title: EpCAM Aptamer-siRNA Chimera Targets and Regress Epithelial Cancer.
abstract: Epithelial cell adhesion molecule (EpCAM), a cancer stem cell (CSC) marker is over expressed in epithelial cancers and in retinoblastoma (RB). We fabricated an EpCAM targeting aptamer-siRNA chimera and investigated its anti-tumor property and EpCAM intracellular domain (EpICD) mediated signaling in epithelial cancer. The anti-tumor efficacy of EpCAM aptamer-siEpCAM chimera (EpApt-siEp) was evaluated by qPCR, northern and Western blotting in WERI-Rb1- RB cell line, primary RB tumor cells and in MCF7- breast cancer cell line. Anti-tumor activity of EpApt-siEp was studied in vivo using epithelial cancer (MCF7) mice xenograft model. The mechanism and pathways involved in the anti-tumor activity was further studied using protein arrays and qPCR. EpApt-siEp chimera was processed in vitro by dicer enzyme. Treatment of the WERI-Rb1 and MCF7 cells with EpApt-siEp revealed statistically significant down regulation of EpCAM expression (P<0.005) and concomitant reduction in cellular proliferation. In primary RB cells cultured from RB tumors, EpApt-siEp silenced EpCAM, significantly inhibited (P<0.01) cell proliferation and induced cytotoxicity. Knockdown of EpICD expressed in RB primary tumors led to repression of pluripotency markers, SOX2, OCT4, NANOG, and CD133. In vivo studies showed complete tumor growth regression without any toxicity in animals (P<0.001) and tumor tissues showed significant downregulation (P<0.05) of EpCAM, MRP1, ABCG2, stathmin, survivin and upregulation of ATM (P<0.05) leading to apoptosis by intrinsic pathway with minor alteration in cytokines. Our results revealed that EpApt-siEp potentially eradicated EpCAM positive cancer cells through CSC marker suppression and apoptosis, while sparing normal EpCAM negative adjacent cells.
authors.affiliation: Department of Nanobiotechnology, Vision Research Foundation, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Chennai, India; Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Centre for Molecular and Medical Research (C-MMR), Faculty of Health, Deakin University, Geelong, Victoria-3217, Australia.
authors.forename: Nithya
authors.initials: N
authors.lastname: Subramanian
authors.affiliation: Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Centre for Molecular and Medical Research (C-MMR), Faculty of Health, Deakin University, Geelong, Victoria-3217, Australia.
authors.forename: Jagat R
authors.initials: JR
authors.lastname: Kanwar
authors.affiliation: Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Centre for Molecular and Medical Research (C-MMR), Faculty of Health, Deakin University, Geelong, Victoria-3217, Australia.
authors.forename: Rupinder K
authors.initials: RK
authors.lastname: Kanwar
authors.affiliation: L & T Ocular Pathology department, Vision Research Foundation, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Chennai, India.
authors.forename: JagadeeshBabu
authors.initials: J
authors.lastname: Sreemanthula
authors.affiliation: L & T Ocular Pathology department, Vision Research Foundation, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Chennai, India.
authors.forename: Jyotirmay
authors.initials: J
authors.lastname: Biswas
authors.affiliation: Department of Ocular Oncology and Vitreoretina, Medical Research Foundation, Sankara Nethralaya, Chennai, India.
authors.forename: Vikas
authors.initials: V
authors.lastname: Khetan
authors.affiliation: Department of Nanobiotechnology, Vision Research Foundation, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Chennai, India; L & T Ocular Pathology department, Vision Research Foundation, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Chennai, India.
authors.forename: Subramanian
authors.initials: S
authors.lastname: Krishnakumar
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000378:metabolism*; D017209:Apoptosis; D052157:Aptamers, Nucleotide / Q000378:metabolism*; D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004705:Endocytosis; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D007150:Immunohistochemistry; D056747:Immunomodulation; D007700:Kinetics; D061986:MCF-7 Cells; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D014411:Neoplastic Stem Cells / Q000378:metabolism; D040081:Protein Array Analysis; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism*; D012175:Retinoblastoma; D043244:Ribonuclease III / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D000951:Antigens, Neoplasm; D052157:Aptamers, Nucleotide; D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D034741:RNA, Small Interfering; D043244:Ribonuclease III