ABSTRACT
Accurate measurement of progranulin (PGRN) in the circulation and in the tumor microenvironment is essential for understanding its role in cancer progression and metastasis. This chapter describes a number of approaches to measure the transcription level of the GRN gene and to detect and analyze PGRN expression in cancer cells and in the local environment of the tumor, in mouse and human samples. These validated protocols are utilized to investigate the functional role of PGRN in cancer. Finally, we discuss strategies to investigate the functions of PGRN in tumors using genetically modified mouse models and gene silencing techniques.
Subject(s)
Biochemistry/methods , Progranulins/metabolism , Tumor Microenvironment , Adoptive Transfer , Animals , Flow Cytometry , Genetic Engineering , Hematopoietic Stem Cells/metabolism , Humans , Mice, Knockout , RNA, Messenger/genetics , RNA, Messenger/metabolism , Staining and LabelingABSTRACT
Alterations in PIK3CA, the gene encoding the p110α subunit of phosphatidylinositol 3-kinase (PI3Kα), are frequent in head and neck squamous cell carcinomas. Inhibitors of PI3Kα show promising activity in various cancer types, but their use is curtailed by dose-limiting side effects such as hyperglycaemia. In the present study, we explore the efficacy, specificity and safety of the targeted delivery of BYL719, a PI3Kα inhibitor currently in clinical development in solid tumours. By encapsulating BYL719 into P-selectin-targeted nanoparticles, we achieve specific accumulation of BYL719 in the tumour milieu. This results in tumour growth inhibition and radiosensitization despite the use of a sevenfold lower dose of BYL719 compared with oral administration. Furthermore, the nanoparticles abrogate acute and chronic metabolic side effects normally observed after BYL719 treatment. These findings offer a novel strategy that could potentially enhance the efficacy of PI3Kα inhibitors while mitigating dose-limiting toxicity in patients with head and neck squamous cell carcinomas.
Subject(s)
Carcinoma, Squamous Cell/drug therapy , Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Head and Neck Neoplasms/drug therapy , Nanoparticles/chemistry , Thiazoles/pharmacology , Xenograft Model Antitumor Assays , Animals , Carcinoma, Squamous Cell/metabolism , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases/metabolism , Drug Delivery Systems/methods , Head and Neck Neoplasms/metabolism , Humans , Mice, Nude , Microscopy, Electron, Scanning , Nanoparticles/ultrastructure , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Thiazoles/administration & dosage , Thiazoles/chemistry , Treatment OutcomeABSTRACT
Disseminated tumors are poorly accessible to nanoscale drug delivery systems because of the vascular barrier, which attenuates extravasation at the tumor site. We investigated P-selectin, a molecule expressed on activated vasculature that facilitates metastasis by arresting tumor cells at the endothelium, for its potential to target metastases by arresting nanomedicines at the tumor endothelium. We found that P-selectin is expressed on cancer cells in many human tumors. To develop a targeted drug delivery platform, we used a fucosylated polysaccharide with nanomolar affinity to P-selectin. The nanoparticles targeted the tumor microenvironment to localize chemotherapeutics and a targeted MEK (mitogen-activated protein kinase kinase) inhibitor at tumor sites in both primary and metastatic models, resulting in superior antitumor efficacy. In tumors devoid of P-selectin, we found that ionizing radiation guided the nanoparticles to the disease site by inducing P-selectin expression. Radiation concomitantly produced an abscopal-like phenomenon wherein P-selectin appeared in unirradiated tumor vasculature, suggesting a potential strategy to target disparate drug classes to almost any tumor.
Subject(s)
Nanoparticles/chemistry , P-Selectin/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Cell Survival/drug effects , Cell Survival/radiation effects , Drug Delivery Systems , Female , Humans , Immunohistochemistry , In Vitro Techniques , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/radiotherapy , Lung Neoplasms/secondary , Melanoma/complications , Melanoma/drug therapy , Melanoma/metabolism , Melanoma/radiotherapy , Mice , Mice, Inbred C57BL , Mice, Nude , Polysaccharides/chemistry , Polysaccharides/therapeutic use , Radiation, Ionizing , Spheroids, Cellular/drug effects , Spheroids, Cellular/radiation effects , Tumor Microenvironment , Xenograft Model Antitumor AssaysABSTRACT
Phosphoinositide-3-kinase (PI3K)-α inhibitors have shown clinical activity in squamous cell carcinomas (SCCs) of head and neck (H&N) bearing PIK3CA mutations or amplification. Studying models of therapeutic resistance, we have observed that SCC cells that become refractory to PI3Kα inhibition maintain PI3K-independent activation of the mammalian target of rapamycin (mTOR). This persistent mTOR activation is mediated by the tyrosine kinase receptor AXL. AXL is overexpressed in resistant tumors from both laboratory models and patients treated with the PI3Kα inhibitor BYL719. AXL dimerizes with and phosphorylates epidermal growth factor receptor (EGFR), resulting in activation of phospholipase Cγ (PLCγ)-protein kinase C (PKC), which, in turn, activates mTOR. Combined treatment with PI3Kα and either EGFR, AXL, or PKC inhibitors reverts this resistance.
