The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation.

Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:

Design and Characterization of Injectable Poly(Lactic-Co-Glycolic Acid) Pastes for Sustained and Local Drug Release.

PURPOSE: We describe the preparation of injectable polymeric paste (IPP) formulations for local and sustained release of drugs. Furthermore, we include the characterization and possible applications of such pastes. Particular attention is paid to characteristics relevant to the successful clinical formulation development, such as viscosity, injectability, degradation, drug release, sterilization, stability performance and pharmacokinetics. METHODS: Paste injectability was characterized using measured viscosity and the Hagen-Poiseuille equation to determine injection forces. Drug degradation, release and formulation stability experiments were performed in vitro and drug levels were quantified using HPLC-UV methods. Pharmacokinetic evaluation of sustained-release lidocaine IPPs used five groups of six rats receiving increasing doses subcutaneously. An anti-cancer formulation was evaluated in a subcutaneous tumor xenograft mouse model. RESULTS: The viscosity and injectability of IPPs could be controlled by changing the polymeric composition. IPPs demonstrated good long-term stability and tunable drug-release with low systemic exposure in vivo in rats. Preliminary data in a subcutaneous tumor model points to a sustained anticancer effect. CONCLUSIONS: These IPPs are tunable platforms for local and sustained delivery of drugs and have potential for further clinical development to treat a number of diseases.

Measuring the turbulence profile in the lower atmospheric boundary layer.

Optical turbulence can have a severe effect on the propagation of laser beams through the atmosphere. In free space optics and directed energy applications, these laser beams quite often propagate along a slant or vertical path. In these cases, the refractive index structure function parameter cannot be assumed constant, since it varies with height. How it varies with height, especially in the first few meters above the ground, is not well behaved. Turbulence height profiles have been measured since the 1970s, mainly for astronomical observations. These profiles are usually measured for the atmospheric boundary layer (the layer of air from the ground up to approx. 1 km during day and 100 m during night) and some kilometers above it. We have measured the temperature fluctuations in the first few meters above ground level using a system containing eight resistance thermometer devices, mounted in a row at different spacings. Measurements were made flying this system under a tethered balloon or mounted on a telescoping mast. The temperature structure function parameter, CT2, can be estimated from the temperature fluctuations measured by the 28 different probe pairs and the unique distances between the two probes. Finally, Cn2 is estimated from this temperature structure function parameter and compared to values predicted by a turbulence profile model.

Mutation of the salt bridge-forming residues in the ETV6-SAM domain interface blocks ETV6-NTRK3-induced cellular transformation.

The ETV6-NTRK3 (EN) chimeric oncogene is expressed in diverse tumor types. EN is generated by a t(12;15) translocation, which fuses the N-terminal SAM (sterile α-motif) domain of the ETV6 (or TEL) transcription factor to the C-terminal PTK (protein-tyrosine kinase) domain of the neurotrophin-3 receptor NTRK3. SAM domain-mediated polymerization of EN leads to constitutive activation of the PTK domain and constitutive signaling of the Ras-MAPK and PI3K-Akt pathways, which are essential for EN oncogenesis. Here we show through complementary biophysical and cellular biological techniques that mutation of Lys-99, which participates in a salt bridge at the SAM polymer interface, reduces self-association of the isolated SAM domain as well as high molecular mass complex formation of EN and abrogates the transformation activity of EN. We also show that mutation of Asp-101, the intermolecular salt bridge partner of Lys-99, similarly blocks transformation of NIH3T3 cells by EN, reduces EN tyrosine phosphorylation, inhibits Akt and Mek1/2 signaling downstream of EN, and abolishes tumor formation in nude mice. In contrast, mutations of Glu-100 and Arg-103, residues in the vicinity of the interdomain Lys-99-Asp-101 salt bridge, have little or no effect on these oncogenic characteristics of EN. Our results underscore the importance of specific electrostatic interactions for SAM polymerization and EN transformation.

A polymeric paste-drug formulation for local treatment of upper tract urothelial carcinoma.

BACKGROUND: Intravesical instillation of chemo- or immunotherapy is commonly used in bladder cancer. Upper tract urothelial carcinoma (UTUC) shares similar pathological features, but current formulations are not suitable for direct instillation to the upper urinary tract. OBJECTIVE: To evaluate in vivo applicability, characteristics and toxicity of ST-UC, a mucoadhesive polymeric paste formulation of gemcitabine, for upper urinary tract instillation. MATERIAL AND METHODS: Three pigs received 10 ml of ST-UC (100 mg/ml gemcitabine) retrogradely into 1 renal pelvis for pharmacokinetic studies. Four days later, a second injection into the contralateral renal pelvis was followed by serial euthanasia of the pigs and nephroureterectomy after 1, 3, and 6 hours. Adverse effects were monitored. Urine, serum, and tissue gemcitabine concentrations were measured, along with histologic examination of the upper urinary tract. RESULTS: Retrograde instillation of ST-UC was well tolerated with mild, completely receding hydronephrosis. Urine gemcitabine concentrations were highest in the first 3-hour collection interval. Hundred percent of gemcitabine was recovered in the urine within 24 hours. Serum peak concentrations (cmax) of gemcitabine were low at 5.5 µg/ml compared to the 10 to 30 µg/ml levels observed after a single intravenous dose of 1,000 mg/m2 gemcitabine. The formulation was still traceable after one hour and gemcitabine tissue concentrations are supportive of this extended drug exposure. No major histopathological changes were observed. The main limitation of this study is the lack of antitumor activity data. CONCLUSION: This preclinical evaluation of ST-UC demonstrated feasible instillation in the renal pelvis, no significant safety concerns, and sustained release of gemcitabine.

A polymeric paste-drug formulation for intratumoral treatment of prostate cancer.

OBJECTIVE: Focal therapy has emerged as a treatment option for low- to intermediate-risk localized prostate cancer (PCa) patients, to balance the risks for urinary and sexual morbidity of radical treatment with the psychological burden of active surveillance. In this context, we developed ST-4PC, an injectable, polymeric paste formulation containing docetaxel (dtx) and bicalutamide (bic) for image-guided focal therapy of PCa. The objective of this work was to evaluate the in vitro characteristics and in vivo toxicity and efficacy of ST-4PC. MATERIAL AND METHODS: In vitro drug release was evaluated using high-performance liquid chromatography. In vivo toxicity of blank- and drug-loaded ST-4PC was assessed in mice and rats. Tumor growth inhibition was evaluated in LNCaP subcutaneous (s.c.) and LNCaP-luc orthotopic xenograft models. Using the s.c. model, mice were monitored weekly for weight loss, tumor volume (TV) and serum PSA. For the orthotopic model, mice were additionally monitored for bioluminescence as measure of tumor growth. RESULTS: ST-4PC demonstrated a sustained and steady release of incorporated drugs with 50% dtx and 20% bic being released after 14 days. While no systemic toxicity was observed, dose-dependent local side effects from dtx developed in the s.c. but not in the orthotopic model, illustrating the limitations of s.c. models for evaluating local cytotoxic therapy. In the s.c. model, 0.1%/4% and 0.25%/4% dtx/bic ST-4PC paste significantly reduced PSA progression, but did not have a significant inhibitory effect on TV. ST-4PC loaded with 1%/4% dtx/bic significantly reduced TV, serum PSA, and bioluminescence in the orthotopic xenograft model. Compared with drugs dissolved in DMSO, ST-4PC significantly delayed tumor growth. CONCLUSION: Image-guided focal therapy using ST-4PC demonstrated promising inhibition of PSA progression and orthotopic tumor growth in vivo without significant toxicity, and warrants further clinical evaluation.

Castration induces autoantibody and T cell responses that correlate with inferior outcomes in an androgen-dependent murine tumor model.

We recently reported that hormone therapy induces antigen-specific autoantibody responses in prostate cancer patients. However, the contribution of autoantibody responses to clinical outcomes is unknown. We used an animal model to test the hypothesis that hormone therapy-induced immune responses may be associated with delayed tumor recurrence. Male DD/S mice bearing established tumors from the androgen-dependent Shionogi carcinoma line were castrated to induce tumor regression. Tumor-specific autoantibody responses were measured by immunoblot, and the underlying antigen was identified by serological screening of a cDNA expression library. T cell responses were assessed by immunohistochemistry and IFN-gamma ELISPOT. Following castration, 97% of mice underwent complete tumor regression. Of these, 72% experienced tumor recurrence 18-79 days postcastration, whereas the remaining 28% remained tumor-free for the duration of the experiment. In 55% of mice, castration induced autoantibody responses to an antigen identified as poly(A) binding protein nuclear 1 (PABPN1). Castration also induced PABPN1-specific T cell responses, which were highly correlated to autoantibody responses, and this was accompanied by dense infiltration of tumors by CD3+ T cells 1-2 weeks after castration. Unexpectedly, mice that developed autoantibody and T cell responses to PABPN1 showed a higher rate and shorter latency of tumor recurrence. In mice with recurrent tumors, T cell responses to PABPN1 were still detectable; however, T cell infiltrates were restricted to the peripheral stroma of tumors. In conclusion, castration-induced immune responses are associated with inferior outcomes in the Shionogi carcinoma model, raising concerns about the influence of treatment-induced immune responses on clinical outcomes in humans.

The insulin-like growth factor I receptor is required for Akt activation and suppression of anoikis in cells transformed by the ETV6-NTRK3 chimeric tyrosine kinase.

Signaling through the insulin-like growth factor I receptor (IGF-IR) axis is essential for transformation by many dominantly acting oncoproteins. However, the mechanism by which IGF-IR contributes to oncogenesis remains unknown. To examine this, we compared transformation properties of the oncogenic ETV6-NTRK3 (EN) chimeric tyrosine kinase in IGF-IR-null R- mouse embryo fibroblasts with R- cells engineered to reexpress IGF-IR (R+ cells). We previously showed that R- cells expressing EN (R- EN cells) are resistant to transformation but that transformation is restored in R+ cells. We now show that while R- EN cells have intact Ras-extracellular signal-regulated kinase signaling and cell cycle progression, they are defective in phosphatidylinositol-3-kinase (PI3K)-Akt activation and undergo detachment-induced apoptosis (anoikis) under anchorage-independent conditions. In contrast, R+ cells expressing EN (R+ EN cells) suppress anoikis and are fully transformed. The requirement for IGF-IR in R- EN cells is overcome by ectopic expression of either activated Akt or a membrane-targeted form of EN. Moreover, compared to R- EN cells, R+ EN cells show a dramatic increase in membrane localization of insulin receptor substrate 1 (IRS-1) in association with EN. Since EN is known to bind IRS-1 as an adaptor protein, our findings suggest that IGF-IR may function to localize EN/IRS-1 complexes to cell membranes, in turn facilitating PI3K-Akt activation and suppression of anoikis.

Establishment of a neuroendocrine prostate cancer model driven by the RNA splicing factor SRRM4.

Neuroendocrine prostate cancer (NEPC) is becoming more prevalent as more potent androgen receptor (AR) pathway inhibitors are applied to patients with metastatic tumors. However, there are limited cell and xenograft models currently available, hindering the investigation of signal pathways involved in regulating NEPC progression and the design of high throughput screening assays for inhibitors to treat NEPC patients. Here, we report an NEPC model, LnNE, that is derived from prostate adenocarcinoma cells and has global similarity in transcription and RNA splicing to tumors from NEPC patients. LnNE xenografts are castrate-resistant and highly aggressive. Its tumor take is ∼3-5 weeks and tumor doubling time is ∼2-3 weeks. LnNE expresses multiple neuroendocrine markers, preserves AR expression, but is PSA negative. Its neuroendocrine phenotype cannot be reversed by androgen treatment. LnNE cells grow as multi-cellular spheroids under 2-dimensional culture conditions similar to the NEPC cell line NCI-H660, but have higher proliferation rate and are easier to be transfected. LnNE cells can also adapt to 3-dimensional culture conditions in a 96-plate format, allowing high throughput screening assays. In summary, the LnNE model is useful to study the mechanisms of NEPC progression and to discover potential therapies for NEPC.

Outcomes of hyoid suspension for the treatment of obstructive sleep apnea.

OBJECTIVE: To examine the efficacy of hyoid myotomy and suspension as a treatment of hypopharyngeal obstruction in obstructive sleep apnea. DESIGN: Prospective, observational study. SETTING: Academic medical center. PATIENTS: Twenty-nine consecutive male patients with suspected hypopharyngeal obstruction INTERVENTIONS: Patients underwent hyoid suspension. Uvulopalatopharyngoplasty with or without tonsillectomy was performed at the same time for those patients who had not undergone this procedure previously. Patients underwent clinical examination and sleep study prior to surgery and approximately 1 year postoperatively. MAIN OUTCOME MEASURES: Primary outcome was a successful surgical result, defined as apnea-hypopnea index lower than 20, 50% or greater decline in apnea-hypopnea index, and no oxygen desaturations below 85% on the postoperative sleep study. Secondary outcomes included daytime sleepiness as determined by the Epworth Sleepiness Scale and the severity of snoring. Postoperative complications were also recorded. RESULTS: Only 5 (17%) of 29 patients achieved a successful outcome. The respiratory disturbance index did not change significantly for the group as a whole, although the lowest oxygen saturation did show some improvement. CONCLUSIONS: Hyoid suspension does not provide results equivalent to those reported for genioglossus advancement or multisession tongue radiofrequency. Hyoid suspension alone is not an efficacious treatment for hypopharyngeal airway obstruction in most patients with obstructive sleep apnea.

Hypoxia in the androgen-dependent Shionogi model for prostate cancer at three stages.

The objective of this study was to investigate a possible relationship between androgen status and hypoxia in the Shionogi murine prostate tumor model, which is widely used to study the effects of androgen withdrawal on hormone resistance and radiation response. Binding of the nitroimidazole hypoxia marker EF5 was assessed using the Cy3-tagged monoclonal antibody ELK3-51. Three hours after injection of EF5 (30 mg/kg), tumors from the following three stages were excised: androgen-dependent, regressed tumors 7 days after castration, and androgen-independent. Half of each tumor was disaggregated for analysis by flow cytometry and the remainder was flash frozen. Statistically significant differences (P < 0.01) were found between androgen-dependent, regressed and androgen-dependent tumors: approximately 30, approximately 2 and approximately 50% hypoxic cells, respectively. Frozen sections from androgen-dependent tumors exhibited highly variable EF5 binding; regressed tumors showed very little or no binding; each section from androgen-dependent tumors showed high levels and uniformly distributed binding of EF5. There was no correlation between the degree of hypoxia and tumor weight (P > 0.1). The results from this preliminary study indicate that hypoxia may play an important role with respect to the timing of irradiation in prostate cancer treatments and possibly may be a useful prognostic tool. In addition, hypoxia may also be relevant to progression in this disease after androgen ablation.

Informed consent in functional endoscopic sinus surgery: the patient's perspective.

OBJECTIVE: Informed consent is essential prior to functional endoscopic sinus surgery (FESS). The content of this discussion is often determined by each surgeon. Many doctors discuss all potential complications. This may provoke anxiety or deter from beneficial surgery. Our goal was to examine the process from the patient's perspective. Study design A list of potential complications was reviewed with FESS patients. Patients were asked questions concerning their consent and the potential complications before and after FESS. RESULTS: Both before and after surgery, patients felt that discussion of most potential complications was important. Vision changes and cerebrospinal fluid leak (CSF) received the highest scores. No significant differences were found between primary and revision cases. CONCLUSION: Patients wanted most potential FESS complications to be discussed. Even though this triggered anxiety, they felt it was important to have a thorough disclosure. Findings presented may impact how surgeons counsel patients prior to FESS.