S Gao

Protein arginine methyltransferase 5 is essential for growth of lung cancer cells.

PRMT5 (protein arginine methyltransferase 5) is an enzyme that catalyses transfer of methyl groups from S-adenosyl methionine to the arginine residues of histones or non-histone proteins and is involved in a variety of cellular processes. Although it is highly expressed in some tumours, its direct role in cancer growth has not been fully investigated. In the present study, in human lung tissue samples we found that PRMT5 was highly expressed in lung cancer cells, whereas its expression was not detectable in benign lung tissues. Silencing PRMT5 expression strongly inhibited proliferation of lung adenocarcinoma A549 cells in tissue culture, and silencing PRMT5 expression in A549 cells also abolished growth of lung A549 xenografts in mice. In vitro and in vivo studies showed that the cell growth arrest induced by loss of PRMT5 expression was partially attributable to down-regulation of fibroblast growth factor receptor signalling. These results suggest that PRMT5 and its methyltransferase activity is essential for proliferation of lung cancer cells and may serve as a novel target for the treatment of lung cancer.

Nuclear Transport Signals Control Cellular Localization and Function of Androgen Receptor Cofactor p44/WDR77

The androgen receptor (AR) cofactor p44/WDR77, which regulates expression of a set of androgen target genes, is required for differentiation of prostate epithelium. Aberrant localization of p44/WDR77 in the cytoplasm is associated with prostate tumorigenesis. Here, we describe studies that used the mouse prostate and human prostate cancer cells as model systems to investigate signals that control subcellular localization of p44/WDR77. We observed distinct subcellular location of p44/WDR77 during prostate development. p44/WDR77 localizes in the cytoplasm at the early stage of prostate development, when prostate epithelial cells are rapidly proliferating, and in the nucleus in adult prostate, when epithelial cells are fully differentiated. Subcellular localization assays designed to span the entire open-reading frame of p44/WDR77 protein revealed the presence of two nuclear exclusion signal (NES) and three nuclear localization signal (NLS) sequences in the p44/WDR77 protein. Site-directed mutagenesis of critical residues within an NLS led to loss of nuclear localization and transcriptional activity of p44/WDR77, suggesting that nuclear localization of p44/WDR77 is essential for its function as a transcriptional cofactor for AR. Three identified NLS were not functional in AR-positive prostate cancer (LNCaP and 22RV1) cells, which led to localization of p44/WDR77 in cytoplasm. The function of NLS in LNCaP cells could be restored by factor(s) from Cos 7 or PC3 cells. Mass spectrometric (MALDI-TOF/TOF) analysis identified proteins associated with an NLS and an NES in prostate cancer cells. These results provide a basis for understanding subcellular transport of p44/WDR77 during prostate development and tumorigenesis.

Altered differentiation and proliferation of prostate epithelium in mice lacking the androgen receptor cofactor p44/WDR77.

Although it has been observed that various cofactors modulate activity of the androgen receptor (AR), the specific relationship between AR cofactors and prostate development and functions has not been well studied. To determine whether AR cofactor p44/WDR77 is important in prostate growth and development, we examined prostate architecture in p44/WDR77-null mice and wild-type (WT) littermates. Prostate glands from p44/WDR77-deficient animals were not only smaller than those from WT mice but also had fewer branches and terminal duct tips and were deficient in production of secretory proteins. The p44/WDR77-null prostate tissue was less differentiated and hyperproliferative relative to WT littermates. In addition, the altered expression of androgen-regulated genes was observed in the p44/WDR77-null prostate. Thus, these results suggest that the AR cofactor p44/WDR77 plays important roles in prostate growth and differentiation by modulating AR-target gene expression.

P44/WDR77 restricts the sensitivity of proliferating cells to TGFβ signaling.

We previously reported that a novel WD-40 domain-containing protein, p44/WDR77, drives quiescent epithelial cells to re-enter the cell cycle and plays an essential role for growth of lung and prostate cancer cells. Transforming growth factor beta (TGFβ) signaling is important in the maintenance of non-transformed cells in the quiescent or slowly cycling stage. However, both non-transformed proliferating cells and human cancer cells are non-responsive to endogenous TGFβ signaling. The mechanism by which proliferating cells become refractory to TGFβ inhibition is not well established. Here, we found that silencing p44/WDR77 increased cellular sensitivity to TGFβ signaling and that this was inversely correlated with decreased cell proliferation. Smad2 or 3 phosphorylation, TGFβ-mediated transcription, and TGFβ2 and TGFβ receptor type II (TβRII) expression were dramatically induced by silencing of p44/WDR77. These data support the hypothesis that p44/WDR77 down-regulates the expression of the TGFβ ligand and its receptor, thereby leading to a cellular non-response to TGFβ signaling. Finally, we found that p44/WDR77 expression was correlated with cell proliferation and decreased TGFβ signaling during lung tumorigenesis. Together, these results suggest that p44/WDR77 expression causes the non-sensitivity of proliferating cells to TGFβ signaling, thereby contributing to cellular proliferation during lung tumorigenesis.

SU‐C‐213CD‐04: Evaluation of IsoCal Imaging Isocenter Calibration System for Varian OBI Machines

Purpose: To evaluate the accuracy and reproducibility of the IsoCal geometric calibration system for Varian OBI kV and EPIDimaging systems. Methods: IsoCal consists of hardware (IsoCal phantom, collimator plate) and software. IsoCal calibration starts by imaging the phantom and collimator plate using MV images with different collimator angles as well as MV and kV images at different gantry angles. The software then identifies objects on collimator plate and in the phantom to determine the location of the treatment isocenter and its relation to MV/kV imaging centers. It calculates offsets of the position of the imaging panels versus the treatment isocenter as function of gantry angle and writes a correction file that can be applied to MV/kV system to correct for offsets in the position of the panels. We performed IsoCal calibration three times in a row on 5 different linacs, each time with an independent setup. We also compared the IsoCal calibrations with an in‐house Winston‐Lutz‐based (WL) system as well as a Varian cube‐based (VC) system. Results: The IsoCal corrections ranged from 1.1 to 2.3 mm for MV and 1.1 to 1.8 mm for kV imagers across the 5 linacs. The variations in the three calibrations for each linac were within ±0.2 mm. The differences between IsoCal and the WL and VC systems were within 0.5 mm and 1.0 mm respectively. Without IsoCal the WL results showed discrepancies between the treatment isocenter and MV/kV imaging center ranging from 0.9 to 1.6 mm (MV) and 0.5 to 1.1 mm (kV), with IsoCal applied they were reduced to 0.2 to 0.4 mm (MV) and 0.3 to 0.4 mm (kV) across the 5 linacs.Conclusions: This study demonstrates that IsoCal is an accurate and consistent method for MV/kV image system calibration and periodic quality assurance.

cGMP-dependent protein kinase Iβ interacts with p44/WDR77 to regulate androgen receptor-driven gene expression.

The androgen receptor (AR) pathway plays critical roles in controlling differentiation and proliferation of prostate epithelial cells. We previously identified a novel AR cofactor, p44/WDR77, which specifically enhances AR transcriptional activity in the prostate gland and prostate cancer. To further elucidate p44/WDR77s role in the AR signaling pathway, we conducted a yeast two-hybrid screening and identified cGMP-dependent protein kinase (PKG) as a p44/WDR77-interacting protein. Further investigation by lusiferase assay and kinase assay demonstrated that PKG-Iβ physically interacted with and phosphorylated both p44 and AR and enhanced AR transactivity in synergy with p44 in an androgen- and cGMP-dependent manner. Furthermore, PKG1β expression promoted p44/WDR77 nuclear translocation and inhibited prostate cancer cell growth via G1 cell cycle arrest. Our findings characterize PKG as a novel regulator of AR-mediated transcription by enhancing AR cofactor p44/WDR77s function, which provide a novel mechanism for the growth regulation of prostate cancer cells by the androgen signaling.

Loss of the Androgen Receptor Cofactor p44/WDR77 Induces Astrogliosis

ABSTRACT Astrogliosis is induced by neuronal damage and is also a pathological feature of the major aging-related neurodegenerative disorders. The mechanisms that control the cascade of astrogliosis have not been well established. In a previous study, we identified a novel androgen receptor (AR)-interacting protein, p44/WDR77, that plays a critical role in the proliferation and differentiation of prostate epithelial cells. In the present study, we found that deletion of the p44/WDR77 gene caused premature death with dramatic astrogliosis in mouse brain. We further found that p44/WDR77 is expressed in astrocytes and that loss of p44/WDR77 expression in astrocytes leads to growth arrest and astrogliosis. The astrocyte activation induced by deletion of the p44/WDR77 gene was associated with upregulation of p21Cip1 expression and NF-κB activation. Silencing p21Cip1 or NF-κB p65 expression with short hairpin RNA (shRNA) abolished astrocyte activation and rescued the astrocyte growth inhibition induced by deletion of the p44/WDR77 gene. Our results reveal a novel role for p44/WDR77 in the control of astrocyte activation through p21Cip1 and NF-κB signaling.

A new respiratory monitoring and processing system based on Wii remote: Proof of principle

PURPOSE To create a patient respiratory management system and patient self-practice tool using the Wii remote, a widely available consumer hardware product. METHODS The Wii remote (Wiimote) (Nintendo, Redmond, WA) contains an infrared (IR) camera that can track up to four spots whose coordinates are reported to a host computer via Bluetooth. The Wiimote is capable of tracking a fiducial box currently used by a commercial monitoring system [Real-time Position Management(TM) (RPM) system, Varian Associates, Palo Alto, CA], if the correct IR source is used. The authors validated the Wiimote tracking by comparing the amplitude and frequency of signals among those reported by Wiimote with known movements from an inhouse servo-driven respiratory simulator, as well as with those measured using the RPM. The simulator comparison was done using standard sinusoid signals with amplitude of 2.0 cm as well as recorded patient respiratory traces. The RPM comparisons were done by simultaneously recording the RPM reflective box position with the Wiimote and the RPM. Timing was compared between these two systems by using the digital beam-on signal from the CT scanner, for the 4DCT to synchronize these acquisitions. RESULTS The data acquisition rate from the Wiimote was 100.0 ± 0.4 Hz with a version 2.1 Bluetooth adaptor. The standard deviation of the height of the motion extrema was 0.06 and 1.1 mm when comparing those measured by the Wiimote and the servomotor encoder for standard sinusoid signal and prerecorded patient respiratory signal, respectively. The standard deviation of the amplitude of motion extrema between the Wiimote and RPM was 0.9 mm and the timing difference was 253 ms. CONCLUSION The performance of Wiimote shows promise for respiratory monitoring for its faster sampling rate as well as the potential optical and GPU abilities. If used with care it can deliver reasonable spatial and temporal accuracy.

SU-G-JeP3-15: Is the Reproducibility with Respect to Bone of Tumor Position at Simulation for Breath Hold CT Scans Correlated to the Reproducibility for Multiple Breath Hold CBCTs at Treatment in SBRT Thoracic Patients?

PURPOSE To evaluate correlation between the reproducibility of tumor position under feedback guided voluntary deep inspiration breath hold gating at simulation and at treatment. METHODS All patients treated with breath hold (BH) have 3-6 BH CTs taken at simulation (sim). In addition, if the relationship between the tumor and nearby bony anatomy on treatment BH CT(or CBCT) is found to be greater than 5 mm different at treatment than it was at sim, a repeat BH CT is taken before treatment. We retrospectively analyzed the sim CTs for 19 patients who received BH SBRT lung treatments and had repeat BH CT on treatment. We evaluated the reproducibility of the tumor position during the simulation CTs and compared this to the reproducibility of the tumor position on the repeat treatment CT with our in-house CT alignment software (CT-Assisted Targeting for Radiotherapy). RESULTS Comparing the tumor position for multiple simulation BH CTs, we calculated: maximum difference (max) = 0.69cm; average difference (x) = 0.28cm; standard deviation (σ) = 0.18cm. Comparing the repeat BH CBCTs on treatment days we calculated: max = 0.44cm; x = 0.16cm; σ = 0.22cm. We also found that for 95% of our BH cases, the absolute variation in tumor position within the same imaging day was within 5mm of the range at the time of simulation and treatment. We found that 75% of the BH cases had less residual tumor motion on treatment days than at simulation. CONCLUSION This suggests that a GTV contour based upon the residual tumor motion in multiple BH datasets plus 2 mm margin should be sufficient to cover the full range of residual tumor motion on treatment days.

SU-F-E-18: Training Monthly QA of Medical Accelerators: Illustrated Instructions for Self-Learning

PURPOSE To develop and test clear illustrated instructions for training of monthly mechanical QA of medical linear accelerators. METHODS Illustrated instructions were created for monthly mechanical QA with tolerance tabulated, and underwent several steps of review and refinement. Testers with zero QA experience were then recruited from our radiotherapy department (1 student, 2 computational scientists and 8 dosimetrists). The following parameters were progressively de-calibrated on a Varian C-series linac: Group A = gantry angle, ceiling laser position, X1 jaw position, couch longitudinal position, physical graticule position (5 testers); Group B = Group A + wall laser position, couch lateral and vertical position, collimator angle (3 testers); Group C = Group B + couch angle, wall laser angle, and optical distance indicator (3 testers). Testers were taught how to use the linac, and then used the instructions to try to identify these errors. A physicist observed each session, giving support on machine operation, as necessary. The instructions were further tested with groups of therapists, graduate students and physics residents at multiple institutions. We have also changed the language of the instructions to simulate using the instructions with non-English speakers. RESULTS Testers were able to follow the instructions. They determined gantry, collimator and couch angle errors within 0.4, 0.3, and 0.9degrees of the actual changed values, respectively. Laser positions were determined within 1mm, and jaw positions within 2mm. Couch position errors were determined within 2 and 3mm for lateral/longitudinal and vertical errors, respectively. Accessory positioning errors were determined within 1mm. ODI errors were determined within 2mm when comparing with distance sticks, and 6mm when using blocks, indicating that distance sticks should be the preferred approach for inexperienced staff. CONCLUSION Inexperienced users were able to follow these instructions, and catch errors within the criteria suggested by AAPM TG142 for linacs used for IMRT.