Prolactin induces up-regulation of its cognate receptor in breast cancer cells via transcriptional activation of its generic promoter by cross-talk between ERα and STAT5

Abstract

Prolactin (PRL) serves a critical role in breast cancer progression via activation of its cognate receptor. These studies reveal up-regulation of PRLR gene expression by PRL in absence of estradiol in MCF-7 and T47D breast cancer cells. PRL/PRLR via activation of STAT5 that binds a GAS-element in the PRLR gene and the participation of ERα stimulates PRLR transcription/expression. PRL/PRLR induces phosphorylation of ERα through the JAK2/PI3K/MAPK/ERK and JAK2/HER2 activated pathways. The increased recruitment of phospho-ERα, induced by PRL to Sp1 and C/EBPβ at PRLR promoter sites is essential for PRL-induced PRLR transcription. This recruitment is prevented by blockade of PRL expression using RNA interference or ERα phosphorylation using specific inhibitors of PI3K and ERK. Direct evidence is provided for local actions of PRL, independent of estradiol, in the up-regulation of PRLR transcription/expression by an activation-loop between STAT5 and the phospho-ERα/Sp1/C/EBPβ complex with requisite participation of signaling mechanisms. PRL's central role in the up-regulation of PRLR maximizes the action of the endogenous hormone. This study offers mechanistically rational basis for invasiveness fueled by prolactin in refractory states to adjuvant therapies in breast cancer.

Figure 1. Prolactin upregulation of its cognate receptor transcription/expression

Requisite participation of transcription factors. (A) Temporal expression of PRLR mRNA in response to PRL in MCF-7 cells. (B) Temporal expression of PRLR protein in response to PRL in MCF-7 cells. (C) Effect of PRL on PRLR transcripts in MCF-7 cells transfected with siRNAs: scramble (Scr), STAT5A, STATB or combination of both STAT5A and STAT5B. (D) A schematic representation of PRLR gene with the generic promoter hPIII (indicated in dotted line) including the non-coding exon-1 (hE1₃); the common non-coding exon 2 and coding exons 3-11. (E) Effect of PRL (100 ng/ml for 6h) on PRLR promoter activity transfected with wild type hPIII/ hE1₃ (−480/−112 bp) or constructs with Sp1 or C/EBPβ or STAT5 binding sites GAS mutated (X) or basic pGL2 vector (control) in MCF-7 cells. (F) Effect of PRL on hPIII promoter activity and mRNA in MCF-7 cells treated with an ERα antagonist, ICI 182,780 for 24 h. Results presented as relative luciferase activities (Rluc) normalized to the activities of co-transfected β-galactosidase (β-gal). (G) Effect of PRL on PRLR mRNA expression in MCF-7 cells treated with an ERα antagonist, ICI 182,780 for 24 h. Results in Figures 1 A, B, C, E, F and G are reported as the mean ± SE of three independent experiments. Asterisks indicate statistically significant increase between treated and untreated groups (P < 0.05). Means with a, b superscripts indicate statistically significant differences (P < 0.05).

Figure 2. Recruitment of endogenous ERα and STAT5 onto hPIII promoter

(A) ChIP assay showing the recruitment of ERα in MCF-7 cells transiently transfected with coding region of siRNAs, siSP1 (left) or C/EBPβ (right) followed by treatment with or without PRL (100 ng/ml). (B) ChIP assay showing the recruitment of ERα (left) and STAT5 (right) in MCF-7 cells transiently transfected with the coding region of SP1 siRNAs followed by treatment with or without PRL (100 ng/ml). (C) ChIP assay showing the recruitment of ERα (left) and STAT5 (right) in MCF-7 cells transiently transfected with the coding region of both STAT5A and STATB siRNAs followed by treatment with or without PRL (100 ng/ml). Scramble siRNA (Scr) was used as the positive control. The promoter region of hPIII containing SP1-C/EBPβ and STAT5 was analyzed by qPCR following immunoprecipitation (IP) with the respective antibodies. IgG was the negative control. Asterisks indicate statistically significant differences between PRL treated and untreated groups (P < 0.05).

Figure 3. Recruitment of ERα and STAT5 and complex formation essential for transcription/expression of PRLR induced by PRL

(A) Re-ChIP assay showing the recruitment and association of endogenous ERα and STAT5 onto the hPIII promoter. Re-ChIP was performed with chromatin from MCF-7 cells followed by sequential IP as indicated, with STAT5 antibody follow by ERα antibody (A, left) and in reverse sequence (A, right). (B) Effect of endogenous depletion of PRL by PRL siRNA on recruitment of endogenous ERα and STAT5 to hPIII promoter by ChIP analysis in MCF-7 cells. Effective depletion of endogenous PRL by PRL siRNA is shown in a Western blot, left versus scramble siRNA (Scr) as control, right. Asterisks indicate statistically significant difference between treated and untreated groups (P < 0.05).

Figure 4. PRL induced ERα phosphorylation and nuclear translocation via the PI3K/MAPK kinase pathway

(A) Western blots showing the temporal phosphorylation status of ERα (ser118 and ser167), ERK1/2 and MEK1/2 upon PRL treatment in MCF-7 cells. Total ERK1/2 and β-actin were used as loading controls. (B) Nuclear and cytoplasmic extracts isolated from MCF-7 after PRL treatment were analyzed by Western blot for p-ERα and total ERα. HDAC-1 and SMAC were monitored to validate the purity of the nuclear and cytoplasmic extracts respectively, and β-actin was used as loading control. (C) Western blots showing the effect of MEK inhibitor (U0126) on phosphorylation of ERα and ERK1/2 in MCF-7 cells treated with PRL at different time points. (D) Phosphorylation status of ERα and ERK1/2 in nuclear and cytoplasm extracts upon PRL treatment of cells for 30 min with or without addition of MEK1/2 inhibitor, U0126. (E) Western blot showing the effect of PI3 Kinase inhibitor (upper panel) and JAK inhibitor (lower panel) on phosphorylation status of ERα and ERK1/2 in MCF-7 cells treated with and without PRL. Total ERK1/2 and β-actin expression were shown as controls. (G) Effect of JAK-2 inhibitor (AG-490) on the phosphorylation of STAT5 (pSTAT5), HER2 (pHER2), AKT (pAKT), p70S6K1 (p-p70S6K1) and EK (pER) induced by PRL. The MCF-7 cells were treated either with inhibitors, U0126 (10 μM) or Wortmannin (0.5 μM) or AG-490 (50 μM) for 2h prior to the addition of PRL. Wort: Wortmannin.

Figure 5. PRL induced up-regulation of hPRLR mRNA and protein expression was abolished by ERK1/2, PI3K and JAK inhibitors in MCF-7 cells

(A) ChIP assay showing recruitment of pERα onto hPIII promoter in response to PRL in MCF-7 cells treated with ERK inhibitor U0126. (B) ChIP assay showing recruitment of pERα onto hPIII promoter in response to PRL in MCF-7 cells treated with PI3K inhibitor Wortmannin. Wort: Wortmannin. (C) Effect of U0126 on temporal expression of PRLR mRNA (above) and protein (below) in MCF-7 cells upon PRL treatment. (D) Effect of Wortmannin on temporal expression of PRLR mRNA (above) and protein (below) in MCF-7 cells upon PRL treatment. Wort: Wortmannin. (E) Effect of AG-490 on temporal expression of hPRLR mRNA (above) and protein (below) in MCF-7 cells upon PRL treatment. PRLR protein levels are expressed as percentage of control (control at 0h time point, 100%). Different superscripts letters indicate statistical significance differences (P < 0.05).

Figure 6. PRL induced ERα phosphorylation and up-regulation of hPRLR protein expression in T47D cells

(A), (B) and (C) Lanes 1-4: -Time course of PRLR induced upregulation of PRLR. (A), (B) and (C) Lanes 4-8: -Effects of inhibitors of (A) MEK (U0126), (B) PI3K (Wortmannin), (C) JAK2 (AG-490) on temporal expression of PRLR protein in T47D cells upon PRL treatment. PRLR protein levels are expressed as percentage of control (control at 0h time point, 100%). Asterisks indicate statistically significant increase in PRLR expression when compared to 0 h time point (control) (P < 0.05). (D) Western blots showing the effect of MEK inhibitor (U0126) and PI3 Kinase inhibitors (Wortmannin) on phosphorylation of ERα and ERK1/2 in T47D cells treated with PRL. (E) Effect of JAK-2 inhibitor (AG-490) on the phosphorylation of STAT5 (pSTAT5), HER2 (pHER2), AKT (pAKT), p70S6K1 (p-p70S6K1) and EK (pER) induced by PRL. Total ERK1/2 and β-actin expression were shown as controls. (F) ChIP assay showing recruitment of pERα onto hPIII promoter in response to PRL in T47D cells treated with ERK inhibitor U0126. (G) ChIP assay showing recruitment of pERα onto hPIII promoter in response to PRL in T47D cells treated with PI3K inhibitor Wortmannin. The T47D cells were treated either with inhibitors, U0126 (10 μM) or Wortmannin (0.5 μM) or AG-490 (50 μM) for 2h prior to the addition of PRL. Wort: Wortmannin. Different superscripts letters indicate significanct differences (P < 0.05).

Figure 7. Proposed mechanism of the upregulation of hPRLR induced by its cognate hormone

PRL produced locally in the normal and tumoral breast activates the JAK2/STAT5 pathway via the long form of the PRLR. The pSTAT5 bound to a GAS element located in the non-coding exon 1 of the PRLR associates with pERα recruited to Sp1 and C/EBPβ bound to their sites at the hPRLR promoter causing increase hPRLR transcription, mRNA and protein. PRL/PRLR, independent of estrogen, induces ERα phosphorylation (via the JAK2/PI3K-MEK-ERK pathway), nuclear translocation, and complex formation by its recruitment to Sp1/C/EBPβ bound to their DNA sites at the promoter and transcriptional activation. PRL/PRLR activates JAK2 which phosphorylates HER2 and activates several pathways which in turn may cause phosphorylation of ERα. The series of molecular events induced by endogenous PRL via PRLR causing up-regulation of its cognate receptor could participate in breast cancer progression and explain resistance to endocrine therapy.