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Estrogen receptor beta inhibits transcriptional activity of hypoxia inducible factor-1 through the downregulation of arylhydrocarbon receptor nuclear translocator
Wonchung Lim, Yeomyung Park, Jungyoon Cho, Choa Park, Joonwoo Park, Young-Kwon Park, Hyunsung Park, YoungJoo Lee
Breast Cancer Research , 2011, DOI: 10.1186/bcr2854
Abstract: Vascular endothelial growth factor (VEGF) was measured in conditioned medium by enzyme-linked immunosorbent assays. Reverse transcription polymerase chain reaction (RT-PCR), Western blotting, immunoprecipitation, luciferase assays and chromatin immunoprecipitation (ChIP) assays were used to ascertain the implication of ERβ on HIF-1 function.In this study, we found that the inhibition of HIF-1 activity by ERβ expression was correlated with ERβ's ability to degrade aryl hydrocarbon receptor nuclear translocator (ARNT) via ubiquitination processes leading to the reduction of active HIF-1α/ARNT complexes. HIF-1 repression by ERβ was rescued by overexpression of ARNT as examined by hypoxia-responsive element (HRE)-driven luciferase assays. We show further that ERβ attenuated the hypoxic induction of VEGF mRNA by directly decreasing HIF-1α binding to the VEGF gene promoter.These results show that ERβ suppresses HIF-1α-mediated transcription via ARNT down-regulation, which may account for the tumour suppressive function of ERβ.Estrogen plays a key role in the pathogenesis of breast cancer [1]. The cellular response to estrogen is mediated by two estrogen receptor (ER) isoforms, ERα and ERβ [2]. ER is the primary target for chemoprevention and endocrine therapy in breast cancer and provides prognostic and predictive information about tumour response to endocrine treatment [3]. A series of reports strongly indicated that estrogens, via ERα, stimulate proliferation and inhibit apoptosis [4,5], whereas ERβ opposes the proliferative effect of ERα in vitro [6,7]. The alteration of the intracellular ERα/ERβ ratio affects the estrogen-induced cellular response [8]. In addition to its role in modulating ERα-mediated regulation, ERβ also has distinct functions. Expression of ERβ significantly reduced cancer cell proliferation and tumour growth in severe combined immunodeficient mice [9]. ERβ inhibited proliferation of colon cancer cells [10]. It was suggested that the loss of ERβ ex
Differential Impact of EGFR-Targeted Therapies on Hypoxia Responses: Implications for Treatment Sensitivity in Triple-Negative Metastatic Breast Cancer  [PDF]
Abderrahim El Guerrab, Rabah Zegrour, Carine-Christiane Nemlin, Flavie Vigier, Anne Cayre, Frederique Penault-Llorca, Fabrice Rossignol, Yves-Jean Bignon
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0025080
Abstract: Background In solid tumors, such as breast cancer, cells are exposed to hypoxia. Cancer cells adapt their metabolism by activating hypoxia-inducible factors (HIFs) that promote the transcription of genes involved in processes such as cell survival, drug resistance and metastasis. HIF-1 is also induced in an oxygen-independent manner through the activation of epidermal growth factor receptor tyrosine kinase (EGFR-TK). Triple-negative breast cancer (TNBC) is a subtype of invasive breast cancer characterized by negative expression of hormonal and HER2 receptors, and this subtype generally overexpresses EGFR. Sensitivity to three EGFR inhibitors (cetuximab, gefitinib and lapatinib, an HER2/EGFR-TK inhibitor) was evaluated in a metastatic TNBC cell model (MDA-MB-231), and the impact of these drugs on the activity and stability of HIF was assessed. Methodology/Principal Findings MDA-MB-231 cells were genetically modified to stably express an enhanced green fluorescent protein (EGFP) induced by hypoxia; the Ca9-GFP cell model reports HIF activity, whereas GFP-P564 reports HIF stability. The reporter signal was monitored by flow cytometry. HIF-1 DNA-binding activity, cell migration and viability were also evaluated in response to EGFR inhibitors. Cell fluorescence signals strongly increased under hypoxic conditions (> 30-fold). Cetuximab and lapatinib did not affect the signal induced by hypoxia, whereas gefitinib sharply reduced its intensity in both cell models and also diminished HIF-1 alpha levels and HIF-1 DNA-binding activity in MDA-MB-231 cells. This gefitinib feature was associated with its ability to inhibit MDA-MB-231 cell migration and to induce cell mortality in a dose-dependent manner. Cetuximab and lapatinib had no effect on cell migration or cell viability. Conclusion Resistance to cetuximab and lapatinib and sensitivity to gefitinib were associated with their ability to modulate HIF activity and stability. In conclusion, downregulation of HIF-1 through EGFR signaling seems to be required for the induction of a positive response to EGFR-targeted therapies in TNBC.
DOK2 Inhibits EGFR-Mutated Lung Adenocarcinoma  [PDF]
Alice H. Berger, Ming Chen, Alessandro Morotti, Justyna A. Janas, Masaru Niki, Roderick T. Bronson, Barry S. Taylor, Marc Ladanyi, Linda Van Aelst, Katerina Politi, Harold E. Varmus, Pier Paolo Pandolfi
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0079526
Abstract: Somatic mutations in the EGFR proto-oncogene occur in ~15% of human lung adenocarcinomas and the importance of EGFR mutations for the initiation and maintenance of lung cancer is well established from mouse models and cancer therapy trials in human lung cancer patients. Recently, we identified DOK2 as a lung adenocarcinoma tumor suppressor gene. Here we show that genomic loss of DOK2 is associated with EGFR mutations in human lung adenocarcinoma, and we hypothesized that loss of DOK2 might therefore cooperate with EGFR mutations to promote lung tumorigenesis. We tested this hypothesis using genetically engineered mouse models and find that loss of Dok2 in the mouse accelerates lung tumorigenesis initiated by oncogenic EGFR, but not that initiated by mutated Kras. Moreover, we find that DOK2 participates in a negative feedback loop that opposes mutated EGFR; EGFR mutation leads to recruitment of DOK2 to EGFR and DOK2-mediated inhibition of downstream activation of RAS. These data identify DOK2 as a tumor suppressor in EGFR-mutant lung adenocarcinoma.
Timing of Whole Brain Radiotherapy on Survival of Patients with EGFR-mutated ?Non-small Cell Lung Cancer and Brain Metastases  [PDF]
Guimei LIU, Xinyong ZHANG, Cuimeng TIAN, Guangrong XIA, Ping LIU, Quan ZHANG, Xi LI, Hui ZHANG, Na QIN, Jinghui WANG, Shucai ZHANG
- , 2016, DOI: : 10.3779/j.issn.1009-3419.2016.08.03
Abstract: Background and objective There is no high-level evidence for the time of whole brain radiotherapy (WBRT) for patients with epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) and brain metastases. The aim of this study is to assess the appropriate timing of WBRT for patients with EGFR-mutated NSCLC and brain metastases (BM). Methods There were 78 patients diagnosed with EGFR-mutated NSCLC and BM in Beijing Chest Hospital between August 2009 and May 2015. 48 untreated patients who received both WBRT and EGFR-tyrosine kinase inhibitors (TKIs) therapy. Prognostic factors of intracranial progression-free survival (PFS) and overall survival (OS) were identified by Cox proportional hazards modeling. Results Intracranial objective response rate was 81.3% and disease control rate was 93.8%. Median intracranial PFS was 10 months. Median OS was 18 months. Multivariate analysis of intracranial PFS revealed that Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0-1 (HR=30.436, 95%CI: 4.721-196.211, P<0.001) and early WBRT (HR=3.663, 95%CI: 1.657-8.098, P=0.001) had a better intracranial PFS. Multivariate analysis of OS revealed that PS 0-1 (HR=57.607, 95%CI: 6.135-540.953, P<0.001), early WBRT (HR=2.757, 95%CI: 1.140-6.669, P=0.024), and stereotactic radiosurgery (HR=5.964, 95%CI: 1.895-18.767, P=0.002) were independent prognostic factors of OS. Conclusion Early WBRT combined with EGFR-TKIs can improve outcomes of patients with EGFR-mutated NSCLC and BM, but it needs to be confirmed by large-sample-size and multicenter prospective clinical trials.
Mutated KRAS Is an Independent Negative Prognostic Factor for Survival in NSCLC Stage III Disease Treated with High-Dose Radiotherapy  [PDF]
A. Hallqvist,F. Enlund,C. Andersson,H. Sj?gren,A. Hussein,E. Holmberg,J. Nyman
Lung Cancer International , 2012, DOI: 10.1155/2012/587424
Abstract: Background. The main attention regarding prognostic and predictive markers in NSCLC directs towards the EGFR-targeted pathway, where the most studied genetic alterations include EGFR mutations, EGFR copy number, and KRAS mutations. We wanted to explore the prognostic impact of mutated KRAS in the stage III setting treated with high-dose radiochemotherapy. Methods. Samples were obtained from patients participating in two prospective studies of locally advanced NSCLC receiving combined radiochemotherapy: the RAKET study, a randomized phase II study where patients were treated with induction chemotherapy (carboplatin/paclitaxel) followed by concurrent radiochemotherapy, and the Satellite trial, a phase II study with induction chemotherapy (cisplatin/docetaxel) followed by radiotherapy concurrent cetuximab. The samples were analysed regarding KRAS mutations, EGFR mutations, and EGFR FISH positivity. Results. Patients with mutated KRAS had a significantly inferior survival, which maintained its significance in a multivariate analysis when other possible prognostic factors were taken into account. The prevalence of KRAS mutations, EGFR mutations, and EGFR FISH positivity were 28.8%, 7.5%, and 19.7%, respectively. Conclusion. Mutated KRAS is an independent negative prognostic factor for survival in NSCLC stage III disease treated with combined radiochemotherapy. The prevalence of KRAS mutations and EGFR mutations are as expected in this Scandinavian population. 1. Introduction The importance of finding prognostic and predictive markers is an ongoing challenge in oncology. The main attention regarding NSCLC directs towards the epidermal growth factor receptor- (EGFR) targeted pathway, where the most studied genetic alterations include EGFR mutations, EGFR copy number, and KRAS mutations. KRAS mutations have been shown to be associated with worse survival in resected patients [1, 2] and have also been shown to be a negative prognostic factor regarding adjuvant chemotherapy [3], whereas the significance in the stage III (locally advanced) or stage IV (metastatic) setting still is unclear. In this study, we analysed tumour tissue samples regarding KRAS mutations, EGFR mutations, and EGFR positivity by FISH—high polysomy and amplification as defined by Cappuzzo et al. [4]—in patients with NSCLC stage III disease. The study population is represented by patients from two prospective trials who have received combined radiochemotherapy with curative intent: The RAKET study, a randomized three-armed phase II study where patients were treated with two cycles of induction
1, 9-Pyrazoloanthrones Downregulate HIF-1α and Sensitize Cancer Cells to Cetuximab-Mediated Anti-EGFR Therapy  [PDF]
Yang Lu,Xinqun Li,Haiquan Lu,Zhen Fan
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015823
Abstract: Cetuximab, a monoclonal antibody that blocks the epidermal growth factor receptor (EGFR), is currently approved for the treatment of several types of solid tumors. We previously showed that cetuximab can inhibit hypoxia-inducible factor-1 alpha (HIF-1α) protein synthesis by inhibiting the activation of EGFR downstream signaling pathways including Erk, Akt, and mTOR. 1, 9-pyrazoloanthrone (1, 9 PA) is an anthrapyrazolone compound best known as SP600125 that specifically inhibits c-jun N-terminal kinase (JNK). Here, we report 1, 9 PA can downregulate HIF-1α independently of its inhibition of JNK. This downregulatory effect was abolished when the oxygen-dependent domain (ODD) of HIF-1α (HIF-1α-ΔODD, the domain responsible for HIF-1α degradation) was experimentally deleted or when the activity of HIF-1α prolyl hydroxylase (PHD) or the 26S proteasomal complex was inhibited, indicating that the 1, 9 PA downregulates HIF-1α by promoting PHD-dependent HIF-1α degradation. We found that the combination of 1, 9 PA and cetuximab worked synergistically to induce apoptosis in cancer cells in which cetuximab or 1, 9 PA alone had no or only weak apoptotic activity. This synergistic effect was substantially decreased in cancer cells transfected with HIF-1α-ΔODD, indicating that downregulation of HIF-1α was the mechanism of this synergistic effect. More importantly, 1, 9 PA can downregulate HIF-1α in cancer cells that are insensitive to cetuximab-induced inhibition of HIF-1α expression due to overexpression of oncogenic Ras (RasG12V). Our findings suggest that 1, 9 PA is a lead compound of a novel class of drugs that may be used to enhance the response of cancer cells to cetuximab through a complementary effect on the downregulation of HIF-1α.
Gemcitabine Overcomes Erlotinib Resistance in EGFR-Overexpressing Cancer Cells through Downregulation of Akt  [cached]
Chandra Bartholomeusz, Fumiyuki Yamasaki, Hitomi Saso, Kaoru Kurisu, Gabriel N. Hortobagyi, Naoto T. Ueno
Journal of Cancer , 2011,
Abstract: A phase III clinical trial showed gemcitabine chemotherapy combined with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib significantly improved overall survival in patients with advanced pancreatic cancer. Therefore, we studied whether addition of gemcitabine to erlotinib in cancer cells having intrinsic or acquired erlotinib resistance could restore chemosensitization in these cells. We studied the synergistic effect of erlotinib and gemcitabine in EGFR-overexpressing A-431 cells with acquired erlotinib resistance and in intrinsic erlotinib-resistant triple negative breast cancer (TNBC) BT-549, MDA-MB-231 and MDA-MB-468 cell lines. Erlotinib and gemcitabine were synergistic in both parental intrinsically erlotinib-sensitive A-431 cells (combination index = 0.69 at the effective dose [ED50]) and in two A-431 cell pools that had acquired erlotinib resistance (combination indices = 0.63 and 0.49 at ED50). The synergistic effect of erlotinib and gemcitabine on cancer cells did not require sensitivity to erlotinib provided that erlotinib can inhibit EGFR. The restoration of sensitivity by gemcitabine occurred through downregulation of phosphorylated Akt (p-Akt), which suggests that PI3K-PTEN-Akt activity is important to the synergism between the two agents. In A-431 parental cells, treatment with gemcitabine followed by erlotinib - but not the reverse sequence - was superior to erlotinib alone. The importance of the order of administration maybe due to the downregulation of p-Akt by gemcitabine in a dose- and time-dependent manner in cells with intrinsic or acquired erlotinib resistance. Our data show that gemcitabine increased the cytotoxic effect of erlotinib by downregulating p-Akt in EGFR-overexpressing cells with either intrinsic or acquired erlotinib resistance.
Genome wide SNP comparative analysis between EGFR and KRAS mutated NSCLC and characterization of two models of oncogenic cooperation in non-small cell lung carcinoma
Hélène Blons, Karine Pallier, Delphine Le Corre, Claire Danel, Maxime Tremblay-Gravel, Claude Houdayer, Elizabeth Fabre-Guillevin, Marc Riquet, Philippe Dessen, Pierre Laurent-Puig
BMC Medical Genomics , 2008, DOI: 10.1186/1755-8794-1-25
Abstract: Patterns of alterations were different between EGFR and KRAS mutated tumors and specific chromosomes alterations were linked to the EGFR mutated group. Indeed chromosome regions 14q21.3 (p = 0.027), 7p21.3-p21.2 (p = 0.032), 7p21.3 (p = 0.042) and 7p21.2-7p15.3 (p = 0.043) were found significantly amplified in EGFR mutated tumors. Within those regions 3 genes are of special interest ITGB8, HDAC9 and TWIST1. Moreover, homozygous deletions at CDKN2A and LOH at RB1 were identified in EGFR mutated tumors. We therefore tested the existence of a link between EGFR mutation, CDKN2A homozygous deletion and cyclin amplification in a larger series of tumors. Indeed, in a series of non-small-cell lung carcinoma (n = 98) we showed that homozygous deletions at CDKN2A were linked to EGFR mutations and absence of smoking whereas cyclin amplifications (CCNE1 and CCND1) were associated to TP53 mutations and smoking habit.All together, our results show that genome wide patterns of alteration differ between EGFR and KRAS mutated lung ADC, describe two models of oncogenic cooperation involving either EGFR mutation and CDKN2A deletion or cyclin amplification and TP53 inactivating mutations and identified new chromosome regions at 7p and 14q associated to EGFR mutations in lung cancer.Lung cancer is the leading cause of cancer-related deaths in the western world [1]. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of the cases and represents a heterogeneous group mainly consisting of adenocarcinoma (ADC), large cell carcinoma (LCC) and squamous cell carcinoma (SCC). The incidence of subtypes has changed in the last decades with increasing incidence of ADC. Moreover, while smoking remains the major risk factor for lung cancer, a subgroup of patients develop lung ADC without smoking history. It is not clear whether lung cancer in non-smokers is increasing in western countries but it is obvious that it has particular clinical and biological features. Population based studie
Expression of DDX3 Is Directly Modulated by Hypoxia Inducible Factor-1 Alpha in Breast Epithelial Cells  [PDF]
Mahendran Botlagunta,Balaji Krishnamachary,Farhad Vesuna,Paul T. Winnard Jr.,Guus M. Bol,Arvind H. Patel,Venu Raman
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0017563
Abstract: DEAD box protein, DDX3, is aberrantly expressed in breast cancer cells ranging from weakly invasive to aggressive phenotypes and functions as an important regulator of cancer cell growth and survival. Here, we demonstrate that hypoxia inducible factor-1α is a transcriptional activator of DDX3 in breast cancer cells. Within the promoter region of the human DDX3 gene, we identified three putative hypoxia inducible factor-1 responsive elements. By luciferase reporter assays in combination with mutated hypoxia inducible factor-1 responsive elements, we determined that the hypoxia inducible factor-1 responsive element at position -153 relative to the translation start site is essential for transcriptional activation of DDX3 under hypoxic conditions. We also demonstrated that hypoxia inducible factor-1 binds to the DDX3 promoter and that the binding is specific, as revealed by siRNA against hypoxia inducible factor-1 and chromatin immunoprecipitation assays. Thus, the activation of DDX3 expression during hypoxia is due to the direct binding of hypoxia inducible factor-1 to hypoxia responsive elements in the DDX3 promoter. In addition, we observed a significant overlap in the protein expression pattern of hypoxia inducible factor-1α and DDX3 in MDA-MB-231 xenograft tumors. Taken together, our results demonstrate, for the first time, the role of DDX3 as a hypoxia-inducible gene that exhibits enhanced expression through the interaction of hypoxia inducible factor-1 with hypoxia inducible factor-1 responsive elements in its promoter region.
Cortactin Is a Substrate of Activated Cdc42-Associated Kinase 1 (ACK1) during Ligand-induced Epidermal Growth Factor Receptor Downregulation  [PDF]
Laura C. Kelley, Scott A. Weed
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0044363
Abstract: Background Epidermal growth factor receptor (EGFR) internalization following ligand binding controls EGFR downstream pathway signaling activity. Internalized EGFR is poly-ubiquitinated by Cbl to promote lysosome-mediated degradation and signal downregulation. ACK1 is a non-receptor tyrosine kinase that interacts with ubiquitinated EGFR to facilitate EGFR degradation. Dynamic reorganization of the cortical actin cytoskeleton controlled by the actin related protein (Arp)2/3 complex is important in regulating EGFR endocytosis and vesicle trafficking. How ACK1-mediated EGFR internalization cooperates with Arp2/3-based actin dynamics during EGFR downregulation is unclear. Methodology/Principal Findings Here we show that ACK1 directly binds and phosphorylates the Arp2/3 regulatory protein cortactin, potentially providing a direct link to Arp2/3-based actin dynamics during EGFR degradation. Co-immunoprecipitation analysis indicates that the cortactin SH3 domain is responsible for binding to ACK1. In vitro kinase assays demonstrate that ACK1 phosphorylates cortactin on key tyrosine residues that create docking sites for adaptor proteins responsible for enhancing Arp2/3 nucleation. Analysis with phosphorylation-specific antibodies determined that EGFR-induced cortactin tyrosine phosphorylation is diminished coincident with EGFR degradation, whereas ERK1/2 cortactin phosphorylation utilized in promoting activation of the Arp2/3 regulator N-WASp is sustained during EGFR downregulation. Cortactin and ACK1 localize to internalized vesicles containing EGF bound to EGFR visualized by confocal microscopy. RNA interference and rescue studies indicate that ACK1 and the cortactin SH3 domain are essential for ligand-mediated EGFR internalization. Conclusions/Significance Cortactin is a direct binding partner and novel substrate of ACK1. Tyrosine phosphorylation of cortactin by ACK1 creates an additional means to amplify Arp2/3 dynamics through N-WASp activation, potentially contributing to the overall necessary tensile and/or propulsive forces utilized during EGFR endocytic internalization and trafficking involved in receptor degradation.
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