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. 2012 Jul 20;14(4):R111.
doi: 10.1186/bcr3232.

Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

Ali Naderi, Michelle Meyer

Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

Ali Naderi et al. Breast Cancer Res. .

Abstract

Introduction: Molecular apocrine is a subtype of estrogen receptor (ER)-negative breast cancer that is characterized by a steroid-response gene signature. We have recently identified a positive feedback loop between androgen receptor (AR) and extracellular signal-regulated kinase (ERK) signaling in this subtype. In this study, we investigated the transcriptional regulation of molecular apocrine genes by the AR-ERK feedback loop.

Methods: The transcriptional effects of AR and ERK inhibition on molecular apocrine genes were assessed in cell lines. The most regulated gene in this process, prolactin-induced protein (PIP), was further studied using immunohistochemistry of breast tumors and xenograft models. The transcriptional regulation of PIP was assessed by luciferase reporter assay and chromatin immunoprecipitation. The functional significance of PIP in cell invasion and viability was assessed using siRNA knockdown experiments and the mechanism of PIP effect on integrin-β1 signaling was studied using immunoblotting and immunoprecipitation.

Results: We found that PIP is the most regulated molecular apocrine gene by the AR-ERK feedback loop and is overexpressed in ER-/AR+ breast tumors. In addition, PIP expression is regulated by AR-ERK signaling in xenograft models. These observations are explained by the fact that PIP is a target gene of the ERK-CREB1 pathway and is also induced by AR activation. Furthermore, we demonstrated that PIP has a significant functional role in maintaining cell invasion and viability of molecular apocrine cells because of a positive regulatory effect on the Integrin-ERK and Integrin-Akt signaling pathways. In fact, PIP-knockdown markedly decreases the phosphorylation of ERK, Akt, and CREB1. Importantly, PIP knockdown leads to a marked reduction of integrin-β1 binding to ILK1 and ErbB2 that can be reversed by the addition of fibronectin fragments.

Conclusions: We have identified a novel feedback loop between PIP and CREB1 mediated through the Integrin signaling pathway. In this process, PIP cleaves fibronectin to release fragments that activate integrin signaling, which in turn increases PIP expression through the ERK-CREB1 pathway. In addition, we demonstrated that PIP expression has a profound effect on cell invasion and the viability of molecular apocrine cells. Therefore, PIP signaling may be a potential therapeutic target in molecular apocrine breast cancer.

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Figures

Figure 1
Figure 1
The regulation of molecular apocrine genes by the AR-ERK feedback loop. (A) Heat map of top ranking molecular apocrine-signature genes following the inhibition of AR-ERK signaling using qPCR data. Heat map shows fold changes for gene expression relative to control in MDA-MB-453 and HCC-1954 cell lines. Treatments were carried out by CI-1040 (CI) at 2 µM and 10 µM concentrations, flutamide (FLU) at 25 nM and 40 nM concentrations, and the combination of flutamide at 25 nM or 40 nM and CI-1040 at 2 µM concentrations. Red and green colors depict up-regulation and down-regulation, respectively. Bar indicates the range of fold changes in gene expression. (B) Box plots to demonstrate relative expression of PIP to control following AR-ERK inhibition in MDA-MB-453 and HCC-1954 cell lines using qPCR. CTL: control. (C) Western blot analysis to assess PIP protein levels following AR-ERK inhibition in MDA-MB-453 and HCC-1954 cell lines. Fold changes (RR) in band densities were measured relative to the control (CTL). AR, androgen receptor; ERK, extracellular signal-regulated kinase; qPCR, quantitative PCR; RR, relative risk.
Figure 2
Figure 2
PIP protein expression in primary breast tumors and in vivo models. (A) Immunohistochemistry (IHC) staining for PIP in ER negative (ER-) breast tumors. AR+ group: ≥20% of cells showing positive AR staining; AR- group: <20% of cells stained for AR. Percentage of cells with positive staining are demonstrated for each group. *P <0.01 is for AR+ versus AR-. Error Bars: ± 2SEM. (B) IHC staining for PIP in AR+ and AR- breast tumors. Magnification is at 60X. (C) IHC staining for PIP in xenograft tumors generated using MDA-MB-453 cell line. Control: a control tumor; FLU: a flutamide-treated tumor; PD: a PD0325901-treated tumor. Magnification is at 60X. (D) IHC for PIP in xenograft tumors. Percentage of cells positive for PIP was assessed using IHC and compared between each treatment group and control (CTL). * P <0.01 is for FLU or PD treatment versus CTL. Error Bars: ± 2SEM. AR, androgen receptor; SEM, standard error of the mean.
Figure 3
Figure 3
Transcriptional regulation of PIP by AR and CREB1. (A) Luciferase reporter assay. The transcriptional activation of PIP promoter by PRLR, AR, CREB1, PRLR + AR, and PRLR + CREB1 expression constructs was assessed using Dual-Luciferase assays in MCF-7 cells and relative response ratios are reported. Co-transfection with the PIP reporter vector and an empty pcDNA vector was used as a control (CTL). *P <0.01, is compared to the control group. (B) Induction of PIP expression following DHT treatment. PIP expression was assessed using qPCR following DHT treatment at 30 minute, 1 hour, 3 hour, 12 hour, 24 hour, and 48 hour time-points in MDA-MB-453 and HCC-1954 cell lines. Fold changes are measured relative to the respective control at each time point. *P <0.03, is compared to the control group (dashed line). Error Bars: ± 2SEM. (C) Putative transcription factor binding sites for CREB1 in 1.5 kb promoter region of PIP. P1 (primer set 1) and P2 (primer set 2) are regions of amplification for ChIP assays. (D) ChIP assay with CREB1 antibody. The results of qPCR amplification for ChIP assays are demonstrated with two sets of primers for PIP promoter. Percentage recovery of input chromatin is shown for each primer set. *, P <0.01 is for CREB1 Ab. versus control Ab. Error Bars: ± 2SEM. (E) Western blot analysis to show CREB1 and PIP protein levels following CREB1-knockdown using siRNA in MDA-MB-453 cell line. Fold changes (RR) in band densities were measured relative to non-targeting siRNA control (CTL). Ab, antibody; AR, androgen receptor; ChIP, chromatin immunoprecipitation; DHT, dihydrotestosterone; qPCR, quantitative PCR; RR, relative risk; SEM, standard error of the mean.
Figure 4
Figure 4
The effect of PIP knockdown on cell invasion and viability. (A) qPCR to demonstrate PIP-knockdown efficiencies with siRNA-duplex1 (D1) and siRNA-duplex2 (D2) in MDA-MB-453 cell line. PIP expression following knockdown was assessed relative to non-targeting siRNA control (CTL) and fold change is shown for each duplex. (B) Western blot analysis to show PIP protein level following PIP-knockdown in MDA-MB-453 cell line as described in (A). Fold changes (RR) in band densities were measured relative to the control (CTL). (C) The effect of PIP expression on cell invasion. Cell invasion assays were carried out after PIP-knockdown with PIP-D1 and PIP-D2 in MDA-MB-453 cell line. Transfection with non-targeting siRNA control (CTL) was used as a control. *, P <0.03 is for each PIP-knockdown versus CTL. Error Bars: ± 2SEM. (D) MTT assay to measure cell viability following PIP-knockdown with PIP-D1 and PIP-D2 in MDA-MB-453 cell line. CTL: non-targeting siRNA control. *, P <0.03 is for each PIP-knockdown versus CTL. Error Bars: ± 2SEM. MTT, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide; qPCR, quantitative PCR; RR, relative risk; SEM, standard error of the mean.
Figure 5
Figure 5
The effect of PIP knockdown on ERK-Akt and integrin-β1signaling. (A) Western blot analysis to measure the levels of phosphorylated (Ph)-ERK, total (T)-ERK, ph-Akt, and T-Akt following PIP-knockdown with siRNA duplex1 (PIP-D1) and duplex2 (PIP-D2) in MDA-MB-453 cell line. Fold changes of Phospho/Total ratios (Ph/T-RR) were assessed relative to non-targeting siRNA control (CTL). (B) Western blot analysis to measure the level of ph-CREB1, T-CREB1, and ILK1 following PIP-knockdown as described in (A). Ph-ATF1 is the phosphorylated form of CREB-related protein that is known to be detected by this antibody. (C) Integrin-β1 immunoprecipitation (IP). IP assays were carried out with Integrin-β1 following PIP-knockdown with PIP-D1 and PIP-D2 in MDA-MB-453 cell line. Non-targeting siRNA was used as control (CTL). Western blot analysis was carried out on IP samples to measure the integrin-β1 binding to ILK1 and ErbB2. Immunoblotting with integrin-β1 antibody was used as a loading control. Fold changes (RR) of ILK1 and ErbB2 following PIP-knockdown were measured relative to that of control-siRNA. (D) Integrin-β1 immunoprecipitation following PIP-knockdown and the addition of fibronectin fragments (Fn-fs). PIP-knockdown with PIP-D1 was carried out as described in (C). Twenty-four hours after PIP-knockdown, cells were treated with α-chymotryptic fibronectin fragment 120K at 100 µg/ml concentration. Control cells were treated with vehicle only. Fold changes (RR) of ILK1 and ErbB2 following PIP-knockdown + Fn-fs were measured relative to the control. (E) The effect of fibronectin fragments on cell invasion following PIP-knockdown. Cell invasion assays were carried out after PIP-knockdown with PIP-D1 in MDA-MB-453 cell line. Transfection with non-targeting siRNA control (CTL) was used as a control. Treatment with fibronectin fragments was carried out as described in (D). Error Bars: ± 2SEM. Δ; is the difference between CTL and PIP-D1+Fn-fs groups. ERK, extracellular signal-regulated kinase; ILK1, integrin-linked kinase 1; RR, relative risk; SEM, standard error of the mean.
Figure 6
Figure 6
Schematic diagram of the PIP signaling pathway in ER-negative breast cancer. Red arrow denotes stimulatory effect. ER, estrogen receptor; Fn, fibronectin; Fn-f, Fibronectin fragment; ITG-β1, integrin-β1.
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