Cytoplasmic p53 couples oncogene-driven glucose metabolism to apoptosis and is a therapeutic target in glioblastoma.

Cross-talk among oncogenic signaling and metabolic pathways may create opportunities for new therapeutic strategies in cancer. Here we show that although acute inhibition of EGFR-driven glucose metabolism induces only minimal cell death, it lowers the apoptotic threshold in a subset of patient-derived glioblastoma (GBM) cells. Mechanistic studies revealed that after attenuated glucose consumption, Bcl-xL blocks cytoplasmic p53 from triggering intrinsic apoptosis. Consequently, targeting of EGFR-driven glucose metabolism in combination with pharmacological stabilization of p53 with the brain-penetrant small molecule idasanutlin resulted in synthetic lethality in orthotopic glioblastoma xenograft models. Notably, neither the degree of EGFR-signaling inhibition nor genetic analysis of EGFR was sufficient to predict sensitivity to this therapeutic combination. However, detection of rapid inhibitory effects on [18F]fluorodeoxyglucose uptake, assessed through noninvasive positron emission tomography, was an effective predictive biomarker of response in vivo. Together, these studies identify a crucial link among oncogene signaling, glucose metabolism, and cytoplasmic p53, which may potentially be exploited for combination therapy in GBM and possibly other malignancies.

Emerging Approaches for Targeting Metabolic Vulnerabilities in Malignant Glioma.

Malignant gliomas are intractable and among the most lethal human malignancies. Like other cancers, metabolic reprogramming is a key feature of glioma and is thought to accommodate the heightened nutrient requirements for tumor cell proliferation, growth, and survival. This metabolic rewiring, driven by oncogenic signaling and molded by the unique environment of the brain, may impose vulnerabilities that could be exploited therapeutically for increased tumor control. In this review, we discuss the prominent metabolic features of malignant glioma, the key pathways regulating glioma metabolism, and the potential therapeutic opportunities for targeting metabolic processes.

Chemical methods for the simultaneous quantitation of metabolites and proteins from single cells.

We describe chemical approaches for integrated metabolic and proteomic assays from single cells. Quantitative assays for intracellular metabolites, including glucose uptake and three other species, are designed as surface-competitive binding assays with fluorescence readouts. This enables integration into a microarray format with functional protein immunoassays, all of which are incorporated into the microchambers of a single-cell barcode chip (SCBC). By using the SCBC, we interrogate the response of human-derived glioblastoma cancer cells to epidermal growth factor receptor inhibition. We report, for the first time, on both the intercellular metabolic heterogeneity as well as the baseline and drug-induced changes in the metabolite-phosphoprotein correlation network.

Quantitative assessments of glycolysis from single cells.

The most common positron emission tomography (PET) radio-labeled probe for molecular diagnostics in patient care and research is the glucose analog, 2-deoxy-2-[F-18]fluoro-D-glucose (18F-FDG). We report on an integrated microfluidics-chip/beta particle imaging system for in vitro18F-FDG radioassays of glycolysis with single cell resolution. We investigated the kinetic responses of single glioblastoma cancer cells to targeted inhibitors of receptor tyrosine kinase signaling. Further, we find a weak positive correlation between cell size and rate of glycolysis.

Two-wave nanotherapy to target the stroma and optimize gemcitabine delivery to a human pancreatic cancer model in mice.

Pancreatic ductal adenocarcinoma (PDAC) elicits a dense stromal response that blocks vascular access because of pericyte coverage of vascular fenestrations. In this way, the PDAC stroma contributes to chemotherapy resistance in addition to causing other problems. In order to improve the delivery of gemcitabine, a first-line chemotherapeutic agent, a PEGylated drug-carrying liposome was developed, using a transmembrane ammonium sulfate gradient to encapsulate the protonated drug up to 20% w/w. However, because the liposome was precluded from entering the xenograft site due to the stromal interference, we developed a first-wave nanocarrier that decreases pericyte coverage of the vasculature through interference in the pericyte recruiting TGF-ß signaling pathway. This was accomplished using a polyethyleneimine (PEI)/polyethylene glycol (PEG)-coated mesoporous silica nanoparticle (MSNP) for molecular complexation to a small molecule TGF-ß inhibitor, LY364947. LY364947 contains a nitrogen atom that attaches, through H-bonding, to PEI amines with a high rate of efficiency. The copolymer coating also facilitates systemic biodistribution and retention at the tumor site. Because of the high loading capacity and pH-dependent LY364947 release from the MSNPs, we achieved rapid entry of IV-injected liposomes and MSNPs at the PDAC tumor site. This two-wave approach provided effective shrinkage of the tumor xenografts beyond 25 days, compared to the treatment with free drug or gemcitabine-loaded liposomes only. Not only does this approach overcome stromal resistance to drug delivery in PDAC, but it also introduces the concept of using a stepwise engineered approach to address a range of biological impediments that interfere in nanocancer therapy in a spectrum of cancers.

Codelivery of an optimal drug/siRNA combination using mesoporous silica nanoparticles to overcome drug resistance in breast cancer in vitro and in vivo.

We used a multifunctional mesoporous silica nanoparticle (MSNP) carrier to overcome doxorubicin (Dox) resistance in a multidrug resistant (MDR) human breast cancer xenograft by codelivering Dox and siRNA that targets the P-glycoprotein (Pgp) drug exporter. The Pgp siRNA selection from among a series of drug resistance targets was achieved by performing high throughput screening in a MDR breast cancer cell line, MCF-7/MDR. Following the establishment of a MCF-7/MDR xenograft model in nude mice, we demonstrated that a 50 nm MSNP, functionalized by a polyethyleneimine-polyethylene glycol (PEI-PEG) copolymer, provides protected delivery of stably bound Dox and Pgp siRNA to the tumor site. The effective biodistribution and reduced reticuloendothelial uptake, as a result of our nanocarrier design, allowed us to achieve an 8% enhanced permeability and retention effect at the tumor site. Compared to free Dox or the carrier loaded with either drug or siRNA alone, the dual delivery system resulted in synergistic inhibition of tumor growth in vivo. Analysis of multiple xenograft biopsies demonstrated significant Pgp knockdown at heterogeneous tumor sites that correspond to the regions where Dox was released intracellularly and induced apoptosis. We emphasize that the heterogeneity originates in the tumor microenvironment, which influences the vascular access, rather than heterogeneous Pgp expression in the MDR cells. Taken together, these data provide proof-of-principle testing of the use of a dual drug/siRNA nanocarrier to overcome Dox resistance in a xenograft. The study also provides the first detailed analysis of the impact of heterogeneity in the tumor microenvironment on the efficacy of siRNA delivery in vivo.

Mesoporous silica nanoparticles: A multifunctional nano therapeutic system.

Efficient and safe drug delivery has always been a challenge in medicine. The use of nanotechnology, such as the development of nano drug delivery systems (DDS), has received great attention with high enthusiasm owing to the potential that nanocarriers can theoretically act as "magic bullets" and selectively target affected organs and cells while sparing normal tissues. The family of nano DDS includes conventional nano drug delivery materials such as lipids and polymers that have been scaled to the nanometer size range. With the rapid development of synthesis and characterization techniques for engineered nanomaterials, new DDS platforms have emerged, including inorganic based nanocarriers, such as mesoporous silica nanoparticles (MSNP). MSNP are able to act as a multifunctional delivery platform that is capable of delivering therapeutic elements to a variety of disease models (especially cancer) at cellular and in vivo levels. Furthermore, MSNP have shown to be exceptional delivery platforms capable of protectively packaging hydrophobic and hydrophilic drug molecules as well as other therapeutic elements for controlled on-demand delivery. In addition, MSNP have demonstrated the capability to image the delivery site for theranostic purposes. These functionalities have led to the development of MSNP as novel multifunctional nanocarriers, and therefore provide them with unique advantages compared to other nanocarriers.