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MicroRNA-25 regulates chemoresistance-associated autophagy in breast cancer cells, a process modulated by the natural autophagy inducer isoliquiritigenin.

Wang Z, Wang N, Liu P, Chen Q, Situ H, Xie T, Zhang J, Peng C, Lin Y, Chen J - Oncotarget (2014)

Bottom Line: Recent findings have revealed that dysregulated miRNAs contribute significantly to autophagy and chemoresistance.More importantly, miRNA 3.0 array experiments identified miR-25 as the main target of ISL in triggering autophagy flux.Subsequent in vivo experiments showed that ISL had chemosensitizing potency, as revealed by an increase in LC3-II staining, the downregulation of ABCG2, a reduction in miR-25 expression and the activation of the miR-25 target ULK1.

View Article: PubMed Central - PubMed

Affiliation: Department of Mammary Disease, Guangdong Provincial Hospital of Chinese Medicine; School of Chinese Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China; These authors contributed equally to this work.

ABSTRACT
Recent findings have revealed that dysregulated miRNAs contribute significantly to autophagy and chemoresistance. Pharmacologically targeting autophagy-related miRNAs is a novel strategy to reverse drug resistance. Here, we report a novel function of isoliquiritigenin (ISL) as a natural inhibitor of autophagy-related miR-25 in killing drug-resistant breast cancer cells. ISL induced chemosensitization, cell cycle arrest and autophagy, but not apoptosis, in MCF-7/ADR cells. ISL also promoted the degradation of the ATP-binding cassette (ABC) protein ABCG2 primarily via the autophagy-lysosome pathway. More importantly, miRNA 3.0 array experiments identified miR-25 as the main target of ISL in triggering autophagy flux. A mechanistic study validated that miR-25 inhibition led to autophagic cell death by directly increasing ULK1 expression, an early regulator in the autophagy induction phase. miR-25 overexpression was demonstrated to block ISL-induced autophagy and chemosensitization. Subsequent in vivo experiments showed that ISL had chemosensitizing potency, as revealed by an increase in LC3-II staining, the downregulation of ABCG2, a reduction in miR-25 expression and the activation of the miR-25 target ULK1. Overall, our results not only indicate that ISL acts as a natural autophagy inducer to increase breast cancer chemosensitivity, but also reveal that miR-25 functions as a novel regulator of autophagy by targeting ULK1.

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ISL reverses breast cancer drug resistance by inducing autophagy miR-25 downregulation(A) ISL synergistically interacted with epirubicin to inhibit the growth of drug-resistant breast cancer xenografts (the values represent the means ± SD, n=6, *P<0.05, **P<0.01 vs. epirubicin group); (B) immunofluorescence results showed that ISL induced the upregulation of LC3-II and the downregulation of ABCG2 in breast cancer tissues; (C) qPCR results confirmed that ISL inhibited miR-25 expression in vivo (the values represent the means ± SD, n=3,**P<0.01); (D) western blotting validated that ISL inhibited ABCG2 expression and activated autophagy markers in vivo, including the upregulation of LC3-II, ULK1 and BECN1.
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Figure 6: ISL reverses breast cancer drug resistance by inducing autophagy miR-25 downregulation(A) ISL synergistically interacted with epirubicin to inhibit the growth of drug-resistant breast cancer xenografts (the values represent the means ± SD, n=6, *P<0.05, **P<0.01 vs. epirubicin group); (B) immunofluorescence results showed that ISL induced the upregulation of LC3-II and the downregulation of ABCG2 in breast cancer tissues; (C) qPCR results confirmed that ISL inhibited miR-25 expression in vivo (the values represent the means ± SD, n=3,**P<0.01); (D) western blotting validated that ISL inhibited ABCG2 expression and activated autophagy markers in vivo, including the upregulation of LC3-II, ULK1 and BECN1.

Mentions: To investigate whether ISL also chemosensitized breast cancer via autophagy induction in vivo, we constructed a drug-resistant breast cancer xenograft by injecting MCF-7/ADR cells into the mammary pads of NOD/SCID mice. The mice were then treated with epirubicin or ISL. The results showed that epirubicin had little effect in limiting cancer growth, whereas ISL alone significantly inhibited breast cancer volume. The inhibitory effects were the highest when ISL and epirubicin were applied together to treat the drug-resistant breast cancer (Figure 6A). The in vivo results were in consistent with our in vitro findings. Immunofluorescence detection revealed that ISL increased the LC3-II expression in breast cancer tissue but decreased the expression of ABCG2 (Figure 6B), indicating that the chemosensitizing effects of ISL in vivo were closely connected to autophagy induction. To validate whether the in vivo activation of autophagy was induced by miR-25 downregulation, we measured the expression of miR-25 in drug-treated groups. The results showed that ISL with or without epirubicin significantly inhibited miR-25 expression in the tumor tissue (Figure 6C); this was accompanied by increases in the expression of LC3-II, ULK1 and BECN1 as well as a decrease in the expression of ABCG2 (Figure 6D). Taken together, our data demonstrated that ISL induced the autophagic cell death of drug-resistant breast cancer cells both in vitro and in vivo and that miR-25 functioned as the primary autophagy-related modulator by targeting ULK1.


MicroRNA-25 regulates chemoresistance-associated autophagy in breast cancer cells, a process modulated by the natural autophagy inducer isoliquiritigenin.

Wang Z, Wang N, Liu P, Chen Q, Situ H, Xie T, Zhang J, Peng C, Lin Y, Chen J - Oncotarget (2014)

ISL reverses breast cancer drug resistance by inducing autophagy miR-25 downregulation(A) ISL synergistically interacted with epirubicin to inhibit the growth of drug-resistant breast cancer xenografts (the values represent the means ± SD, n=6, *P<0.05, **P<0.01 vs. epirubicin group); (B) immunofluorescence results showed that ISL induced the upregulation of LC3-II and the downregulation of ABCG2 in breast cancer tissues; (C) qPCR results confirmed that ISL inhibited miR-25 expression in vivo (the values represent the means ± SD, n=3,**P<0.01); (D) western blotting validated that ISL inhibited ABCG2 expression and activated autophagy markers in vivo, including the upregulation of LC3-II, ULK1 and BECN1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4196180&req=5

Figure 6: ISL reverses breast cancer drug resistance by inducing autophagy miR-25 downregulation(A) ISL synergistically interacted with epirubicin to inhibit the growth of drug-resistant breast cancer xenografts (the values represent the means ± SD, n=6, *P<0.05, **P<0.01 vs. epirubicin group); (B) immunofluorescence results showed that ISL induced the upregulation of LC3-II and the downregulation of ABCG2 in breast cancer tissues; (C) qPCR results confirmed that ISL inhibited miR-25 expression in vivo (the values represent the means ± SD, n=3,**P<0.01); (D) western blotting validated that ISL inhibited ABCG2 expression and activated autophagy markers in vivo, including the upregulation of LC3-II, ULK1 and BECN1.
Mentions: To investigate whether ISL also chemosensitized breast cancer via autophagy induction in vivo, we constructed a drug-resistant breast cancer xenograft by injecting MCF-7/ADR cells into the mammary pads of NOD/SCID mice. The mice were then treated with epirubicin or ISL. The results showed that epirubicin had little effect in limiting cancer growth, whereas ISL alone significantly inhibited breast cancer volume. The inhibitory effects were the highest when ISL and epirubicin were applied together to treat the drug-resistant breast cancer (Figure 6A). The in vivo results were in consistent with our in vitro findings. Immunofluorescence detection revealed that ISL increased the LC3-II expression in breast cancer tissue but decreased the expression of ABCG2 (Figure 6B), indicating that the chemosensitizing effects of ISL in vivo were closely connected to autophagy induction. To validate whether the in vivo activation of autophagy was induced by miR-25 downregulation, we measured the expression of miR-25 in drug-treated groups. The results showed that ISL with or without epirubicin significantly inhibited miR-25 expression in the tumor tissue (Figure 6C); this was accompanied by increases in the expression of LC3-II, ULK1 and BECN1 as well as a decrease in the expression of ABCG2 (Figure 6D). Taken together, our data demonstrated that ISL induced the autophagic cell death of drug-resistant breast cancer cells both in vitro and in vivo and that miR-25 functioned as the primary autophagy-related modulator by targeting ULK1.

Bottom Line: Recent findings have revealed that dysregulated miRNAs contribute significantly to autophagy and chemoresistance.More importantly, miRNA 3.0 array experiments identified miR-25 as the main target of ISL in triggering autophagy flux.Subsequent in vivo experiments showed that ISL had chemosensitizing potency, as revealed by an increase in LC3-II staining, the downregulation of ABCG2, a reduction in miR-25 expression and the activation of the miR-25 target ULK1.

View Article: PubMed Central - PubMed

Affiliation: Department of Mammary Disease, Guangdong Provincial Hospital of Chinese Medicine; School of Chinese Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China; These authors contributed equally to this work.

ABSTRACT
Recent findings have revealed that dysregulated miRNAs contribute significantly to autophagy and chemoresistance. Pharmacologically targeting autophagy-related miRNAs is a novel strategy to reverse drug resistance. Here, we report a novel function of isoliquiritigenin (ISL) as a natural inhibitor of autophagy-related miR-25 in killing drug-resistant breast cancer cells. ISL induced chemosensitization, cell cycle arrest and autophagy, but not apoptosis, in MCF-7/ADR cells. ISL also promoted the degradation of the ATP-binding cassette (ABC) protein ABCG2 primarily via the autophagy-lysosome pathway. More importantly, miRNA 3.0 array experiments identified miR-25 as the main target of ISL in triggering autophagy flux. A mechanistic study validated that miR-25 inhibition led to autophagic cell death by directly increasing ULK1 expression, an early regulator in the autophagy induction phase. miR-25 overexpression was demonstrated to block ISL-induced autophagy and chemosensitization. Subsequent in vivo experiments showed that ISL had chemosensitizing potency, as revealed by an increase in LC3-II staining, the downregulation of ABCG2, a reduction in miR-25 expression and the activation of the miR-25 target ULK1. Overall, our results not only indicate that ISL acts as a natural autophagy inducer to increase breast cancer chemosensitivity, but also reveal that miR-25 functions as a novel regulator of autophagy by targeting ULK1.

Show MeSH
Related in: MedlinePlus