Sarah Crunkhorn,
Associate Editor, Nature Reviews Drug Discovery

"Therapy: microRNA suppresses liver cancer"

Hepatocellular carcinoma is a particularly lethal form of cancer, owing to typically late diagnosis and a lack of effective therapies. Now, writing in Cell, Kota and colleagues demonstrate that reinstating the expression of a single microRNA (miRNA) that is specifically downregulated in liver cancer cells suppresses tumour progression, unveiling a new potential anticancer strategy.

Abnormal expression of specific miRNAs has been implicated in the pathogenesis of various human cancers, and miRNA expression profiles have proved useful in tumour classification, prognosis and predicting response to therapy. As most miRNAs are downregulated in tumours, Kota and colleagues proposed that replacing an miRNA that is diminished in cancer cells, but highly expressed and therefore tolerated in normal cells, may provide a safe and effective treatment approach.

To explore their theory, they first set out to identify miRNAs that fitted this profile. miR-26a was found to be the most dramatically downregulated miRNA in cancer cells from a mouse liver tumour model, as compared with normal liver cells. This finding was confirmed in human liver-derived hepatocellular carcinoma cells.

Next, the authors investigated whether miR-26a exhibited antiproliferative properties. Following retroviral-mediated enforced expression of miR-26a in HepG2 cells (to achieve a level similar to that observed in normal liver tissue), flow cytometry revealed that there were fewer cells in S phase and more cells in G1 than in controls. This indicated that miR-26a induced a G1 cell cycle arrest. Prediction using the Targetscan algorithm, followed by in vitro confirmation, demonstrated that miR-26a induced this G1 arrest through the repression of cyclin D2 and cyclin E2.

To evaluate the therapeutic potential of miR-26a, Kota and colleagues first developed an adeno-associated virus (AAV) vector system for in vivo delivery, which was targeted to the liver following systemic administration. A single tail vein injection of the miR-26a AAV vector into mice led to high miR-26a liver expression without any toxic effects when assessed 3 weeks later.

They then assessed the vector in the mouse model of liver cancer at 11 weeks of age — a time when animals typically have multiple small- to medium-sized tumours. After 3 weeks, 6 out of 8 control mice had developed fulminant disease, whereas 8 out of 10 treated mice were protected from disease progression and exhibited only small tumours or a complete absence of tumours. In addition, the lack of response in the other two treated mice seemed to be due to technical failures. Further studies revealed that miR-26a-mediated tumour suppression is associated with sustained inhibition of proliferation and specific induction of apoptosis.

Overall, this study identifies a new target for liver cancer treatment and provides proof-of-concept for the systemic delivery of tumour-suppressing miRNAs as an anticancer approach. The reported deregulation of miR-26a in various other tumours suggests that this strategy might have a broad application.

"Therapeutic microRNA Delivery Suppresses Tumorigenesis in a Murine Liver Cancer Model",
Cell, Volume 137, Issue 6, 1005-1017, 12 June 2009, doi:10.1016/j.cell.2009.04.021
http://www.cell.com/retrieve/pii/S0092867409004462

Summary

Therapeutic strategies based on modulation of microRNA (miRNA) activity hold great promise due to the ability of these small RNAs to potently influence cellular behavior. In this study, we investigated the efficacy of a miRNA replacement therapy for liver cancer. We demonstrate that hepatocellular carcinoma (HCC) cells exhibit reduced expression of miR-26a, a miRNA that is normally expressed at high levels in diverse tissues. Expression of this miRNA in liver cancer cells in vitro induces cell-cycle arrest associated with direct targeting of cyclins D2 and E2. Systemic administration of this miRNA ina mouse model of HCC using adeno-associated virus (AAV) results in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression without toxicity. These findings suggest that delivery of miRNAs that are highly expressed and therefore tolerated in normal tissues but lost in disease cells may provide a general strategy for miRNA replacement therapies.

Article Outline:

Introduction
Results

Downregulation of Putative Antitumorigenic miRNAs in Myc-Induced Liver Tumors
miR-26a Expression Induces a G1 Arrest in Human Liver Cancer Cells
miR-26a Directly Represses Expression of Cyclin D2 and Cyclin E2
MYC Is Not a Target of miR-26a
Development of an AAV Vector System to Simultaneously Express a miRNA and eGFP
Therapeutic Delivery of miR-26a Suppresses Tumorigenesis in tet-o-MYC; LAP-tTA Mice
Delivery of miR-26a Reduces Cancer Cell Proliferation and Induces Tumor-Specific Apoptosis

Discussion
Experimental Procedures

Cell Culture
RNA Isolation, Northern Blotting, and qPCR
Cell-Cycle Profiling
Western Blotting
Luciferase Reporter Assays
Vector Delivery
Analysis of Liver Function Tests
Immunohistochemistry

Acknowledgements
Supplemental Data
References

Summary:
The loss of expression of particular microRNAs can contribute to tumorigenesis. Kota et al. (2009) now explore in a mouse model a promising new approach for the treatment of liver cancer re-establishing the expression of an miRNA using a viral vector.

http://www.cell.com/abstract/S0092-8674(09)00643-6

1: Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model.
Kota J, Chivukula RR, O'Donnell KA, Wentzel EA, Montgomery CL, Hwang HW, Chang TC, Vivekanandan P, Torbenson M, Clark KR, Mendell JR, Mendell JT.

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PMID: 19524505 [PubMed - indexed for MEDLINE]

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