Expression Of p-EGFR Research Articles

Abstract NTOC-115: ASSOCIATION BETWEEN NUCLEAR SYK PHOSPHORYLATION AND EGFR/ERBB2 PATHWAY AND POOR SURVIVAL IN HIGH GRADE SEROUS OVARIAN CANCER

Abstract BACKGROUND: Spleen tyrosine kinase (SYK) has emerged as a cancer associated kinase of which aberrant activity contributes to tumor development and drug resistance in several types of human cancer including ovarian carcinoma. To better understand the mechanism how the SYK pathway participates in tumor progression, we studied the biochemistry and transcriptional regulation of SYK in high-grade ovarian serous carcinoma. METHODS: Immunohistochemical analyses were performed for phosphorylated SYK (Y525/526) and total SYK levels in 42 primary serous ovarian carcinomas and 112 tumor ascites formalin-fixed paraffin embedded tissues. The relative expression of SYK long and short isoforms in peritoneal ascites and cell lines were examined by reverse transcriptase-PCR. Transcriptome analysis of gene regulated by SYK inhibition was carried out using Illumina Human HT-12 v4 arrays. Results from array was analyzed for upstream regulators. IHC data was correlated with pEGFR (Y1197) and pERBB2 (Y877) nuclear expression. Association with survival was determined by Kaplan-Meier analysis and the log-rank test. RESULTS: We observed that active phosphorylated SYK (p-SYK, Y525/526) is mainly localized in the nucleus and cytoskeleton in cell lines and tissues. Investigation of SYK mRNA spliced variants in ascites tumors showed predominant SYK long isoform expression, consistent with its nuclear localization. Survival analyses revealed that intense nuclear p-SYK correlated with poor overall survival in two ovarian carcinoma cohorts. Patients with relatively high p-SYK in the nucleus had approximately half the median survival compared to those with undetectable or low nuclear p-SYK. SYK inhibition affected changes in gene expression that are downstream of ERBB2 and EGFR signaling. Correlation analyses in tissues suggested a relationship between p-SYK and pERBB2 or pEGFR signaling. CONCLUSIONS: Our results suggest that activation of the SYK pathway leads to its phosphorylation and localization in cell nucleus. SYK activity plays a role in regulating EGFR and ERBB2 phosphorylation and activation. Thus, SYK may regulate tumor-promoting gene transcription through cross-talks with the EGFR/ERBB2 pathways. Citation Format: Yu, Yu; Ardighieri, Laura; Suryo Rahmanto, Yohan; Chen, Lin Y.; Tessarollo, Nayara G.; Mizushima, Taichi; Ayhan, Ayse; Davidson, Ben; Wang, Tian L., and Shih Ie M. ASSOCIATION BETWEEN NUCLEAR SYK PHOSPHORYLATION AND EGFR/ERBB2 PATHWAY AND POOR SURVIVAL IN HIGH GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-115.

Significance of Phosphorylated Epidermal Growth Factor Receptor and Its Signal Transducers in Human Soft Tissue Sarcoma.

Previous studies have shown that total epidermal growth factor receptor (EGFR) protein is highly expressed in soft tissue sarcoma (STS). We aimed to investigate the significance of phosphorylated-EGFR (pEGFR) and its activated-downstream signal transducers in STS tissue samples. A tissue microarray comprising 87 STS samples was assessed for total EGFR, pEGFR and its phosphorylated signal transducers and expression was correlated with clinicopathlogical parameters including patient outcome. Although the expression of total EGFR was significantly associated with adverse STS histologic grade (p = 0.004) and clinical stage (p = 0.012) similar to pEGFR, phosphorylated protein kinase B (pAkt) and phosphorylated extracellular signal regulated kinase (pERK), it is not a prognostic factor for survival. By contrast, the expression of pEGFR is an independent factor for cancer specific survival, while pERK is an independent prognostic factor for both overall and cancer specific survival in STS (p < 0.05, Cox proportional hazard model and log-rank test) in addition to the recognised factors of tumour grade and clinical stage. pERK and pEGFR are new independent prognostic factors for overall and/or cancer specific survival in STS. The expression of EGFR/pEGFR, and their associated downstream signal transducers, was associated with STS progression, suggesting that EGFR downstream signalling pathways may jointly support STS cell survival.

Design, synthesis and evaluation of azaacridine derivatives as dual-target EGFR and Src kinase inhibitors for antitumor treatment

Overexpression of EGFR is often associated with advanced stage disease and poor prognosis. In certain cancers, Src works synergistically with EGFR to promote proliferation, survival, invasion and metastasis. Development of dual-target drugs against EGFR and Src is of therapeutic advantage against these cancers. Based on molecular docking and our previous studies, we rationally designed a new series of azaacridine derivatives as potent EGFR and Src dual inhibitors. Most of the synthesized azaacridines displayed good antiproliferative activity against K562 and A549 cells. The representative compound 13b showed nM IC50 values against K562 and A549 cells, and inhibited EGFR at inhibition rate of 33.53% at 10 μM and Src at inhibition rate of 72.12% at 1 μM. Furthermore, compound 13b could inhibit the expression of EGFR, p-EGFR, Src and p-Src. Moreover, 13b efficiently inhibited the invasion of tumor cells and induced cancer cells apoptosis. Our study suggested that azaacridine scaffold can be developed as novel multi-target kinase inhibitors for cancer therapy.

UBE2J2 promotes hepatocellular carcinoma cell epithelial-mesenchymal transition and invasion in vitro.

Ubiquitin-conjugating enzyme E2 J2 (UBE2J2) is an ubiquitin proteasome component that responds to proteotoxic stress. We found that UBE2J2 was highly expressed in cellular protrusions of HCCLM3 metastatic hepatocellular carcinoma (HC) cells. Immunohistochemical analyses showed that UBE2J2 was expressed at higher levels in HC patient tissues than in corresponding non-tumor tissues. Because cellular protrusions are important for cell invasion, we hypothesized that UBE2J2 promotes HC cell invasion. We used chip-based surface plasmon resonance (SPR) to assess possible mechanisms of UBE2J2-regulated HCCLM3 cell invasion. We found that p-EGFR interacted with UBE2J2, and this finding was confirmed by co-immunoprecipitation analysis. UBE2J2 overexpression activated endothelial-mesenchymal transition in the non-invasive SMMC7721 HC cell line, and promoted invasion. UBE2J2 silencing reduced HCCLM3 cell invasion and endocytosis, and downregulated p-EGFR expression. p-EGFR inhibition by lapatinib reduced UBE2J2-promoted cell invasion, suggesting p-EGFR is important for UBE2J2-mediated HCCLM3 cell invasion. These findings demonstrate that endocytosis by HC cells is closely related to invasion, and may provide new anti-HC therapeutic targets. UBE2J2 may also be a novel biomarker for clinical HC diagnosis.

Casticin impairs cell migration and invasion of mouse melanoma B16F10 cells via PI3K/AKT and NF-κB signaling pathways.

Casticin, a polymethoxyflavone, is one of the major active components obtained from Fructus viticis, which have been shown to have anticancer activities including induce cell apoptosis in human cancer cells. The aim of this study was to investigate the molecular mechanisms by which casticin inhibits cell migration and invasion of mouse melanoma B16F10 cells. Cell viability was examined by MTT assay and the results indicated that casticin decreased the total percentages of viable cells in dose-dependent manners. Casticin affected cell migration and invasion in B16F10 cells were examined by wound healing mobility assay and Boyden chamber migration and invasion assay and results indicated that casticin inhibited cell migration and invasion in dose-dependent manners. Western blotting was used to examine the protein expression of B16F10 cells after exposed to casticin and the results showed that casticin decreased the expressions of MMP-9, MMP-2, MMP-1, FAK, 14-3-3, GRB2, Akt, NF-κB p65, SOS-1, p-EGFR, p-JNK 1/2, uPA, and Rho A in B16F10 cells. Furthermore, cDNA microarray assay was used to show that casticin affected associated gene expression of cell migration and invasion and the results indicated that casticin affected some of the gene expression such as increased SCN1B (cell adhesion molecule 1) and TIMP2 (TIMP metallopeptidase inhibitor 2) and decreased NDUFS4 (NADH dehydrogenase (ubiquinone) Fe-S protein4), VEGFA (vascular endothelial growth factor A), and DDIT3 (DNA-damage-inducible transcript 3) which associated cell migration and invasion in B16F10 cells. Based on those observations, we suggest that casticin could be used as a novel anticancer metastasis of melanoma cancer in the future.

A prospective, randomized study of low molecular weight as supplemental therapy in metastatic colorectal cancer patients treated with FOLFIRI regimen and target agents.

746 Background: Fucoidan is recently studied for its anti-cancer and anti-inflammatory effects. It also has been used as food supplements in Asia for many years. This study is the first prospective, randomized trial to compare the clinical outcomes and adverse events in metastatic colorectal cancer (mCRC) patients, who are treated with FOLFIRI regimen and target agents as the first line setting with or without low molecular weight Fucoidan (LMF). Additionally, the study also aimed to evaluate the inhibitory effect of LMF in EGFR-KRAS-BRAF-ERK signaling pathway. Methods: A total of 54 patients with mCRC were randomly divided into a study group (28 cases, 4g BID oral low molecular weight Fucoidan powder) and a control group (26 cases, 4g BID oral placebo powder). The supplements were given for 6 months during the therapeutic period. The primary endpoint was the disease-control rate (DCR), and the secondary endpoints were overall response rate (ORR), progression-free survival (PFS), overall survival (OS) and adverse effects between the two groups. MTT cell viability assay and western blotting analysis were used to evaluate synergic effect of LMF plus 5-FU or Cetuximab on Caco-2, SW620 and HCT-15. Results: 54 mCRC patients were enrolled, including 28 patients in the study group and 26 patients in the control group. The DCR was 92.8% vs. 69.2% in study and control group respectively ( P = 0.026) in median follow up time of 11.5 months. The PFS was 15.93 ± 1.20 vs. 10.80 ± 1.06 months ( P = 0.075), ORR was 60.7% vs. 46.2% ( P = 0.284) and OS was 18.04 ± 0.91 vs. 12.96 ± 0.83 months ( P= 0.092). There were no statistical differences in adverse effects between two groups. In vitrostudy, LMF plus 5-FU or Cetuximab showed synergic cytotoxic effect (maximal 24% increase) through the inhibition of the expression of p-EGFR, p-ERK and p-Akt. Conclusions: The preliminary results show that LMF as supplemental therapy in mCRC patients may improve DCR. In vitro, Fucoidan showed the synergic cytotoxic effect with 5-FU or Cetuximab. The possible mechanism may involve the inhibition effect of Fucoidan in phosphorylation of EGFR-KRAS-BRAF-ERK signaling pathway.

Cetuximab sensitivity of head and neck squamous cell carcinoma xenografts is associated with treatment-induced reduction in EGFR, pEGFR, and pSrc.

The aims of this study were to validate in vitro drug sensitivity testing of head and neck squamous cell carcinoma (HNSCC) cell lines in an in vivo xenograft model and to identify treatment-induced changes in the epidermal growth factor receptor (EGFR) signaling pathway that could be used as markers for cetuximab treatment response. The in vitro and in vivo cetuximab sensitivity of two HNSCC cell lines, UT-SCC-14 and UT-SCC-45, was assessed using a crystal violet assay and xenografts in nude mice, respectively. The expression of EGFR, phosphorylated EGFR (pEGFR), phosphorylated Src (pSrc), and Ki-67 was investigated by immunohistochemistry. To verify these results, the in vitro expression of EGFR and pEGFR was analyzed with ELISA in a panel of 10 HNSCC cell lines. A close correlation was found between in vitro and in vivo cetuximab sensitivity data in the two investigated HNSCC cell lines. In treatment sensitive UT-SCC-14 xenografts, there was a decrease in EGFR, pEGFR, and pSrc upon cetuximab treatment. Interestingly, in insensitive UT-SCC-45 xenografts, an increased expression of these three proteins was found. The change in EGFR and pEGFR expression in vivo was confirmed in cetuximab-sensitive and cetuximab-insensitive HNSCC cell lines using ELISA. High sensitivity to cetuximab was strongly associated with a treatment-induced reduction in pEGFR both in vivo and in vitro in a panel of HNSCC cell lines, suggesting that EGFR and pEGFR dynamics could be used as a predictive biomarker for cetuximab treatment response.

Absence of miR-146a in Podocytes Increases Risk of Diabetic Glomerulopathy via Up-regulation of ErbB4 and Notch-1

Podocyte injury is an early event in diabetic kidney disease and is a hallmark of glomerulopathy. MicroRNA-146a (miR-146a) is highly expressed in many cell types under homeostatic conditions, and plays an important anti-inflammatory role in myeloid cells. However, its role in podocytes is unclear. Here, we show that miR-146a expression levels decrease in the glomeruli of patients with type 2 diabetes (T2D), which correlates with increased albuminuria and glomerular damage. miR-146a levels are also significantly reduced in the glomeruli of albuminuric BTBR ob/ob mice, indicating its significant role in maintaining podocyte health. miR-146a-deficient mice (miR-146a-/-) showed accelerated development of glomerulopathy and albuminuria upon streptozotocin (STZ)-induced hyperglycemia. The miR-146a targets, Notch-1 and ErbB4, were also significantly up-regulated in the glomeruli of diabetic patients and mice, suggesting induction of the downstream TGFβ signaling. Treatment with a pan-ErbB kinase inhibitor erlotinib with nanomolar activity against ErbB4 significantly suppressed diabetic glomerular injury and albuminuria in both WT and miR-146a-/- animals. Treatment of podocytes in vitro with TGF-β1 resulted in increased expression of Notch-1, ErbB4, pErbB4, and pEGFR, the heterodimerization partner of ErbB4, suggesting increased ErbB4/EGFR signaling. TGF-β1 also increased levels of inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and MCP-1 induced protein-1 (MCPIP1), a suppressor of miR-146a, suggesting an autocrine loop. Inhibition of ErbB4/EGFR with erlotinib co-treatment of podocytes suppressed this signaling. Our findings suggest a novel role for miR-146a in protecting against diabetic glomerulopathy and podocyte injury. They also point to ErbB4/EGFR as a novel, druggable target for therapeutic intervention, especially because several pan-ErbB inhibitors are clinically available.

Efficacy of afatinib, an irreversible ErbB family blocker, in the treatment of intracerebral metastases of non-small cell lung cancer in mice.

Few effective therapeutic options are currently available for the treatment of non-small cell lung cancer (NSCLC) with brain metastases (BM). Recent evidence shows that NSCLC patients with BMs respond well to afatinib, but little is known about the underlying mechanisms. In this study, we evaluated the efficacy of afatinib in treatment of BMs in mice and investigated whether afatinib could actively penetrate the brain-blood barrier and bind to its target. NSCLC BM model was established in nude mice by intracerebral injection of PC-9.luc cells. The tumors were measured weekly using in vivo quantitative bioluminescence. The mice are administrated afatinib (15, 30 mg·kg-1·d-1, ig) for 14 d. The antitumor efficacy of afatinib was determined by tumor growth inhibition (TGI), which was calculated as [1-(change of tumor volume in treatment group/control group)×100]. Pharmacokinetic characteristics were measure in mice receiving a single dose of afatinib (30 mg/kg, ig). Pharmacodynamics of afatinib was also assessed by detecting the expression of pEGFR (Tyr1068) in brain tumor foci using immunohistochemistry. Administration of afatinib (15, 30 mg·kg-1·d-1) dose-dependently inhibited PC-9 tumor growth in the brain with a TGI of 90.2% and 105%, respectively, on d 14. After administration of afatinib (30 mg/kg), the plasma concentration of afatinib was 91.4±31.2 nmol/L at 0.5 h, reached a peak (417.1±119.9 nmol/L) at 1 h, and was still detected after 24 h. The cerebrospinal fluid (CSF) concentrations followed a similar pattern. The T1/2 values of afatinib in plasma and CSF were 5.0 and 3.7 h, respectively. The AUC(0-24 h) values for plasma and CSF were 2375.5 and 29.1 nmol/h, respectively. The plasma and CSF concentrations were correlated (r=0.844, P<0.01). Pharmacodynamics study showed that the expression levels of pEGFR were reduced by 90% 1 h after afatinib administration. The Emax was 86.5%, and the EC50 was 0.26 nmol/L. A positive correlation between CSF concentrations and pEGFR modulation was revealed. Afatinib penetrates the BBB in NSCLC BM mice and contributes to the brain tumor response. The CSF exposure level is correlated with the plasma level, which in turn is correlated with the modulation of pEGFR in the tumor tissues. The results support for the potential application of afatinib in NSCLC patients with BMs.

Effects and its mechanism of Nimotuzumab on radiosensitivity of esophageal carcinoma ECA-109 and TE-13 cell lines

Objective: To investigate the effects of nimotuzumab on radiosensitivity of ECA-109 and TE-13 esophageal carcinoma cell lines and explore its possible mechanism. Methods: The ECA-109 and TE-13 cells were divided into control group, irradiation group, medicine group, and combined group (irradiation + medicine). In the combined group, ECA-109 and TE-13 cells were treated with nimotuzumab for 24 h before irradiation, and the cells were collected 2 h after irradiation. The radiosensitizing effects of nimotuzumab on ECA-109 and TE-13 cells were evaluated by clone formation assay. Cell apoptosis was detected by flow cytometry. Western blotting was used to evaluate the expression of EGFR, p-EGFR, DNA-PKcs, p-DNA-PKcs and γH2AX. Results: The values of Dq (quasithreshold dose), D0(mean lethal dose)and SF2 (surviving fraction at 2 Gy) of ECA-109 and TE-13 cells in the combined group were significantly lower than those of the radiation group (for ECA-109 cells, 1.11 vs. 1.72, 1.40 vs. 2.14, 0.42 vs. 0.66, respectively; for TE-13 cells, 0.41 vs. 0.46, 0.43 vs. 0.65, 0.40 vs. 0.71, respectively (all P<0.05). The sensitivity enhancement ratio (SER) of ECA-109 and TE-13 cells were 1.35 and 1.43, respectively. Flow cytometry showed that the apoptosis rate of ECA-109 and TE-13 cells in the combined group were significantly higher than those of the radiation group [for ECA-109 cells, (41.31±1.52)% vs. (9.54±0.52)%; for TE-13 cells, (46.28±0.28)% vs. (11.32±0.31)%, both P<0.01]. Western blotting showed that the expression levels of EGFR and DNA-PKcs were not significantly different in all groups (all P>0.05). Compared with those of the control group, p-EGFR and p-DNA-PKcs of the radiation group were significantly higher in both cell lines (P<0.05), and the γH2AX levels in the radiation group and medicine group were significantly higher than that of the control group (P<0.05). Compared with those of the radiation group and medicine group, p-EGFR and p-DNA-PKcs protein expression in the combined group were decreased significantly (P<0.05), while γH2AX protein expression was significantly increased (P<0.05). Conclusions: Nimotuzumab can enhance the radiosensitivity of esophageal cancer ECA-109 and TE-13 cells. The potential mechanism may be related to the inhibition of EGFR phosphorylation and down-regulation of DNA damage repair proteins. The radiosensitizing effect of nimotuzumab is greater on poorly differentiated esophageal cancer cells.

Fucosyltransferase-4 and Oligosaccharide Lewis Y Antigen as potentially Correlative Biomarkers of Helicobacter pylori CagA Associated Gastric Cancer.

H. pylori cytotoxin associated antigen A (CagA) plays a significant role in the progression of gastric cancer but their effect on fucosylation to develop gastric cancer is unknown. Fucosyltransferase IV (FUT4) is the key enzyme for synthesis of LewisY (LeY) carried by glycoproteins and glycolipids on the cell membrane. Herein, we compare the expression of CagA, p-EGFR, FUT4 and LeY in gastritis (n=128, 176), gastric ulcer (n=174, 213), and gastric cancer (n=323, 261) tissue and serum samples, respectively by IHC and ELISA. Moreover, we investigated the potential correlation of CagA with FUT4 and LeY overexpression through EGFR activation. IHC and ELISA results showed higher positive cases of H. pylori CagA (83, 86 %), p-EGFR (81, 72 %), FUT4 (91, 97%) and LeY (93, 92 %) in gastric cancer, compared to gastritis and gastric ulcer, H. pylori CagA (58, 67 & 59, 73 %), p-EGFR (52, 63 & 35, 47 %), FUT4 (68, 78 & 67, 82 %) and LeY (62,76 & 65, 85 %), respectively. We found a significant high expression (H-Value) of CagA (1.79, 1.66), p-EGFR (1.53, 1.58), FUT4 (2.14, 1.66) and LeY (1.69, 1.61) in gastric cancer tissues and serum, respectively as compared to chronic gastritis and gastric ulcers, CagA (0.64,1.14), p-EGFR (0.856, 0.678), FUT4 (0.949,1.197) and LeY (0.68,1.008) (P<0.0001), respectively. Furthermore, H. pylori CagA showed significant correlation with p-EGFR (R-0.62, -0.74), FUT4 (R-0.81, -0.76) and LeY (R-0.82, -0.70) in gastric tissues and serum (P<0.0001). H. pylori CagA plays key role in the development of gastric cancer with overexpression of FUT4/LeY, serve as potentially correlative biomarkers of H. pylori CagA associated gastric cancer.

Gold nanoparticle–conjugated quercetin inhibits epithelial–mesenchymal transition, angiogenesis and invasiveness via EGFR/VEGFR‐2‐mediated pathway in breast cancer

Epidermal growth factor plays a critical role in breast malignancies by enhancing cell proliferation, invasion, angiogenesis and metastasis. Epithelial-mesenchymal transition (EMT) is a crucial process by which epithelial cells lose polarity and acquire migratory mesenchymal properties. Gold nanoparticles are an efficient drug delivery vehicle for carrying chemotherapeutic agents to target cancer cells and quercetin is an anti-oxidative flavonoid known with potent anti-malignant cell activity. Cell viability was assessed by MTT assay, and protein expression was examined by Western blotting and immunocytochemistry. Cell invasion was monitored using invasion chambers, and cell migration was analysed by scratch wound-healing assay. In vitro and ex vivo angiogenesis studies were performed by capillary-like tube formation assay and chick embryo angiogenesis assay (CEA). 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary carcinoma in Sprague-Dawley rats. We observed a significant reduction in protein expression of vimentin, N-cadherin, Snail, Slug, Twist, MMP-2, MMP-9, p-EGFR, VEGFR-2, p-PI3K, Akt and p-GSK3β, and enhanced E-cadherin protein expression in response to AuNPs-Qu-5 treatment. AuNPs-Qu-5 inhibited migration and invasion of MCF-7 and MDA-MB-231 cells compared to free quercetin. AuNPs-Qu-5-treated HUVECs had reduced cell viability and capillary-like tube formation. In vitro and in vivo angiogenesis assays showed that AuNPs-Qu-5 suppressed tube and new blood vessel formation. Treatment with AuNPs-Qu-5 impeded tumour growth in DMBA-induced mammary carcinoma in SD rats compared to treatment with free quercetin. Our results suggest that AuNPs-Qu-5 inhibited EMT, angiogenesis and metastasis of the breast cancer cells tested by targeting the EGFR/VEGFR-2 signalling pathway.

Phosphorylated Epidermal Growth Factor Receptor Expression Is Associated With Clinicopathologic Parameters and Patient Survival in Mobile Tongue Squamous Cell Carcinoma

Phosphorylated epidermal growth factor receptor (pEGFR) activates several signaling pathways, resulting in tumor-promoting cellular activities, and has been implicated in malignant transformation and disease progression. The present study evaluated the clinical significance of pEGFR protein expression in mobile tongue squamous cell carcinoma (SCC). The present cohort study included 48 patients with mobile tongue SCC. We evaluated whether pEGFR immunohistochemical protein expression is associated with clinical variables and patient outcome. Of the 48 patients included in the present cohort study, 25 were men and 23 were women. The median patient age was 60years (interquartile range 53 to 72). pEGFR protein expression was significantly increased in well-differentiated tumors compared with poorly differentiated tumors (P=.001). Elevated pEGFR protein expression was significantly more frequently observed in mobile tongue SCC cases with a well-defined tumor shape and an earlier disease stage (P= .010 and P=.019, respectively). Patients with mobile tongue SCC presenting with elevated pEGFR expression had longer overall and disease-free survival times compared with those with low pEGFR expression (P= .015 and P= .006, respectively; log-rank test). On multivariate analysis, pEGFR expression proved to be an independent prognostic factor of both overall and disease-free survival (P= .008 and P= .044, respectively; Cox regression analysis). The results of the present study support evidence that the pEGFR signaling pathway might be implicated in the malignant transformation of mobile tongue SCC. Additional studies are recommended to validate whether pEGFR could be used as a potential biomarker and therapeutic target in mobile tongue SCC.

PDIA6 regulation of ADAM17 shedding activity and EGFR-mediated migration and invasion of glioblastoma cells.

OBJECTIVE In patients with glioblastoma, local invasion of tumor cells causes recurrence and shortens survival. The goal of this study was to determine whether protein disulfide isomerase (PDI) A6 regulates migration and invasion of glioblastoma cells and the associated factors. METHODS U87MG cells were treated with either PDIA6 or ADAM17 small interfering RNA (siRNA) fragments or with both types of siRNA fragments, and expression was confirmed by reverse transcription-polymerase chain reaction and Western blot. Migration and invasion were assessed using a wound-healing assay, a Matrigel assay, and an organotypic culture system. After the U87MG cells were treated with siRNAs and epidermal growth factor receptor (EGFR) inhibitors, the expression of matrix metalloproteinase-2 (MMP-2), membrane Type 1-matrix metalloproteinase (MT1-MMP), integrin, phosphorylated focal adhesion kinase (pFAK), and phosphorylated EGFR (pEGFR) was detected by Western blotting and zymography. RESULTS U87MG cell migration and invasion increased significantly after inhibition of PDIA6. The MMP-2 activation ratio and ADAM17 activity (as a sheddase of the proligand) increased, and expression of pEGFR, pFAK, integrin α5β3, and MT1-MMP was induced, compared with control levels. Furthermore, heparin-binding epidermal growth factor (EGFR signaling ligand) was highly expressed in PDIA6-knockdown cells. After siPDIA6-transfected U87MG cells were treated with EGFR signaling inhibitors, expression of pFAK, MMP-2, and MT1-MMP decreased and invasion decreased significantly. Simultaneous double-knockdown of PDIA6 and ADAM17 reduced pEGFR and pFAK expression, compared with control levels. CONCLUSIONS The authors propose that inhibiting PDIA6 could transduce EGFR signaling by activating and inducing ADAM17 during migration and invasion of U87MG glioblastoma cells. The results of this study suggest that PDIA6 is an important component of EGFR-mediated migration and invasion of U87MG cells. This is the first report of the effects of PDIA6 on migration and invasion in glioblastoma.

Synergistic inhibitory effect of cetuximab and tectochrysin on human colon cancer cell growth via inhibition of EGFR signal.

The purpose of this study was to evaluate the enhancing potency of tectochrysin, a flavonoid isolated from Alpinia oxyphylla Miquel by combining cetuximab, an anti-EGFR monoclonal antibody, on human colon cancer cell growth through further inhibition of EGFR pathway. HCT116 and SW480 colon cancer cells were treated with cetuximab (30μg/mL, 1/10 of IC50), tectochrysin (5μg/mL, 1/3 of IC50), or the combination of both agents. The growth inhibitory effect was examined using the MTT assay while apoptotic cell death was performed using TUNEL staining assays. The DNA binding activity of NF-kappa B and AP-1 was investigated by electrophoretic mobility shift assay. Protein expression was determined by Western blot. Cell proliferation was significantly inhibited by the combination of cetuximab and tectochrysin than treatment with cetuximab or tectochrysin alone (combination index: 0.572 and 0.533, respectively). Combination treatment of cells with cetuximab and tectochrysin significantly reduced the expressions of p-EGFR and COX-2 in both cell lines. Combination treatment also significantly inhibited activities of NF-kB and AP-1 compared to the single agent treatment. Our results indicate that combined therapy with lower concentration of cetuximab and tectochrysin could significantly enhance the cancer cell growth inhibitory effect through the inhibition of EGFR signaling.

GW627368X inhibits proliferation and induces apoptosis in cervical cancer by interfering with EP4/EGFR interactive signaling.

PGE2, the major product of cyclooxygenases implicated in carcinogenesis, is significantly upregulated in cervical cancer. PGE2 via prostanoid receptor EP4 stimulates proliferation and motility while inhibiting apoptosis and immune surveillance. It promotes angiogenesis by stimulating the production of pro-angiogenic factors. The present study demonstrates GW627368X, a highly selective competitive EP4 antagonist, which hinders cervical cancer progression by inhibiting EP4/epithelial growth factor receptor (EGFR) interactive signaling. GW627368X reduced protein kinase A (PKA) phosphorylation which in turn leads to decreased cAMP response element-binding protein (CREB) activation. Decreased PKA phosphorylation also directly enhanced Bax activity and in part reduced glycogen synthase kinase 3 (GSK3)β phosphorylation. Owing to the interactive signaling between EP4 and EGFR, GW627368X lowered EGFR phosphorylation in turn reducing Akt, mitogen-activated protein kinase (MAPK) and GSK3β activity significantly. Sublethal dose of GW627368X was found to reduce the nuclear translocation of β-catenin in a time dependent manner along with time-dependent decrease in cytoplasmic as well as whole-cell β-catenin. Decreased CREB and β-catenin transcriptional activity restricts the aberrant transcription of key genes like EP4, cyclooxygenase (COX)-2, vascular endothelial growth factor and c-myc, which ultimately control cell survival, proliferation and angiogenesis. Reduced activity of EGFR resulted in enhanced expression of 15-hydroxyprostaglandin dehydrogenase increasing PGE2 degradation thereby blocking a positive feedback loop. In xenograft model, dose-dependent decrease in cancer proliferation was observed characterized by reduction in tumor mass and volume and a marked decrease in Ki67 expression. A diminished CD31 specific staining signified decreased tumor angiogenesis. Reduced expression of pAkt, pMAPK, pEGFR and COX-2 validated in vitro results. GW627368X therefore effectively inhibits tumor survival, motility, proliferation and angiogenesis by blocking EP4/EGFR interactive signaling. EP4 is a potent therapeutic target in cervical cancer and can be explored in combination with conventional therapies to attain superior outcomes and to overcome complications associated with organ toxicities, therapeutic resistance and disease relapse.

Protective Effect of Electro-Acupuncture on Neural Myelin Sheaths Mediated through Activation of Epidermal Growth Factor Receptor after Compressed Spinal Cord Injury

Electro acupuncture has been widely used to treat demyelinating diseases, such as multiple sclerosis (MS), acute haemorrhagic leukoencephalitis (AHLE), compressed spinal cord injury (CSCI). However, the protective effect of electro acupuncture (EA) on neural myelin sheaths remains controversy attributed to the dimness of its therapeutic mechanism. In this study, we tried to explore the protective mechanism of EA in a custom-designed model of CSCI. Zusanli (ST36) and Huantiao (GB30) acupoints were stimulated by EA. The motor functions were monitored by Basso, Beattie and Bresnahanloco motor rating scale. The pathological changes in axonal myelinated fibers were estimated by luxol fast blue (LFB) and transmission electron microscopy (TEM). Epidermal growth factor receptor (EGFR), oligodendrocyte transcription factor 2 (Olig2), caspase-3 and phosphorylated Akt1 (pAkt1) were detected by immunofluorescence and western blot assays. After 7-day treatment of EA, the expression of p-EGFR, pAkt1 and Olig2 was significantly up-regulated which was consistent with changes of locomotor skill and ultrastructure of myelin sheath. By contrast, the expression of active caspase-3 was obviously down-regulated. Our results indicated that the protective effect of EA on neural myelin sheaths might be mediated via activation of EGFR after CSCI.

Preclinical In Vitro, In Vivo, and Pharmacokinetic Evaluations of FLLL12 for the Prevention and Treatment of Head and Neck Cancers.

Despite its high promise for cancer prevention and therapy, the potential utility of curcumin in cancer is compromised by its low bioavailability and weak potency. The purpose of the current study was to assess the in vitro and in vivo efficacy and pharmacokinetic parameters of the potent curcumin analogue FLLL12 in SCCHN and identify the mechanisms of its antitumor effect. IC50 values against a panel of one premalignant and eight malignant head and neck cancer cell lines as well as apoptosis assay results suggested that FLLL12 is 10- to 24-fold more potent than natural curcumin depending on the cell line and induces mitochondria-mediated apoptosis. In vivo efficacy (xenograft) and pharmacokinetic studies also suggested that FLLL12 is significantly more potent and has more favorable pharmacokinetic properties than curcumin. FLLL12 strongly inhibited the expression of p-EGFR, EGFR, p-AKT, AKT, Bcl-2, and Bid and increased the expression of Bim. Overexpression of constitutively active AKT or Bcl-2 or ablation of Bim or Bid significantly inhibited FLLL12-induced apoptosis. Further mechanistic studies revealed that FLLL12 regulated EGFR and AKT at transcriptional levels, whereas Bcl-2 was regulated at the translational level. Finally, FLLL12 strongly inhibited the AKT downstream targets mTOR and FOXO1a and 3a. Taken together, our results strongly suggest that FLLL12 is a potent curcumin analogue with more favorable pharmacokinetic properties that induces apoptosis of head and neck cancer cell lines by inhibition of survival proteins including EGFR, AKT, and Bcl-2 and increasing of the proapoptotic protein Bim.

Predictive efficacy of (11)C-PD153035 PET imaging for EGFR-tyrosine kinase inhibitor sensitivity in non-small cell lung cancer patients.

To determine the correlation of (11)C-PD153035 uptake with epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) sensitivity and phosphorylated EGFR (pEGFR) expression in non-small cell lung cancer (NSCLC) cell lines with different EGFR-TKI sensitivities and in their corresponding xenografts. Four human NSCLC cell lines (HCC827, PC9, A549, and H1975) in the logarithmic phase were co-incubated with (11)C-PD153035 to analyze the correlation of (11)C-PD153035 uptake with EGFR-TKI sensitivity, and EGFR/pEGFR expression. Nude mice xenograft models bearing the four NSCLCs were prepared. (11)C-PD153035 positron-emission tomography (PET)-computed tomography (CT) was used to image the xenografts and observe radioactive uptakes. Correlation of the in vivo uptakes with EGFR-TKI sensitivity, and EGFR/pEGFR expression was analyzed. HCC827 and PC9 cells, which were highly sensitive to EGFR-TKIs, exhibited higher (11)C-PD153035 uptakes than the other cells. A549 cells, which were moderately sensitive to EGFR-TKIs, showed higher uptake than the EGFR-TKI-resistant H1975 cells, which showed little or no uptake. Radioactive uptakes were positively correlated with pEGFR expression in all cells. PET-CT showed that radioactivity was highest in HCC827 xenografts. The radioactivity in PC9 xenografts was higher than that in A549 and H1975 xenografts. Tumor vs. non-tumor tissue ratio values were positively correlated with pEGFR expression in HCC827 and PC9 xenografts, but not in A549 and H1975 xenografts. In conclusion, (11)C-PD153035 can serve as an EGFR imaging agent in vitro and in vivo, and predicts sensitivity to EGFR-TKIs. This will provide an experimental basis for clinical applications of (11)C-PD153035 and individualized NSCLC therapy.

Coexpression of receptor tyrosine kinase AXL and EGFR in human primary lung adenocarcinomas

AXL has been identified as a tyrosine kinase switch that causes resistance to inhibitors targeting epidermal growth factor receptor (EGFR) signaling in non-small cell lung cancer (NSCLC). However, the relationship between 2 receptor tyrosine kinases, AXL and EGFR, and the relevance of AXL expression with EGFR mutation status in treatment-naive human NSCLCs remain uncertain. In this study, we evaluated the coexpression pattern of AXL, EGFR, and pEGFR(1068) in 109 lung adenocarcinoma patients with or without an EGFR mutation. There were 68 (62.4%) patients with tumors harboring EGFR mutations such as 19 del and/or L858R; 2 patients were T790M positive. The expression of AXL, EGFR, and pEGFR(1068) was detected in 60 (55%), 68 (62.4%), and 57 (52.3%) of 109 patients, respectively. The positive rates of EGFR and pEGFR(1068) were associated with the L858R mutation alone or with the 19 del and L858R mutation status. Further analysis indicated that the percentage of AXL(+)/EGFR(+)/pEGFR(1068) coexpression in 68 EGFR-activating mutations patients was significantly higher than that in 39 EGFR wild-type patients (30.9% versus 10.3%, P=.015). Furthermore, in the subgroup of AXL(+) patients (35 mutation(+) and 23 wild-type patients), the coexpression rates of AXL(+)/pEGFR(1068+) and AXL(+)/EGFR(+)/pEGFR(1068+) in patients with EGFR mutations were significantly higher compared with those in wild-type patients (both P<.05). Our study emphasized that the AXL and EGFR receptor tyrosine kinases were coexpressed in a subgroup of treatment-naive lung adenocarcinomas with or without EGFR mutations. Anti-AXL therapeutics delivered up front in combination with an EGFR inhibitor might prevent or delay resistance in patients with AXL-positive, EGFR-mutant, or wild-type NSCLC.