David Kozono

From structure to disease : the evolving tale of aquaporin biology

Our understanding of the movement of water through cell membranes has been greatly advanced by the discovery of a family of water-specific, membrane-channel proteins — the aquaporins. These proteins are present in organisms at all levels of life, and their unique permeability characteristics and distribution in numerous tissues indicate diverse roles in the regulation of water homeostasis. The recognition of aquaporins has stimulated a reconsideration of membrane water permeability by investigators across a wide range of disciplines.

Aquaporin water channels: molecular mechanisms for human diseases1

Although water is the major component of all biological fluids, the molecular pathways for water transport across cell membranes eluded identification until the discovery of the aquaporin family of water channels. The atomic structure of mammalian AQP1 illustrates how this family of proteins is freely permeated by water but not protons (hydronium ions, H3O+). Definition of the subcellular sites of expression predicted their physiological functions and potential clinical disorders. Analysis of several human disease states has confirmed that aquaporins are involved in multiple different illnesses including abnormalities of kidney function, loss of vision, onset of brain edema, starvation, and arsenic toxicity.

Aquaglyceroporin AQP9: solute permeation and metabolic control of expression in liver

Aquaglyceroporins form the subset of the aquaporin water channel family that is permeable to glycerol and certain small, uncharged solutes. AQP9 has unusually broad solute permeability and is expressed in hepatocyte plasma membranes. Proteoliposomes reconstituted with expressed, purified rat AQP9 protein were compared with simple liposomes for solute permeability. At pH 7.5, AQP9 proteoliposomes exhibited Hg2+-inhibitible glycerol and urea permeabilities that were increased 63-fold and 90-fold over background. β-Hydroxybutyrate permeability was not increased above background, and osmotic water permeability was only minimally elevated. During starvation, the liver takes up glycerol for gluconeogenesis. Expression of AQP9 in liver was induced up to 20-fold in rats fasted for 24–96 h, and the AQP9 level gradually declined after refeeding. No changes in liver AQP9 levels were observed in rats fed ketogenic diets or high-protein diets, but AQP9 levels were elevated in livers of rats made diabetic by streptozotocin injection. When blood glucose levels of the diabetic rats were restored to normal by insulin treatments, the AQP9 levels returned to baseline. Confocal immunofluorescence revealed AQP9 immunostaining on the sinusoidal surfaces of hepatocyte plates throughout the livers of control rats. Denser immunostaining was observed in the same distribution in livers of fasted and streptozotocin-treated rats. We conclude that AQP9 serves as membrane channel in hepatocytes for glycerol and urea at physiological pH, but not for β-hydroxybutyrate. In addition, levels of AQP9 expression fluctuate depending on the nutritional status of the subject and the circulating insulin levels.

Characterization of Aquaporin-6 as a Nitrate Channel in Mammalian Cells REQUIREMENT OF PORE-LINING RESIDUE THREONINE 63

Aquaporins (AQP) were originally regarded as plasma membrane channels that are freely permeated by water or small uncharged solutes but not by ions. Unlike other aquaporins, AQP6 overexpressed in Xenopus laevis oocytes was previously found to exhibit Hg2+ or pH-activated ion conductance. AQP6 could not be analyzed electrophysiologically in mammalian cells, however, because the protein is restricted to intracellular vesicles. Here we report that addition of a green fluorescence protein (GFP) tag to the N terminus of rat AQP6 (GFP-AQP6) redirects the protein to the plasma membranes of transfected mammalian cells. This permitted measurement of rapid, reversible, pH-induced anion currents by GFP-AQP6 in human embryonic kidney 293 cells. Surprisingly, anion selectivity relative to Cl− revealed high nitrate permeability even at pH 7.4;P NO3 /P Cl > 9.8. Site-directed mutation of a pore-lining threonine to isoleucine at position 63 at the midpoint of the channel reduced NO3 −/Cl− selectivity. Moreover, no anomalous mole-fraction behavior was observed with NO3 −/Cl− mixtures, suggesting a single ion-binding pore in each subunit. Our studies indicate that AQP6 exhibits a new form of anion permeation with marked specificity for nitrate conferred by a specific pore-lining residue, observations that imply that the primary role of AQP6 may be in cellular regulation rather than simple fluid transport.

A DNA Repair Pathway–Focused Score for Prediction of Outcomes in Ovarian Cancer Treated With Platinum-Based Chemotherapy

Background New tools are needed to predict outcomes of ovarian cancer patients treated with platinum-based chemotherapy. We hypothesized that a molecular score based on expression of genes that are involved in platinum-induced DNA damage repair could provide such prognostic information. Methods Gene expression data was extracted from The Cancer Genome Atlas (TCGA) database for 151 DNA repair genes from tumors of serous ovarian cystadenocarcinoma patients (n = 511). A molecular score was generated based on the expression of 23 genes involved in platinum-induced DNA damage repair pathways. Patients were divided into low (scores 0–10) and high (scores 11–20) score groups, and overall survival (OS) was analyzed by Kaplan–Meier method. Results were validated in two gene expression microarray datasets. Association of the score with OS was compared with known clinical factors (age, stage, grade, and extent of surgical debulking) using univariate and multivariable Cox proportional hazards models. Score performance was evaluated by receiver operating characteristic (ROC) curve analysis. Correlations between the score and likelihood of complete response, recurrence-free survival, and progression-free survival were assessed. Statistical tests were two-sided. Results Improved survival was associated with being in the high-scoring group (high vs low scores: 5-year OS, 40% vs 17%, P < .001), and results were reproduced in the validation datasets (P < .05). The score was the only pretreatment factor that showed a statistically significant association with OS (high vs low scores, hazard ratio of death = 0.40, 95% confidence interval = 0.32 to 0.66, P < .001). ROC curves indicated that the score outperformed the known clinical factors (score in a validation dataset vs clinical factors, area under the curve = 0.65 vs 0.52). The score positively correlated with complete response rate, recurrence-free survival, and progression-free survival (Pearson correlation coefficient [r2] = 0.60, 0.84, and 0.80, respectively; P < .001 for all). Conclusion The DNA repair pathway–focused score can be used to predict outcomes and response to platinum therapy in ovarian cancer patients.

Ion permeation of AQP6 water channel protein. Single channel recordings after Hg2+ activation.

Aquaporin-6 (AQP6) has recently been identified as an intracellular vesicle water channel with anion permeability that is activated by low pH or HgCl2. Here we present direct evidence of AQP6 channel gating using patch clamp techniques. Cell-attached patch recordings of AQP6 expressed inXenopus laevis oocytes indicated that AQP6 is a gated channel with intermediate conductance (49 picosiemens in 100 mm NaCl) induced by 10 μmHgCl2. Current-voltage relationships were linear, and open probability was fairly constant at any given voltage, indicating that Hg2+-induced AQP6 conductance is voltage-independent. The excised outside-out patch recording revealed rapid activation of AQP6 channels immediately after application of 10 μmHgCl2. Reduction of both Na+ and Cl− concentrations from 100 to 30 mm did not shift the reversal potential of the Hg2+-induced AQP6 current, suggesting that Na+ is as permeable as Cl−. The Na+ permeability of Hg2+-induced AQP6 current was further demonstrated by22Na+ influx measurements. Site-directed mutagenesis identified Cys-155 and Cys-190 residues as the sites of Hg2+ activation both for water permeability and ion conductance. The Hill coefficient from the concentration-response curve for Hg2+-induced conductance was 1.1 ± 0.3. These data provide the first evidence of AQP6 channel gating at a single-channel level and suggest that each monomer contains the pore region for ions based on the number of Hg2+-binding sites and the kinetics of Hg2+-activation of the channel.

Targeting EGFR Induced Oxidative Stress by PARP1 Inhibition in Glioblastoma Therapy

Despite the critical role of Epidermal Growth Factor Receptor (EGFR) in glioblastoma pathogenesis [1], [2], EGFR targeted therapies have achieved limited clinical efficacy [3]. Here we propose an alternate therapeutic strategy based on the conceptual framework of non-oncogene addiction [4], [5]. A directed RNAi screen revealed that glioblastoma cells over-expressing EGFRvIII [6], an oncogenic variant of EGFR, become hyper-dependent on a variety of DNA repair genes. Among these, there was an enrichment of Base Excision Repair (BER) genes required for the repair of Reactive Oxygen Species (ROS)-induced DNA damage, including poly-ADP ribose polymerase 1 (PARP1). Subsequent studies revealed that EGFRvIII over-expression in glioblastoma cells caused increased levels of ROS, DNA strand break accumulation, and genome instability. In a panel of primary glioblastoma lines, sensitivity to PARP1 inhibition correlated with the levels of EGFR activation and oxidative stress. Gene expression analysis indicated that reduced expression of BER genes in glioblastomas with high EGFR expression correlated with improved patient survival. These observations suggest that oxidative stress secondary to EGFR hyper-activation necessitates increased cellular reliance on PARP1 mediated BER, and offer critical insights into clinical trial design.

Necroptosis is a novel mechanism of radiation-induced cell death in anaplastic thyroid and adrenocortical cancers

BACKGROUND Necroptosis is a recently described mechanism of programmed cellular death. We hypothesize that necroptosis plays an important role in radiation-induced cell death in endocrine cancers. METHODS Thyroid and adrenocortical carcinoma cell lines were exposed to increasing doses of radiation in the presence of necroptosis inhibitor Nec-1 and/or apoptosis-inhibitor zVAD. H295R cells deficient in receptor interacting protein 1 (RIP1), an essential kinase for necroptosis, were used as controls. Survival curves were generated at increasing doses of radiation. RESULTS Nec-1 and zVAD increased cellular survival with increasing doses of radiotherapy in 8505c, TPC-1, and SW13. Both inhibitors used together had an additive effect. At 6 Gy, 8505c, TPC-1, and SW13 cell survival was significantly increased compared to controls by 40%, 33%, and 31% with Nec-1 treatment, by 53%, 47%, and 44% with zVAD treatment, and by 80%, 70%, and 65% with both compounds, respectively (P < .05). H295R showed no change in survival with Nec-1 treatment. The radiobiologic parameter quasithreshold dose was significantly increased in 8505c, TPC-1, and SW13 cells when both Nec-1 and zVAD were used in combination to inhibit necroptosis and apoptosis together, revealing resistance to standard doses of fractionated therapeutic radiation. CONCLUSION Necroptosis contributes to radiation-induced cell death. Future studies should investigate ways to promote the activation of necroptosis to improve radiosensitivity.

Aggressive therapy for patients with non-small cell lung carcinoma and synchronous brain-only oligometastatic disease is associated with long-term survival.

OBJECTIVES Optimal therapy for patients with non-small cell lung carcinoma (NSCLC) presenting with synchronous brain-only oligometastases (SBO) is not well defined. We sought to analyze the effect of differing therapeutic paradigms in this subpopulation. MATERIALS AND METHODS We retrospectively analyzed NSCLC patients with 1-4 SBO diagnosed between 1/2000 and 1/2011 at our institution. Patients with T0 tumors or documented Karnofsky Performance Status <70 were excluded. Aggressive thoracic therapy (ATT) was defined as resection of the primary disease or chemoradiotherapy whose total radiation dose exceeded 45 Gy. Cox proportional hazards and competing risks models were used to analyze factors affecting survival and first recurrence in the brain. RESULTS Sixty-six patients were included. Median follow-up was 31.9 months. Intrathoracic disease extent included 9 stage I, 10 stage II and 47 stage III patients. Thirty-eight patients received ATT, 28 did not. Patients receiving ATT were younger (median age 55 vs. 60.5 years, p=0.027) but were otherwise similar to those who did not. Receipt of ATT was associated with prolonged median overall survival (OS) (26.4 vs. 10.5 months; p<0.001) with actuarial 2-year rates of 54% vs. 26%. ATT remained associated with OS after controlling for age, thoracic stage, performance status and initial brain therapy (HR 0.40, p=0.009). On multivariate analysis, the risk of first failure in the brain was associated with receipt of ATT (HR 3.62, p=0.032) and initial combined modality brain therapy (HR 0.34, p=0.046). CONCLUSION Aggressive management of thoracic disease in NSCLC patients with SBO is associated with improved survival. Careful management of brain disease remains important, especially for those treated aggressively.

Updated patterns of failure after multimodality therapy for malignant pleural mesothelioma

OBJECTIVE We have previously described patterns of failure after extrapleural pneumonectomy and multimodality therapy for malignant pleural mesothelioma and sought to update our results with a larger cohort of recent patients. METHODS A total of 169 patients underwent extrapleural pneumonectomy without preoperative chemotherapy between 2001 and 2010. Data for treatment, recurrence, and survival were determined from medical records. A thoracic radiologist reviewed postoperative computed tomography or positron emission tomography computed tomography scans to determine sites of recurrence. Time to recurrence was estimated by the Kaplan-Meier method. Rates were compared using the Fisher exact test. RESULTS The median age of patients was 62 years. Histology on final pathology was epithelial for 104 patients (62%) and nonepithelial for 65 patients (38%). A total of 132 patients (78%) received heated intraoperative chemotherapy; 77 patients (45%) received adjuvant chemotherapy, and 71 patients (42%) received adjuvant radiation therapy. Most chemotherapy regimens included platinum or pemetrexed. Median radiation therapy dose was 54 Gy. Among 158 evaluable patients, a recurrence developed in 118 (75%). Median follow-up was 83 months, median time to recurrence was 13.1 months, and median survival was 15 months. Sites of first recurrence were in the ipsilateral hemithorax or mediastinum for 54% of patients, in the abdomen for 39% of patients, in the contralateral hemithorax for 28% of patients, and in other distant sites for 5% of patients. Some patients had simultaneous recurrences in multiple sites. CONCLUSIONS The most common site of recurrence after extrapleural pneumonectomy and planned multimodality therapy remains the ipsilateral hemithorax (including mediastinum), and true distant failure (other than the abdomen or contralateral hemithorax) remains unusual. The distribution of recurrences is strikingly similar to our prior report.