Inhibition Of Epidermal Growth Factor Receptor Signaling Research Articles

Inhibition of progression of PanIN through antagonizing EGFR.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely malignant tumor with high lethality in humans. Pancreatic intraepithelial neoplasia (PanIN) is the predominant precancerous lesion for PDAC. Although PanIN is frequently detected in the normal and inflamed pancreas, only a few of PanIN eventually progress into PDAC. Thus, inhibition of PanIN-to-PDAC conversion is critical for preventing the occurrence of PDAC. Here, we evaluated the effect of inhibition of epidermal growth factor receptor (EGFR) signaling on the progression of low-grade PanIN into high-grade PDAC in an established mouse PDAC model (Ptf1a-Cre; K-rasG12D). We found that intraductal infusion of EGFR inhibitors at 12 weeks of age, which induced sustained inhibition of EGFR signaling in the pancreas, significantly decreased the incidence of high-grade PanIN in these mice at 24 weeks of age. Thus, our study suggests that inhibition of EGFR signaling may prevent development of PDAC.

Ginsenoside Rh2 inhibits growth of glioblastoma multiforme through mTor.

Being the most malignant primary brain tumor in humans, glioblastoma multiforme (GBM) has a fairly poor patient survival after current combined treatment with chemotherapy, radiation, and surgery. Ginsenoside Rh2 (GRh2) has been reported to have a therapeutic effect on some tumors, and we recently reported its inhibitory effect on GBM growth in vitro and in vivo, possibly through an epidermal growth factor receptor (EGFR) signaling pathway. Here, using specific inhibitors, we found that the activation of EGFR signaling promoted GBM growth through PI3k/Akt/mTor signaling pathways. Moreover, GRh2 efficiently inhibited activation of this pathway at the receptor level. Together with our previous findings, these data suggest that GRh2 may suppress GBM growth through its competition with EGFR ligands for binding to the EGFR, and binding to EGFR by GRh2 does not lead to receptor phosphorylation. Thus, our data highlight a previous unappreciated role for GRh2 to inhibit EGFR signaling. GRh2 thus appears to be a promising therapy for cancers that require EGFR signaling to growth.

Abstract 3361: EGFR signaling regulates aerobic glycolysis in EGFR-mutated lung adenocarcinoma

Abstract Recent accumulating evidences suggest that genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancerous cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer metabolism has not yet been clearly elucidated. In this study, we measured extracellular lactate production, glucose consumption and the glucose-induced extracellular acidification rate (ECAR) after the inhibition of EGFR signaling in LAD cell culture. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolomic techniques, the major intracellular metabolites in EGFR-mutated LAD cells were quantified in the absence or presence of the tyrosine kinase inhibitor (TKIs) erlotinib. The molecular profile regulating the metabolic enzymes at the transcriptional and post-translational levels was characterized by RT-PCR, Western blotting and flow cytometry. We demonstrated that EGFR signaling was required for lactate production, glucose consumption and glucose-induced ECAR, indicating that EGFR signaling promoted the Warburg effect in LAD cells. Moreover, comprehensive metabolomic analysis revealed that the levels of key intermediate metabolites in glycolysis and the pentose phosphate pathway (PPP) were decreased after treatment with EGFR inhibitors. The glucose transport carried out by GLUT1 and GLUT3 was the most rapid and critically regulated function of glucose metabolism linked to EGFR signaling, although the expression of MYC-dependent glycolytic genes and the phosphorylation of pyruvate kinase muscle (PKM2) were down-regulated by EGFR inhibition at a later time point. A modest down-regulation of both total and membrane-bound GLUT3 was also observed after EGFR inhibition. Despite an equivalent total amount of GLUT1 in LAD cells, the levels of membrane-bound GLUT1 was decreased by EGFR-TKI treatment, suggesting that EGFR signaling controlled GLUT1 function through post-translational modification. We concluded that EGFR signaling regulated aerobic glycolysis in EGFR-mutated LAD cells. Our data suggests that the glycolytic pathway plays a central role in the pathogenesis of EGFR-mutated lung cancer. This regulatory pathway could be an attractive target for the development of targeted therapies to better treat patients with EGFR-mutated LAD. Citation Format: Hideki Makinoshima, Masahiro Takita, Shingo Matsumoto, Atsushi Yagishita, Satoshi Owada, Hiroyasu Esumi, Katsuya Tsuchihara. EGFR signaling regulates aerobic glycolysis in EGFR-mutated lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3361. doi:10.1158/1538-7445.AM2014-3361

MiRNA-34a inhibits EGFR-signaling-dependent MMP7 activation in gastric cancer.

The molecular mechanism underlying cancer invasiveness and metastasis of gastric carcinoma remains elusive. Here, we reported significant decrease in microRNA (miRNA)-34a and significant increase in phosphorylated epidermal growth factor receptor (EGFR) and matrix metalloproteinase-7 (MMP7) in the resected gastric carcinoma from the patients, compared with adjacent normal tissue. Moreover, strong correlation was detected among these three factors. To examine whether a causal link exists, we used two human gastric carcinoma lines, SNU-5 and HGC27, to study the molecular basis of miRNA-34a, EGFR signaling, and MMP7 activation. We found that EGF-induced EGFR phosphorylation in SNU-5 or HGC27 cells activated MMP7 and consequently cancer invasiveness. Both an inhibitor for EGFR and an inhibitor for Akt significantly inhibited the EGF-induced activation of MMP7, suggesting a phosphatidylinositol 3-kinase (PI3K) signaling cascade dependent pathway. Moreover, miRNA-34a levels were not affected by EGF-induced EGFR phosphorylation. However, overexpression of miRNA-34a antagonized EGF-induced MMP7 activation without affecting EGFR phosphorylation in SNU-5 or HGC27 cells. Taken together, our data suggest that miRNA-34 inhibits EGFR signaling via downstream PI3K signaling cascades to regulate MMP7 expression in gastric carcinoma. Thus, miRNA-34a, EGFR, and MMP7 appear to be promising therapeutic targets for preventing the metastasis of gastric carcinoma.

The Moraxella catarrhalis-induced pro-inflammatory immune response is enhanced by the activation of the epidermal growth factor receptor in human pulmonary epithelial cells

BackgroundChronic lower airway inflammation is considered to be a major cause of pathogenesis and disease progression in chronic obstructive pulmonary disease (COPD). Moraxella catarrhalis is a COPD-associated pathogen causing exacerbations and bacterial colonization in the lower airways of patients, which may contribute to chronic inflammation. Increasing evidence suggests that the epidermal growth factor receptor (EGFR) modulates inflammatory processes in the human airways. The goal of this study was to investigate the role of EGFR in the M. catarrhalis-induced pro-inflammatory immune response in airway epithelial cells. MethodsThe effects of inhibition and gene silencing of EGFR on M. catarrhalis-dependent pro-inflammatory cytokine expression in human primary bronchial epithelial cells (NHBEs), as well as the pulmonary epithelial cell lines BEAS-2B and A549 were analyzed. We also assessed the involvement of EGFR-dependent ERK and NF-κB signaling pathways. ResultsThe M. catarrhalis-induced pro-inflammatory immune response depends, at least in part, on the phosphorylation and activation of the EGF receptor. Interaction of M. catarrhalis with EGFR increases the secretion of pro-inflammatory cytokines, which is mediated via ERK and NF-κB activation. ConclusionThe interaction between M. catarrhalis and EGFR increases airway inflammation caused by this pathogen. Our data suggest that the inhibition of EGFR signaling in COPD could be an interesting target for reducing M. catarrhalis-induced airway inflammation.

Epidermal growth factor receptor inhibition attenuates liver fibrosis and development of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most rapidly increasing cause of cancer-related mortality in the United States. Because of the lack of viable treatment options for HCC, prevention in high-risk patients has been proposed as an alternative strategy. The main risk factor for HCC is cirrhosis and several lines of evidence implicate epidermal growth factor (EGF) in the progression of cirrhosis and development of HCC. We therefore examined the effects of the EGF receptor (EGFR) inhibitor erlotinib on liver fibrogenesis and hepatocellular transformation in three different animal models of progressive cirrhosis: a rat model induced by repeated, low-dose injections of diethylnitrosamine (DEN), a mouse model induced by carbon tetrachloride (CCl4 ), and a rat model induced by bile duct ligation (BDL). Erlotinib reduced EGFR phosphorylation in hepatic stellate cells (HSC) and reduced the total number of activated HSC. Erlotinib also decreased hepatocyte proliferation and liver injury. Consistent with all these findings, pharmacological inhibition of EGFR signaling effectively prevented the progression of cirrhosis and regressed fibrosis in some animals. Moreover, by alleviating the underlying liver disease, erlotinib blocked the development of HCC and its therapeutic efficacy could be monitored with a previously reported gene expression signature predictive of HCC risk in human cirrhosis patients. These data suggest that EGFR inhibition using Food and Drug Administration-approved inhibitors provides a promising therapeutic approach for reduction of fibrogenesis and prevention of HCC in high-risk cirrhosis patients who can be identified and monitored by gene expression signatures.

EGFR signaling-dependent inhibition of glioblastoma growth by ginsenoside Rh2

Glioblastoma is the most common and most aggressive malignant primary brain tumor in humans, accounting for 52 % of all functional tissue brain tumor cases and 20 % of all intracranial tumors. The typical treatment involves a combination of chemotherapy, radiation, and surgery, whereas it still achieves fairly poor patient survival. Ginsenoside Rh2 has been reported to have a therapeutic effect on some tumors, but its effect on glioblastoma has not been extensively evaluated. Here, we show that ginsenoside Rh2 can substantially inhibit the growth of glioblastoma in vitro and in vivo in a mouse model. Moreover, the inhibition of the tumor growth appears to result from combined effects on decreased tumor cell proliferation and increased tumor cell apoptosis. Further analyses suggest that ginsenoside Rh2 may have its antiglioblastoma effect through inhibition of the epidermal growth factor receptor (EGFR) signaling pathway in tumor cells. In a lose-of-function experiment, recombinant EGFR was given together with ginsenoside Rh2 to the tumor cells in vitro and in vivo, which completely blocked the antitumor effects of ginsenoside Rh2. Thus, our data not only reveal an anti-glioblastoma effect of ginsenoside Rh2 but also demonstrate that this effect may function via inhibition of EGFR signaling in glioblastoma cells.

Epidermal growth factor receptor inhibitor ameliorates excessive astrogliosis and improves the regeneration microenvironment and functional recovery in adult rats following spinal cord injury

BackgroundAstrogliosis is a common phenomenon after spinal cord injury (SCI). Although this process exerts positive effects on axonal regeneration, excessive astrogliosis imparts negative effects on neuronal repair and recovery. Epidermal growth factor receptor (EGFR) pathway is critical to the regulation of reactive astrogliosis, and therefore is a potential target of therapeutics to better control the response. In this report, we aim to investigate whether blocking EGFR signaling using an EGFR tyrosine kinase specific inhibitor can attenuate reactive astrogliosis and promote functional recovery after a traumatic SCI.MethodThe astrocyte scratch injury model in vitro and the weight-drop SCI model in vivo were used as model systems. PD168393 was used to inhibit EGFR signaling activation. Astrocytic activation and phosphorylated EGFR (pEGFR) were observed after immunofluorescence staining and Western blot analysis. The rate of proliferation was determined by immunofluorescence detection of BrdU-incorporating cells located next to the wound. The levels of TNF-α, iNOS, COX-2 and IL-1β in the culture medium under different conditions were assayed by ELISA. Western blot was performed to semi-quantify the expression of EGFR/pEGFR, glial fibrillary acid protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs). Myelin was stained by Luxol Fast Blue Staining. Cresyl violet eosin staining was performed to analyze the lesion cavity volume and neuronal survival following injury. Finally, functional scoring and residual urine recording were performed to show the rats’ recovery.ResultsEGFR phosphorylation was found to parallel astrocyte activation, and EGFR inhibitor PD168393 potently inhibited scratch-induced reactive astrogliosis and proinflammatory cytokine/mediator secretion of reactive astrocytes in vitro. Moreover, local administration of PD168393 in the injured area suppressed CSPGs production and glial scar formation, and resulted in reduced demyelination and neuronal loss, which correlated with remarkable hindlimb motor function and bladder improvement in SCI rats.ConclusionsThe specific EGFR inhibitor PD168393 can ameliorate excessive reactive astrogliosis and facilitate a more favorable environment for axonal regeneration after SCI. As such, EGFR inhibitor may be a promising therapeutic intervention in CNS injury.

Apigenin and Wogonin Regulate Epidermal Growth Factor Receptor Signaling Pathway Involved in MUC5AC Mucin Gene Expression and Production from Cultured Airway Epithelial Cells.

BackgroundWe investigated whether wogonin and apigenin significantly affect the epidermal growth factor receptor (EGFR) signaling pathway involved in MUC5AC mucin gene expression, and production from cultured airway epithelial cells; this was based on our previous report that apigenin and wogonin suppressed MUC5AC mucin gene expression and production from human airway epithelial cells.MethodsConfluent NCI-H292 cells were pretreated with wogonin or apigenin for 15 minutes or 24 hours and then stimulated with epidermal growth factor (EGF) for 24 hours or the indicated periods.ResultsWe found that incubation of NCI-H292 cells with wogonin or apigenin inhibited the phosphorylation of EGFR. The downstream signals of EGFR such as phosphorylation of MEK1/2 and ERK1/2 were also inhibited by wogonin or apigenin.ConclusionThe results suggest that wogonin and apigenin inhibits EGFR signaling pathway, which may explain how they inhibit MUC5AC mucin gene expression and production induced by EGF.

Intranasal epidermal growth factor treatment rescues neonatal brain injury.

There are no clinically relevant treatments available that improve function in the growing population of very preterm infants (<32 weeks gestation) with neonatal brain injury. Diffuse white matter injury (DWMI) is a common finding in these children and results in chronic neurodevelopmental impairments1,2. As shown recently, failure in oligodendrocyte progenitor cell maturation contributes to DWMI3. In a previous study, we demonstrated that epidermal growth factor receptor (EGFR) plays an important role in oligodendrocyte development4. Here, we examine whether enhanced epidermal growth factor receptor (EGFR) signaling stimulates the endogenous response of EGFR-expressing progenitor cells during a critical period after brain injury, and promotes cellular and behavioral recovery in the developing brain. Using an established model of very preterm brain injury, we demonstrate that selective overexpression of human (h)EGFR in oligodendrocyte lineage cells or the administration of intranasal heparin binding EGF immediately after injury decreases oligodendroglia death, enhances generation of new oligodendrocytes from progenitor cells (OPCs) and promotes functional recovery. Furthermore, these interventions diminish ultrastructural abnormalities and alleviate behavioral deficits on white matter-specific paradigms. Inhibition of EGFR signaling with a molecularly targeted agent used for cancer therapy demonstrates that EGFR activation is an important contributor to oligodendrocyte regeneration and functional recovery after DWMI. Thus, our study provides direct evidence that targeting EGFR in OPCs at a specific time after injury is clinically feasible and applicable for the treatment of premature children with white matter injury.

GPR30 as an initiator of tamoxifen resistance in hormone-dependent breast cancer

IntroductionTamoxifen is widely used to treat hormone-dependent breast cancer, but its therapeutic benefit is limited by the development of drug resistance. Here, we investigated the role of estrogen G-protein coupled receptor 30 (GPR30) on Tamoxifen resistance in breast cancer.MethodsPrimary tumors (PTs) of breast cancer and corresponding metastases (MTs) were used to evaluate the expression of GPR30 and epidermal growth factor receptor (EGFR) immunohistochemically. Tamoxifen-resistant (TAM-R) subclones derived from parent MCF-7 cells were used to investigate the role of GPR30 in the development of tamoxifen resistance, using MTT assay, western blot, RT-PCR, immunofluorescence, ELISA and flow cytometry. TAM-R xenografts were established to assess anti-tumor effects of combination therapy with GPR30 antagonist G15 plus 4-hydroxytamoxifen (Tam), using tumor volume measurement and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).ResultsIn 53 human breast cancer specimens, GPR30 expression in MTs increased compared to matched PTs; in MTs, the expression patterns of GPR30 and EGFR were closely related. Compared to parent MCF-7 cells, TAM-R cells had greater growth responses to 17β-estradiol (E2), GPR30 agonist G1 and Tam, and significantly higher activation of Mitogen-activated protein (MAP) kinases; but this increased activity was abolished by G15 or AG1478. In TAM-R cells, GPR30 cell-surface translocation facilitated crosstalk with EGFR, and reduced cAMP generation, attenuating inhibition of EGFR signaling. Combination therapy both promoted apoptosis in TAM-R cells and decreased drug-resistant tumor progression.ConclusionsLong-term endocrine treatment facilitates the translocation of GPR30 to cell surfaces, which interferes with the EGFR signaling pathway; GPR30 also attenuates the inhibition of MAP kinases. These factors contribute to tamoxifen resistance development in breast cancer. Combination therapy with GPR30 inhibitors and tamoxifen may provide a new therapeutic option for drug-resistant breast cancer.

Differentiation of glioblastoma multiforme stem-like cells leads to downregulation of EGFR and EGFRvIII and decreased tumorigenic and stem-like cell potential

Glioblastoma multiforme (GBM) is the most common and devastating primary brain tumor among adults. Despite recent treatment progress, most patients succumb to their disease within 2 years of diagnosis. Current research has highlighted the importance of a subpopulation of cells, assigned brain cancer stem-like cells (bCSC), to play a pivotal role in GBM malignancy. bCSC are identified by their resemblance to normal neural stem cells (NSC), and it is speculated that the bCSC have to be targeted in order to improve treatment outcome for GBM patients. One hallmark of GBM is aberrant expression and activation of the epidermal growth factor receptor (EGFR) and expression of a deletion variant EGFRvIII. In the normal brain, EGFR is expressed in neurogenic areas where also NSC are located and it has been shown that EGFR is involved in regulation of NSC proliferation, migration, and differentiation. This led us to speculate if EGFR and EGFRvIII are involved in the regulation of bCSC. In this study we use GBM neurosphere cultures, known to preserve bCSC features. We demonstrate that EGFR and EGFRvIII are downregulated upon differentiation and moreover that when EGFR signaling is abrogated, differentiation is induced. Furthermore, we show that differentiation leads to decreased tumorigenic and stem cell-like potential of the neurosphere cultures and that by specifically inhibiting EGFR signaling it is possible to target the bCSC population. Our results suggest that differentiation therapy, possibly along with anti-EGFR treatment would be a feasible treatment option for patients with GBM, by targeting the bCSC population.

Abstract CN07-01: A phase IIa randomized, double-blind trial of erlotinib in inhibiting EGF receptor signaling in aberrant crypt foci of the colon

Abstract Colorectal cancer (CRC) progresses through multiple distinct stages that are potentially amenable to chemopreventative intervention. Epidermal Growth Factor Receptor (EGFR) inhibitors can shrink advanced tumors including CRC. There is significant evidence that EGFR also plays important roles in CRC initiation, and that EGFR inhibitors block tumorigenesis. We therefore performed a double-blind randomized trial (N=45) to test whether the EGFR inhibitor erlotinib (25mg, 50mg and 100mg doses) given for up to 28 days had an acceptable safety and efficacy profile to reduce EGFR signaling biomarkers in colorectal aberrant crypt foci (ACF), a subset of which progress to CRC, and normal rectal tissue. Participants were stratified by NSAID dose. Erlotinib was well tolerated and there were no unanticipated adverse events. Colorectal ACF and normal tissue EGFR signaling changes with erlotinib were modest and there was no clear dose response. However, individuals with high baseline EGFR signaling may potentially constitute a subset that could benefit from this strategy. Overall, our results support the safety of erlotinib in the chemoprevention setting, but efficacy in colorectal EGFR signaling inhibition is likely insufficient to merit further studies without additional pre-screening stratification. Citation Format: Steven Lipkin, Asmita Bhattacharya, Timothy Morgan, Jason Zell, Daniel Gillen, Vanessa Wong, Jinah Chung, Wen Pin Chen, Rachel Gonzalez, Frank Meyskens, Luz Rodriguez, Eva Szabo, Robert Carroll, Richard Benya. A phase IIa randomized, double-blind trial of erlotinib in inhibiting EGF receptor signaling in aberrant crypt foci of the colon. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr CN07-01.

Epidermal growth factor receptor signaling impairs the antiviral activity of interferon-alpha

Interferon-alpha (IFN-α) exhibits its antiviral activity through signal transducer and activator of transcription protein (STAT) signaling and the expression of IFN response genes (IRGs). Viral infection has been shown to result in activation of epidermal growth factor receptor (EGFR)-a host cell entry factor used by several viruses, including hepatitis C virus. However, the effect of EGFR activation for cellular antiviral responses is unknown. Here, we uncover cross-talk between EGFR and IFN-α signaling that has a therapeutic effect on IFN-α-based therapies and functional relevance for viral evasion and IFN resistance. We show that combining IFN-α with the EGFR inhibitor, erlotinib, potentiates the antiviral effect of each compound in a highly synergistic manner. The extent of the synergy correlated with reduced STAT3 phosphorylation in the presence of erlotinib, whereas STAT1 phosphorylation was not affected. Furthermore, reduced STAT3 phosphorylation correlated with enhanced expression of suppressors of cytokine signaling 3 (SOCS3) in the presence of erlotinib and enhanced expression of the IRGs, radical S-adenosyl methionine domain containing 2 and myxovirus resistance protein 1. Moreover, EGFR stimulation reduced STAT1 dimerization, but not phosphorylation, indicating that EGFR cross-talk with IFN signaling acts on the STATs at the level of binding DNA. Our results support a model where inhibition of EGFR signaling impairs STAT3 phosphorylation, leading to enhanced IRG expression and antiviral activity. These data uncover a novel role of EGFR signaling in the antiviral activity of IFN-α and open new avenues of improving the efficacy of IFN-α-based antiviral therapies.

Aberrantly activated AREG–EGFR signaling is required for the growth and survival of CRTC1–MAML2 fusion-positive mucoepidermoid carcinoma cells

Salivary gland tumors (SGT) are a group of highly heterogeneous head and neck malignancies with widely varied clinical outcomes and no standard effective treatments. The CRTC1-MAML2 fusion oncogene, encoded by a recurring chromosomal translocation t(11;19)(q14-21;p12-13), is a frequent genetic alteration found in >50% of mucoepidermoid carcinomas (MEC), the most common malignant SGT. In this study, we aimed to define the role of the CRTC1-MAML2 oncogene in the maintenance of MEC tumor growth and to investigate critical downstream target genes and pathways for therapeutic targeting of MEC. By performing gene expression analyses and functional studies via RNA interference and pharmacological modulation, we determined the importance of the CRTC1-MAML2 fusion gene and its downstream AREG-EGFR signaling in human MEC cancer cell growth and survival in vitro and in vivo using human MEC xenograft models. We found that CRTC1-MAML2 fusion oncogene was required for the growth and survival of fusion-positive human MEC cancer cells in vitro and in vivo. The CRTC1-MAML2 oncoprotein induced the upregulation of the epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) by co-activating the transcription factor CREB, and AREG subsequently activated EGFR signaling in an autocrine manner that promoted MEC cell growth and survival. Importantly, CRTC1-MAML2-positive MEC cells were highly sensitive to EGFR signaling inhibition. Therefore, our study revealed that aberrantly activated AREG-EGFR signaling is required for CRTC1-MAML2-positive MEC cell growth and survival, suggesting that EGFR-targeted therapies will benefit patients with advanced, unresectable CRTC1-MAML2-positive MEC.

The Autophagy Inhibitor Chloroquine Overcomes the Innate Resistance of Wild-Type EGFR Non-Small-Cell Lung Cancer Cells to Erlotinib

The epidermal growth factor receptor (EGFR) inhibitor erlotinib is much less effective in non-small-cell lung cancer (NSCLC) tumors with wild-type EGFR, than in tumors with activating EGFR mutations. Autophagy is a tightly regulated lysosomal self-digestion process, which may alternatively promote cell survival or type II cell death. This study assessed the role of autophagy in erlotinib-mediated cytotoxicity. We used wild-type EGFR erlotinib-sensitive and erlotinib-resistant NSCLC cell lines to determine whether inhibiting autophagy by a therapeutic agent potentiated the antitumor activity of erlotinib in vitro and in vivo. Erlotinib at a clinically relevant concentration (2 μM) induced autophagy in NSCLC cells with wild-type EGFR, and the degree of induction was greater in cells that were resistant than sensitive, suggesting that autophagy is cytoprotective. This was confirmed by knockdown of the autophagy-related gene Atg-5, and by using the autophagy inhibitor chloroquine (CQ), both of which increased the cytotoxicity of erlotinib. The synergistic activity of CQ was not because of the potentiation of erlotinib's effects on autophagy, cell-cycle arrest, and inhibition of both EGFR or downstream signaling of EGFR. Rather, CQ markedly activated apoptosis in the cells. The ability of CQ to potentiate the antitumor activity of erlotinib was also seen in mice bearing NSCLC tumor xenografts. The ability to adapt to anti-EGFR therapy by triggering autophagy may be a key determinant for resistance to erlotinib in wild-type EGFR NSCLC. Inhibition of autophagy by CQ represents a novel strategy to broaden the spectrum of erlotinib efficacy in wild-type EGFR NSCLC tumors.

Nitric oxide donating anilinopyrimidines: Synthesis and biological evaluation as EGFR inhibitors

To search for potent nitric oxide (NO) donating epidermal growth factor receptor (EGFR) inhibitors, a series of phenylsulfonylfuroxan-based anilinopyrimidines 10a–h were synthesized and biologically evaluated. Compounds 10f–h exhibited potent inhibitory activity against EGFR L858R/T790M and were as potent as WZ4002 in inhibition of H1975 cells harboring EGFR L858R/T790M. Additionally, 10h produced high levels of NO in H1975 cells but not in normal human cells, and its antiproliferative activity was diminished by hemoglobin, an NO scavenger. Furthermore, 10h inhibited EGFR activation and downstream signaling in H1975 cells. These results suggest that the strong antiproliferative activity of 10h could be attributed to the synergic effects of high levels of NO production and inhibition of EGFR and downstream signaling in the cancer cells.

De-Repression of PDGFRβ Transcription Promotes Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in Glioblastoma Patients

Acquired resistance to tyrosine kinase inhibitors (TKI) represents a major challenge for personalized cancer therapy. Multiple genetic mechanisms of acquired TKI resistance have been identified in several types of human cancer. However, the possibility that cancer cells may also evade treatment by co-opting physiologically regulated receptors has not been addressed. Here, we show the first example of this alternate mechanism in brain tumors by showing that EGF receptor (EGFR)-mutant glioblastomas (GBMs) evade EGFR TKIs by transcriptionally de-repressing platelet-derived growth factor receptor β (PDGFRβ). Mechanistic studies show that EGFRvIII signaling actively suppresses PDGFRβ transcription in an mTORC1- and extracellular signal-regulated kinase-dependent manner. Genetic or pharmacologic inhibition of oncogenic EGFR renders GBMs dependent on the consequently de-repressed PDGFRβ signaling for growth and survival. Importantly, combined inhibition of EGFR and PDGFRβ signaling potently suppresses tumor growth in vivo. These data identify a novel, nongenetic TKI resistance mechanism in brain tumors and provide compelling rationale for combination therapy. These results provide the fi rst clinical and biologic evidence for receptor tyrosinekinase (RTK) "switching" as a mechanism of resistance to EGFR inhibitors in GBM and provide a molecular explanation of how tumors can become "addicted" to a non amplified, nonmutated, physiologically regulated RTK to evade targeted treatment.

Propagation of Human Prostate Cancer Stem-Like Cells Occurs through EGFR-Mediated ERK Activation

Prostate cancer stem-like cells (PCSCs) are being intensely investigated largely owing to their contributions towards prostate tumorigenesis, however, our understanding of PCSC biology, including their critical pathways, remains incompletely understood. While epidermal growth factor (EGF) is widely used in maintaining PCSC cells in vitro, the importance of EGF-dependent signaling and its downstream pathways in PCSC self-renewal are not well characterized. By investigating DU145 sphere cells, a population of prostate cancer cells with stem-like properties, we report here that epidermal growth factor receptor (EGFR) signaling plays a critical role in the propagation of DU145 PCSCs. Activation of EGFR signaling via addition of EGF and ectopic expression of a constitutively-active EGFR mutant (EGFRvIII) increased sphere formation. Conversely, inhibition of EGFR signaling by using EGFR inhibitors (AG1478 and PD168393) and knockdown of EGFR significantly inhibited PCSC self-renewal. Consistent with the MEK-ERK pathway being a major target of EGFR signaling, activation of the MEK-ERK pathway contributed to EGFR-facilitated PCSC propagation. Modulation of EGFR signaling affected extracellular signal-related kinase (ERK) activation. Inhibition of ERK activation through multiple approaches, including treatment with the MEK inhibitor U0126, ectopic expression of dominant-negative MEK1(K97M), and knockdown of either ERK1 or ERK2 resulted in a robust reduction in PCSC propagation. Collectively, the present study provides evidence that EGFR signaling promotes PCSC self-renewal, in part, by activating the MEK-ERK pathway.

Abstract 530: Inhibition of amphiregulin-mediated EGFR signaling suppresses tumorigenicity in breast cancer cells.

Abstract Approximately half of all basal type breast cancers show evidence of autocrine activation of the epidermal growth factor receptor (EGFR) by amphiregulin (AREG). In addition, AREG expression is associated with aggressive and chemoresistant forms of breast cancer. Studies in our laboratory show that when AREG is the activating ligand, EGFR accumulates at the cell surface, resulting in altered receptor trafficking and downstream signaling. To investigate the effect of AREG-mediated activation of EGFR on the phenotype of AREG-expressing cancers, AREG expression was knocked-down in SUM149 cells, a highly tumorigenic and metastatic cell line isolated from a basal subtype inflammatory breast cancer, which expresses high levels of AREG and overexpresses EGFR. Microarray analysis of AREG knockdown cells revealed a number of relevant pathways affected by AREG-EGFR signaling. These pathways include Notch signaling, MAPK activation, IL-1 signaling and multiple components of the Wnt signaling pathway. In particular, knockdown of AREG or inhibition of EGFR signaling in AREG stimulated cells resulted in the upregulation of DKK-1, an antagonist of canonical Wnt signaling, and the expression of several WNT target genes. Flow cytometric analysis of these cells demonstrated that AREG knockdown resulted in a significant reduction in CD44+ cells while simultaneously increasing the non-stem cell fraction of CD44-/CD24+ cells. Because CD44hi/CD24lo is characteristic of undifferentiated cells that have the potential to initiate tumor formation, the tumorigenicity of these cells was investigated by transplanting parental and AREG knock-down SUM149 cells into the mammary fat pads of NOD-SCID mice. The tumorigenic activity of the AREG knock-down derivatives was significantly reduced compared to the parental SUM149 cells. In particular, the AREG knockdown clone that expressed the lowest level of AREG expression and the highest level of DKK-1 expression failed to form tumors in NOD-SCID mice. To investigate the link between AREG-mediated activation of EGFR, receptor trafficking, and downstream signaling pathways that are responsible for changes in expression of WNT pathway genes, we are investigating differences in EGFR phosphorylation and downstream signaling stimulated by AREG versus EGF. In conjunction with our previous findings that AREG-mediated EGFR signaling results in activation of NF-κβ and the upregulation of IL1α/β, our current results suggest a link between these pathways, WNT signaling, and tumorigenicity in SUM-149 cells. Citation Format: Christiana S. Kappler, Stephen P. Ethier. Inhibition of amphiregulin-mediated EGFR signaling suppresses tumorigenicity in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 530. doi:10.1158/1538-7445.AM2013-530