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Carolyn K Harrod,1 Brenda Y Hernandez,2 Courtney Yates,3 Robert Harrod1 1Department of Biological Sciences and The Dedman College Center for Drug Discovery, Design & Delivery, Southern Methodist University, Dallas, TX, USA; 2Hawaii Tumor Registry, University of Hawaii Cancer Center, Honolulu, HI, USA; 3Laboratory Animal Resource Center, Southern Methodist University, Dallas, TX, USACorrespondence: Robert Harrod, Department of Biological Sciences and The Dedman College Center for Drug Discovery, Design & Delivery, Southern Methodist University, 6501 Airline Drive, 334-DLS, Dallas, TX, 75275-0376, USA, Tel +1 214 768 3864, Fax +1 214 768 3955, Email rharrod@smu.eduIntroduction: The TP53-induced glycolysis and apoptosis regulator (TIGAR) is upregulated in many cancers and often correlates with poor clinical prognoses and serves as a key determinant of therapy-responsiveness. The TIGAR protein is structurally nearly identical to the phosphatase subunit of the bifunctional, fructose-6-phosphokinase/fructose-2,6-bisphosphatase, which has likely hindered efforts to date to develop small-molecule pharmacological inhibitors of TIGAR.Purpose: The objective of this study was to investigate the efficacy of a siRNA-tigar-biopolymer, TI6752 (Tituxistatin), to therapeutically inhibit tumorigenesis in an in vivo xenograft model of colorectal cancer.Materials and Methods: The overexpression of TIGAR within K-Ras+ tumor cells and the infiltration of PECAM-1+ endothelial progenitors in primary colorectal carcinoma clinical samples were detected by immunoconfocal microscopy. Immunodeficient NIH III-nude mice were subcutaneously engrafted with HCT116 colon cancer cells and then treated with three doses of TI6752 (1 mg/kg bw) or a Vehicle control, administered intravenously at weekly intervals. The animals were humanely sacrificed and HCT116 cells within the tumor tissues were visualized using an Anti-human Ki67 primary antibody. The accumulation of biotin-labeled TI6752 within preexisting HCT116 tumor tissues, compared to other secondary organs (heart, liver, kidneys), was visualized using an AlexaFluor488-conjugated Anti-Biotin primary antibody.Results: We have shown that TIGAR is highly expressed in K-Ras+ colorectal carcinoma clinical samples and correlates with robust angiogenesis. Using a preclinical HCT116 xenograft model of colorectal carcinoma, we have demonstrated that therapeutic IV-administration of a pegylated siRNA-biopolymer, TI6752, inhibited tumor growth and reduced the infiltration of PECAM-1+ endothelial progenitors into xenograft tumor tissues without causing any adverse secondary effects.Conclusion: This study has demonstrated that IV-delivery of a pegylated siRNA-biopolymer, TI6752, targeted against tigar mRNA transcripts, effectively inhibited tumor growth and angiogenesis in an HCT116 xenograft model of colorectal carcinoma. TI6752 could represent a effective therapeutic approach to target TIGAR’s pro-oncogenic functions in human cancers.Plain Language Summary: The TIGAR protein is upregulated in many human cancers and has been shown to confer a protective pro-survival advantage to tumor cells associated with aggressive cell proliferation, invasiveness/metastasis, and refractory therapy-resistance that often correlates with poor clinical prognoses. Nevertheless, its significant structural homology to a metabolic enzyme (fructose-6-phosphokinase/fructose-2,6-bisphosphatase) required for cellular energy production suggests the TIGAR protein may be an undruggable anti-oncological target. This recent era has highlighted the expanding applications of RNA therapeutics and, therefore, we have tested the efficacy of a siRNA-based biopolymer, TI6752, that inhibits tigar gene expression in an in vivo preclinical HCT116 xenograft model of colorectal cancer. These translational studies revealed that IV-administration of TI6752 prevented the growth of HCT116 colorectal carcinoma tumors in treated experimental animals without causing any observed adverse secondary effects. We also demonstrated that biotin-labeled TI6752 accumulated within the tumor parenchyma but was not detectable in other tissues, including the heart or liver. Our findings indicate that TI6752 could represent an effective approach to therapeutically target the expression and pro-oncogenic functions of TIGAR in refractory cancers that do not otherwise respond to conventional anticancer therapies.Keywords: TIGAR, RNA interference therapy, anti-oncological, K-Ras, angiogenesis, colorectal cancer