Lihong Wang-Bishop1, Zheng Chen2, Ahmed Gomaa2, Albert Craig Lockhart3, Safia Salaria4, Jialiang Wang4, Keeli B Lewis4, Jeffrey Ecsedy5, Kay Washington4, Robert Daniel Beauchamp6, Wael El-Rifai7. 1. Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, Tennessee. 2. Department of Surgery, Miller School of Medicine, University of Miami, Miami, Florida. 3. Division of Medical Oncology, Miller School of Medicine, University of Miami, Miami, Florida; Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, Florida. 4. Section of Surgical Sciences, Vanderbilt University Medical Center, Nashville, Tennessee. 5. Translational Medicine, Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts. 6. Section of Surgical Sciences, Vanderbilt University Medical Center, Nashville, Tennessee; Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee. 7. Department of Surgery, Miller School of Medicine, University of Miami, Miami, Florida; Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, Florida; Department of Veterans Affairs, Miami VA Healthcare system, Miami, Florida. Electronic address: welrifai@med.miami.edu.
Abstract
BACKGROUND & AIMS: Activation of KRAS signaling and overexpression of the aurora kinase A (AURKA) are often detected in luminal gastrointestinal cancers. We investigated regulation of ribosomal protein S6 kinase B1 (RPS6KB1) by AURKA and the effects of alisertib, an AURKA inhibitor, in mice xenograft tumors grown from human gastrointestinal cancer cells with mutant, activated forms of KRAS. METHODS: We tested the effects of alisertib or AURKA overexpression or knockdown in 10 upper gastrointestinal or colon cancer cell lines with KRAS mutations or amplifications using the CellTiter-Glo luminescence and clonogenic cell survival assays. We used the proximity ligation in situ assay to evaluate protein co-localization and immunoprecipitation to study protein interactions. Nude mice with xenograft tumors grown from HCT116, SNU-601, SW480, or SNU-1 cells were given oral alisertib (40 mg/kg, 5 times/wk) for 4 weeks. Tumor samples were collected and analyzed by immunoblots and immunohistochemistry. Tissue microarrays from 151 paraffin-embedded human colon tumors, with adjacent normal and adenoma tissues, were analyzed by immunohistochemistry for levels of AURKA. RESULTS: Alisertib reduced proliferation and survival of the cell lines tested. AURKA knockdown or inhibition with alisertib reduced levels of phosphorylated RPS6KB1 (at T389) and increased levels of proteins that induce apoptosis, including BIM, cleaved PARP, and cleaved caspase 3. AURKA co-localized and interacted with RPS6KB1, mediating RPS6KB1 phosphorylation at T389. We detected AURKA-dependent phosphorylation of RPS6KB1 in cell lines with mutations in KRAS but not in cells with wild-type KRAS. Administration of alisertib to mice with xenograft tumors significantly reduced tumor volumes (P < .001). Alisertib reduced phosphorylation of RPS6KB1 and Ki-67 and increased levels of cleaved caspase 3 in tumor tissues. In analyses of tissue microarrays, we found significant overexpression of AURKA in gastrointestinal tumor tissues compared with non-tumor tissues (P = .0003). CONCLUSION: In studies of gastrointestinal cancer cell lines with activated KRAS, we found AURKA to phosphorylate RPS6KB1, promoting cell proliferation and survival and growth of xenograft tumors in mice. Agents that inhibit AURKA might slow the growth of gastrointestinal tumors with activation of KRAS.
BACKGROUND & AIMS: Activation of KRAS signaling and overexpression of the aurora kinase A (AURKA) are often detected in luminal gastrointestinal cancers. We investigated regulation of ribosomal protein S6 kinase B1 (RPS6KB1) by AURKA and the effects of alisertib, an AURKA inhibitor, in micexenograft tumors grown from humangastrointestinal cancer cells with mutant, activated forms of KRAS. METHODS: We tested the effects of alisertib or AURKA overexpression or knockdown in 10 upper gastrointestinal or colon cancer cell lines with KRAS mutations or amplifications using the CellTiter-Glo luminescence and clonogenic cell survival assays. We used the proximity ligation in situ assay to evaluate protein co-localization and immunoprecipitation to study protein interactions. Nude mice with xenograft tumors grown from HCT116, SNU-601, SW480, or SNU-1 cells were given oral alisertib (40 mg/kg, 5 times/wk) for 4 weeks. Tumor samples were collected and analyzed by immunoblots and immunohistochemistry. Tissue microarrays from 151 paraffin-embedded humancolon tumors, with adjacent normal and adenoma tissues, were analyzed by immunohistochemistry for levels of AURKA. RESULTS:Alisertib reduced proliferation and survival of the cell lines tested. AURKA knockdown or inhibition with alisertib reduced levels of phosphorylated RPS6KB1 (at T389) and increased levels of proteins that induce apoptosis, including BIM, cleaved PARP, and cleaved caspase 3. AURKA co-localized and interacted with RPS6KB1, mediating RPS6KB1 phosphorylation at T389. We detected AURKA-dependent phosphorylation of RPS6KB1 in cell lines with mutations in KRAS but not in cells with wild-type KRAS. Administration of alisertib to mice with xenograft tumors significantly reduced tumor volumes (P < .001). Alisertib reduced phosphorylation of RPS6KB1 and Ki-67 and increased levels of cleaved caspase 3 in tumor tissues. In analyses of tissue microarrays, we found significant overexpression of AURKA in gastrointestinal tumor tissues compared with non-tumor tissues (P = .0003). CONCLUSION: In studies of gastrointestinal cancer cell lines with activated KRAS, we found AURKA to phosphorylate RPS6KB1, promoting cell proliferation and survival and growth of xenograft tumors in mice. Agents that inhibit AURKA might slow the growth of gastrointestinal tumors with activation of KRAS.
Authors: Ahmed Katsha; Mohammed Soutto; Vikas Sehdev; Dunfa Peng; M Kay Washington; M Blanca Piazuelo; Mohammed N Tantawy; H Charles Manning; Pengcheng Lu; Yu Shyr; Jeffrey Ecsedy; Abbes Belkhiri; Wael El-Rifai Journal: Gastroenterology Date: 2013-08-29 Impact factor: 22.682
Authors: Robert W Schnepp; Priya Khurana; Edward F Attiyeh; Pichai Raman; Sara E Chodosh; Derek A Oldridge; Maria E Gagliardi; Karina L Conkrite; Shahab Asgharzadeh; Robert C Seeger; Blair B Madison; Anil K Rustgi; John M Maris; Sharon J Diskin Journal: Cancer Cell Date: 2015-10-17 Impact factor: 31.743
Authors: Hyun Min Koh; Bo Geun Jang; Chang Lim Hyun; Young Sill Kim; Jin Won Hyun; Weon Young Chang; Young Hee Maeng Journal: J Pathol Transl Med Date: 2016-12-25