- Email: [email protected]
The Prognostic Role and Regulatory Signaling Pathway of Hepatoma-Derived Growth Factor in Oral Cancer
E604
International Journal of Radiation Oncology Biology Physics
3437
Purpose/Objective(s): Glioblastoma (GBM) is the most common form of brain cancer and among the most lethal of human cancers overall. DNA damage response (DDR) proteins such as ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNApk) are being investigated as therapeutic targets for radiosensitization of GBM. Previously, (1aminoethyl)iminobis[N-(oleicylcysteinyl-1-amino-ethyl)-propionamide (ECO) NPs conjugated to Arg-Gly-Asp (RGD) and polyethylene glycol (PEG) were shown to effectively deliver siRNA targeting pathways involved with tumor growth and metastasis. We hypothesize that RGDPEG-ECO NPs will provide an effective platform to deliver siRNA targeting ATM and DNApk to enhance radiation response in glioma and glioma stem cell populations. Materials/Methods: Normal astrocytes, glioma (U251), and glioma stem cells (NSC11, GBMJ1) were used for in vitro studies. Cellular uptake of NPs loaded with fluorescent siRNA (5nM) was assessed using flow cytometry. Protein analyses elucidated the timing and degree of silencing using NP/siRNA (40nM) targeting ATM and DNApk. The effects of siRNA delivery on radiation response was evaluated using clonogenic cell survival and gH2AX foci assays. Treatment with each siRNA alone (40nM) and in combination (20nM each) were evaluated. Pilot studies were performed to assess the ability of NPs to deliver siRNA in vivo. A cranial guide screw model was used in nude mice for orthotopic implantation of U251 tumor cells bearing the luciferase gene and intratumoral injection of NPs carrying siRNA targeting luciferase. The bioluminescence signal was recorded daily to evaluate the degree of silencing. Results: Exposure with either ATM or DNApk siRNA containing NPs was shown to increase radiosensitivity in all tumor cell lines evaluated. At 24 hours following radiation, gH2AX studies showed significantly impaired DSB repair in the U251 cell line with DNApk and ATM/DNApk combination treatment. Further, the combination treatment resulted in significantly higher levels of gH2AX in both glioma stem cell lines. Importantly, each NP/siRNA treatment showed no significant effect on DSB repair in normal astrocytes. In accordance with gH2AX studies, the dose enhancement factors at a surviving fraction of 0.1 for ATM, DNApkcs, and combination treatments were 1.24, 1.43, and 1.34 for U251, 1.34, 1.35, and 1.48 for NSC11, and 1.35, 1.36, and 1.51 for GBMJ1. In vivo studies are ongoing. Conclusion: RGD-PEG-ECO NPs effectively delivered siRNA targeting ATM and DNApkcs to both glioma and glioma stem cells demonstrating the potential to target and treat a heterogeneous GBM cell population. The subsequent enhancement of radiation response in vitro coupled with in vivo observations of bioluminescence silencing provide the basis for future investigation into the use of RGD-PEG-ECO/siRNA complexes to enhance the radiation response of GBM in vivo. Author Disclosure: J.A. Lee: None. N. Ayat: None. Z. Sun: None. Z. Lu: None. K.A. Camphausen: None.
Optimizing Tumor Control Probability in Radiation Therapy Treatment Design e Application to HDR Cervical Cancer E.K. Lee,1 F. Yuan,1 C. Yu,1 A. Templeton,2 R. Yao,2 K.D. Kiel,3 and J.C.H. Chu3; 1Georgia Institute of Technology, Atlanta, GA, 2Rush University School of Medicine, Chicago, IL, 3Rush University Medical Center, Chicago, IL Purpose/Objective(s): The ultimate goal of radiotherapy treatment is to find a treatment that will yield a high tumor control probability (TCP) with an acceptable normal tissue complication probability (NTCP). Yet most treatment planning today is not based upon optimization of TCPs and NTCPs, but rather upon meeting physical dose and volume constraints defined by the planner. It has been suggested that treatment planning evaluation and optimization would be more effective if they were biologically and not dose/volume based. We design treatment plans that optimize TCP directly and contrast them with the clinical dose-based plans. PET image is incorporated to evaluate gain in TCP for dose escalation. Materials/Methods: We build a nonlinear mixed integer programming optimization model that maximizes TCP directly while satisfying the dose requirements on the targeted organ and healthy tissues. For HDR, the TCP equation at the end of the treatment according to the model developed by Zaider and Minerbo. The optimization model maximizes the TCP and satisfies to the dose requirement under physician’s instruction. We employ PET-image to facilitate our treatment planning by identifying the dense pockets of cancer cells. The volume delineated by PET-image defines the boost target volume (BTV). We incorporate partial dose volume constraints on the maximum/minimum dose to PTVBTV and BTV and the maximum dose to RECTUM, BLADDER, and BOWEL. For example, a constraint of the maximum dose to RECTUMcan be that 95% of the voxels must receive no more than 90% of the prescriptive dose and 100% of the voxels must receive no more than 120% of the prescriptive dose. The solution strategy first fits the TCP function with a piecewise-linear approximation, then solves the problem that maximizes the piecewise linear approximation of TCP, and finally performs a local neighborhood search to improve the TCP value. To gauge the feasibility, characteristics, and potential benefit of PET-image guided dose escalation, initial validation consists of fifteen cervical cancer HDR patient cases. These patients have all received prior 45Gy of external radiation dose. For both escalated strategies, we consider 35Gy PTV-dose, and two variations (37Gy-boost to BTV vs 40Gy-boost) to PET-image-pockets. Results: TCP for standard clinical plans range from 59.4% - 63.6%. TCP for dose-based PET-guided escalated-dose-plan ranges from 63.8%-98.6% for all patients; whereas TCP-optimized plans achieves over 91% for all patients. There is marginal difference in TCP among those with 37Gyboosted vs 40Gy-boosted. There is no increase in rectum and bladder dose among all plans. Conclusion: Optimizing TCP directly results in highly conformed doseescalated treatment plans. The TCP-optimized plan is individualized based on the biological PET-image of the patients. The TCP-optimization framework is generalizable and has been applied successfully to other external-beam delivery modalities. A clinical trial is on-going to gauge the clinical significance. Author Disclosure: E.K. Lee: None. F. Yuan: None. C. Yu: None. A. Templeton: None. R. Yao: None. K.D. Kiel: None. J.C. Chu: None.
3438 Improving Radiation Response in Glioblastoma Using ECO Nanoparticle Delivery of siRNA Targeting DNA Damage Repair J.A. Lee,1 N. Ayat,2 Z. Sun,2 Z.R. Lu,2 and K.A. Camphausen1; 1Radiation Oncology Branch, National Cancer Institute, NIH, Bethesda, MD, 2 Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH
3439 The Prognostic Role and Regulatory Signaling Pathway of Hepatoma-Derived Growth Factor in Oral Cancer Y.W. Lin,1,2 J.C. Wu,3 and M.H. Tai2,4; 1Department of Radiation Oncology, Chi Mei Medical Center, Tainan, Taiwan, 2Institute of Biomedical Science, National Sun Yat-sen University, Kaohsiung, Taiwan, 3 Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung, Taiwan, 4Center for Neuroscience, National Sun Yat-sen University, Kaohsiung, Taiwan Purpose/Objective(s): Hepatoma-derived growth factor (HDGF) participates in oncogenic progression and represents a prognostic factor in several types of cancer. The current study aimed to elucidate the correlation between HDGF expression, vascular endothelial growth factor (VEGF) expression, clinic-pathologic parameters, and the possible regulatory mechanism between HDGF and VEGF. Materials/Methods: Immunohistochemical analysis using tissue microarray consisting of surgically resected samples from 95 patients with oral cancer patients were performed to delineate the correlation between HDGF, clinic-pathologic parameters and survival. SCC4 cells were treated
Volume 99 Number 2S Supplement 2017 with recombinant HDGF protein in a dose dependent manner. Gene expression was analyzed by Western blot and RT-PCR. Secreted VEGF was also measured by ELISA assay. HIF-1a, NFkB and STAT3 signaling pathway were also analyzed. The nucleolin neutralizing antibody, HIF-1a inhibitor, and NFkB inhibitor were applied to SCC4 cells with or without recombinant HDGF to investigate their effects on signaling pathway between HDGF and VEGF. Results: Expression of nuclear HDGF and VEGF was significantly correlated with primary T stage (PZ 0.004 and PZ 0.038, respectively) and histological grade (PZ 0.013 and PZ 0.017, respectively) in the oral cancer patients. VEGF expression also associated with nodal stage (PZ 0.021). Expression of nuclear HDGF and VEGF was highly correlated (PZ 0.006). Multivariate analysis indicated that the expression level of nuclear HDGF level is an independent prognostic factor for disease-specific and local recurrence-free survival. High expression of VEGF and adjuvant concurrent chemoradiotherapy are independent factors in diseasespecific, local recurrence-free, and metastasis-free survival. HDGF significantly increased about 1.5 fold of VEGF gene expression compared with the control group in SCC4 cells. Protein levels of VEGF were also increased by HDGF stimuli in a dose-dependent manner. HDGF also stimulated more secreted VEGF protein in culture medium than the control group. HDGF at low dose (1 ng/ml) had potent to enhance the phosphorylation levels of AKT and IkB and the protein level of HIF-1a and NFkB, but not modulated the phosphorylation levels of STAT3 even at high dose (100ng/ml). The nucleolin-neutralizing antibody abolished HDGF-stimulated HIF-1a, NFkB and VEGF protein expression in SCC4 cells. HIF-1a inhibitor (Chetomin) can antagonize the HDGF-induced VEGF gene expression and protein level but not the NFkB inhibitor. Conclusion: These results demonstrated that HDGF upregulated VEGF gene expression and protein level via HIF-1a signaling pathway in nonhypoxic condition through interacting with nucleolin and led to poor prognosis in oral cancer patients. The findings indicated that HDGF might be a candidate target for the development of therapeutic strategies for cancer treatment. Author Disclosure: Y. Lin: None. J. Wu: None. M. Tai: None.
3440 MicroRNA-29c Functions as a Tumor Suppressor by Targeting VEGFA in Lung Adenocarcinoma L. Liu,1 N. BI,1 L. Wu,2 X. Ding,3 Y. Men,1 W. Zhou,2 L. Li,1 W. Zhang,2 S. Shi,1 Y. Song,2 and L. Wang4; 1National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China, 2National Cancer Center / Cancer Institute & Hospital, Chinese Academic of Medical Sciences, Peking Union Medical College, Beijing, China, 3Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China, 4Department of Radiation Oncology, National Cancer Center / Cancer Institute & Hospital, Chinese Academic of Medical Sciences, Peking Union Medical College, Beijing, China Purpose/Objective(s): Lung adenocarcinoma (LAD) is considered to be a highly aggressive disease with heterogeneous prognosis and the molecular mechanisms underlying tumor progression remain elusive. Growing evidence demonstrates that the dysregulation of microRNAs (miRNAs) plays an important role in various tumor processes. The aim of this study is to discover prognostic miRNA and investigate its role involved in progression of LAD. Materials/Methods: Prognosis related miRNA was detected by miRNA microarray using formalin-fixed paraffin-embedded (FFPE) specimens from 87 patients with IIIA-N2 LAD. The cell proliferation was evaluated by Cell Titer 96 AQueous One Solution Cell Proliferation Assay (MTS), and the migration/invasion was evaluated by transwell assay. The bioinformatics methods and luciferase reporter assay were applied to detect the relationship between miRNA and its target. The mRNA and protein levels
Poster Viewing E605 of miRNA target were determined by quantitative real time polymerase chain reaction (qRT-PCR) analysis, western blot and enzyme-linked immunosorbent assay (ELISA). Changes of angiogenesis induced by miRNA was evaluated by human umbilical vein endothelial cell (HUVEC) tube formation assay. Immunohistochemistry (IHC) analysis was performed in FFPE specimens of patients to evaluate the correlation between miR-29c with microvessel density (MVD) and VEGFA expression. Results: MiR-29c expression downregulation was significantly associated with unfavorable prognosis in IIIA-N2 LAD. MiR-29c inhibited cell proliferation, migration and invasion in cell lines. Integrated analysis revealed that vascular endothelial growth factor A (VEGFA) was a direct target of miR-29c. MiR-29c reduced the capability of tumor cells to promote HUVEC tube formation. The compromised cell proliferation, migration/invasion and angiogenesis induced by miR-29c mimic transfection were reversed by transfection of VEGFA expression plasmid. Furthermore, the correlation of miR-29c with MVD and VEGFA was confirmed in patients’ samples. Conclusion: MiR-29c acts as a tumor suppressor by targeting VEGFA and may represent a promising prognostic biomarker as well as a potential therapeutic target for LAD. Author Disclosure: L. Liu: None. N. BI: None. L. Wu: None. X. Ding: None. Y. Men: None. W. Zhou: None. L. Li: None. W. Zhang: None. S. Shi: None. Y. Song: None. L. Wang: None.
3441 Correlation of Molecular Features With Clinical Outcomes in Glioblastoma Treated with Chemoradiation C. Liveringhouse,1 G.D. Grass,2 K.A. Ahmed,3 A.O. Naghavi,2 R.J. Macaulay,4 A.B. Etame,3 N. Tran,5 P. Forsyth,5 S. Sahebjam,5 P.A.S. Johnstone,6 T.J. Robinson,7 and M. Yu8; 1USF Morsani College of Medicine, Tampa, FL, 2H. Lee Moffitt Cancer Center and Research Institute, Department of Radiation Oncology, Tampa, FL, 3H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 4H. Lee Moffitt Cancer Center and Research Institute, Department of Anatomic Pathology, Tampa, FL, 5H. Lee Moffitt Cancer Center and Research Institute, Department of Neuro-oncology, Tampa, FL, 6Department of Radiation Oncology, Moffitt Cancer Center, Department of Radiation Oncology, Tampa, FL, 7 Department of Radiation Oncology, Moffitt Cancer Center, Tampa, FL, 8 Moffitt Cancer Center, Tampa, FL Purpose/Objective(s): Glioblastoma (GBM) remains a difficult disease to treat and carries a very poor prognosis. Recent research demonstrated molecular markers can stratify patient outcomes. To further assess the value of using biomarkers, we retrospectively evaluated patients treated with chemoradiation (CRT) and compared outcomes based on clinical and molecular characteristics. Materials/Methods: 81 primary GBM patients treated with CRT from 2010 to 2016 with available molecular information were retrospectively analyzed. Details of clinical characteristics, treatments and outcomes were abstracted from clinical chart review and radiologic examination. Available molecular profiling (MGMT, IDH1, EGFR, PTEN, ATRX, BRAF, 1p/ 19q, and ki67) was collected. Overall survival (OS) and time to progression (TTP) were estimated from time of diagnosis with Kaplan-Meier methods and compared with log-rank tests. Cox proportional hazards method was used for univariate (UVA) and multivariate (MVA) analyses. Decision tree modeling was used to cluster molecular groups to determine groups with optimal OS. Results: The median age at diagnosis was 57 years (range: 21-83), the majority were male (60%; nZ49) and 83% had a KPS 70 (range: 60-100). 57% were treated with gross total resection (GTR), subtotal resection (37%) or biopsy (6%) at diagnosis. All patients were treated with CRT and 99% received concurrent temozolomide (TMZ). The median RT dose was 60 Gy (range: 45-72). 77% of patients received maintenance TMZ and 43%
International Journal of Radiation Oncology Biology Physics
3437
Purpose/Objective(s): Glioblastoma (GBM) is the most common form of brain cancer and among the most lethal of human cancers overall. DNA damage response (DDR) proteins such as ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNApk) are being investigated as therapeutic targets for radiosensitization of GBM. Previously, (1aminoethyl)iminobis[N-(oleicylcysteinyl-1-amino-ethyl)-propionamide (ECO) NPs conjugated to Arg-Gly-Asp (RGD) and polyethylene glycol (PEG) were shown to effectively deliver siRNA targeting pathways involved with tumor growth and metastasis. We hypothesize that RGDPEG-ECO NPs will provide an effective platform to deliver siRNA targeting ATM and DNApk to enhance radiation response in glioma and glioma stem cell populations. Materials/Methods: Normal astrocytes, glioma (U251), and glioma stem cells (NSC11, GBMJ1) were used for in vitro studies. Cellular uptake of NPs loaded with fluorescent siRNA (5nM) was assessed using flow cytometry. Protein analyses elucidated the timing and degree of silencing using NP/siRNA (40nM) targeting ATM and DNApk. The effects of siRNA delivery on radiation response was evaluated using clonogenic cell survival and gH2AX foci assays. Treatment with each siRNA alone (40nM) and in combination (20nM each) were evaluated. Pilot studies were performed to assess the ability of NPs to deliver siRNA in vivo. A cranial guide screw model was used in nude mice for orthotopic implantation of U251 tumor cells bearing the luciferase gene and intratumoral injection of NPs carrying siRNA targeting luciferase. The bioluminescence signal was recorded daily to evaluate the degree of silencing. Results: Exposure with either ATM or DNApk siRNA containing NPs was shown to increase radiosensitivity in all tumor cell lines evaluated. At 24 hours following radiation, gH2AX studies showed significantly impaired DSB repair in the U251 cell line with DNApk and ATM/DNApk combination treatment. Further, the combination treatment resulted in significantly higher levels of gH2AX in both glioma stem cell lines. Importantly, each NP/siRNA treatment showed no significant effect on DSB repair in normal astrocytes. In accordance with gH2AX studies, the dose enhancement factors at a surviving fraction of 0.1 for ATM, DNApkcs, and combination treatments were 1.24, 1.43, and 1.34 for U251, 1.34, 1.35, and 1.48 for NSC11, and 1.35, 1.36, and 1.51 for GBMJ1. In vivo studies are ongoing. Conclusion: RGD-PEG-ECO NPs effectively delivered siRNA targeting ATM and DNApkcs to both glioma and glioma stem cells demonstrating the potential to target and treat a heterogeneous GBM cell population. The subsequent enhancement of radiation response in vitro coupled with in vivo observations of bioluminescence silencing provide the basis for future investigation into the use of RGD-PEG-ECO/siRNA complexes to enhance the radiation response of GBM in vivo. Author Disclosure: J.A. Lee: None. N. Ayat: None. Z. Sun: None. Z. Lu: None. K.A. Camphausen: None.
Optimizing Tumor Control Probability in Radiation Therapy Treatment Design e Application to HDR Cervical Cancer E.K. Lee,1 F. Yuan,1 C. Yu,1 A. Templeton,2 R. Yao,2 K.D. Kiel,3 and J.C.H. Chu3; 1Georgia Institute of Technology, Atlanta, GA, 2Rush University School of Medicine, Chicago, IL, 3Rush University Medical Center, Chicago, IL Purpose/Objective(s): The ultimate goal of radiotherapy treatment is to find a treatment that will yield a high tumor control probability (TCP) with an acceptable normal tissue complication probability (NTCP). Yet most treatment planning today is not based upon optimization of TCPs and NTCPs, but rather upon meeting physical dose and volume constraints defined by the planner. It has been suggested that treatment planning evaluation and optimization would be more effective if they were biologically and not dose/volume based. We design treatment plans that optimize TCP directly and contrast them with the clinical dose-based plans. PET image is incorporated to evaluate gain in TCP for dose escalation. Materials/Methods: We build a nonlinear mixed integer programming optimization model that maximizes TCP directly while satisfying the dose requirements on the targeted organ and healthy tissues. For HDR, the TCP equation at the end of the treatment according to the model developed by Zaider and Minerbo. The optimization model maximizes the TCP and satisfies to the dose requirement under physician’s instruction. We employ PET-image to facilitate our treatment planning by identifying the dense pockets of cancer cells. The volume delineated by PET-image defines the boost target volume (BTV). We incorporate partial dose volume constraints on the maximum/minimum dose to PTVBTV and BTV and the maximum dose to RECTUM, BLADDER, and BOWEL. For example, a constraint of the maximum dose to RECTUMcan be that 95% of the voxels must receive no more than 90% of the prescriptive dose and 100% of the voxels must receive no more than 120% of the prescriptive dose. The solution strategy first fits the TCP function with a piecewise-linear approximation, then solves the problem that maximizes the piecewise linear approximation of TCP, and finally performs a local neighborhood search to improve the TCP value. To gauge the feasibility, characteristics, and potential benefit of PET-image guided dose escalation, initial validation consists of fifteen cervical cancer HDR patient cases. These patients have all received prior 45Gy of external radiation dose. For both escalated strategies, we consider 35Gy PTV-dose, and two variations (37Gy-boost to BTV vs 40Gy-boost) to PET-image-pockets. Results: TCP for standard clinical plans range from 59.4% - 63.6%. TCP for dose-based PET-guided escalated-dose-plan ranges from 63.8%-98.6% for all patients; whereas TCP-optimized plans achieves over 91% for all patients. There is marginal difference in TCP among those with 37Gyboosted vs 40Gy-boosted. There is no increase in rectum and bladder dose among all plans. Conclusion: Optimizing TCP directly results in highly conformed doseescalated treatment plans. The TCP-optimized plan is individualized based on the biological PET-image of the patients. The TCP-optimization framework is generalizable and has been applied successfully to other external-beam delivery modalities. A clinical trial is on-going to gauge the clinical significance. Author Disclosure: E.K. Lee: None. F. Yuan: None. C. Yu: None. A. Templeton: None. R. Yao: None. K.D. Kiel: None. J.C. Chu: None.
3438 Improving Radiation Response in Glioblastoma Using ECO Nanoparticle Delivery of siRNA Targeting DNA Damage Repair J.A. Lee,1 N. Ayat,2 Z. Sun,2 Z.R. Lu,2 and K.A. Camphausen1; 1Radiation Oncology Branch, National Cancer Institute, NIH, Bethesda, MD, 2 Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH
3439 The Prognostic Role and Regulatory Signaling Pathway of Hepatoma-Derived Growth Factor in Oral Cancer Y.W. Lin,1,2 J.C. Wu,3 and M.H. Tai2,4; 1Department of Radiation Oncology, Chi Mei Medical Center, Tainan, Taiwan, 2Institute of Biomedical Science, National Sun Yat-sen University, Kaohsiung, Taiwan, 3 Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung, Taiwan, 4Center for Neuroscience, National Sun Yat-sen University, Kaohsiung, Taiwan Purpose/Objective(s): Hepatoma-derived growth factor (HDGF) participates in oncogenic progression and represents a prognostic factor in several types of cancer. The current study aimed to elucidate the correlation between HDGF expression, vascular endothelial growth factor (VEGF) expression, clinic-pathologic parameters, and the possible regulatory mechanism between HDGF and VEGF. Materials/Methods: Immunohistochemical analysis using tissue microarray consisting of surgically resected samples from 95 patients with oral cancer patients were performed to delineate the correlation between HDGF, clinic-pathologic parameters and survival. SCC4 cells were treated
Volume 99 Number 2S Supplement 2017 with recombinant HDGF protein in a dose dependent manner. Gene expression was analyzed by Western blot and RT-PCR. Secreted VEGF was also measured by ELISA assay. HIF-1a, NFkB and STAT3 signaling pathway were also analyzed. The nucleolin neutralizing antibody, HIF-1a inhibitor, and NFkB inhibitor were applied to SCC4 cells with or without recombinant HDGF to investigate their effects on signaling pathway between HDGF and VEGF. Results: Expression of nuclear HDGF and VEGF was significantly correlated with primary T stage (PZ 0.004 and PZ 0.038, respectively) and histological grade (PZ 0.013 and PZ 0.017, respectively) in the oral cancer patients. VEGF expression also associated with nodal stage (PZ 0.021). Expression of nuclear HDGF and VEGF was highly correlated (PZ 0.006). Multivariate analysis indicated that the expression level of nuclear HDGF level is an independent prognostic factor for disease-specific and local recurrence-free survival. High expression of VEGF and adjuvant concurrent chemoradiotherapy are independent factors in diseasespecific, local recurrence-free, and metastasis-free survival. HDGF significantly increased about 1.5 fold of VEGF gene expression compared with the control group in SCC4 cells. Protein levels of VEGF were also increased by HDGF stimuli in a dose-dependent manner. HDGF also stimulated more secreted VEGF protein in culture medium than the control group. HDGF at low dose (1 ng/ml) had potent to enhance the phosphorylation levels of AKT and IkB and the protein level of HIF-1a and NFkB, but not modulated the phosphorylation levels of STAT3 even at high dose (100ng/ml). The nucleolin-neutralizing antibody abolished HDGF-stimulated HIF-1a, NFkB and VEGF protein expression in SCC4 cells. HIF-1a inhibitor (Chetomin) can antagonize the HDGF-induced VEGF gene expression and protein level but not the NFkB inhibitor. Conclusion: These results demonstrated that HDGF upregulated VEGF gene expression and protein level via HIF-1a signaling pathway in nonhypoxic condition through interacting with nucleolin and led to poor prognosis in oral cancer patients. The findings indicated that HDGF might be a candidate target for the development of therapeutic strategies for cancer treatment. Author Disclosure: Y. Lin: None. J. Wu: None. M. Tai: None.
3440 MicroRNA-29c Functions as a Tumor Suppressor by Targeting VEGFA in Lung Adenocarcinoma L. Liu,1 N. BI,1 L. Wu,2 X. Ding,3 Y. Men,1 W. Zhou,2 L. Li,1 W. Zhang,2 S. Shi,1 Y. Song,2 and L. Wang4; 1National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China, 2National Cancer Center / Cancer Institute & Hospital, Chinese Academic of Medical Sciences, Peking Union Medical College, Beijing, China, 3Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China, 4Department of Radiation Oncology, National Cancer Center / Cancer Institute & Hospital, Chinese Academic of Medical Sciences, Peking Union Medical College, Beijing, China Purpose/Objective(s): Lung adenocarcinoma (LAD) is considered to be a highly aggressive disease with heterogeneous prognosis and the molecular mechanisms underlying tumor progression remain elusive. Growing evidence demonstrates that the dysregulation of microRNAs (miRNAs) plays an important role in various tumor processes. The aim of this study is to discover prognostic miRNA and investigate its role involved in progression of LAD. Materials/Methods: Prognosis related miRNA was detected by miRNA microarray using formalin-fixed paraffin-embedded (FFPE) specimens from 87 patients with IIIA-N2 LAD. The cell proliferation was evaluated by Cell Titer 96 AQueous One Solution Cell Proliferation Assay (MTS), and the migration/invasion was evaluated by transwell assay. The bioinformatics methods and luciferase reporter assay were applied to detect the relationship between miRNA and its target. The mRNA and protein levels
Poster Viewing E605 of miRNA target were determined by quantitative real time polymerase chain reaction (qRT-PCR) analysis, western blot and enzyme-linked immunosorbent assay (ELISA). Changes of angiogenesis induced by miRNA was evaluated by human umbilical vein endothelial cell (HUVEC) tube formation assay. Immunohistochemistry (IHC) analysis was performed in FFPE specimens of patients to evaluate the correlation between miR-29c with microvessel density (MVD) and VEGFA expression. Results: MiR-29c expression downregulation was significantly associated with unfavorable prognosis in IIIA-N2 LAD. MiR-29c inhibited cell proliferation, migration and invasion in cell lines. Integrated analysis revealed that vascular endothelial growth factor A (VEGFA) was a direct target of miR-29c. MiR-29c reduced the capability of tumor cells to promote HUVEC tube formation. The compromised cell proliferation, migration/invasion and angiogenesis induced by miR-29c mimic transfection were reversed by transfection of VEGFA expression plasmid. Furthermore, the correlation of miR-29c with MVD and VEGFA was confirmed in patients’ samples. Conclusion: MiR-29c acts as a tumor suppressor by targeting VEGFA and may represent a promising prognostic biomarker as well as a potential therapeutic target for LAD. Author Disclosure: L. Liu: None. N. BI: None. L. Wu: None. X. Ding: None. Y. Men: None. W. Zhou: None. L. Li: None. W. Zhang: None. S. Shi: None. Y. Song: None. L. Wang: None.
3441 Correlation of Molecular Features With Clinical Outcomes in Glioblastoma Treated with Chemoradiation C. Liveringhouse,1 G.D. Grass,2 K.A. Ahmed,3 A.O. Naghavi,2 R.J. Macaulay,4 A.B. Etame,3 N. Tran,5 P. Forsyth,5 S. Sahebjam,5 P.A.S. Johnstone,6 T.J. Robinson,7 and M. Yu8; 1USF Morsani College of Medicine, Tampa, FL, 2H. Lee Moffitt Cancer Center and Research Institute, Department of Radiation Oncology, Tampa, FL, 3H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 4H. Lee Moffitt Cancer Center and Research Institute, Department of Anatomic Pathology, Tampa, FL, 5H. Lee Moffitt Cancer Center and Research Institute, Department of Neuro-oncology, Tampa, FL, 6Department of Radiation Oncology, Moffitt Cancer Center, Department of Radiation Oncology, Tampa, FL, 7 Department of Radiation Oncology, Moffitt Cancer Center, Tampa, FL, 8 Moffitt Cancer Center, Tampa, FL Purpose/Objective(s): Glioblastoma (GBM) remains a difficult disease to treat and carries a very poor prognosis. Recent research demonstrated molecular markers can stratify patient outcomes. To further assess the value of using biomarkers, we retrospectively evaluated patients treated with chemoradiation (CRT) and compared outcomes based on clinical and molecular characteristics. Materials/Methods: 81 primary GBM patients treated with CRT from 2010 to 2016 with available molecular information were retrospectively analyzed. Details of clinical characteristics, treatments and outcomes were abstracted from clinical chart review and radiologic examination. Available molecular profiling (MGMT, IDH1, EGFR, PTEN, ATRX, BRAF, 1p/ 19q, and ki67) was collected. Overall survival (OS) and time to progression (TTP) were estimated from time of diagnosis with Kaplan-Meier methods and compared with log-rank tests. Cox proportional hazards method was used for univariate (UVA) and multivariate (MVA) analyses. Decision tree modeling was used to cluster molecular groups to determine groups with optimal OS. Results: The median age at diagnosis was 57 years (range: 21-83), the majority were male (60%; nZ49) and 83% had a KPS 70 (range: 60-100). 57% were treated with gross total resection (GTR), subtotal resection (37%) or biopsy (6%) at diagnosis. All patients were treated with CRT and 99% received concurrent temozolomide (TMZ). The median RT dose was 60 Gy (range: 45-72). 77% of patients received maintenance TMZ and 43%