Betulinic acid targets YY1 and ErbB2 through cannabinoid receptor-dependent disruption of microRNA-27a:ZBTB10 in breast cancer

Abstract

Treatment of ErbB2-overexpressing BT474 and MDA-MB-453 breast cancer cells with 1 to 10 μmol/L betulinic acid inhibited cell growth, induced apoptosis, downregulated specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and decreased expression of ErbB2. Individual or combined knockdown of Sp1, Sp3, Sp4 by RNA interference also decreased expression of ErbB2 and this response was because of repression of YY1, an Sp-regulated gene. Betulinic acid-dependent repression of Sp1, Sp3, Sp4, and Sp-regulated genes was due, in part, to induction of the Sp repressor ZBTB10 and downregulation of microRNA-27a (miR-27a), which constitutively inhibits ZBTB10 expression, and we show for the first time that the effects of betulinic acid on the miR-27a:ZBTB10-Sp transcription factor axis were cannabinoid 1 (CB1) and CB2 receptor-dependent, thus identifying a new cellular target for this anticancer agent.

Figure 1

Effects of betulinic acid (BA) on cell proliferation and apoptosis. A, structure of BA. B, BA-mediated inhibition of BT474 and MDA-MB-453 cell growth. Cells were treated with different concentrations of BA for up to 4 days and the number of cells in each treatment group was determined as described in Materials and Methods. *, significant (P < 0.05) growth inhibition is indicated. Results are expressed as means ± SE for at least 3 replicate determinations for each treatment group. C, effects of BA on cleaved (c) PARP and survivin. Cells were treated with 10 μmol/L BA for 48 hours and whole-cell lysates were analyzed by Western blotting as described in Materials and Methods. D, BA induces apoptosis in cancer cells. Cells were treated with DMSO or 10 μmol/L BA for 24 hours and analyzed with a TUNEL assay as described in Materials and Methods. DAPI, 4′,6—diamidino— 2—phenylindole; FITC, fluorescein isothiocyanate.

Figure 2

Effects of betulinic acid (BA) on Sp1, Sp3, Sp4, YY1, ErbB2, and ErbB2-dependent proteins. A, BA decreases Sp protein and survivin levels in BT474 and MDA-MB-453 cells. Cells were treated with DMSO (D), 10 μmol/L BA alone or in combination with 1 μmol/L lactacystin (Lac) for 48 hours, and whole-cell lysates were analyzed by Western blotting as described in Materials and Methods. B, BA decreases mRNA levels of Sp proteins. Cells were treated with 10 μmol/L BA for 16 hours, and mRNA levels were determined as described in Materials and Methods. Results are expressed as means ± SE for 3 replicate determinations for each treatment group and significant (P < 0.05) decreases are indicated (*). C, BA decreases protein levels of ErbB2 and ErbB2-dependent proteins. Cells were treated with DMSO (D), 10 μmol/L BA alone or in combination with 1 μmol/L lactacystin for 48 hours, and whole-cell lysates were analyzed by Western blotting as described in Materials and Methods. D, BA decreases YY1 promoter activity. MDA-MB-453 cells were transfected with empty vector (PGL2), the YY1 p-277-luc, or the YY1 p-1729-luc construct. Cells were then treated with 5 or 10 μmol/L BA for 24 hours. Luciferase activity was determined as described in Materials and Methods. Results are means ± SE for 3 separate determinations and significant (P < 0.05) induction of luciferase activity by BA is indicated (*).

Figure 3

Effects of cannabinoid and vanilloid receptor antagonists on betulinic acid (BA)-induced responses. Effects of AM251, AM630, and capsazepine (Cap) on BA-mediated repression of Sps and survivin proteins in BT474 (A) and MDA-MB-453 (B) cells. Effects of AM251, AM630, and capsazepine on BA-mediated downregulation of ErbB2 and ErbB2-regulated kinases in BT474 (C) and MDA-MB-453 (D) cells and expression of CB receptors (D). Cells were pretreated with or without 6 μmol/L AM251, 6 μmol/L AM630, or 2 μmol/L capsazepine for 1 hour, and then DMSO (D) or 10 μmol/L BA were added to the medium for 48 hours, and whole-cell lysates were analyzed by Western blotting as described in Materials and Methods.

Figure 4

Betulinic acid (BA) is a CB receptor agonist. Specific binding of BA to CB receptors (A) and binding affinities (B). The specific binding and binding affinities of BA to CB1 and CB2 receptors were determined as described in Materials and Methods. C, knockdown of CB receptors by RNA interference. BT474 cells were transfected with iLamin (control) or iCB1 receptor or iCB2 receptor (oligonucleotides), and whole-cell lysates were analyzed by Western blotting as described in Materials and Methods. D, effects of FAAH knockdown or CAY10401 on Sp proteins. MDA-MB-453 cells were transfected with siFAAH or BT474 cells were treated with DMSO or CAY10401 for 24 hours, and whole-cell lysates were analyzed by Western blotting as described in Materials and Methods.

Figure 5

Effects of betulinic acid (BA) on miR-27a and ZBTB10, and the role of cannabinoid receptors on BA-mediated effects. A, downregulation of miR-27a. Cells were pretreated with or without 6 μmol/L AM251 or 6 μmol/L AM630 for 1 hour, DMSO or 5 or 10 μmol/L BA were added to the medium for 24 hours, and miR-27a levels were determined as described in Materials and Methods. Results are expressed as means ± SE for 3 replicate determinations for each treatment group and significant (P < 0.05) inhibition of miR-27a (**) and inhibition by the antagonists are indicated (*). B, induction of ZBTB10. Cells were treated and processed as described in A, and significant (P < 0.05) induction by BA (*) and inhibition by the antagonists (**) are indicated. C, effects of ZBTB10 overexpression and antisense miR-27a on Sp protein levels, YY1, and ErbB2 proteins. Cells were transfected with 1 μg pCMV6-XL4-ZBTB10 plasmid or empty vector, 50 nmol/L antisense miR-27a (as-miR-27a), or control, and whole-cell lysates were analyzed by Western blotting as described in Materials and Methods. D, effects of miR-27a mimic or as-miR-27a on luciferase activity in ZBTB10 3′UTR-luc construct transfected cells. MiR-27a mimic (50 nmol/L) or as-miR-27a were transfected into BT474 and MDA-MB-453 cells as described in Materials and Methods, and a dual luciferase reporter assay was conducted according to the manufacturer’s instructions. Results are expressed as means ± SE for 3 replicate determinations for each treatment group and significant (P < 0.05) decreases or induction are indicated (*).

Figure 6

Betulinic acid (BA) inhibits tumor growth in BT474 xenografts. Inhibition of tumor size (A) and weight (B). Athymic nude mice bearing BT474 cells as xenografts were treated with BA (20 mg/kg/d), and tumor sizes and weights were determined as described in Materials and Methods. Significantly (P < 0.05) decreased tumor sizes and weights are indicated (*). C, BA decreases expression of Sp1, Sp3, and Sp4 proteins in tumors. Whole-cell lysates from corn oil and BA-treated tumors were analyzed by Western blotting as described in Materials and Methods. D, immunostaining for ErbB2 and Sp1. Fixed tumor tissue from corn oil-and BA-treated mice were stained with ErbB2 and Sp1 antibodies as described in Materials and Methods.