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Targeting a Metabolic Phenotype in EMT-like Prostate Cancer Cells to Improve Radiation Therapy
E516
International Journal of Radiation Oncology Biology Physics
3292
Purpose/Objective(s): Sporadic breast cancer accounts for 80-85% of all breast cancer. A subset of sporadic breast cancers is associated with functional inactivation of BRCA1 and displays a basal-like phenotype similar to hereditary BRCA1-associated breast cancers. The BRCA1 inactivation phenotype is sometimes referred to as “BRCAness.” Association of BRCAness with specific chemosensitivity has been suggested, but is not well defined in clinical response data. Our goal is to identify and characterize the BRCAness phenotype among sporadic breast cancer cell lines by examining homologous recombination (HR) proficiency. We hypothesize that tumors showing deficiency in HR can have normal BRCA1 protein expression, but fail to recruit BRCA1 to sites of DNA damage. Materials/Methods: HR deficiency was initially detected by formation of RAD51 foci and further characterized by colony survival and HR functional assay using flow cytometry. For RAD51 focus formation, DNA double-strand breaks (DSB) were induced by irradiation, cells were stained for RAD51, and foci were counted using confocal microscopy. For colony survival assay, interstrand crosslinks (ICLs) were induced with mitomycin-C; colony formation was assessed after 2 week incubation. To measure HR, the DR-GFP plasmid was transfected into cells, DNA DSB was induced, and flow cytometry was used to assess the extent of induced GFP expression. DR-GFP was assayed +/-BRCA1 siRNA knockdown to evaluate whether HR was influenced by additional BRCA1 protein depletion. Results: The MCF7 and MDA-MB-231 cell lines each demonstrated >2-fold induction of RAD51 foci in irradiated cells over baseline. HCC202 showed no significant RAD51 foci induction, while HCC1428 were only weakly inducing suggesting a functional RAD51 defect. Colony survival showed relative insensitivity of MDA-MB-231 to interstrand cross linking; MCF7 was intermediately sensitive to ICL formation while HCC1428 and HCC202 were highly sensitive. Preliminary flow cytometry suggested increased GFP expression among MCF7 and MDA-MB231 cells after DSB induction, while HCC1428 showed no increase in GFP expression. Conclusion: MCF7 and MDA-MB-231 cell lines are HR-proficient as shown by RAD51 recruitment to nuclear foci, response to ICL formation, and the DR-GRP assay. The cell lines HCC1428 and HCC202 are HR-defective as indicated by failure to recruit RAD51 foci to sites of DNA DSB, marked sensitivity to ICL formation, and reduced HR by DR-GFP. This characterization of HR-deficiency in breast cancer cell lines with normal BRCA1 protein expression will allow for further exploration into the mechanism of BRCAness. In addition, these experiments support the use of HR-deficiency targeting agents for sporadic cancers exhibiting BRCAness, which may be as prevalent as w25% of breast cancer. Author Disclosure: J. Haseltine: None. M. Thompson: None. R.W. Mutter: None. R. Delsite: None. S.N. Powell: None.
Targeting a Metabolic Phenotype in EMT-like Prostate Cancer Cells to Improve Radiation Therapy D.L. Mitchell,1 M. Buchakjian,1 D.C. DeZwaan,2 B. Wagner,2 D. Spitz,2 T.D. Rutkowski,2 and M.D. Henry2; 1University of Iowa Hospitals and Clinics, Iowa City, IA, 2University of Iowa Carver College of Medicine, Iowa City, IA Purpose/Objective(s): Prostate cancer mortality has decreased; however our progress is tempered by metastatic disease resistant to available therapies. Epithelial-to-mesenchymal transition (EMT) has been implicated in both prostate cancer metastasis and therapeutic resistance. Mechanisms by which EMT contributes to therapeutic resistance, including radiation therapy are not well understood. One possibility is that EMT-like cancer cells are metabolically reprogrammed in a manner that enables radioresistance. Understanding the metabolic differences in EMT-like prostate cancer cells may provide new targets for metastatic disease and/or disease resistant to current therapies. In this proposal we investigate the hypothesis that EMT-like prostate cancer cells differentially rely on fatty acid oxidation as means of facilitating radioresistance Materials/Methods: Prostate cancer cell lines (PC3-E epithelial variant; TEM4-18 mesenchymal variant) were maintained in DMEM/F12 + 10% FBS, 5% NAA, and G418. Oxygen consumption rate and extracellular acidification rate were measured. Western blot analysis was done to measure the indicated protein concentrations. Cells were irradiated with increasing amounts of radiation or at (2Gy) in the presence and absence of 1mM of etomoxir followed by assays of clonogenic survival. Fatty acid oxidation was done using radiolabeled palmitic acid in low glucose (5.55 mM) DMEM and scintillation counts were normalized to protein concentration. Bio-energetic metabolites were measured via the Albert Einstein Metabolomics Core. Results: The mesenchymal prostate cancer cells demonstrated radioresistance relative to the epithelial PC3-E cells (p < 0.0001, 2 and 4 Gy), and a different metabolic profile as indicated by higher levels of phosphorylated AMP Kinase, fatty acid oxidation, phosphorylated acetyl-CoA carboxylase, ATP, and NADPH (p < 0.03, 0.004, 0.01, 0.0001, 0.001 respectively). They consumed less oxygen (p < 0.001), while concurrently producing less lactate and fructose 1.6 bisphosphate (both indicators of glycolysis). Inhibition of fatty acid oxidation with etomoxir sensitized cells to radiation at 2 Gy (p < 0.001). Conclusion: Cells in an EMT-like state have a metabolic profile more dependent on fatty acid oxidation which confers a survival benefit in glucose low environments and resistance to radiation therapy. Our data are consistent with previous findings that blockade of fatty acid oxidation inhibits prostate cancer growth. Fatty acid oxidation is a plausible therapeutic target that can potentially sensitize a resistant sub-population to radiation therapy thereby improving definitive therapy, decreasing metastatic disease, and improving overall survival. Author Disclosure: D.L. Mitchell: None. M. Buchakjian: None. D.C. DeZwaan: None. B. Wagner: None. D. Spitz: None. T.D. Rutkowski: None. M.D. Henry: None.
3293 Sporadic Breast Cancer Cell Lines Show Homologous Recombination Deficiency From BRCA1 Pathway Inactivation With Normal BRCA1 Protein and Are Sensitive to Crosslinking Agents J. Haseltine,1 M. Thompson,2 R.W. Mutter,3 R. Delsite,4 and S.N. Powell4; 1 Weill Cornell Medical College, New York City, NY, 2Emory University Hospital, Atlanta, GA, 3Mayo Clinic, Rochester, MN, 4Memorial Sloan Kettering Cancer Center, New York, NY
3294 Thoracolumbar Spinal Cord Tolerance to High-Dose Conformal Proton-Photon Radiation Therapy V.K. Chowdhry,1 S. Goldberg,1 L. Liu,1 J.A. Adams,1 K. Bernstein,1 N.J. Liebsch,2 A. Niemierko,1 and T.F. DeLaney1; 1Massachusetts General Hospital, Boston, MA, 2Massachusetts General Hospital, Harvard Medical School, Boston, MA Purpose/Objective(s): To evaluate and understand the tolerance of the thoracolumbar spinal cord using equivalent uniform dose (EUD) and dosevolume histogram (DVH) analysis after combined high dose photon-proton radiation therapy. Materials/Methods: A total of 94 patients were identified as having high dose radiation therapy, 5900 cGy (RBE), to the thoracolumbar spine.
International Journal of Radiation Oncology Biology Physics
3292
Purpose/Objective(s): Sporadic breast cancer accounts for 80-85% of all breast cancer. A subset of sporadic breast cancers is associated with functional inactivation of BRCA1 and displays a basal-like phenotype similar to hereditary BRCA1-associated breast cancers. The BRCA1 inactivation phenotype is sometimes referred to as “BRCAness.” Association of BRCAness with specific chemosensitivity has been suggested, but is not well defined in clinical response data. Our goal is to identify and characterize the BRCAness phenotype among sporadic breast cancer cell lines by examining homologous recombination (HR) proficiency. We hypothesize that tumors showing deficiency in HR can have normal BRCA1 protein expression, but fail to recruit BRCA1 to sites of DNA damage. Materials/Methods: HR deficiency was initially detected by formation of RAD51 foci and further characterized by colony survival and HR functional assay using flow cytometry. For RAD51 focus formation, DNA double-strand breaks (DSB) were induced by irradiation, cells were stained for RAD51, and foci were counted using confocal microscopy. For colony survival assay, interstrand crosslinks (ICLs) were induced with mitomycin-C; colony formation was assessed after 2 week incubation. To measure HR, the DR-GFP plasmid was transfected into cells, DNA DSB was induced, and flow cytometry was used to assess the extent of induced GFP expression. DR-GFP was assayed +/-BRCA1 siRNA knockdown to evaluate whether HR was influenced by additional BRCA1 protein depletion. Results: The MCF7 and MDA-MB-231 cell lines each demonstrated >2-fold induction of RAD51 foci in irradiated cells over baseline. HCC202 showed no significant RAD51 foci induction, while HCC1428 were only weakly inducing suggesting a functional RAD51 defect. Colony survival showed relative insensitivity of MDA-MB-231 to interstrand cross linking; MCF7 was intermediately sensitive to ICL formation while HCC1428 and HCC202 were highly sensitive. Preliminary flow cytometry suggested increased GFP expression among MCF7 and MDA-MB231 cells after DSB induction, while HCC1428 showed no increase in GFP expression. Conclusion: MCF7 and MDA-MB-231 cell lines are HR-proficient as shown by RAD51 recruitment to nuclear foci, response to ICL formation, and the DR-GRP assay. The cell lines HCC1428 and HCC202 are HR-defective as indicated by failure to recruit RAD51 foci to sites of DNA DSB, marked sensitivity to ICL formation, and reduced HR by DR-GFP. This characterization of HR-deficiency in breast cancer cell lines with normal BRCA1 protein expression will allow for further exploration into the mechanism of BRCAness. In addition, these experiments support the use of HR-deficiency targeting agents for sporadic cancers exhibiting BRCAness, which may be as prevalent as w25% of breast cancer. Author Disclosure: J. Haseltine: None. M. Thompson: None. R.W. Mutter: None. R. Delsite: None. S.N. Powell: None.
Targeting a Metabolic Phenotype in EMT-like Prostate Cancer Cells to Improve Radiation Therapy D.L. Mitchell,1 M. Buchakjian,1 D.C. DeZwaan,2 B. Wagner,2 D. Spitz,2 T.D. Rutkowski,2 and M.D. Henry2; 1University of Iowa Hospitals and Clinics, Iowa City, IA, 2University of Iowa Carver College of Medicine, Iowa City, IA Purpose/Objective(s): Prostate cancer mortality has decreased; however our progress is tempered by metastatic disease resistant to available therapies. Epithelial-to-mesenchymal transition (EMT) has been implicated in both prostate cancer metastasis and therapeutic resistance. Mechanisms by which EMT contributes to therapeutic resistance, including radiation therapy are not well understood. One possibility is that EMT-like cancer cells are metabolically reprogrammed in a manner that enables radioresistance. Understanding the metabolic differences in EMT-like prostate cancer cells may provide new targets for metastatic disease and/or disease resistant to current therapies. In this proposal we investigate the hypothesis that EMT-like prostate cancer cells differentially rely on fatty acid oxidation as means of facilitating radioresistance Materials/Methods: Prostate cancer cell lines (PC3-E epithelial variant; TEM4-18 mesenchymal variant) were maintained in DMEM/F12 + 10% FBS, 5% NAA, and G418. Oxygen consumption rate and extracellular acidification rate were measured. Western blot analysis was done to measure the indicated protein concentrations. Cells were irradiated with increasing amounts of radiation or at (2Gy) in the presence and absence of 1mM of etomoxir followed by assays of clonogenic survival. Fatty acid oxidation was done using radiolabeled palmitic acid in low glucose (5.55 mM) DMEM and scintillation counts were normalized to protein concentration. Bio-energetic metabolites were measured via the Albert Einstein Metabolomics Core. Results: The mesenchymal prostate cancer cells demonstrated radioresistance relative to the epithelial PC3-E cells (p < 0.0001, 2 and 4 Gy), and a different metabolic profile as indicated by higher levels of phosphorylated AMP Kinase, fatty acid oxidation, phosphorylated acetyl-CoA carboxylase, ATP, and NADPH (p < 0.03, 0.004, 0.01, 0.0001, 0.001 respectively). They consumed less oxygen (p < 0.001), while concurrently producing less lactate and fructose 1.6 bisphosphate (both indicators of glycolysis). Inhibition of fatty acid oxidation with etomoxir sensitized cells to radiation at 2 Gy (p < 0.001). Conclusion: Cells in an EMT-like state have a metabolic profile more dependent on fatty acid oxidation which confers a survival benefit in glucose low environments and resistance to radiation therapy. Our data are consistent with previous findings that blockade of fatty acid oxidation inhibits prostate cancer growth. Fatty acid oxidation is a plausible therapeutic target that can potentially sensitize a resistant sub-population to radiation therapy thereby improving definitive therapy, decreasing metastatic disease, and improving overall survival. Author Disclosure: D.L. Mitchell: None. M. Buchakjian: None. D.C. DeZwaan: None. B. Wagner: None. D. Spitz: None. T.D. Rutkowski: None. M.D. Henry: None.
3293 Sporadic Breast Cancer Cell Lines Show Homologous Recombination Deficiency From BRCA1 Pathway Inactivation With Normal BRCA1 Protein and Are Sensitive to Crosslinking Agents J. Haseltine,1 M. Thompson,2 R.W. Mutter,3 R. Delsite,4 and S.N. Powell4; 1 Weill Cornell Medical College, New York City, NY, 2Emory University Hospital, Atlanta, GA, 3Mayo Clinic, Rochester, MN, 4Memorial Sloan Kettering Cancer Center, New York, NY
3294 Thoracolumbar Spinal Cord Tolerance to High-Dose Conformal Proton-Photon Radiation Therapy V.K. Chowdhry,1 S. Goldberg,1 L. Liu,1 J.A. Adams,1 K. Bernstein,1 N.J. Liebsch,2 A. Niemierko,1 and T.F. DeLaney1; 1Massachusetts General Hospital, Boston, MA, 2Massachusetts General Hospital, Harvard Medical School, Boston, MA Purpose/Objective(s): To evaluate and understand the tolerance of the thoracolumbar spinal cord using equivalent uniform dose (EUD) and dosevolume histogram (DVH) analysis after combined high dose photon-proton radiation therapy. Materials/Methods: A total of 94 patients were identified as having high dose radiation therapy, 5900 cGy (RBE), to the thoracolumbar spine.