title: Unbiased analysis of pancreatic cancer radiation resistance reveals cholesterol biosynthesis as a novel target for radiosensitisation.
abstract: BACKGROUND
  Despite its promise as a highly useful therapy for pancreatic cancer (PC), the addition of external beam radiation therapy to PC treatment has shown varying success in clinical trials. Understanding PC radioresistance and discovery of methods to sensitise PC to radiation will increase patient survival and improve quality of life. In this study, we identified PC radioresistance-associated pathways using global, unbiased techniques.
  
  
  METHODS
  Radioresistant cells were generated by sequential irradiation and recovery, and global genome cDNA microarray analysis was performed to identify differentially expressed genes in radiosensitive and radioresistant cells. Ingenuity pathway analysis was performed to discover cellular pathways and functions associated with differential radioresponse and identify potential small-molecule inhibitors for radiosensitisation. The expression of FDPS, one of the most differentially expressed genes, was determined in human PC tissues by IHC and the impact of its pharmacological inhibition with zoledronic acid (ZOL, Zometa) on radiosensitivity was determined by colony-forming assays. The radiosensitising effect of Zol in vivo was determined using allograft transplantation mouse model.
  
  
  RESULTS
  Microarray analysis indicated that 11 genes (FDPS, ACAT2, AG2, CLDN7, DHCR7, ELFN2, FASN, SC4MOL, SIX6, SLC12A2, and SQLE) were consistently associated with radioresistance in the cell lines, a majority of which are involved in cholesterol biosynthesis. We demonstrated that knockdown of farnesyl diphosphate synthase (FDPS), a branchpoint enzyme of the cholesterol synthesis pathway, radiosensitised PC cells. FDPS was significantly overexpressed in human PC tumour tissues compared with healthy pancreas samples. Also, pharmacologic inhibition of FDPS by ZOL radiosensitised PC cell lines, with a radiation enhancement ratio between 1.26 and 1.5. Further, ZOL treatment resulted in radiosensitisation of PC tumours in an allograft mouse model.
  
  
  CONCLUSIONS
  Unbiased pathway analysis of radioresistance allowed for the discovery of novel pathways associated with resistance to ionising radiation in PC. Specifically, our analysis indicates the importance of the cholesterol synthesis pathway in PC radioresistance. Further, a novel radiosensitiser, ZOL, showed promising results and warrants further study into the universality of these findings in PC, as well as the true potential of this drug as a clinical radiosensitiser.
authors.affiliation: Fred & Pamela Buffet Cancer Center, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Souchek
authors.affiliation: Fred & Pamela Buffet Cancer Center, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Baine
authors.affiliation: Department of Radiation Oncology, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: C
authors.initials: C
authors.lastname: Lin
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: S
authors.initials: S
authors.lastname: Rachagani
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: S
authors.initials: S
authors.lastname: Gupta
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: S
authors.initials: S
authors.lastname: Kaur
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: K
authors.initials: K
authors.lastname: Lester
authors.affiliation: Department of Radiation Oncology, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: D
authors.initials: D
authors.lastname: Zheng
authors.affiliation: Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: S
authors.initials: S
authors.lastname: Chen
authors.affiliation: Department of Biostatistics, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: L
authors.initials: L
authors.lastname: Smith
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: A
authors.initials: A
authors.lastname: Lazenby
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Johansson
authors.affiliation: 1] Fred & Pamela Buffet Cancer Center, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA [2] Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: M
authors.initials: M
authors.lastname: Jain
authors.affiliation: 1] Fred & Pamela Buffet Cancer Center, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA [2] Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: S K
authors.initials: SK
authors.lastname: Batra
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000532:radiotherapy*; D000818:Animals; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000032:analysis; D004164:Diphosphonates / Q000494:pharmacology* / Q000627:therapeutic use; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D051228:Geranyltranstransferase / Q000032:analysis / Q000235:genetics*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology* / Q000627:therapeutic use; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D020411:Oligonucleotide Array Sequence Analysis; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000532:radiotherapy*; D011836:Radiation Tolerance / Q000187:drug effects* / Q000235:genetics; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology* / Q000627:therapeutic use; D000077211:Zoledronic Acid
chemical_list: D018076:DNA, Complementary; D004164:Diphosphonates; D007093:Imidazoles; D011838:Radiation-Sensitizing Agents; D000077211:Zoledronic Acid; D002784:Cholesterol; D051228:Geranyltranstransferase