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MARCKS Regulates Growth, Radiation Sensitivity, and Is a Novel Prognostic Factor for Glioblastoma
Poster Viewing Abstracts S715
Volume 84 Number 3S Supplement 2012
3323 Predictive Biomarkers for Gemcitabine as a Chemoradiosensitizer in Bladder Cancer M. Kerr and A.E. Kiltie; University of Oxford, Oxford, United Kingdom Purpose/Objective(s): Muscle invasive bladder cancer is treated by cystectomy or radiation therapy-based approaches, and both neoadjuvant chemotherapy and concurrent chemoradiation improve survival rates modestly. Tailoring which chemotherapeutic drug is used in combination by use of predictive biomarkers may improve clinical outcome further. The antimetabolite gemcitabine is an effective radiosensitizer in many cancers, including bladder. We aim to investigate proteins key in the uptake and metabolism of gemcitabine as potential predictive biomarkers for response to this drug in bladder cancer cell lines: hENT1 is a transporter involved in cellular entry of gemcitabine; ribonucleoside-diphosphate reductase subunits 1 and 2 (RRM1 and RRM2) can be inhibited by gemcitabine, thus decreasing endogenous levels of deoxyribonucleotides needed for DNA replication; cytidine deaminase (CDA) can convert gemcitabine to an inactive uridine form; and deoxycytidine kinase (dCK) is a kinase critical in the conversion of gemcitabine from prodrug to active antimetabolite. Materials/Methods: RT112 and CAL29 bladder cancer cell lines were treated with gemcitabine for 24 h prior to replating at appropriate cell density and irradiated with 0- 8 Gy for clonogenic survival assay. Cells were stained with propidium iodide for flow cytometry analysis of the cell cycle. Transient knockdown of hENT1, RRM1, CDA and dCK was achieved using a standard siRNA transfection protocol. Western blots and qPCR were performed to establish protein and mRNA levels, respectively. Results: Gemcitabine radiosensitized RT112 and CAL29 bladder cancer cell lines after 24 h exposure to 20 nM drug, with sensitizer enhancement ratios of 1.36 and 1.45, respectively. Radiosensitization corresponds to an accumulation of greater than 85 % cells in S phase. Knockdown of hENT1, RRM1 and CDA has no significant impact on gemcitabine radiosensitivity. However, cells that have reduced dCK levels by siRNA display resistance to gemcitabine. Furthermore, cells conditioned to become resistant to 10 M gemcitabine had a striking reduction in dCK, both at the protein level (undetectable on western blot) and 50% reduction of mRNA. Sensitivity to gemcitabine can be restored by transient overexpression of dCK in CAL29 cells, but gemcitabine-resistant RT112 cells appear not to be resensitized with transient overexpression of dCK. We aim to test RT112 cells that have stable overexpression of dCK. Conclusion: Gemcitabine is an effective radiosensitizer in bladder cancer cell lines. Knockdown of dCK confers resistance to gemcitabine, consistent with reduction of dCK in cells with acquired gemcitabine resistance. Sensitivity is restored by dCK overexpression in one resistant cell line. We aim to further investigate dCK as a potential biomarker for gemcitabine response. Author Disclosure: M. Kerr: None. A.E. Kiltie: None.
3324 MARCKS Regulates Growth, Radiation Sensitivity, and Is a Novel Prognostic Factor for Glioblastoma J. Jarboe, J.C. Anderson, C.W. Duarte, T. Mehta, S. Nowsheen, T.D. Rohrbach, R.O. McCubrey, G.Y. Gillespie, E.S. Yang, and C.D. Willey; University of Alabama at Birmingham, Birmingham, AL Purpose/Objective(s): The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is crucial to many aspects of cell growth and survival in glioblastoma (GBM). Phosphatidyl inositol bisphosphate (PIP2) is converted to the triphosphate (PIP3) by PI3K which leads to Akt activation. Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) is capable of sequestering PIP2 at the membrane via electrostatic interactions. We thus hypothesized that the MARCKS protein could regulate proliferation and radioresistance via modulation of the PI3K/Akt pathway; and therefore would have a positive impact on clinical outcome for GBM patients. Materials/Methods: Knockdown and over-expression of the MARCKS protein were achieved with lentiviral mediated shRNA or lentiviral mediated over-expression. Cell proliferation assays were performed.
Radiation sensitivity was assessed with clonogenic assays. Immunoblotting was performed using anti-MARCKS antibody, anti-pAkt Ser473, antiCaspase 3 and anti-phospho DNA-PKcs. Gene expression data was downloaded for 192 out of the 202 patients that did not have prior glioma. Cox proportional hazards models were fit using the coxph routine. KaplanMeier curves for MARCKS quartiles or other expression categories were generated by predicting the survival times from the Cox model at the median age value using the survfit function from the survival package. Results: Genetic silencing of the MARCKS protein in U251 GBM cells promoted increased proliferation (p<0.01) and increased radiation resistance (DER Z 0.80). This correlated with a 40% increase in activation of the PI3K/Akt pathway. The mechanism of increased radiation resistance was an increase in non-homologous end joining (NHEJ) DNA repair as indicated by increased DNA-PK activation, and decreased apoptosis as indicated by decreases in cleaved caspase 3 after irradiation. Conversely, over-expression of the MARCKS protein in U87 GBM cells lead to decreases in proliferation (p<0.001), however; the cells unexpectedly adopted a senescent state (p<0.0001). Age-adjusted MARCKS gene expression inversely correlated with overall survival in GBM patients in the TCGA database (p Z 0.004). This effect was found to be specific to the Proneural molecular subtype (median survival of 47.2 months vs. 12.2 months, p<0.0002), and most pronounced in tumors with unmethylated MGMT status (median survival 65.3 months vs. 10.7 months, p<0.001). Conclusions: We have demonstrated that MARCKS can regulate proliferation, radiation sensitivity and is associated with improved clinical outcomes. These findings suggest MARCKS as a novel target and biomarker for prognosis in the Proneural subtype of GBM. Author Disclosure: J. Jarboe: None. J.C. Anderson: None. C.W. Duarte: None. T. Mehta: None. S. Nowsheen: None. T.D. Rohrbach: None. R.O. McCubrey: None. G.Y. Gillespie: None. E.S. Yang: None. C.D. Willey: None.
3325 A New Tool for Studying Tumor- and Treatment-associated Exosomes and Microparticles J.C. Jones,1,2 H. Cao,2 M. Limaye,2 A. Koong,2 and S. Knox2; 1Dana Farber Cancer Institute, Boston, MA, 2Stanford University, Stanford, CA Purpose/Objective(s): Sensitive and specific biomarkers of disease status and treatment response are needed in order to optimize personalized and adaptive therapies for our patients. Exosomes (40-200 nm), microparticles (150-500 nm), apoptotic bodies (>800 nm), and other submicron particles released by tumor cells and normal tissues into the circulation carry proteins, miRNA, and other molecules that can be used as biomarkers for monitoring treatment responses and customizing therapies. In order to be able to study the sizes, compositions, and functions of subsets of these submicron particles, we developed a nano-scale cytometric method for analysis and sorting. Here, we report the first patient treatment response profiles obtained with the method. Materials/Methods: We examined plasma collected and archived as part of a prospective phase II clinical trial investigating the use of single fraction stereotactic ablative body radiosurgery (SABR) for locally advanced, unresectable (stage T4) pancreatic carcinoma. Duplicate samples from 9 patients (including pre-SABR, 4-6 week, and 3 month time points) and from 3 age-matched healthy individuals were analyzed. The developed method was used to demonstrate the abundance and distributions of exosomes and microparticles in the plasma samples at each time point. Nanosight nanoparticle tracking analysis was also used to determine size and concentration parameters for each sample. Paired Student’s t-tests were used to evaluate the difference between pre-treatment and follow-up exosome and microparticle population compositions. Results: The developed method demonstrated that the plasma microparticle concentration was increased significantly (p Z 0.039) at 3 months, but not at 4-6 weeks, after completion of SABR. No significant increase in the large exosome proportion of the submicron population was observed by the developed method after SABR.
Volume 84 Number 3S Supplement 2012
3323 Predictive Biomarkers for Gemcitabine as a Chemoradiosensitizer in Bladder Cancer M. Kerr and A.E. Kiltie; University of Oxford, Oxford, United Kingdom Purpose/Objective(s): Muscle invasive bladder cancer is treated by cystectomy or radiation therapy-based approaches, and both neoadjuvant chemotherapy and concurrent chemoradiation improve survival rates modestly. Tailoring which chemotherapeutic drug is used in combination by use of predictive biomarkers may improve clinical outcome further. The antimetabolite gemcitabine is an effective radiosensitizer in many cancers, including bladder. We aim to investigate proteins key in the uptake and metabolism of gemcitabine as potential predictive biomarkers for response to this drug in bladder cancer cell lines: hENT1 is a transporter involved in cellular entry of gemcitabine; ribonucleoside-diphosphate reductase subunits 1 and 2 (RRM1 and RRM2) can be inhibited by gemcitabine, thus decreasing endogenous levels of deoxyribonucleotides needed for DNA replication; cytidine deaminase (CDA) can convert gemcitabine to an inactive uridine form; and deoxycytidine kinase (dCK) is a kinase critical in the conversion of gemcitabine from prodrug to active antimetabolite. Materials/Methods: RT112 and CAL29 bladder cancer cell lines were treated with gemcitabine for 24 h prior to replating at appropriate cell density and irradiated with 0- 8 Gy for clonogenic survival assay. Cells were stained with propidium iodide for flow cytometry analysis of the cell cycle. Transient knockdown of hENT1, RRM1, CDA and dCK was achieved using a standard siRNA transfection protocol. Western blots and qPCR were performed to establish protein and mRNA levels, respectively. Results: Gemcitabine radiosensitized RT112 and CAL29 bladder cancer cell lines after 24 h exposure to 20 nM drug, with sensitizer enhancement ratios of 1.36 and 1.45, respectively. Radiosensitization corresponds to an accumulation of greater than 85 % cells in S phase. Knockdown of hENT1, RRM1 and CDA has no significant impact on gemcitabine radiosensitivity. However, cells that have reduced dCK levels by siRNA display resistance to gemcitabine. Furthermore, cells conditioned to become resistant to 10 M gemcitabine had a striking reduction in dCK, both at the protein level (undetectable on western blot) and 50% reduction of mRNA. Sensitivity to gemcitabine can be restored by transient overexpression of dCK in CAL29 cells, but gemcitabine-resistant RT112 cells appear not to be resensitized with transient overexpression of dCK. We aim to test RT112 cells that have stable overexpression of dCK. Conclusion: Gemcitabine is an effective radiosensitizer in bladder cancer cell lines. Knockdown of dCK confers resistance to gemcitabine, consistent with reduction of dCK in cells with acquired gemcitabine resistance. Sensitivity is restored by dCK overexpression in one resistant cell line. We aim to further investigate dCK as a potential biomarker for gemcitabine response. Author Disclosure: M. Kerr: None. A.E. Kiltie: None.
3324 MARCKS Regulates Growth, Radiation Sensitivity, and Is a Novel Prognostic Factor for Glioblastoma J. Jarboe, J.C. Anderson, C.W. Duarte, T. Mehta, S. Nowsheen, T.D. Rohrbach, R.O. McCubrey, G.Y. Gillespie, E.S. Yang, and C.D. Willey; University of Alabama at Birmingham, Birmingham, AL Purpose/Objective(s): The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is crucial to many aspects of cell growth and survival in glioblastoma (GBM). Phosphatidyl inositol bisphosphate (PIP2) is converted to the triphosphate (PIP3) by PI3K which leads to Akt activation. Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) is capable of sequestering PIP2 at the membrane via electrostatic interactions. We thus hypothesized that the MARCKS protein could regulate proliferation and radioresistance via modulation of the PI3K/Akt pathway; and therefore would have a positive impact on clinical outcome for GBM patients. Materials/Methods: Knockdown and over-expression of the MARCKS protein were achieved with lentiviral mediated shRNA or lentiviral mediated over-expression. Cell proliferation assays were performed.
Radiation sensitivity was assessed with clonogenic assays. Immunoblotting was performed using anti-MARCKS antibody, anti-pAkt Ser473, antiCaspase 3 and anti-phospho DNA-PKcs. Gene expression data was downloaded for 192 out of the 202 patients that did not have prior glioma. Cox proportional hazards models were fit using the coxph routine. KaplanMeier curves for MARCKS quartiles or other expression categories were generated by predicting the survival times from the Cox model at the median age value using the survfit function from the survival package. Results: Genetic silencing of the MARCKS protein in U251 GBM cells promoted increased proliferation (p<0.01) and increased radiation resistance (DER Z 0.80). This correlated with a 40% increase in activation of the PI3K/Akt pathway. The mechanism of increased radiation resistance was an increase in non-homologous end joining (NHEJ) DNA repair as indicated by increased DNA-PK activation, and decreased apoptosis as indicated by decreases in cleaved caspase 3 after irradiation. Conversely, over-expression of the MARCKS protein in U87 GBM cells lead to decreases in proliferation (p<0.001), however; the cells unexpectedly adopted a senescent state (p<0.0001). Age-adjusted MARCKS gene expression inversely correlated with overall survival in GBM patients in the TCGA database (p Z 0.004). This effect was found to be specific to the Proneural molecular subtype (median survival of 47.2 months vs. 12.2 months, p<0.0002), and most pronounced in tumors with unmethylated MGMT status (median survival 65.3 months vs. 10.7 months, p<0.001). Conclusions: We have demonstrated that MARCKS can regulate proliferation, radiation sensitivity and is associated with improved clinical outcomes. These findings suggest MARCKS as a novel target and biomarker for prognosis in the Proneural subtype of GBM. Author Disclosure: J. Jarboe: None. J.C. Anderson: None. C.W. Duarte: None. T. Mehta: None. S. Nowsheen: None. T.D. Rohrbach: None. R.O. McCubrey: None. G.Y. Gillespie: None. E.S. Yang: None. C.D. Willey: None.
3325 A New Tool for Studying Tumor- and Treatment-associated Exosomes and Microparticles J.C. Jones,1,2 H. Cao,2 M. Limaye,2 A. Koong,2 and S. Knox2; 1Dana Farber Cancer Institute, Boston, MA, 2Stanford University, Stanford, CA Purpose/Objective(s): Sensitive and specific biomarkers of disease status and treatment response are needed in order to optimize personalized and adaptive therapies for our patients. Exosomes (40-200 nm), microparticles (150-500 nm), apoptotic bodies (>800 nm), and other submicron particles released by tumor cells and normal tissues into the circulation carry proteins, miRNA, and other molecules that can be used as biomarkers for monitoring treatment responses and customizing therapies. In order to be able to study the sizes, compositions, and functions of subsets of these submicron particles, we developed a nano-scale cytometric method for analysis and sorting. Here, we report the first patient treatment response profiles obtained with the method. Materials/Methods: We examined plasma collected and archived as part of a prospective phase II clinical trial investigating the use of single fraction stereotactic ablative body radiosurgery (SABR) for locally advanced, unresectable (stage T4) pancreatic carcinoma. Duplicate samples from 9 patients (including pre-SABR, 4-6 week, and 3 month time points) and from 3 age-matched healthy individuals were analyzed. The developed method was used to demonstrate the abundance and distributions of exosomes and microparticles in the plasma samples at each time point. Nanosight nanoparticle tracking analysis was also used to determine size and concentration parameters for each sample. Paired Student’s t-tests were used to evaluate the difference between pre-treatment and follow-up exosome and microparticle population compositions. Results: The developed method demonstrated that the plasma microparticle concentration was increased significantly (p Z 0.039) at 3 months, but not at 4-6 weeks, after completion of SABR. No significant increase in the large exosome proportion of the submicron population was observed by the developed method after SABR.