EpCAM Aptamer-siRNA Chimera Targets and Regress Epithelial Cancer

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.

[1]  B. Giepmans,et al.  EpCAM proteolysis: new fragments with distinct functions? , 2013, Bioscience reports.

[2]  M. Rots,et al.  EpCAM in carcinogenesis: the good, the bad or the ugly. , 2010, Carcinogenesis.

[3]  Markus Munz,et al.  The emerging role of EpCAM in cancer and stem cell signaling. , 2009, Cancer research.

[4]  J. Rossi,et al.  Aptamer-targeted cell-specific RNA interference , 2010, Silence.

[5]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[6]  K. Hioki,et al.  Immunohistochemical localization of epithelial glycoprotein EGP-2 and carcinoembryonic antigen in normal colonic mucosa and colorectal tumors. , 1998, Anticancer research.

[7]  Y. Hannun,et al.  EpCAM Is Overexpressed in Breast Cancer and Is a Potential Target for Breast Cancer Gene Therapy , 2004, Cancer Research.

[8]  All in the RNA family , 2006, Nature Biotechnology.

[9]  U. Krishnan,et al.  EpCAM is a putative stem marker in retinoblastoma and an effective target for T-cell-mediated immunotherapy , 2012, Molecular vision.

[10]  L. Dirix,et al.  An open-label, randomized phase II study of adecatumumab, a fully human anti-EpCAM antibody, as monotherapy in patients with metastatic breast cancer. , 2010, Annals of oncology : official journal of the European Society for Medical Oncology.

[11]  Rebecca SY Wong,et al.  Apoptosis in cancer: from pathogenesis to treatment , 2011, Journal of experimental & clinical cancer research : CR.

[12]  W. Duan,et al.  Selection of DNA aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture. , 2013, Analytical chemistry.

[13]  Xiaohu Gao,et al.  siRNA-aptamer chimeras on nanoparticles: preserving targeting functionality for effective gene silencing. , 2011, ACS nano.

[14]  Brigitte Mack,et al.  Nuclear signalling by tumour-associated antigen EpCAM , 2009, Nature Cell Biology.

[15]  O. Gires,et al.  On the abundance of EpCAM on cancer stem cells , 2009, Nature Reviews Cancer.

[16]  S. Krishnakumar,et al.  Genome-wide changes accompanying the knockdown of Ep-CAM in retinoblastoma , 2010, Molecular vision.

[17]  R. Kanwar,et al.  Chimeric aptamers in cancer cell-targeted drug delivery , 2011, Critical reviews in biochemistry and molecular biology.

[18]  A. Reynolds,et al.  The contributions of dsRNA structure to Dicer specificity and efficiency. , 2005, RNA.

[19]  EpCAM aptamer mediated cancer cell specific delivery of EpCAM siRNA using polymeric nanocomplex , 2015, Journal of Biomedical Science.

[20]  Cristina Romero-López,et al.  In Vitro and Ex Vivo Selection Procedures for Identifying Potentially Therapeutic DNA and RNA Molecules , 2010, Molecules.

[21]  I. Treilleux,et al.  Abnormal expression of the ATM and TP53 genes in sporadic breast carcinomas. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[22]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[23]  B. Mack,et al.  EpCAM Is Involved in Maintenance of the Murine Embryonic Stem Cell Phenotype , 2009, Stem cells.

[24]  Anton P. McCaffrey,et al.  Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors , 2009, Nature Biotechnology.

[25]  Liyu Li,et al.  Nucleolin-targeting liposomes guided by aptamer AS1411 for the delivery of siRNA for the treatment of malignant melanomas. , 2014, Biomaterials.

[26]  M. Ferrari,et al.  Combinatorial selection of DNA thioaptamers targeted to the HA binding domain of human CD44. , 2010, Biochemistry.

[27]  S. Krishnakumar,et al.  Oncogenic microRNA 17-92 cluster is regulated by epithelial cell adhesion molecule and could be a potential therapeutic target in retinoblastoma , 2012, Molecular vision.

[28]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[29]  D. Coppola,et al.  EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer , 2012, Molecular Cancer.

[30]  K. Tsao,et al.  Serum TIMP-1 in gastric cancer patients: a potential prognostic biomarker. , 2006, Annals of clinical and laboratory science.

[31]  R. Kanwar,et al.  Target-specific delivery of doxorubicin to retinoblastoma using epithelial cell adhesion molecule aptamer , 2012, Molecular vision.

[32]  J. Shankaranarayanan,et al.  Aptamer-based therapeutics of the past, present and future: from the perspective of eye-related diseases. , 2014, Drug discovery today.

[33]  David H. Mathews,et al.  RNAstructure: web servers for RNA secondary structure prediction and analysis , 2013, Nucleic Acids Res..

[34]  G. Dranoff,et al.  Cytokines in cancer pathogenesis and cancer therapy , 2004, Nature Reviews Cancer.

[35]  John Yu,et al.  Epithelial Cell Adhesion Molecule Regulation Is Associated with the Maintenance of the Undifferentiated Phenotype of Human Embryonic Stem Cells* , 2010, The Journal of Biological Chemistry.

[36]  A. Plückthun,et al.  Epithelial cell adhesion molecule-targeted drug delivery for cancer therapy , 2013, Expert opinion on drug delivery.

[37]  Libing Song,et al.  miR-18a Impairs DNA Damage Response through Downregulation of Ataxia Telangiectasia Mutated (ATM) Kinase , 2011, PloS one.

[38]  S. Ross,et al.  Is low-molecular-weight heparin safe for venous thromboembolism prophylaxis in patients with traumatic brain injury? A Western Trauma Association multicenter study , 2012, The journal of trauma and acute care surgery.

[39]  S. B. Atienza-Samols,et al.  With Contributions by , 1978 .

[40]  Xiaoting Zhang,et al.  RNA aptamers and their therapeutic and diagnostic applications. , 2013, International journal of biochemistry and molecular biology.

[41]  V. Smits,et al.  Cleavage and degradation of Claspin during apoptosis by caspases and the proteasome , 2007, Cell Death and Differentiation.

[42]  Seonghwan Lee,et al.  Aptamers and Their Biological Applications , 2012, Sensors.

[43]  O. Gires,et al.  EpCAM (CD326) finding its role in cancer , 2007, British Journal of Cancer.

[44]  Tao Wang,et al.  RNA aptamers targeting cancer stem cell marker CD133. , 2013, Cancer letters.

[45]  A. Mohan,et al.  EpCAM expression in retinoblastoma: a novel molecular target for therapy. , 2004, Investigative ophthalmology & visual science.

[46]  J. Kanwar,et al.  A strain-promoted alkyne-azide cycloaddition (SPAAC) reaction of a novel EpCAM aptamer-fluorescent conjugate for imaging of cancer cells. , 2014, Chemical communications.

[47]  D. Fan,et al.  EpCAM is overexpressed in gastric cancer and its downregulation suppresses proliferation of gastric cancer , 2009, Journal of Cancer Research and Clinical Oncology.

[48]  J. Bruno.  A Review of Therapeutic Aptamer Conjugates with Emphasis on New Approaches , 2013, Pharmaceuticals.

[49]  Xiaohu Gao,et al.  A Universal Protein Tag for Delivery of SiRNA-Aptamer Chimeras , 2013, Scientific Reports.

[50]  W. Duan,et al.  RNA aptamer against a cancer stem cell marker epithelial cell adhesion molecule , 2011, Cancer science.