Differential Impact of EGFR-Targeted Therapies on Hypoxia Responses: Implications for Treatment Sensitivity in Triple-Negative Metastatic Breast Cancer

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.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.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

Comparison of various grafting chemistries for the development of antibody-based biochips for planetary exploration

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Comparison of various grafting chemistries for the development of antibody-based biochips for planetary exploration

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Presentation of the Biochip experiment on the EXPOSE-R2 facility aboard the International Space Station

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Presentation of the Biochip experiment on the EXPOSE-R2 facility aboard the International Space Station

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Photochemistry on the Space Station—Antibody Resistance to Space Conditions after Exposure Outside the International Space Station

Antibody-based analytical instruments are under development to detect signatures of life on planetary bodies.Antibodies are molecular recognition reagents able to detect their target at sub-nanomolar concentrations, withhigh affinity and specificity. Studying antibody binding performances under space conditions is mandatory toconvince space agencies of the adequacy of this promising tool for planetary exploration.To complement previous ground-based experiments on antibody resistance to simulated irradiation, weevaluate in this paper the effects of antibody exposure to real space conditions during the EXPOSE-R2 missionoutside the International Space Station. The absorbed dose of ionizing radiation recorded during the 588 days ofthis mission (220 mGy) corresponded to the absorbed dose expected during a mission to Mars. Moreover,samples faced, at the same time as irradiation, thermal cycles, launch constraints, and long-term storage. Amodel biochip was used in this study with antibodies in freeze-dried form and under two formats: free orcovalently grafted to a solid surface.We found that antibody-binding performances were not significantly affected by cosmic radiation, and morethan 40% of the exposed antibody, independent of its format, was still functional during all this experiment. Weconclude that antibody-based instruments are well suited for in situ analysis on planetary bodies. Key Words:Astrobiology—Cosmic rays—Biochip—Antibody—Planetary exploration. Astrobiology 19, 1053–1062

Validation of the GFP-P564 and Ca9-GFP hypoxia-inducible reporters.

<p>Hypoxic stimuli induced HIF-1 alpha accumulation in MDA-MB-231 cells and increased fluorescence in the GFP-P564 and Ca9-GFP cell models. Cells were treated with 200 µM cobalt (Co), 200 µM DFO, or 1 mM dimethyloxalylglycine (DMOG) or exposed to 1% O₂ for 24 H (hx). Untreated cells were incubated under normoxic conditions (nx) and used as controls. (A) Fluorescence was evaluated by flow cytometry, and the signal significantly increased (p<0.05) under hypoxic conditions. DFO induced (B) time- and (C) dose-dependent fluorescence, as assessed by flow cytometry in GFP-P564 and Ca9-GFP cells. (D) Total HIF-1 alpha was quantified in whole MDA-MB-231 cells using an ELISA assay. (E) HIF-1 alpha protein levels in MDA-MB-231 cells were analyzed by western blot. (F) HIF-1 alpha DNA-binding activity was analyzed in MDA-MB-231 nuclear extracts using a 96-well ELISA assay; the absorbance was assessed at 450 nm. Each well was coated with a dsDNA sequence containing the HIF-1 alpha response element. The data represent three independent experiments (average mean ± SEM).</p

Effect of anti-EGFR-targeted therapies on MDA-MB-231 cell migration and viability.

<p>(A) Cells were treated with 75 µg/mL cetuximab (cx), 10 µg/mL lapatinib (lp) or 10 µg/mL gefitinib (gf) and allowed to migrate through inserts (8 µm) under normoxia and hypoxia (1% O2) for 24 H. Cell migration is expressed as the percentage of unmigrated cells. Only gefitinib inhibited MDA-MB-231 cell migration under both conditions. (B) Cells were treated with increasing concentrations of cetuximab (C0:0, C1:25, C2:75, C3:100, C4:150 µg/mL), lapatinib and gefitinib (C0:0, C1:3, C2:10, C3:20, C4:40 µg/mL) under normoxic conditions. Viability was assessed using green calcein-AM labeling and fluorometry (ex: 490/em: 520 nm). Cetuximab and lapatinib did not affect cell viability, whereas gefitinib induced mortality in a dose-dependent manner. (C) A viability assay was performed in a 96-well plate by measuring drug effects under hypoxia in a CMV-GFP cell model that constitutively expressed EGFP. The fluorescence of each well was measured by fluorometry (ex: 488/em: 507 nm), and the signal intensity was proportional to the number of viable cells. Gefitinib strongly induced mortality. The results are representative of at least three independent experiments. Statistical significance was determined by unpaired t-test between treated cells and controls (* p<0.05).</p

Effect of EGFR inhibitors on hypoxia-induced responses in GFP-P564 and Ca9-GFP cells.

<p>Cells were treated with 75 µg/mL cetuximab (cx), 10 µg/mL lapatinib (lp) or 10 µg/mL gefitinib (gf) under hypoxic conditions and stimulated with 200 µM DFO, 5 µM MG132 or 1 mM DMOG or exposed to 1% O₂ (hx) for 24 H. Fluorescence intensity was assessed by flow cytometry. As controls (ct), cells were incubated under normoxic conditions and left untreated. (A) Cetuximab and lapatinib did not affect the induction of fluorescence by DFO, whereas gefitinib suppressed it in both models. (B-C) Gefitinib also inhibited the induction of fluorescence in GFP-P564 and Ca9-GFP cells in the presence of DMOG and under hypoxia. (D) MG132 is a proteasome inhibitor that induces the stabilization of HIF-1 alpha. Gefitinib diminished the MG132-mediated stabilization of HIF-1 alpha. (E) Cetuximab, lapatinib and gefitinib did not affect the constitutive fluorescence of MDA-MB-231 cells transfected with pEGFP-C1 (CMV-GFP model). Data are shown as the mean ± SEM (n = 3). Statistical differences were assessed using unpaired t-tests; p<0.05 was considered to be significant (* p<0.05).</p