Survivin repression by p53, Rb and E2F2 in normal human melanocytes

Abstract

The inhibitor of apoptosis protein survivin is a dual mediator of apoptosis resistance and cell cycle progression and is highly expressed in cancer. We have shown previously that survivin is up-regulated in melanoma compared with normal melanocytes, is required for melanoma cell viability, and that melanocyte expression of survivin predisposes mice to ultraviolet-induced melanoma and metastasis. The mechanism of survivin up-regulation in the course of melanocyte transformation and its repression in normal melanocytes, however, has not been clearly defined. We show here that p53 and retinoblastoma (Rb), at basal levels and in the absence of any activating stimuli, are both required to repress survivin transcription in normal human melanocytes. Survivin repression in melanocytes does not involve alterations in protein stability or promoter methylation. p53 and Rb (via E2Fs) regulate survivin expression by direct binding to the survivin promoter; p53 also affects survivin expression by activating p21. We demonstrate a novel role for E2F2 in the negative regulation of survivin expression. In addition, we identify a novel E2F-binding site in the survivin promoter and show that mutation of either the p53- or E2F-binding sites is sufficient to increase promoter activity. These studies suggest that compromise of either p53 or Rb pathways during melanocyte transformation leads to up-regulation of survivin expression in melanoma.

Figure 1

Survivin expression is negatively regulated by p53 and Rb in human melanocytes. (a) Cell lysates were obtained from normal human melanocytes (Mel), human melanocyte lines infected with retrovirus expressing SV40ER (Mel-SV40ER); SV40ER and hTERT (Mel-STV); SV40ER, HRasG12V and hTERT (Mel-STR); and SV40ER, TPR-Met and hTERT (Mel-STM); and human metastatic melanoma lines YUSAC2, YUGEN8, and LOX. Lysates were subjected to SDS-PAGE and analyzed by Western blotting with antibodies against Survivin and Actin. (b) Knockdown of p53 or Rb leads to upregulation of Survivin expression. Melanocytes were transfected with control scrambled siRNA (Scr) or siRNA targeting p53 or Rb. Cell lysates were prepared 24, 48, or 72 h following transfection, and blotted for p53, Rb, Survivin, and Actin as indicated. (c) RNA was prepared from melanocytes 24 h following transfection with control scrambled siRNA (Scr) or siRNA targeting p53 or Rb, and used for RT-PCR with primers specific for Survivin or Actin.

Figure 2

Methylation-independent regulation of Survivin. (a) Schematic depicting survivin promoter and proximal exonic sequence, and location of HpaII (open circles) and HhaI (filled circles) sites, and binding sites for p53 (open box) and E2F factors (shaded boxes) relative to the translational (ATG) start site (+1, arrow). PCR primers designed to amplify the −650 to +130 and −216 to +555 regions of the promoter are respectively indicated by filled and open arrowheads. (b) Genomic DNA from melanocytes and melanoma lines YUSAC2 and YUGEN8 was digested with methylation-sensitive enzymes, then subjected to PCR using primers specific for survivin promoter and actin sequences as indicated. (c) Melanocytes were cultured in the absence or presence of 0.5 μM 5-aza for 96 h, then cell lysates were blotted for Survivin, Maspin, or Actin as indicated.

Figure 3

Cell cycle analysis of Survivin expression and activity of p53 and Rb. (a) Melanocytes (Mel) were untreated (control) or treated with mimosine, thymidine, or nocodazole to arrest cells in the G1, S, and G2M phases, respectively. After 24 h, cells were stained with propidium iodide and analyzed by flow cytometry. (b) Lysates were prepared from untreated melanocytes (Con) and melanocytes treated with mimosine (MIM, G1 block), thymidine (THY, S block), or nocodazole (NOC, G2M block) as in (a), and blotted for Survivin, p53, p21, Rb, phosphorylated Rb species, and Actin.

Figure 4

Lack of interaction of p53 and Rb pathways in Survivin regulation. (a) Melanocytes were infected with lentiviruses expressing control scrambled siRNA (Scr), p53 siRNA, Rb siRNA, or a combination of both p53 and Rb siRNA-expressing lentiviruses. After 72 h, cell lysates were prepared and blotted for Survivin, Actin, p53, p21, total Rb, and various phosphorylated Rb species. (b) Melanocytes were transfected with control scrambled siRNA (Scr) or siRNA targeting p21. After 24–48 h, cell lysates were prepared and blotted for Survivin, Actin, p21, total Rb, and various phosphorylated Rb species as indicated.

Figure 5

Binding of p53 and E2F factors negatively regulates the survivin promoter. (a) Melanocyte nuclear extracts were incubated with ³²P-labeled duplexes corresponding to the consensus binding site for p53 in the survivin promoter and anti-p53 antibody pAb 421 (p53 Ab), in the absence or presence of 100-fold excess cold wild-type (WT) or non-specific (NS) competitor, as indicated. Asterisks indicate non-specific bands. (b) Melanocyte nuclear extracts were incubated with duplexes corresponding to the E2F-binding site in the survivin promoter, in the absence or presence of 100-fold excess cold wild-type (WT) or mutant (MUT) competitor, as indicated. (c) YUSAC2 cells were transfected with luciferase reporter constructs driven by wild-type (WT) survivin promoter sequences (open bar) or WT sequences containing mutations in the E2F and/or p53 binding sites (shaded bars). Firefly fluorescence was normalized to Renilla. Error bars represent SEM of triplicate determinations. P values for comparisons between each mutant construct and the WT construct are indicated by asterisks (*, p=0.01; **, p<0.001; ***, p=0.002). (d) YUGEN8 cells were transfected with the same constructs as in (c). Error bars represent SEM of triplicate determinations. P values for comparisons between each mutant construct and the WT construct are indicated by asterisks (*, p < 0.01; **, p = 0.01).

Figure 6

E2F2 is a negative regulator of Survivin. (a) Lysates from human melanocytes and YUSAC2 melanoma cells were blotted for E2F2, E2F4 and Actin as indicated. (b) Melanocytes were transfected with 20 nM scrambled siRNA (Scr) or siRNA targeting E2F2. Cell lysates were prepared 48 h following transfection, and blotted for E2F2, Survivin, and Actin as indicated. (c) HeLa cells were transfected with 50 nM scrambled siRNA or siRNA targeting E2F2. Cell lysates were prepared at 48 hrs following transfection, and blotted for E2F2, Survivin, and Actin.