Mechanisms of resistance to cetuximab and influence of treatment combinations in HNSCC cell lines

Dans le traitement des cancers des voies aérodigestives supérieures (VADS), une approche biologique par des anti-EGFR (Epidermal Growth Factor Receptor) comme le cetuximab (Erbitux®) a récemment été proposée. Le cetuximab est un anticorps monoclonal chimérique qui se lie spécifiquement au domaine extracellulaire de l’EGFR, régulateur central de la prolifération et de la différenciation dans les cancers. Par cette liaison, le cetuximab entre en compétition avec les ligands du récepteur et empêche son activation, induit son internalisation et bloque la transduction du signal vers les voies de signalisation en aval. Même si cette approche thérapeutique est rationnelle puisque l’EGFR est surexprimé dans la plupart des cancers et notamment dans les cancers des VADS, certains types de cancers présentent une résistance à cet anticorps. Parmi les molécules qui ciblent EGFR il existe également des inhibiteurs de l’activité tyrosine kinase intracellulaire de l’EGFR comme le gefitinib (Iressa®), mais ce dernier n’est actuellement pas prescrit dans le traitement des cancers des VADS.Le but de notre travail a été d’étudier les mécanismes de résistance au cetuximab dans des lignées cellulaires de cancers des VADS puis de proposer des associations thérapeutiques pouvant pallier à cette résistance.Nous avons choisi deux lignées cellulaires de cancers des VADS, CAL33 et SQ20B en comparaison à la lignée épidermoïde A431 sur exprimant EGFR et sensible au cetuximab. Nous avons pu mettre en évidence que CAL33 et SQ20B étaient résistantes au cetuximab mais de manière surprenante sensibles au gefitinib. Nous avons montré que l’absence d’inhibition de phosphorylation d’AKT et qu’une altération de l’internalisation de l’EGFR par le cetuximab étaient responsables en partie de la résistance au cetuximab dans ces modèles cellulaires.Afin de pallier à cette résistance nous avons alors étudié les conséquences biologiques de l’association du cetuximab avec (i) des inhibiteurs de la voie PI3K/AKT par différentes approches et avec (ii) les radiations ionisantes. Dans un premier temps, nous avons étudié l’influence de l’inhibition de la voie AKT par un inhibiteur de PI3K ou un siRNA ciblant AKT. Nous avons démontré que l’inhibition de la phosphorylation d’AKT par l’inhibiteur LY294002 sensibilisait au cetuximab la lignée CAL33 porteuse d’une mutation activatrice du gène PIK3CA codant pour la sous-unité catalytique p110 de la protéine PI3K. Nous avons montré que la persistance de l’activation d’AKT dans la lignée CAL33 prévenait l’effet anti tumoral du cetuximab, tandis que la résistance au cetuximab dans la lignée SQ20B ne semblait pas dépendante de la voie AKT.Une association de traitement du cetuximab avec les radiations ionisantes est déjà proposée en clinique dans le traitement des cancers des VADS. Nous avons donc dans un second temps déterminé les effets de cette association de traitement dans les lignées SQ20B et CAL33 respectivement sauvage et mutée dans la voie de signalisation AKT et dans la lignée contrôle A431. Nous avons montré que l’association du cetuximab aux radiations ionisantes potentialisait l’effet du cetuximab sur l’inhibition de prolifération de la lignée A431 alors que nous n’avons observé aucune potentialisation de l’effet du cetuximab sur la prolifération dans les lignées résistantes CAL33 et SQ20B. Dans ce travail, nous montrons que la voie AKT apparaît donc comme un élément central dans la réponse au cetuximab dans la lignée CAL33 et que l’association du cetuximab avec un inhibiteur de la voie PI3K/AKT pourrait être une bonne option thérapeutique dans le traitement des cancers des VADS mutés pour PIK3CA.In the treatment of HNSCC (Head and Neck Squamous Cell Carcinoma), a biological approach by anti-EGFR (Epidermal Growth Factor Receptor) such as cetuximab (Erbitux ®) has been recently proposed. Cetuximab is a chimeric monoclonal antibody that specifically binds the extracellular domain of EGFR, a central regulator of proliferation and differentiation in cancer. For this binding, cetuximab is in competition with EGFR ligands, prevents receptor activation, induces its internalization and blocks the transduction and the downstream signaling pathways. Although this therapeutic approach is rational because EGFR is overexpressed in most of cancers, some cancers are resistant to this antibody. Among the molecules that target EGFR, there are also inhibitors of intracellular tyrosine kinase activity of EGFR such as gefitinib (Iressa ®), but these molecules are not currently used for the treatment of HNSCC cancers. The aim of our study was to characterize resistance mechanisms to cetuximab in a panel of HNSCC cell lines and to propose the treatment combination able to overcome this resistance.We chose two cell lines of HNSCC, CAL33 and SQ20B in comparison with A431 cell line which over express EGFR and which is sensitive to cetuximab. We had shown that CAL33 and SQ20B were resistant to cetuximab but sensitive to gefitinib. This study showed that the absence of inhibition of phosphorylation of AKT by cetuximab and the alteration of the EGFR internalization were responsible in part of the cetuximab resistance in these cell models. Then we studied the biological effects of the combination of cetuximab with (i) inhibition of PI3K/AKT by different approaches and (ii) ionizing radiation.We initially studied the influence of inhibition of AKT pathway by a PI3K inhibitor or siRNA targeting Akt. We demonstrated that inhibition of AKT phosphorylation by LY294002 sensitized CAL33 to cetuximab. This cell line carries an activating mutation of the PIK3CA gene encoding the catalytic subunit p110 of PI3K protein. We have shown that persistent activation of AKT in line CAL33 warned antitumor effect of cetuximab, while resistance to cetuximab in line SQ20B did not seem dependent on the AKT pathway. A combination therapy of cetuximab with ionizing radiation is already proposed in the clinical treatment of these cancers. We therefore determined n the second step, the effects of this combination treatment in lines CAL33 and SQ20B respectively in the wild and mutant AKT signaling pathway and the control line A431. We have shown that the combination of cetuximab to ionizing radiation potentiated the effect of cetuximab on the inhibition of proliferation of line A431, whereas we observed no potentiation of the effect of cetuximab on the proliferation in the resistant lines CAL33 and SQ20B. In this work, we show that the AKT pathway is therefore a central element in the response to cetuximab in line CAL33 and the combination of cetuximab with an inhibitor of the PI3K/AKT pathway might be a good therapeutic option in the treatment of these cancers with a PIK3CA mutation

miRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1

BACKGROUND: Tumor hypoxia is one of the features of tumor microenvironment that contributes to chemoresistance. miRNAs have recently been shown to play important roles in tumorigenesis and drug resistance. Moreover, hypoxia also regulates the expression of a series of miRNAs. However, the interaction between chemoresistance, hypoxia and miRNAs has not been explored yet. The aim of this study is to understand the mechanisms activated/inhibited by miRNAs under hypoxia that induce resistance to chemotherapy-induced apoptosis. METHODS: TaqMan low-density array was used to identify changes in miRNA expression when cells were exposed to etoposide under hypoxia or normoxia. The effects of miR-196b overexpression on apoptosis and cell proliferation were studied in HepG2 cells. miR-196b target mRNAs were identified by proteomic analysis, luciferase activity assay, RT-qPCR and western blot analysis. RESULTS: Results showed that hypoxia down-regulated miR-196b expression that was induced by etoposide. miR-196b overexpression increased the etoposide-induced apoptosis and reversed the protection of cell death observed under hypoxia. By a proteomic approach combined with bioinformatics analyses, we identified IGF2BP1 as a potential target of miR-196b. Indeed, miR-196b overexpression decreased IGF2BP1 RNA expression and protein level. The IGF2BP1 down-regulation by either miR-196b or IGF2BP1 siRNA led to an increase in apoptosis and a decrease in cell viability and proliferation in normal culture conditions. However, IGF2BP1 silencing did not modify the chemoresistance induced by hypoxia, probably because it is not the only target of miR-196b involved in the regulation of apoptosis. CONCLUSIONS: In conclusion, for the first time, we identified IGF2BP1 as a direct and functional target of miR-196b and showed that miR-196b overexpression reverses the chemoresistance induced by hypoxia. These results emphasize that the chemoresistance induced by hypoxia is a complex mechanism. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12943-015-0349-6) contains supplementary material, which is available to authorized users

Correlations with proliferation status of tumours. a.

<p>Box plot corresponding to p53 mRNA expression depending on proliferation status. <b>b.</b> Box plot corresponding to proliferation rate depending on <i>TP53</i> mutation status. <b>c.</b> Box plot corresponding to proliferation rate depending on TP53 mutation status coupled with level of p53 mRNA expression. <b>d.</b> Box plot corresponding to proliferation rate depending on <i>LOH</i> in 17p13. <b>e.</b> Box plot corresponding to p53 mRNA expression depending on <i>TP53</i> mutation status. <b>f.</b> Box plot corresponding to p53 mRNA expression depending on <i>LOH</i> in 17p13. <i>p</i><0.05 was considered significant.</p

Schematic representation of P53 protein.

<p>Its 3 domains and corresponding coding exons are represented. Mutations of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055103#pone-0055103-t002" target="_blank">Table 2</a> appear at the top of coding exons.</p

Detail of observed <i>TP53</i> gene mutations.

<p>Detail of observed <i>TP53</i> gene mutations.</p

Clinical details of studied population.

<p>NA: Not Available.</p

Influence of <i>TP53</i> mutations on outcomes. a.

<p>Kaplan-Meier curves of disease-free (left) and overall (right) survival in patients categorized on different categories of p53 mRNA expression. <b>b.</b> Kaplan-Meier curves of disease-free (left) and overall (right) survival in patients categorized on different categories of LOH in 17p13. <b>c.</b> Kaplan-Meier curves of disease-free (left) and overall (right) survival in patients categorized on <i>TP53</i> gene mutation status. Dotted red lines correspond to median disease-free or overall survival. <b>d.</b> Kaplan-Meier curves of disease-free (left) and overall (right) survival in patients categorized on different kinds and location of <i>TP53</i> mutation. Dotted red lines correspond to median disease-free or overall survival. <i>p</i>-values were calculated in relation to wild-type population (green: <i>vs.</i> missense mutation in the 2^nd domain, blue: <i>vs.</i> missense mutation in the 3^rd domain; red: <i>vs.</i> mutation inducing a loss of the 2^nd and 3^rd domains). Censored patients are represented on the curves by black crosses. Number at risk are presented below graphs. Only <i>p</i><0.05 was considered significant.</p

Mechanisms underlying resistance to cetuximab in the HNSCC cell line: role of AKT inhibition in bypassing this resistance.

EGFR is frequently overexpressed in head and neck squamous cell cancer (HNSCC). Cetuximab is a monoclonal antibody designed to interact with EGFR, block its activation, reduce the downstream signaling pathways and induce EGFR internalization. This study aims to investigate the role of the EGFR signaling pathway and EGFR internalization in a cetuximab-resistant cell line and to propose a new therapeutic strategy to optimize treatment of HNSCC. The HNSCC cell line, CAL33 was sensitive to gefitinib but resistant to cetuximab. Cetuximab induces an unexpected EGFR phosphorylation in CAL33 cells similarly to EGF but this EGFR activation does not trigger EGFR internalization/degradation, the process currently implicated in the response to cetuximab. Cetuximab inhibits ERK and AKT phosphorylation in cetuximab-sensitive A431 cells, whereas the level of AKT phosphorylation is unmodified in cetuximab-resistant cells. Interestingly, CAL33 cells harbor a PIK3CA mutation. The treatment of CAL33 cells with PI3K inhibitor and cetuximab restores the inhibition of AKT phosphorylation and induces growth inhibition. Our results indicate that EGFR internalization is impaired by cetuximab treatment in CAL33 cells and that the AKT pathway is a central element in cetuximab resistance. The combination of cetuximab with a PI3K inhibitor could be a good therapeutic option in PIK3CA-mutated HNSCC