W Hu

NF-κB and Enhancer-binding CREB Protein Scaffolded by CREB-binding Protein (CBP)/p300 Proteins Regulate CD59 Protein Expression to Protect Cells from Complement Attack

Background: CD59 is the sole membrane complement regulatory protein in restricting membrane attack complex assembly. Results: CD59 gene produces eight transcripts that share three transcriptional initiation sites but the same open reading frame. Conclusion: NF-κB and CREB (as an enhancer-binding protein) bridged by CBP/p300 are responsible for the inducible expression of CD59. Significance: CD59 regulation mechanism suggests potential drug targets for controlling various complement-related human diseases. The complement system can be activated spontaneously for immune surveillance or induced to clear invading pathogens, in which the membrane attack complex (MAC, C5b-9) plays a critical role. CD59 is the sole membrane complement regulatory protein (mCRP) that restricts MAC assembly. CD59, therefore, protects innocent host cells from attacks by the complement system, and host cells require the constitutive and inducible expression of CD59 to protect themselves from deleterious destruction by complement. However, the mechanisms that underlie CD59 regulation remain largely unknown. In this study we demonstrate that the widely expressed transcription factor Sp1 may regulate the constitutive expression of CD59, whereas CREB-binding protein (CBP)/p300 bridge NF-κB and CREB, which surprisingly functions as an enhancer-binding protein to induce the up-regulation of CD59 during in lipopolysaccharide (LPS)-triggered complement activation, thus conferring host defense against further MAC-mediated destruction. Moreover, individual treatment with LPS, TNF-α, and the complement activation products (sublytic MAC (SC5b-9) and C5a) could increase the expression of CD59 mainly by activating NF-κB and CREB signaling pathways. Together, our findings identify a novel gene regulation mechanism involving CBP/p300, NF-κB, and CREB; this mechanism suggests potential drug targets for controlling various complement-related human diseases.

A novel CRIg-targeted complement inhibitor protects cells from complement damage

The inappropriate activation of complement may contribute to various immune diseases. The alternative pathway (AP) predominates during complement activation regardless of the initiating pathways. Hence, the main AP regulator factor H (FH) holds great potential as an attractive therapeutic intervention. In addition, complement receptor of the immunoglobulin superfamily (CRIg) has been demonstrated to inhibit AP and, more notably, still specifically binds to C3b/iC3b. We thus developed novel CRIg‐targeted complement inhibitors by connecting the functional domains of CRIg and FH, which we termed CRIg‐FH and CRIg‐L‐FH. CRIg‐L‐FH, slightly more potent than CRIg‐FH, considerably inhibited both AP‐ and also classical pathway (CP)‐mediated hemolysis and successfully eliminated the deposition of C3b/iC3b. Kinetic analysis further revealed that the binding affinity constant (KD) of CRIg/FH was in the micromolar range, consistent with its long‐lasting binding to complement‐attacked cells. CRIg‐L‐FH efficiently protected aberrant erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH) from AP‐and CP‐mediated complement damage (IC50 was 22.43 and 64.69 nM, respectively). Moreover, CRIg‐L‐FH was found to inhibit complement activation induced by the anti‐Thy1 antibody in a mesangiopro‐liferative glomerulonephritis (MPGN) rat model. Hence, CRIg‐L‐FH protects glomerular mesangial cells (GMCs) from complement‐mediated injury and proliferative lesions. These findings strongly suggest that CRIg/FH is a potential therapeutic drug candidate for a range of complement‐mediated diseases.—Qiao, Q., Teng, X., Wang, N., Lu, R., Guo, L., Zhang, X., Du, Y., Wang, W., Chen, S., Wu, Q., He, G., Wang, Y., and Hu, W., A novel CRIg‐targeted complement inhibitor protects cells from complement damage. FASEB J. 28, 4986–4999 (2014). www.fasebj.org

Potential impact of cardiac dose-volume on acute cardiac toxicity following concurrent trastuzumab and radiotherapy.

PURPOSE To analyse the risk factors associated with acute cardiotoxicity in HER2-positive breast cancer patients receiving concurrent trastuzumab and radiotherapy. PATIENTS AND METHODS Medical records of 45 breast cancer patients treated between 02/2009 and 02/2011 by concurrent trastuzumab and radiotherapy were collected. Radiation was delivered to the conserved breast or chest wall with or without regional nodes. Dose prescription was 50Gy in 25 fractions over five weeks with a tumor bed boost of 10Gy in 5 fractions in breast conservation. Acute cardiotoxicity (grade≥1) was defined using NCI-CTC v2.0. Doses to the heart and left ventricle were quantified. RESULTS Median follow-up of left ventricular ejection fraction and clinical assessment from the completion of radiotherapy was 10 months (range: 3-27 months) and 25 months (range: 13-40 months), respectively. Ten (22.2%) and one (2.2%) of the 45 patients developed grade 1 and grade 2 cardiotoxicity, respectively. For 24 left-sided patients, the mean heart dose was significantly higher in those with cardiac events compared to those without (10.14 and 6.27Gy, respectively; P<0.05). A continuous increase of D17-D57 and V10-V15 of the heart and increase of D40-D80 and V5-V7 of the left ventricle were statistically significant in left-sided patients who developed cardiotoxicity compared with those who did not (P<0.05). No significant relationship of dosimetric parameters of cardiac structures and cardiac events was found in right-sided patients. CONCLUSIONS Left-sided irradiation with increased low dose-volume and mean heart dose were associated with increased but reversible low-grade early cardiac toxicity after use of concurrent trastuzumab.

CD59 Regulation by SOX2 Is Required for Epithelial Cancer Stem Cells to Evade Complement Surveillance

Summary Cancer stem cells (CSCs) are highly associated with therapy resistance and metastasis. Interplay between CSCs and various immune components is required for tumor survival. However, the response of CSCs to complement surveillance remains unknown. Herein, using stem-like sphere-forming cells prepared from a mammary tumor and a lung adenocarcinoma cell line, we found that CD59 was upregulated to protect CSCs from complement-dependent cytotoxicity. CD59 silencing significantly enhanced complement destruction and completely suppressed tumorigenesis in CSC-xenografted nude mice. Furthermore, we identified that SOX2 upregulates CD59 in epithelial CSCs. In addition, we revealed that SOX2 regulates the transcription of mCd59b, leading to selective mCD59b abundance in murine testis spermatogonial stem cells. Therefore, we demonstrated that CD59 regulation by SOX2 is required for stem cell evasion of complement surveillance. This finding highlights the importance of complement surveillance in eliminating CSCs and may suggest CD59 as a potential target for cancer therapy.

The membrane complement regulatory protein CD59 promotes tumor growth and predicts poor prognosis in breast cancer

Breast cancer is the most prevalent type of cancer among women. CD59, a membrane complement regulatory protein, has been demonstrated to be overexpressed in most solid tumors, where it facilitates tumor cell escape from complement surveillance. However, the role of CD59 in breast cancer growth and clinical prognosis is not fully revealed. To investigate the role of CD59 in breast cancer growth and prognostic significance, we knocked down CD59 in a breast cancer cell line that is highly metastatic to the lungs, MDA-MB‑231-HM. Cell growth was measured in vitro and in vivo using a xenograft model. In addition, clinical data on a cohort of 120 patients with or without lung metastasis was analyzed based on CD59 expression, which was detected by immunohistochemistry. Knockdown of CD59 significantly inhibited MDA-MB‑231-HM cell growth both in vitro and in vivo. An analysis of clinical data on 120 patients revealed that patients with CD59 overexpression may have a worse prognosis. CD59 may therefore be a prognostic biomarker for poor outcome in breast cancer patients.

SU-E-T-59: A Novel Multi-Beam Dynamic IMRT with Fixed-Jaw Technique for Left Breast Cancer Patients with Regional Lymph Nodes Radiotherapy

Purpose: This study was to investigate the dosimetric benefit of a novel intensity modulated radiation therapy (IMRT) technique for irradiating the left breast and regional lymph node (RLN). Methods: The breast and RLN (internal mammary node and periclavicular node) and normal tissue were contoured for 16 consecutive left-sided breast cancer patients previously treated with RT after lumpectomy. Nine equi-spaced fields IMRT (9 -field IMRT), tangential multi-beam IMRT (tangential-IMRT) and IMRT with fixed-jaw technique (FJT-IMRT) were developed and compared with three-dimensional conformal RT (3DCRT). Prescribed dose was 50 Gy in 25 fractions. Dose distributions and dose volume histograms were used to evaluate plans. Results: All IMRTs achieved similar target coverage and substantially reduced heart V30 and V20 compared to the 3DCRT. The average heart mean dose had different changes, which were 9.0Gy for 9-field IMRT, 5.7Gy for tangential-IMRT and 4.2Gy for FJT-IMRT. For the contralateral lung and breast, the 9-field IMRT has the highest mean dose; and the FJT-IMRT and tangential-IMRT had similar lower value. For the thyroid, both 9-field IMRT and FJT-IMRT had similar V30 (20% and 22%) and were significantly lower than that of 3DCRT (34%) and tangential-IMRT (46%). Moreover, the thyroid mean dose of FJT-IMRT is the lowest. For cervical esophagus and humeral head, the FJT-IMRT also had the best sparing. Conclusion: All 9-field IMRT, tangential-IMRT and FJT-IMRT had superiority for targets coverage and substantially reduced the heart volume of high dose irradiation. The FJT-IMRT showed advantages of avoiding the contralateral breast and lung irradiation and decreasing the thyroid, humeral head and cervical esophagus radiation dose at the expense of a slight monitor units (MUs) increasing.

The Association Between VEGF +936C/T and −634G/C Polymorphisms and Breast Cancer Susceptibility, Tumor Growth, and Metastases: Evidence From 20,728 Subjects

The relation between the polymorphisms of vascular endothelial growth factor (VEGF) and breast cancer remains inconclusive. In our meta-analysis based on 10,340 breast cancer patients and 10,388 controls, we found breast cancer susceptibility was elevated in individuals carrying the VEGF +936C allele, especially in Asians, and the +936CC increases tumor growth. The G allele of –634G/C polymorphism reduces breast cancer susceptibility in Asians, and breast cancer patients of –634GG genotype has decreased tumor growth. These results suggest that both the VEGF +936C/T and –634G/C polymorphisms influence breast cancer susceptibility and tumor growth, instead of metastasis.

Delineation of the Cardiac Substructures Based on PET-CT and Contrast-Enhanced CT in Patients with Left Breast Cancer Treated with Postoperative Radiotherapy:

The aim of this study is to evaluate the volume differences between contrast-enhanced CT-based left ventricle (LV) and PET-CT-based LV and assess the impact of dose on the substructure volume differences in patients with left breast cancer treated with adjuvant radiotherapy. From October 2008 to February 2009, 14 patients with post-operatively confirmed left breast cancer were enrolled in the current study. The patients were scanned using contrast-enhanced CT for simulation, and 18F-FDG PET-CT was employed to display the structure of the left ventricle of each before radiotherapy (RT). The LV was delineated based on both contrast-enhanced CT and PET-CT. And other substructures, such as the left anterior descending coronary artery (LAD), were contoured in each patient, with the six-field simple intensity modulated radiotherapy (sIMRT) technique created for all. The mean volumes of the left ventricle based on contrast-enhanced CT (LV-CT) and PET-CT (LV-PET) were found to be 107.296 cm3 and 112.931 cm3, respectively (p = 0.06). The volume of LV receiving ≥50% prescription dose was significantly correlated with the volume of the heart receiving the same dosage (γ = 0.869). There was less correlation between the volume of LAD and that of the heart under the same condition (γ = 0.22). As a conclusion, the left ventricle can be delineated effectively based on the image of PET-CT, the contrast-enhanced CT based LV can serve as an appropriate alternative. Moreover, the volume of LV receiving high dose in RT closely correlated with the volume of the heart using sIMRT technique, which may pave the way for further exploring radiation-induced cardiac injuries in patients with left breast cancer.

SU-F-R-33: Can CT and CBCT Be Used Simultaneously for Radiomics Analysis

PURPOSE To investigate whether CBCT and CT can be used in radiomics analysis simultaneously. To establish a batch correction method for radiomics in two similar image modalities. METHODS Four sites including rectum, bladder, femoral head and lung were considered as region of interest (ROI) in this study. For each site, 10 treatment planning CT images were collected. And 10 CBCT images which came from same site of same patient were acquired at first radiotherapy fraction. 253 radiomics features, which were selected by our test-retest study at rectum cancer CT (ICC>0.8), were calculated for both CBCT and CT images in MATLAB. Simple scaling (z-score) and nonlinear correction methods were applied to the CBCT radiomics features. The Pearson Correlation Coefficient was calculated to analyze the correlation between radiomics features of CT and CBCT images before and after correction. Cluster analysis of mixed data (for each site, 5 CT and 5 CBCT data are randomly selected) was implemented to validate the feasibility to merge radiomics data from CBCT and CT. The consistency of clustering result and site grouping was verified by a chi-square test for different datasets respectively. RESULTS For simple scaling, 234 of the 253 features have correlation coefficient ρ>0.8 among which 154 features haveρ>0.9 . For radiomics data after nonlinear correction, 240 of the 253 features have ρ>0.8 among which 220 features have ρ>0.9. Cluster analysis of mixed data shows that data of four sites was almost precisely separated for simple scaling(p=1.29 * 10-7 , χ2 test) and nonlinear correction (p=5.98 * 10-7 , χ2 test), which is similar to the cluster result of CT data (p=4.52 * 10-8 , χ2 test). CONCLUSION Radiomics data from CBCT can be merged with those from CT by simple scaling or nonlinear correction for radiomics analysis.

SU-F-T-251: The Quality Assurance for the Heavy Patient Load Department in the Developing Country: The Primary Experience of An Entire Workflow QA Process Management in Radiotherapy

PURPOSE To implement an entire workflow quality assurance (QA) process in the radiotherapy department and to reduce the error rates of radiotherapy based on the entire workflow management in the developing country. METHODS The entire workflow QA process management starts from patient registration to the end of last treatment including all steps through the entire radiotherapy process. Error rate of chartcheck is used to evaluate the the entire workflow QA process. Two to three qualified senior medical physicists checked the documents before the first treatment fraction of every patient. Random check of the treatment history during treatment was also performed. A total of around 6000 patients treatment data before and after implementing the entire workflow QA process were compared from May, 2014 to December, 2015. RESULTS A systemic checklist was established. It mainly includes patients registration, treatment plan QA, information exporting to OIS(Oncology Information System), documents of treatment QAand QA of the treatment history. The error rate derived from the chart check decreases from 1.7% to 0.9% after our the entire workflow QA process. All checked errors before the first treatment fraction were corrected as soon as oncologist re-confirmed them and reinforce staff training was accordingly followed to prevent those errors. CONCLUSION The entire workflow QA process improved the safety, quality of radiotherapy in our department and we consider that our QA experience can be applicable for the heavily-loaded radiotherapy departments in developing country.