Role of the tumor suppressive microRNA, miR-34a, in the anti-cancer effects of plant based therapeutics

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 292-325-Breast & Prostate Cancer
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-314
Kris Hargraves*1, Lin He1 and Gary L Firestone2
1University of California at Berkeley, 2University of California at Berkeley, Berkeley, CA
MicroRNA are small, non-coding RNAs that post-transcriptionally regulate as much as 30% of the transcribed human genome and could be potential targets of anti-cancer therapeutics (1, 2.)

The microRNA, miR-34a, is a component of the p53 tumor suppressor pathway and has been shown to mediate induction of cell cycle arrest, senescence, and apoptosis in cancer cells (3, 4.) Indole-3-carbinol (I3C) derived from cruciferous vegetables and artemisinin isolated from the sweet wormwood plant, Artemisia annua, effect components of the p53 pathway to growth arrest human breast cancer cells, implicating a potential role for miR-34a in their anti-proliferative effects (5, 6.) Both compounds are currently undergoing clinical trials for the treatment of reproductive cancers (7, 8.)

To investigate the possibility of miR-34a regulation by I3C and artemisinin, human breast cancer cells containing wild-type or mutant p53 were treated with either compound under conditions of cell cycle arrest as detected by flow cytometry. Quantitative PCR analysis of mature microRNA levels revealed a time and dose-dependent upregulation of miR-34a that correlated with induction of functional p53 by I3C and decreases in the mRNA and protein levels of CDK4 and CDK6, cyclin-dependant kinases that are known targets of miR-34a inhibition.

Luciferase assays in which cells were transfected with the miR-34a binding site of CDK6 or CDK4 mRNA attached to the firefly luciferase reporter gene confirmed miR-34a reduces CDK6 and CDK4 levels in cells growth arrested by I3C or artemisinin.

miR-34a also appears critical for the anti-proliferative effects of both compounds as cells in which miR-34a levels have been reduced by transfection of non-translatable miR-34a targets did not growth arrest upon treatment with either drug. Levels of CDK6 and CDK4 also remained unchanged in treated miR-34a knockdown cells, suggesting miR-34a is a crucial component of CDK regulation by both phytochemicals.

Interestingly, transfection of dominant negative p53 prevented I3C upregulation of miR-34a in growth arrested cells containing wild-type p53 yet had no effect on artemisinin regulation of miR-34a, indicating a p53-indepedent mechanism of miR-34a regulation. Artemisinin also upregulates miR-34a in breast cancer cell lines containing non-functional p53.

All of these data suggest that miR-34a plays a critical role in the anti-proliferative effects of artemisinin and indole-3-carbinol in human breast cancer cells. Such evidence further elucidates the therapeutic potential of either drug to ectopically express tumor suppressive microRNA while implicating the use of miR-34a expression levels to determine the efficacy of phytochemical treatment.

(1) Lim et al. Nature (2005.) Microarray analysis shows that some microRNAs down regulate large numbers of target mRNAs. 433, 769-773. (2) Croce, C.M. and George Adrian Calin. Cancer Research (2006). MicroRNA-Cancer Connection: The beginning of a new tale. 66, 7390-7394.  (3) He et al. Nature (2007). A microRNA component of the p53 tumor suppressor network. 447, 1130-1134. (4) Hermeking, H. Cell Death and Differentiation. (2009.) The miR-34 family in cancer and  apoptosis. 17, 193-199. (5) Brew, et al. International Journal of Cancer (2006). Indole-3-carbinol activates the ATM  signaling pathway independent of DNA damage to stabilize p53 and induce G1 arrest of  human mammary epithelial cells. 118, 857-868. (6) Firestone, GL and Shyam N. Sundar. Expert Reviews in Molecular Medicine (2009).  Anticancer activities of artemisinin and its bioactive derivatives. doi: 10.1017/S1462399409001239.  (7) Aggarwal, B.B and Haruyo Ichikawa. Cell Cycle (2005). Molecular targets and anti-cancer potential of indole-3-carbinol. 4, 1201-1215. (8) Crespo-Ortiz MP and MQ Wei .Journal of Biomedicine and Biotechnology (2012). Antitumor activity of artemisinin and its derivatives: from a well-known anti-malarial agent to a potential anticancer drug. doi: 10.1155/2012/247597.

Nothing to Disclose: KH, LH, GLF

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