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. 2010 Jun;44(6):319-29.
doi: 10.1677/JME-09-0140. Epub 2010 Mar 17.

Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells

Feng Fang  1 , Jiamao Zheng, Traci L Galbaugh, Alyson A Fiorillo, Elizabeth E Hjort, Xianke Zeng, Charles V Clevenger
Affiliations

Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells

Feng Fang et al. J Mol Endocrinol. 2010 Jun.

Abstract

The effects of prolactin (PRL) during the pathogenesis of breast cancer are mediated in part though Stat5 activity enhanced by its interaction with its transcriptional inducer, the prolyl isomerase cyclophilin B (CypB). We have demonstrated that knockdown of CypB decreases cell growth, proliferation, and migration, and CypB expression is associated with malignant progression of breast cancer. In this study, we examined the effect of CypB knockdown on PRL signaling in breast cancer cells. CypB knockdown with two independent siRNAs was shown to impair PRL-induced reporter expression in breast cancer cell line. cDNA microarray analysis was performed on these cells to assess the effect of CypB reduction, and revealed a significant decrease in PRL-induced endogenous gene expression in two breast cancer cell lines. Parallel functional assays revealed corresponding alterations of both anchorage-independent cell growth and cell motility of breast cancer cells. Our results demonstrate that CypB expression levels significantly modulate PRL-induced function in breast cancer cells ultimately resulting in enhanced levels of PRL-responsive gene expression, cell growth, and migration. Given the increasingly appreciated role of PRL in the pathogenesis of breast cancer, the actions of CypB detailed here are of biological significance.

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Conflict of interest statement

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Figures

Figure 1
Figure 1
Microarray analysis demonstrates the effects of PRL on global gene expression. (A) Volcano map demonstrates the relationship between the observed fold change in gene expression and the P value significance of such changes in PRL-treated cells. The dotted lines represent the P value and fold change cutoffs. The red dots represent the selected genes filtered by criteria (fold change ≥1.3 up or down, P<0.05, FDR<0.05). (B) Heat map analysis reveals a global view of genes up- and down-regulated in PRL-treated cells. (C and D) Real-time PCR validates microarray results for BCL3 (C) and BCL6 (D). Statistical analysis was performed using Student's t-test. (E) The biological functional categories were obtained from the molecular and cellular function in the IPA database. (F) The top interaction networks generated using IPA analysis included genes associated with ‘cancer, hematological disease, and cell cycle’. The color indicates up-regulation (red), down-regulation (green), and complexes (gray) of genes. ***P<0.001.
Figure 2
Figure 2
The effects of CypB overexpression and CypB knockdown on a Stat5-responsive reporter. (A) Luciferase assay using pGL4-CISH reporter. Cells were transfected with pGL4-CISH reporter, the renilla luciferase control (pGL4.73), and pcDNA3.1-CypB expression vector. Transfectants were cultured in the minimal defined medium for 24 h, followed by 24 h of PRL stimulation prior to luminescence assay. (B and C) CypB knockdown in T47D cells confirmed by real-time PCR and microarray (B), and transient transfection (C). (D and E) Luciferase assay using pGL4-CISH (D) and pGL4-LHRE (E). T47D parental cells (wt) or si-CypB cells were co-transfected with 100 ng pGL4-CISH (D) or pGL4-LHRE (E), along with 2 ng renilla luciferase control (pGL4.73) and 400 ng pcDNA3.1-CypB expression vector, and maintained in the FBS-containing growth medium overnight. Transfectants were then starved in the FBS-free minimal defined medium for 24 h, followed by 24 h of PRL (10 ng/ml for pGL4-CISH and 100 ng/ml for pGL4-LHRE) stimulation prior to luminescence assay. Statistical analysis was performed using two-way ANOVA. **P<0.01; ***P<0.001.
Figure 3
Figure 3
The characterization of PRL-related genes affected by CypB knockdown. (A) Heat map showed down-regulation of PRLR, S100A6, and PIP in si-CypB cells compared to si-Luc control cells. (B and C) Real-time PCR (B) and western blot (C) confirmed the PRLR down-regulation in si-CypB cells. Statistical analysis was performed using Student's t-test. **P<0.01.
Figure 4
Figure 4
Real-time PCR validated the impairment of PRL-induced gene expression by CypB knockdown in T47D si-CypB stable cells (A–F) and in MCF7 cells (G–H) with transient transfect of si-CypB-T. The y-axis label ‘fold change’ is defined in the Materials and methods section. Statistical analysis was performed using Student's t-test. *P<0.05; **P<0.01.
Figure 5
Figure 5
CypB knockdown results in the decreased PRL-induced soft agar growth and cell motility. Cells were grown on soft agar for 2 weeks, and the pictures were taken under phase contrast microscopy. (A) si-Luc without PRL treatment, (B) si-Luc with PRL treatment (200 ng/ml), (C) si-CypB without PRL treatment, (D) si-CypB with PRL treatment (200 ng/ml), (E) colony number on the soft agar, and (F) the total colony area on the soft agar. **P<0.01; ***P<0.001.
Figure 6
Figure 6
CypB knockdown results in the decreased PRL-induced cell motility. Cell motility was assayed using Boyden chamber migration assay. The inserts were coated with collagen I overnight. T47D cells were arrested in the FBS-free medium and placed in the inserts. Cells were cultured for 20 h, and the migrated cells were counted under a microscope. Statistical analysis was performed using two-way ANOVA. ***P<0.001.
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