MDM4 expression as an indicator of TP53 reactivation by combined targeting of MDM2 and MDM4 in cancer cells without TP53 mutation

Abstract

MDM2 and MDM4, a structurally related MDM2 homolog, negatively regulates expression and functions of TP53 tumor suppressor gene. To explore the precise expression patterns and function of MDM2 and MDM4 in wild-type (wt) TP53 cancer cells, we analyzed 11 various cancer cell lines with wt TP53. All cell lines exhibited deregulated expression of MDM2 and MDM4, and were divided into two distinct types; the one expressing high levels of MDM4 and another expressing low levels of MDM4. The low MDM4 type expressed higher MDM2 levels than the high MDM4 type. In cells with high MDM4 expression, knockdown of MDM4 or MDM2 reactivated TP53, and simultaneous knockdown of MDM2 and MDM4 synergistically reactivated TP53. In contrast, in cells with low MDM4 expression, knockdown of only MDM2 reactivated TP53. These results suggest that both MDM2 and MDM4 are closely involved in TP53 inactivation in cancer cells with high MDM4 expression, whereas only MDM2, and not MDM4, is a regulator of TP53 in cells with low MDM4 expression. MDM4 expression in wt TP53-tumors is a potential indicator for TP53 reactivation cancer therapy by simultaneous targeting of MDM4 and MDM2. Specific knockdown of MDM2 and MDM4 might be applicable for TP53 restoration therapy.

Keywords: MDM2; MDM4; TP53-reactivation; knockdown; p53; siRNA.

Figure 1. Expression levels of p53, MDM2, and MDM4 in cancer cell lines

Expression levels of p53, MDM2, and MDM4 were examined in 14 cancer cell lines (11 wt TP53 cell lines and 3 mt TP53 cell lines) by immunoblotting. SE, short exposure; LE, long exposure.

Figure 2. Effects of siRNAs targeting MDM2 and their dsRDC forms on MDM2 expression and cell growth

(a) Two previously reported (396 and 851) and 17 new siMDM2s were analyzed for their effects on MDM2 expression in SJSA-1 cells by immunoblotting. SJSA-1 cells were transfected with mock, control siRNA (siCtrl), control-R siRNA (siCtrl-R), and siMDM2s at 1 nM for 48 h and then examined for MDM2 expression by immunoblotting. (b) Control siRNA, control-R siRNA, and eight effective siMDM2s, including two previously reported and six new were converted to dsRDC forms (chiCtrl, chiCtrl-R, and chiMDM2s), and examined for MDM2 knockdown activity in SJSA-1 cells 48 h after transfection at 1 nM. (c) Effect of chiMDM2s on growth of SJSA-1 cells were examined. The cells were transfected with control dsRDC (chiCtrl) or eight chiMDM2s at 1 nM for 5 days and then assayed for relative viable cell number using the WST-8 assay (mean ± SD; n = 3). (d) The effects of two highly effective chiMDM2s (1068 and 1489) on growth of mt TP53-cancer cells (KATOIII, NUCG-3, and DLD-1) after transfection at 1 nM for 5 days were examined using the WST-8 assay. Viable cell numbers relative to those transfected with control dsRDC (chiCtrl) are shown (mean ± SD; n = 3; *p < 0.05; Dunnett's test).

Figure 3. Effects of siRNAs targeting MDM4 and their dsRDC forms on MDM4 expression and cell growth

(a) The effects of siMDM4s on MDM4 expression were analyzed in MCF-7 cells by immunoblotting 48 h after transfection with mock, control siRNA (siCtrl), or 10 siMDM4s at 1 nM. SE, short exposure; LE, long exposure. (b) Seven effective siMDM4s and a control siRNA were converted to dsRDC forms (chiMDM4s and chiCtrl), and analyzed for MDM4 knockdown in MCF-7 cells 48 h after transfection at 1 nM. (c) The effect of chiMDM4s on the growth of MCF-7 cells was examined. The cells were transfected with control dsRDC (chiCtrl) or seven chiMDM4s at 1 nM for 5 days and then assayed for relative viable cell number using the WST-8 assay (mean ± SD; n = 3). (d) The effects of six highly effective chiMDM4s on the growth of mt TP53-cancer cells (KATOIII, NUCG-3, and DLD-1) after transfecting at 1 nM for 5 days were examined using the WST-8 assay. Viable cell numbers relative to those transfected with control dsRDC (chiCtrl) are shown (mean ± SD.; n = 3; *p < 0.05; Dunnett's test).

Figure 4. Effect of MDM2 and MDM4 knockdown on the growth of wt TP53 cell lines

dsRDCs targeting MDM2 (chiMDM2-1068 and chiMDM2-1489) (a), MDM4 (chiMDM4-452 and chiMDM4-1036) (b) and control dsRDC (chiCtrl) were transfected into seven cell lines with high MDM4 expression levels (MCF-7, A375, SNU-1, HCT116, NUCG-4, LoVo, and A549) and four cell lines with low MDM4 expression levels (SJSA-1, HepG2, HuH-6, and C32TG) at 1 nM. Five days after transfection, cell viability was determined using the WST-8 assay. Viable cell numbers relative to those transfected with chiCtrl are shown (mean ± SD; n = 3; *p < 0.05; Dunnett's test).

Figure 5. Effect of MDM2 knockdown on expression levels of MDM2, MDM4, p53 and p21

Mock, control dsRDC (Ctrl), and two dsRDCs targeting MDM2 (chiMDM2-1068, chiMDM2-1489) were transfected into seven cells lines with high MDM4 expression, four cell lines with low MDM4 expression and three mt TP53 cell lines at 1 nM. Expression levels of MDM2, MDM4, p53, and p21 were analyzed by immunoblotting 2 days after transfection.

Figure 6. Effect of MDM4 knockdown on expression levels of MDM4, MDM2, p53, and p21

Mock, control dsRDC (Ctrl), and two dsRDCs targeting MDM4 (chiMDM4-452 and -1036) were transfected into seven cell lines with high MDM4 expression, four cell lines with high MDM2 expression and three mt TP53 cell lines at 1 nM. Expression levels of MDM2, MDM4, p53, and p21 were analyzed by immunoblotting 2 days after transfection.

Figure 7. Combined knockdown of MDM2 and MDM4 in wt TP53 cell lines with high and low MDM4 expression

Effects of individual and simultaneous knockdown of MDM2 and MDM4 on cell growth (a) and expression of p53 and p21 (b) were examined in two cell lines with high MDM4 expression (MCF-7 and A375) and two cell lines with low MDM4 expression (SJSA-1 and C32TG). Cells were transfected with MDM4 dsRDC (chiMDM4-452) alone, MDM2 dsRDC (chiMDM2-1489) alone, or both. The total amount of dsRDCs was adjusted to 2 nM by adding control dsRDC (chiCtrl). Cell viability was determined 5 days after transfection using the WST-8 assay. Viable cell numbers of chiCtrl (2 nM) transfected cells was defined as 100% (mean ± SD; n = 3). Levels of MDM2, MDM4, p53, and p21 were analyzed by immunoblotting 2 days after transfection. In panel b, + and ++ indicates 1 nM and 2 nM of dsRDCs, respectively.