Epidermal Growth Factor Receptor mRNA Research Articles

Epidermal growth factor receptor expression affects proliferation and apoptosis in non-small cell lung cancer cells via the extracellular signal-regulated kinase/microRNA 200a signaling pathway.

The present study assessed the function of epidermal growth factor receptor (EGFR) and its molecular targets in non-small cell lung cancer. The results of the present study demonstrated that EGFR protein and mRNA expression in the normal adjacent tissue specimens was decreased compared with that in the lung cancer tissue samples. Compared with the BEAS-2B normal bronchial epithelial cells, EGFR and phosphorylated (p)-extracellular signal-regulated kinase (ERK) protein expression in the SW-900 and A549 lung cancer cells was increased and microRNA (miR)200a expression in the SW-900 and A549 cells was inhibited compared with the BEAS-2B cells. Downregulating miR200a expression significantly suppressed proliferation and promoted apoptosis and caspase (CASP)3 and CASP9 function in the A549 cells and significantly inhibited EGFR and p-ERK protein expression in the A549 cells, compared with the BEAS-2B cells. The results of the present study indicated that downregulating miR200a significantly suppressed proliferation and promoted apoptosis in A549 cells via the regulation of the EGFR and ERK 1/2 signaling pathways.

MiR-539 acts as a tumor suppressor by targeting epidermal growth factor receptor in breast cancer

Breast cancer is the most frequently diagnosed malignancy and the leading cause of cancer-associated death in women worldwide. microRNAs (miRNAs) play critical roles in the cellular processes of breast cancer. However, the crucial roles and underlying mechanisms of miR-539 in breast cancer remain unclear. By RT-qPCR, we found that expression of miR-539 was markedly down-regulated in breast cancer tissues and cell lines compared with that in paired adjacent normal tissues and normal cell lines. The low level of miR-539 expression was positively associated with lymph node metastasis. Furthermore, forced expression of miR-539 inhibited proliferation and migration of breast cancer MDA-MB-231 and MCF7 cells in vitro and suppressed tumor growth in vivo. Moreover, bioinformatics analysis and luciferase reporter assays indicated that epidermal growth factor receptor (EGFR) was a direct target of miR-539. Over-expression of miR-539 decreased the EGFR mRNA and protein levels in MDA-MB-231 and MCF7 cells. In addition, ectopic over-expression of EGFR partly reversed miR-539-inhibited proliferation as well as migration of MDA-MB-231 and MCF7 cells. Taken together, our results demonstrate that miR-539 functions as a tumor suppressor in breast cancer by downregulating EGFR, supporting the targeting of the novel miR-539/EGFR axis as a potentially effective therapeutic approach for breast cancer.

Dual effects of a CpG-DNAzyme targeting mutant EGFR transcripts in lung cancer cells: TLR9 activation and EGFR downregulation.

Non-small-cell lung cancer (NSCLC) is commonly caused by a mutation in the epidermal growth factor receptor (EGFR) and subsequent aberrant EGFR signaling with uncontrolled kinase activity. A deletion mutation in EGFR exon 19 is frequently observed in EGFR gene mutations. We designed a DNAzyme to suppress the expression of mutant EGFR by cleaving the mutant EGFR mRNA. The DNAzyme (named Ex19del Dz) specifically cleaved target RNA and decreased cancer cell viability when transfected into gefitinib-resistant lung cancer cells harboring EGFR exon 19 deletions. The DNAzyme decreased EGFR expression and inhibited its downstream signaling pathway. In addition to EGFR downregulation, Ex19del Dz containing CpG sites activated Toll-like receptor 9 (TLR9) and its downstream signaling pathway via p38 kinase, causing an immunostimulatory effect on EGFR-mutated NSCLC cells. Thus, dual effects of this DNAzyme harboring the CpG site, such as TLR9 activation and EGFR downregulation, leads to apoptosis of EGFR-mutated NSCLC cells.

DNA/RNA markers for colorectal cancer risk in preserved stool specimens: a pilot study.

Exfoliated cells in human stool offer excellent opportunities to non-invasively detect molecular markers associated with colorectal tumorigenesis, and to evaluate the effects of exposures to exogenous and endogenous carcinogenic or chemopreventive substances. This pilot study investigated the feasibility of determining DNA methylation and RNA expression simultaneously in stool specimens treated with a single type of nucleic acid preservatives. Stool specimens from 56 volunteers that were preserved up to a week with RNA later were used in this study. Bisulfite sequencing was used to determine methylation at 27 CpG loci on the estrogen receptor 1 (ESR1) promoter. Taqman assay was used for quantitative reverse transcription polymerase chain reactions to measure cyclooxygenase 2 (COX2) and epidermal growth factor receptor (EGFR) mRNA expression. Subjects' basic demographic and other selected risk factors for colorectal cancer were captured through questionnaires and correlated with the levels of these markers. Less than 10% of the samples failed in individual assays. Overall, 24.0% of the CpG loci on the ESR1 promoter were methylated. COX2 expression and alcohol use were positively correlated; an inverse association was present between EGFR expression and cigarette smoking; and subjects using anti-diabetic medication had higher ESR1 methylation. In addition, higher EGFR expression levels were marginally associated with history of polyps and family history of colorectal cancer. The present study demonstrates that simultaneous analyses for DNA and RNA markers are feasible in stool samples treated with a single type of nucleotide preservatives. Among several associations observed, the association between EGFR expression and polyps deserves further investigation as a potential target for colorectal cancer screening. Larger studies are warranted to confirm some of our observations.

FHL2 interacts with EGFR to promote glioblastoma growth.

Four-and-a-half LIM protein2 (FHL2) is a member of the LIM-only protein family, which plays a critical role in tumorigenesis. We previously reported that FHL2 is upregulated and plays an oncogenic role in glioblastoma (GBM), the most common and aggressive brain tumor. GBM is also marked by amplification of the epidermal growth factor receptor (EGFR) gene and its mutations, of which EGFRvIII is the most common and functionally significant. Here we report that FHL2 physically interacts with the wild-type EGFR and its mutated EGFRvIII form in GBM cells. Expression of FHL2 caused increased EGFR and EGFRvIII protein levels and this was due to an increase in protein stability rather than an increase in EGFR mRNA expression. In contrast, FHL2 knockdown using RNA interference reduced EGFR and EGFRvIII protein expression and the phosphorylation levels of EGFR and AKT. Consistent with these features, EGFR expression was significantly lower in mouse FHL2-null astrocytes, where reintroduction of FHL2 was able to restore EGFR levels. Using established GBM cell lines and patient-derived neurosphere lines, FHL2 silencing markedly induced cell apoptosis in EGFRvIII-positive cells. Targeting FHL2 significantly prevented EGFRvIII-positive GBM tumor growth in vivo. FHL2 expression also positively correlated with EGFR expression in GBM samples from patients. Taken together, our results demonstrate that FHL2 interacts with EGFR and EGFRvIII to increase their levels and this promotes glioma growth, representing a novel mechanism that may be therapeutically targetable.

Abstract A028: Biomarker analysis in a phase 1 study of the antibody-drug conjugate ABBV-221 in patients with solid tumors likely to overexpress the epidermal growth factor receptor (EGFR)

Abstract Background: Aberrant EGFR plays a vital role in the oncogenesis of multiple tumor types. ABBV-221 is a 2nd-generation antibody-drug conjugate (ADC) targeting EGFR, based on the 1st-generation ADC depatuxizumab mafodotin (depatux-m, formerly ABT-414). Depatux-m demonstrates efficacy in glioblastoma (GBM) patients (pts) with EGFR amplification in ongoing studies. ABBV-221 is an affinity matured monoclonal antibody against EGFR linked to the microtubule toxin monomethylauristatin E. ABBV-221 has higher affinity for EGFR than depatux-m, potentially allowing it to target a broader range of tumor types. To understand the population most suitable for ABBV-221, molecular assays characterizing EGFR pathway status, including EGFR protein, EGFR mRNA, and EGFR ligand mRNA expression, were evaluated from available tumor tissue of enrolled patients. The results of each assay were correlated with patient outcome to determine which marker may be best associated with prognosis. Methods: In this phase 1, multicenter study of ABBV-221, eligible pts have solid tumors that are likely to overexpress EGFR. EGFR protein expression in tumor tissue samples was analyzed with immunohistochemistry (IHC) using the Dako pharmDx IHC assay. EGFR and mRNA expression from the EGFR ligands epiregulin (EREG) and amphiregulin (AREG) were measured using next-generation RNA sequencing (RNAseq). Results: As of 29 March 2017, 42 pts were treated (13 colon, 5 head & neck [H&N] cancer, 5 non-small cell lung cancer, 5 GBM, 2 breast, 12 other). Ten dose escalation cohorts have been completed with the last cleared dose 4.5 mg/kg per cycle. The most common adverse events were infusion reactions in 19/42 pts (45%) and fatigue in 17/42 pts (41%). Sixteen pts (38%) had stable disease (SD), including 4 pts who remained on study longer than 6 months. All markers are expressed in a wide range across enrolled pts. The pts with response had high EGFR and EGFR ligand. One pt with head and neck cancer who received 5 cycles of ABBV-221 had a partial response and elevated levels of both EGFR (3-fold higher than median for all pts) and EGFR ligand (8-fold higher AREG and 19-fold higher EREG than median for all pts). One colorectal cancer pt with durable SD >6 months had high levels of ligand (EREG) but not receptor, and one H&N pt with SD >6 months had high levels of EGFR receptor but not ligand. Conclusions: Multiple EGFR pathway status biomarkers were evaluated and correlated with clinical outcomes observed in this phase 1 trial of ABBV-221. Patients who observed clinical benefit had high levels of EGFR pathway markers. The duration of SD in pts with refractory solid tumors is encouraging. Clinical trial information: NCT02365662 Citation Format: Emiliano Calvo, James M. Cleary, Victor Moreno, Maryella Gifford, Lisa Roberts-Rapp, Peter J. Ansell, Ho-Jin Lee, Beibei Hu, David Barch, Christopher Ocampo, Anthony W. Tolcher. Biomarker analysis in a phase 1 study of the antibody-drug conjugate ABBV-221 in patients with solid tumors likely to overexpress the epidermal growth factor receptor (EGFR) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A028.

Investigation on the regulatory effect of PGE2 on ESCC cells through the trans-activation of EGFR by EP2 and the relevant mechanism.

To determine whether the combination of prostaglandin E2 (PGE2) and EP2, the subtype receptor of PGE2, could trans-activate the epidermal growth factor receptor (EGFR). In this experiment, we selected epithelial cells from normal esophageal mucosa as the negative control group, and the ESCC EC109 and TE-1 cell strain as the observation group. Real-time PCR and Western-blotting were used to detect the expression of EP2, EGFR and phosphorylated EGFR (p-EGFR). The pre-treatment of ESCC cell strains was carried out using Butaprost (special agonist of PGE2 and EP2) and RNAi of EP2, and we observed the expression of EP2, EGFR, and p-EGFR. WST-8 (CCK-8) was applied for the detection of the cell proliferation rate. The transwell invasion experiment was conducted for the detection of the invasion capability of cells. The expression of MMP-9 (matrix metalloproteinase-9), VEGF (vascular endothelial growth factor), pro-inflammatory factors (IL-6 and TNF-α) in the cell supernatant were detected using ELISA. The high mRNA and protein expression of EP2, EGFR, and p-EGFR were found in the EC109 and TE-1 cell strains in the observation group, which were higher than those in the control group (p < 0.05). After the intervention of PGE2, EP2 expression was decreased and the p-EGFR expression was increased (p < 0.05). There was no variation found in the expression of EGFR (p > 0.05). After cells were intervened using Butaprost, the expressions of EP2 and p-EGFR were increased (p < 0.05), and there were no changes identified in the expression of EGFR (p > 0.05). After the intervention of RNAi, the expression of EP2 and p-EGFR was decreased (p < .05), and no changes were identified in the expression of EGFR (p > 0.05). After the intervention of PGE2 and Butaprost, great increases were seen in the cell proliferation rate, invasion capability, and the expression of MMP-9, VEGF, IL-6, and TNF-α in EC109 and TE-1 cell strains (p < 0.05), however, the intervention of RNAi could reduce above indexes (p < 0.05). Through cell experiments, we verified that the combination of prostaglandin E2 (PGE2) and EP2, the subtype receptor of PGE2, could trans-activate the epidermal growth factor receptor (EGFR) to regulate the proliferation and invasion capability of esophageal squamous cell carcinoma (ESCC) cells, and secrete and express multiple cytokines, thus discovering the pathological mechanism of inflammation to carcinoma transition in the occurrence of ESCC, and providing the experimental evidence for the search of new target in the treatment of ESCC. ESCC cells can highly express the receptor subtype EP2 of PGE2 that can transactivate the EGFR, through which PGE2 is involved in the transition mechanism from inflammation to cancer.

MicroRNA-370 inhibits the growth and metastasis of lung cancer by down-regulating epidermal growth factor receptor expression.

Abnormal microRNA-370 (miR-370) expression has been frequently reported in several types of cancers, including lung cancer. However, the role and molecular mechanisms of miR-370 in regulating the growth and metastasis of lung cancer have not been clarified. Here, we show higher levels of epidermal growth factor receptor (EGFR), but lower levels of miR-370 expression in most human lung cancer cells and non-tumor cells. Induction of miR-370 over-expression significantly reduced the levels of EGFR expression and the EGFR 3′untranslated region (UTR)-regulated luciferase activity in XWLC-05 and H157 cells, suggesting that miR-370 may bind to the 3′UTR of EGFR mRNA. Compared with the control cells, induction of miR370 overexpression significantly inhibited the proliferation, clone formation capacity, migration and invasion of XWLC-05 and H157 cells while miR-370 inhibitor over-expression enhanced their tumor behaviors in vitro. Furthermore, miR-370 over-expression down-regulated the EGFR and hypoxia-inducible factor (HIF)-1α expression, and attenuated the extracellular single-regulated kinase (ERK)1/2 and AKT phosphorylation in XWLC-05 and H157 cells. In contrast, miR370 inhibitor over-expression increased the EGFR and HIF-1α expression as well as the ERK1/2 and AKT phosphorylation in XWLC-05 and H157 cells. Moreover, miR-370 over-expression significantly reduced the levels of EGFR and CD31 expression and inhibited the growth and lung metastasis of xenograft NSCLC tumors in mice. Our study indicates that miR-370 may bind to the 3′UTR of EGFR to inhibit EGFR expression and the growth, angiogenesis and metastasis of non-small cell lung cancer by down-regulating the ERK1/2 and AKT signaling.

Increased EGFR expression induced by a novel oncogene, CUG2, confers resistance to doxorubicin through Stat1-HDAC4 signaling

Previously, it has been found that the cancer upregulated gene 2 (CUG2) and the epidermal growth factor receptor (EGFR) both contribute to drug resistance of cancer cells. Here, we explored whether CUG2 may exert its anticancer drug resistance by increasing the expression of EGFR. EGFR expression wasassessed using Western blotting, immunofluorescence and capacitanceassays in A549 lung cancer and immortalized bronchial BEAS-2B cells, respectively, stablytransfected with aCUG2expression vector (A549-CUG2; BEAS-CUG2) oran empty control vector (A549-Vec; BEAS-Vec). AftersiRNA-mediated EGFR, Stat1 andHDAC4silencing, antioxidant and multidrug resistance proteinand mRNA levels wereassessedusing Western blottingand RT-PCR.In addition, the respective cells were treated with doxorubicinafter which apoptosis and reactive oxygen species (ROS)levels weremeasured.Stat1 acetylation was assessed by immunoprecipitation. We found that exogenous CUG2 overexpression induced EGFR upregulation in A549 and BEAS-2B cells, whereas EGFRsilencing sensitized these cells to doxorubicin-induced apoptosis. In addition, we found that exogenous CUG2 overexpression reduced the formation of ROS during doxorubicin treatment by enhancing the expression of antioxidant and multidrug resistant proteins such as MnSOD, Foxo1, Foxo4, MRP2 and BCRP, whereas EGFR silencing congruently increased the levels of ROS by decreasing the expression of these proteins. We also found that EGFR silencing and its concomitant Akt, ERK, JNK and p38 MAPK inhibition resulted in a decreased Stat1 phosphorylation and, thus, a decreased activation. Since also acetylation can affect Stat1 activation via a phospho-acetyl switch, HDAC inhibition may sensitize cells to doxorubicin-induced apoptosis. Interestingly, we found that exogenous CUG2 overexpression upregulated HDAC4, but not HDAC2 or HDAC3. Conversely, we found that HDAC4 silencing sensitized the cells to doxorubicin resistance by decreasing Stat1 phosphorylation and EGFR expression, thus indicating an interplay between HDAC4, Stat1 and EGFR. Taken together, we conclude that CUG2-induced EGFR upregulation confers doxorubicin resistance to lung (cancer) cells through Stat1-HDAC4 signaling.

RANK and EGFR in invasive breast carcinoma

Breast cancer is the most common malignancy, affecting one in eight women in North America and Europe. The human epidermal growth factor receptor (EGFR) protein comprises a major determinant of normal development but also cancer. RANK receptor (Receptor Activator of Nuclear factor-κB) is a tumor necrosis superfamily member and a binding partner for RANKL, which was recently implicated in breast cancer initiation, progression and metastasis. Here we provide preliminary evidence of a possible interplay between RANK and EGFR signaling in breast cancer. TCGA (cancergenome.nih.gov) publicly available data for EGFR and TNFRSF11A (RANK) genes from breast cancer patients and breast cancer cell lines were retrieved and analyzed. RANK mRNA showed a statistically significant positive correlation (p <0.001) with the mRNA and protein expression of EGFR, but not with ERBB2/3/4. Further analyses of survival data of a group of breast cancer patients (n = 248) from TCGA, revealed an EGFRhi/RANKhi subpopulation that showed a statistically significant (p = 0.001) reduced overall survival when compared to EGFRlow/RANKlow group of patients. Finally, EGFR and RANK combinatorial in vitro analyses revealed a significant upregulation of AKT and ERK signaling after EGF stimulation in cell lines and also an increase of breast cancer cell invasiveness.

Expressions of annexins and epidermal growth factor receptor in pediatric middle ear cholesteatoma

Objective To explore the expressions of annexin and epidermal growth factor receptor(EGFR) in pediatric middle ear cholesteatoma. Methods Twenty-three children with middle ear cholesteatoma and 26 children with normal skin of external auditory canal(control group) were selected from the children enrolled in the First Affiliated Hospital of Zhengzhou University from August 2014 to March 2016.The expressions of annexin A1 (AnxA1), AnxA2 and EGFR mRNA in cholesteatoma and normal tissues were analyzed by quantitative real-time PCR (qPCR). Protein expressions of AnxA1, AnxA2 and EGFR were evaluated by using Western blot and immunohistochemistry methods. Results The expressions of AnxA1, AnxA2 and EGFR mRNA were significantly increased in cholesteatoma compared with the control group(AnxA1: 4.68±1.77 vs.2.65±0.96, U=111.5, P<0.001; AnxA2: 3.89±1.00 vs.2.47±0.81, U=84.5, P<0.001; EGFR: 4.97±1.85 vs.3.50±0.95, U=153.5, P=0.004). AnxA1 and AnxA2 mRNA expressions were positively correlated with EGFR mRNA (AnxA1 and EGFR: r2=0.283 2, P=0.009; AnxA2 and EGFR: r2=0.213 5, P=0.027). Compared with the control group, protein expressions of AnxA1, AnxA2 and EGFR were markedly enhanced (AnxA1: 0.450±0.031 vs.0.320±0.026, U=102.4, P<0.001; AnxA2: 0.568±0.024 vs.0.365±0.028, U=94.6, P<0.001; EGFR: 0.397±0.021 vs.0.228±0.017, U=128.4, P<0.001). The results of immunohistochemistry analysis showed that the ratio of AnxA1, AnxA2 and EGFR positive cells were higher than those in the control group(AnxA1: 65.22% vs.38.46%, χ2=9.296, P=0.026; AnxA2: 69.57% vs.46.15%, χ2=8.378, P=0.039; EGFR: 69.57% vs.50.00%, χ2=10.574, P=0.014). Conclusions The expressions of AnxA1, AnxA2 and EGFR are upregulated in pediatric cholesteatoma, with AnxA1 and AnxA2 expressions positively correlated with EGFR. Key words: Annexin A1; Annexin A2; Epidermal growth factor receptor; Cholesteatoma; Child

Abstract 1032: Identification of Ran binding protein 6 as a novel negative regulator of EGFR and candidate tumor suppressor in glioblastoma

Abstract Amplification and overexpression of the epidermal growth factor receptor (EGFR) are common in glioblastoma (GBM) and frequently associated with silencing of the phosphatase and tensin homologue (PTEN) tumor suppressor. PTEN silencing has been associated with clinical resistance to EGFR tyrosine kinase inhibitors, in part by raising EGFR levels. Here, we investigated the effect of PTEN on the EGFR signaling complex by EGFR affinity immunopurification and mass spectrometry with and without PTEN knockdown. We identified Ran binding protein 6 (RanBP6), a 125-kDa protein of previously unknown functions, as EGFR interacting protein in PTEN expressing, but not PTEN knockdown cells. Further studies of the effect of RanBP6 on EGFR revealed that RanBP6 depletion by shRNA or CRISPR/Cas9-mediated gene silencing resulted in increased EGFR mRNA levels and upregulation of EGFR promoter activity. Consistent with a model of a negative EGFR regulation by RanBP6, we observed an inverse correlation between RanBP6 and EGFR mRNA levels in PTEN wildtype but not PTEN altered cancer cells in a large panel of human cancer cell lines (Cancer Cell Line Encyclopedia). To further understand the mechanism of how RanBP6 negatively regulates EGFR mRNA level, we found that RanBP6 interacted with nuclear Ran-GTPase and repressed EGFR transcription by promoting nuclear import of Signal transducer and activator of transcription 3 (STAT3). Lastly, RanBP6 appeared to be frequently deleted on chromosome 9p in GBM. We showed that RanBP6 silencing raised EGFR levels and signal output and accelerated in-vivo glioma growth. Our results establish a novel function of RanBP6 as a link between EGFR signaling and the Ran-mediated nuclear import pathway, and identify RanBP6 as candidate tumor suppressor on chromosome 9p. Citation Format: Wan-Ying Hsieh, Barbara Oldrini, Hediye Erdjument-Bromage, Paolo Codega, Maria S. Carro, Igor Vivanco, Dan Rohle, Carl Campos, Craig Bielski, Barry Taylor, Paul Tempst, Massimo Squatrito, Ingo K. Mellinghoff. Identification of Ran binding protein 6 as a novel negative regulator of EGFR and candidate tumor suppressor in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1032. doi:10.1158/1538-7445.AM2017-1032

Inhibition of N-acetylglucosaminyltransferase V enhances the cetuximab-induced radiosensitivity of nasopharyngeal carcinoma cells likely through EGFR N-glycan alterations.

N-acetylglucosaminyltransferase V (GnT-V), an enzyme that catalyzes the formation of the N-linked β-1-6 branching of oligosaccharides, is related to the radiosensitivity of nasopharyngeal carcinoma (NPC). Cetuximab (C225) is an epidermal growth factor receptor (EGFR) inhibitor used as a radiosensitizer in the treatment of NPC. In this study, we used GnT-V as a molecular target to further sensitize cetuximab-treated NPC cells to radiation. The results from two NPC cell lines (CNE1 and CNE2) revealed that the silencing of GnT-V enhanced cetuximab-induced radiosensitivity by decreasing the β-1-6 branching of oligosaccharides on the EGFR. GnT-V down-regulation combined with cetuximab decreased the survival fraction, healing rate and cell viability and increased the apoptosis rate. Concomitantly, the combination of cetuximab and irradiation did not change the EGFR mRNA and protein levels and decreased the β-1-6 branching on the EGFR. Subsequently, we further explored the signaling downstream of EGF, particularly the PI3K/Akt signaling pathway, and discovered that treatment consisting of GnT-V down-regulation, irradiation and cetuximab was negatively correlated with phospho-Akt and phspho-PI3K. Finally, an in vivo experiment with radiotherapy revealed that the combination of GnT-V down-regulation and cetuximab decelerated tumor growth. In summary, our study demonstrated that the combination of decreased GnT-V activity and cetuximab enhanced NPC radiosensitivity, and the possible mechanism underlying this effect might involve the N-linked β1-6 branching of the EGFR.

Precision knockdown of EGFR gene expression using radio frequency electromagnetic energy.

Electromagnetic fields (EMF) in the radio frequency energy (RFE) range can affect cells at the molecular level. Here we report a technology that can record the specific RFE signal of a given molecule, in this case the siRNA of epidermal growth factor receptor (EGFR). We demonstrate that cells exposed to this EGFR siRNA RFE signal have a 30-70% reduction of EGFR mRNA expression and ~60% reduction in EGFR protein expression vs. control treated cells. Specificity for EGFR siRNA effect was confirmed via RNA microarray and antibody dot blot array. The EGFR siRNA RFE decreased cell viability, as measured by Calcein-AM measures, LDH release and Caspase 3 cleavage, and increased orthotopic xenograft survival. The outcomes of this study demonstrate that an RFE signal can induce a specific siRNA-like effect on cells. This technology opens vast possibilities of targeting a broader range of molecules with applications in medicine, agriculture and other areas.

EGFR gene status predicts response and survival benefit in a preclinical gastric cancer trial treating patient‑derived xenografts with cetuximab.

Cetuximab has been evaluated as a first-line treatment with conflicting results. The aim of the present study was to investigate the relationship between epidermal growth factor receptor (EGFR) status, and response and survival benefit following cetuximab treatment in gastric cancer (GC). Using 20patient-derived GC xenograft (PDX) models, the mice (10mice/model) were randomly assigned into two groups. The control group and treatment group were treated with PBS and cetuximab, respectively. The drug response was evaluated by monitoring tumor growth. Survival benefit was evaluated by comparing the survival curves corresponding to the time for the tumors to reach 600mm3. Our results revealed that the PDX models treated with cetuximab had better survival than that noted for the non-treated group (P<0.05). The EGFR status was measured by FISH, qPCR, RNAish and immunohistochemistry, respectively. Four cases in the treated group were identified as responsive to cetuximab. EGFR mRNA and protein overexpression were associated with the response to cetuximab (P<0.05). EGFR amplification, mRNA and protein overexpression were associated with prolonged survival in the cetuximab-treated PDX models. Moreover, in the PDX models with EGFR amplification, mRNA or protein overexpression, cetuximab treatment was associated with a better survival compared with that noted in the untreated group in the PDX models (P<0.05), while the survival was not statistically different in the other cases (P>0.05). In conclusion, cetuximab provided survival benefit in the trial. The level of EGFR amplification and overexpression significantly predicted response and survival benefit, particularly the mRNA and protein expression level. A combination of mRNA and protein expression may predict efficacy of cetuximab more efficiently.

Genetic Delivery of RNA Molecules to Alter Expression of EGFR in Brain Cancer Cells

Glioblastoma multiforme (GBM) is an incurable and aggressive type of brain tumor. It is the most common central nervous system malignancy with a median survival of only 14 months. GBM is characterized by increased activation of one or more types of cell membrane receptors that drive the proliferation of tumor cells. One receptor in particular, epidermal growth factor receptor (EGFR), is dysregulated in about 60% of GBM tumors. The amplification and overexpression of the EGFR gene makes it a primary target for therapy. Although a great deal is known about the biology of EGFR‐activated GBM tumors, therapies against its biologic processes are limited by the blood‐brain barrier. Therapeutic strategies targeting splicing of the pre‐mRNA transcript, before the mRNA is translated into an active peptide, show great promise. In our current approach, we have designed and cloned a therapeutic gene that encodes a pre‐trans‐splicing RNA (PTR). Using anti‐sense targeting, the therapeutic PTR delivers an early stop codon and polyadenylation signal into the EGFR pre‐mRNA transcript, leading to a short, non‐membrane bound soluble peptide. To optimize the PTR we are testing alternative antisense binding sequences, as well as distinct splicing elements along the EGFR transcript. We make use of multiple GBM cell lines and RT‐PCR to measure changes in expression of the EGFR transcript. In addition, we are developing a novel approach using a fluorescent PTR (fPTR) to quickly measure changes in expression. Results show that our vectors were expressed at high levels with subsequent changes in EGFR mRNA expression. Our approach to deliver the DNA encoding anti‐EGFR PTRs to alter pre‐mRNA splicing of the EGFR mRNA transcript in GBM cells has the potential to work from the inside‐out, bypassing systemic barriers.Support or Funding InformationMonmouth University School of Science Research Program, Bristol‐Myers Squibb, Independent College Fund of New Jersey, Johnson &amp; Johnson

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.

MiR-370 regulates cell proliferation and migration by targeting EGFR in gastric cancer.

Epidermal growth factor receptor (EGFR) is known to be overexpressed in ~30% of gastric cancer (GC) cases, and may serve as an effective biomarker for predicting the clinical benefit of anti-EGFR therapy. However, the mechanism underlying the regulation of EGFR expression remains unknown. Evidence indicates that post-transcriptional regulation may exist in the process of EGFR expression. In the present study, we aimed to explore whether miR-370 is involved in this process, and how it impacts the biological behaviors of GC cells. In the present study, we first determined the role of EGFR in GC by means of an EGFR overexpression plasmid and siRNAs. Then, the expression levels of EGFR protein and mRNA in GC tissues were analyzed through immunohistochemistry and quantitative RT-PCR (RT-qPCR). Bioinformatics tools and dual-luciferase assay were applied to predict and validate the relevant miRNA targeting EGFR. Finally, human GC MGC-803 cells were selected to explore the effect of miRNA on cell proliferation and migration. We found that suppression of EGFR inhibited the proliferation and migration of GC cells. In addition, the levels of EGFR protein in the GC tissues were ~4times higher than that in the corresponding paired non-cancerous tissues while the mRNA levels of EGFR were only ~2-fold as high as that in the adjacent non-cancerous tissues. Bioinformatics tools predicted that miRNA-370 was a regulator of EGFR and dual‑luciferase assay validated that miR-370 could directly bind to the 3'-untranslated region (3'-UTR) of EGFR mRNA. Meanwhile, an inverse correlation between miR-370 and EGFR was found in the GC tissues. Overexpression of miR-370 suppressed the proliferation and migration of GC cells, while downregulation of miR-370 promoted proliferation and migration. The present study may provide new information for understanding the molecular mechanism underlying the regulation of EGFR protein expression in GC and may be of important clinical significance to guide targeted therapy.

MiR-455 inhibits cell proliferation and migration via negative regulation of EGFR in human gastric cancer.

Epidermal growth factor receptor (EGFR) plays an important role in various types of cancer. However, the therapeutic agents that target EGFR have not produced favorable results in gastric cancer. miRNAs are known to regulate gene expression at the post-transcriptional level. We wondered if miRNAs could be potential therapeutic agents for the targeted therapy against EGFR. In this study, we found an increase in the copies of EGFR mRNA and the upregulated expression of the EGFR protein in gastric cancer tissues compared with normal gastric tissues. Both bioinformatic analysis and luciferase reporter assay revealed that miR-455 could directly bind with the 3'-untranslated region (3'UTR) of EGFR mRNA. Subsequently, invitro studies were conducted to identify the effects of miR-455 on cell proliferation and migration and further confirm the cancer-promoting role of EGFR in gastric cancer. The results revealed that miR-455 negatively regulated EGFR expression at the post-transcriptional level, thus suppressing cell growth and migration. To conclude, our results offer a potential targeted therapeutic method against EGFR in gastric cancer mediated by miR-455.

A tumor-targeting cRGD-EGFR siRNA conjugate and its anti-tumor effect on glioblastoma in vitro and in vivo

The epidermal growth factor receptor (EGFR) is an important anti-tumor target. The development of novel molecular-targeted anti-tumor drugs that can target the interior of tumor cells and specifically silence EGFR expression is valuable and promising. In this work, a promising anti-tumor conjugate comprising methoxy-modified EGFR siRNA and cyclic arginine-glycine-aspartic acid (cRGD) peptides, which selectively bind to αvβ3 integrins, was synthesized and examined. To prepare cRGD-EGFR siRNA (cRGD-siEGFR), cRGD was covalently conjugated to the 5′-end of an siRNA sense strand using a thiol-maleimide linker. The cellular uptake and cytotoxicity of cRGD-siEGFR in vitro were tested using an αvβ3-positive U87MG cell line. In vivo bio-distribution, anti-tumor activity, immunogenicity and toxicity were investigated in a nude mouse tumor model through repeated i.v. administration of cRGD-siEGFR (7 times over a 48 h interval). Analyses of in vitro data showed that cRGD-siEGFR silenced EGFR expression effectively, with high tumor targeting ability. Administration of cRGD-siEGFR to tumor-bearing nude mice led to significant inhibition of tumor growth, obvious reduction of EGFR expression and down-regulation of EGFR mRNA and protein in tumor tissue. Furthermore, serum biochemistry and pathological section evaluation did not indicate any serious toxicity of cRGD-siEGFR in vivo. cRGD-siEGFR is likely a promising candidate with high targeting ability, substantial anti-tumor effects and low toxicity in vitro and in vivo.