Therapeutic miRNA Delivery Suppresses Tumorigenesis in a Murine Liver Cancer Model

Janaiah Kota,1 Raghu R. Chivukula,2 Kathryn A. O’Donnell,3,4 Erik A. Wentzel,2 Chrystal LMontgomery,1Hun-Way Hwang,2 Tsung-Cheng Chang,2 Perumal Vivekanandan,5 MichaelTorbenson,5 K. Reed Clark,1,7Jerry R. Mendell,1,7,8,* and Joshua T. Mendell2,4,6 ,*1Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, OH 43205, USA2The McKusick-Nathans Institute of Genetic Medicine3The High Throughput Biology Center4Department of Molecular Biology and Genetics5Department of Pathology6Department of Pediatrics Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA7Department of Pediatrics8Department of Neurology The Ohio State University, Columbus, OH 43210, USA

Cell 137, 1005–1017, 2009

Cell 137, 1005–1017, 2009

microRNA-Regulators of our genome

www.microrna.ic.cz/obr/image003.png

miRNA and Cancer All examined tumor types

Abnormal miRNA expression patterns

• miRNA profiles Tumor classification Prognosis Response to therapy

• Functional Studies Potent pro- and anti- tumorigenic activity of

specific miRNA both in-vitro and in-vivo.

miRNA based Cancer Therapeutics

Two Approaches

Inhibition of oncogenic miRNA

Replacement of tumor suppressor miRNA

Progress In The Field

• Anti-sense mediated inhibition of oncogenic miRNAs (Love et al.,2008; Stenvang et al.,2008).

• Global miRNA repression enhances cellular transformation and tumorigenesis in both in vitro and in vivo models (Kumar et al., 2007)

• Myc activation in B cell lymphoma leads to widespread miRNA repression. (Mendel & Chang et.al)

• Enforced expression of these specific miRNAs dramatically suppresses lymphomagenesis.

• Re-expression of even a single miRNA could provide significant anti-cancer therapeutics.

• Viral delivery of let-7 miRNA ( Tumour Suppressor ) suppressed tumor growth in a mouse model of adenocarcenoma (Esquela-Kerscher et al., 2008; Kumar et al., 2008) .

• let-7 directly targets KRAS,( an Oncogene) the initiating oncogene in this tumor model.

• Thus, the utility of miRNA therapeutics in situations where they do not target the initiating oncogene remains to be studied.

HYPOTHESIS

• miRNAs can be directly used as general anticancer therapeutics even when they do not target the initiating oncogene.

• REASONING: The most therapeutically useful miRNAs would be expressed at low levels in tumors but would be highly expressed in normal tissues.

Selection of Therapeutic miRNA

Along with mouse,miR-26a expressed at lower levels in human HCC.

Therapeutic Potential of miR-26a

• INITIAL TEST:- MSCV derived retroviral construct to enforce expression of miR-26a in HepG2 (Hepatocarcinoma Cell Lines) cells.

• Cell cycle profiles- PI staining and Flow Cytometry

Cell cycle profiles after Nocodazole treatment

miR26a expression induces G1 arrest in Human Liver Cancer Cells

miR-26a Directly Represses Expression of Cyclins D2 and E2

• Predicted targets of miR-26a examined by Targetscan algorithm. Cyclin E1, cyclin E2, cyclin D2 and CDK6

Required in G1-S transition

Verification of direct regulation of transcripts by miR-26a

• Reporter plasmids with predicted binding sites cloned downstream of luciferase ORF.

MYC is Not a Target of miR-26a

• Potent therapeutic role of miR-26a proved in vitro.

• Can systemic delivery of miR-26a be used as a therapeutic strategy for this tumor type in vivo?

• First confirmed that miR-26a does not regulate MYC itself.

• MYC not a predicted target of miR-26a by common algorithms –Targetscan, miRanda and PicTar.

• Western blotting confirms retroviral expression of miR-26a in HepG2 cells doesn’t affect MYC protein abundance.

AAV Vector System to express miR-26a and eGFP• CONSTRUCTS

VECTOR DELIVERY• Single tail-vein injection.• Liver tissue harvested after 3 weeks: >90% Transduction.

• Previous study-AAV mediated delivery of shRNA induced acute liver toxicity probably due to inhibition of miRNA pathway.

Suppression of Tumorigenesis by miR-26a

• Timeline:-

6/8 8/10

2/10 treated mice- aggressive tumor- lower transduction efficiency

Technical failure rather than resistance to miR-26a therapeutics.

EFFECT ON PROLIFERATION AND TUMOR-SPECIFIC APOPTOSIS

CELLULAR PROLIFERATION- Ki67 staining

TUMOR-SPECIFIC APOPTOSIS- TUNEL staining

Thank You