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MDA-MB-231 Dendra2 tumors followed by intravital microscopy after 8 days of Pyr1 treatment

RPPA results of tubulin acetylation, tubulin detyrosination and cofilin phosphorylation levels in tumors of mice treated with vehicle, Pyr1 and PTX

LIM kinases (LIMK) are emerging targets for cancer therapy, and they function as network hubs to coordinate actin and microtubule dynamics. When LIMKs are inhibited, actin microfilaments are disorganized and microtubules are stabilized. Owing to their stabilizing effect on microtubules, LIMK inhibitors may provide a therapeutic strategy to treat taxane-resistant cancers. In this study, we investigated the effect of LIMK inhibition on breast tumor development and on paclitaxel-resistant tumors, using a novel selective LIMK inhibitor termed Pyr1. Treatment of breast cancer cells, including paclitaxel-resistant cells, blocked their invasion and proliferation in vitro and their growth in vivo in tumor xenograft assays. The tumor-invasive properties of Pyr1 were investigated in vivo by intravital microscopy of tumor xenografts. A striking change of cell morphology was observed with a rounded phenotype arising in a subpopulation of cells, while other cells remained elongated. Notably, although Pyr1 decreased the motility of elongated cells, it increased the motility of rounded cells in the tumor. Pyr1 administration prevented the growth of metastasis but not their spread. Overall, our results provided a preclinical proof of concept concerning how a small-molecule inhibitor of LIMK may offer a strategy to treat taxane-resistant breast tumors and metastases. Cancer Res; 76(12); 3541–52. ©2016 AACR.

Effect of PTX on the viability of breast cell lines.

Pharmacokinetic analysis of Pyr1 and its metabolite M1

Weight and behavior monitoring of mice used in the TS/A-pGL3 xenografts experiment

Supplementary information, i.e. suppplementary table, supplemenatry figures and movies legends and supplementary materiel and methods

Analysis of LIMK1 expression in different breast cancer cell lines.

FRAP analysis of actin dynamics in invadopodia treated with vehicle or Pyr1

Pyr1 effect on tumor cell density

MDA-MB-231 Dendra2 tumors followed by intravital microscopy after 8 days of vehicle treatment

In vivo analysis of the Pyr1 effect on LIMKs activity markers

International audience; Alterations in glycosylation cause the emergence of tumor-associated carbohydrate antigens (TACAs) during tumorigenesis. Truncation of O-glycans reveals the Thomsen nouveau (Tn) antigen, an N-acetylgalactosamine (GalNAc) frequently attached to serine or threonine amino acids, that is accessible on the surface of cancer cells but not on healthy cells. Interestingly, GalNac can be recognized by macrophage galactose lectin (MGL), a type C lectin receptor expressed in immune cells. In this study, recombinant MGL fragments were tested in vitro for their cancer cell-targeting efficiency by flow cytometry and confocal microscopy and in vivo after administration of fluorescent MGL to tumor-bearing mice. Our results demonstrate the ability of MGL to target Tn-positive human tumors without inducing toxicity. This outcome makes MGL, a fragment of a normal human protein, the first vector candidate for in vivo diagnosis and imaging of human tumors and, possibly, for therapeutic applications.

Celine Bouclier and Matthieu Simon contributed equally ; Amandine Hurbin and May C. Morris contributed equally.; International audience; CDK4/cyclin D kinase constitutes an attractive pharmacological target for development of anticancer therapeutics, in particular in KRAS-mutant lung cancer patients, who have a poor prognosis and no targeted therapy available yet. Although several ATP-competitive inhibitors of CDK4 have been developed for anticancer therapeutics, they suffer from limited specificity and efficacy. Methods: As an alternative to ATP-competitive inhibitors we have designed a stapled peptide to target the main interface between CDK4 and cyclin D, and have characterized its physico-chemical properties and affinity to bind cyclin D1. Results: We have validated a positive correlation between CDK4/cyclin D level and KRAS mutation in lung cancer patients. The stapled peptide enters cells rapidly and efficiently, and inhibits CDK4 kinase activity and proliferation in lung cancer cells. Its intrapulmonary administration in mice enables its retention in orthotopic lung tumours and complete inhibition of their growth when co-administered with Abemaciclib. Conclusion: The stapled peptide targeting the main interface between CDK4 and cyclin D provides promising therapeutic perspectives for patients with lung cancer.

International audience; Many Receptor Tyrosine Kinases translocate from the cell surface to the nucleus in normal and pathological conditions, including cancer. Here we report the nuclear expression of insulin-like growth factor-1 receptor (IGF1R) in primary human lung tumours. Using lung cancer cell lines and lung tumour xeno-grafts, we demonstrate that the epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) gefitinib induces the nuclear accumulation of IGF1R in mucinous lung adenocarcinoma by a mechanism involving the intracellular re-localization of the growth factor amphiregulin. Amphiregulin allows the binding of IGF1R to importin-b1 and promotes its nuclear transport. The nuclear accumulation of IGF1R by amphiregulin induces cell cycle arrest through p21 WAF1/CIP1 upregulation, and prevents the induction of apoptosis in response to gefitinib. These results identify amphiregulin as the first nuclear localization signal-containing protein that interacts with IGF1R and allows its nuclear translocation. Furthermore they indicate that nuclear expression of IGF1R contributes to EGFR-TKI resistance in lung cancer.

Advanced ovarian cancer is the most lethal gynecological cancer, with a high rate of chemoresistance and relapse. Photodynamic therapy offers new prospects for ovarian cancer treatment, but current photosensitizers lack tumor specificity, resulting in low efficacy and significant side-effects. In the present work, the clinically approved photosensitizer verteporfin was encapsulated within nanostructured lipid carriers (NLC) for targeted photodynamic therapy of ovarian cancer. Cellular uptake and phototoxicity of free verteporfin and NLC-verteporfin were studied in vitro in human ovarian cancer cell lines cultured in 2D and 3D-spheroids, and biodistribution and photodynamic therapy were evaluated in vivo in mice. Both molecules were internalized in ovarian cancer cells and strongly inhibited tumor cells viability when exposed to laser light only. In vivo biodistribution and pharmacokinetic studies evidenced a long circulation time of NLC associated with efficient tumor uptake. Administration of 2 mg.kg&minus, 1 free verteporfin induced severe phototoxic adverse effects leading to the death of 5 out of 8 mice. In contrast, laser light exposure of tumors after intravenous administration of NLC-verteporfin (8 mg.kg&minus, 1) significantly inhibited tumor growth without visible toxicity. NLC-verteporfin thus led to efficient verteporfin vectorization to the tumor site and protection from side-effects, providing promising therapeutic prospects for photodynamic therapy of cancer.

International audience; Standard chemotherapies that interfere with microtubule dynamics are a chemotherapeutic option used for the patients with advanced malignancies that invariably relapse after targeted therapies. However, major efforts are needed to reduce their toxicity, optimize their efficacy, and reduce cancer chemoresistance to these agents. We previously identified a pyrrolo[2,3d]pyrimidine-based microtubule-depolymerizing agent (PP-13) that binds to the colchicine site of β-tubulin and exhibits anticancer properties in solid human cancer cells, including che-moresistant subtypes. Here, we investigated the therapeutic potential of PP-13 in vitro and in vivo. PP-13 induced a mitotic blockade and apoptosis in several cancer cells cultured in two-dimensions or three-dimensions spheroids, in conjunction with reduced cell proliferation. Capillary-like tube formation assays using HUVECs showed that PP-13 displayed antiangiogenic properties. It also inhibited cancer cell motility and invasion, in in vitro wound-healing and transwell migration assays. Low concentration PP-13 (130 nmol.L −1) treatment significantly reduced the metastatic invasiveness of human cancer cells engrafts on chicken chorioallantoic membrane. In nude mice, 0.5 or 1 mg.kg −1 PP-13 intraperitoneally administered three-times a week reduced the sizes of paclitaxel-refractory orthotopic breast tumors, delayed the progression of metastasis, and decreased the global metastatic load compared to 0.5 mg.kg −1 paclitaxel or vehicle alone. PP-13 did not show any apparent early adverse effect in vivo. These data suggest that PP-13 is a promising alternative to standard chemotherapy in antimitotic drug-refractory tumors, especially through its impact on metastasis.

International audience; KDAC inhibitors (KDACi) overcome gefitinib primary resistance in non-small cell lung cancer (NSCLC) including mutant-KRAS lung adenocarcinoma. To identify which proteins are involved in the restoration of this sensitivity and to provide new therapeutic targets for mutant-KRAS lung adenocarcinoma, we performed an iTRAQ quantitative proteomic analysis after subcellular fractionation of H358-NSCLC treated with gefitinib and KDACi (TSA/NAM) versus gefitinib alone. The 86 proteins found to have been significantly dysregulated between the two conditions, were mainly involved in cellular metabolism and cell transcription processes. As expected, the pathway related to histone modifications was affected by the KDACi. Pathways known for controlling tumor development and (chemo)-resistance (miRNA biogenesis/glutathione metabolism) were affected by the KDACi/gefitinib treatment. Moreover, 57 dysregulated proteins were upstream of apoptosis (such as eEF1A2 and STAT1) and hence provide potential therapeutic targets. The inhibition by siRNA of eEF1A2 expression resulted in a slight decrease in H358-NSCLC viability. In addition, eEF1A2 and STAT1 siRNA transfections suggested that both STAT1 and eEF1A2 prevent AKT phosphorylation known for enhancing gefitinib resistance in NSCLC. Therefore, altogether our data provide new insights into proteome regulations in the context of overcoming the NSCLC resistance to gefitinib through KDACi in H358 KRAS mutated and amphiregulin-overexpressing NSCLC cells.

Resume Objectifs Etudier le role de l’imagerie de fluorescence proche infrarouge dans la detection et la resection des adenopathies cervicales metastatiques des cancers des VADS. Materiel et methodes Des cellules de cancer des VADS d’origine humaine CAL33 ont ete implantees au niveau de la cavite buccale de la souris nue. Les souris ont ete suivies apres la resection tumorale afin de detecter le developpement de metastases ganglionnaires. Un traceur fluorescent specifique a l’integrine αvβ3 , qui est exprime par les cellules CAL33, a ete injecte en intraveineux chez les souris survivantes entre le deuxieme et le quatrieme mois post-explantation tumorale. Une camera d’imagerie de fluorescence proche infrarouge a permis de detecter la captation du traceur fluorescent au sein des adenopathies cervicales metastatiques et de guider le geste d’exerese des adenopathies qui ont ete par la suite analysees histologiquement. Resultats Des adenopathies metastatiques ont ete observees chez 42,8 % des souris survivantes entre le deuxieme et le quatrieme mois post-explantation des tumeurs orthotopiques. L’imagerie de fluorescence proche infrarouge a permis de detecter en temps reel les adenopathies metastatiques cliniques et infracliniques pendant le geste de l’exerese chirurgicale. Ces resultats ont ete confirmes histologiquement. Conclusion L’imagerie de fluorescence proche infrarouge offre un contraste en temps reel entre le tissu sain et le tissu cancereux, ce qui permet de detecter les adenopathies metastatiques en peroperatoire. Cette etape preclinique est indispensable avant de tester cette technique chez l’etre humain.

International audience; Keywords: Near-infrared fluorescence imaging-guided surgery Head and neck squamous cell carcinoma Metastatic adenopathy vˇ3 integrin RAFT-c(RGD)4 a b s t r a c t Objectives: To study the role of near-infrared fluorescence imaging in the detection and resection of metastatic cervical lymph nodes in head and neck cancer. Materials and methods: CAL33 head and neck cancer cells of human origin were implanted in the oral cavity of nude mice. The mice were followed up after tumor resection to detect the development of lymph node metastases. A specific fluorescent tracer for ˛vˇ3 integrin expressed by CAL33 cells was injected intravenously in the surviving mice between the second and the fourth month following tumor resection. A near-infrared fluorescence-imaging camera was used to detect tracer uptake in metastatic cervical lymph nodes, to guide of lymph-node resection for histological analysis. Results: Lymph node metastases were observed in 42.8% of surviving mice between the second and the fourth month following orthotopic tumor resection. Near-infrared fluorescence imaging provided real-time intraoperative detection of clinical and subclinical lymph node metastases. These results were confirmed histologically. Conclusion: Near infrared fluorescence imaging provides real-time contrast between normal and malignant tissue, allowing intraoperative detection of metastatic lymph nodes. This preclinical stage is essential before testing the technique in humans.

Simple Summary Peritoneal carcinomatosis, the formation of wide-spread metastases throughout the abdominal cavity, remains challenging to diagnose and treat. Photodynamic diagnosis and photodynamic therapy are promising approaches for the diagnosis and treatment of peritoneal carcinomatosis, which use photosensitizers for fluorescence detection or photochemical treatment of (micro) metastases. With the aim of highlighting the potential of this theranostic approach, this review outlines the clinical state of the art in the use of photodynamic diagnosis and therapy for peritoneal carcinomatosis, identifies the major challenges, and provides emerging perspectives from preclinical studies to address these challenges. We conclude that the development of novel illumination strategies and targeted photonanomedicines may aid in achieving more efficient cytoreductive surgery. In addition to combination treatments with chemo-, and radiotherapy, such approaches hold significant promise to improve the outlook of patients with peritoneal carcinomatosis. Abstract Peritoneal carcinomatosis occurs frequently in patients with advanced stage gastrointestinal and gynecological cancers. The wide-spread peritoneal micrometastases indicate a poor outlook, as the tumors are difficult to diagnose and challenging to completely eradicate with cytoreductive surgery and chemotherapeutics. Photodynamic diagnosis (PDD) and therapy (PDT), modalities that use photosensitizers for fluorescence detection or photochemical treatment of cancer, are promising theranostic approaches for peritoneal carcinomatosis. This review discusses the leading clinical trials, identifies the major challenges, and presents potential solutions to advance the use of PDD and PDT for the treatment of peritoneal carcinomatosis. While PDD for fluorescence-guided surgery is practically feasible and has achieved clinical success, large randomized trials are required to better evaluate the survival benefits. Although PDT is feasible and combines well with clinically used chemotherapeutics, poor tumor specificity has been associated with severe morbidity. The major challenges for both modalities are to increase the tumor specificity of the photosensitizers, to efficiently treat peritoneal microtumors regardless of their phenotypes, and to improve the ability of the excitation light to reach the cancer tissues. Substantial progress has been achieved in (1) the development of targeted photosensitizers and nanocarriers to improve tumor selectivity, (2) the design of biomodulation strategies to reduce treatment heterogeneity, and (3) the development of novel light application strategies. The use of X-ray-activated PDT during whole abdomen radiotherapy may also be considered to overcome the limited tissue penetration of light. Integrated approaches that take advantage of PDD, cytoreductive surgery, chemotherapies, PDT, and potentially radiotherapy, are likely to achieve the most effective improvement in the management of peritoneal carcinomatosis.

International audience; Combinations of therapeutic agents could synergistically enhance the response of lung cancer cells. Co-delivery systems capable of transporting chemotherapeutics with different physicochemical properties and with the simultaneous release of drugs remain elusive. Here, we assess the ability of nanoparticles of 30-nm diameter obtained from the self-assembly of hyaluronan-based copolymer targeting CD44 receptors to encapsulate both gefitinib and vorinostat for effective combinational lung cancer treatment. Drug loading was performed by nanoprecipitation. Drug release experiments showed a slow release of both drugs after 5 days. Using two- and three-dimensional lung adenocarcinoma cell cultures, we observed that the nanoparticles were mostly found at the periphery of the CD44-expressing spheroids. These drug-loaded nanoparticles were as cytotoxic as free drugs in the two- and three-dimensional systems and toxicity was due to apoptosis induction. In mouse models, intravenous injection of hyaluronan-based nanoparticles showed a selective delivery to subcutaneous CD44-overexpressing tumors, despite a significant liver capture. In addition, the systemic toxicity of the free drugs was reduced by their co-delivery using the nanoparticles. Finally, intrapulmonary administration of drug-loaded nanoparticles, to avoid a possible hepatic toxicity due to their accumulation in the liver, showed a stronger inhibition of orthotopic lung tumor growth compared to free drugs. In conclusion, hyaluronan-based nanoparticles provide active targeting partially mediated by CD44, less-toxic drug release and improved antitumor efficiency.

Background Appropriate animal models are required to test novel therapeutics for head and neck squamous cell carcinoma (HNSCC) such as near-infrared (NIR) imaging-guided surgery. Methods We developed an optimized animal model of orthotopic HNSCC (in female athymic NMRI (Naval Medical Research Institute) nude mice) with a prolonged survival time. Resection of the orthotopic tumors was performed 30 days after implantation with or without the aid of a miniaturized clinical grade NIR optical imaging device, after systemic administration of a fluorescent RGD-based probe that targets αvβ3 integrin. Results NIR optical imaging-guided surgery increased the recurrence-free survival rate by 50% through the detection of fluorescent cancer residues as small as 185 µm; these fragments could remain unidentified if resection was performed exclusively under unaided visual guidance. Conclusion NIR optical imaging-guided surgery showed an improved HNSCC tumor resection quality in our optimized orthotopic animal model. © 2015 Wiley Periodicals, Inc. Head Neck, 2015

Melanoma is a cutaneous cancer with an increasing worldwide prevalence and high mortality due to unresectable or metastatic stages. Mutations inBRAF,NRAS, orKITare present in more than 60% of melanoma cases, but a useful blood-based biomarker for the clinical monitoring of melanoma patients is still lacking. Thus, the analysis of circulating tumor cells (CTCs) and/or cell-free circulating tumor DNA (ctDNA) analysis from blood (liquid biopsies) appears to be a promising noninvasive, repeatable, and systemic sampling tool for detecting and monitoring melanoma. Here, we review the molecular biology-based strategies used for ctDNA quantification in melanoma patients, as well as their main clinical applications. Droplet digital PCR (ddPCR) and next generation sequencing (NGS) technologies appear to be two versatile and complementary strategies to study rare variant mutations for the detection and monitoring of melanoma progression. Among the different clinical uses of ctDNA, we highlight the assessment of molecular heterogeneity and the identification of genetic determinants for targeted therapy as well as the analysis of acquired resistance. Importantly, ctDNA quantification might also be a novel biomarker with a prognostic value for melanoma patients.

International audience; Despite the emergence of targeted therapies and immunotherapy, chemotherapy remains the gold-standard for the treatment of most patients with solid malignancies. Spindle poisons that interfere with microtubule dynamics are commonly used in chemotherapy drug combinations. However, their troublesome side effects and the emergence of chemoresistance highlight the need for identifying alternative agents. We performed a high throughput cell-based screening and selected a pyrrolopyrimidine molecule (named PP-13). In the present study, we evaluated its anticancer properties in vitro and in vivo. We showed that PP-13 exerted cytotoxic effects on various cancer cells, including those resistant to current targeted therapies and chemotherapies. PP-13 induced a transient mitotic blockade by interfering with both mitotic spindle organization and microtubule dynamics and finally led to mitotic slippage, aneuploidy and direct apoptotic death. PP-13 was identified as a microtubule-targeting agent that binds directly to the colchicine site in β-tubulin. Interestingly, PP-13 overcame the multidrug-resistant cancer cell phenotype and significantly reduced tumour growth and metastatic invasiveness without any noticeable toxicity for the chicken embryo in vivo. Overall, PP-13 appears to be a novel synthetic microtubule inhibitor with interesting anticancer properties and could be further investigated as a potent alternative for the management of malignancies including chemoresistant ones. In the last two decades, advances in the understanding of carcinogenesis have revolutionized the management of cancer patients with the development of targeted therapies and immunotherapy. Identification of genetic alterations in subsets of cancers leading to different subcellular signals of tumour growth and progression elicited the clinical use of oncogene-targeted therapies for patients harbouring such anomalies 1-4. In lung cancers with EGFR-activating mutations, anti-EGFR therapies, including gefitinib, erlotinib, and afatinib, have been shown to improve progression-free survival and were approved as first-line options 5. However, despite the initial response, resistance mechanisms almost inevitably ensue and limit the long-term potency of targeted therapies 6, 7. Moreover, efforts are still needed for the management of patients without targetable oncogenic driver

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.

Victor Jeannot,1,2 Benoit Busser,1–3 Laetitia Vanwonterghem,1,2 Sophie Michallet,1,2 Sana Ferroudj,1,2 Murat Cokol,4 Jean-Luc Coll,1,2 Mehmet Ozturk,1,2,5 Amandine Hurbin1,2 1INSERM U1209, Department Cancer Targets and Experimental Therapeutics, Grenoble, France; 2University Grenoble Alpes, Institute for Advanced Biosciences, Grenoble, France; 3Department of Biochemistry, Toxicology and Pharmacology, Grenoble University Hospital, Grenoble, France; 4Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey; 5Faculty of Medicine, Dokuz Eyul University, Izmir Biomedicine and Genome Center, Izmir, Turkey Abstract: Development of drug resistance limits the efficacy of targeted therapies. Alternative approaches using different combinations of therapeutic agents to inhibit several pathways could be a more effective strategy for treating cancer. The effects of the approved epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (gefitinib) or a multi-targeted kinase inhibitor (sorafenib) in combination with a histone deacetylase inhibitor (vorinostat) on cell proliferation, cell cycle distribution, apoptosis, and signaling pathway activation in human lung adenocarcinoma and hepatocarcinoma cells with wild-type EGFR and mutant KRAS were investigated. The effects of the synergistic drug combinations were also studied in human lung adenocarcinoma and hepatocarcinoma cells in vivo. The combination of gefitinib and vorinostat synergistically reduced cell growth and strongly induced apoptosis through inhibition of the insulin-like growth factor-1 receptor/protein kinase B (IGF-1R/AKT)-dependent signaling pathway. Moreover, the gefitinib and vorinostat combination strongly inhibited tumor growth in mice with lung adenocarcinoma or hepatocarcinoma tumor xenografts. In contrast, the combination of sorafenib and vorinostat did not inhibit cell proliferation compared to a single treatment and induced G2/M cell cycle arrest without apoptosis. The sorafenib and vorinostat combination sustained the IGF-1R-, AKT-, and mitogen-activated protein kinase-dependent signaling pathways. These results showed that there was synergistic cytotoxicity when vorinostat was combined with gefitinib for both lung adenocarcinoma and hepatocarcinoma with mutant KRAS in vitro and in vivo but that the combination of vorinostat with sorafenib did not show any benefit. These findings highlight the important role of the IGF-1R/AKT pathway in the resistance to targeted therapies and support the use of histone deacetylase inhibitors in combination with EGFR-tyrosine kinase inhibitors, especially for treating patients with mutant KRAS resistant to other treatments. Keywords: targeted therapy, combined treatments, non-small cell lung cancer, hepatocarcinoma

International audience; Complete resection of head and neck cancers with negative surgical margins improves the prognosis of the disease and decreases the recurrence rate. Near-infrared fluorescence-guided surgery of head and neck cancer is a rapidly evolving field that represents an invaluable tool for tumor detection and resection. Here, we present a literature review of the principles of near-infrared fluorescence imaging and its use in head and neck cancer surgery. We discuss important studies in both animal models and humans that have been carried out up to this point. We also outline the important fluorescent molecules and devices used in head and neck fluorescence imaging-guided surgery. Although near-infrared fluorescence-guided surgery for head and neck cancers showed efficacy in animal models, its use in humans is limited by the small number of fluorescent probes that are approved for clinical use. However, it is considered as a novel surgical aid that helps delineate tumor margins preoperatively and could spare patients from the added morbidity that is associated with additional surgery or chemoradiation. In addition, it is a useful tool to detect sentinel lymph nodes as well as met-astatic lymph nodes.

International audience; New approaches that are more efficient and able to specifically reach lung tumors are needed. We developed new hyaluronan-based nanoparticles targeting CD44 receptors of two different sizes and compared their lung cancer cells targeting efficacy in vitro and in vivo. The nanoparticles' cellular uptake was dose-dependent, and specific to hyaluronan receptors, particularly CD44. The binding and internalization differed according to nanoparticle size. In vivo biodistribution studies in two orthotopic lung tumor models showed that intrapulmonary nebulized nanoparticles accumulated in lungs, but not in the tumor nodules. In contrast, despite a significant liver capture, intravenous injection led to a better accumulation of the nanoparticles in the lung tumors compared with the surrounding healthy lung tissues. We demonstrated that the hyaluronan-based nanoparticles size plays significant role in cellular uptake and biodistribution. Small nanoparticles showed active targeting of CD44-overexpressing tumors, suggesting that they could be used as drug-delivery system. From the Clinical Editor: Combating cancers remains an important goal in clinical medicine. In this study, the authors investigated the ability of two hyaluronan-based nanoparticles targeting CD44 receptors to home in on lung cancer cells in an in-vivo orthotropic model. The preferential uptake of smaller sized nanoparticles via intravenous route has further enhanced the existing knowledge of future drug designs

Near-infrared fluorescence image-guided surgery, FIGS, has lately shown a huge potential in oncologic and lymphatic related surgeries. In some indications such as liver or heart surgery, fluorescence-reachable anatomic structures are limited by the access to the surgical field. Nevertheless, most of the systems available on the market are too large to image the sides of cavities. Small devices are clearly required to improve workability of fluorescence imaging systems. The current work describes the evaluation of Fluostick a CE med certified instrument dedicated to narrow area imaging. This small size device is made of an optical head connected to a control box. We tested this instrumentation at the preclinical level for the optical-guided surgery of head and neck tumors.

Appropriate animal models are required to test novel therapeutics for head and neck squamous cell carcinoma (HNSCC) such as near-infrared (NIR) imaging-guided surgery.We developed an optimized animal model of orthotopic HNSCC (in female athymic NMRI (Naval Medical Research Institute) nude mice) with a prolonged survival time. Resection of the orthotopic tumors was performed 30 days after implantation with or without the aid of a miniaturized clinical grade NIR optical imaging device, after systemic administration of a fluorescent RGD-based probe that targets αv β3 integrin.NIR optical imaging-guided surgery increased the recurrence-free survival rate by 50% through the detection of fluorescent cancer residues as small as 185 µm; these fragments could remain unidentified if resection was performed exclusively under unaided visual guidance.NIR optical imaging-guided surgery showed an improved HNSCC tumor resection quality in our optimized orthotopic animal model. © 2015 Wiley Periodicals, Inc. Head Neck 38: E246-E255, 2016.

International audience; Amphiregulin (AR) and insulin-like growth factor-1 (IGF1) are growth factors known to promote non-small cell lung cancer (NSCLC) survival. We have previously published that 1) AR and IGF1, secreted by H358 NSCLC cells, cooperate to protect those cells and H322 NSCLC cells from serum-starved apoptosis; 2) H358 cells resist Bax-induced apoptosis through an inhibition of Bax conformational change. We show here that the antiapoptotic activity of the AR/IGF1 combination is specifically abolished by the PKC inhibitors calphostin C and staurosporine, but not by the MAPK and phosphatidylinositol 3-kinase inhibitors PD98059 and wortmannin, suggesting the involvement of a PKC-dependent and MAPK- and phosphatidylinositol 3-kinase-independent survival pathway. The PKCdelta inhibitor rottlerin restores apoptosis induced by serum deprivation. In addition, phosphorylation of PKCdelta and PKCzeta/lambda, but not of PKCalpha/beta(II), increases in serum-starved H358 cells and in H322 cells treated with an AR/IGF1 combination and is blocked by calphostin C. The combination of AR and IGF1 increases p90(rsk) and Bad phosphorylation as well as inhibiting the conformational change of Bax by a PKC-dependent mechanism. Finally, PKCdelta, PKCzeta, or p90(rsk) small interfering RNAs block the antiapoptotic activity of AR/IGF1 combination but have no effect on partial apoptosis inhibition observed with each factor used alone. Constitutively active PKC expression inhibits serum deprivation-induced apoptosis, whereas a catalytically inactive form of p90(rsk) restores it. Thus, AR and IGF1 cooperate to prevent apoptosis by activating a specific PKC-p90(rsk)-dependent pathway, which leads to Bad and Bax inactivation. This signaling pathway is different to that used by single factor.

Activity of the magnocellular neurons that synthesize vasopressin in the supraoptic and paraventricular nuclei of the hypothalamus is modulated by local release of the neuropeptide within the nuclei. V1a and V1b vasopressin receptor genes are expressed in these cells. The present study reports the localization of V1a and V1b receptors using multiple labeling immunocytochemistry. Both receptors are mainly located in vasopressinergic magnocellular neurons and colocalized with vasopressin in cytoplasmic vesicles dispersed throughout the cell. Possible functional modifications of the mRNA and protein levels of the V1a receptor, the major isoform, were also investigated by semiquantitative in situ hybridization and immunocytochemistry in rats submitted to reduced or increased water intake. V1a mRNA and receptor levels varied with water balance. V1a mRNA level dropped in rats submitted to high water intake. Conversely, dehydration up-regulated the V1a receptor content. These observations suggest that the pathways that regulate the expression of the genes encoding vasopressin and the V1a receptor are linked, which fits the present findings that the two partners are colocalized in cytoplasmic vesicles. Colocalization might explain how V1 autoreceptors are controlled by cell activity and/or local concentration of vasopressin (released locally by the neurons themselves), allowing fine adjustment of magnocellular neuron activity.

Introduction Les inhibiteurs de l’activite tyrosine kinase (TK) de l’EGFR (EGFR-TKI) sont utilises pour le traitement des cancers du poumon mais de nombreux mecanismes de resistance existent. Jusqu’a present, l’exploration des mecanismes de resistance s’est focalisee sur les reseaux de signalisation traditionnellement actives a partir de la membrane plasmique. Or, il est connu que les RTK sont capables de migrer dans le noyau ou ils exercent des fonctions propres. L’expression nucleaire de RTK, dont l’EGFR et l’IGF-1R a ete impliquee dans la progression tumorale et la reponse therapeutique. Nous avons observe que le gefitinib favorise une accumulation specifique de l’IGF-1R dans le noyau de certaines cellules d’adenocarcinome pulmonaire resistantes aux EGFR-TKI. Notre hypothese est que l’expression nucleaire de l’IGF-1R contribue a la resistance des tumeurs pulmonaires aux EGFR-TKI. Materiels et methodes H358, H441, H322 et A549 sont des lignees d’adenocarcinomes pulmonaires exprimant un EGFR de type sauvage et resistantes aux EGFR-TKI. H358 et H441 ont un haut niveau d’expression d’amphireguline (Areg), un ligand specifique de l’EGFR, alors que H322 et A549 expriment un tres faible niveau d’Areg. Les complexes proteiques sont etudies par co-immunoprecipitation et Proximity Ligation Assays. L’expression de l’Areg est neutralisee par des siRNA. L’endocytose est bloquee en utilisant le dynasore. L’apoptose est etudiee par marquage de la caspase-3 active et analyse au FACS. Resultats Nous decrivons pour la premiere fois l’expression nucleaire de l’IGF-1R dans des lignees cellulaires de cancer du poumon non a petites cellules et dans des tumeurs primaires. Nous montrons que le gefitinib induit l’endocytose et le transport nucleaire de l’IGF-1R dans des lignees d’adenocarcinomes mucineux par un mecanisme impliquant son association avec l’importine-β1. La neutralisation de l’Areg bloque la formation du complexe importine-β1/IGF-1R induit par le gefitinib et inhibe l’accumulation nucleaire de l’IGF-1R dans des lignees cellulaires. Ces resultats sont confirmes dans des xenogreffes chez la souris nude. Le gefitinib inhibe la secretion de l’Areg et induit sa re-localisation intracellulaire permettant la formation d’un complexe importine-β1/Areg/IGF-1R. L’accumulation nucleaire de l’IGF-1R mediee par l’Areg en reponse au gefitinib est associee a une augmentation du marquage Ki67 dans les xenogreffes tumorales. Conclusion Ces resultats identifient l’expression nucleaire de l’IGF-1R comme un nouveau determinant de la reponse aux EGFR-TKI et suggerent qu’une voie de proliferation Areg/IGF-1R contribue a la resistance des adenocarcinomes mucineux au gefitinib.

To characterize the volume-sensitive, osmolyte permeable anion channels responsible for the osmodependent release of taurine from supraoptic nucleus (SON) astrocytes, we investigated the pharmacological properties of the [(3)H]-taurine efflux from acutely isolated SON. Taurine release induced by hypotonic stimulus (250 mosmol l(-1)) was not antagonized by the taurine transporter blocker guanidinoethyl sulphonate, confirming the lack of implication of the transporter. The osmodependent release of taurine was blocked by a variety of Cl(-) channel inhibitors with the order of potency: NPPB>niflumic acid>DPC>DIDS>ATP. On the other hand, release of taurine was only weakly affected by other compounds (dideoxyforskolin, 4-bromophenacyl bromide, mibefradil) known to block volume-activated anion channels in other cell preparations, and was completely insensitive to tamoxifen, a broad inhibitor of these channels. Although the molecular identity of volume-sensitive anion channels is not firmly established, a few genes have been postulated as potential candidates to encode such channels. We checked the expression in the SON of three of them, ClC(3), phospholemman and VDAC(1), and found that the transcripts of these genes are found in SON neurons, but not in astrocytes. Similar observation was previously reported for ClC(2). In conclusion, the osmodependent taurine permeable channels of SON astrocytes display a particular pharmacological profile, suggesting the expression of a particular type or subtype of volume-sensitive anion channel, which is likely to be formed by yet unidentified proteins.

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.

Introduction Il n’existe actuellement pas de biomarqueur valide pour le suivi des patients atteints de melanome. Le suivi par dosage plasmatique quantitatif de l’ADN circulant mute BRAF et NRAS pourrait permettre un suivi etroit de l’evolution de la maladie et des reponses aux traitements chez des patients dont les tumeurs portent ces mutations. Ce dosage pourrait en effet refleter la charge tumorale presente chez le patient. Nous presentons ici la correlation entre les cinetiques d’ADN mute circulant et l’evolution clinique de 3 patients atteints de melanome metastatique mutes BRAF V600E. Patients et methodes Trois dosages mensuels consecutifs d’ADN BRAF mute V600E et sauvage ont ete realises a partir des plasmas de patients atteints de melanome metastatique, apres signature d’un consentement eclaire. L’analyse a ete realisee par droplet digital PCR (QX200, BioRad). Les resultats sont exprimes en nombre de copies du variant mute et en ratio normalise (ratio entre nombre de copies de variants BRAF mutes et nombre de variants BRAF sauvages). Resultats Patient 1 : stabilisation de la maladie chez un patient atteint de melanome de stade IV M1c traite par vemurafenib puis progression de la maladie concomitante a une augmentation du nombre de copies d’ADN BRAF mute V600E. Patient 2 : stabilisation de la maladie chez un patient atteint de melanome de stade IV M1c avec metastases viscerales et cerebrales traite par dabrafenib, puis progression de la maladie concomitante a une augmentation du nombre de copies d’ADN BRAF mute V600E. Patient 3 : patient mute BRAF avec metastases cerebrales en echappement apres protocole ipilimumab + radiotherapie et premier cycle de vemurefanib. Decision d’un traitement sequentiel alternant temozolidine 3 mois puis vemurafenib 3 mois. Objectivation d’une reprise de reponse au vemurafenib avec diminution de la charge en ADN circulant apres reintroduction de la therapie ciblee anti-BRAF. Pour les 3 patients, les courbes d’ADN en nombre absolu de copies detectees et le ratio d’ADN tumoral circulant BRAF mute/BRAF sauvage evoluaient de facon identique dans le temps. Discussion Cette etude preliminaire demontre l’interet de quantifier de l’ADN circulant mute dans le plasma des patients. Ce type de monitoring est simple a realiser, il est peu invasif et generalement bien accepte par les malades. De plus, il existe une correlation entre les cinetiques d’ADN mute et l’evolution clinique de la maladie. Conclusion A l’ere des biopsies liquides, le monitoring de l’ADN tumoral circulant pourrait facilement etre realise en routine et contribuer a optimiser le suivi des patients en objectivant l’evolution de la maladie et la reponse aux traitements, quelle que soit la strategie therapeutique.

International audience; 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.

International audience; RGD peptides recognize the α(v)β(3) 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.

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 (p0.0001) and amphiregulin (p = 0.004) with a tendency for more frequent KRAS mutations, in contrast to non-mucinous tumours that overexpressed EGFR (p0.0001) and TTF-1 (p0.0001) 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.

International audience; 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.

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.

Breast cancer is the second cause of death in Europe with an incidence of 464.00 new cases in 2012. Chemotherapy is frequently used to treat severe breast cancer. Current drugs like taxanes and anthracycline used alone or in combination are efficient. Moreover the use of biomarkers, like HER2 and BRCA status is now helping in the choice of the most adapted chemotherapy to the patient. De novo or acquired resistance limits, however, the clinical usefulness of drugs used in current chemotherapy. The development of new therapeutics effective against drug-resistant cancers thus still represents an important challenge. Our team has discovered a new LIM Kinases (LIMK1/2) inhibitor, “LIM-Pyr1”. LIMK1/2 are situated at a crossroads of several signaling pathways mainly activated by tyrosine kinase receptors and regulate both actin and microtubules dynamics. LIMK1/2 regulate actin dynamics through cofilin phosphorylation. Cofilin is an actin-depolymerizing factor and its phosphorylation inactivates its actin severing activity. LIMK1/2 also regulate microtubule dynamics through a mechanism unknown yet. LIMK1/2 inhibition induce microtubules stabilization (1). Moreover, LIMK1/2 are overexpressed in many invasive cancers and appear to be a relevant target for anticancer therapy (2, 3). We have shown that LIM-Pyr1 is toxic on cell lines resistant to conventional chemotherapy (4) and tested LIM-Pyr1 therapeutic activity on different breast cancer models (xenografts in mice). We found that LIM-Pyr1 shows a potent antitumor activity both on primary and secondary tumors, with no detectable undesirable side effects. The antitumor effect is effective on paclitaxel resistant xenografts. Finally, intravital microscopy analysis indicates that a LIM-Pyr1 treatment induces a strong morphological change of tumor cells inside the tumors and reduces their migration. LIM-Pyr1 and its derivatives could thus represent a pharmacological alternative to overcome resistances often observed when tumors are treated with microtubule targeting agents. (1) O. Bernard, LIM Kinases, regulators of actin dynamics, Int. Journals of Biochemistry and cell biology (2007) 1071-1076 (2) F. Manetti, Recent finding confirm LIM Domain Kinases as emerging target candidates for cancer therapy, Curr. Cancer Drug Targets (2012) 12,543-560 (3) W. Wang, R. Eddy, J. Condeelis, The cofilin pathway in breast cancer invasion and metastasis, Nat. Cancer Reviews (2007) (4) R. Prudent, E. Vassal-Stermann, C-H Nguyen et al., Pharmacological inhibition of LIM Kinases stabilizes microtubules, Cancer Research (2012) Citation Format: Chloé Prunier, Julien Vollaire, Véronique Josserand, Anoek Zomer, Amandine Hurbin, Renaud Prudent, Pascale Cohen, Jacco van Rheenen, Jean-Luc Coll, Marc Billaud, Laurence Lafanechère. Anti-cancer activity of a new LIM-Kinases inhibitor: “LIM-Pyr1”. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5399. doi:10.1158/1538-7445.AM2015-5399

Introduction Lung cancer is the leading cause of mortality by cancer in the world. There is an urgent need to propose new therapeutic approaches in addition to conventional therapy. To be efficient and to decrease side effects, treatments must specifically reach tumors. In this context, this work aims to develop a new nanocarrier, able to target tumors and to measure its benefit in cancer therapy. The nanoparticles (Np) we used here combine a biodegradable polypeptide, and the Hyaluronan (Hya), the natural ligand of the CD44 receptors upregulated on cancer cells. Methods The ability of Hya fragments with different molecular weights (5000 and 35000 Da), or Np with different sizes (30 and 300 nm), to target the CD44 receptors was studied in vitro using lung cancer cell lines (H358, H460, A549, H322), in which CD44 expression level was measured. We investigated Hya and Np accumulation in tumor in vivo using an orthotopic lung cancer model, after administration by systemic route (IV injections) or pulmonary route (nebulization). We also analysed the accumulation of an anti-CD44 antibody in vivo. Noninvasive optical imaging techniques (2D and 3D fluorescence) allowed the real-time follow-up of the Np in mice. Results Binding of Hya fragments on cell lines was dose-dependent, with a higher affinity for the low molecular weight Hya. Np bound lung cancer cells and were internalized at 37 °C. This interaction was partially dependent to CD44 expression level. In vivo, we observed a better accumulation of the anti-CD44 antibody administered by nebulization, in tumor-bearing mice, compared to healthy mice. In contrast, no specific accumulation of Hya or Np was observed in tumors after nebulization. The nebulized Np showed long time retention in lungs. We observed a higher accumulation of Np, but not Hya, in the lungs of tumor-bearing mice compared to healthy mice, after IV injection. Conclusions We show that CD44 receptor is a pertinent target for lung cancer treatment. Despite the promising results obtained after anti-CD44 antibody nebulization, this way of administration seems less efficient to target tumor with Np. Our results will allow us to select the best Np formulation to load anticancer drugs. We will then evaluate the potential antitumor activity of drug-loaded Np using lung cancer model in mice.

Introduction Lung cancers are the fourth most common diagnosed cancer in the world and the main cause of cancer death. A large majority of lung cancers are classified as poor prognosis non-small cell lung cancers (NSCLC) with 5-year survival rate less than 15%. Most NSCLC cells resist to apoptosis and targeted therapies. To identify new molecules able to restore apoptosis in resistant NSCLC cells, a molecular high throughput screening was performed on NSCLC cells resistant to serum starvation. Seven thousand and five hundred molecules were screened at 2.5 μmol/L, and 71 compounds restoring apoptosis after primary and validation screenings on NSCLC cells, were selected. Among these molecules, we identified a family of compounds with a strong structural homology, known to inhibit kinase activity. Our goal is to develop new therapeutic strategies able to overcome resistance of NSCLC cells. Methods We selected 15 molecules of interest to identify the potential target(s) of these molecules by western blotting, RT-qPCR and immunofluorescence. We also assessed the effects of these compounds on NSCLC cells viability (MTS assay), apoptosis (Hoechst staining), and cell cycle (flow cytometry analysis). Results The 15 compounds showed IC50 ranging from 0.1 to 50 μmol/L. After 24 h treatment, the molecules induced G2/M cell cycle arrest, except the molecule 11, which strongly induced subG1 accumulation. The 15 molecules significantly restored serum deprivation-induced apoptosis in H358 NSCLC cells at 96 h. They also reduced cell viability in presence of serum, except the molecules 9 and 11. Most of the molecules decreased the expression level of EGFR and IGF1R and the phosphorylation level of Src, PKCδ, ERK and AKT. These 6 proteins are known as participating in apoptosis signalling pathways. Surprisingly, all compounds also reduced the level of tubulin and modified its network conformation. Conclusions The family of compounds that we identified is able to restore apoptosis in resistant NSCLC cells and could be used as new therapeutic approaches in NSCLC. We will assess the effects of combination therapies on resistant NSCLC cells, and determine the target(s) of these molecules by kinome array. This project will identify the best drug-candidate to counteract the NSCLC cells resistance, alone or in combination with targeted therapies.

International audience; 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 am-phiregulin 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 an-ti-apoptotic activity of H358-conditioned medium is completely abolished after incubation with anti-amphi-regulin neutralizing antibody and only partially with anti-IGF1 neutralizing antibody. H358-conditioned medium and amphiregulin induce IGF1 receptor phospho-rylation in H322 cells, which is prevented by anti-amphi-regulin 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.