EZH2 is required for breast and pancreatic cancer stem cell maintenance and can be used as a functional cancer stem cell reporter.

Although cancer is largely seen as a disease stemming from genetic mutations, evidence has implicated epigenetic regulation of gene expression as a driving force for tumorigenesis. Epigenetic regulation by histone modification, specifically through polycomb group (PcG) proteins such as EZH2 and BMI-1, is a major driver in stem cell biology and is found to be correlated with poor prognosis in many tumor types. This suggests a role for PcG proteins in cancer stem cells (CSCs). We hypothesized that epigenetic modification by EZH2, specifically, helps maintain the CSC phenotype and that in turn this epigenetic modifier can be used as a reporter for CSC activity in an in vitro high-throughput screening assay. CSCs isolated from pancreatic and breast cancer lines had elevated EZH2 levels over non-CSCs. Moreover, EZH2 knockdown by RNA interference significantly reduced the frequency of CSCs in all models tested, confirming the role of EZH2 in maintenance of the CSC population. Interestingly, genes affected by EZH2 loss, and therefore CSC loss, were inversely correlated with genes identified by CSC enrichment, further supporting the function of EZH2 CSC regulation. We translated these results into a novel assay whereby elevated EZH2 staining was used as a reporter for CSCs. Data confirmed that this assay could effectively measure changes, both inhibition and enrichment, in the CSC population, providing a novel approach to look at CSC activity. This assay provides a unique, rapid way to facilitate CSC screening across several tumor types to aid in further CSC-related research.

The role of epigenetic regulation in stem cell and cancer biology.

Normal development and homeostasis requires a carefully coordinated gene expression program. Appropriate transcriptional regulation is maintained, in part, through epigenetic modifications of both DNA and histones. It is now apparent that the epigenetic landscape is complex and carefully controlled to both silence and activate gene transcription and that these states remain exquisitely poised for reversal. The deregulation of epigenetics in cancer is common and results in both the activation of oncogenes and the silencing of tumor suppressors. A tremendous amount of research corroborates the existence in many tumor types of a cancer stem cell that is both the origin and cell type responsible for resistance of tumors to current therapies. As our understanding of cancer stem cell biology continues, it is apparent that these cells are also under the influence of epigenetic regulation. We will discuss the cancer stem cell hypothesis and the role of epigenetics in both normal and cancer stem cell biology.

Augmentation of therapeutic efficacy in drug-resistant tumor models using ceramide coadministration in temporal-controlled polymer-blend nanoparticle delivery systems.

The development of multidrug resistance (MDR) is a major hindrance to cancer eradication as it renders tumors unresponsive to most chemotherapeutic treatments and is associated with cancer resurgence. This study describes a novel mechanism to overcome MDR through a polymer-blend nanoparticle platform that delivers a combination therapy of C6-ceramide (CER), a synthetic analog of an endogenously occurring apoptotic modulator, together with the chemotherapeutic drug paclitaxel (PTX), in a single formulation. The PTX/CER combination therapy circumvents another cellular mechanism whereby MDR develops, by lowering the threshold for apoptotic signaling. In vivo studies in a resistant subcutaneous SKOV3 human ovarian and in an orthotopic MCF7 human breast adenocarcinoma xenograft showed that the PTX and CER nanoparticle combination therapy reduced the final tumor volume at least twofold over treatment with the standard PTX therapy alone. The study also revealed that the cotherapy accomplished this enhanced efficacy by generating an enhancement in apoptotic signaling in both tumor types. Additionally, acute evaluation of safety with the combination therapy did not show significant changes in body weight, white blood cell counts, or liver enzyme levels. The temporal-controlled nanoparticle delivery system presented in this study allows for a simultaneous delivery of PTX + CER in breast and ovarian tumor model drug, leading to a modulation of the apoptotic threshold. This strategy has tremendous potential for effective treatment of refractory disease in cancer patients.

Evaluations of combination MDR-1 gene silencing and paclitaxel administration in biodegradable polymeric nanoparticle formulations to overcome multidrug resistance in cancer cells.

In this study, the effect of MDR-1 gene silencing, using small interfering RNA (siRNA), and paclitaxel (PTX) co-therapy in overcoming tumor multidrug resistance was examined. Poly(ethylene oxide)-modified poly(beta-amino ester) (PEO-PbAE) and PEO-modified poly(epsilon-caprolactone) (PEO-PCL) nanoparticles were formulated to efficiently encapsulate MDR-1 silencing siRNA and PTX, respectively. Upon administration in multidrug resistant SKOV3(TR) human ovarian adenocarcinoma cells, siRNA-mediated MDR-1 gene silencing was evident at 100 nM dose. Combination of MDR-1 gene silencing and nanoparticle-mediated delivery significantly influenced the cytotoxic activity of PTX in SKOV3(TR) cells similar to what was observed in drug sensitive SKOV3 cells. We speculate that the enhancement in cytotoxicity was due to an increase in intracellular drug accumulation upon MDR-1 gene silencing leading to an apoptotic cell-kill effect. Taken together, these preliminary results are highly encouraging for the development of combination nano-therapeutic strategies that combine gene silencing and drug delivery to provide more potent therapeutic effect, especially in refractory tumors.

Biodistribution and pharmacokinetic analysis of Paclitaxel and ceramide administered in multifunctional polymer-blend nanoparticles in drug resistant breast cancer model.

In this study, we have investigated the biodistribution and pharmacokinetic analysis of paclitaxel (PTX) and the apoptotic signaling molecule, C6-ceramide (CER), when administered in a multifunctional polymer-blend nanoparticle formulation to female nude mice bearing an orthotopic drug sensitive MCF7 and multidrug resistant MCF7 TR (MDR-1 positive) human breast adenocarcinoma. A polymer-blend nanoparticle system was engineered to incorporate temporally controlled sequential release of the combination drug payload. Hereby, PTX was encapsulated in the pH-responsive rapid releasing polymer, poly(beta-amino ester) (PbAE), while CER was present in the slow releasing polymer, poly(D,L-lactide-co-glycolide) (PLGA) within these blend nanoparticles. When particle formulations were administered intravenously to MCF7 and MCF7 TR tumor bearing mice, higher concentrations of PTX were found in the blood due to longer retention time and an enhanced tumor accumulation relative to administration of free drug. In addition, the PLGA/PbAE blend nanoparticles were effective in enhancing the residence time of both drugs at the tumor site by reducing systemic clearance. Overall, these results are highly encouraging for development of multifunctional polymer-blend nanoparticle formulations that can be used for temporal-controlled administration of two drugs from a single formulation.

Multi-functional nanocarriers for targeted delivery of drugs and genes.

In this review article, we describe the different nano-platforms developed in our laboratory at Northeastern University in Boston, MA for the targeted delivery of drugs and genes. Special emphasis is placed on nano-platforms that offer opportunities for multi-functionalization to allow for targeted stimuli-responsive and/or simultaneous strategic delivery of multiple drugs, genes, as well as the combination of therapeutic systems with image contrast enhancers. Polymeric and lipid-based nanocarriers can provide versatile platforms for the delivery of multiple pharmacological agents, specifically to enhance therapeutic effect and overcome drug resistance in cancer. In addition, polymeric nanoparticles and nanoparticles-in-microsphere oral system (NiMOS) are useful for systemic and oral gene therapy, respectively.

Multi-functional nanocarriers to overcome tumor drug resistance.

The development of resistance to variety of chemotherapeutic agents is one of the major challenges in effective cancer treatment. Tumor cells are able to generate a multi-drug resistance (MDR) phenotype due to microenvironmental selection pressures. This review addresses the use of nanotechnology-based delivery systems to overcome MDR in solid tumors. Our own work along with evidence from the literature illustrates the development of various types of engineered nanocarriers specifically designed to enhance tumor-targeted delivery through passive and active targeting strategies. Additionally, multi-functional nanocarriers are developed to enhance drug delivery and overcome MDR by either simultaneous or sequential delivery of resistance modulators (e.g., with P-glycoprotein substrates), agents that regulate intracellular pH, agents that lower the apoptotic threshold (e.g., with ceramide), or in combination with energy delivery (e.g., sound, heat, and light) to enhance the effectiveness of anticancer agents in refractory tumors. In preclinical studies, the use of multi-functional nanocarriers has shown significant promise in enhancing cancer therapy, especially against MDR tumors.

Modulation of intracellular ceramide using polymeric nanoparticles to overcome multidrug resistance in cancer.

Although multidrug resistance (MDR) is known to develop through a variety of molecular mechanisms within the tumor cell, many tend to converge toward the alteration of apoptotic signaling. The enzyme glucosylceramide synthase (GCS), responsible for bioactivation of the proapoptotic mediator ceramide to a nonfunctional moiety glucosylceramide, is overexpressed in many MDR tumor types and has been implicated in cell survival in the presence of chemotherapy. The purpose of this study was to investigate the therapeutic strategy of coadministering ceramide with paclitaxel, a commonly used chemotherapeutic agent, in an attempt to restore apoptotic signaling and overcome MDR in the human ovarian cancer cell line SKOV3. Poly(ethylene oxide)-modified poly(epsilon-caprolactone) (PEO-PCL) nanoparticles were used to encapsulate and deliver the therapeutic agents for enhanced efficacy. Results show that indeed the cotherapy eradicates the complete population of MDR cancer cells when they are treated at their IC(50) dose of paclitaxel. More interestingly, when the cotherapy was combined with the properties of nanoparticle drug delivery, the MDR cells can be resensitized to a dose of paclitaxel near the IC(50) of non-MDR (drug sensitive) cells, indicating a 100-fold increase in chemosensitization via this approach. Molecular analysis of activity verified the hypothesis that the efficacy of this therapeutic approach is indeed due to a restoration in apoptotic signaling, although the beneficial properties of PEO-PCL nanoparticle delivery seemed to enhance the therapeutic success even further, showing the promising potential for the clinical use of this therapeutic strategy to overcome MDR.

Poly(ethylene glycol)-modified nanocarriers for tumor-targeted and intracellular delivery.

The success of anti-cancer therapies largely depends on the ability of the therapeutics to reach their designated cellular and intracellular target sites, while minimizing accumulation and action at non-specific sites. Surface modification of nanoparticulate carriers with poly(ethylene glycol) (PEG)/poly(ethylene oxide) (PEO) has emerged as a strategy to enhance solubility of hydrophobic drugs, prolong circulation time, minimize non-specific uptake, and allow for specific tumor-targeting through the enhanced permeability and retention effect. Furthermore, PEG/PEO modification has emerged as a platform for incorporation of active targeting ligands, thereby providing the drug and gene carriers with specific tumor-targeting properties through a flexible tether. This review focuses on the recent developments surrounding such PEG/PEO-surface modification of polymeric nanocarriers to promote tumor-targeting capabilities, thereby enhancing efficacy of anti-cancer therapeutic strategies.

Multi-functional polymeric nanoparticles for tumour-targeted drug delivery.

The use of nanoparticles as drug delivery vehicles for anticancer therapeutics has great potential to revolutionise the future of cancer therapy. As tumour architecture causes nanoparticles to preferentially accumulate at the tumour site, their use as drug delivery vectors results in the localisation of a greater amount of the drug load at the tumour site; thus improving cancer therapy and reducing the harmful nonspecific side effects of chemotherapeutics. In addition, formulation of these nanoparticles with imaging contrast agents provides a very efficient system for cancer diagnostics. Given the exhaustive possibilities available to polymeric nanoparticle chemistry, research has quickly been directed at multi-functional nanoparticles, combining tumour targeting, tumour therapy and tumour imaging in an all-in-one system, providing a useful multi-modal approach in the battle against cancer. This review will discuss the properties of nanoparticles that allow for such multiple functionality, as well as recent scientific advances in the area of multi-functional nanoparticles for cancer therapeutics.

NF-kappaB participates in the corticotropin-releasing, hormone-induced regulation of the pituitary proopiomelanocortin gene.

Corticotropin-releasing hormone is a main regulator of mammalian stress response by stimulating pituitary proopiomelanocortin (POMC) gene expression, and thus adrenocorticotropic hormone (ACTH) secretion, which then causes glucocorticoid release from the adrenal. In a recent study in the pituitary corticotroph cell line AtT20, oxidative stress stimulated the activity of nuclear transcription factor B (NF-kappaB), whereas corticotropin-releasing hormone (CRH) inhibited both the constitutive and the oxidative stress-induced NF-kappaB DNA-binding activity. To further investigate the role of NF-kappaB on the CRH-induced pituitary POMC gene activation, AtT20 cells were transiently transfected with a POMC-luciferase construct mutated at an NF-kappaB binding site. After treatment with CRH, intracellular POMC-luciferase activity was significantly higher from the stimulation observed with transfection of the parental POMC-luciferase construct. In agreement with a previous report, CRH inhibited the constitutive NF-kappaB DNA-binding activity in AtT20 cells, as shown by electrophoretic mobility-shift assay, as soon as within 15 min of treatment. These effects of CRH were blocked by the CRH-R1 antagonist CP154,256. Our findings provide evidence that the regulation of corticotroph NF-kappaB activity by CRH is related to the activation of the pituitary POMC gene and, thus, may play an important role in stress response.