Role of ornithine decarboxylase in regulation of estrogen receptor alpha expression and growth in human breast cancer cells

Abstract

Our previous studies demonstrated that specific polyamine analogues, oligoamines, down-regulated the activity of a key polyamine biosynthesis enzyme, ornithine decarboxylase (ODC), and suppressed expression of estrogen receptor alpha (ERα) in human breast cancer cells. However, the mechanism underlying the potential regulation of ERα expression by polyamine metabolism has not been explored. Here, we demonstrated that RNAi-mediated knockdown of ODC (ODC KD) down-regulated the polyamine pool, and hindered growth in ERα-positive MCF7 and T47D and ERα-negative MDA-MB-231 breast cancer cells. ODC KD significantly induced the expression and activity of the key polyamine catabolism enzymes, spermine oxidase (SMO) and spermidine/spermine N (1)-acetyltransferase (SSAT). However, ODC KD-induced growth inhibition could not be reversed by exogenous spermidine or overexpression of antizyme inhibitor (AZI), suggesting that regulation of ODC on cell proliferation may involve the signaling pathways independent of polyamine metabolism. In MCF7 and T47D cells, ODC KD, but not DFMO treatment, diminished the mRNA and protein expression of ERα. Overexpression of antizyme (AZ), an ODC inhibitory protein, suppressed ERα expression, suggesting that ODC plays an important role in regulation of ERα expression. Decrease of ERα expression by ODC siRNA altered the mRNA expression of a subset of ERα response genes. Our previous analysis showed that oligoamines disrupt the binding of Sp1 family members to an ERα minimal promoter element containing GC/CA-rich boxes. By using DNA affinity precipitation and mass spectrometry analysis, we identified ZBTB7A, MeCP2, PARP-1, AP2, and MAZ as co-factors of Sp1 family members that are associated with the ERα minimal promoter element. Taken together, these data provide insight into a novel antiestrogenic mechanism for polyamine biosynthesis enzymes in breast cancer.

Fig. 1

ODC siRNA down-regulated polyamine biosynthesis. a Cells were transfected with ODC siRNA or treated with 5 mM DFMO for 24 h. qPCR was performed to measure the mRNA level of ODC. b After transfection, cells were harvested and analyzed by immunoblots for expression of ODC. c ODC activity was measured after siRNA transfection or 5 mM DFMO treatment. d After siRNA transfection, intracellular polyamine levels were measured

Fig. 2

ODC KD-induced expression and activity of SMO and SSAT. SMO (a) and SSAT (b) mRNA levels in MCF7 and T47D cells were measured by qPCR after ODC siRNA transfection. c Western blotting analysis of SMO in MCF7 and T47D cells. d SMO activity was measured using methods as described in “Materials and methods”

Fig. 3

ODC KD inhibits cell growth and induces apoptosis. a After transient siRNA treatment for 48 h, cells were detached by trypsinization and counted. b Equal amounts (20 µg/well) of whole-cell extracts were fractionated and immunoblotted with antibodies for apoptosis related proteins as indicated

Fig. 4

Effects of ODC KD on expression and activity of ERα. Cells were transfected with scramble control or ODC siRNA or treated with 5 mM DFMO. a Immunoblot with anti-ERα antibody was performed and analyzed. b Immunoblot analysis of the protein expression level of RARβ and VDR. c Immunoblot analysis of the expression level of PR and cyclin D1 proteins. d The mRNA expression of ERα response genes in scramble and ODC siRNA treated cells was analyzed using PCR array

Fig. 5

Role of antizyme in ODC regulated ERα expression. a Immunoblot analysis of AZ protein in MCF7 and T47D cells transiently transfected with ODC siRNA. b Flag-tagged AZ was stably expressed in MCF7 cells. ERα protein level was detected by immunoblot

Fig. 6

Identification of multi-protein complex at ERα minimal promoter element. a MCF7 cells were harvested and DNA affinity precipitation assay (DAPA) was performed. b The mass spectrometry analysis was carried out to identify factors that bind to ERα minimal promoter element

Fig. 7

Effect of AZI on ODC KD-mediated growth inhibition. a MDA-MB-231 cells were transiently transfected with scramble or ODC siRNA or treated with 5 mM DFMO with or without 2 µM spermidine and 1 mM aminoguanidine (AG). Total cell numbers were counted at the indicated time points. b MDA-MB231 cells were stably transfected with pEFIRES-P (vector) or pEFIRES-AZI plasmids. Single clones were analyzed for AZI and AZ expression by immunoblots. c Vector control and AZI overexpressing clone AZI-20 were transiently transfected with ODC siRNA or treated with 5 mM DFMO. Total cell numbers were counted in the presence or absence of spermidine and AG. d Vector control and AZI-20 cells were transiently transfected with ODC siRNA. Cell lysate was analyzed for cyclin D1, AZ1 and AZI expression by immunoblots. Numbers 1, 2, 3, and 4 represent untreated, scramble siRNA transfected, hODC siRNA transfected and DFMO (5 mM)-treated cells respectively. e Histograms represent the mean protein expression levels of three determinations relative to actin ± SD as determined by quantitative immunoblotting