Inhibitionof cyclin D1 expression by androgen receptor in breast cancer cells-identification of a novel androgen response element
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(555) Results. RESULTS DHT administration inhibits serum-induced MCF-7 cells proliferation We previously demonstrated that MCF-7 cells are androgen responsive and that DHT treatment induces a transient increase in AR protein levels (27), similar to those seen in other cell types (57). To investigate the role of activated AR on breast cancer cell proliferation, the response of MCF-7 cells to the non aromatizable androgen DHT and/or the AR antagonist OHFl was measured after 24, 48 and 72 h of treatment in PRF DMEM implemented with 2.5% of steroid-depleted serum (PRF–CT). DHT concentration was chosen based on previous studies demonstrating dosedependent inhibitory effects of DHT on MCF-7 cells proliferation (28,30). As expected, cells grown in presence of PRF-CT proliferated; DHT treatment instead inhibited serum-induced MCF-7 cells proliferation and, by the end of the treatment, the mean number of DHT-treated cells was ~30% of respective controls. Addition of the AR antagonist OHFl (or, bicalutamide, data not shown) effectively reversed the inhibitory effect of DHT on MCF-7 cells proliferation, suggesting that it was mediated by the AR (Figure 1A). A similar pattern of DHT-dependent effect on MCF-7 cells proliferation was obtained by simultaneously performed MTT colorimetric assay (Figure 1B). These data well correlated with cell-cycle analysis showing an increase of the percentage of cells in G0/G1 phase and a concurrent decrease in the S phase, following 72 h of DHT treatment. At this time point the presence of sub-G1 apoptotic cells was undetectable (Figure 1C).. 22.
(556) Results. Figure 1. Proliferative response of MCF-7 cells to DHT. Serum starved MCF-7 cells were grown in PRF–CT in absence or presence of 10 -7 M DHT and/or 10-6 M OH-Fl. The effects of the various drugs on cell proliferation were measured 0, 24, 48 and 72 h following initial exposure to treatments by: ( A) cell counting using a Burker’s Chamber, with cell viability determined by trypan blue dye exclusion or ( B) the MTT assay as described in ‘Materials and Methods’ section. ( B) Serum starved MCF-7 cells were grown in PRF–CT in absence or presence of 10 -7 M DHT for 72 h and then subjected to cell-cycle analysis as described in ‘Materials and Methods’ section. Data, representing a mean±SD of three independent experiments, each in triplicate, were statistically analysed by Student’s t-test, *P<0.05 versus untreated.. 23.
(557) Results. Activated AR decreases cyclin D1 expression and promoter activity’ Since DHT administration reduces the G1/S phase transition in MCF-7 cells (28,30), we inquired whether DHT-induced decrease of MCF-7 cells proliferation might be consequent to the modulation of cyclin D1 expression, whose induction represents a key rate-limiting event in mitogenic signalling leading to S-phase entry. To this aim, serum starved MCF-7 cells were left untreated or treated with 10 -7 M DHT for 24 h, 48 h or 72 h and cyclin D1 expression was assessed by real- time RT–PCR and WB analysis. As shown in Figure 2A, MCF-7 cells exhibited a decrease in the serum-dependent levels of cyclin D1 mRNA (Figure 2A) and protein (Figure 2B) following 48 and 72 h of DHT treatment. The involvement of the AR in the negative regulation of cyclin D1 expression was ascertained by silencing AR expression in MCF-7 cells (Figure 2B). To test whether activated AR might negatively modulate cyclin D1 promoter activity, MCF-7 cells were transiently transfected with a cyclin D1 promoter reporter plasmid (D1!-2996) and left untreated or treated with 10-7 M DHT. The cyclin D1 promoter was induced by serum, while DHT treatment inhibited basal cyclin D1 promoter activity, decreasing the serum-induced signal by ~50% OHFl addition reversed this effect, suggesting that it was due to AR activation (Figure 2C). To substantiate the AR involvement in the modulation of CCND1 expression, we evaluated the effects of ectopic AR expression on the transcriptional activity of the cyclin D1 promoter. HEK-293 cells, which do not express AR, were co-transfected with the D1!-2996 plasmid and increasing amounts of a full length AR encoding plasmid. In the absence of exogenous AR expression, DHT treatment cannot influence cyclin D1 24.
(558) Results. promoter activity. On the contrary, in the presence of ectopic AR, a dosedependent decrease in the serum-induced cyclin D1 promoter activity was observed upon DHT administration (Figure 2D). To investigate whether the effect of AR on cyclin D1 promoter activity is dependent on its transactivation properties, luciferase assay was performed in AR-negative HEK-293 cells transfected with an expression plasmid encoding an AR carrying a mutation (AR (Cys-574!Arg)) in the DBD, which disrupts its ability to bind target DNA sequences (27,51). In these circumstances, no decrease in cyclin D1 promoter activity could be detected (Figure 2D), suggesting the existence, in the cyclin D1 promoter, of putative androgen responsive region(s).. 25.
(559) Results. Figure 2. CCND1 expression and promoter activity are inhibited by DHT-activated AR. (A) Serum-starved MCF-7 cells were treated or left untreated with 10 -7 M DHT for 24, 48 or 72 h in PRF–CT and total RNA was extracted. The abundance of cyclin D1 mRNA was detected by real-time reverse transcription-PCR, as described in ‘Materials and Methods’ section. (B) Total proteins were isolated from MCF-7 cells transfected with 0.5 µg pSiAR or 0.5 µg pSiCon scrambled control and treated for 24, 48 and 72 h with 10-7 M DHT or left untreated in PRF–CT. A 50 µg of protein lysates were analysed by WB to evaluate the expression of cyclin D1 and AR. The expression of "-actin was assessed as control of protein loading. Results are representative of three independent experiments. (C) Serum-starved MCF-7 cells were transiently transfected with D1!2960 reporter plasmid (0.25 µg/well). Upon transfection, cells were treated with 10-7 M DHT and/or 10-6 M OH-Fl, or left untreated in PRF–CT for 24 h. Firefly luciferase activity was detected and expressed as percentage of Relative Luciferase Activity. Activation of the reporter gene D1!-2960 in the presence of PRF–CT is arbitrarily set at 26.
(560) Results 100%. Results represent the means±SD of five separate experiments each in triplicate. Data were statistically analysed by student’s t-test, *P<0.01 versus untreated. (D) AR negative HEK 293 cells were co-transfected with D1 !-2960 (0.25 µg/well) plus increasing quantities (µg) of pcDNA3-AR (AR) or CMVP881 (AR (Cys-574!Arg) 0.5 µg/ well) as indicated. Upon transfection, cells were treated with 10 -7M DHT, or left untreated in PRF–CT for 24 h. Firefly luciferase activity was detected and expressed as percentage of relative luciferase activity. Activation of the reporter gene D1 !-2960 in the presence of PRF–CT is arbitrarily set at 100%. Linear relation between transfected AR plasmid and expressed AR protein quantity was evaluated by WB. Results represent the mean±SD from five independent experiments. Data were statistically analysed by Student’s t-test, *P<0.05 and °P<0.01 versus untreated.. Characterization of functional androgen responsive region(s) in the cyclin D1 promoter To define the AR responsive region(s) of the cyclin D1 promoter, a series of 5'-promoter-deleted mutants were used and tested for both androgen sensitivity and promoter activity, MCF-7 cells. The constructs D1 !-2996, D1!-944 and D1!-848, which include 2.960 kb, 0.944 kb and 0.848 kb of the cyclin D1 promoter fragment, respectively, showed a decreased transcriptional activity upon DHT stimulation with respect to untreated controls. A weaker inhibition of the cyclin D1 promoter signal was evidenced using the D1!-254 and D1!-136 constructs, while the D1!-96 plasmid, failed to respond to DHT (Figure 3).. 27.
(561) Results. Figure 3. Functional analysis of the promoter regions involved in AR-mediated modulation of the cyclin D1 promoter. (Left panel) Schematic representation of human cyclinD1 promoter fragments used in this study. All of the promoter constructs contain the same 3'-boundary (+142). The 5 '-boundaries of the promoter fragments varied from 2960 to 96. (Right panel) Transcriptional activity of cyclin D1 promoter constructs following DHT stimulation is shown. Serum-starved MCF-7 cells were transiently transfected with the indicated mutated plasmid (0.25 µg/well), as reported in ‘Materials and Methods’ section. Upon transfection, cells were treated with 10-7 M DHT or left untreated in PRF–CT for 24 h. Firefly luciferase activity was detected and expressed as percentage of Relative Luciferase Activity. Activation of reporter plasmids in the presence of PRF–CT only is arbitrarily set at 100. Results represent the means±SD of three separate experiments each in triplicate. Data were statistically analysed by Student’s t-test, *P<0.05 and °P<0.01 versus untreated.. Sequence analysis of the -848 bp to -254 bp cyclin D1 promoter DNA fragment revealed a likely ARE sequence (TGCTAAattAGTTCT) located at the -570 bp position of the promoter. This putative ARE ( CCND1-ARE) is homologous to the AREs found in the promoters of the sc (sc-ARE1.2) 28.
(562) Results. and slp (slp-HRE2) genes (58) (Figure 4A). To assess the relative importance of this sequence we used site-directed mutagenesis to alter it. Nucleotide substitutions were introduced into the 570-bp to 556-bp fragment of D1!-944 and the mutant promoter D1!-944-mARE was assayed in parallel with the unmutated D1!-944 promoter. Disruption of the putative ARE consensus site resulted in a significant loss of the inhibitory effect of DHT on cyclin D1 promoter activity (Figure 4B). These results address the CCND1- ARE as a crucial sequence in mediating cyclin D1 promoter inhibition upon DHT exposure. Nevertheless, mutations at the putative ARE did not completely abolish cyclin D1 promoter responsiveness to DHT, as indicated by the persistence of a weak DHT-induced decrease in the luciferase activity of the D1 !-136 construct, containing two Sp1 sites. To investigate whether this additional cis-element might be involved in the androgen response, as previously demonstrated for other AR target genes (57,58), site-directed mutagenesis was used to introduce nucleotide substitutions into the -117-bp to -101-bp fragment of D1!-944 (D1!-944-mSp1) or D1!-944-mARE (D1!-944mARE/ Sp1) and the mutant promoters were assayed in functional analysis. As depicted in Figure 4B, DHT treatment is able to induce only an about 30% inhibition of D1!-944-mSp1 promoter activity, indicating that Sp1 sites are important for an AR full inhibitory effect on the cyclin D1 promoter. Consistent with these observations, mutation of both ARE and Sp1 motifs completely abolishes DHT responsiveness of the cyclin D1 promoter further suggesting that its regulation occurs through a functional interaction of the AR with the CCND1 ARE and Sp1 responsive sequences.. 29.
(563) Results. Figure 4. Mutations of the putative ARE site or the Sp1 sites reduce the ability of AR to down-regulate cyclin D1 transcriptional activity . (A) Representation of the putative ARE site location within the cyclin D1 promoter. A list of known high affinity AR-specific ARE sequences is listed in the lower panel. (B) Site-directed mutagenesis was performed as described in ‘Materials and Methods’ section. MCF-7 cells were cotransfected, as reported in ‘Materials and Methods’ section, by using the mutated promoter constructs, schematically represented in (C). Upon transfection, cells were treated with 10-7 M DHT or left untreated in PRF–CT for 24 h. Firefly luciferase activity was detected and expressed as percentage of Relative Luciferase Activity. Activation of reporter gene in the presence of PRF–CT is arbitrarily set at 100. Results 30.
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