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Signaling and Regulation

PAX5α Enhances the Epithelial Behavior of Human Mammary Carcinoma Cells

Laurent J-P Vidal, Jo K. Perry, Cecile M. Vouyovitch, Vijay Pandey, Severine E. Brunet-Dunand, Hichem C. Mertani, Dong-Xu Liu and Peter E. Lobie
DOI: 10.1158/1541-7786.MCR-09-0368 Published March 2010

Abstract

Deregulated PAX5 expression has been associated with metastatic mammary carcinoma, although the precise role of PAX5 in cancer progression is unclear. Stable forced expression of PAX5α in the mammary carcinoma cell lines MCF-7 and MDA-MB-231 reduced cell cycle progression, cell survival, and anchorage-independent cell growth. In xenograft studies, forced expression of PAX5α was associated with a significant reduction in tumor volume. Furthermore, forced expression of PAX5α in mammary carcinoma cells resulted in altered cell morphology with resultant enhancement of epithelial cell characteristics. Morphologic changes were associated with localization of β-CATENIN at cell-cell junctions and with altered mRNA expression of mesenchymal markers in mammary carcinoma cells. In addition, forced expression of PAX5α in MCF-7 and MDA-MB-231 cells significantly reduced cell migration and invasion. Concomitantly, small interfering RNA–mediated depletion of PAX5α increased MCF-7 total cell number, cell motility, migration, and invasion. These studies show that PAX5α enhances the epithelial characteristics of mammary carcinoma cells, reminiscent of mesenchymal to epithelial transition. Mol Cancer Res; 8(3); 444–56

Keywords
  • PAX5
  • mammary carcinoma
  • mesenchymal-epithelial transition

This article is featured in Highlights of This Issue, p. 293

Introduction

It is widely accepted that tumorigenesis is an aberrant form of organogenesis and that both normal developmental and neoplastic processes progress through common pathways (1). This is illustrated by the large number of developmentally regulated genes and signal transduction pathways with demonstrated roles in neoplastic progression. The paired box (PAX) gene family are one such example, with family members implicated in both developmental and neoplastic processes (2). The PAX family of genes belongs to the homeobox gene superfamily and is composed of nine developmentally regulated genes encoding transcription factors that have key roles in organ development and tissue differentiation during embryogenesis. PAX transcription factors share a highly conserved 128-amino-acid DNA binding domain, known as the paired box domain, that recognizes specific DNA sequences (3, 4). Among these nine PAX genes, PAX2, PAX5, and PAX8 form a subgroup of genes that contain the paired box domain as well as a partial homeodomain (5).

PAX5 influences B-cell differentiation and development by promoting lineage commitment while also inhibiting later differentiation events. Consequently, PAX5 upregulates expression of B-cell lineage commitment genes while also inhibiting genes involved in B-cell terminal differentiation during the early stages of development (6). PAX5 is expressed at all stages of B-cell development, from the earliest B-lineage–committed precursor cell up to the mature B-cell stage. However, PAX5 expression is absent in terminally differentiated plasma cells (7). PAX5 mRNA expression has also been reported in the developing central nervous system and adult mouse testis (8) as well as in human adult brain tissue and some tissues of the male and female genital tract (9).

The human PAX5 gene is located on chromosome 9p13 and encodes a 50-kDa transcription factor. In addition to the paired box domain, PAX5 contains a partial one-helixed homeodomain and an 8-amino-acid octapeptide motif that functions as a transcriptional inhibitory motif (4). A number of alternatively spliced mRNA transcript variants have been described for both human and mouse PAX5, which result in both NH2-terminal and COOH-terminal protein variations (10, 11). Two developmentally regulated alternative PAX5 splice variants containing distinct 5′-exons (exon 1A and 1B), which encode two isoforms of PAX5 (PAX5α and PAX5β), were originally described by Busslinger et al. (11). Another study describes three additional murine alternative transcripts/isoforms: Pax5a, 5b, 5d and 5e. Pax5a is the full length Pax5 isoform; Pax5b and Pax5e have a partial deletion of exon 2, whereas isoforms Pax5d and Pax5e also differ from Pax5a in their COOH-terminal region (10). Human PAX5β, also known as B-cell lineage–specific activator protein (BSAP; ref. 12), is essential for B-cell lineage commitment and is required for progression of B-cell development beyond the early pro-B (pre-BI) cell stage (13). However, little is known about PAX5α.

Whereas increased PAX5 expression has been reported for a variety of malignancies (4, 11, 14-16), the precise role of PAX5 in cancer remains unclear, and conflicting evidence exists suggestive of both tumor-promoting and tumor suppressor functions (14, 17). Furthermore, functional investigations rarely distinguish between PAX5α and PAX5β isoforms. In a recent study, PAX5 was identified as one of the 51-gene expression signature that defines metastatic breast cancer with a >100-fold increase in PAX5 mRNA expression observed in tumors metastasized to the lymph nodes. Here, we show that only PAX5α mRNA is expressed in the human mammary carcinoma cell line MCF-7. We also report here that forced expression of PAX5α in mammary carcinoma cells decreases cell proliferation, survival, anchorage independence, and tumor formation in a xenograft model, consistent with a potential tumor suppressor role. In addition, forced expression of PAX5α in mammary carcinoma cells promoted an epithelial cell morphology and reduced cell migration and invasion. Thus, PAX5α enhances epithelial characteristics in human mammary carcinoma cells.

Materials and Methods

Plasmid Constructs

The coding sequence for human PAX5 transcript variant α, which uses exon 1A (GenBank accession no. NM_016734), was cloned into the mammalian expression vector pIRESneo3 (Invitrogen), designated pIRESneo3-PAX5α. For PAX5 small interfering RNA (siRNA)–mediated depletion, two oligonucleotides (DNA sequence A: 5′-AATCGCTGAATATAAACGCCA-3′ or DNA sequence B: 5′-AACCAGTCCCAGCTTCCAGTC-3′), specific to PAX5 both transcripts α and β, were cloned into the siRNA expression vector pSilencer2.1-U6hygro (Ambion) according to the manufacturer's instructions. The resultant vectors were designated pSilencer-SiA-P5 and pSilencer-SiB-P5. The negative control siRNA plasmid pSilencer-CK contains a siRNA that has no significant sequence similarity to any human gene sequence (Ambion).

Cell Lines and Cell Transfection

Human mammary carcinoma cell lines MCF-7 and MDA-MB-231 were obtained from the American Type Culture Collection and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L l-glutamine at 37°C in a humidified 5% CO2 incubator. For PAX5α expression, cells were transiently or stably transfected with pIRESneo3-PAX5α or an empty pIRESneo3 vector as a control using Saint Mix (Synvolux Therapeutics B.V.) according to the manufacturer's instructions. For PAX5 depletion, cells were stably transfected with pSilencer-SiA-P5, pSilencer-SiB-P5, or pSilencer-scrambled as a control. These cell lines were designated MCF7-PAX5α-SiA, MCF7-PAX5α-SiB, and MCF7-pSi, respectively. Pooled stable transfectants were selected in medium containing 800 μg/mL G418 (for pIRESneo3) or 100 μg/mL hygromycin B (for pSilencer2.1-U6hygro) for 21 d.

Preparation of Total RNA and Reverse Transcription-PCR

Total RNA was isolated from cells using TRIzol (Invitrogen). Semiquantitative reverse transcription-PCR (RT-PCR) was done using a OneStep RT-PCR kit (Qiagen). Primers used for RT-PCR are listed in Supplementary Table S2. Amplified PCR products were visualized on a 1.5% agarose gel.

Luciferase Reporter Assay for p53 Promoter

MCF7-PAX5 or MCF7-VEC cells were cultured to 50% to 70% confluence in six-well plates. Transient transfection was performed using Saint Mix. The p53 reporter plasmid pGL3-p53pro (18) and a β-galactosidase expression vector were transfected (0.2 μg each per well) in serum-free RPMI medium. After 24 h, cells were washed with PBS and luciferase assays were performed using a Steady-Glo luciferase assay system (Promega) according to the manufacturer's instructions. Results were normalized to the level of β-galactosidase activity and protein concentration in the samples.

Western Blot Analysis

Protein extraction and Western blot analysis were performed as described (19) using the following antibodies: goat anti-human PAX5 (1:200; Santa Cruz Biotechnology), which does not distinguish between PAX5α and PAX5β isoforms, and rabbit anti-goat antibody conjugated with peroxidase (1:2,000; DAKO). Blots were stripped and reprobed with mouse anti–β-actin (Santa Cruz Biotechnology).

Immunofluorescence

Following fixation with 4% paraformaldehyde in PBS, cells were incubated with mouse anti–β-catenin (1:200; Santa Cruz Biotechnology). Secondary antibody (1:500) was sheep anti-mouse rhodamine (Jackson ImmunoReserch). Cells were examined with an inverted Olympus IX71 microscope containing a U-RFL-T fluorescence light source and a DP70 digital camera.

Immunohistochemistry

Immunohistochemistry was carried out as previously described (20, 21) on seven cases of mammary ductal carcinoma in situ, eight cases of invasive ductal carcinoma, and six normal breast samples. Briefly, 5-μm-thick paraffin sections of tumors were mounted on slides, deparaffinized, and rehydrated. The slides were treated with 3% H2O2 for 30 min and incubated in PBS-10% bovine serum albumin before incubation with the primary antibody. The sections were washed with PBS and incubated with the secondary antibody conjugated with peroxidase (1:2,000; DAKO). The slides were washed with PBS, and a diaminobenzidine-based peroxidase substrate (ImPact DAB, Vector Laboratories) was added for detection. Antibodies used in the xenograft studies were VIMENTIN (Vim 3B4, LabVision/Neomarkers; 1:100) or β-CATENIN (Santa Cruz Biotechnology; 1:300) with the secondary antibodies biotinylated antirabbit IgG (BA-1000) and antimouse IgG (BA-9200; 1:300), respectively.

Total Cell Number

Cells were seeded in six-well plates at a density of 50,000 per well and cultured in 10% FBS–supplemented medium. Cells were counted 24 h after seeding and every 2nd day for 6 d.

In vitro Cell Migration and Invasion Assays

Estimation of cell migration or invasion was performed using Transwell invasion chambers (Corning) with porous filters (8-μm pores for MCF-7 cells; 12-μm pores for MDA-MB-231 cells), as described (22). For cell invasion assays, chambers were coated with BD Matrigel diluted in serum-free medium (1:30 for MCF-7; 1:10 for MDA-MB-231). Cells (1 × 105/mL) were plated in the upper chamber. Following 36-h incubation for MCF-7 cells and 24-h incubation for MDA-MB-231 cells, filters were fixed with paraformaldehyde and stained, and migrated/invaded cells were counted. For the wound healing migration assay, confluent monolayers of cells were scraped with pipette tips, washed with PBS, and incubated in culture medium supplemented with 5% FBS for 75 h.

5′-Bromo-2′-Deoxyuridine Incorporation Assay and Measurement of Apoptosis

Mitogenesis was directly assayed by measuring the incorporation of 5′-bromo-2′-deoxyuridine (BrdUrd; ref. 23). Apoptotic cell death was measured by fluorescent microscopic analysis of cell DNA staining patterns with Hoechst 33258 as previously described (23).

Soft-Agar Colony Formation Assay

Anchorage-independent growth assays were done as previously described (24). Cell lines (1 × 105 cells per well) were cultured in six-well plates with a 0.35% agar layer. The plates were incubated for 21 d, after which the cultures were inspected and photographed.

Xenograft Analysis

MCF7-PAX5 or MCF7-VEC cells (5 × 106) were suspended in 100 μL of Matrigel and injected into the first mammary fat pad of five 3- to 4-wk-old female athymic mice (Harlan Laboratory, Italy), which simultaneously received a 60-d-release pellet containing 0.72 mg of 17β-estradiol (Innovative Research of America), as described (25). The tumor volumes were measured every 3 to 4 d and tumors were harvested 6 wk after inoculation. All experiments were carried out under a protocol approved by the Université Claude Bernard administrative committee on laboratory animal care.

Statistical Analysis

All data are expressed as mean ± SD unless specified otherwise. Data were analyzed using unpaired two-tailed t test. All experiments are averages of at least triplicate determinants and were repeated at least three times. A single representative figure is shown.

Results

Relevance of PAX5 Expression to Human Mammary Carcinoma

To determine the clinical relevance of PAX5 expression in mammary neoplasia, we analyzed the gene expression database Oncomine, premium research edition, version 4.2,5 for the expression profile of PAX5 in human mammary carcinoma (Supplementary Table S1). Although PAX5 expression did not differ significantly between normal tissue and breast cancer, a positive correlation was observed between increased PAX5 mRNA expression and breast carcinoma grade in three studies (P ≤ 0.049; Supplementary Table S1; refs. 26, 27), whereas an inverse correlation was observed between increased PAX5 mRNA expression and breast carcinoma grade in two studies (28, 29). Furthermore, increased PAX5 expression was positively correlated with metastasis at both 3 and 5 years in three studies (P ≤ 0.045; refs. 28, 30, 31; Supplementary Table S1). However, decreased PAX5 expression was associated with increased survival after 5 years in one study (P ≤ 0.039; ref. 32). Two studies observed increased PAX5 expression in estrogen receptor–positive compared with estrogen receptor–negative mammary carcinomas (P ≤ 0.039; refs. 33, 34), whereas two additional studies observed decreased PAX5 expression in estrogen receptor–positive compared with estrogen receptor–negative mammary carcinomas (refs. 26, 35; Supplementary Table S1). No distinction between the α and β isoforms of PAX5 was made in these studies.

Forced Expression and siRNA-Mediated Depletion of PAX5α in Mammary Carcinoma Cells

The PAX5 gene gives rise to two alternative transcripts (PAX5α and PAX5β), which are transcribed from two distinct promoters, resulting in the use of two alternative first exons (12). Semiquantitative RT-PCR with transcript-specific primers (12) showed that only PAX5α mRNA was expressed in MCF-7 cells (Fig. 1A) consistent with a previous report showing methylation of the PAX5β gene promoter associated with transcriptional silencing in MCF-7 cells (12).

FIGURE 1.
FIGURE 1.

PAX5 expression in human mammary carcinoma cells. A, PAX5α and PAX5β transcript–specific primers were used to identify the presence of PAX5α mRNA and the absence of PAX5β mRNA in MCF-7 cells under serum-free conditions by semiquantitative RT-PCR. Confirmation of forced expression of PAX5α in MCF7-PAX5α cells (PAX5α) or siRNA-mediated depletion of PAX5α (PAX5α-SiA and PAX5α-SiB) in stably transfected MCF-7 cell lines by RT-PCR (B) and Western blot analysis (C) when compared with the control cell lines MCF7-VEC (VEC) and MCF7-pSi (pSi). D, ratio of Western blot band densities. Columns, mean; bars, SD.

To define the potential role of PAX5α in mammary neoplastic progression, a cell model system was established in the mammary carcinoma cell line MCF-7. MCF-7 cells were stably transfected with the PAX5α expression vector pIRESneo3-PAX5α (designated MCF7-PAX5α) or an empty vector as a control (designated MCF7-VEC). In addition, the effect of decreased PAX5α expression was determined through siRNA-mediated depletion of PAX5α. Two stable MCF-7 cell lines (designated MCF7-PAX5α-SiA and MCF7-PAX5α-SiB) were established by stable transfection of MCF-7 cells with two different PAX5-specific siRNA plasmids, pSilencer-SiA-P5 and pSilencer-SiB-P5, respectively. A control cell line was generated by stable transfection of the negative control pSilencer plasmid (designated MCF7-pSi). Pooled stable transfectants were used to minimize any effect of potential clonal selection.

Forced expression of PAX5α in MCF7-PAX5α and depletion of endogenous PAX5α in MCF7-PAX5α-SiA and MCF7-PAX5α-SiB cell lines were confirmed by RT-PCR (Fig. 1B) and Western blot analysis (Fig. 1C and D). Increased PAX5α activity in MCF7-PAX5α or decreased activity in MCF7-PAX5α-SiA and MCF7-PAX5α-SiB cells was also confirmed by luciferase assay using a CD19 promoter-reporter construct (data not shown), a known target gene of PAX5 (12, 36).

Stable cell lines were similarly established in a second mammary carcinoma cell line, MDA-MB-231. These cell lines were designated MB231-PAX5α and MB231-VEC, respectively. Forced expression of PAX5α in MB231-PAX5α cell lines was confirmed by RT-PCR and Western blot analysis (data not shown). Semiquantitative RT-PCR also showed that MDA-MB-231 cells do not express detectable endogenous levels of PAX5α or PAX5β mRNA (data not shown), which is consistent with a previous report showing methylation of both PAX5α and PAX5β gene promoters associated with transcriptional silencing in MDA-MB-231 cells (12).

Forced Expression of PAX5α in Mammary Carcinoma Cells Reduces Total Cell Number and Cell Cycle Progression and Promotes Apoptotic Cell Death

One hallmark of cancer is the concomitant deregulation of cell proliferation and apoptotic cell death (37). We therefore investigated the effect of forced expression of PAX5α on total mammary carcinoma cell number, cell proliferation, and apoptotic cell death. Forced expression of PAX5α in MCF7 and MDA-MB-231 cells significantly decreased total cell number when compared with the control cell lines MCF7-VEC and MDA-MB-VEC over a period of 6 days in serum-replete conditions (Fig. 2A and B). Concordantly, siRNA-mediated depletion of PAX5α increased total cell number in MCF7-PAX5α-SiA and MCF7-PAX5α-SiB cell lines compared with the control cell line MCF7-pSi over a period of 6 days under serum-replete conditions (Fig. 2C).

FIGURE 2.
FIGURE 2.

PAX5α decreases total cell number, decreases cell cycle progression, and promotes apoptotic cell death in mammary carcinoma cells. A to C, total cell number conducted in 10% FBS medium with MCF7-VEC and MCF7-PAX5α cell lines (A), MDA-MB231-VEC and MDA-MB231-PAX5α cell lines (B), or MCF7-Si, MCF7-PAX5α-SiA, and MCF7-PAX5α-SiB cell lines (C). BrdUrd nuclear incorporation assay (D and E) and apoptotic cell death as determined by nuclear Hoechst staining (F and G) performed in stably transfected MCF-7 and MDA-MB-231 cells. Semiquantitative RT-PCR analysis of selected genes involved in cell proliferation and apoptotic cell death (H and I) and p53 promoter-luciferase assay of PAX5α (J) performed in stably transfected MCF-7 cells (PAX5α) compared with MCF7-VEC control cells (VEC). Columns, mean; bars, SD. *, P ≤ 0.05; **, P ≤ 0.005.

We next determined entry to S phase in cells using a nuclear BrdUrd incorporation assay. Forced expression of PAX5α decreased entry into S phase by 25% in MCF-7 cells (Fig. 2D) and 70% in MDA-MB-231 cells (Fig. 2E) in serum-free conditions compared with the control cell lines MCF7-VEC and MDA-MB-VEC. In addition, Hoechst staining showed that forced expression of PAX5α increased apoptotic cell death in MCF-7 by 40% (Fig. 2F) and in MDA-MB-231 by 84% (Fig. 2G) in serum-free conditions when compared with the control cell lines. Consistent with the observed antiproliferative effect of forced expression of PAX5α in MCF-7 cells, semiquantitative RT-PCR analysis showed that forced expression of PAX5α decreased mRNA levels of the cell cycle progression gene CYCLIND1 (Fig. 2H). In addition, forced expression of PAX5α increased mRNA levels of the tumor suppressor gene p53 and reduced mRNA levels of the prosurvival genes BCL-xL and BCL-2 (Fig. 2H and I). Thus, PAX5α decreases total cell number in mammary carcinoma cells as a consequence of decreased cell cycle progression and increased apoptotic cell death.

In contrast to the observed increased level of p53 mRNA in MCF7-PAX5α cells (Fig. 2H), a previous study has described the transcriptional downregulation of p53 by PAX5 (38). To verify our findings, we performed a p53 promoter-luciferase assay in which MCF7-VEC and MCF7-PAX5α cells were transiently transfected with a p53 promoter-luciferase construct. Forced expression of PAX5α in MCF-7 cells transcriptionally upregulated p53 expression (Fig. 2J), thus confirming our observation that PAX5α increases p53 mRNA levels.

Forced Expression of PAX5α in Mammary Carcinoma Cells Represses Anchorage-Independent Growth

Resistance to anoikis, or anchorage-independent growth, is one characteristic of oncogenically transformed cells. In a soft-agar colony formation assay, forced expression of PAX5α in MCF7-PAX5α cells reduced anchorage-independent growth by 80% when compared with the control cell line MCF7-VEC (Fig. 3A). Colony formation was also determined in the mammary carcinoma cell line, MDA-MB-231. Forced expression of PAX5α in MDA-MB231-PAX5α cells resulted in a small but significant decrease in colony formation when compared with MDA-MB231-VEC cells (Fig. 3B). In addition, forced expression of PAX5α in MDA-MB-231 cells decreased the average size of colonies formed in soft agar by 25% compared with colonies formed by cells transfected with a control plasmid (Fig. 3C).

FIGURE 3.
FIGURE 3.

Forced expression of PAX5α in mammary carcinoma cells represses anchorage-independent growth and reduces tumor formation in vivo. A, forced expression of PAX5α abrogates the ability of MCF-7 cells to form colonies in soft agar. B and C, forced expression of PAX5α reduces the ability of MDA-MB-231 cells to form colonies in soft agar (B) and reduces the size of colonies formed (C), as shown on the representative light microscopy image (×20). Columns, mean; bars, SD. D, PAX5α forced expression in MCF-7 cells decreases the growth of mammary tumors in a xenograft model of human mammary carcinoma. Points, mean; bars, SD. *, P ≤ 0.05; **, P ≤ 0.005.

Forced Expression of PAX5α Reduces Tumor Growth In vivo

Given that forced expression of PAX5α decreased cell proliferation, survival, and anchorage-independent growth, we investigated the effect of the forced expression of PAX5α on xenograft growth in immunodeficient mice. Two groups of mice (n = 12 per group) were injected with 5 × 106 MCF7-PAX5α or MCF7-VEC cells into the first mammary fat pad. Mice were monitored every 3 to 4 days for tumor formation over a 6-week period. Palpable tumors developed in all mice by day 15 post-injection. The rate of tumor formation by MCF7-PAX5α or MCF7-VEC cells did not significantly differ until day 29 (Fig. 3D). However, from day 29 to 40, MCF7-VEC tumors grew significantly faster than MCF7-PAX5α tumors (Fig. 3D). The retarded growth of MCF7-PAX5α tumors continued until day 40 when the mice were sacrificed. Maintenance of PAX5α expression in carcinoma cells was confirmed by immunohistochemistry on histologic sections derived from the tumors (data not shown). Together, these results show that forced expression of PAX5α significantly reduces mammary tumor growth in vivo.

Forced Expression of PAX5α Restores Epithelial Phenotype in Mammary Carcinoma Cells

During neoplastic progression, cells with epithelial characteristics may acquire a mesenchymal phenotype in a process termed epithelial-mesenchymal transition (EMT; refs. 39, 40). We examined MCF7-VEC and PAX5α cells to determine whether forced expression of PAX5α affected MCF-7 cell morphology. MCF7-VEC cell morphology was similar to that of the parental MCF-7 cell line: a predominantly cobblestone appearance with few scattered cells and few cytoplasmic extensions (Fig. 4A). Forced expression of PAX5α increased the epithelial characteristics of MCF7-PAX5α cells: cells were cuboidal and grew in strictly structured groups (Fig. 4A). Immunofluorescent cell labeling of β-CATENIN showed a diffuse distribution of this protein in MCF7-VEC cells with limited localization at cell-cell junctions in both serum-free and serum-replete conditions (Fig. 4B). In contrast, β-CATENIN was almost exclusively localized at cell-cell junctions in MCF7-PAX5α cells (Fig. 4B). Similar localization of β-CATENIN was also observed in MDA-MB231-PAX5α, in contrast to MDA-MB231-VEC cells (Fig. 4C). Semiquantitative RT-PCR showed that forced expression of PAX5α in MCF-7 cells increased mRNA levels of the mammary differentiation marker β-CASEIN (Fig. 4D), whereas there was no change in mRNA levels of the epithelial cell markers E-CADHERIN, α-CATENIN, and OCCLUDIN (Fig. 4D). However, forced expression of PAX5 reduced mRNA levels of the mesenchymal markers VIMENTIN and FIBRONECTIN and the matrix metalloproteinase MMP-2 in MCF-7 cells (Fig. 4D). Similarly, forced expression of PAX5α in MDA-MB-231 cells increased mRNA levels of β-CASEIN, α-CATENIN, and β-CATENIN and decreased mRNA levels of FIBRONECTIN and MMP-2 (Fig. 4E).

FIGURE 4.
FIGURE 4.

Forced expression of PAX5α promotes an epithelial phenotype in mammary carcinoma cells. A, the morphology of MCF7-VEC and MCF7-PAX5α cells cultured on plastic was examined by phase-contrast microscopy under ×400 magnification. MCF7-PAX5α cells were more cuboidal in appearance and grew in a homogenous structured monolayer. B, immunofluorescent localization of β-CATENIN in MCF7-VEC and MCF7-PAX5α cells under serum-free and 10% FBS conditions. Bar, 20 μm. C, immunofluorescent localization of β-CATENIN in MDA-MB-VEC and MDA-MB-PAX5α cells under serum-free and 10% FBS conditions. Bar, 20 μm. D and E, semiquantitative RT-PCR analysis of epithelial markers (E-CADHERIN, α-CATENIN, and OCCLUDIN), cell adhesion molecule (β-CATENIN), mesenchymal markers (VIMENTIN and FIBRONECTIN), matrix metalloproteinase (MMP-2), and mammary differentiation marker (β-CASEIN) in MCF7-VEC (VEC) and MCF7-PAX5α (PAX5α) cells (D) and in MDA-MB-VEC (VEC) and MDA-MB-PAX5α (PAX5α) cells (E). F, immunohistochemical localization of VIMENTIN and β-CATENIN proteins in MCF7-VEC and MCF7-PAX5α tumor sections.

We also determined the protein localization of β-CATENIN and the mesenchymal marker VIMENTIN in MCF7-VEC– and MCF7-PAX5α–derived tumors by immunohistochemistry (Fig. 4F). Increased β-CATENIN immunoreactivity was observed in sections taken from MCF7-PAX5α tumors when compared with MCF7-VEC tumors, with increased localization observed at the intercellular junctions, consistent with the earlier in vitro immunofluorescence studies (Fig. 4F). In addition, we observed decreased VIMENTIN immunoreactivity in sections taken from MCF7-PAX5α tumors when compared with MCF7-VEC tumors (Fig. 4F). Combined, these studies showed that forced expression of PAX5α promotes a more differentiated epithelial cell morphology in MCF-7 and MDA-MB-231 cells.

Forced Expression of PAX5α in Mammary Carcinoma Cells Represses Cell Motility, Migration, and Invasion

The observed promotion of epithelial cell characteristics in human mammary carcinoma cells expressing PAX5α was suggestive of a decreased potential for migration and invasion. Because MCF-7 cells are only weakly motile and invasive, we did not observe significant effects of forced PAX5α expression on MCF-7 cell migration and invasion (data not shown). However, siRNA-mediated depletion of endogenous PAX5α in MCF7-PAX5α-SiB and MCF7-PAX5α-SiA cells resulted in a marked increase in cell migration compared with the control cell line MCF7-pSi in wound healing assay (Fig. 5A). In addition, siRNA-mediated depletion of PAX5α in MCF7-PAX5α-SiB and MCF7-PAX5α-SiA cells promoted MCF-7 cell migration and invasion in Transwell chamber assays when compared with MCF7-pSi cells (Fig. 5B and C).

FIGURE 5.
FIGURE 5.

Depletion of PAX5α expression in MCF-7 cells stimulates cell motility, migration, and invasion. A, wound healing assay. The wounded areas were examined under ×20 magnification after 75 h. Migration assay (B) and invasion assay (C) following siRNA-mediated depletion of endogenous PAX5α in MCF7-PAX5α-SiA (PAX5α-SiA) and MCF7-PAX5α-SiB (PAX5α-SiB) cell lines when compared with the control cell line MCF7-pSi (pSi; *, P ≤ 0.05). Results are presented as the ratio of migrated or invaded cells compared with the control (control cell line = 1). Columns, mean; bars, SD.

We also investigated the effect of forced expression of PAX5α in the highly motile and invasive mammary carcinoma cell line MDA-MB-231. MDA-MB-231 cells were transiently transfected with the PAX5α expression plasmid (pIRESneo3-PAX5α), and PAX5α mRNA expression was confirmed by RT-PCR (Fig. 6A). Wound healing assays showed that transient expression of PAX5α decreased MDA-MB-231 cell migration when compared with cells transfected with a control plasmid (Fig. 6B). In Transwell chamber assays, PAX5α transient expression also dramatically reduced MDA-MB-231 cell migration by 65% (Fig. 6C) and cell invasion by 75% (Fig. 6D) when compared with controls.

FIGURE 6.
FIGURE 6.

Transient forced expression of PAX5α in MDA-MB-231 cells represses cell motility, migration, and invasion. A, semiquantitative RT-PCR detection of PAX5α mRNA expression following transient transfection of MDA-MB-231 cells with either pIRESneo3-PAX5α or an empty vector (pIRESneo3) as a control. Total RNA was isolated 5 d after transfection. Transient forced expression of PAX5α in MDA-MD-231 cells reduced wound healing (B), cell migration (C), and cell invasion (D). *, P ≤ 0.025. B, wound healing assays were examined under ×10 magnification after 75 h. C and D, Transwell membranes of invasion and migration assays were observed under ×20 magnification. Results are presented as the ratio of migrated or invaded cells compared with the control transfection (control transfection = 1). Columns, mean; bars, SD.

Discussion

EMT, or epithelial to mesenchymal cell transition, is a critical process in normal development and has been implicated in cancer progression and metastatic dissemination (39, 40). In the reverse process, mesenchymal-epithelial transition (MET), mesenchymal cells revert to a more epithelial cell type (41-43). This is accompanied by loss of expression of mesenchymal markers, as well as decreased cell motility and decreased ability of cells to invade through the extracellular matrix. Another characteristic of MET is the formation of epithelial cell characteristics such as tight junctions containing OCCLUDIN and adherens junctions formed by β-CATENIN, E-CADHERIN, and α-CATENIN (44). We have identified herein that PAX5α promotes epithelial behavior in MCF-7 cells, reminiscent of MET. Forced expression of PAX5α promoted an epithelial cell morphology in MCF-7 cells with reduced mRNA levels of the mesenchymal markers VIMENTIN and FIBRONECTIN. This was accompanied by relocalization of β-CATENIN to the sites of cell-cell junctions. In addition, reduced VIMENTIN protein localization and localization of β-CATENIN to the sites of cell-cell junctions were also observed in vivo in sections taken from MCF7-PAX5α tumors. Forced expression of PAX5α in MCF-7 and MDA-MB-231 cells reduced cell migration and invasion. Conversely, siRNA-mediated depletion of PAX5α enhanced mesenchymal features such as cell motility and invasiveness in MCF-7 cells. Interestingly, elevated levels of β-CASEIN mRNA indicate that forced PAX5α expression promotes MCF-7 cell differentiation, analogous to the role of PAX5 in B-lymphocyte commitment and identity (45, 46). Similarly, other PAX genes have previously been implicated in such phenotypic transitions (47-50). PAX6 has recently been shown to inhibit glioblastoma cell invasiveness through repression of MMP-2 expression (47). In addition, PAX2 has been reported to be expressed during MET in the human kidney and is downregulated during terminal differentiation (48, 49). Similarly, PAX3 has been shown to promote MET in the mesenchymal mammalian cell line Soas-2 (50). Interestingly, PAX3-induced MET was also associated with the relocalization of β-CATENIN to the sites of cell-cell contact (50), consistent with the observed relocation of β-CATENIN in our present report resulting from forced expression of PAX5α in MCF-7 cells.

Clinical correlations of PAX5 expression in mammary carcinoma have been contradictory and require further investigation. A recent study by Ellsworth et al. (51) has identified increased PAX5 expression in tumors metastasized to the lymph nodes compared with the corresponding primary tumor. This is supported by expression data from the gene expression database Oncomine, which indicated that PAX5 expression is positively correlated with breast cancer metastasis in three studies (28, 30, 31). In contrast, an immunohistochemical study conducted by Mhawech-Fauceglia et al. (52) detected PAX5 in only one case out of 164 benign and malignant tumor samples. However, Mhawech-Fauceglia et al. also failed to observe expression of PAX5 in cases of astrocytomas, medulloblastomas, and neuroblastomas, whereas several other studies have shown expression of PAX5 mRNA in these three pathologies (14-16).

The postulated general principle of tumor suppression proposes the loss of expression of tumor suppressor genes during tumor progression (53). Here, we have described a potential inhibitory role for PAX5α in mammary carcinoma. However, paradoxically, PAX5 mRNA expression is correlated with tumor progression (Supplementary Table S1). Interestingly, similar contradictory observations have been previously made with other PAX family members. Indeed, expression of PAX2 has been described in more than 50% of mammary tumors examined (54). However, PAX2 has recently been shown to inhibit the proliferation of MCF-7 cells through repression of human epidermal growth factor receptor (HER2/ERBB2) gene expression (55). Thus, like PAX5, PAX2 is expressed in mammary tumors where it inhibits mammary carcinoma cell proliferation. In contrast, PAX2 has also been reported to be involved in the stimulation of endometrial carcinogenesis (56). Hence, PAX2 also displays contradictory effects depending on the tissue of expression. Similarly, we reported herein the inhibition of mammary neoplastic progression by PAX5α, whereas Baumann Kubetzko et al. (14) showed that PAX5 promoted tumorigenicity of a benign “S-type” neuroblastoma cell line. However, the authors did not identify which PAX5 isoform they were investigating. Therefore, the tissue or cell type of expression and the isoform studied may affect the role of paired box transcription factors such as PAX5 and PAX2. Moreover, although little is known about the endocrine regulation of PAX genes, both transcription factors have been described to be regulated by hormones. PAX2 has been shown to be activated by estrogen (56), whereas the expression of PAX5 has been reported to be regulated by autocrine human growth hormone in mammary carcinoma cells (57). Both hormones are characterized by their pleiotropic effects in tissue targets (58, 59). Such pleiotropic effects may be dependent on the tissue of expression.

A tumorigenic role for β-CATENIN has previously been shown in the murine mammary gland (60, 61). Accordingly, the accumulation of β-CATENIN in the cytoplasm and nuclei has been reported to occur in 50% of human mammary carcinoma (62). Furthermore, β-CATENIN has been shown to regulate gene expression through recruitment of nuclear cofactors such as LEF and TCF family members in a variety of cancers (63). The relocalization of β-CATENIN to the sites of cell-cell contacts prevents its accumulation in the cytoplasm and nuclei. Therefore, PAX5α may impair the formation of the oncogenic complex β-CATENIN/LEF/TCF by promoting relocalization of β-CATENIN. Interestingly, increased expression of β-CATENIN has been shown to reduce PAX5 expression in lymphoid progenitors (64), which, when combined with our observations, strongly suggests functional interactions between PAX5 and β-CATENIN. However, the nature of these interactions, as well as the precise role of PAX5 in β-CATENIN function, remains to be determined.

MET is important developmentally, and mesenchymal-epithelial reverting transitions have also been implicated in colonization of metastatic seed cancer cells at distant metastatic sites in numerous tumor systems including breast cancer (41-43, 65). Metastasizing cancer cells are thought to progress through several stages: EMT, which supports migration/invasion from the primary tumor site; intravasation through the vascular wall; survival in circulation; and extravasation at a secondary site (48, 66). Very few circulating tumor cells will establish micrometastases at a distal site, and even less will establish themselves sufficiently to form secondary tumors. MET is thought to play a critical role in tumor establishment at a distal site ensuring successful metastatic colonization (43, 48). If PAX5α does in fact promote MET in metastasizing breast cancer cells, it would be expected that PAX5α expression would correlate with metastasis in breast cancer, and indeed, this seems to be the case. A recent study has shown that PAX5 mRNA expression was elevated 100-fold in tumors metastasized to the lymph nodes from 20 patients when compared with the corresponding primary mammary tumors (51). Furthermore, PAX5α has been identified as a target gene of metastasis-associated protein 1 (MTA1) and may contribute to the high incidence of spontaneous B-cell lymphomas in MTA-transgenic mice (67). Thus, PAX5α may be an example of a metastasis virulence gene, a gene that exerts its functions primarily at the site of metastatic colonization facilitating tumor establishment at a secondary site and ensuring successful colonization (66).

Through its cell cycle arrest and apoptotic activities, p53 pivotally controls cell behavior. In contrast to a previous study (38), we observed a transcriptional upregulation of the tumor suppressor gene p53 by PAX5α in MCF-7 cells, whereas Stuart et al. (38) showed that PAX5 transcriptionally inactivated p53 through binding to a PAX-binding site within its untranslated first exon in mouse NIH 3T3 fibroblasts. This group also described an inverse relationship between PAX5 expression and p53 protein expression in human astrocytomas (16). However, Nutt et al. (68) found that expression of the p53 gene was not affected in pre-BI cells derived from Pax5-mutant mice (−/−), whereas a recent study by Norhany et al. (69) failed to find a negative correlation between PAX5 expression and p53 protein expression in oral cell carcinoma. In this context, the authors suggested that the evidence reported by Stuart et al. (38) may be ambiguous because the absence of p53 immunohistochemical positivity cannot differentiate physiologic conditions related to the presence of wild-type p53 and conditions with repressed p53 activity (69). However, differential regulation of p53 by PAX5, as reported in the literature, may result from cell or tissue specificity. Torlakovic et al. (9) previously proposed that transcription factors exhibit their effect with regard to the environment they are expressed in, and therefore the interpretation of their effects requires prudence, in particular in a genetically unstable environment such as neoplasms. Such functional variability may also be the result of PAX5 alternative splicing. Palmisano et al. (12) described a variation in the promoter methylation state of PAX5α and PAX5β in a variety of cell lines, suggestive of epigenetic regulation. They showed that PAX5α and PAX5β can be expressed alternatively or simultaneously. However, few previous PAX5 studies have determined which PAX5 isoform was under investigation. Therefore, the conflicting functions that have been allocated to one PAX5 only may instead be played by two distinct transcription factors, PAX5α and PAX5β, respectively.

In conclusion, we have described a role for the PAX5α isoform in mammary carcinoma. Of the two alternative PAX5α and PAX5β transcripts, only PAX5α mRNA was expressed in MCF-7 cells. Forced expression of PAX5α in mammary carcinoma cells promoted an epithelial phenotype in these cells. Further investigation will elucidate the role of PAX5 in different neoplastic models. In the literature, contradictory roles have been described for PAX5 in a neoplastic context. As discussed earlier, previous functional investigations rarely distinguished between PAX5 isoforms, which may have contributed to this confusion. In addition, little attention has been dedicated to the potential endocrine regulation of PAX5 alternative splicing. Further investigation will elucidate whether endocrine regulation influences PAX5 alternative splicing and function in a neoplastic context.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Grant Support: The Marsden Fund, The Royal Society of New Zealand, The Breast Cancer Research Trust (NZ), The Foundation of Research Science and Technology (NZ), and The New Zealand Breast Cancer Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

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Molecular Cancer Research: 8 (3)
March 2010
Volume 8, Issue 3

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PAX5α Enhances the Epithelial Behavior of Human Mammary Carcinoma Cells
Laurent J-P Vidal, Jo K. Perry, Cecile M. Vouyovitch, Vijay Pandey, Severine E. Brunet-Dunand, Hichem C. Mertani, Dong-Xu Liu and Peter E. Lobie
Mol Cancer Res March 1 2010 (8) (3) 444-456; DOI: 10.1158/1541-7786.MCR-09-0368
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