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. 2012 Aug 15;11(16):3067-78.
doi: 10.4161/cc.21476. Epub 2012 Aug 8.

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Vijay Alla  1 , Bhavani S Kowtharapu, David Engelmann, Stephan Emmrich, Ulf Schmitz, Marc Steder, Brigitte M Pützer
Affiliations

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Vijay Alla et al. Cell Cycle. .

Abstract

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.

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Figures

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Figure 1. p73 induces miR-205 expression. (A) Expression of miR-205 in SK-Mel-29 cells infected with Adp53, Adp73 or AdGFP (set as 1) was determined by TaqMan qRT-PCR. (B) Scheme of the miR-205 promoter (top). Three putative p73 binding sites (BS) are indicated as BS1, BS2 and BS3 relative to the pre-miR sequence. In vivo binding of p73 to miR-205 promoter regions was detected by ChIP using anti-p73 (ER-15) or control IgG (bottom). PCR primers for the p21(WAF1/CIP1) promoter were used as positive control. Input lane, 10% of total chromatin. (C) Biotin-labeled miR-205 p73-binding sites were incubated with nuclear extract and a 50-fold molar excess of cold double-stranded self-competitor or an equimolar amount of mutant site competitor. The arrow indicates a specific band competed by cognate competitor. Lane 1, without nuclear extract; NE, nuclear extract; C, competitor; MC, mutant competitor. (D) Luciferase assay of SK-Mel-29 cotransfected with 1 µg miR-205 promoter, BS1Del and BS2,3Del deletion constructs and 0.5 µg of p73 expression plasmid (black bars) or pcDNA (gray bars). The p73-responsive human Bax promoter construct was used as positive control. Luciferase activities are expressed as the fold activation relative to cotransfection of the promoter construct with empty vector following normalization to total protein concentrations in the cell extract at 36 h after transfection. Error bars indicate the mean ± SD. E, Luciferase activities measured after cotransfection of the miR-205 promoter with increasing amounts (0.25, 0.50, 1 µg) of p73 (left) and p53 (right) expression plasmid or 1 µg pcDNA that served as control. Results are shown as fold increase compared with pGL3basic which was set as 1. Expression levels of p73, p53 and actin in different samples are indicated by western blot.
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Figure 2. Inhibition of miR-205 stimulation by DNp73. Luciferase assay of SK-Mel-29 cells co-transfected with p73-responsive miR-205 reporter construct and (A) 1 µg E2F1 expression plasmid in the presence of 0.1 and 0.5 µM of ASO116 or (B) 1 µg pcDNAp73 with 0.1, 0.5 and 1 µg of DNp73 construct. pcDNA served as control. Promoter activity of the control was normalized to 1. Standard deviation of the mean is indicated by error bars. Protein expression including DNp73 knockdown was validated by immunoblot using actin for equal loading.
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Figure 3. Downregulation of miR-205 in metastatic melanoma tissues and cell lines correlates with high levels of p73/DNp73. qRT-PCR analysis of endogenous miR-205, p73 and DNp73 expression levels in (A) melanocytes, non-metastatic SK-Mel-29 or metastatic SK-Mel-147 w/o ASO116 treatment and (B) in a cohort of primary vs. metastatic melanoma tissues (*, miR-205, p < 0.001; p73, p = 0.001; DNp73, p = 0.026).
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Figure 4. MiR-205 mediates the DNA damage response of p73. (A) Relative expression of miR-205 in cisplatin treated SK-Mel-147 cells with and without p73 knockdown compared with undamaged cells that express shRNA-control (set as 1). (B and C) The percentage of apoptotic SK-Mel-147 after cisplatin treatment was measured by FACS analysis following overexpression of p73 and antagomiR-205 (B), shp73 and miR-205 (C, left) or ASO116, DNp73 and miR-205 (C, center). Lentiviral expression of miR-205 was confirmed by qPCR compared with miR-control transduced cells (C, right). Bars represent the mean ± SD of three independent experiments. Statistical significance was determined by Student’s t-test. P-values of less than 0.05 were deemed statistically significant. (D) Tumors established in nude mice by subcutaneous injection of 5 x 106 SK-Mel-147 cells stably expressing miR-205 or miR-control and infected ex vivo with Adshp73 were treated with cisplatin (arrows) and monitored for tumor growth. Each group, n = 5. Graphs represent the tumor volume at indicated time points after tumor cell injection. AdshGFP-infected cells were used as control. Student’s t-test (two-sided) indicates statistically significant differences between the groups 6 weeks post-injection. (shp73 vs. shp73-miR-205, p = 0.008); (shp73-miRNA-control vs. shp73-miR-205, p = 0.004); (shp73 vs. shGFP, p = 0.016).
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Figure 5. E2F1 induces chemoresistance via DNp73-mediated reduction of miR-205. (A) Protein levels of endogenous E2F1, p73 and DNp73 in cisplatin-treated SK-Mel-147 cells expressing shE2F1 or control shGFP compared with untreated cells. Actin was used for equal loading. (B) The percentage of apoptotic cells shown in (A) following knockdown of E2F1 and/or miR-205 in response to cisplatin was measured by FACS analysis (top and bottom panel). shE2F1-dependent changes in miR-205 expression are indicated (central panel). Statistical significance was determined by Student’ s t-test (p < 0.05). (C) Relative miR-205 expression in cisplatin-resistant SK-Mel-147 clones compared with parental cells set as 1 (right). Bar graphs indicate the mean ± SD of three independent experiments. E2F1 levels were analyzed by western blot (bottom).
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Figure 6. Bcl-2 and the ATP-binding cassette transporters ABCA2 and ABCA5 are direct targets of miR-205. (A) qRT-PCR analysis of ABCA2, ABCA5, Bcl-2, E2F1 and ZEB1 in SK-Mel-147 infected with lentivirus expressing miR-205 (gray bars). Values are calculated after normalization with mean expression levels in cells overexpressing miR-control (set as 1; black bars). (B, upper panel) Reporter contructs containing predicted miR-205 binding sites in the Bcl-2, ABCA2 and ABCA5 3′UTR were assayed. E2F1 3′UTR was used as positive control. The miR-205 seed sequence is conserved in six different species (lower panel). (C) The expression levels of E2F1 and miR-205 target proteins in stable miR-205 or miR-control SK-Mel-147 cells (left) as well as in SK-Mel-29 cells at 48 h after knockdown of miR-205 compared with cells treated with antagomiR-control (right). Actin was used for equal loading. (D-E) E2F1, Bcl-2, ABCA2 and ABCA5 protein levels at 48 h after knockdown of E2F1 in SK-Mel-147 cells (D) or in primary tumors growing 6 weeks after cell injection in the xenograft model shown in Figure 4D. (E) Using actin as loading control. (F and G) Bioinformatic analysis of published microarray data showed a correlation of E2F1 and ABCA2 or Bcl-2 transcript levels in malignant melanomas (n = 24) vs. benign nevi (n = 18) (D) and prostate cancer metastases (n = 24) vs. primary tumors (n = 64) (E). In each graph, the solid lines within the boxes represent the median value and boxes show the 25th to 75th percentile range of mRNA levels. Bars represent 95% confidence intervals, with circles representing outliers. All statistical tests were one-sided.
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