Grb2-assosiated binder (Gab) family proteins are docking molecules that can interact with receptor tyrosine kinases (RTKs) and cytokine receptors and bind several downstream signalling proteins. ...Studies in several cell types have shown that Gab1 may have a role in signalling mediated by the two RTKs epidermal growth factor (EGF) receptor (EGFR) and Met, the receptor for hepatocyte growth factor (HGF), but the involvement of Gab1 in EGFR and Met signalling has not been directly compared in the same cell. We have studied mechanisms of activation and role in mitogenic signalling of Gab1 in response to EGF and HGF in cultured rat hepatocytes. Gab1, but not Gab2, was expressed in the hepatocytes and was phosphorylated upon stimulation with EGF or HGF. Depletion of Gab1, using siRNA, decreased the ERK and Akt activation, cyclin D1 expression, and DNA synthesis in response to both EGF and HGF. Studies of mechanisms of recruitment to the receptors showed that HGF induced co-precipitation of Gab1 and Met while EGF induced binding of Gab1 to Grb2 but not to EGFR. Gab1 activation in response to both EGF and HGF was dependent on PI3K. While EGF activated Gab1 and Shc equally, within the same concentration range, HGF very potently and almost exclusively activated Gab1, having only a minimal effect on Shc. Collectively, our results strongly suggest that although Gab1 interacts differently with EGFR and Met, it is involved in mitogenic signalling mediated by both these growth factor receptors in hepatocytes.
•Gab1 is involved in EGF- and HGF-induced proliferation in rat hepatocytes.•Gab1 is recruited directly to Met by HGF but indirectly to EGFR by EGF.•Activation of Gab1 in response to both EGF and HGF is dependent on PI3K activity.•HGF/Met preferentially uses Gab1, rather than Shc, in downstream signalling.
Aims: Early diagnosis and treatment of chronic kidney disease (CKD) may delay disease progression since 45% of patients are not diagnosed until stage 3. We examine the Kidney Disease Improving Global ...Outcomes (KDIGO) clinical guidelines that define rules based on the longitudinal progression of urine albumin creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) for flagging CKD in people with type 2 diabetes (T2-PwD).
Methods: EHR data from over 400,000 T2-PwDs in the Indiana Network for Patient Care (INPC) database were used for this analysis. The KDIGO clinical practice guidelines defines CKD as persistently elevated urine albumin excretion (>=30 mg/g), or persistently reduced eGFR (< 60 ml/min per 1.73m2), or both, for more than 3 months. Our automated system scanned each patient’s longitudinal record and identified those patients with abnormal UACR and eGFR values in the cases where sufficient values were available. A flag associated with either UACR or eGFR or both and the date of flagging is indicated by the system if abnormal marker values persist for more than 3 months. CKD is indicated if one or both flags are triggered.
Results: Out of 422,089 patients 35,573 (8%) were identified as diagnosed with CKD. Only 3% of these patients had at least 2 UACR readings and, therefore, the automated system considered primarily eGFR as the marker for flagging CKD. Out of 35,573 patients with CKD diagnosis, 16,342 (46%) had an eGFR warning flag that predated the CKD diagnosis date. The system flagged a potential identification as much as 599 days (median value) in advance of the diagnosis of CKD in the dataset.
Conclusion: Our results show that earlier identification of CKD onset can be automated to flag patients wherein a positive diagnosis of CKD is likely. This flag can then prompt a confirmatory diagnosis of CKD, allowing better treatment interventions and management of CKD, reducing the burden of disease, and potentially decreasing costs associated with treatments in later CKD stages.
Disclosure
D. Malagarriga: None. S. Chittajallu: Employee; Self; Roche Diabetes Care. P. J. Galley: Employee; Self; Roche Diabetes Care. I. Singh: Employee; Self; Roche Diabetes Care. J. T. Odegard: Employee; Self; Roche Diabetes Care.
Objectives: While a large proportion of chronic kidney disease (CKD) cost in the U.S. is borne by Medicare, most CKD patients have commercial coverage, either as primary insurance or as a Medicare ...supplement. This study examines the economic impact of CKD on commercial payers in the U.S.
Methods: A retrospective cohort study using IBM MarketScan® data from January 2010 - December 2018 was conducted. Patients with ≥2 outpatient (≥30 days apart) and/or ≥1 inpatient claims containing a diagnosis code for CKD were eligible for analysis. The earliest CKD diagnosis date served as the index date. Included patients were continuously enrolled for ≥6 months pre- and post-index. Annualized progression rates and direct medical costs per patient per year (PPPY) by CKD stage were analyzed for the overall CKD population and patients with pre-existing type 1 or type 2 diabetes.
Results: Of 753,097 CKD patients identified, 310,837 (41.3%) had diabetes. More CKD patients with pre-existing diabetes had hypertension (81.7% vs. 75.7% for overall CKD). Approximately 45% of patients in both cohorts were diagnosed at Stage 3. PPPY costs for Stages 1-5 were $19,970, $26,248, $32,687, $46,091, and $61,492, respectively. For CKD patients with pre-existing diabetes, PPPY costs were $21,028, $29,941, $37,835, $50,614, and $67,160 for Stages 1-5. The increase in Stage 5 cost over Stage 1 cost was 208% and 219% for CKD overall and CKD with pre-existing diabetes, respecitvely. Annualized CKD progression was higher for CKD patients with pre-existing diabetes compared to CKD patients overall (162 vs. 130 progressions per 1000 patients in a given year).
Conclusions: Annualized rates of CKD progression are high, with the burden of CKD increasing more than 2-fold from Stage 1 to Stage 5. Earlier identification and treatment management of CKD are needed to reduce the burden of disease.
Disclosure
T. Kauf: Consultant; Self; Roche Diabetes Care. W. Wang: Consultant; Self; Roche Diabetes Care. A. Dillon: Consultant; Self; Roche Diabetes Care. I. Singh: Employee; Self; Roche Diabetes Care. J.T. Odegard: None. C. Ringemann: Employee; Self; Roche Diabetes Care. S. Haldrup: Employee; Self; F. Hoffmann-La Roche Ltd.
Background: People with diabetes in general have a higher risk of developing Chronic Kidney Disease (CKD). The early detection of CKD in people with diabetes helps their health care providers to ...adjust their therapy to slow down the disease progression to higher stages, prevent complications, and reduce cardiovascular-related conditions. It also takes a great financial burden off the insurance companies in the long run by way of future treatment costs. We have developed a model that uses the historical data from the past 2 years of people with diabetes (PwD) and predicts the chance of them having CKD in the next 3 years.
Methods: Our model employed the XGBoost (eXtreme Gradient Boosting) algorithm. It used more than 860,000 PwD data from the IBM EHR (Electronic Health Records) database for training and optimizing the model parameters. It was then independently verified using more than 500,000 PwD data from the CPRD (Clinical Praxis Research Datalink) database from the UK, and close to 140,000 PwD data from the INPC (Indiana Network for Patient Care) database from the US. We considered an incident of CKD based on the appearance of a relevant ICD code. The major benefit of the XGBoost algorithm in the context of risk prediction based on EHR data is that there is no need for imputation of the missing values.
Results: We evaluated the performance of the Roche XGBoost model for CKD in terms of the area under the receiver operating characteristic curve (AUC). While we obtain comparable results for the US-based data sets (prediction model: AUCExplorys=0.83, AUCINPC=0.84), we observe a performance decrease for the validation on CPRD data (AUCCPRD=0.74). However, on all data sources our XGBoost model shows a superior performance when compared to appropriate benchmark algorithms from literature.
Conclusions: The XGBoost model for CKD disease prediction has a superior performance over benchmark models from literature.
Disclosure
N. Afshar: Employee; Self; Roche Diabetes Care. I. Singh: Employee; Self; Roche Diabetes Care. T. Huschto: Employee; Self; Roche Diabetes Care. M. Andorrà: Advisory Panel; Self; Attune Neurosciences Inc., Employee; Self; Roche Diabetes Care, Stock/Shareholder; Self; Bionure, S. L., Goodgut, S. L. S. Chittajallu: Employee; Self; Roche Diabetes Care. C. Ringemann: Employee; Self; Roche Diabetes Care. J. T. Odegard: Employee; Self; Roche Diabetes Care. H. Mikulski: None. V. N. Babinsky: Employee; Self; Roche Diabetes Care. H. König: Employee; Self; Roche Diabetes Care.
Lysophosphatidic acid (LPA) is a small glycerophospholipid ubiquitously present in tissues and plasma. It acts through receptors belonging to the G-protein-coupled receptor (GPCR) family, is involved ...in several biological processes, and is strongly implicated in different cancers. In this paper, we have investigated the effects of LPA on DNA synthesis and migration in a panel of pancreatic and colon cancer cells, with particular focus on the involvement of the epidermal growth factor (EGF) receptor (EGFR) in LPA-induced signaling. LPA stimulated DNA synthesis and/or migration in all the cell lines included in this study. In five of the six cell lines, LPA induced phosphorylation of the EGFR, and the effects on EGFR and Akt, and in some of the cells also ERK, were sensitive to the EGFR tyrosine kinase inhibitor gefitinib, strongly suggesting LPA-induced EGFR transactivation in these cells. In contrast, in one of the pancreatic carcinoma cell lines (Panc-1), we found no evidence of transactivation of the EGFR. In the pancreatic carcinoma cell lines where transactivation took place (BxPC3, AsPC1, HPAFII), gefitinib reduced LPA-induced DNA synthesis and/or migration. However, we also found evidence of transactivation in the two colon carcinoma cell lines (HT29, HCT116) although gefitinib did not inhibit LPA-induced DNA synthesis or migration in these cells. Taken together, the data indicate that in many gastrointestinal carcinoma cells, LPA uses EGFR transactivation as a mechanism when exerting such effects as stimulation of cell proliferation and migration, but EGFR-independent pathways may be involved instead of, or in concerted action with, the EGFR transactivation.
Neurotensin has been found to promote colon carcinogenesis in rats and mice, and proliferation of human colon carcinoma cell lines, but the mechanisms involved are not clear. We have examined ...signalling pathways activated by neurotensin in colorectal and pancreatic carcinoma cells.
Colon carcinoma cell lines HCT116 and HT29 and pancreatic adenocarcinoma cell line Panc-1 were cultured and stimulated with neurotensin or epidermal growth factor (EGF). DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA. Levels and phosphorylation of proteins in signalling pathways were assessed by Western blotting.
Neurotensin stimulated the phosphorylation of both extracellular signal-regulated kinase (ERK) and Akt in all three cell lines, but apparently did so through different pathways. In Panc-1 cells, neurotensin-induced phosphorylation of ERK, but not Akt, was dependent on protein kinase C (PKC), whereas an inhibitor of the β-isoform of phosphoinositide 3-kinase (PI3K), TGX221, abolished neurotensin-induced Akt phosphorylation in these cells, and there was no evidence of EGF receptor (EGFR) transactivation. In HT29 cells, in contrast, the EGFR tyrosine kinase inhibitor gefitinib blocked neurotensin-stimulated phosphorylation of both ERK and Akt, indicating transactivation of EGFR, independently of PKC. In HCT116 cells, neurotensin induced both a PKC-dependent phosphorylation of ERK and a metalloproteinase-mediated transactivation of EGFR that was associated with a gefitinib-sensitive phosphorylation of the downstream adaptor protein Shc. The activation of Akt was also inhibited by gefitinib, but only partly, suggesting a mechanism in addition to EGFR transactivation. Inhibition of PKC blocked neurotensin-induced DNA synthesis in HCT116 cells.
While acting predominantly through PKC in Panc-1 cells and via EGFR transactivation in HT29 cells, neurotensin used both these pathways in HCT116 cells. In these cells, neurotensin-induced activation of ERK and stimulation of DNA synthesis was PKC-dependent, whereas activation of the PI3K/Akt pathway was mediated by stimulation of metalloproteinases and subsequent transactivation of the EGFR. Thus, the data show that the signalling mechanisms mediating the effects of neurotensin involve multiple pathways and are cell-dependent.
Oral squamous cell carcinoma is an aggressive neoplasm with serious morbidity and mortality, which typically spreads through local invasive growth. Lysophosphatidic acid (LPA) is involved in a number ...of biological processes, and may have a role in cancer cell migration and invasiveness. LPA is present in most tissues and can activate cells through six different LPA receptors (LPAR1-6). Although LPA is predominantly promigratory, some of the receptors may have antimigratory effects in certain cells. The signalling mechanisms of LPA are not fully understood, and in oral carcinoma cells the specific receptors and pathways involved in LPA-stimulated migration are unknown.
The oral carcinoma cell lines E10, SCC-9, and D2 were investigated. Cell migration was studied in a scratch wound assay, and invasion was demonstrated in organotypic three dimensional co-cultures. Protein and mRNA expression of LPA receptors was studied with Western blotting and qRT-PCR. Activation of signalling proteins was examined with Western blotting and isoelectric focusing, and signalling mechanisms were further explored using pharmacological agents and siRNA directed at specific receptors and pathways.
LPA stimulated cell migration in the two oral carcinoma cell lines E10 and SCC-9, but was slightly inhibitory in D2. The receptor expression profile and the effects of specific pharmacological antagonist and agonists indicated that LPA-stimulated cell migration was mediated through LPAR3 in E10 and SCC-9. Furthermore, in both these cell lines, the stimulation by LPA was dependent on PKC activity. However, while LPA induced transactivation of EGFR and the stimulated migration was blocked by EGFR inhibitors in E10 cells, LPA did not induce EGFR transactivation in SCC-9 cells. In D2 cells, LPA induced EGFR transactivation, but this was associated with slowing of a very high inherent migration rate in these cells.
The results demonstrate LPA-stimulated migration in oral carcinoma cells through LPAR3, mediated further by PKC, which acts either in concert with or independently of EGFR transactivation.
It is important to understand the mechanisms by which the cells integrate signals from different receptors. Several lines of evidence implicate epidermal growth factor (EGF) receptor (EGFR) in the ...pathophysiology of hepatocarcinomas. Data also suggest a role of prostaglandins in some of these tumours, through their receptors of the G protein-coupled receptor (GPCR) family. In this study we have investigated mechanisms of interaction between signalling from prostaglandin receptors and EGFR in hepatocarcinoma cells.
The rat hepatocarcinoma cell line MH1C1 and normal rat hepatocytes in primary culture were stimulated with EGF or prostaglandin E2 (PGE2) and in some experiments also PGF2α. DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA, phosphorylation of proteins in signalling pathways was assessed by Western blotting, mRNA expression of prostaglandin receptors was determined using qRT-PCR, accumulation of inositol phosphates was measured by incorporation of radiolabelled inositol, and cAMP was determined by radioimmunoassay.
In the MH1C1 hepatocarcinoma cells, stimulation with PGE2 or PGF2α caused phosphorylation of the EGFR, Akt, and ERK, which could be blocked by the EGFR tyrosine kinase inhibitor gefitinib. This did not occur in primary hepatocytes. qRT-PCR revealed expression of EP1, EP4, and FP receptor mRNA in MH1C1 cells. PGE2 stimulated accumulation of inositol phosphates but not cAMP in these cells, suggesting signalling via PLCβ. While pretreatment with EP1 and EP4 receptor antagonists did not inhibit the effect of PGE2, pretreatment with an FP receptor antagonist blocked the phosphorylation of EGFR, Akt and ERK. Further studies suggested that the PGE2-induced signal was mediated via Ca2+ release and not PKC activation, and that it proceeded through Src and shedding of membrane-bound EGFR ligand precursors by proteinases of the ADAM family.
The results indicate that in MH1C1 cells, unlike normal hepatocytes, PGE2 activates the MEK/ERK and PI3K/Akt pathways by transactivation of the EGFR, thus diversifying the GPCR-mediated signal. The data also suggest that the underlying mechanisms in these cells involve FP receptors, PLCβ, Ca2+, Src, and proteinase-mediated release of membrane-associated EGFR ligand(s).
The receptor tyrosine kinases EGFR and Met induce phosphorylation of the docking protein Gab1, and there is evidence that Gab1 may have a role in the signaling from these receptors. Studying ...hepatocytes, we previously found that although Gab1 mechanistically interacted in different ways with EGFR and Met, it was involved in mitogenic signaling induced by both EGF and HGF. It has been reported that in EGFR, Gab1 is required particularly at a low dose of EGF. Whether this also applies to HGF/Met signaling has not been investigated. We have studied the role of Gab1 in activation of the Akt and ERK pathways at low‐ and high‐intensity stimulation with EGF and HGF in cultured hepatocytes. In cells where Gab1 was depleted by a specific Gab1‐directed siRNA, the EGF‐induced phosphorylation of ERK was lowered and HGF‐induced phosphorylation of both ERK and Akt was substantially reduced. These effects were more marked at low‐dose HGF stimulation. The inhibitory consequence of Gab1 depletion was particularly pronounced for HGF‐induced Akt phosphorylation. The results suggest that Gab1 is an important signal amplifier for low‐intensity stimulation by HGF.
PDF
