were randomized 1 to 1 to receive bilateral continuous Ipu infusion of liatermin 15 microg/putamen/day or placebo. The primary end point was the change in Unified Parkinson Disease Rating Scale (UPDRS) motor score in the practically defined off condition at 6 months. Secondary end points included other UPDRS scores, motor tests, dyskinesia ratings, patient diaries, and (18)F-dopa uptake. RESULTS: At 6 months, mean percentage changes in "off" UPDRS motor score were -10.0% and -4.5% in the liatermin and placebo groups, respectively. This treatment difference was not significant (95% confidence interval, -23.0 to 12.0, p = 0.53). Secondary end point results were similar between the groups. A 32.5% treatment difference favoring liatermin in mean (18)F-dopa influx constant (p = 0.019) was observed. Serious, device-related adverse events required surgical repositioning of catheters in two patients and removal of devices in another. Neutralizing antiliatermin antibodies were detected in three patients (one on-study and two in the open-label extension). INTERPRETATION: Liatermin did not confer the predetermined level of clinical benefit to patients with PD despite increased (18)F-dopa uptake. It is uncertain whether technical differences between this trial and positive open-label studies contributed in any way this negative outcome.

Adult↗
PubMed2006

Neurostimulation for chronic noncancer pain: an evaluation of the clinical evidence and recommendations for future trial designs.

OBJECT: Neurostimulation to treat chronic pain includes approved and investigational therapies directed at the spinal cord, thalamus, periaqueductal or periventricular gray matter, motor cortex, and peripheral nerves. Persistent pain after surgery and work-related or neural injuries are common indications for such treatments. In light of the risks, efforts, costs, and expectations associated with neurostimulation therapies, a careful reexamination of the methods used to gather evidence for this treatment's long-term efficacy is in order. METHODS: The authors combed English-language publications to determine the nature of the evidence supporting the efficacy of neurostimulation therapies for chronic noncancer pain. To formulate recommendations for the design of future studies, the results of their analysis were compared with established guidelines for the evaluation of medical evidence. Evidence supporting the efficacy of neurostimulation has been collected predominantly from retrospective series or from prospective studies whose design or methods of analysis make them subject to limited interpretation. To date, there has been no successful clinical study focused on establishing the efficacy of neurostimulation for pain and incorporating sufficient numbers of participants, matched control groups, sham stimulation, randomization, prospectively defined end points, and methods for controlling experimental bias. Currently available data provide little support for the common practices of psychological or pharmacological screening or trial stimulation to predict and/or improve long-term results. CONCLUSIONS: These findings do not diminish the value of previous investigations or positive patient experiences and do not mean that the treatments are ineffective; rather, they reveal that new data are required to answer the questions raised in and by previous study data. Future analyses of emerging neurostimulation modalities for pain should, whenever feasible, require unambiguous diagnoses as an entry criterion and should involve the use of randomization, parallel control groups that receive sham stimulation, and blinding of patients, investigators, and device programmers. Given the chronicity of patient symptoms and stimulation therapies, efficacy should be studied for 1 year or longer after device implantation. Meticulous study methods are especially important to evaluate new therapies like motor cortex and occipital nerve stimulation.

Chronic Disease↗
PubMed2005

Randomized phase II trial of the clinical and biological effects of two dose levels of gefitinib in patients with recurrent colorectal adenocarcinoma.

PURPOSE: The clinical objective of this trial was to evaluate gefitinib in patients with metastatic colorectal cancer that had progressed despite prior treatment. Serial tumor biopsies were performed when possible and analyzed for activation of the epidermal growth factor receptor (EGFR) signaling pathway. Serial serum samples were measured for amphiregulin and transforming growth factor-alpha (TGFalpha). PATIENTS AND METHODS: One hundred fifteen patients were randomly assigned to receive gefitinib 250 or 500 mg orally once a day. One hundred ten patients were assessable for clinical efficacy. Biologic evaluation was performed on paired tumor samples from 28 patients and correlated with clinical outcome. RESULTS: Median progression-free survival was 1.9 months (95% CI, 1.8 to 2.1 months) and 4-month progression-free survival rate was 13% +/- 5%. One patient achieved a radiographic partial response (RR = 1%; 95% CI, 0.01% to 5%). Median survival was 6.3 months (95% CI, 5.1 to 8.2 months). The most common adverse events were skin rash, diarrhea, and fatigue. In the biopsy cohort, expression of total or activated EGFR, activated Akt, activated MAP-kinase, or Ki67 did not decrease following 1 week of gefitinib. However, a trend toward decreased post-treatment levels of activated Akt and Ki67 was observed in patients with a PFS higher than the median, although these did not reach the .05 level of significance. CONCLUSION: Gefitinib is inactive as a single agent in patients with previously treated colorectal cancer. In tumor samples, gefitinib did not inhibit activation of its proximal target, EGFR. Trends were observed for inhibition of downstream regulators of cellular survival and proliferation in patients achieving longer progression-free survival.

Adenocarcinoma↗
PubMed2005

Differential effects of amphiregulin and TGF-alpha on the morphology of MDCK cells.

Although both amphiregulin and TGF-alpha are known to exert their effects through the EGF receptor, we find that concentrations of recombinant human amphiregulin and TGF-alpha that are equipotent in EGF receptor activation and mitogenesis exhibit markedly different effects on MDCK cell morphology. Amphiregulin induces a spindle-like morphology that is associated with a redistribution of E-cadherin from a Triton-insoluble to Triton-soluble pool. TGF-alpha does not affect epithelial morphology nor does it affect the distribution of the Triton-soluble or -insoluble pool of E-cadherin. The effects of amphiregulin on E-cadherin are associated with actin rearrangement. The morphological and biochemical effects of amphiregulin are prevented by EGF receptor blockade but require Src-family kinase activity and MAPK signaling. These results identify an action of amphiregulin that is distinct from TGF-alpha that may contribute to amphiregulin's participation in the pathogenesis of inflammatory disorders like psoriasis and cancer.

Amphiregulin↗
PubMed2005

Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates.

Epithelial metaplasia occurs when one predominant cell type in a tissue is replaced by another, and is frequently associated with an increased risk of subsequent neoplasia. In both mouse and human pancreas, acinar-to-ductal metaplasia has been implicated in the generation of cancer precursors. We show that pancreatic epithelial explants undergo spontaneous acinar-to-ductal metaplasia in response to EGFR signaling, and that this change in epithelial character is associated with the appearance of nestin-positive transitional cells. Lineage tracing involving Cre/lox-mediated genetic cell labeling reveals that acinar-to-ductal metaplasia represents a true transdifferentiation event, mediated by initial dedifferentiation of mature exocrine cells to generate a population of nestin-positive precursors, similar to those observed during early pancreatic development. These results demonstrate that a latent precursor potential resides within mature exocrine cells, and that this potential is regulated by EGF receptor signaling. In addition, these observations provide a novel example of rigorously documented transdifferentiation within mature mammalian epithelium, and suggest that plasticity of mature cell types may play a role in the generation of neoplastic precursors.

Animals↗
PubMed2005

Identification of MAGI-3 as a transforming growth factor-alpha tail binding protein.

The cytoplasmic domain of the transforming growth factor-alpha precursor (proTGFalpha) contains a C-terminal PSD-95/SAP90, Discs Large, and Zona Occludens-1 (PDZ) recognition motif (TVV). By yeast two-hybrid screening of a mouse embryo library, we have found that a third member of a family of PDZ-containing proteins, membrane associated guanylate kinase inverted-3 (MAGI-3), binds to TGFalpha's TVV. MAGI-3 is widely expressed in multiple mouse tissues, including brain. Immunolocalization showed that MAGI-3 and TGFalpha were colocalized in neurons in the cortex and dentate gyrus, as well as in ependymal cells and some astrocytes. In vitro, proTGFalpha bound the PDZ-1 domain of MAGI-3 and MAGI-2, but not MAGI-1. MAGI-3 and the 17-kDa cell surface form of proTGFalpha interact transiently in MDCK cells stably transfected with both MAGI-3 and human proTGFalpha cDNAs. MAGI-3 and wild-type proTGFalpha colocalize at the cell surface. In contrast, MAGI-3 forms a stable complex with membrane-fixed TGFalpha early in the secretory pathway and interacts with immature and cell surface forms of membrane-fixed TGFalpha. Overexpression of MAGI-3 resulted in increased levels of TGFalpha in the basolateral medium of polarized MDCK cells, suggesting that MAGI-3 has a role in efficient trafficking of TGFalpha to the cell surface in polarized epithelial cells.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Gene expression profile analysis of mouse colon embryonic development.

During late embryogenesis, the mouse colon develops from a pseudostratified, undifferentiated endoderm to a single-layered columnar epithelium with accompanying mesenchymal maturation. To identify regulatory genetic programs underlying these morphological changes, we profiled gene expression of the developing mouse colon by microarray from embryonic day (E)13.5 to E18.5. Unbiased cluster analysis of 13,484 cDNA elements revealed two distinct groups of genes whose expression changes reflect the dynamic morphological events of the epithelium and mesenchyme during this period. Additional analyses revealed two subsets of genes whose expression is either upregulated or downregulated over the same developmental period. Of those genes whose expression increases from E13.5 to E18.5 (n = 158), known functions include acquisition and/or maintenance of colonic differentiation. Genes whose transcription is downregulated over this period (n = 49) have demonstrated roles in nuclear organization, transcriptional regulation, and cell proliferation. These results provide the basis for a molecular portrait of colonic development during late embryogenesis and should be a valuable resource for investigators interested in colonic development and neoplasia, as well as comparative organogenesis.

Animals↗
PubMed2005

Keratin 19 gene drives Cre recombinase expression throughout the early postimplantation mouse embryo.

The development of Cre-lox technology has created new opportunities for studying the tissue-specific functions of genes in vivo during development and disease. We analyzed the spatial and temporal activity of Cre recombinase whose coding sequence was inserted into the endogenous locus for keratin 19. Rather than providing epithelial-specific recombination during organogenesis, this K19cre allele allows unexpected recombination in early embryonic development, resulting in recombination of a loxP-flanked allele throughout all tissues of the mouse, but with sparing of the extraembryonic endoderm, including the anterior visceral endoderm.

Animals↗
PubMed2005

E-cadherin is regulated by the transcriptional repressor SLUG during Ras-mediated transformation of intestinal epithelial cells.

BACKGROUND: Loss of the cell membrane protein E-cadherin is a critical event during Ras-mediated transformation of intestinal epithelial cells. The purpose of our study is to determine if activation of the transcriptional repressor SLUG is an important component of the mechanism of Ras-induced loss of E-cadherin. METHODS: Rat intestinal epithelial (RIE) cells were engineered to express mutated human Ha-Ras(Val12) complementary DNA (H-Ras cells). Cell morphology was examined by light microscopy. RNA and protein expression were measured by semiquantitative polymerase chain reaction and Western blot analyses, respectively. Short interfering RNA with 2 different oligos was used to knock down the expression of SLUG. RESULTS: Oncogenic ras induces upregulation of the transcriptional repressor SLUG and subsequent downregulation of the junctional protein E-cadherin. Gene silencing of SLUG by short interfering RNA allows E-cadherin to be reexpressed. E-cadherin protein reexpression allows partial rescue of the transformed phenotype. CONCLUSION: These data suggest a mechanism whereby Ras signaling causes an upregulation of transcriptional repressors and subsequent downregulation of E-cadherin as a malignant phenotype is propagated.

Animals↗
PubMed2005

Ligand-dependent activation of the epidermal growth factor receptor by secondary bile acids in polarizing colon cancer cells.

BACKGROUND: Secondary bile acids such as deoxycholic acid (DCA) are known to promote colorectal cancer (CRC). Increasing evidence suggests that DCA-induced signaling is mediated by activation of the epidermal growth factor receptor (EGFR). We have shown that activation of the EGFR induces up-regulation of cyclooxygenase 2, basolateral release of prostaglandins (PGs), and mitogenesis in a polarizing human colon cancer cell line, HCA-7. The purpose of this study was to determine the mechanism by which DCA activates EGFR in human polarizing CRC cell lines HCA-7 and HCT-8. METHODS: A primary, non-tumor-promoting bile acid (cholic acid [CA]) and a secondary, tumor-promoting bile acid, DCA, were added to the apical and basolateral compartment of polarized HCA-7 and HCT-8 cells. These cells were pretreated with monoclonal antibody 528, a monoclonal antibody that inhibits ligand binding to EGFR, or with WAY-022, a selective inhibitor of tumor necrosis factor-alpha converting enzyme/a disintegrin and metalloprotease-17 (TACE/ADAM-17), which cleaves amphiregulin (AR) to its mature, soluble form from the basolateral cell membrane. AR levels were measured in the apical and basolateral medium and cell lysates by radioimmunoassay. PGs were measured in the apical and basolateral medium by gas chromatography/mass spectrometry. RESULTS: Basolateral delivery of DCA, but not CA, preferentially stimulated release of AR into the basolateral medium compared with cell lysates of polarized HCA-7 and HCT-8 cells. Basolateral delivery of DCA resulted in increased basolateral PGE2 levels (P < .05), and this effect was attenuated by pretreatment with monoclonal antibody 528 (P < .05). Inhibiting cell surface cleavage of AR with WAY-022 before DCA treatment reduced AR (P < .05) and PGE2 (P < .05) levels in the basolateral medium. CONCLUSION: DCA, but not CA, results in compartment-specific, ligand-dependent activation of EGFR and subsequent increased basolateral PGE2 levels. The mechanism of DCA-induced EGFR activation is ligand-dependent and is controlled, at least in part, at the level of AR release from the basolateral cell membrane.

Amphiregulin↗
PubMed2005

Chronic treatment of Ménétrier's disease with Erbitux: clinical efficacy and insight into pathophysiology.

BACKGROUND & AIMS: Ménétrier's disease is a rare premalignant hypertrophic gastropathy characterized by large rugal folds, foveolar hyperplasia with glandular atrophy, hypochlorhydria, and hypoalbuminemia. Patients with severe disease often exhibit refractory nausea and vomiting and require gastrectomy. Evidence from both mice and human beings suggests a critical role for epidermal growth factor receptor (EGFR) signaling in the pathogenesis of this disease. We previously reported significant clinical and biochemical improvement of a single patient treated for 1 month with Erbitux, a monoclonal antibody that blocks ligand binding to EGFR. METHODS/RESULTS: We describe 2 patients who were given longer-term treatment with Erbitux as an alternative to gastrectomy. The first patient presented with nausea, hypoalbuminemia, and peripheral edema that required total parenteral nutrition (TPN) and infusions of albumin. On institution of Erbitux, there was rapid improvement in nausea and vomiting and stabilization of serum albumin with discontinuation of TPN and albumin infusions. Serum albumin remained stable during a 1-year course of Erbitux without supplemental protein. Application before and after Erbitux of the radiopaque dye ruthenium red to biopsies of the gastric oxyntic gland mucosa demonstrated prompt and persistent closure of tight junctions by electron microscopy. The second patient presented with chronic gastric bleeding that required bimonthly blood transfusions. During a 4-month course of Erbitux, his hematocrit stabilized, and transfusion requirements were eliminated. CONCLUSIONS: The present report demonstrates the efficacy of prolonged Erbitux therapy in patients with different presentations of severe Ménétrier's disease and also provides insight into the pathophysiology of the protein-losing gastropathy.

Antibodies, Monoclonal↗