Reprogramming of endothelial gene expression by tamoxifen inhibits angiogenesis and ERα-negative tumor growth.

Rationale: 17ß-estradiol (E2) can directly promote the growth of ERα-negative cancer cells through activation of endothelial ERα in the tumor microenvironment, thereby increasing a normalized tumor angiogenesis. ERα acts as a transcription factor through its nuclear transcriptional AF-1 and AF-2 transactivation functions, but membrane ERα plays also an important role in endothelium. The present study aims to decipher the respective roles of these two pathways in ERα-negative tumor growth. Moreover, we delineate the actions of tamoxifen, a Selective Estrogen Receptor Modulator (SERM) in ERα-negative tumors growth and angiogenesis, since we recently demonstrated that tamoxifen impacts vasculature functions through complex modulation of ERα activity. Methods: ERα-negative B16K1 cancer cells were grafted into immunocompetent mice mutated for ERα-subfunctions and tumor growths were analyzed in these different models in response to E2 and/or tamoxifen treatment. Furthermore, RNA sequencings were analyzed in endothelial cells in response to these different treatments and validated by RT-qPCR and western blot. Results: We demonstrate that both nuclear and membrane ERα actions are required for the pro-tumoral effects of E2, while tamoxifen totally abrogates the E2-induced in vivo tumor growth, through inhibition of angiogenesis but promotion of vessel normalization. RNA sequencing indicates that tamoxifen inhibits the E2-induced genes, but also initiates a specific transcriptional program that especially regulates angiogenic genes and differentially regulates glycolysis, oxidative phosphorylation and inflammatory responses in endothelial cells. Conclusion: These findings provide evidence that tamoxifen specifically inhibits angiogenesis through a reprogramming of endothelial gene expression via regulation of some transcription factors, that could open new promising strategies to manage cancer therapies affecting the tumor microenvironment of ERα-negative tumors.

Comparison of Estetrol Exposure between Women and Mice to Model Preclinical Experiments and Anticipate Human Treatment.

Estetrol (E4) is a natural estrogen with promising therapeutic applications in humans. The European Medicines Agency and the Food and Drug Administration have approved the use of 15 mg E4/3 mg drospirenone for contraceptive indication. Phase III clinical trials with 15-20 mg E4 for the relief of climacteric complaints are currently running. Relevant data from preclinical animal models are needed to characterize the molecular mechanisms and the pharmacological effects of E4 and possibly to reveal new therapeutic applications and to anticipate potential adverse effects. Therefore, it is important to design experimental procedures in rodents that closely mimic or anticipate human E4 exposure. In this study, we compared the effects of E4 exposure after acute or chronic administration in women and mice. Women who received chronic E4 treatment per os at a dose of 15 mg once daily reached a steady state within 6 to 8 days, with a mean plasma concentration of 3.20 ng/mL. Importantly, with subcutaneous, intraperitoneal or oral administration of E4 in mice, a stable concentration over time that would mimic human pharmacokinetics could not be achieved. The use of osmotic minipumps continuously releasing E4 for several weeks provided an exposure profile mimicking chronic oral administration in women. Measurements of the circulating concentration of E4 in mice revealed that the mouse equivalent dose necessary to mimic human treatment does not fit with the allometric prediction. In conclusion, this study highlights the importance of precise definition of the most appropriate dose and route of administration to utilize when developing predictive preclinical animal models to mimic or anticipate specific human treatment.

Use of liposome-encapsulated estetrol for treatment of neonatal hypoxic-ischemic encephalopathy.

Estetrol (E4) is a natural estrogen synthesized only during pregnancy. It has strong neuroprotective and antioxidative activities. The aim of the present study was to define the neuroprotective potency of E4 encapsulated either in liposome (Lipo-E4) or in drug-in cyclodextrin (HP-ß-CD) in liposome (DCL) system, and compare them with a single use of E4. In vitro studies were performed in an oxidative stress model of primary hippocampal neuronal cell cultures, followed by the lactate dehydrogenase activity and cell proliferation assays. In vivo studies were conducted by using a model of neonatal hypoxic-ischemic encephalopathy in immature rat pups. Brain samples were studied by (immuno)histochemistry for the detection of survived cells, expression of microtubule-associated protein-2, myelin basic protein, doublecortin and vascular-endothelial growth factor. Concentrations of glial fibrillary acidic protein in blood serum were studied by ELISA. In vitro, cell proliferation was significantly up-regulated in cultures treated either by DCL-E4 or E4 compared to the control cells, whereas DCL-E4 treated cells had significantly higher survival rate than the cells treated by E4 alone. Evaluation of brain samples showed that DCL-E4 and a high dose of E4 alone significantly preserve the grey and the white matter loses, and diminish GFAP expression in blood. Although DCL-E4 and E4 have similar effect on neurogenesis in the hippocampus and the cortex, DCL-E4 treatment significantly up-regulates angiogenesis in the hippocampus compared to a single use of E4. Present work reveals for the first time that liposome-encapsulated E4 might be a better alternative to a single use of E4.

Sex-Based Differences in the Tumor Microenvironment.

Cancers are heterogeneous multifactorial diseases consisting of a major public health issue worldwide. Sex disparities are evidenced in cancer incidence, mortality, expression of prognosis factor, response to treatment, and survival. For both sexes, an interplay of intrinsic and environmental factors influences cancer cells and tumor microenvironment (TME) components. The TME cumulates both supportive and communicative functions, contributing to cancer development, progression, and metastasis dissemination. The frontline topics of this chapter are focused on the contribution of sex, via steroid hormones, such as estrogens and androgens, on the following components of the TME: cancer-associated fibroblasts (CAFs), extracellular matrix (ECM), blood and lymphatic endothelial cells, and immunity/inflammatory system.

Estetrol and Mammary Gland: Friends or Foes?

Estrogens have pleiotropic effects on many reproductive and non-reproductive tissues and organs including the mammary gland, uterus, ovaries, vagina, and endothelium. Estrogen receptor α functions as the principal mediator of estrogenic action in most of these tissues. Estetrol (E4) is a native fetal estrogen with selective tissue actions that is currently approved for use as the estrogen component in a combined oral contraceptive and is being developed as a menopause hormone therapy (MHT, also known as hormone replacement therapy). However, exogenous hormonal treatments, in particular MHTs, have been shown to promote the growth of preexisting breast cancers and are associated with a variable risk of breast cancer depending on the treatment modality. Therefore, evaluating the safety of E4-based formulations on the breast forms a crucial part of the clinical development process. This review highlights preclinical and clinical studies that have assessed the effects of E4 and E4-progestogen combinations on the mammary gland and breast cancer, focusing in particular on the estrogenic and anti-estrogenic properties of E4. We discuss the potential advantages of E4 over current available estrogen-formulations as a contraceptive and for the treatment of symptoms due to menopause. We also consider the potential of E4 for the treatment of endocrine-resistant breast cancer.

Estetrol Combined to Progestogen for Menopause or Contraception Indication Is Neutral on Breast Cancer.

Given the unequivocal benefits of menopause hormone therapies (MHT) and combined oral contraceptives (COC), there is a clinical need for new formulations devoid of any risk of breast cancer promotion. Accumulating data from preclinical and clinical studies support that estetrol (E4) is a promising natural estrogen for MHT and COC. Nevertheless, we report here that E4 remains active on the endometrium, even under a dose that is neutral on breast cancer growth and lung metastasis dissemination. This implies that a progestogen should be combined with E4 to protect the endometrium of non-hysterectomized women from hyperplasia and cancer. Through in vivo observations and transcriptomic analyses, our work provides evidence that combining a progestogen to E4 is neutral on breast cancer growth and dissemination, with very limited transcriptional impact. The assessment of breast cancer risk in patients during the development of new MHT or COC is not possible given the requirement of long-term studies in large populations. This translational preclinical research provides new evidence that a therapeutic dose of E4 for MHT or COC, combined with progesterone or drospirenone, may provide a better benefit/risk profile towards breast cancer risk compared to hormonal treatments currently available for patients.

Auditory function and dysfunction: estrogen makes a difference.

Estrogen is the major female hormone involved in reproductive functions, but it also exerts a variety of additional roles in non-reproductive organs. In this review, we highlight the preclinical and clinical studies that have pointed out sex differences and estrogenic influence on audition. We also describe the experimental evidences supporting a protective role of estrogen towards acquired forms of hearing loss. Although a high level of endogenous estrogen is associated with a better hearing function, hormonal treatments at menopause have provided contradictory outcomes. The various factors that are likely to explain these discrepancies include the treatment regimen as well as the hormonal status and responsiveness of the patients. The complexity of estrogen signaling is being untangled and many downstream effectors of its genomic and non-genomic actions have been identified in other systems. Based on these advances and on the common physio-pathological events that underlie age-related, drug or noise-induced hearing loss, we discuss potential mechanisms for their protective actions in the cochlea.

Liposomes and drug-in-cyclodextrin-in-liposomes formulations encapsulating 17ß-estradiol: An innovative drug delivery system that prevents the activation of the membrane-initiated steroid signaling (MISS) of estrogen receptor α.

The encapsulation into liposomes of several types of molecules presents the advantages to protect the activity of these molecules and to target specific tissues. Nevertheless, a major obstacle remains the incomplete understanding of nano-bio interactions. Specifically, the impact that inclusion of drug into liposomes or of drug-in-cyclodextrin-in liposomes (DCL) could have on the molecular and cellular mechanism of drug action is largely unknown. As a proof of concept, we evaluated the impact of 17ß-estradiol (E2) included into liposomes or DCL on estrogen receptor (ER)α signaling pathways. Indeed, ERα relays the pleiotropic actions of E2 in physiology and pathophysiology through two major pathways: (1) the genomic/nuclear effects associated to the transcriptional activity of the ERα and (2) the rapid/nongenomic/membrane-initiated steroid signaling (MISS) effects related to the induction of fast signaling pathways occurring when ERα is anchored to the plasma membrane. We evidenced that the inclusion of E2 into liposomes (Lipo-E2) or into DCL (DCL-E2) prevented the activation of the rapid/nongenomic/extranuclear/MISS pathway of ERα, while the activation of the genomic/nuclear pathway was maintained. These results support that Lipo-E2 and DCL-E2 could be a useful tool to delineate the complex molecular mechanisms associated to ERα. In conclusion, this study supports the notion that inclusion of drugs into liposomes or DCL could modify some specific pathways of their molecular and cellular mechanisms of action. These results emphasized that attention should be paid to nano-bio interactions induced by the use of nanovectors in medicine.

Stromal integrin α11 regulates PDGFR-ß signaling and promotes breast cancer progression.

Cancer-associated fibroblasts (CAFs) are key actors in modulating the progression of many solid tumors such as breast cancer (BC). Herein, we identify an integrin α11/PDGFRß+ CAF subset displaying tumor-promoting features in BC. In the preclinical MMTV-PyMT mouse model, integrin α11-deficiency led to a drastic reduction of tumor progression and metastasis. A clear association between integrin α11 and PDGFRß was found at both transcriptional and histological levels in BC specimens. High stromal integrin α11/PDGFRß expression was associated with high grades and poorer clinical outcome in human BC patients. Functional assays using five CAF subpopulations (one murine, four human) revealed that integrin α11 promotes CAF invasion and CAF-induced tumor cell invasion upon PDGF-BB stimulation. Mechanistically, integrin α11 pro-invasive activity relies on its ability to interact with PDGFRß in a ligand-dependent manner and to promote its downstream JNK activation, leading to the production of tenascin C, a pro-invasive matricellular protein. Pharmacological inhibition of PDGFRß and JNK impaired tumor cell invasion induced by integrin α11-positive CAFs. Collectively, our study uncovers an integrin α11-positive subset of pro-tumoral CAFs that exploits PDGFRß/JNK signalling axis to promote tumor invasiveness in BC.

Ozone-primed neutrophils promote early steps of tumour cell metastasis to lungs by enhancing their NET production.

BACKGROUND: Air pollution, including particulates and gazes such as ozone (O3), is detrimental for patient's health and has repeatedly been correlated to increased morbidity and mortality in industrialised countries. Although studies have described a link between ambient particulate matter and increased lung cancer morbidity, no direct relation has yet been established between O3 exposure and metastatic dissemination to lungs. OBJECTIVES: To outline the mechanisms through which pulmonary O3 exposure modulates metastasis kinetics in an experimental mouse model of O3 exposure. METHODS: Metastatic responses to pulmonary O3 exposure were assessed using a reliable experimental mouse model of concomitant pulmonary O3 exposure and tumour cell injection. Roles of neutrophils in O3-induced lung metastasis were highlighted using blocking anti-Ly6G antibodies; moreover, the implication of neutrophil extracellular traps (NETs) in metastatic processes was evaluated using MRP8cre-Pad4lox/lox mice or by treating mice with DNase I. RESULTS: Pulmonary O3 exposure strongly facilitates the establishment of lung metastasis by (1) Inducing a pulmonary injury and neutrophilic inflammation, (2) Influencing very early steps of metastasis, (3) Priming neutrophils' phenotype to release NETs that favour tumour cell colonisation in lungs. The ability of O3-primed neutrophils to enhance lung colonisation by tumour cells was proven after their adoptive transfer in Balb/c mice unexposed to O3. CONCLUSIONS: Pulmonary neutrophils induced by O3 promote metastatic dissemination to lungs by producing NETs. These findings open new perspectives to improve treatment and prevention strategies in patients affected by metastatic diseases.

Lymph/angiogenesis contributes to sex differences in lung cancer through oestrogen receptor alpha signalling.

Oestrogen signalling pathways are emerging targets for lung cancer therapy. Unravelling the contribution of oestrogens in lung cancer development is a pre-requisite to support the development of sex-based treatments and identify patients who could potentially benefit from anti-oestrogen treatments. In this study, we highlight the contribution of lymphatic and blood endothelia in the sex-dependent modulation of lung cancer. The orthotopic graft of syngeneic lung cancer cells into immunocompetent mice showed that lung tumours grow faster in female mice than in males. Moreover, oestradiol (E2) promoted tumour development, increased lymph/angiogenesis and VEGFA and bFGF levels in lung tumours of females through an oestrogen receptor (ER) alpha-dependent pathway. Furthermore, while treatment with ERb antagonist was inefficient, ERa antagonist (MPP) and tamoxifen decreased lung tumour volumes, altered blood and lymphatic vasculature and reduced VEGFA and bFGF levels in females, but not in males. Finally, the quantification of lymphatic and blood vasculature of lung adenocarcinoma biopsies from patients aged between 35 and 55 years revealed more extensive lymphangiogenesis and angiogenesis in tumour samples issued from women than from men. In conclusion, our findings highlight an E2/ERa-dependent modulation of lymphatic and blood vascular components of lung tumour microenvironment. Our study has potential clinical implication in a personalised medicine perspective by pointing to the importance of oestrogen status or supplementation on lung cancer development that should be considered to adapt therapeutic strategies.

Whole blood microsampling for the quantitation of estetrol without derivatization by liquid chromatography-tandem mass spectrometry.

Quantitative bioanalysis and especially pharmacokinetic studies are challenging since only low volumes of biological material are available and low concentrations (ng/ml) are often expected. In this context, volumetric absorptive microsampling (VAMS) devices were developed to accurately collect 10 or 20µl of whole blood from tested subjects. In this study, we present the development and validation of ultra-high performance liquid chromatography coupled to tandem mass spectrometry method after VAMS sampling for the quantitation of estetrol (E4), a potentially new medicine for hormone replacement, contraception and osteoporosis therapies. Interestingly, a very simple sample preparation procedure was developed without any derivatization step. Even if lack of sensitivity is a common consideration when using negative ionization mode, we demonstrated in this work that an excellent sensitivity could be reached by carefully optimizing the nature and concentration of the mobile phase additive. After the optimization of every experimental parameter, the stability, selectivity, trueness, precision and accuracy of the final method were successfully demonstrated. In addition, the excellent performances of the method were confirmed by two independent proof-of-concept pharmacokinetic studies of E4 after VAMS collection in a murine model.

Accurate Control of 17ß-Estradiol Long-Term Release Increases Reliability and Reproducibility of Preclinical Animal Studies.

Estrogens are the subject of intensive researches aiming to elucidate their mechanism of action on the various tissues they target and especially on mammary gland and breast cancer. The use of ready-to-use slow releasing devices to administer steroids, especially estrogens, to small experimental animals remains the method of choice in terms of animal well-being and of safety for both the researcher and the animal. In this study, we evaluated and compared, in vitro and in vivo, the release kinetic of estradiol (E2) over sixty days from two different slow-releasing systems: the matrix pellet (MP) and the reservoir implant (RI). We compared the impact of these systems in three E2-sensitive mouse models : mammary gland development, human MCF7 adenocarcinoma xenograft and mouse melanoma progression. The real amount of E2 that is released from both types of devices could differ from manufacturer specifications due to inadequate release for MP and initial burst effect for RI. Compared to MP, the interindividual variability was reduced with RI thanks to a superior control of the E2 release. Depending on the dose-dependent sensitivity of the physiological or pathological readout studied, this could lead to an improvement of the statistical power of in vivo experiments and thus to a reduction of the required animal number. Altogether, our data draw attention on the importance to adequately select the slow-releasing device that is the most appropriated to a specific experiment to better fulfill the 3Rs rule (Replacement, Reduction, Refinement) related to animal welfare and protection.

Estrogen receptors and estetrol-dependent neuroprotective actions: a pilot study.

Estetrol (E4) has strong antioxidative, neurogenic and angiogenic effects in neural system resulting in the attenuation of neonatal hypoxic-ischemic encephalopathy. We aimed to define the role of estrogen receptors in E4-dependent actions in neuronal cell cultures and prove the promyelinating effect of E4. In vitro the antioxidative and cell survival/proliferating effects of E4 on H2O2-induced oxidative stress in primary hippocampal cell cultures were studied using different combinations of specific inhibitors for ERα (MPP dihydrochloride), ERß (PHTTP), GPR30 (G15) and palmytoilation (2-BR). LDH activity and cell survival assays were performed. In vivo the promyelinating role of different concentrations of E4 (1 mg/kg/day, 5 mg/kg/day, 10 mg/kg/day, 50 mg/kg/day) was investigated using the hypoxic-ischemic brain damage model in the 7-day-old immature rats before/after the induction of hypoxic-ischemic insult. Myelin basic protein (MBP) immunostaining was performed on brain coronal sections. Our results show that LDH activity is significantly upregulated in cell cultures where the E4's effect was completely blocked by concomitant treatment either with ERα and ERß inhibitors (MPP and PHTPP, respectively), or ERα and ERß inhibitors combined with 2-BR. Cell survival is significantly downregulated in cell cultures where the effect of E4 was blocked by ERß inhibitor (PHTTP) alone. The blockage of GRP30 receptor did affect neither LDH activity nor cell survival. MBP immunostaining is significantly upregulated in E4-pretreated groups at a concentration of 5 mg/kg/day and 50 mg/kg/day E4, whereas the MBP-positive area OD ratio is significantly increased in all the E4-treated groups. E4's antioxidative actions mostly depend on ERα and ERß, whereas neurogenesis and possibly promyelinating activities might be realized through ERß.

Use of estetrol with other steroids for attenuation of neonatal hypoxic-ischemic brain injury: to combine or not to combine?

Estetrol (E4), estradiol (E2) and progesterone (P4) have important antioxidative and neuroprotective effects in neuronal system. We aimed to study the consequence of combined steroid therapy in neonatal hypoxic-ischemic encephalopathy (HIE). In vitro the effect of E4 combined with other steroids on oxidative stress and the cell viability in primary hippocampal cultures was evaluated by lactate dehydrogenase and cell survival assays. In vivo neuroprotective and therapeutic efficacy of E4 combined with other steroids was studied in HIE model of immature rats. The rat pups rectal temperature, body and brain weights were evaluated.The hippocampus and the cortex were investigated by histo/immunohistochemistry: intact cell number counting, expressions of markers for early gray matter lose, neuro- and angiogenesis were studied. Glial fibrillary acidic protein was evaluated by ELISA in blood samples. In vitro E4 and combinations of high doses of E4 with P4 and/or E2 significantly diminished the LDH activity and upregulated the cell survival.In vivopretreatment or treatment by different combinations of E4 with other steroids had unalike effects on body and brain weight, neuro- and angiogenesis, and GFAP expression in blood. The combined use of E4 with other steroids has no benefit over the single use of E4.

Quantitative Assessment of Mouse Mammary Gland Morphology Using Automated Digital Image Processing and TEB Detection.

The assessment of rodent mammary gland morphology is largely used to study the molecular mechanisms driving breast development and to analyze the impact of various endocrine disruptors with putative pathological implications. In this work, we propose a methodology relying on fully automated digital image analysis methods including image processing and quantification of the whole ductal tree and of the terminal end buds as well. It allows to accurately and objectively measure both growth parameters and fine morphological glandular structures. Mammary gland elongation was characterized by 2 parameters: the length and the epithelial area of the ductal tree. Ductal tree fine structures were characterized by: 1) branch end-point density, 2) branching density, and 3) branch length distribution. The proposed methodology was compared with quantification methods classically used in the literature. This procedure can be transposed to several software and thus largely used by scientists studying rodent mammary gland morphology.

Mesenchymal Stem Cells Shed Amphiregulin at the Surface of Lung Carcinoma Cells in a Juxtacrine Manner.

Solid tumors comprise cancer cells and different supportive stromal cells, including mesenchymal stem cells (MSCs), which have recently been shown to enhance tumor growth and metastasis. We provide new mechanistic insights into how bone marrow (BM)-derived MSCs co-injected with Lewis lung carcinoma cells promote tumor growth and metastasis in mice. The proinvasive effect of BM-MSCs exerted on tumor cells relies on an unprecedented juxtacrine action of BM-MSC, leading to the trans-shedding of amphiregulin (AREG) from the tumor cell membrane by tumor necrosis factor-α-converting enzyme carried by the BM-MSC plasma membrane. The released soluble AREG activates cancer cells and promotes their invasiveness. This novel concept is supported by the exploitation of different 2D and 3D culture systems and by pharmacological approaches using a tumor necrosis factor-α-converting enzyme inhibitor and AREG-blocking antibodies. Altogether, we here assign a new function to BM-MSC in tumor progression and establish an uncovered link between AREG and BM-MSC.

Combined estrogenic and anti-estrogenic properties of estetrol on breast cancer may provide a safe therapeutic window for the treatment of menopausal symptoms.

Increased risk of breast cancer is a critical side effect associated with the use of a menopausal hormone therapy (MHT). Estetrol (E4) is a natural estrogen produced by the human fetal liver and is a promising compound for clinical use in MHT. However, its impact on breast cancer is controversial and poorly defined. In this preclinical study, we show that E4 acts as a weak estrogen by stimulating the growth of hormone-dependent breast cancer only at concentrations exceeding menopausal therapeutic needs. E4 presents also an antitumor activity by decreasing the strong proliferative effect of estradiol (E2). While estrogen receptor alpha (ERα) is the predominant receptor mediating its effects, the dual weak-estrogenic/anti-estrogenic feature of E4 results from differential signaling pathways activation. Both nuclear and rapid extra-nuclear signaling pathway are necessary for a complete estrogenic effect of E4. However, the antitumor action of E4 is not due to a capacity to antagonize E2-induced nuclear activity. Altogether, our results highlight that E4 has a limited impact on breast cancer and may offer a safe therapeutic window for the treatment of menopausal symptoms.

Bone marrow-derived mesenchymal stem cells drive lymphangiogenesis.

It is now well accepted that multipotent Bone-Marrow Mesenchymal Stem Cells (BM-MSC) contribute to cancer progression through several mechanisms including angiogenesis. However, their involvement during the lymphangiogenic process is poorly described. Using BM-MSC isolated from mice of two different backgrounds, we demonstrate a paracrine lymphangiogenic action of BM-MSC both in vivo and in vitro. Co-injection of BM-MSC and tumor cells in mice increased the in vivo tumor growth and intratumoral lymphatic vessel density. In addition, BM-MSC or their conditioned medium stimulated the recruitment of lymphatic vessels in vivo in an ear sponge assay, and ex vivo in the lymphatic ring assay (LRA). In vitro, MSC conditioned medium also increased the proliferation rate and the migration of both primary lymphatic endothelial cells (LEC) and an immortalized lymphatic endothelial cell line. Mechanistically, these pro-lymphangiogenic effects relied on the secretion of Vascular Endothelial Growth Factor (VEGF)-A by BM-MSC that activates VEGF Receptor (VEGFR)-2 pathway on LEC. Indeed, the trapping of VEGF-A in MSC conditioned medium by soluble VEGF Receptors (sVEGFR)-1, -2 or the inhibition of VEGFR-2 activity by a specific inhibitor (ZM 323881) both decreased LEC proliferation, migration and the phosphorylation of their main downstream target ERK1/2. This study provides direct unprecedented evidence for a paracrine lymphangiogenic action of BM-MSC via the production of VEGF-A which acts on LEC VEGFR-2.