Amandine Hurbin

Side-effects of a systemic injection of linear polyethylenimine–DNA complexes

Systemic administration of linear polyethylenimine–DNA complexes (L‐PEI/DNA) results in transient expression of the transgene in the lung. This study analyzes the side‐effects associated with L‐PEI‐mediated transfection.

The multiple roles of amphiregulin in human cancer.

Amphiregulin (AREG) is one of the ligands of the epidermal growth factor receptor (EGFR). AREG plays a central role in mammary gland development and branching morphogenesis in organs and is expressed both in physiological and in cancerous tissues. Various studies have highlighted the functional role of AREG in several aspects of tumorigenesis, including self-sufficiency in generating growth signals, limitless replicative potential, tissue invasion and metastasis, angiogenesis, and resistance to apoptosis. The oncogenic activity of AREG has already been described in the most common human epithelial malignancies, such as lung, breast, colorectal, ovary and prostate carcinomas, as well as in some hematological and mesenchymal cancers. Furthermore, AREG is also involved in resistance to several cancer treatments. In this review, we describe the various roles of AREG in oncogenesis and discuss its translational potential, such as the development of anti-AREG treatments, based on AREG activity. In the last decade, independent groups have reported successful but sometimes contradictory results in relation to the potential of AREG to serve as a prognostic and/or predictive marker for oncology, especially with regard to anti-EGFR therapies. Thus, we also discuss the potential usefulness of using AREG as a therapeutic target and validated biomarker for predicting cancer outcomes or treatment efficacy.

Inhibition of Apoptosis by Amphiregulin via an Insulin-like Growth Factor-1 Receptor-dependent Pathway in Non-small Cell Lung Cancer Cell Lines

Several abnormalities in the insulin-like growth factor-1 (IGF1) and erbB receptors pathways stimulate the growth and survival of lung cancer cells, but their mechanisms of action and cooperation are poorly understood. In this report, we have identified a new mechanism of apoptosis inhibition by amphiregulin through an IGF1-dependent survival pathway in non-small cell lung cancer (NSCLC) cells: amphiregulin activates the IGF1 receptor that in turn induces the secretion of amphiregulin and IGF1. In the absence of serum, the NSCLC cell line H358 resists apoptosis and secretes factors protecting the NSCLC cell line H322 from serum deprivation apoptosis. IGF1 receptor inhibitor AG1024 as well as epidermal growth factor receptor inhibitors AG556 and ZD1839 restore apoptosis in H322 cells cultured in H358-conditioned medium. Accordingly, the anti-apoptotic activity of H358-conditioned medium is completely abolished after incubation with anti-amphiregulin neutralizing antibody and only partially with anti-IGF1 neutralizing antibody. H358-conditioned medium and amphiregulin induce IGF1 receptor phosphorylation in H322 cells, which is prevented by anti-amphiregulin neutralizing antibody but not by AG556 or ZD1839. H358 cells secrete a high level of amphiregulin that, in combination with IGF1, prevents serum deprivation apoptosis. Finally, IGF1 receptor inhibitor blocks amphiregulin and IGF1 release by H358 cells.

Caffeine Sensitizes Human H358 Cell Line to p53-mediated Apoptosis by Inducing Mitochondrial Translocation and Conformational Change of BAX Protein

The mechanisms involved in p53-mediated cell death remain controversial. In the present study, we investigated this cell death pathway by stably transfecting the p53-null H358 cell line with a tetracycline-dependent wild type p53-expressing vector. Restoration of p53 triggered a G2/M cell cycle arrest and enhanced BAX protein expression, without inducing apoptosis or potentiating the cytotoxic effect of etoposide, vincristine, and cis-platinum. Accordingly, overexpression of BAX in H358 cells, through stable transfection of a tetracycline-regulated expression vector, did not induce cell death. Interestingly, the methylxanthine caffeine (4 mm) promoted the translocation of BAX from the cytosol to the mitochondria. In the setting of an overexpression of BAX, caffeine induced a conformational change of the protein and apoptosis. The consequences of caffeine were independent of its cell cycle-related activities. All together, caffeine synergizes with p53 for inducing cell death through a cell cycle-independent mechanism, involving mitochondrial translocation and conformational change of BAX protein.

Amphiregulin Promotes BAX Inhibition and Resistance to Gefitinib in Non-small-cell Lung Cancers

Molecular resistance mechanisms affecting the efficiency of receptor tyrosine kinase inhibitors such as gefitinib in non-small-cell lung cancer (NSCLC) cells are not fully understood. Amphiregulin (Areg) overexpression has been proposed to predict NSCLC resistance to gefitinib and we have established that Areg-overexpressing H358 NSCLC cells resist apoptosis. Here, we demonstrate that Areg prevents gefitinib-induced apoptosis in NSCLC cells. We show that H358 cells are resistant to gefitinib in contrast to H322 cells, which do not overexpress Areg. Inhibition of Areg expression by small-interfering RNAs (siRNAs) restores gefitinib sensitivity in H358 cells, whereas addition of recombinant Areg confers resistance in H322 cells. Areg knockdown overcomes resistance to gefitinib and induced apoptosis in NSCLC H358 cells in vitro and in vivo. Under gefitinib treatment, Areg decreases the expression of the proapoptotic protein BAX, inhibits its conformational change and its mitochondrial translocation. Thus, in the presence of Areg, gefitinib-mediated apoptosis is reduced because BAX is sequestered in the cytoplasm. This suggests that treatments using epidermal growth factor receptor (EGFR) inhibitors may be poorly efficient in patients with elevated levels of Areg. These findings indicate the need for inhibition of Areg to enhance the efficiency of the EGFR inhibitors in patients suffering NSCLC.

Inhibition of Apoptosis by Amphiregulin via an Insulin‐like Growth Factor‐1 Receptor—Dependent Pathway in Non‐Small Cell Lung Cancer Cell Lines

Abstract: The reciprocal activation of amphiregulin (AR) and insulin‐like growth factor‐1 (IGF1) pathways has been shown to induce inhibition of serum deprivation apoptosis in non‐small cell lung cancer (NSCLC) cell lines H358 and H322. We demonstrated that AR activated the IGF1 receptor (IGF1‐R), which in turn induced the secretion of AR and IGF1. Transactivation of the IGF1‐R by AR is independent of its binding to EGFR. Thus, AR can inhibit apoptosis in NSCLC cells through an IGF1‐R‐dependent pathway.

Insulin-like growth factor-1 receptor inhibition overcomes gefitinib resistance in mucinous lung adenocarcinoma

The appropriate selection of patients is a major challenge in the treatment of non‐small cell lung cancer (NSCLC) with epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs). Prospective trials in adenocarcinoma demonstrated that the mucinous subtype presents a poorer outcome under EGFR‐TKI treatment than the non‐mucinous subtype. Our aim was to determine the molecular characteristics associated with resistance to EGFR‐TKIs in mucinous and non‐mucinous adenocarcinoma. Eighty adenocarcinoma samples, including 34 tumours from patients treated with gefitinib in a phase II clinical trial (IFCT0401), were classified as mucinous (n = 32) or non‐mucinous (n = 48) adenocarcinoma. We demonstrated that four biological markers were differentially expressed between the two subtypes: mucinous tumours that overexpressed IGF1R (p < 0.0001) and amphiregulin (p = 0.004) with a tendency for more frequent KRAS mutations, in contrast to non‐mucinous tumours that overexpressed EGFR (p < 0.0001) and TTF‐1 (p < 0.0001) with more frequent EGFR mutations (p = 0.037). Higher IGF1R (p = 0.02) and lower TTF‐1 (p = 0.02) expression was associated with disease progression under gefitinib treatment. We observed in vitro cross‐talk between EGFR and IGF1R signalling pathways in gefitinib‐resistant H358 mucinous cells. Anti‐amphiregulin siRNAs and anti‐IGF1R treatments sensitized the H358 cells to gefitinib‐induced apoptosis with additive effects, suggesting that these treatments could overcome the resistance of mucinous tumours to EGFR‐TKIs, including those with KRAS mutation. Our results highlighted that mucinous and non‐mucinous adenocarcinoma subtypes are different entities with different therapeutic responses to EGFR‐TKIs. These data will foster the development of therapeutic strategies for treating adenocarcinoma with mucinous component. Copyright

The PI3K/AKT pathway promotes gefitinib resistance in mutant KRAS lung adenocarcinoma by a deacetylase-dependent mechanism

To select the appropriate patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs), it is important to gain a better understanding of the intracellular pathways leading to EGFR‐TKI resistance, which is a common problem in patients with lung cancer. We recently reported that mutant KRAS adenocarcinoma is resistant to gefitinib as a result of amphiregulin and insulin‐like growth factor‐1 receptor overexpression. This resistance leads to inhibition of Ku70 acetylation, thus enhancing the BAX/Ku70 interaction and preventing apoptosis. Here, we determined the intracellular pathways involved in gefitinib resistance in lung cancers and explored the impact of their inhibition. We analyzed the activation of the phosphatidyl inositol‐3‐kinase (PI3K)/AKT pathway and the mitogen‐activated protein kinase/extracellular‐signal regulated kinase (MAPK/ERK) pathway in lung tumors. The activation of AKT was associated with disease progression in tumors with wild‐type EGFR from patients treated with gefitinib (phase II clinical trial IFCT0401). The administration of IGF1R‐TKI or amphiregulin‐directed shRNA decreased AKT signaling and restored gefitinib sensitivity in mutant KRAS cells. The combination of PI3K/AKT inhibition with gefitinib restored apoptosis via Ku70 downregulation and BAX release from Ku70. Deacetylase inhibitors, which decreased the BAX/Ku70 interaction, inhibited AKT signaling and induced gefitinib‐dependent apoptosis. The PI3K/AKT pathway is thus a major pathway contributing to gefitinib resistance in lung tumors with KRAS mutation, through the regulation of the BAX/Ku70 interaction. This finding suggests that combined treatments could improve the outcomes for this subset of lung cancer patients, who have a poor prognosis.

Targeted delivery of a proapoptotic peptide to tumors in vivo.

RGD peptides recognize the αvβ3 integrin, a receptor that is overexpressed on the surface of both tumor blood vessels and cancerous cells. These peptides are powerful tools that act as single antiangiogenic molecules, but recently also have been used for tumor imaging and drug targeting. We designed the molecule RAFT-(c[-RGDfK-])4, a constrained and chemically defined entity that can be produced at clinical-grade quality. This scaffold was covalently coupled via a labile bridge to the proapoptotic peptide (KLAKLAK)2 (RAFT-RGD-KLA). A fluorescent, activatable probe was also introduced, allowing intracellular localization. At 2.5 µM, this molecule induced the intracellular release of an active KLA peptide, which in turn caused mitochondrial depolarization and cell death in vitro in tumor cells. In a mouse model, the RAFT-RGD-KLA peptide was found to prevent the growth of remote subcutaneous tumors. This study demonstrated that the antitumor peptide is capable of killing tumor cells in an RGD-dependent manner, thus lowering the nonspecific cytotoxic effects expected to occur when using cationic cytotoxic peptides. Thus, this chemistry is suitable for the design of complex, multifunctional molecules that can be used for both imaging and therapeutics, representing the next generation of perfectly controlled, targeted drug-delivery systems.

Amphiregulin Promotes Resistance to Gefitinib in NonSmall Cell Lung Cancer Cells by Regulating Ku70 Acetylation

Multiple molecular resistance mechanisms reduce the efficiency of receptor tyrosine kinase inhibitors such as gefitinib in non-small cell lung cancer (NSCLC). We previously demonstrated that amphiregulin (Areg) inhibits gefitinib-induced apoptosis in NSCLC cells by inactivating the proapoptotic protein BAX. In this part of the investigation, we studied the molecular mechanisms leading to BAX inactivation. We show that Areg prevents gefitinib-mediated acetylation of Ku70. This augments the BAX-Ku70 interaction and therefore prevents BAX-mediated apoptosis. Accordingly, Areg or Ku70 knock down restore BAX activation and apoptosis in gefitinib-treated H358 cells in vitro. In addition, overexpression of the histone acetyltransferase (HAT) CREB-binding protein (CBP) or treatments with histone deacetylase (HDAC) inhibitors sensitize H358 cells to gefitinib. Moreover, a treatment with vorinostat, a HDAC inhibitor strongly sensitized tumors to gefitinib in vivo. These findings suggest new prospects in combining both HDAC and epidermal growth factor receptor inhibitors for the treatment of NSCLC.