Down-regulation Of Epidermal Growth Factor Receptor Research Articles

Endocytosis separates EGF receptors from endogenous fluorescently labeled HRas and diminishes receptor signaling to MAP kinases in endosomes.

Signaling from epidermal growth factor receptor (EGFR) to extracellular-stimuli-regulated protein kinase 1/2 (ERK1/2) is proposed to be transduced not only from the cell surface but also from endosomes, although the role of endocytosis in this signaling pathway is controversial. Ras is the only membrane-anchored component in the EGFR-ERK signaling axis, and therefore, its location determines intracellular sites of downstream signaling. Hence, we labeled endogenous H-Ras (HRas) with mVenus fluorescent protein using gene editing in HeLa cells. mVenus-HRas was primarily located at the plasma membrane, and in small amounts in tubular recycling endosomes and associated vesicles. EGF stimulation resulted in fast but transient activation of mVenus-HRas. Although EGF:EGFR complexes were rapidly accumulated in endosomes together with the Grb2 adaptor, very little, if any, mVenus-HRas was detected in these endosomes. Interestingly, the activities of MEK1/2 and ERK1/2 remained high beyond the point of the physical separation of HRas from EGF:EGFR complexes and down-regulation of Ras activity. Paradoxically, this sustained MEK1/2 and ERK1/2 activation was dependent on the active EGFR kinase. Cell surface biotinylation and selective inactivation of surface EGFRs suggested that a small fraction of active EGFRs remaining in the plasma membrane is responsible for continuous signaling to MEK1/2 and ERK1/2. We propose that, under physiological conditions of cell stimulation, EGFR endocytosis serves to spatially separate EGFR-Grb2 complexes and Ras, thus terminating Ras-mediated signaling. However, sustained minimal activation of Ras by a small pool of active EGFRs in the plasma membrane is sufficient for extending MEK1/2 and ERK1/2 activities.

High mannose-binding Pseudomonas fluorescens lectin (PFL) downregulates cell surface integrin/EGFR and induces autophagy in gastric cancer cells.

BackgroundPseudomonas fluorescens lectin (PFL) belongs to a recently discovered anti-HIV lectin family and induces anoikis-like cell death of MKN28 gastric cancer cells by causing α2 integrin internalization through recognition of high mannose glycans; however, the detailed anti-cancer mechanism is not fully elucidated.MethodsCell adherence potency of MKN28 upon PFL treatment was assessed using a colorimetric assay. Cell surface molecules to which PFL bound were identified by peptide mass finger printing with Matrix Assisted Laser Desorption/Ionization-time of flight mass spectrometry and their cellular localization determined by immunofluorescence microscopy. Gene and protein expression in PFL-treated MKN28 cells were evaluated by microarray analysis and western blot, and the function of these genes was evaluated by siRNA knock-down. A proliferation assay measured the sensitivity of PFL-treated cancer cells to anti-cancer drugs. The effect of PFL on subcutaneous MKN28 tumor growth and hepatic tumor formation in BALB/c nude mice was evaluated.ResultsThe strength of MKN28 cell adherence in vitro to the extracellular matrix was impaired by PFL treatment, consistent with the observation that PFL induces rapid downregulation of surface integrins. PFL also was found to bind to cell surface epidermal growth factor receptor (EGFR). Surface EGFR molecules were endocytosed following PFL binding, and were degraded in a time-dependent fashion. This degradation process was largely the result of autophagy, as revealed by the increased expression of autophagic proteins. PFL-induced EGFR degradation was partly inhibited by RAB7 siRNA as well as LC3 siRNA, and internalized EGFR colocalized with ATG9 at 48 h post-PFL treatment, suggesting that these proteins contribute to dynamic degradation induced by PFL. PFL-induced decrease in surface EGFR rendered MKN28 cells susceptible to gefitinib, a selective inhibitor of EGFR tyrosine kinase. In vivo experiments showed that PFL-treated MKN28-EGFP cells injected in the portal vein of BALB/c nude mice failed to form tumor colonies on the liver, and intratumoral injection of PFL significantly inhibited tumor growth.ConclusionPFL-mediated downregulation of integrin and EGFR contributes to the inhibition of tumor growth in vitro and in vivo. This novel anti-cancer mechanism of PFL suggests that this lectin would be useful as an anti-cancer drug or an adjuvant for other drugs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2099-2) contains supplementary material, which is available to authorized users.

MIIP accelerates epidermal growth factor receptor protein turnover and attenuates proliferation in non-small cell lung cancer.

The migration and invasion inhibitory protein (MIIP) has been discovered recently to have inhibitory functions in cell proliferation and migration. Overexpression of MIIP reduced the intracellular steady-state level of epidermal growth factor receptor (EGFR) protein in lung cancer cells with no effect on EGFR mRNA expression compared to that in the control cells. This MIIP-promoted EGFR protein degradation was reversed by proteasome and lysosome inhibitors, suggesting the involvement of both proteasomal and lysosomal pathways in this degradation. This finding was further validated by pulse-chase experiments using 35S-methionine metabolic labeling. We found that MIIP accelerates EGFR protein turnover via proteasomal degradation in the endoplasmic reticulum and then via the lysosomal pathway after its entry into endocytic trafficking. MIIP-stimulated downregulation of EGFR inhibits downstream activation of Ras and blocks the MEK signal transduction pathway, resulting in inhibition of cell proliferation. The negative correlation between MIIP and EGFR protein expression was validated in lung adenocarcinoma samples. Furthermore, the higher MIIP protein expression predicts a better overall survival of Stage IA-IIIA lung adenocarcinoma patients who underwent radical surgery. These findings reveal a new mechanism by which MIIP inhibits cell proliferation.

Genome-Wide Transcriptional Analysis Reveals the Protection against Hypoxia-Induced Oxidative Injury in the Intestine of Tibetans via the Inhibition of GRB2/EGFR/PTPN11 Pathways.

The molecular mechanisms for hypoxic environment causing the injury of intestinal mucosal barrier (IMB) are widely unknown. To address the issue, Han Chinese from 100 m altitude and Tibetans from high altitude (more than 3650 m) were recruited. Histological and transcriptome analyses were performed. The results showed intestinal villi were reduced and appeared irregular, and glandular epithelium was destroyed in the IMB of Tibetans when compared with Han Chinese. Transcriptome analysis revealed 2573 genes with altered expression. The levels of 1137 genes increased and 1436 genes decreased in Tibetans when compared with Han Chinese. Gene ontology (GO) analysis indicated most immunological responses were reduced in the IMB of Tibetans when compared with Han Chinese. Gene microarray showed that there were 25-, 22-, and 18-fold downregulation for growth factor receptor-bound protein 2 (GRB2), epidermal growth factor receptor (EGFR), and tyrosine-protein phosphatase nonreceptor type 11 (PTPN11) in the IMB of Tibetans when compared with Han Chinese. The downregulation of EGFR, GRB2, and PTPN11 will reduce the production of reactive oxygen species and protect against oxidative stress-induced injury for intestine. Thus, the transcriptome analysis showed the protecting functions of IMB patients against hypoxia-induced oxidative injury in the intestine of Tibetans via affecting GRB2/EGFR/PTPN11 pathways.

Hydroxytyrosol, a product from olive oil, reduces colon cancer growth by enhancing epidermal growth factor receptor degradation.

We studied the effects and mechanism of 2-(3,4-dihydroxyphenil)ethanol (or hydroxytyrosol, HT), a polyphenol from extra virgin olive oil, investigating the regulation of epidermal growth factor receptor (EGFR) expression in colon tumour cells. We demonstrate that HT significantly downregulates EGFR expression in human colorectal adenocarcinoma cells HT-29, CaCo2, and WiDr, and in HT-29 xenografts. HT accelerates EGFR degradation by reducing its half-life. Specifically, HT induces EGFR ubiquitination that is mediated by phosphorylation at pY1045, the docking site for Cbl, thereby enabling receptor ubiquitination and degradation. Pretreatment with either the lysosomal inhibitor chloroquine, or the proteasomal inhibitor MG132 blocks HT-induced EGFR downregulation. In colon cancer cells, EGFR downregulation by HT is associated with reduced cell proliferation. Tumour growth and EGFR expression levels are also decreased by HT treatment in HT-29 xenograft. We conclude that HT downregulates EGFR expression via lysosomal and proteasomal degradation, activated by HT-induced EGFR phosphorylation at pY1045 and increased Cbl activity. Cbl activation induces, in turn, EGFR ubiquitination. Our results reveal a new mechanism for HT's antitumour effects that may be important for colon tumour prevention and treatment.

Phosphatidic Acid Increases Epidermal Growth Factor Receptor Expression by Stabilizing mRNA Decay and by Inhibiting Lysosomal and Proteasomal Degradation of the Internalized Receptor.

Overexpression of epidermal growth factor receptor (EGFR) is one of the frequent mechanisms implicated in cancer progression, and so is the overexpression of the enzyme phospholipase D (PLD) and its reaction product, phosphatidic acid (PA). However, an understanding of how these signaling molecules interact at the level of gene expression is lacking. Catalytically active PLD enhanced expression of EGFR in human breast cancer cells. Overexpression of the PLD2 isoform increased EGFR mRNA and protein expression. It also negated an EGFR downregulation mediated by small interfering RNA targeting EGFR (siEGFR). Several mechanisms contributed to the alteration in EGFR expression. First was the stabilization of EGFR transcripts as PLD2 delayed mRNA decay, which prolonged their half-lives. Second, RNase enzymatic activity was inhibited by PA. Third, protein stabilization also occurred, as indicated by PLD resistance to cycloheximide-induced EGFR protein degradation. Fourth, PA inhibited lysosomal and proteasomal degradation of internalized EGFR. PLD2 and EGFR colocalized at the cell membrane, and JAK3 phosphorylation at Tyr980/Tyr981 followed receptor endocytosis. Further, the presence of PLD2 increased stabilization of intracellular EGFR in large recycling vesicles at ∼15 min of EGF stimulation. Thus, PLD2-mediated production of PA contributed to the control of EGFR exposure to ligand through a multipronged transcriptional and posttranscriptional program during the out-of-control accumulation of EGFR signaling in cancer cells.

Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells.

Ubiquitination of the epidermal growth factor receptor (EGFR) that occurs when Cbl and Grb2 bind to three phosphotyrosine residues (pY1045, pY1068 and pY1086) on the receptor displays a sharp threshold effect as a function of EGF concentration. Here we use a simple modelling approach together with experiments to show that the establishment of the threshold requires both the multiplicity of binding sites and cooperative binding of Cbl and Grb2 to the EGFR. While the threshold is remarkably robust, a more sophisticated model predicted that it could be modulated as a function of EGFR levels on the cell surface. We confirmed experimentally that the system has evolved to perform optimally at physiological levels of EGFR. As a consequence, this system displays an intrinsic weakness that causes—at the supraphysiological levels of receptor and/or ligand associated with cancer—uncoupling of the mechanisms leading to signalling through phosphorylation and attenuation through ubiquitination.

Presenilin 1 is necessary for neuronal, but not glial, EGFR expression and neuroprotectionviaγ-secretase-independent transcriptional mechanisms

Epidermal growth factor receptor (EGFR) plays pivotal roles in cell proliferation, differentiation, and tissue development, while EGFs protect neurons from toxic insults by binding EGFR and stimulating survival signaling. Furthermore, recent evidence implicates this receptor in neurometabolic disorders like Alzheimer disease and aging. Here we show that absence of presenilin 1 (PS1) results in dramatic decrease (>95%) of neuronal EGFR and that PS1-null (PS1(-/-)) brains have reduced amounts of this receptor. PS1(-/-) cortical neurons contain little EGFR and show no epidermal growth factor-induced survival signaling or protection against excitotoxicity, but exogenous EGFR rescues both functions even in absence of PS1. EGFR mRNA is greatly reduced (>95%) in PS1(-/-) neurons, and PS1(-/-) brains contain decreased amounts of this mRNA, although PS1 affects the stability of neither EGFR nor its mRNA. Exogenous PS1 increases neuronal EGFR mRNA, while down-regulation of PS1 decreases this mRNA. These effects are neuron specific, as PS1 affects the EGFR of neither glial nor fibroblast cells. In addition, PS1 controls EGFR through novel mechanisms shared with neither γ-secretase nor PS2. Our data reveal that PS1 functions as a positive transcriptional regulator of neuronal EGFR controlling its expression in a cell-specific manner. Severe downregulation of EGFR may contribute to developmental abnormalities and lethal phenotype found in PS1, but not PS2, null mice. Furthermore, PS1 may affect neuroprotection and Alzheimer disease by controlling survival signaling of neuronal EGFR.

EGFR over-expression in non-small cell lung cancers harboring EGFR mutations is associated with marked down-regulation of CD82

Epidermal growth factor receptor (EGFR) gene mutations are strongly associated with lung adenocarcinoma and favorable response to EGFR tyrosine kinase inhibitor. The mutated EGFR proteins (EGFRs) are hyper-phosphorylated and refractory to receptor down-regulation. To address the discrepancy between hyper-phosphorylation and lack of down-regulation of mutant EGFRs, we have examined the expression of EGFR negative regulators in non-small cell lung cancer (NSCLC) cell lines. We found that NSCLC cell lines expressing mutant EGFRs often had low expression of various negative regulators for EGFR. Among them, tumor suppressor CD82 was up-regulated by wild type (WT) EGFR but down-regulated by mutant EGFRs. Reconstitution of CD82 exerted stronger suppressive effects on mutant EGFRs than on WT EGFR. Active exportation of CD82 through the exosome was one of the mechanisms involved in achieving the overall CD82 down-regulation in mutant EGFR-expressing lung cancer cell lines. Over-expression of mutant EGFR protein frequently occurred in the lung cancer tissues of mutant EGFR-transgenic mice and also associated with CD82 down-regulation. Immunoblot analyses on the tumor tissues from 23 lung adenocarcinoma patients (12 with WT EGFR, and 11 with mutant EGFRs) also identified significantly stronger down-regulation of CD82 in tumors with mutant EGFRs than WT. Our data indicate that CD82 down-regulation could be a critical step involved in the EGFR over-expression and the stronger tumorigenic activity triggered by EGFR mutations. Up-regulation of the CD82 level may become a promising new treatment strategy for lung adenocarcinoma.

COX-2 and EGFR: Partners in Crime Split by Aspirin

COX-2 and EGFR: Partners in Crime Split by Aspirin

Nimotuzumab increases the anti-tumor effect of photodynamic therapy in an oral tumor model.

Oral squamous cell carcinoma (OSCC) represents 90% of all oral cancers and is characterized with poor prognosis and low survival rate. Epidermal growth factor receptor (EGFR) is highly expressed in oral cancer and is a target for cancer therapy and prevention. In this present work, we evaluate the efficacy of photodynamic therapy (PDT) in combination with an EGFR inhibitor, nimotuzumab in oral cancer cell lines and OSCC xenograft tumor model. PDT is a promising and minimally invasive treatment modality that involves the interaction of a photosensitizer, molecular oxygen and light to destroy tumors. We demonstrated that EGFR inhibitors nimotuzumab and cetuximab exhibits anti-angiogenic properties by inhibiting the migration and invasion of oral cancer cell lines and human endothelial cells. The EGFR inhibitors also significantly reduced tube formation of endothelial cells. Chlorin e6-PDT in combination with nimotuzumab and cetuximab reduced cell proliferation in different oral cancer and endothelial cells. Furthermore, our in vivo studies showed that the combination therapy of PDT and nimotuzumab synergistically delayed tumor growth when compared with control and PDT treated tumors. Downregulation of EGFR, Ki-67 and CD31 was observed in the tumors treated with combination therapy. Analysis of the liver and kidney function markers showed no treatment related toxicity. In conclusion, PDT outcome of oral cancer can be improved when combined with EGFR inhibitor nimotuzumab.

EGFR/ERBB receptors differentially modulate sebaceous lipogenesis

The roles of the epidermal growth factor receptor (EGFR) in sebaceous glands remain poorly explored. We show that human sebocytes express EGFR and lower levels of ERBB2 and ERBB3, all receptors being downregulated after the induction of lipid synthesis. Nile red staining showed that siRNA-mediated downregulation of EGFR or ERBB3 increases lipid accumulation, whereas ERBB2 downregulation has no effect. Spectrometry confirmed induction of triglycerides after EGFR or ERBB3 downregulation and revealed induction of cholesteryl esters after downregulation of EGFR, ERBB2 or ERBB3. Thus, EGFR/ERBB receptors differentially modulate sebaceous lipogenesis, a key feature of sebaceous gland physiology and of several skin diseases.

Berberine induces senescence of human glioblastoma cells by downregulating the EGFR-MEK-ERK signaling pathway.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor and has a poor prognosis. We, here, report a potent antitumor effect of berberine, an isoquinoline alkaloid, on GBM. Berberine was found to have an IC50 that is much lower than temozolomide in vitro in U87, U251, and U118 glioblastoma cells. Although previous studies showed that berberine primarily exerts its anticancer effect by inducing cell-cycle arrest, apoptosis, and autophagy, we observed that the antitumor effect of berberine on glioblastoma cells was primarily achieved through induction of cellular senescence. In glioblastoma cells treated with berberine, the level of epidermal growth factor receptor (EGFR) was greatly reduced. Examination of the activities of the kinases downstream of EGFR revealed that the RAF-MEK-ERK signaling pathway was remarkably inhibited, whereas AKT phosphorylation was not altered. Pharmacologic inhibition or RNA interference of EGFR similarly induced cellular senescence of glioblastoma cells. Furthermore, the cellular senescence induced by berberine could be rescued by introduction of a constitutive active MKK. Berberine also potently inhibited the growth of tumor xenografts, which was accompanied by downregulation of EGFR and induction of senescence. Our findings thus revealed a new route by which berberine exerts its anticancer activity. Because EGFR is commonly upregulated in glioblastoma, the demonstration of effective inhibition of EGFR by berberine points to the possibility of using berberine in the treatment of patients with glioblastoma.

A phase 3 open-label, randomized, multicenter study of Imprime PGG in combination with cetuximab in patients with KRAS wild type metastatic colorectal cancer.

TPS787 Background: Imprime PGG (Imprime) is a novel immune modulator (complex carbohydrate biologic), which harnesses innate immune cells to enhance killing of antibody-targeted tumor cells. In a phase 2 single-arm clinical trial in mCRC, the combination of Imprime with cetuximab resulted in 24% ORR, 62% disease control rate (DCR), and median time to progression (TTP) of 12 wks (Tamayo ME, Ann Onc 2010), representing approximate 100% increases vs historical control (Cunningham, NEJM 2004). ORR was 45%, DCR, 82% and TTP, 24 wks in pts with KRAS WT tumors (post hoc analysis). Single-agent cetuximab has been shown to improve objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) in patients (pts) with epidermal growth factor receptor (EGFR) expressing, KRAS wild-type (WT) metastatic colorectal cancer (mCRC) who failed oxaliplatin- and irinotecan-based therapy or are intolerant to irinotecan. The mechanism of action of cetuximab is thought to rely on competitive blockade of endogenous ligand binding and downstream signaling, internalization and down regulation of EGFR, as well as antibody-dependent cellular cytotoxicity (ADCC) (Erbitux SmPC). The current trial, sponsored by Biothera (registered with ClinicalTrials.gov NCT01309126) is to confirm these findings in phase 3. Methods: Eligible pts will have had prior oxaliplatin- and irinotecan-based therapy or be intolerant to irinotecan, and will meet key inclusion criteria including measurable disease and ECOG performance status of 0 or 1. In a 2:1 randomization, stratified by geographic region, prior chemotherapy and site, approximately 795 pts will receive weekly open-label Imprime plus cetuximab or cetuximab alone. The primary endpoint of the study is OS and primary analysis will occur when ~709 deaths have occurred. Secondary endpoints include PFS, ORR (based on RECIST 1.1), quality of life, safety and pharmacokinetics. Exploratory endpoints include biomarker analyses. Pt screening and enrollment is underway in the United States and Europe. Clinical trial information: NCT01309126.

Plumbagin Ameliorates CCl 4 -Induced Hepatic Fibrosis in Rats via the Epidermal Growth Factor Receptor Signaling Pathway.

Epidermal growth factor (EGF) and its signaling molecules, EGFreceptor (EGFR) and signal transducer and activator of transcription factor 3 (STAT3), have been considered to play a role in liver fibrosis and cirrhosis. Plumbagin (PL) is an extracted component from the plant and has been used to treat different kinds of cancer. However, its role in regulation of EGFR and STAT3 during liver fibrosis has not been investigated. In this study, the effects of PL on the regulation of EGFR and STAT3 were investigated in carbon tetrachloride (CCl4) induced liver fibrosis and hepatic stellate cells (HSC-T6). PL significantly attenuated liver injury and fibrosis in CCl4 treated rats. At concentrations of 2 to 6 μM, PL did not induce significant cytotoxicity of HSC-T6 cells. Moreover, PL reduced phosphorylation of EGFR and STAT3 in both fibrotic liver and heparin-binding EGF-like growth factor (HB-EGF) treated HSC-T6 cells. Furthermore, PL reduced the expression of α-SMA, EGFR, and STAT3 in both fibrotic liver and HB-EGF treated HSC-T6 cells. In conclusion, plumbagin could ameliorate the development of hepatic fibrosis through its downregulation of EGFR and STAT3 in the liver, especially in hepatic stellate cells.

The Antitumorigenic Function of EGFR in Metastatic Breast Cancer is Regulated by Expression of Mig6

Numerous studies by our lab and others demonstrate that epidermal growth factor receptor (EGFR) plays critical roles in primary breast cancer (BC) initiation, growth and dissemination. However, clinical trials targeting EGFR function in BC have lead to disappointing results. In the current study we sought to identify the mechanisms responsible for this disparity by investigating the function of EGFR across the continuum of the metastatic cascade. We previously established that overexpression of EGFR is sufficient for formation of in situ primary tumors by otherwise nontransformed murine mammary gland cells. Induction of epithelial-mesenchymal transition (EMT) is sufficient to drive the metastasis of these EGFR-transformed tumors. Examining growth factor receptor expression across this and other models revealed a potent downregulation of EGFR through metastatic progression. Consistent with diminution of EGFR following EMT and metastasis EGF stimulation changes from a proliferative to an apoptotic response in in situ versus metastatic tumor cells, respectively. Furthermore, overexpression of EGFR in metastatic MDA-MB-231 BC cells promoted their antitumorigenic response to EGF in three dimensional (3D) metastatic outgrowth assays. In line with the paradoxical function of EGFR through EMT and metastasis we demonstrate that the EGFR inhibitory molecule, Mitogen Induced Gene-6 (Mig6), is tumor suppressive in in situ tumor cells. However, Mig6 expression is absolutely required for prevention of apoptosis and ultimate metastasis of MDA-MB-231 cells. Further understanding of the paradoxical function of EGFR between primary and metastatic tumors will be essential for application of its targeted molecular therapies in BC.

A new tumour suppression mechanism by p27Kip1: EGFR down-regulation mediated by JNK/c-Jun pathway inhibition

p27Kip1 is a potent inhibitor of cyclin-dependent kinases that drive G1-to-S cell-cycle transition. Reduced p27Kip1 expression is prevalent in a wide range of human tumours; however, the exact mechanism(s) of p27Kip1-mediated tumour suppression remains obscure. In the present study, we identified a close inverse relationship between p27Kip1 and EGFR (epidermal growth factor receptor) expression: the parental T24 human bladder cancer cells had high p27Kip1 expression but low EGFR expression and, in striking contrast, the metastatic derivative of T24 (T24T) had low p27Kip1 expression but high EGFR expression. This relationship was also found in various human cancer tissues, and was not only just correlative but also causal; depletion of p27Kip1 in MEF (mouse embryonic fibroblast) cells resulted in markedly elevated EGFR expression, a result reproducible with an Egfr promoter-luciferase reporter in both T24 and MEF cells, suggesting transcriptional repression of EGFR by p27Kip1. Indeed, p27Kip1 was found to regulate EGFR expression via the JNK (c-Jun N-terminal kinase)/c-Jun transcription factor: p27Kip1 deficiency activated JNK/c-Jun, whereas inhibition of JNK/c-Jun by dominant-negative mutants dramatically repressed Egfr transcription. Furthermore, the proximal promoter of the Egfr gene was crucial for its transcription, where the recruiting activity of c-Jun was much greater in p27Kip1−/− cells than in p27Kip1+/+ cells. Introduction of GFP–p27Kip1 into T24T cells suppressed JNK/c-Jun activation, EGFR expression and anchorage-independent growth. The results of the present study demonstrate that p27Kip1 suppresses JNK/c-Jun activation and EGFR expression in MEFs and human bladder cancer cells, and the results obtained are consistent with those from human cancer specimens. The present study provides new insights into p27Kip1 suppression of cancer cell growth, migration and metastasis.

MiR-491-5p-induced apoptosis in ovarian carcinoma depends on the direct inhibition of both BCL-XL and EGFR leading to BIM activation.

We sought to identify miRNAs that can efficiently induce apoptosis in ovarian cancer cells by overcoming BCL-XL and MCL1 anti-apoptotic activity, using combined computational and experimental approaches. We found that miR-491-5p efficiently induces apoptosis in IGROV1-R10 cells by directly inhibiting BCL-XL expression and by inducing BIM accumulation in its dephosphorylated form. This latter effect is due to direct targeting of epidermal growth factor receptor (EGFR) by miR-491-5p and consequent inhibition of downstream AKT and MAPK signalling pathways. Induction of apoptosis by miR-491-5p in this cell line is mimicked by a combination of EGFR inhibition together with a BH3-mimetic molecule. In contrast, SKOV3 cells treated with miR-491-5p maintain AKT and MAPK activity, do not induce BIM and do not undergo cell death despite BCL-XL and EGFR downregulation. In this cell line, sensitivity to miR-491-5p is restored by inhibition of both AKT and MAPK signalling pathways. Altogether, this work highlights the potential of miRNA functional studies to decipher cell signalling pathways or major regulatory hubs involved in cell survival to finally propose the rationale design of new strategies on the basis of pharmacological combinations.

Abstract LB-104: Reverting the angiogenic switch of glioblastoma with a nanopolyplex based on the molecular fingerprint of tumor dormancy

Abstract Small,microscopic, avascular and therefore asymptomatic tumors can remain in their dormant stage for a considerable period of time depending on numerous processes. One crucial mechanism underlying the transformation from a dormant to a fast-growing phenotype is the ability of tumor cells to induce angiogenesis, a phenomenon termed “angiogenic switch”. We used the aggressive tumor-forming U-87 MG human glioblastoma cell line to identify and isolate a clone which generates dormant microscopic tumors (1). This clone was isolated using single-cell clone and identified by gene expression signature of dormant tumors (2). While both cell lines share a similar growth rate in vitro, we found profound differences in tumor growth patterns when injected into mice. Furthermore, both cell lines exhibit major differences in their angiogenic potential and in expression patterns of genes involved in angiogenesis regulation. Two of the major dissimilarities were found in thrombospondin-1 (TSP-1) and epidermal growth factor receptor (EGFR) expression levels. The dormant avascular tumor-generating cell line (U-87-D) expresses significantly higher levels of TSP-1 and lower levels of EGFR compared to the fast-growing angiogenic tumor-generating parental cell line (U-87-F). It has been previously demonstrated that TSP-1 is a key endogenous angiogenesis inhibitor, whose expression is lost during the malignant transformation. EGFR is a major modulator of tumorigenicity in glioblastoma and therefore, is considered an attractive potential target for glioblastoma therapy. Following the identification of a tumor dormancy gene signature, we induced the upregulation of TSP-1 signaling, using a TSP-1 peptidomimetic (TSP-1 PM), and the downregulation of EGFR, using a dendritic nanocarrier entrapping siRNA (polyglycerol amine (PG-NH2)-siEGFR). We evaluated the ability of this combination therapy to reverse the fast-growing angiogenic phenotype of U-87-F to the dormant avascular phenotype of U-87-D. Mice bearing established U-87-F tumors (50 mm3) received TSP-1 PM (50 mg/kg/day every day) and PG-NH2-siEGFR (2 mg/kg twice a week) for 14 days. This combination therapy exhibited anti-angiogenic and anti-tumorigenic activity. It remarkably decreased tumor volume by 99.5% compared with the control on day 25 post treatment initiation, to a volume of ∼1 mm3.Immunohistochemistry analysis of TSP-1 PM-treated tumors revealed reduced abnormal vasculature, increased αSMA expression and decreased VEGF expression. We concluded that TSP-1 PM in combination with EGFR-siRNA present a promising treatment for advanced glioblastoma promoting a dormant phenotype.

A Phase 3 Open-Label, Randomized, Multicenter Study of Imprime Pgg in Combination with Cetuximab in Patients with Kras Wild Type Metastatic Colorectal Cancer

ABSTRACT Background: For patients (pts) with epidermal growth factor receptor (EGFR) expressing, KRAS wild-type (WT) metastatic colorectal cancer (mCRC) who failed oxaliplatin- and irinotecan-based therapy or who are intolerant to irinotecan, single-agent cetuximab has been shown to improve objective response rate (ORR), progression-free survival (PFS) and overall survival (OS). The mechanism of action of cetuximab is thought to rely on competitive blockade of endogenous ligand binding and downstream signaling, internalization and downregulation of EGFR, as well as antibody-dependent cellular cytotoxicity (ADCC) (Erbitux SmPC). Imprime PGG (Imprime) is a novel immune modulator (complex carbohydrate biologic), which harnesses innate immune cells to enhance killing of antibody-targeted tumor cells. In a phase 2 single-arm clinical trial in mCRC, the combination of Imprime with cetuximab resulted in 24% ORR, 62% disease control rate (DCR), and median time to progression (TTP) of 12 wks (Tamayo ME, Ann Onc 2010), representing approximate 100% increases vs historical control (Cunningham, NEJM 2004). ORR was 45%, DCR 82% and TTP 24 wks in pts with KRAS WT tumors (post hoc analysis). The current trial, sponsored by Biothera and registered with ClinicalTrials.gov NCT01309126, EudraCT 2010-023562-51, is to confirm these findings in phase 3. Trial design: Approximately 795 pts with previously treated, KRAS WT mCRC will be randomized 2:1 to weekly open-label Imprime plus cetuximab or cetuximab alone, until disease progression. Randomization will be stratified by geographic region, prior chemotherapy and site. The primary endpoint of the study is OS. Secondary endpoints are PFS, ORR (based on RECIST 1.1), quality of life, safety and pharmacokinetics. Exploratory endpoints include biomarker analyses. The primary analysis will occur when ∼709 deaths have occurred. Eligible pts have measurable disease, an ECOG performance status of 0 or 1 and have received prior oxaliplatin- and irinotecan-based therapy or are intolerant to irinotecan. Pts with known hypersensitivity to cetuximab or yeast are excluded. Pt screening and enrollment is underway in Europe and the United States. Disclosure: M.L. Patchen, M. Gargano, M. Grady, J. Lowe, R.D. Huhn and A. Braun: Author is a Biothera employee and has stock options and/or grants.