title: Regulation of oncogenic KRAS signaling via a novel KRAS-integrin-linked kinase-hnRNPA1 regulatory loop in human pancreatic cancer cells.
abstract: Integrin-linked kinase (ILK) is a mediator of aggressive phenotype in pancreatic cancer. On the basis of our finding that knockdown of either KRAS or ILK has a reciprocal effect on the other's expression, we hypothesized the presence of an ILK-KRAS regulatory loop that enables pancreatic cancer cells to regulate KRAS expression. This study aimed to elucidate the mechanism by which this regulatory circuitry is regulated and to investigate the translational potential of targeting ILK to suppress oncogenic KRAS signaling in pancreatic cancer. Interplay between KRAS and ILK and the roles of E2F1, c-Myc and heterogeneous nuclear ribonucleoprotein as intermediary effectors in this feedback loop was interrogated by genetic manipulations through small interfering RNA/short hairpin RNA knockdown and ectopic expression, western blotting, PCR, promoter-luciferase reporter assays, chromatin immunoprecipitation and pull-down analyses. In vivo efficacy of ILK inhibition was evaluated in two murine xenograft models. Our data show that KRAS regulated the expression of ILK through E2F1-mediated transcriptional activation, which, in turn, controlled KRAS gene expression via hnRNPA1-mediated destabilization of the G-quadruplex on the KRAS promoter. Moreover, ILK inhibition blocked KRAS-driven epithelial-mesenchymal transition and growth factor-stimulated KRAS expression. The knockdown or pharmacological inhibition of ILK suppressed pancreatic tumor growth, in part, by suppressing KRAS signaling. These studies suggest that this KRAS-E2F1-ILK-hnRNPA1 regulatory loop enables pancreatic cancer cells to promote oncogenic KRAS signaling and to interact with the tumor microenvironment to promote aggressive phenotypes. This regulatory loop provides a mechanistic rationale for targeting ILK to suppress oncogenic KRAS signaling, which might foster new therapeutic strategies for pancreatic cancer.
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA.|Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan.|Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors.forename: P-C
authors.initials: PC
authors.lastname: Chu
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
authors.forename: M-C
authors.initials: MC
authors.lastname: Yang
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
authors.forename: S K
authors.initials: SK
authors.lastname: Kulp
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
authors.forename: S B
authors.initials: SB
authors.lastname: Salunke
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA.|Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA.
authors.forename: L E
authors.initials: LE
authors.lastname: Himmel
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
authors.forename: C-S
authors.initials: CS
authors.lastname: Fang
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Jadhav
authors.affiliation: Department of Surgery, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Y-S
authors.initials: YS
authors.lastname: Shan
authors.affiliation: Department of Pathology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: C-T
authors.initials: CT
authors.lastname: Lee
authors.affiliation: Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: M-D
authors.initials: MD
authors.lastname: Lai
authors.affiliation: Department of Surgery, College of Medicine, The Ohio State University, Columbus, OH, USA.
authors.forename: L A
authors.initials: LA
authors.lastname: Shirley
authors.affiliation: Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH, USA.
authors.forename: T
authors.initials: T
authors.lastname: Bekaii-Saab
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA.|Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan.|Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors.forename: C-S
authors.initials: CS
authors.lastname: Chen
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D050687:E2F1 Transcription Factor / Q000502:physiology; D058750:Epithelial-Mesenchymal Transition; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000502:physiology*; D006801:Humans; D051379:Mice; D010190:Pancreatic Neoplasms / Q000473:pathology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D016271:Proto-Oncogene Proteins c-myc / Q000502:physiology; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D015533:Transcriptional Activation; D000099200:Scaffold Protein ILK
chemical_list: D050687:E2F1 Transcription Factor; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D017346:Protein Serine-Threonine Kinases; D016271:Proto-Oncogene Proteins c-myc; D016283:Proto-Oncogene Proteins p21(ras); D000099200:Scaffold Protein ILK; C493753:E2F1 protein, human; C000619657:Hnrnpa1 protein, mouse; C117307:KRAS protein, human; C489427:MYC protein, human; C000619656:hnRNPA1 protein, human