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. Author manuscript; available in PMC: 2013 May 1.
Published in final edited form as: J Cell Physiol. 2012 May;227(5):2276–2282. doi: 10.1002/jcp.22966

Runx2 Controls a Feed-forward loop between Androgen and Prolactin-induced Protein (PIP) in Stimulating T47D Cell Proliferation

Sanjeev K Baniwal 1,3,*, Gillian H Little 2,3, Nyam-Osor Chimge 2,3, Baruch Frenkel 1,2,3,*
PMCID: PMC3376385  NIHMSID: NIHMS314584  PMID: 21809344

Abstract

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 breast and prostate cancer 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 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 ~11kb 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.

Keywords: Runx2, PIP, AR, Breast cancer marker, Transcription regulation

Introduction

The three members of the mammalian Runt-related family of transcription factors play divergent roles in hematopoiesis (Runx1), skeletal development (Runx2), and maintenance of the gastric epithelium (Runx3). Additionally, these proteins have been implicated in context dependent negative and positive control of cancer progression (Blyth et al., 2005). Runx2 in particular was detected in various advanced tumors including those originating from breast and prostate epithelia (Akech et al., 2009; Blyth et al., 2005; Javed et al., 2005; Kayed et al., 2007; Shore, 2005). The ectopic expression of Runx2 in these cells promotes invasiveness and stimulates related genes involved in epithelial-mesenchymal transformation such as SNAIL, Sox9, and SMAD3 (Akech et al., 2009; Baniwal et al., 2010). Ablation of Runx2 in MDA-231 breast cancer (BCa) and in PC3 PCa cells inhibited their growth within the bone microenvironment and the associated osteolysis (Akech et al., 2009; Javed et al., 2005). Finally, high Runx2 expression in BCa tumors has been associated with poor prognosis (Onodera et al., 2010).

PIP, a.k.a. Gross Cystic Disease Fluid Protein-15 (GCDFP-15) is a ~16 kDa, 146-amino acid glycoprotein independently identified first in human gross cystic disease fluid (Haagensen et al., 1980) and later in the culture supernatant of T47D BCa cell cultures treated with either prolactin or glucocorticoids (Shiu and Iwasiow, 1985). PIP is biosynthesized by apocrine cells and found in body fluids such as saliva, tear, milk, and seminal fluid (Hassan et al., 2009). Clinically, PIP is a specific and sensitive serum marker of advanced breast cancer (BCa), highly expressed by >70% of tumors of apocrine origin (Clark et al., 1999; Haagensen et al., 1990). PIP is also associated with PCa, where its expression is significantly higher than in normal prostate tissue (Tian etal., 2004).

Little is known about the role of PIP in health or disease (Hassan et al., 2009). Possibly, seminal fluid PIP supports spermatozoa survival by neutralizing anti-sperm IgG antibodies (Chiu and Chamley, 2003). However, mice lacking PIP develop normally, have normal life expectancy, and exhibit no overt reproductive or other abnormalities (Blanchard et al., 2009). Based on PIP’s amino acid sequence, Caputo et al found that PIP is an aspartyl protease capable of cleaving fibronectin in vitro (Caputo et al., 2000). Such enzymatic activity may play a role in degradation of the extra cellular matrix and cancer metastasis. PIP may also promote cancer-associated inflammation via its high affinity binding to CD4 and the resulting inhibition of T-cell apoptosis (Gaubin et al., 1999). Finally, purified PIP is mitogenic for various BCa and immortal mammary cell lines (Cassoni et al., 1995).

Androgens stimulate PIP expression in various BCa cell lines including T47D, ZR-75, and MDA-MB453 (Ellison et al., 2002; Murphy et al., 1987). In T47D cells, androgens were most potent at physiological concentrations and induced robust PIP expression at levels 3–4 orders of magnitude lower than glucocorticoids (Haagensen et al., 1990; Murphy et al., 1987). Furthermore, immunohistochemical staining of breast tumors suggested a strong correlation between PIP expression and androgen receptor (AR) activity, as well as between PIP and PSA, a classical AR-regulated gene (Hall et al., 1998). Two functional half androgen response elements have been identified ~1.3-kb upstream of the PIP transcription start site, and the androgen response of PIP in ZR-75 cells required functional AR and Stat5, as well as the presence of prolactin in the culture medium (Carsol et al., 2002).

Using whole genome microarray analysis of PCa cells, we recently reported that PIP was one of the genes most highly stimulated by Runx2 (Baniwal et al., 2010). Because PIP expression is highly sensitive to androgens and because Runx2 interacts with AR physically and functionally, we investigated their crosstalk in regulating PIP expression. We further addressed the functional role of PIP in T47D breast cancer cells. We found that AR and Runx2 synergistically stimulate PIP transcription, and that PIP is exquisitely required for T47D breast cancer cell proliferation.

Experimental Methods

Cell culture reagents and antibodies

C4-2B cells were obtained from ViroMed Laboratories (Minneapolis, MN). LNCaP, T47D, and MDA-231 cells were from ATCC (Rockville, MD, USA), and were kindly provided by USC’s Drs. Gerhard A Coetzee, Michael Stallcup, and Graham Casey, respectively. The cells were maintained in RPMI-1640 medium supplemented with 10% Tet System Approved FBS™ from Clontech, CA, USA. Hygromycin B was purchased from Invitrogen, Carlsbad, CA, USA, and added to the growth medium at 50 μg/ml. Dox from Calbiochem, La Jolla, CA, USA was used at 0.5 μg/ml, and an equal volume of distilled water was used as vehicle control. Puromycin, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and DHT were obtained from Sigma, St Louis, MO, USA. DHT was used at 100 nM and equal volume of ethanol was added as vehicle control. Mouse ANTI-FLAG® M2 monoclonal antibody was purchased from Sigma. Mouse anti-Runx2 was from Invitrogen. The anti-PIP antibody (ab 62363) was purchased from abcam Inc., Cambridge, MA; The mouse monoclonal anti-Tubulin antibody, developed by Dr. Charles Walsh, was obtained from the Developmental Studies Hybridoma Bank under the auspices of the NICHD and The University of Iowa, Department of Biological Sciences, Iowa City, USA.

Plasmids

The dox-inducible lentiviral expression and knock-down plasmids were based on the pSLIK (single lentivector for inducible knockdown) vector (Shin et al., 2006). The pSLIK-Flag-Runx2 plasmid was described earlier (Baniwal et al., 2010). DNA sequences encoding shRNAs for Runx2 and PIP were designed using the RNAiCodex program (http://katahdin.cshl.org/html/scripts/resources.pl). Oligonucleotides used for cloning are listed in Table S1. The shRNA-coding oligonucleotides were initially cloned into the lentiviral entry vector pEN_TmiRc3 (ATCC® catalog: MBA-248), and the resulting plasmid was recombined using Gateway® LR Clonase® II enzyme mix (Invitrogen) with the pSLIK destination vector carrying a hygromycin resistance gene (ATCC® catalog: MBA-237). The entry and destination vectors were kindly provided by USC’s Dr. Elizabeth Lowler (Childrens Hospital Los Angeles). Constitutively expressing shRNA lentiviral plasmids targeting either a non-specific sequence or distinct PIP-specific sequences were purchased from Sigma (Table S1).

Lentivirus production and infection

For packaging, the lentiviral expression plasmids were cotransfected by the calcium chloride method into HEK293T cells along with helper plasmids pMD.G1 and pCMVR8.91 (Kim et al., 2008; Phillips and Garcia, 2008). Culture media containing viral particles were harvested after 48–72 hours and used for transduction of the indicated cells inthe presence of 8 μg/ml Polybrene (Millipore Corp., MA, USA). The transduced cells were selected with either 50 μg/ml of Hygromycin or 3 μg/mL of Puromycin.

RT-qPCR

Total RNA was isolated using Aurum Total RNA kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) following the manufacturer’s recommendations and 1 μg was reverse transcribed using the qScript™ cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD, USA). The cDNA was subjected to real-time qPCR amplification using RT-qPCR was performed using the CFX96™ RT-PCR system, the iQ™ SYBR® Green Supermix (both from Bio-Rad, Hercules, CA, USA) and the primers listed in Table S1.

Proliferation and cell cycle analyses

These assays were performed as described previously (Baniwal et al., 2010). Briefly, cells were incubated at 37°C with 0.5 mg/mL of MTT dissolved in PBS for 2 hours. Cells were then lysed using DMSO and the development of color was quantified at 595 nM using Victor3V™ from PerkinElmer, Shelton, CT, USA. For cell cycle analysis, propidium iodide (PI)-stained cells were subjected to fluorescence-activated cell sorting (FACScaliber, Becton Dickinson, MA, USA) and each cell was assigned to the G1, S, G2 or M phase of the cell cycle based on the PI intensity and using the Multicycle v3.0 software (Phoenix Flow Systems, San Diego, CA, USA).

Cell extract preparation and western blot analysis

Whole cell extracts were prepared by lysing 1×105 – 2×105 cells in 200 μL of extraction buffer [100 mM Tris (pH 7.4), 500 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% Nonidet® P-40] supplemented with Complete™ protease inhibitor mix (Roche Diagnostics, Indianapolis, IN, USA) 40 μg protein was mixed with an equal volume of Laemmli buffer followed by SDS-PAGE, transferred to Amersham Hybon™-P PVDF (GE Healthcare, Piscataway, NJ, USA) membranes, and visualization using specific antibodies and the Western Lightning™ Plus-ECL kit (PerkinElmer Inc, Waltham, MA, USA). Cytoplasmic and nuclear extracts were obtained using extraction buffer containing 5 mM and 500 mM NaCl, respectively, and proportional volumes were subjected to Western blot analysis as described above.

Chromatin-immunoprecipitation (ChIP) assays

AR and Runx2 ChIP assays were performed essentially as described previously (Jia et al., 2008). For the Runx2 ChIP assay, cells were sonicated in a buffer containing 0.1% SDS, 2mM EDTA and 50mM Tris pH8.0, and the chromatin was incubated with Flag M2 antibody (0.5μg) overnight at 4°C, followed by pulling down of the Runx2 precipitates with protein A dynabeads (Invitrogen). The DNA was purified using Qiagen PCR cleanup kit and quantified using qPCR for regions of interest using primers listed in Table S1.

Results

Runx2 stimulates PIP expression in prostate and breast cancer cells

The LNCaP prostate cancer (PCa) cell line and its derivative C4-2B express the pro-metastatic transcription factor Runx2 and the metastatic marker PIP at barely detectable levels (Baniwal et al., 2010). Using a doxycycline (dox)-inducible lentiviral system, we conditionally expressed Runx2dox in these cells to levels seen in more highly metastatic PCa cells (Baniwal et al., 2010; Shin et al., 2006). Microarray analysis of the C4-2B/Rx2dox cells identified PIP as one of the genes most responsive to Runx2, and RT-qPCR confirmed elevated PIP mRNA levels in both cell lines in three independent experiments (Figure 1A and S1) (Baniwal et al., 2010). Moreover, very low expression levels of Runx2 were sufficient to effectively enhance PIP expression (Figure 1A). Western blot analysis of C4-2B/Rx2dox cells detected the PIP protein only after Runx2 induction by dox (Figure 1B).

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

Figure 1

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A–B, Assessment of PIP expression by RT-qPCR (A) and western blotting (B) after induction of Runx2 expression in C4-2B/Rx2dox 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/shRx2dox 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.

Both PIP and Runx2 are endogenously expressed in the T47D breast cancer cell line (Murphy et al., 1987; Ning and Robins, 1999). To investigate the requirement of Runx2 for PIP expression in T47D cells, we transduced them with lentiviruses encoding a dox-inducible shRNA targeting Runx2 (shRx2, Table S1). Gradual Runx2 knockdown in the T47D/shRx2dox cells dose-dependently diminished PIP mRNA levels (Figure 1C), and 50% Runx2 mRNA knockdown inhibited PIP protein expression almost completely (Figure 1D). As control, dox-induced non-specific shRNA altered neither Runx2 nor PIP expression (Figure S2). The robust induction of PIP in response to Runx2 in C4-2B cells and its striking inhibition upon Runx2 knockdown in T47D cells suggest that Runx2 is a crucial regulator of PIP expression.

Runx2 is required for hormone-stimulated PIP expression

PIP transcription in T47D cells is regulated by multiple hormones including prolactin, growth hormone (GH), glucocorticoids, and androgens (Murphy et al., 1987). Among these, DHT is most potent, effectively stimulating PIP expression at physiological concentrations of 1 to 100 pM (Murphy et al., 1987). The glucocorticoid receptor (GR) and the androgen receptor (AR) have been shown to physically interact with Runx2 and modulate its activity (Baniwal et al., 2009; Ning and Robins, 1999). We therefore postulated that Runx2 may play a role in hormone-stimulated PIP expression, and initially tested this postulate using DHT-treated T47D/shRx2dox cultures. Cells were maintained in growth medium supplemented with charcoal-stripped serum with or without dihydrotestosterone (DHT), and simultaneously treated with either vehicle or dox to silence Runx2. As shown by RT-qPCR and western blot analyses, PIP mRNA and protein were barely detectable in untreated cells (Figure 2A, lane 1), suggesting that serum ingredient(s) stripped by charcoal were required for PIP expression. PIP mRNA and protein levels remained basal upon Runx2 silencing (Figure 2A, lane 2). DHT-treatment dramatically stimulated PIP mRNA and protein expression (Figure 2A, lane 3). Remarkably, the DHT-mediated stimulation of PIP expression did not occur after Runx2 silencing (Figure 2A, lane 4). The diminished DHT-stimulated PIP expression upon Runx2 silencing suggests an essential regulatory role of Runx2 in the androgenic response of PIP expression. Similarly, Runx2 silencing abrogated the stimulation of PIP expression by GH and the glucocorticoid dexamethasone (Figure S3). Thus, Runx2 is necessary for hormone stimulated PIP expression in breast cancer cells.

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

Figure 2

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A, T47D/shRunx2dox 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/Rx2dox 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).

The crosstalk between Runx2 and AR in regulating PIP expression was further investigated in the C4-2B/Rx2dox cells. RT-qPCR and western blot analyses demonstrated very low levels for PIP mRNA and protein in untreated cells (Figure 2C, D; lane 1). Treatment with either dox, to induce Runx2, or DHT moderately increased PIP mRNA and protein levels (Figure 2C, D, lanes 2, 3). However, co-treatment with both dox and DHT resulted in synergistic up-regulation of PIP mRNA and protein (Figure 2C, D lane 4). Thus, loss of hormonal stimulation of PIP expression upon Runx2 silencing in T47D cells, and the synergistic stimulation of PIP expression by Runx2 and DHT in C4-2B cells suggest a pivotal role for Runx2 in the hormonal response of PIP in both breast and prostate cancer cells.

Runx2 augments AR recruitment to a novel PIP upstream enhancer

To investigate the mechanism of Runx2-mediated PIP transcription, and since AR and Runx2 synergistically stimulated PIP expression, we investigated their association with sequences upstream of the PIP transcription start site (TSS). In silico analysis using JASPAR (Sandelin et al., 2004) revealed four regions, designated R-I (−0.9-kb), R-II (−2.4-kb), R-III (−9.4-kb) and R-IV (−11-kb), with either multiple Runx motifs or a combination of Runx and AR motifs (Figure 3A). Association of AR and Runx2 to these regions was investigated using Chromatin-immunoprecipitation (ChIP) assays. C4-2B/Rx2dox cells were treated for 16 hours with either dox to induce Runx2 expression or vehicle as control, followed by 4 hours of treatment with DHT or vehicle. ChIP analysis revealed that DHT mediated robust occupancy of the R-IV region by AR (Figure 3B). More interesting was the influence of Runx2 on AR recruitment to the R-IV region: in contrast to the 5-fold stimulation of AR recruitment by DHT in the absence of Runx2, AR recruitment to this region was 30-fold higher than control in the presence Runx2 (Figure 3B). Runx2 also enhanced, to a lesser extent, AR recruitment to the R-I, R-II and R-III regions. That R-I and R-II do not contain consensus AR-binding sites suggest that AR is recruited to these sites via interaction with Runx2 or other factors. ChIP assay of Runx2 occupancy showed strong recruitment to R-IV and to a lesser extent to R-I, but not to RII or RIII (Figure 3C). Unlike the influence of Runx2 on AR recruitment, Runx2 recruitment to R-IV was not influenced by DHT (Figure 3C). These results demonstrate co-occupancy of the R-IV PIP enhancer by AR and Runx2 in living cells. The enhanced AR recruitment to the R-IV region in the presence of Runx2 very likely accounts for the synergistic up-regulation of PIP by Runx2 androgen signaling.

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

Figure 3

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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/Rx2dox 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.

PIP is required for serum- and androgen-stimulated T47D cell proliferation

In order to gain insight into the function of PIP, we specifically knocked down its expression in T47D cells by transducing them with lentiviruses encoding shRNAs targeting different regions within the PIP open reading frame (Table S1). We derived three T47D sub-lines that expressed shPIP either constitutively (T47D/shPIP1 and T47D/shPIP2) or conditionally after dox treatment (T47D/shPIPdox). Each of these three distinct shRNAs effectively reduced PIP expression levels (Figure S4A and 4C). In all cases, microscopic observations and MTT-based cell growth assays indicated a marked decrease in cell proliferation after PIP knockdown (Figure 4A and S4B, C). The same viruses had no effect on the growth of MDA-231 breast cancer cells, which do not express PIP (Clark et al., 1999) (Figure S4D). As additional control, T47D/NSdox cells where dox-treatment induces expression of a non-specific shRNA affected neither PIP nor Runx2 expression levels (Figure S2) also showed no effect on cell proliferation (Figure S4E). In the T47D/shPIPdox cultures, the block of cell growth was evident within 48 hours of dox-mediated shPIP induction, and persisted till day 12, as long as the PIP knockdown lasted. However, dox withdrawal on day 6 resulted in gradual resumption of cell growth by day 10 (Figure 4A). These results suggest that PIP plays an essential role in T47D cell proliferation, and that a lack of PIP induces reversible quiescence in these cells.

Figure 4. PIP is required for T47D cell proliferation.

Figure 4

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A, T47D/shPIPdox 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/shPIPdox 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/shPIPdox 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/shPIPdox cells treated for 6 hours with DHT after knocking down PIP as in (C).

Next we used T47D/shPIPdox cells to investigate the role of PIP in DHT-stimulated cell proliferation (Migliaccio et al., 2000). Cells were maintained in growth medium supplemented with charcoal-stripped serum and treated with DHT and/or dox to knockdown PIP. Cell growth was determined by performing MTT assays every 24 hours for six days. The T47D cultures maintained with charcoal-stripped serum exhibited slow growth, and were completely arrested after dox-induced PIP knockdown (Figure 4B). More impressively, dox abolished the fast growth observed in the DHT-treated cultures (Figure 4B). This occurred without compromising the DHT-mediated stabilization of AR (Figure 4C). To further delineate the effect of PIP knockdown on T47D cell proliferation, we assessed its effects on cell cycle progression under the same conditions as in Figure 4B. Fluorescence-activated cell sorting (FACS) analysis revealed a 2-fold decrease in the percentage of cells in the S/G2/M-phases of the cell cycle after dox-induced PIP knockdown in the absence of DHT (Figure 4D). DHT treatment of T47D/shPIPdox cells for 6 hours promoted cell cycle progression, increasing the fraction of S/G2/M-phase cells from 8% to 19.5% (Figure 4D). dox-mediated PIP knockdown reduced the fraction of S/G2/M-phase cells to 7.4% (Figure 4D). In contrast, neither DHT nor dox affected T47D cell apoptosis (data not shown). Taken together, our results suggest that PIP is required for T47D cell proliferation. In particular, DHT-stimulated cell proliferation is completely dependent on PIP.

PIP is required for AR-mediated transcriptional stimulation

We investigated the potential role of PIP in androgen signaling by measuring the influence of PIP knockdown on DHT-stimulated gene expression in T47D/shPIPdox cells. Cultures were treated for 48 hours with either dox, to silence PIP, or water vehicle, followed by 24-hour of treatment with either DHT or ethanol vehicle. Expression levels of the classical AR target genes FKBP5, PSA, FASN and SGK, as well as PIP and AR, were determined by RT-qPCR. As shown in Figure 5A, PIP knockdown dramatically compromised DHT stimulated expression of all AR target genes tested, and the DHT responses including that of PIP itself. As control, dox treatment of T47D/shPIPdox cells remarkably reduced both basal and DHT-induced PIP expression, and PIP silencing had no or minimal effect on AR mRNA levels (Figure 4C, 5A). Thus, PIP is not only an AR target gene (Figures 2, 3 and 4C), but its expression feeds forward to support androgen signaling in T47D cells.

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

Figure 5

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A, RT-qPCR analysis of PIP, AR and four AR-regulated genes, FKBP1, PSA, FASN, and SGK, in T47D/shPIPdox cells treated with either DHT or dox or their indicated combinations. B, T47D/shPIPdox 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.

We further investigated the requirement of PIP for androgen signaling by analyzing the nucleo-cytoplasmic distribution of AR in T47D/shPIPdox cells following treatments with DHT or vehicle in the presence or absence of dox. In the absence of dox, AR was detectable in both the cytoplasmic and nuclear fractions, and DHT treatment resulted in preferential nuclear localization (Figure 5B). dox-mediated PIP knockdown reduced AR localization in the nucleus, a phenomenon that was dramatic in DHT-treated cells, but was also evident in the ethanol/vehicle-treated sample (Figure 5B). Noteworthy, PIP knockdown did not affect AR or Runx2 expression (Figure 4C, 5B). Thus, the inhibition of DHT responsiveness by PIP knockdown is associated with abrogation of AR nuclear localization.

Discussion

Although PIP/GCDFP-15 is used as a clinical marker for breast cancer (Haagensen et al., 1980; Murphy et al., 1987; Wick et al., 1989) including metastatic disease, little is known about its function in tumor progression. That PIP silencing resulted in cell cycle and growth arrest in the T47D culture model suggests that this protease may be required for cell proliferation at least in a subset of breast cancer and other PIP-positive tumors. This renders PIP a potential therapeutic target in addition to its current diagnostic role in the management of BCa.

PIP may mediate the oncogenic effect of androgens in BCa (Chia et al., 2011; Doane et al., 2006; Naderi et al., 2011). Indeed, the majority of BCa tumors express both AR and PIP (Bundred et al., 1990; Gonzalez et al., 2008) and here we show that PIP knock-down inhibits androgen-stimulated T47D cell proliferation. However, PIP knock-down also inhibited serum-stimulated cell proliferation, implicating its role in BCa progression beyond the context of androgen signaling. Since PIP expression is regulated by a variety of other hormones including prolactin, GH, and glucocorticoids (Murphy et al., 1987; Shiu and Iwasiow, 1985), it is well positioned as a putative central node orchestrating their growth related functions in cancer.

Our interest in PIP was triggered by its robust responsiveness to the transcription factor Runx2 in prostate cancer cells (Baniwal, 2010). Runx2, best known for its role in osteoblast differentiation and bone formation (Ducy et al., 1997; Komori et al., 1997; Otto et al., 1997), also controls cell cycle progression, either positively or negatively (Bae and Choi, 2004; Blyth et al., 2005; Cameron and Neil, 2004; Kilbey et al., 2007; Pratap et al., 2003; Thomas et al., 2004; Zaidi et al., 2007), and plays a role in cancer metastasis (Akech et al., 2009; Blyth et al., 2010; Chua et al., 2009; Javed et al., 2005; Pratap et al., 2005; Pratap et al., 2006; Shore, 2005). The roles of PIP downstream of Runx2 in cancer remain to be investigated. Be that as it may, the Runx2 target PIP offers a more attractive therapeutic target than Runx2 itself not only because PIP is an accessible extracellular protease, but also because inhibition of Runx2 is expected to results in loss of both the pro- and anti-tumorigenic activities of this transcription factor (Akech et al., 2009; Blyth et al., 2005; Pratap et al., 2006).

We observed strong synergism between AR and Runx2 in stimulating PIP expression. This is reminiscent of the synergism between the glucocorticoid receptor and Runx2 in stimulating the Slp gene (Ning and Robins, 1999). Unique sequence parameters of the −11-kb PIP enhancer, containing the R-IV Runx2 binding site and two flanking AR binding sites, likely facilitate productive physical interaction between AR, Runx2 and the DNA, as demonstrated by the enhanced recruitment of AR to this region in the presence of both its ligand and Ruxn2. In remarkable contrast to the synergistic stimulation of PIP by AR and Runx2, the interaction between these two transcription factors results in loss of their DNA-binding activity in other contexts [(Baniwal et al., 2009) and additional unpublished data]. To decipher rules that dictate the locus-specific outcomes of the interactions between AR and Runx2, we are in the process of genome-wide analyses of the DNA sequences that support either synergy between AR and Runx2 or their mutual inhibition.

The mechanism of action of PIP in permitting cell cycle progression remains a major challenge. We show that PIP is necessary for DHT-mediated nuclear localization of AR, and for activation of its target genes. Probably, however, this is only one aspect of PIP signaling because its silencing arrested growth in cultures that were maintained with complete serum, where mitogens other than androgens likely predominated. We speculate that the protease activity of PIP at the cell surface is necessary to facilitate multiple signaling pathways, only some of which are related to AR function. The same or other PIP-dependent signals may be required for cell cycle progression in general. In favor of this view, our unpublished preliminary screen of 71 receptor tyrosine kinases suggested that PIP silencing decreased the tyrosine phosporylation of FAK, Fyn, EphB3, and Hck receptors (data not shown). Possibly, PIP-mediated cleavage of fibronectin (Caputo et al., 2000) is necessary for proper signaling from the extracellular matrix through integrin receptors to activate these tyrosine kinases. The detailed analysis of PIP-dependent cellular signaling in breast and other cancer cells is under investigation. However, it is interesting to note that PIP is not always required for cell proliferation as demonstrated by the normal development of PIP knockout mice (Blanchard et al., 2009). Future anti-PIP therapeutics is therefore expected to benefit a subset of patients where cancer cell proliferation depends on PIP, and such therapies will likely be well tolerated.

Supplementary Material

Supp FigS1-S4

Figure S1, Microsoft PowerPoint, Runx2 stimulates PIP expression in LNCaP Prostate Cancer cells.

Figure S2, Microsoft PowerPoint, Expression of non-specific shRNA does not affect PIP and Runx2 expression.

Figure S3, Microsoft PowerPoint, Runx2 is required for Growth Hormone (GH)- and Dexamethasone (Dex)-induced PIP gene expression.

Figure S4, Microsoft PowerPoint, PIP knock-down compromises T47D cell proliferation.

Supp Table S1

Acknowledgments

Contract Grant Sponsor: National Institute of Health RO1 grants [DK071122, DK071122S1, CA 109147, and AR047052]. BF holds the J. Harold and Edna L. LaBriola Chair in Genetic Orthopedic Research at USC

We thank Ms. Yunfan Shi for her expert technical assistance.

Abbreviations

PIP

Prolactin-induced Protein

GCDFP-15

Gross cystic disease fluid protein-15

Dox

doxycycline

DHT

dihydrotestosterone

AR

Androgen receptor

PCa

Prostate Cancer

BCa

Breast Cancer

MTT

(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, tetrazole)

Footnotes

The authors declare no conflict of interest.

References

  1. Akech J, Wixtted JJ, Bedard K, van der Deen M, Hussain S, Guise TA, van Wijnen AJ, Stein JL, Languino LR, Altieri DC, Pratap J, Keller E, Stein GS, Lian JB. Runx2 association with progression of prostate cancer in patients: mechanisms mediating bone osteolysis and osteoblastic metastatic lesions. Oncogene. 2009;(29):811–821. doi: 10.1038/onc.2009.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bae SC, Choi JK. Tumor suppressor activity of RUNX3. Oncogene. 2004;23(24):4336–4340. doi: 10.1038/sj.onc.1207286. [DOI] [PubMed] [Google Scholar]
  3. Baniwal KO, Gabet Y, Shah R, Purcell DJ, Mav D, Gabet A, Shi Y, Coetzee GA, Frenkel B. Runx2 Transcriptome of Prostate Cancer Cells: Insights into Invasiveness and Bone Metastasis. Molecular Cancer. 2010;9:258. doi: 10.1186/1476-4598-9-258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baniwal SK, Khalid O, Gabet Y, Shah RR, Purcell DJ, Mav D, Kohn-Gabet AE, Shi Y, Coetzee GA, Frenkel B. Runx2 transcriptome of prostate cancer cells: insights into invasiveness and bone metastasis. Mol Cancer. 2010;9:258. doi: 10.1186/1476-4598-9-258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baniwal SK, Khalid O, Sir D, Buchanan G, Coetzee GA, Frenkel B. Repression of Runx2 by androgen receptor (AR) in osteoblasts and prostate cancer cells: AR binds Runx2 and abrogates its recruitment to DNA. Mol Endocrinol. 2009;23(8):1203–1214. doi: 10.1210/me.2008-0470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blanchard A, Nistor A, Castaneda FE, Martin D, Hicks GG, Amara F, Shiu RP, Myal Y. Generation and initial characterization of the prolactin-inducible protein (PIP) null mouse: accompanying global changes in gene expression in the submandibular gland. Can J Physiol Pharmacol. 2009;87(10):859–872. doi: 10.1139/Y09-077. [DOI] [PubMed] [Google Scholar]
  7. Blyth K, Cameron ER, Neil JC. The RUNX genes: gain or loss of function in cancer. Nat Rev Cancer. 2005;5(5):376–387. doi: 10.1038/nrc1607. [DOI] [PubMed] [Google Scholar]
  8. Blyth K, Vaillant F, Jenkins A, McDonald L, Pringle MA, Huser C, Stein T, Neil J, Cameron ER. Runx2 in normal tissues and cancer cells: A developing story. Blood Cells Mol Dis. 2010;45(2):117–123. doi: 10.1016/j.bcmd.2010.05.007. [DOI] [PubMed] [Google Scholar]
  9. Bundred NJ, Stewart HJ, Shaw DA, Forrest AP, Miller WR. Relation between apocrine differentiation and receptor status, prognosis and hormonal response in breast cancer. Eur J Cancer. 1990;26(11–12):1145–1147. doi: 10.1016/0277-5379(90)90273-v. [DOI] [PubMed] [Google Scholar]
  10. Cameron ER, Neil JC. The Runx genes: lineage-specific oncogenes and tumorsuppressors. Oncogene. 2004;23(24):4308–4314. doi: 10.1038/sj.onc.1207130. [DOI] [PubMed] [Google Scholar]
  11. Caputo E, Manco G, Mandrich L, Guardiola J. A novel aspartyl proteinase from apocrine epithelia and breast tumors. J Biol Chem. 2000;275(11):7935–7941. doi: 10.1074/jbc.275.11.7935. [DOI] [PubMed] [Google Scholar]
  12. Carsol JL, Gingras S, Simard J. Synergistic action of prolactin (PRL) and androgen on PRL-inducible protein gene expression in human breast cancer cells: a unique model for functional cooperation between signal transducer and activator of transcription-5 and androgen receptor. Mol Endocrinol. 2002;16(7):1696–1710. doi: 10.1210/mend.16.7.0875. [DOI] [PubMed] [Google Scholar]
  13. Cassoni P, Sapino A, Haagensen DE, Naldoni C, Bussolati G. Mitogenic effect of the 15-kDa gross cystic disease fluid protein (GCDFP-15) on breast-cancer cell lines and on immortal mammary cells. Int J Cancer. 1995;60(2):216–220. doi: 10.1002/ijc.2910600215. [DOI] [PubMed] [Google Scholar]
  14. Chia KM, Liu J, Francis GD, Naderi A. A Feedback Loop between Androgen Receptor and ERK Signaling in Estrogen Receptor-Negative Breast Cancer. Neoplasia. 2011;13(2):154–166. doi: 10.1593/neo.101324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Chiu WW, Chamley LW. Human seminal plasma prolactin-inducible protein is an immunoglobulin G-binding protein. J Reprod Immunol. 2003;60(2):97–111. doi: 10.1016/s0165-0378(03)00084-6. [DOI] [PubMed] [Google Scholar]
  16. Chua CW, Chiu YT, Yuen HF, Chan KW, Man K, Wang X, Ling MT, Wong YC. Suppression of androgen-independent prostate cancer cell aggressiveness by FTY720: validating Runx2 as a potential antimetastatic drug screening platform. Clin Cancer Res. 2009;15(13):4322–4335. doi: 10.1158/1078-0432.CCR-08-3157. [DOI] [PubMed] [Google Scholar]
  17. Clark JW, Snell L, Shiu RP, Orr FW, Maitre N, Vary CP, Cole DJ, Watson PH. The potential role for prolactin-inducible protein (PIP) as a marker of human breast cancer micrometastasis. Br J Cancer. 1999;81(6):1002–1008. doi: 10.1038/sj.bjc.6690799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Doane AS, Danso M, Lal P, Donaton M, Zhang L, Hudis C, Gerald WL. An estrogen receptor-negative breast cancer subset characterized by a hormonally regulated transcriptional program and response to androgen. Oncogene. 2006;25(28):3994–4008. doi: 10.1038/sj.onc.1209415. [DOI] [PubMed] [Google Scholar]
  19. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. 1997;89(5):747–754. doi: 10.1016/s0092-8674(00)80257-3. [DOI] [PubMed] [Google Scholar]
  20. Ellison G, Klinowska T, Westwood RF, Docter E, French T, Fox JC. Further evidence to support the melanocytic origin of MDA-MB-435. Mol Pathol. 2002;55(5):294–299. doi: 10.1136/mp.55.5.294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gaubin M, Autiero M, Basmaciogullari S, Metivier D, Mis hal Z, Culerrier R, Oudin A, Guardiola J, Piatier-Tonneau D. Potent inhibition of CD4/TCR-mediated T cell apoptosis by a CD4-binding glycoprotein secreted from breast tumor and seminal vesicle cells. J Immunol. 1999;162(5):2631–2638. [PubMed] [Google Scholar]
  22. Gonzalez LO, Corte MD, Vazquez J, Junquera S, Sanchez R, Alvarez AC, Rodriguez JC, Lamelas ML, Vizoso FJ. Androgen receptor expresion in breast cancer: relationship with clinicopathological characteristics of the tumors, prognosis, and expression of metalloproteases and their inhibitors. BMC Cancer. 2008;8:149. doi: 10.1186/1471-2407-8-149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Haagensen DE, Jr, Dilley WG, Mazoujian G, Wells SA., Jr Review of GCDFP-15. An apocrine marker protein. Ann N Y Acad Sci. 1990;586:161–173. doi: 10.1111/j.1749-6632.1990.tb17804.x. [DOI] [PubMed] [Google Scholar]
  24. Haagensen DE, Jr, Gall SA, Brazy JE, Giannola J, Wells SA., Jr Analysis of amniotic fluid, maternal plasma, and cord blood for a human breast gross cystic disease fluid protein. Am J Obstet Gynecol. 1980;138(1):25–32. doi: 10.1016/0002-9378(80)90007-1. [DOI] [PubMed] [Google Scholar]
  25. Hall RE, Clements JA, Birrell SN, Tilley WD. Prostate-specific antigen and gross cystic disease fluid protein-15 are co-expressed in androgen receptor-positive breast tumours. Br J Cancer. 1998;78(3):360–365. doi: 10.1038/bjc.1998.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hassan MI, Waheed A, Yadav S, Singh TP, Ahmad F. Prolactin inducible protein in cancer, fertility and immunoregulation: structure, function and its clinical implications. Cell Mol Life Sci. 2009;66(3):447–459. doi: 10.1007/s00018-008-8463-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Javed A, Barnes GL, Pratap J, Antkowiak T, Gerstenfeld LC, van Wijnen AJ, Stein JL, Lian JB, Stein GS. Impaired intranuclear trafficking of Runx2 (AML3/CBFA1) transcription factors in breast cancer cells inhibits osteolysis in vivo. Proc Natl Acad Sci U S A. 2005;102(5):1454–1459. doi: 10.1073/pnas.0409121102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jia L, Berman BP, Jariwala U, Yan X, Cogan JP, Walters A, Chen T, Buchanan G, Frenkel B, Coetzee GA. Genomic androgen receptor-occupied regions with different functions, defined by histone acetylation, coregulators and transcriptional capacity. PLoS One. 2008;3(11):e3645. doi: 10.1371/journal.pone.0003645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kayed H, Jiang X, Keleg S, Jesnowski R, Giese T, Berger MR, Esposito I, Lohr M, Friess H, Kleeff J. Regulation and functional role of the Runt-related transcription factor-2 in pancreatic cancer. Br J Cancer. 2007;97(8):1106–1115. doi: 10.1038/sj.bjc.6603984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kilbey A, Blyth K, Wotton S, Terry A, Jenkins A, Bell M, Hanlon L, Cameron ER, Neil JC. Runx2 disruption promotes immortalization and confers resistance to oncogene-induced senescence in primary murine fibroblasts. Cancer Res. 2007;67(23):11263–11271. doi: 10.1158/0008-5472.CAN-07-3016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kim JH, Yang CK, Heo K, Roeder RG, An W, Stallcup MR. CCAR1, a key regulator of mediator complex recruitment to nuclear receptor transcription complexes. Mol Cell. 2008;31(4):510–519. doi: 10.1016/j.molcel.2008.08.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 1997;89(5):755–764. doi: 10.1016/s0092-8674(00)80258-5. [DOI] [PubMed] [Google Scholar]
  33. Migliaccio A, Castoria G, Di Domenico M, de Falco A, Bilancio A, Lombardi M, Barone MV, Ametrano D, Zannini MS, Abbondanza C, Auricchio F. Steroid-induced androgen receptor-oestradiol receptor beta-Src complex triggers prostate cancer cell proliferation. Embo J. 2000;19(20):5406–5417. doi: 10.1093/emboj/19.20.5406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Murphy LC, Tsuyuki D, Myal Y, Shiu RP. Isolation and sequencing of a cDNA clone for a prolactin-inducible protein (PIP). Regulation of PIP gene expression in the human breast cancer cell line, T-47D. J Biol Chem. 1987;262(31):15236–15241. [PubMed] [Google Scholar]
  35. Naderi A, Chia KM, Liu J. Synergy between inhibitors of androgen receptor and MEK has therapeutic implications in estrogen receptor-negative breast cancer. Breast Cancer Res. 2011;13(2):R36. doi: 10.1186/bcr2858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ning YM, Robins DM. AML3/CBFalpha1 is required for androgen-specific activation of the enhancer of the mouse sex-limited protein (Slp) gene. J Biol Chem. 1999;274(43):30624–30630. doi: 10.1074/jbc.274.43.30624. [DOI] [PubMed] [Google Scholar]
  37. Onodera Y, Miki Y, Suzuki T, Takagi K, Akahira J, Sakyu T, Watanabe M, Inoue S, Ishida T, Ohuchi N, Sasano H. Runx2 in human breast carcinoma: its potential roles in cancer progression. Cancer Sci. 2010;101(12):2670–2675. doi: 10.1111/j.1349-7006.2010.01742.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell. 1997;89(5):765–771. doi: 10.1016/s0092-8674(00)80259-7. [DOI] [PubMed] [Google Scholar]
  39. Phillips JE, Garcia AJ. Retroviral-mediated gene therapy for the differentiation of primary cells into a mineralizing osteoblastic phenotype. Methods Mol Biol. 2008;433:333–354. doi: 10.1007/978-1-59745-237-3_20. [DOI] [PubMed] [Google Scholar]
  40. Pratap J, Galindo M, Zaidi SK, Vradii D, Bhat BM, Robinson JA, Choi JY, Komori T, Stein JL, Lian JB, Stein GS, van Wijnen AJ. Cell growth regulatory role of Runx2 during proliferative expansion of preosteoblasts. Cancer Res. 2003;63(17):5357–5362. [PubMed] [Google Scholar]
  41. Pratap J, Javed A, Languino LR, van Wijnen AJ, Stein JL, Stein GS, Lian JB. The Runx2 osteogenic transcription factor regulates matrix metalloproteinase 9 in bone metastatic cancer cells and controls cell invasion. Mol Cell Biol. 2005;25(19):8581–8591. doi: 10.1128/MCB.25.19.8581-8591.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pratap J, Lian JB, Javed A, Barnes GL, van Wijnen AJ, Stein JL, Stein GS. Regulatory roles of Runx2 in metastatic tumor and cancer cell interactions with bone. Cancer Metastasis Rev. 2006;25(4):589–600. doi: 10.1007/s10555-006-9032-0. [DOI] [PubMed] [Google Scholar]
  43. Sandelin A, Alkema W, Engstrom P, Wasserman WW, Lenhard B. JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res. 2004;32(Database issue):D91–94. doi: 10.1093/nar/gkh012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Shin KJ, Wall EA, Zavzavadjian JR, Santat LA, Liu J, Hwang JI, Rebres R, Roach T, Seaman W, Simon MI, Fraser ID. A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. Proc Natl Acad Sci U S A. 2006;103(37):13759–13764. doi: 10.1073/pnas.0606179103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shiu RP, Iwasiow BM. Prolactin-inducible proteins in human breast cancer cells. J Biol Chem. 1985;260(20):11307–11313. [PubMed] [Google Scholar]
  46. Shore P. A role for Runx2 in normal mammary gland and breast cancer bone metastasis. J Cell Biochem. 2005;96(3):484–489. doi: 10.1002/jcb.20557. [DOI] [PubMed] [Google Scholar]
  47. Thomas DM, Johnson SA, Sims NA, Trivett MK, Slavin JL, Rubin BP, Waring P, McArthur GA, Walkley CR, Holloway AJ, Diyagama D, Grim JE, Clurman BE, Bowtell DD, Lee JS, Gutierrez GM, Piscopo DM, Carty SA, Hinds PW. Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma. J Cell Biol. 2004;167(5):925–934. doi: 10.1083/jcb.200409187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tian W, Osawa M, Horiuchi H, Tomita Y. Expression of the prolactin-inducible protein (PIP/GCDFP15) gene in benign epithelium and adenocarcinoma of the prostate. Cancer Sci. 2004;95(6):491–495. doi: 10.1111/j.1349-7006.2004.tb03238.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wick MR, Lillemoe TJ, Copland GT, Swanson PE, Manivel JC, Kiang DT. Gross cystic disease fluid protein-15 as a marker for breast cancer: immunohistochemical analysis of 690 human neoplasms and comparison with alpha-lactalbumin. Hum Pathol. 1989;20(3):281–287. doi: 10.1016/0046-8177(89)90137-8. [DOI] [PubMed] [Google Scholar]
  50. Zaidi SK, Pande S, Pratap J, Gaur T, Grigoriu S, Ali SA, Stein JL, Lian JB, van Wijnen AJ, Stein GS. Runx2 deficiency and defective subnuclear targeting bypass senescence to promote immortalization and tumorigenic potential. Proc Natl Acad Sci U S A. 2007;104(50):19861–19866. doi: 10.1073/pnas.0709650104. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp FigS1-S4

Figure S1, Microsoft PowerPoint, Runx2 stimulates PIP expression in LNCaP Prostate Cancer cells.

Figure S2, Microsoft PowerPoint, Expression of non-specific shRNA does not affect PIP and Runx2 expression.

Figure S3, Microsoft PowerPoint, Runx2 is required for Growth Hormone (GH)- and Dexamethasone (Dex)-induced PIP gene expression.

Figure S4, Microsoft PowerPoint, PIP knock-down compromises T47D cell proliferation.

NIHMS314584-supplement-Supp_FigS1-S4.pdf (319.7KB, pdf)
Supp Table S1
NIHMS314584-supplement-Supp_Table_S1.doc (38.5KB, doc)

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