RNAi Screening of the Glucose-Regulated Chaperones in Cancer with Self-Assembled siRNA Nanostructures.
The emerging field of RNA nanotechnology has been used to design well-programmed, self-assembled nanostructures for applications in chemistry, biology, and medicine. At the forefront of its utility in cancer is the unrestricted ability to self-assemble multiple siRNAs within a single nanostructure formulation for the RNAi screening of a wide range of oncogenes while potentiating the gene therapy of malignant tumors. In our RNAi nanotechnology approach, V- and Y-shape RNA templates were designed and constructed for the self-assembly of discrete, higher-ordered siRNA nanostructures targeting the oncogenic glucose regulated chaperones. The GRP78-targeting siRNAs self-assembled into genetically encoded spheres, triangles, squares, pentagons and hexagons of discrete sizes and shapes according to TEM imaging. Furthermore, gel electrophoresis, thermal denaturation, and CD spectroscopy validated the prerequisite siRNA hybrids for their RNAi application. In a 24 sample siRNA screen conducted within the AN3CA endometrial cancer cells known to overexpress oncogenic GRP78 activity, the self-assembled siRNAs targeting multiple sites of GRP78 expression demonstrated more potent and long-lasting anticancer activity relative to their linear controls. Extending the scope of our RNAi screening approach, the self-assembled siRNA hybrids (5 nM) targeting of GRP-75, 78, and 94 resulted in significant (50-95%) knockdown of the glucose regulated chaperones, which led to synergistic effects in tumor cell cycle arrest (50-80%) and death (50-60%) within endometrial (AN3CA), cervical (HeLa), and breast (MDA-MB-231) cancer cell lines. Interestingly, a nontumorigenic lung (MRC5) cell line displaying normal glucose regulated chaperone levels was found to tolerate siRNA treatment and demonstrated less toxicity (5-20%) relative to the cancer cells that were found to be addicted to glucose regulated chaperones. These remarkable self-assembled siRNA nanostructures may thus encompass a new class of potent siRNAs that may be useful in screening important oncogene targets while improving siRNA therapeutic efficacy and specificity in cancer.
Program in Chemical Biology and Department of Medicine, Memorial Sloan Kettering Cancer Center , New York, New York 10065, United States.|Department of Chemistry and Biochemistry, Seton Hall University , South Orange, New Jersey 07079, United States.
Mayurbhai R
MR
Patel
Department of Chemistry and Biochemistry, Seton Hall University , South Orange, New Jersey 07079, United States.
Stephen D
SD
Kozuch
Department of Chemistry and Biochemistry, Seton Hall University , South Orange, New Jersey 07079, United States.
Christopher N
CN
Cultrara
Department of Chemistry and Biochemistry, Queens College, City University of New York , 65-30 Kissena Blvd., Flushing, New York 11367, United States.|Ph.D. Programs in Chemistry and Biochemistry, The Graduate Center of the City University of New York , New York City, New York 10016, United States.
Reeta
R
Yadav
Department of Chemistry and Biochemistry, Queens College, City University of New York , 65-30 Kissena Blvd., Flushing, New York 11367, United States.|Ph.D. Programs in Chemistry and Biochemistry, The Graduate Center of the City University of New York , New York City, New York 10016, United States.
Suiying
S
Huang
Department of Chemistry and Biochemistry, Queens College, City University of New York , 65-30 Kissena Blvd., Flushing, New York 11367, United States.|Ph.D. Programs in Chemistry and Biochemistry, The Graduate Center of the City University of New York , New York City, New York 10016, United States.
Uri
U
Samuni
Program in Chemical Biology and Department of Medicine, Memorial Sloan Kettering Cancer Center , New York, New York 10065, United States.
John
J
Koren
Program in Chemical Biology and Department of Medicine, Memorial Sloan Kettering Cancer Center , New York, New York 10065, United States.
Gabriela
G
Chiosis
Department of Chemistry and Biochemistry, Seton Hall University , South Orange, New Jersey 07079, United States.
David
D
Sabatino
GRP; RNAi nanotechnology; cancer gene therapy; cervical and breast cancer; chaperones; endometrial; glucose regulated proteins; siRNA nanostructures
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