Suppression Of Epidermal Growth Factor Receptor Expression Research Articles

Human Dermcidin Protects Mice Against Hepatic Ischemia-Reperfusion-Induced Local and Remote Inflammatory Injury.

BackgroundHepatic ischemia and reperfusion (I/R) injury is commonly associated with surgical liver resection or transplantation, and represents a major cause of liver damage and graft failure. Currently, there are no effective therapies to prevent hepatic I/R injury other than ischemic preconditioning and some preventative strategies. Previously, we have revealed the anti-inflammatory activity of a sweat gland-derived peptide, dermcidin (DCD), in macrophage/monocyte cultures. Here, we sought to explore its therapeutic potential and protective mechanisms in a murine model of hepatic I/R.MethodsMale C57BL/6 mice were subjected to hepatic ischemia by clamping the hepatic artery and portal vein for 60 min, which was then removed to initiate reperfusion. At the beginning of reperfusion, 0.2 ml saline control or solution of DCD (0.5 mg/kg BW) or DCD-C34S analog (0.25 or 0.5 mg/kg BW) containing a Cys (C)→Ser (S) substitution at residue 34 was injected via the internal jugular vein. For survival experiments, mice were subjected to additional resection to remove non-ischemic liver lobes, and animal survival was monitored for 10 days. For mechanistic studies, blood and tissue samples were collected at 24 h after the onset of reperfusion, and subjected to measurements of various markers of inflammation and tissue injury by real-time RT-PCR, immunoassays, and histological analysis.ResultsRecombinant DCD or DCD-C34S analog conferred a significant protection against lethal hepatic I/R when given intravenously at the beginning of reperfusion. This protection was associated with a significant reduction in hepatic injury, neutrophilic CXC chemokine (Mip-2) expression, neutrophil infiltration, and associated inflammation. Furthermore, the administration of DCD also resulted in a significant attenuation of remote lung inflammatory injury. Mechanistically, DCD interacted with epidermal growth factor receptor (EGFR), a key regulator of liver inflammation, and significantly inhibited hepatic I/R-induced phosphorylation of EGFR as well as a downstream signaling molecule, protein kinase B (AKT). The suppression of EGFR expression by transducing Egfr-specific shRNA plasmid into macrophages abrogated the DCD-mediated inhibition of nitric oxide (NO) production induced by a damage-associated molecular pattern (DAMP), cold-inducible RNA-binding protein, CIRP.ConclusionsThe present study suggests that human DCD and its analog may be developed as novel therapeutics to attenuate hepatic I/R-induced inflammatory injury possibly by impairing EGFR signaling.

Heterogeneous delivery across the blood-brain barrier limits the efficacy of an EGFR-targeting antibody drug conjugate in glioblastoma.

Antibody drug conjugates (ADCs) targeting the epidermal growth factor receptor (EGFR), such as depatuxizumab mafodotin (Depatux-M), is a promising therapeutic strategy for glioblastoma (GBM) but recent clinical trials did not demonstrate a survival benefit. Understanding the mechanisms of failure for this promising strategy is critically important. PDX models were employed to study efficacy of systemic vs intracranial delivery of Depatux-M. Immunofluorescence and MALDI-MSI were performed to detect drug levels in the brain. EGFR levels and compensatory pathways were studied using quantitative flow cytometry, Western blots, RNAseq, FISH, and phosphoproteomics. Systemic delivery of Depatux-M was highly effective in nine of 10 EGFR-amplified heterotopic PDXs with survival extending beyond one year in eight PDXs. Acquired resistance in two PDXs (GBM12 and GBM46) was driven by suppression of EGFR expression or emergence of a novel short-variant of EGFR lacking the epitope for the Depatux-M antibody. In contrast to the profound benefit observed in heterotopic tumors, only two of seven intrinsically sensitive PDXs were responsive to Depatux-M as intracranial tumors. Poor efficacy in orthotopic PDXs was associated with limited and heterogeneous distribution of Depatux-M into tumor tissues, and artificial disruption of the BBB or bypass of the BBB by direct intracranial injection of Depatux-M into orthotopic tumors markedly enhanced the efficacy of drug treatment. Despite profound intrinsic sensitivity to Depatux-M, limited drug delivery into brain tumor may have been a key contributor to lack of efficacy in recently failed clinical trials.

MicroR-545 mediates colorectal cancer cells proliferation through up-regulating epidermal growth factor receptor expression in HOTAIR long non-coding RNA dependent.

The functional impact of recently discovered miRNAs in human cancer remains to be clarified. One miRNA in colorectal cancer which has attracted attention is miR-545. In this study, we examined the function of miR-545 in proliferation of colorectal cancer cells. Expressions of HOTAIR, miRNA-545, and epidermal growth factor receptor (EGFR) mRNA were measured in 100 paired cancerous and non-cancerous tissues as well as in SW480 and LOVO colorectal cancer cell (CRC) lines by quantitative RT-PCR. The relative protein level of EGFR was measured using western blotting. Effects of miRNA-545 and HOTAIR on gastric cancer cells were studied by overexpression and RNA interference approaches. Insight of mechanism of promotion cancer by miR-545 was gained from luciferase reporter assay and gene expression analysis. CRC proliferation was evaluated using clone formation and MTT assay. Differential expressions of HOTAIR, miR-545, and EGFR were observed in cancerous tissues in comparison to non-cancerous tissues. By expressional management of miR-545, we observed that miR-545 negatively regulated cell proliferation. Also luciferase reporter assay revealed that miR-545 inhibited regulated EGFR expression by affecting its 3'-UTR activity. In addition, miR-545 expression was suppressed by HOTAIR overexpression whereas enhanced by HOTAIR silence. Suppression of EGFR expression by miR-545 mimic was abrogated by HOTAIR overexpression. Monitoring of tumor growth in mice showed that miR-545 overexpression suppressed LOVO tumor growth. Our data suggested that HOTAIR long non-coding RNA mediates microR-545 regulating colorectal cancer cells proliferation.

RNA interference for epidermal growth factor receptor enhances the radiosensitivity of esophageal squamous cell carcinoma cell line Eca109.

The present study investigated the effects of small interfering RNAs (siRNAs) specific to the epidermal growth factor receptor (EGFR) gene, on the radiosensitivity of esophageal squamous cell carcinoma cells. EGFR gene siRNAs (EGFR-siRNA) were introduced into esophageal cancer Eca109 cells using Lipofectamine® 2000. The EGFR messenger (m)RNA expression levels, EGFR protein expression and cell growth were assessed using reverse transcription-polymerase chain reaction analysis, western blot analysis and a Cell Counting Kit-8 (CCK-8), respectively. In addition, colony assays were used to determine the inhibitory effects of X-ray radiation on EGFR-silenced cells. EGFR mRNA and protein levels were reduced in the Eca109 cells transfected with EGFR-siRNA. The relative EGFR mRNA expression levels were reduced to 26.74, 9.52 and 4.61% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These mRNA levels were significantly reduced compared with the those of the control group (42.44%; P<0.0001). Transfection with siRNA3 resulted in the greatest reduction in EGFR mRNA expression, with an inhibition rate of 85%. The relative EGFR protein expression levels were reduced to 24.05, 34.91 and 34.14% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These protein levels were significantly reduced compared with those of the control group (78.57%; P<0.0001). Transfection with siRNA1 resulted in the greatest reduction in EGFR protein expression, with an inhibition rate of 72.84%. This reduction in EGFR expression inhibited the proliferation of Eca109 cells, which was identified using the CCK-8 assay. The proliferation inhibition ratio was 28.2%. The cells treated with irradiation in addition to EGFR-siRNA, demonstrated reduced radiobiological parameters (D0, Dq and SF2) compared with those of cells treated with irradiation only, with a sensitization enhancing ratio of 1.5. In conclusion, suppression of EGFR expression may enhance the radiosensitivity of esophageal cancer Eca109 cells and therefore may represent a promising approach for future clinical practice.

Oncogenic KRAS Impairs EGFR Antibodies' Efficiency by C/EBPβ-Dependent Suppression of EGFR Expression

Oncogenic KRAS mutations in colorectal cancer (CRC) are associated with lack of benefit from epidermal growth factor receptor (EGFR)-directed antibody (Ab) therapy. However, the mechanisms by which constitutively activated KRAS (KRAS(G12V)) impairs effector mechanisms of EGFR-Abs are incompletely understood. Here, we established isogenic cell line models to systematically investigate the impact of KRAS(G12V) on tumor growth in mouse A431 xenograft models as well as on various modes of action triggered by EGFR-Abs in vitro. KRAS(G12V) impaired EGFR-Ab-mediated growth inhibition by stimulating receptor-independent downstream signaling. KRAS(G12V) also rendered tumor cells less responsive to Fc-mediated effector mechanisms of EGFR-Abs-such as complement-dependent cytotoxicity (CDC) and Ab-dependent cell-mediated cytotoxicity (ADCC). Impaired CDC and ADCC activities could be linked to reduced EGFR expression in KRAS-mutated versus wild-type (wt) cells, which was restored by small interfering RNA (siRNA)-mediated knockdown of KRAS4b. Immunohistochemistry experiments also revealed lower EGFR expression in KRAS-mutated versus KRAS-wt harboring CRC samples. Analyses of potential mechanisms by which KRAS(G12V) downregulated EGFR expression demonstrated significantly decreased activity of six distinct transcription factors. Additional experiments suggested the CCAAT/enhancer-binding protein (C/EBP) family to be implicated in the regulation of EGFR promoter activity in KRAS-mutated tumor cells by suppressing EGFR transcription through up-regulation of the inhibitory family member C/EBPβ-LIP. Thus, siRNA-mediated knockdown of C/EBPβ led to enhanced EGFR expression and Ab-mediated cytotoxicity against KRAS-mutated cells. Together, these results demonstrate that KRAS(G12V) signaling induced C/EBPβ-dependent suppression of EGFR expression, thereby impairing Fc-mediated effector mechanisms of EGFR-Abs and rendering KRAS-mutated tumor cells less sensitive to these therapeutic agents.

Suppression of the Epidermal Growth Factor Receptor Inhibits Epithelial–Mesenchymal Transition in Human Pancreatic Cancer PANC-1 Cells

Aberrant expression of epidermal growth factor receptor (EGFR) has been detected in pancreatic cancer; however, the mechanisms of EGFR in inducing pancreatic cancer development have not been adequately elucidated. The objective of this study was to determine the role of EGFR in mediating epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. Pancreatic cancer cell line PANC-1 was transfected with small interfering RNA of EGFR by use of a lentiviral expression vector to establish an EGFR-knockdown cell line (si-PANC-1). PANC-1 cells transfected with lentiviral vector expressing negative control sequence were used as negative control (NC-PANC-1). Scratch assay and transwell study were used to analyze cell migration and invasion. Real-time PCR and Western blotting were used to detect the expression of EMT markers E-cadherin, N-cadherin, vimentin, and fibronectin and transcription factors snail, slug, twist1, and sip1 in PANC-1, NC-PANC-1, and si-PANC-1 cells. Immunofluorescent staining with these antibodies and confocal microscopy were used to observe their cellular location and morphologic changes. After RNA interference of EGFR, the migration and invasion ability of si-PANC-1 cells decreased significantly. The expression of epithelial phenotype marker E-cadherin increased and the expression of mesenchymal phenotype markers N-cadherin, vimentin, and fibronectin decreased, indicating reversion of EMT. We also observed intracellular translocation of E-cadherin. Expression of transcription factors snail and slug in si-PANC-1 cells decreased significantly. Suppression of EGFR expression can significantly inhibit EMT of pancreatic cancer PANC-1 cells. The mechanism may be related with the down-regulation of the expression of transcription factors snail and slug.

Ganoderma tsugae extract inhibits expression of epidermal growth factor receptor and angiogenesis in human epidermoid carcinoma cells: In vitro and in vivo

We examined the anti-angiogenic effects of Ganoderma tsugae methanol extract (GTME) on human epidermoid carcinoma A-431 cells. Our data indicate that GTME inhibits the expression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) in vitro and in vivo, and also inhibits the capillary tube formation of human umbilical vein endothelial cells (HUVECs). We also show that the suppression of VEGF expression by GTME can be restored by treatment with EGF. These results suggest that GTME inhibits VEGF expression via the suppression of EGFR expression, resulting in the downregulation of VEGF secretion from epidermoid carcinoma A-431 cells. These findings reveal a novel role for G. tsugae in inhibiting EGFR and VEGF expression, which are important for tumor angiogenesis and growth. Thus, GTME may provide a potential therapeutic approach for anti-tumor treatment.

NEAP causes down‐regulation of EGFR, subsequently induces the suppression of NGF‐induced differentiation in PC12 cells

Neuroendocrine-associated phosphatase (NEAP), an atypical dual specificity phosphatase is preferentially expressed in neuroendocrine cells. In this study we found that NEAP, but not NEAP-(C152S) mutant, evidently reduced epidermal growth factor (EGF) receptor (EGFR) downstream signaling, and impaired cell growth in response to EGF stimulation in PC12 cells. These phenomena were associated with NEAP-mediated down-regulation of EGFR mRNA and protein. NEAP had no significant effect on ErbB2/3 expression and phosphorylation levels in response to heregulin, indicating that the negative effect of NEAP on EGFR was selective. We showed that NEAP suppressed EGFR expression via decreasing the EGFR promoter activity and this was mediated through down-regulations of the Akt pathway and Wilms' tumor gene product (WT1). Consistent with these results, expression of WT1 reversed the suppressive effect of NEAP on EGFR promoter activity. Additionally, NEAP knockdown by RNA interference enhanced EGFR protein expression and nerve growth factor-induced differentiation, and an EGFR-specific inhibitor could reverse the later event. Taken together, our study indicated that NEAP modulates PC12 differentiation via suppression of EGFR expression and signaling.

Suppression of EGFR expression by antisense or small interference RNA inhibits U251 glioma cell growth in vitro and in vivo

Epidermal growth factor receptor (EGFR) had been reported as one of the major responsible genes for malignant progression and phenotype reversion of gliomas, and has been used as one of the most important therapeutic targets. In the present study, small interference RNA (siRNA) and antisense EGFR expression constructs, which target sequences of human EGFR catalytic domain (2400-2420) and the 3'-coding region, respectively, were used to examine the growth inhibition effects on U251 glioma cells. Cell growth was significantly inhibited and G2/M arrest was observed in antisense- and siRNA-treated groups. Matrigel matrix demonstrated spotted cell clustering pattern in antisense- and siRNA-transfected U251 cells, indicating poor cell growth activities. In addition, the tumor volumes in U251 subcutaneous mice model treated with antisense and siRNA were significantly smaller than those treated with control siRNA and phosphate-buffered saline. Also, glial fibrillary acidic protein expression was upregulated in antisense- and siRNA-treated groups than the control groups. Our results demonstrated that antisense- or siRNA-targeting intracellular region of EGFR can inhibit EGFR expression, exerted growth inhibition effect on U251 glioma cells in vitro and in vivo. Consequently, siRNA expression plasmid-mediated gene therapy would be a new strategy in treatment of gliomas.

Id-1 expression induces androgen-independent prostate cancer cell growth through activation of epidermal growth factor receptor (EGF-R)

The failure of prostate cancer treatment is largely due to the development of androgen independence, since the androgen depletion therapy remains the front-line option for this cancer. Previously, we reported that over-expression of the helix-loop-helix protein Id-1 was associated with progression of prostate cancer and ectopic expression of Id-1 induced serum-independent proliferation in prostate cancer cells. In the present study, we investigated if exogenous Id-1 expression in the androgen sensitive LNCaP cells had any effect on androgen-dependent cell growth and studied the molecular mechanisms involved. Using stable Id-1 transfectants, we found that expression of Id-1 was able to reduce androgen-stimulated growth and S phase fraction of the cell cycle in LNCaP cells, indicating that Id-1 may be involved in the development of androgen independence in these cells. The Id-1-induced androgen-independent prostate cancer cell growth was correlated with up-regulation of EGF-R (epidermal growth factor-receptor) and PSA (prostate specific antigen) expression, as confirmed by western blotting analysis and luciferase assays. In contrast, down-regulation of Id-1 in androgen-independent DU145 cells by its antisense oligonucleotides resulted in suppression of EGF-R expression at both transcriptional and protein levels. In addition, the results from immunohistochemistry study showed that Id-1 expression was significantly elevated in hormone refractory prostate cancer tissues when compared with the hormone-dependent tumours. Our results suggest that up-regulation of Id-1 in prostate cancer cells may be one of the mechanisms responsible for developing androgen independence and this process may be regulated through induction of EGF-R expression. Inactivation of Id-1 may provide a potential therapeutic strategy leading to inhibition of androgen-independent prostate cancer cell growth.