Runx2 controls a feed-forward loop between androgen and prolactin-induced protein (PIP) in stimulating T47D cell proliferation

Abstract

Prolactin-Induced Protein (PIP) is a small polypeptide expressed by breast and prostate cancer (BCa, PCa) cells. However, both the regulation of PIP expression and its function in cancer cells are poorly understood. Using BCa and PCa cells, we found that Runx2, a pro-metastatic transcription factor, functionally interacts with the Androgen Receptor (AR) to regulate PIP expression. Runx2 expression in C4-2B PCa cells synergized with AR to promote PIP expression, whereas its knockdown in T47D BCa cells abrogated basal as well as hormone stimulated PIP expression. Chromatin immunoprecipitation (ChIP) assays showed that Runx2 and AR co-occupied an enhancer element located ∼11 kb upstream of the PIP open reading frame, and that Runx2 facilitated AR recruitment to the enhancer. PIP knockdown in T47D cells compromised DHT-stimulated expression of multiple AR target genes including PSA, FKBP5, FASN, and SGK1. The inhibition of AR activity due to loss of PIP was attributable at least in part to abrogation of its nuclear translocation. PIP knockdown also suppressed T47D cell proliferation driven by either serum growth factors or dihydrotestosterone (DHT). Our data suggest that Runx2 controls a positive feedback loop between androgen signaling and PIP, and pharmacological inhibition of PIP may be useful to treat PIP positive tumors.

Figure 1. Runx2 stimulates PIP expression in Prostate and Breast Cancer cells

A–B, Assessment of PIP expression by RT-qPCR (A) and western blotting (B) after induction of Runx2 expression in C4-2B/Rx2^dox PCa cells by treatment with the indicated dox concentrations. Anti-Flag antibody was used to detect Runx2. C–D, Assessment of PIP expression in T47D/shRx2^dox BCa cells by RT-qPCR (C) and western blotting (D) after shRNA-mediated silencing of Runx2 by the indicated concentrations of dox. Tubulin was used as loading control in B and D.

Figure 2. Androgen and Runx2 signaling cooperate to regulate PIP expression

A, T47D/shRunx2^dox were treated with dox to silence Runx2 in the presence or absence of DHT and PIP mRNA levels were quantitated by RT-qPCR. B, Western blot analysis of PIP, Runx2, AR and Tubulin under the same conditions as in (A). C, C4-2B/Rx2^dox cells were treated with dox to express Runx2 in the presence or absence of DHT, and subjected to RT-qPCR analysis of PIP mRNA. D, Western blot analysis of PIP, Runx2, AR and Tubulin (loading control) under the same conditions as in (C).

Figure 3. Runx2 and AR are co-recruited to a novel -11-kb PIP enhancer

A, Schematic depiction of DNA sequences upstream of the PIP transcription start site, with predicted binding sites for AR and Runx2. Arrows indicate binding sites for four primer pairs designed to flank four regions of interest, designated R-I through R-IV. B–C, C4-2B/Rx2^dox cells were pre-treated with dox or water vehicle for 16 hours, followed by 10 nM DHT or ethanol vehicle for 4 hours. Quantitative ChIP assays were performed using primers amplifying the indicated regions after immunoprecipition of AR (B) or Runx2 (C). Primers used to amplify regions R-I through R-IV, as well as an intergenic control region, are listed in Table S1.

Figure 4. PIP is required for T47D cell proliferation

A, T47D/shPIP^dox cells were maintained in medium supplemented with complete serum and containing either dox or Vehicle. Cell proliferation was assessed using MTT assays at the indicated time points. Treatment was from day 2 till the MTT assay, except in the indicated group, where dox was withdrawn on Day 6. B, T47D/shPIP^dox cells were maintained for two days in medium supplemented with charcoal-stripped serum. DHT and/or dox were then added to the medium and MTT assays were performed at the indicated time points. C, T47D/shPIP^dox cells were maintained and treated as in B, followed by Western blot analysis of whole cell extracts from day 1 cultures using antibodies for AR, PIP and Tubulin. D FACS based cell cycle analysis of T47D/shPIP^dox cells treated for 6 hours with DHT after knocking down PIP as in (C).

Figure 5. PIP regulates the transcriptional activity and the subcellular distribution of AR

A, RT-qPCR analysis of PIP, AR and four AR-regulated genes, FKBP1, PSA, FASN, and SGK, in T47D/shPIP^dox cells treated with either DHT or dox or their indicated combinations. B, T47D/shPIP^dox cells were treated with DHT or vehicle in the presence or absence of dox, and then subjected to cellular fractionation. The cytoplasmic and nuclear fractions were subjected to western blot analysis of AR, Runx2, and PIP. Tubulin and di-methyl-Histone-4 (H4) specific were detected as cytoplasmic and nuclear markers to validate the purity of the respective fractions.