Zinc Transporter 4 (ZIP4): A Predictive Biomarker for Decreasing Radiosensitivity and Promoting Tumor Migration and Metastasis Through TGF-β–Dependent Manner in Nasopharyngeal Carcinoma (NPC)

Volume 90  Number 1S  Supplement 2014

Oral Scientific Sessions

S89

Purpose/Objective(s): Although the majority of prostate cancer (PCa) can be effectively treated with radiation therapy, approximately 50% of men with high risk PCa will have recurrence of the disease. A number of mechanisms, including DNA repair efficiency, can influence cellular sensitivity to radiation and chemotherapy. The role of microRNAs (miRNAs) in these processes has not been fully elucidated. miRNAs are small non-coding RNAs which post-transcriptionally regulate gene expression and are predicted to regulate over 30% of human genes. Here we investigate the impact of 810 different miRNAs on PCa cell biology and sensitivity to radiation therapy. We anticipate that these miRNAs may be targets or therapeutics for enhancing the radiation therapy of PCa. Materials/Methods: We have constructed a reporter vector with the Metridia Luciferase (MLuc) gene under the control of the human b-actin promoter and enhancer. MLuc activity of stably transfected PCa cells with this vector (LNCaP-MLuc,PC3-MLuc) was highly correlated with viable cell number. On day 0, 810 human miRNA mimics were transfected into LNCaP-MLuc cells. On day 2, one group of cells was irradiated with 4 Gy, while the other group remained untreated. On day 13, MLuc activity was examined to quantify cell viability and therapeutic effect, normalizing to the control miRNA. Toxic miRNAs, which increased cell death by over 50% in the absence of irradiation, were excluded from the analysis. Radiosensitizing or radioprotective miRNAs were defined as those which increased cell death by over 80% or increased cell survival by over 2 fold, respectively (cut off p < 0.05). Radiosensitizing miRNAs were further validated in multiple PCa cell models. Results: The miRNA viability and radiation sensitizing screens in LNCaPMLuc cells were performed in duplicates, and were highly reproducible (R2  0.75). Among 810 miRNAs, 127 were found to be toxic. One hundred twenty-two were identified as radiosensitizing, and 65 were identified as radioprotective (p < 0.05). The miRNAs belonging to the same families induced matched radiosensitization phenotypes. For example, miRNAs from the miR-15/16, miR-34/449 and miR-1/133 families were each observed to be radiation sensitizing, whereas miRNAs from the miR-106b family were found to be radioprotective. Over half of the top radiosensitizing miRNAs were confirmed to sensitize PC3-MLuc cells to radiation therapy. Additional miRNAs were validated by clonogenic survival assay in DU145 cells (with the DMF0.1 of 1.17 to 1.44). Conclusions: Through a series of high-throughput functional screening we have identified miRNAs capable of regulating PCa cell radiosensitivity. These miRNAs may have therapeutic potential in the treatment of PCa with radiation therapy. Author Disclosure: M. Hedayati: None. K. Hatano: None. B. Kumar: None. W. Chowdhury: None. R. Rodriguez: None. Y. Zhang: None. T. DeWeese: None. S. Lupold: None.

hematological analysis (results for 91 patients included). The overall change from D1 to Df in fatigue, insomnia, and the blood parameters was assessed by mixed model regression analyses. The impact of prior adjuvant chemotherapy on the change in fatigue and CD34+ counts was assessed using interaction terms. Correlations between fatigue endpoints and blood parameters were estimated using Pearson correlation coefficients. Results: General fatigue levels peaked at the end of treatment, and returned to baseline at M1. The worsening in general fatigue from D1 to Df, adjusted for anxiety, depression and insomnia, was statistically significant (p Z 0.0001). CD34+, CD45+, total WBC, lymphocytes and platelet counts all decreased over the course of RT, with the lowest level observed at Df (p < 0.001 for all). There was a significant inverse correlation between worsening mental fatigue with lower CD34+ (p Z 0.011), WBC (p Z 0.047), lymphocytes (p Z 0.040), and hemoglobin (p Z 0.007). Similar correlations were found between increasing insomnia and reduced CD34+ (p Z 0.001), CD45+ (p Z 0.002), WBC (p Z 0.001), lymphocytes (p Z 0.003), and hemoglobin (p Z 0.040). Higher hemoglobin levels associated with lower general and physical fatigue (both p < 0.001), and improved activity and motivation scores (p < 0.001 and p Z 0.025, respectively). Patients who received prior chemotherapy (39) reported significantly higher fatigue, depression and insomnia scores (all p < 0.05), and had lower CD34+, CD45+, WBC, lymphocytes, and hemoglobin (all p < 0.001) at D1 compared to those without chemotherapy. The rate of decline in CD34+ counts remained the same, whereas the rate of increase in fatigue was greater in no chemotherapy vs chemotherapy patients. Conclusions: Worsening mental fatigue and increasing insomnia observed during RT corresponded with reductions in circulating CD34+ and other hematologic parameters. Elucidating the mechanisms underlying this relationship between the blood, the bone marrow (CD34+), and the brain could provide important insights into these cancer patient symptoms. Author Disclosure: K. Han: None. T. Lymberiou: None. M. Li: None. W. Shi: None. X. Shen: None. W. Xu: None. P. Catton: None. A. Fyles: None. R. Sutherland: None. R. Carlson: None. M. Yap: None. M. Minden: None. F. Liu: None.

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Purpose/Objective(s): Metastasis is one of the most lethal attributes of human malignancy. Aberrant expression of zinc transporter4 (ZIP4) leading to altered intracellular zinc levels and promoting extracellular uptake are involved in the malignant behavior such as metastasis in multiple cancers. However, it is still unclear whether the ZIP4 expression is related to prognosis or radiosensitivity in human nasopharyngeal carcinoma (NPC). The purpose of this study is to investigate the relation between expression of ZIP4 and malignant behavior in nasopharyngeal carcinoma and identify the mechanism. Materials/Methods: We examined the expressions of ZIP4 using immunohistochemistry in biopsy specimens of 102 NPC patients and analyzed the correlation with clinic characteristics and prognosis. The radiosensitivity of ZIP4-knocked down NPC cells (CNE1 and CNE2) were analyzed by MTT, clone formation and cell cycle assays, as well as the expression of radiosensitivity biomarkers (PI3K, DNA-PKS, Cyclin B, CDK1 and Histone H2A.X ) by Western Blotting and immunofluorescence. In addition, ZIP4 overexpression or down-regulated nasopharyngeal carcinoma cells (CNE1 and CNE2) were analyzed for their ability to migration by Transwell, as well as for the expression of the components of TGF-b dependent epithelial mesenchymal transition (EMT) using Western Blotting and immunofluorescence method. For better monitoring of cell migration behavior, the metastatic potential of ZIP4-knocked down CM-

The Relationship Between Circulating CD34+ Cells With Mental Fatigue and Insomnia During Adjuvant Breast Cancer Radiation Therapy (RT) K. Han,1 T. Lymberiou,1 M. Li,1 W. Shi,2 X. Shen,1 W. Xu,1 P. Catton,1 A. Fyles,1 R. Sutherland,3 R. Carlson,1 M. Yap,1 M. Minden,1 and F. Liu1; 1 Princess Margaret Cancer Centre, Toronto, ON, Canada, 2Ontario Cancer Institute, Princess Margaret Cancer Centre, Toronto, ON, Canada, 3 Toronto General Hospital, Toronto, ON, Canada Purpose/Objective(s): We had previously demonstrated that local RT induced “homing” of hematopoietic stem cells (HSCs) to the site of treatment, and that increasing fatigue observed during the first week of RT was inversely related to the number of circulating HSCs (CD34+). The objective of the current study is to investigate this relationship in a larger, independent cohort of women undergoing adjuvant breast cancer RT. Materials/Methods: One hundred eight patients completed the Multidimensional Fatigue Inventory (MFI-20), Hospital Anxiety and Depression Score (HADS), and Insomnia Severity Index (ISI) immediately prior to the 1st, 2nd, 5th, and final fractions of breast RT (D1, D2, D5, Df; respectively), and 1 month after completion of RT (M1). At these time points, patients also underwent phlebotomies for CD34+, CD45+, and full

192 Zinc Transporter 4 (ZIP4): A Predictive Biomarker for Decreasing Radiosensitivity and Promoting Tumor Migration and Metastasis Through TGF-beDependent Manner in Nasopharyngeal Carcinoma (NPC) P. Zhang, R. Liu, and J. Lang; Department of Radiation Oncology, Sichuan Cancer Hospital, Chengdu, China

S90

International Journal of Radiation Oncology  Biology  Physics

Dil-labeled CNE2 cells was evaluated in vivo using a zebra fish xenotransplantation model. Results: We found that ZIP4 expression was positive correlation with tumor stages (p Z 0.005), negative correlation with patient survival rates (p Z 0.0005) and distant metastasis free survival rates (p < 0.0001). Then, MTT assays indicated that knocking down of ZIP4 inhibited cell proliferation after radiation therapy (p Z 0.0014) and inhibited cell clone formation of 40% in nasopharyngeal carcinoma cells (CNE1 and CNE2). Cell cycle assays proved that G2/M phase increased from 1.33% to 27.60% after knocking down of ZIP4. Furthermore, Western Blotting and immunofluorescence assays for radiosensitivity biomarkers also indicated that down-regulated ZIP4 could increase the radiosensitivity in NPC cells. Subsequently, Our study identified that the ZIP4 promoted cell’s ability to migration and changed TGF-b expression. ZIP4 could promote the process of EMT through TGF-b dependent manner in NPC cells and in zebra fish animal models. Conclusions: Our results confirmed that ZIP4 could affect radiosensitivity and promote metastasis in NPC. ZIP4 would be a predictive biomarker for tumor metastasis and the outcome of patients with NPC and a novel cancer therapy target. Author Disclosure: P. Zhang: None. R. Liu: None. J. Lang: None.

regulated by cMyc, and cMyc-regulated genes, including CDKN2B. We found that PCAT-1 did not alter cMyc mRNA levels, suggesting posttranscriptional regulation. Luciferase with the cMyc 3’-UTR had increased luciferase activity upon PCAT-1 expression, consistent with PCAT-1 preventing miRNA translational repression. Indeed, PCAT-1 prevented translational downregulation of cMyc by its inhibitory miRNA miR-34a. Interestingly, this PCAT-1-derepression could be reversed by miR-36673p, which is predicted to bind PCAT-1. Conclusions: We demonstrate novel regulation of the cMyc oncogene and consequent proliferation by an ncRNA functional network composed of the lncRNA PCAT-1 and 2 miRNAs. This mechanism suggests additional therapeutic strategies for cMyc-driven tumors through targeting or mimicking these ncRNAs. Myc also affects radiation response, and therefore these are potential radiosensitizing strategies as well. Author Disclosure: J.R. Evans: None. J. Prensner: Q. Patent/License Fee/Copyright; PCAT-1. W. Chen: None. M. Ljungman: None. T. Lawrence: None. A. Chinnaiyan: Q. Patent/License Fee/Copyright; PCAT-1. F. Feng: E. Research Grant; Celgene. G. Consultant; Myriad Genetics. J. In-kind Donation; GenomeDx Biosciences. K. Advisory Board; Medivation, Astellas, NanoString Technologies.

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193 The Long Noncoding RNA PCAT-1 Promotes Prostate Cell Proliferation Through Posttranscriptional Stabilization of the cMyc Oncogene J.R. Evans, J. Prensner, W. Chen, M. Ljungman, T. Lawrence, A. Chinnaiyan, and F. Feng; University of Michigan Radiation Oncology, Ann Arbor, MI Purpose/Objective(s): Long noncoding RNAs (lncRNAs) are a novel layer of biology that have numerous important functions in cancer. We identified the lncRNA PCAT-1 as an outlier lncRNA in high-grade localized and metastatic prostate cancer. PCAT-1 promotes proliferation and exerts some of its activity through functional interaction with the polycomb repressor chromatin-modifying complex PRC2. We were also intrigued by the location of PCAT-1 725kb upstream of the cMyc oncogene. Given that many lncRNAs act in cis to modulate gene function, we hypothesized that PCAT-1 may be modulating cMyc function. Materials/Methods: Lentivirus and transfection were used for RNAi knockdown and overexpression stably and transiently, respectively. Experiments were performed in the LNCaP prostate cancer cell line, which endogenously express PCAT-1, and the DU145 prostate cancer and RWPE immortalized nontransformed prostate epithelium cell lines, which have very low PCAT-1 expression. Cell proliferation was measured by counting typan blue-stained cells. BrdU pulse-chase labeling followed by anti-BrdU pulldown and qPCR was used to measure mRNA synthesis and stability. The cMyc 3’-UTR sequence was appended to the luciferase ORF, and luciferase assays performed to measure translational regulation of the cMyc 3’-UTR. Western blot was used to measure protein levels. Gene expression was analyzed by microarray and for enrichment patterns with the DAVID bioinformatics platform. MicroRNA binding was predicted with miRanda, mirBase, and TargetScan. Results: PCAT-1 increased proliferation and cMyc protein levels in DU145 and RWPE cells, and this proliferation was blocked by cMyc RNAi. Conversely, PCAT-1 RNAi decreased proliferation and cMyc protein levels in LNCaP cells. PCAT-1-induced gene expression changes are enriched for protein biosynthesis and transcriptional elongation, which are

Oral Scientific Abstract 194; Table

A Volumetric Analysis of GTVD and HR CTV as Defined by the GEC ESTRO Recommendations in FIGO Stage IIB Cervical Cancer Patients Treated With IGABT in a Prospective Multicentric Trial N.T. Jastaniyah,1 K. Yoshida,2 K. Tanderup,3 J. Lindegaard,3 F. Patel,4 P. Petric,5 B. Segedin,6 C. Haie-Meder,7 R. Cooper,8 and R. Po¨tter9; 1King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia, 2 Kobe University Graduate School of Medicine, Kobe, Japan, 3Aarhus University Hospital, Aarhus, Denmark, 4Post Graduate Institute of Medical Education and Research, Chandigarh, India, 5National Center for Cancer Care and Research, Doha, Qatar, 6Institute of Oncology, Ljubljana, Slovenia, 7Institut Gustave Roussy, Paris, France, 8St. James’s Institute of Oncology, Leeds, United Kingdom, 9Medical University of Vienna, Vienna, Austria Purpose/Objective(s): To quantify gross tumor volume at diagnosis (GTVD) and high-risk clinical target volume (HR CTV) at brachytherapy (BT) and to describe subgroups with different patterns of HR CTV in relation to GTVD in patients with FIGO stage IIB cervical cancer treated with image-guided adaptive brachytherapy (IGABT). Secondly, to evaluate the feasibility of IGABT in target coverage with various target volumes and regression patterns. Materials/Methods: A total of 345 patients with FIGO stage IIB cervical cancer enrolled in the EMBRACE study and with available information on the GTVD and HR CTV were analyzed. T2-weighted MRI scans were obtained for all patients at the time of diagnosis and at the time of BT. GTVD and HR CTV were defined as per the GEC ESTRO recommendations. A stepwise approach was used to categorize the patients into four groups. First, the relationship of GTVD was compared to the mean GTVD of the entire cohort ( mean GTVD vs > mean GTVD). Then, four patterns of tumor regression were determined based on: the relationship of HR CTV to GTVD for each patient (HR CTV < GTVD vs HR CTV  GTVD) and the extent of parametrial disease (PMD) at time of BT (no PMD vs proximal vs distal). The clinical, pathological and treatment characteristics were then compared among the four groups. Results: The mean (SD) GTVD and HR CTV were 43.6 (32.8) cm3 and 31.6 (16.1) cm3, respectively. The characteristics of the four groups are

Description of the stratification criteria among the 4 groups

Group 1 (n Z 118)

Group 2 (n Z 75)

Group 3 (n Z 133)

Group 4 (n Z 19)

Small tumors (mean GTVD 16.8 cm3). HR CTV  GTVD (mean HR CTV 27.8 cm3). Minimal or no PMD at BT.

Intermediate size tumors (mean GTVD 44.5 cm3). HR CTV < GTVD (mean HR CTV 25.6 cm3). No PMD at BT.

Large tumors (mean GTVD 64.4 cm3). HR CTV < GTVD (mean HR CTV 34.8 cm3). Residual proximal PMD at BT.

Large tumors (mean GTVD 61.0 cm3). HR CTV  GTVD (mean HR CTV 55.1cm3). Residual distal PMD at BT.