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Carcinogenesis Advance Access originally published online on July 13, 2006
Carcinogenesis 2007 28(2):259-266; doi:10.1093/carcin/bgl122
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Inhibition of breast cancer growth and invasion by single-minded 2s

Hyeong-Il Kwak1, Tanya Gustafson1, Richard P. Metz1, Brian Laffin1, Pepper Schedin2 and Weston W. Porter1,*

1 Department of Integrated Biosciences, College of Veterinary Medicine Texas A&M University, College Station, TX 77843-4458, USA
2 Division of Medical Oncology, University of Colorado Health Sciences Center CO, USA

*To whom correspondence and requests for reprints should be addressed. Tel: +979 845 0733; Fax: +979 862 4929; Email: wporter{at}cvm.tamu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Single-minded 2 (SIM2) is a member of the bHLH-PAS family of transcription factors. SIM2 was initially identified by positional cloning on chromosome 21 and is thought to contribute to the etiology of trisomy-21 [Down syndrome (DS)]. In addition to the physical and mental deficiencies associated with this genetic disease, it has become apparent that women with DS are 10–25times less likely to die from breast cancer in comparison with age-matched normal populations. This is thought to be a result of gene dosage effect of tumor suppressor genes on chromosome 21. Here, we report that a splice variant of SIM2, SIM2 short (SIM2s), is differentially expressed in normal breast and breast cancer-derived cell lines and is downregulated in human breast cancer samples. Re-establishment of SIM2s in MDA-MB-435 breast cancer cells significantly reduced proliferation, anchorage-independent growth and invasive potential. Consistent with its role as a transcriptional repressor, SIM2s directly decreased expression of matrix metalloprotease-3, a known mediator of breast cancer metastasis. These results suggest that SIM2s has breast tumor suppressive activity.

Abbreviations: bHLH, basic helix–loop–helix; CAV1, caveolin-1; ChIP, chromatin immunoprecipitation; DS, Down syndrome; ECM, extracellular matrix; MMP, matrix metalloproteases; PAS, Per-Arnt-SIM; PMA, phorbol 12-myristate 13-acetate; RT–PCR, reverse transcription–polymerase chain reaction; SE, standard error; SIM2, single-minded 2; SIM2s, SIM2 short


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Down syndrome (DS), which results from trisomy 21, is the most common chromosomal abnormality and is a leading cause of mental retardation. In addition to facial abnormalities and learning deficiencies, people with DS are prone to a multitude of physiological conditions including congenital heart disease, early onset of Alzheimer's disease and diabetes (1). However, owing to improvements in medical care, DS patients are living longer, healthier lives than was possible just a few decades ago. As a result, it has become apparent that the incidence of cancer in individuals with DS is unique. Most notably, people with DS are more susceptible to childhood leukemias and germ cell cancers, but are >50% less likely to develop solid tumors, including cancers of the lung, colon, skin, head and neck (1–7). Even more striking is the observation that the incidence of breast cancer is significantly decreased (1–6) and that mortality from this disease (10- to 25-fold) is reduced (1,4–8) in women with DS as compared with age-matched controls. These observations have led to the hypothesis that one or more tumor suppressor genes are present on chromosome 21.

Attempts to identify genes on chromosome 21 that contribute to DS resulted in the isolation of single-minded 2 (SIM2), one of two human orthologs of the Drosophila single-minded (sim) gene (9,10). SIM1 and SIM2 are members of the basic helix–loop–helix/Per-Arnt-SIM (bHLH/PAS) family of transcription factors, which includes genes responsible for maintenance of circadian rhythms (period), sensors of hypoxia (hypoxia inducible factors) and environmental contaminants (aryl hydrocarbon receptor) (11). SIM2 differs from sim and most mammalian member of the bHLH/PAS family by functioning as a transcriptional repressor (10,12). Transgenic mice that overexpress Sim2 display many of the mental characteristics seen in DS patients, including impaired learning ability and reduced fear responses, implying that SIM2 plays a key role in DS (13,14). A splice variant of human and mouse SIM2, designated SIM2 short (SIM2s), has also been identified (15,16). Although the significance of this splice variant is presently unknown, it has been proposed that SIM2s plays a role in cancer progression (17–19).

Despite the fact that SIM2 may contribute to many of the physiological abnormalities associated with DS, very little is known about its role in development outside the CNS, or in breast cancer susceptibility. To determine if SIM2 plays a role in breast cancer, we assayed a panel of human breast- and breast cancer-derived cell lines for SIM2 expression. We found that SIM2 is expressed at relatively high levels in non-transformed breast, and non-invasive breast cancer cells in comparison with the more invasive breast cancer-derived cells. Surprisingly, SIM2s was the most prevalent isoform of SIM2 expressed with full-length SIM2 only detectable in MCF10A cells. Immunohistochemical analyses of normal human breast and breast tumor tissue sections indicated that SIM2s is downregulated in the majority of human breast cancers. Re-expression of SIM2s in highly invasive MDA-MB-435 breast cancer cells resulted in decreased proliferation, inhibition of growth in soft agar, loss of invasive potential and downregulation of matrix metalloprotease-3 (MMP3) gene expression. The effect of SIM2s on MMP3 expression were regulated at the promoter level and is consistent with its ability to function as a transcriptional suppressor. These results suggest that SIM2s is a breast tumor suppressor gene and provide insight into possible mechanisms of SIM2s-mediated inhibition of breast cancer progression.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Plasmids
The SIM2s coding region was amplified by Hi Fidelity Taq polymerase (Roche, Indinapolis, IN) from MCF7 cell cDNA using the primers 5'-ATG AAG GAG AAG TCC AAG AAT GCG GCC AAG-3' and 5'-CTA CTT AGA AGC AGA AAG AGG GC-3'. The polymerase chain reaction (PCR) product was cloned into pCR2.1 TOPO (Invitrogen, Carlsbad, CA), and individual clones were sequenced at least twice in both directions to confirm identity. pLNCX2-SIM2s was made by subcloning SIM2s cDNA into the EcoRI site of the pLNCX2 retroviral expression vector (Clontech, Moutain View, CA). pcDNA3-SIM2s was made by cloning the entire SIM2s insert into the EcoRI site of pcDNA3.1. The insert from the largest human MMP3-luc construct (–2264 to +37) was amplified from human genomic DNA using the primers 5'-CCT GTT TGA CAT TTG CTA TG-3' and 5'-TTG TCT CTA TGC CTT GCT G-3' and cloned into pCR-2.1 TOPO (Invitrogen). The entire insert was removed and cloned into pGL3-Basic.

Cell lines
The cell lines used in these studies were derived from our collection or obtained from American Type Culture Collection (ATCC, Manassas, VA). The 16N normal breast epithelial cells and 21T breast cancer cells were a gift from Dr Heide Ford at the University of Colorado Health Science Center and were grown as described previously (20). HEK-293t Ampho-Phoenix packaging cells were obtained from ATCC with permission from Dr Gary Nolan at Stanford University and maintained as recommended. All cells were grown in 5% CO2 at 37°C.

Transient transfections and chromatin immunoprecipitation (ChIP)
Cells were seeded at 4 x 104 cells per well in 24-well plates Invitrogen the day before transfection. The following morning, cells were co-transfected with a normalizing plasmid (pß-Gal) and various amounts of test plasmids using Lipofectamine and Plus reagent (Invitrogen). After 3 h, the transfection medium was replaced with growth medium and cells were allowed to recover overnight. The following day, cells were exposed to vehicle [dimethyl sulfoxide (DMSO)] or 50 µg/ml phorbol 12-myristate 13-acetate (PMA) for 24 h. Cells were harvested, and cell lysates were assayed for luciferase and ß-gal activity using the dual luciferase assay and Galacto-Light. Luciferase activities were normalized to ß-gal internal control values and Applied Biosystem, Foster City, CA are represented as the means ± standard error (SE) for three wells per condition. Significant differences were determined using Student's t-test. ChIP assays were performed as described previously (16) using the following primers: for endogenous MMP3 ChIP, F1—CTG CTG CCA TTT GGA TGA AA and R1—GCT CAA GCA TTC TAT GTG GGT; for transfected MMP3, F—AAT TAG AGC TGC CAC AGC TTC and R—TGA AGA TGC CCA CAC AGT TGA; for the E-cadherin promoter, F—AAA AGC CCT TTC TGA TCC CA and R—TGG AGT CTG AAC TGA CTT CCG.

Reverse transcription (RT) and real-time PCR
Total RNA was isolated using RNeasy Mini kits (Qiagen, Valencia, CA) along with the RNase-free DNase Set (Qiagen) to remove genomic DNA. One microgram total RNA was reverse-transcribed into cDNA using oligo (dT) and Superscript II Reverse Transcriptase (Invitrogen). RT–PCR reactions were performed with Taq DNA Polymerase (Invitrogen) in a total volume of 25 µl. Primers used for RT–PCR amplification were total (pan) SIM2 (FP: 5'-TGA AAT GTG TCT TGG CGA AAA-3', RP: 5'-GCG TAG CGG AGG TGG AAC-3'), SIM2s (FP: 5'-GCT GAG AAC AAA CCC TTA CC-3', RP: 5'-GAA GCA GAA AGA GGG CAA GTT-3'), SIM2 long form (FP: 5'-ACA GCT CGT TCC AAA TGG AC-3', RP: 5'-TAG TGG CCG CAG CTC GGG AA-3'), SIM1 (FP: 5'-GCT GGT GGA AGA GAG GCA TT-3', RP: 5'-TGG AGA ACT GAC CAC ACT AT-3') and GAPDH (FP: 5'-AAT CCC ATC ACC ATC TTCC CA-3', RP: 5'-GTC ATC ATA TTT GGC AGG TT-3'). Real-time PCR was performed using SYBR Green Master Mix (Applied Biosystems, Foster City, CA). Primers for analyzing total SIM2 by real-time PCR were FP: 5'-AGA CAA AGC TGA GAA CAA ACC CTT A-3' and RP: 5'-CCG CAT TCC AGT TTG TCC AT-3'. MMP3 was analyzed using the primers FP: 5'-TTC CTG ATG TTG GTC ACT TCA GA-3' and RP: 5'-TCC TGT ATG TAA GGT GGG TTT TCC-3'. TBP was used as a normalizing gene and was assayed using the primers FP: 5'-TGC ACA GGA GCC AAG AGT GAA-3' and RP: 5'-CAC ATC ACA GCT CCC CAC CA-3'. Product specificity was determined by dissociation curve analyses after each run, and product identities were confirmed by sequencing. Real-time PCR data were analyzed by the {Delta}{Delta}CT method (21). The relative positions of the SIM2-specific primers used are indicated in Figure 1A.


Figure 1
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  		Fig. 1 Single-minded expression in breast epithelial- and cancer-derived cell lines. (A) Schematic representation of SIM2 and SIM2s mRNAs with relative positions of PCR primers used in these studies indicated. Exons are indicated by numbered boxes. Structural motifs are represented by bars: bHLH (basic helix–loop–helix domain, black bar), PAS (Per-Arnt-Sim domains, stripped bars), Pro/Ser (proline/serine-rich, white bar) and Pro/Ala (proline/alanine-rich, gray bar) regions implicated in harboring SIM2 repression domains. PCR primer locations are indicated by name and arrows. (B) Analyses of total SIM2 (pan SIM2), SIM2s, SIM2 long form (SIM2L) and SIM1 message levels in normal human breast and breast cancer-derived cell lines by RT–PCR. Cell lines are indicated at the top of the figure. (C) Quantification of total SIM2 expression in human breast and breast cancer cells by real-time RT–PCR. Data were quantified by the {Delta}{Delta}CT method using TATA binding protein mRNA as the normalizer. (D) Western analysis of SIM2s expression in representative breast cancer cells using a SIM2s-specific antibody.

 
Retroviral transduction
For retroviral transduction, SIM2s cloned into the pLNCX2 vector (Clontech) was transfected into Phoenix packaging cells for 6–8 h with 25 µl of lipofectamine and Plus reagents (Invitrogen). The medium was replaced 24 h later and virus-containing supernatants were collected after 48 h. Supernatants were filtered through a 0.45 µm filter syringe and added to MDA-MB-435 cells in 6-well plates in the presence of 8 µg/ml polybrene (Sigma, St. Louis, MO). The plates were then centrifuged at 1000 x g at room temperature for 45 min. Following a 24 h incubation, the medium was replaced and stable transduced cells were selected in the presence of puromycin. The cells were used within the first 10–12 passages and critical experiments were performed within the first few passages. At least three independent infections were utilized and similar results were obtained. SIM2s expression was confirmed by RT–PCR, real-time RT–PCR and immunofluorescence.

Immunostaining, immunofluorescence and western blot
The following primary antibodies were used: anti-SIM2 (Chemicon, Temecula, CA; catalog number AB4145), anti-SIM2s (Santa Cruz Invitrogen, catalog number sc-8715) and MMP3 (Chemicon). Secondary antibodies were FITC anti–rabbit and Texas red anti–rabbit Invitrogen. Human breast and breast tumor tissue arrays were purchased from Chemicon. The sections were baked at 55oC for 15 min and then dehydrated by sequential washes in xylene and a series of graded ethanol washes. Antigen retrieval was performed for 20 min at 98°C in 0.01 mol/l sodium citrate buffer, pH 6.4, in a microwave oven. For immunostaining, sections were incubated in 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. After 30 min block in 5% milk, the sections were incubated with 1 : 100 dilution of primary antibody 1 h at room temperature. The sections were washed in phosphate-buffered saline containing 0.1% tween-20 and then incubated with either biotinylated donkey anti-goat (Vector Laboratories, Burlingame, CA) followed by avidin peroxidase using the Vectastain ABC elite kit (Vector Laboratories) or Alexa-conjugated secondary Invitrogen. The chromogenic reaction was carried out with 3–3' diaminobenzidine (Sigma). The slides were mounted using Permount (Sigma) or Vectashield containing DAPI (Vector Laboratories) and evaluated under a microscope.

Zymography
To determine MMP3 activity, conditioned medium from treated cells was concentrated ~20-fold using Centricon 10 spin concentrators Millipore, Brillerica, MA. Samples were quantified by Bradford analysis and equal amounts of protein were mixed with Laemmli sample buffer without reducing agents, incubated for 15 min at 37°C and separated on precast gradient SDS-polyacrylamide slab gels containing 1 mg/ml casein (Invitrogen). Following electrophoresis, gels were placed in 2.5% Triton X-100 for 30 min and then incubated at 37°C in 50 mM Tris–HCl, pH 7.4, containing 5 mM CaCl2 for 18 h. MMP3 activity was visualized by Coomassie blue staining. A non-specific band at 77 000 Kd was used as a loading control.

Soft agar and invasion
Control or SIM2s-transduced MDA-MB-435 cells (5 x 103 cells per plate) in 2 ml of medium [Dulbecco-modified essential medium (DMEM) + 10% fetal bovine serum (FBS)] supplemented with 0.35% agarose were layered onto a 1.5 ml base of medium containing 0.5% agarose in 35 mm dishes. Media was changed daily, and after 14 days of growth, cells were stained with 0.005% crystal violet, photographed and colonies > 200 µm were counted. Data presented are the summary of three separate experiments. For cell invasion assays, 1 x 105 cells in 0.5 ml of serum-free DMEM were plated in Boyden chambers containing 8 µm pore membranes coated with Matrigel (Becton Dickinson). The lower chamber contained 10% FBS in DMEM to serve as a chemo-attractant. Following a 24 h incubation, cells that had not left the top chamber were removed from the membrane by scraping. Cells that had migrated to the bottom surface of the membrane were stained with Diff-Quik reagent (American Scientific Products, McGaw Park, IL) and examined under a bright-field microscope. Values are the average number of cells per five fields per membrane and analyzed by Student's t-test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
SIM2s is downregulated in breast cancer cell lines and primary breast cancer samples
To determine if SIM2 plays a role in breast cancer, we examined a panel of human breast- and breast cancer-derived cell lines for SIM gene expression by standard and real-time RT–PCR. The relative positions of the SIM2-specific primers used in these analyses are indicated in Figure 1A. The 21T cell line series was established from normal breast (16N) and primary (21NT and 21PT) and metastatic (21MT1 and 21MT2) breast tumor specimens from a single patient with infiltrating ductal and intraductal carcinoma (20,22,23). Together with other human breast- and breast cancer-derived cell lines (i.e. MCF10A, MCF7, ZR75, T47D, MDA-MB-231, MDA-MB-453 and MDA-MB-435 cells), these cells provide a useful model to evaluate gene expression in a simulated tumor progression series. Total SIM2 mRNA was detectable at the highest levels in MCF10A and 16N normal breast epithelial cells (Figure 1B). SIM2 expression was markedly decreased in the 21T breast cancer progression series and in the other breast cancer cell lines examined. RT–PCR analyses using primers to discern between the short and long forms of SIM2 indicated that SIM2-positive cell lines expressed the SIM2s isoform, with the exception of MCF10A cells, which expressed only the long form of SIM2 (Figure 1B). In contrast, SIM1 mRNA was not detected in any of the breast cells, but was abundant in kidney-derived HEK 293 cells. Quantification of relative SIM2 expression in these cells by real-time RT–PCR corroborated the RT–PCR results and confirmed that SIM2 expression is highest in non-transformed MCF10A and 16N cells, is much lower in the non-invasive, estrogen receptor positive MCF-7, T47D and ZR75 breast cancer cells and is dramatically decreased in the highly invasive estrogen receptor negative MDA-MB-453 and MDA-MB-435 cell lines (Figure 1C). A subset of these cell lines was analyzed for SIM2s protein levels by western blot (Figure 1D). In agreement with the PCR data, SIM2s protein was only detectable in MCF-7 and T47D cells. Interestingly, SIM2s protein was not detected in MDA-MB-231 cells despite having observable levels of SIM2s message. These results suggest that SIM2s is the most prevalent isoform of SIM2 expressed in human breast-derived cell lines. This was confirmed by immunohistochemical staining of normal breast and primary breast tumor tissues using a SIM2s-specific antibody. SIM2s protein was readily detectable in the ductal epithelium of normal breast tissue (Figure 2A), whereas SIM2s was not present in 18 out of 25 (72%) breast tumor samples (Figure 2B–D, representative data shown).


Figure 2
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  		Fig. 2 Immunohistochemical analysis of SIM2s protein levels in normal human breast and breast tumor tissues. A human breast cancer tissue array (Chemicon) containing 5 normal and 25 breast tumor tissue sections (in duplicate) was analyzed for SIM2s protein levels by immunohistochemistry as described in Materials and methods. (A) Representative image of a normal human breast tissue sample showing ductal epithelial cells with high levels of SIM2s staining. (B) Representative image of a SIM2s-positive breast tumor sample. Note that the majority of breast tumor samples (18 out of 25, or 72%) were SIM2s negative. When present in tumor samples, SIM2s staining was considerably less intense than that observed in the normal breast tissues. (C–D) Representative images of SIM2s-negative human breast tumor sections.

 
Inhibition of breast cancer growth by SIM2s
Our data indicate that SIM2s expression is lost during breast cancer progression. To determine if SIM2s has tumor suppressor activity, MDA-MB-435 breast cancer cells were transduced with a recombinant retrovirus expressing SIM2s. Re-establishment of SIM2s expression was confirmed by RT–PCR (Figure 3A) and immunofluorescence (Figure 3B) and was similar to the levels observed in MCF10A cells (data not shown). Quantification of colonies after 2 weeks of selection showed that SIM2s significantly inhibited colony growth in MDA-MB-435 cells (Figure 3C). Subsequently, a 7-day proliferation assay confirmed that stable expression of SIM2s blocked MDA-MB-435 cell growth (Figure 3D). In addition to pooled samples, individual colonies of control- and SIM2s-transduced cells were isolated by serial dilution; however, these clones lost SIM2s expression after a few weeks of selection (data not shown), further supporting a role for SIM2s in inhibiting tumor cell growth.


Figure 3
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  		Fig. 3 SIM2s suppresses growth of MDA-MB-435 breast cancer cells. MDA-MB-435 cells were infected with recombinant retroviruses expressing SIM2s or Puro only (Control vector). Two days after infection, puromycin was added to the cell medium and selection was continued for 5 days. (A) Confirmation of SIM2s expression in SIM2s-infected MDA-MB-435 cells by RT–PCR. (B) Confirmation of SIM2s protein expression by immunofluorescence using a SIM2s polyclonal antibody. (C) Re-introduction of SIM2s inhibits expansion of MDA-MB-435 colonies under puromycin selection in comparison with vector-only cells. The number of colonies formed after 2 weeks of puromycin selection was significantly lower (*P < 0.0001) in SIM2s-transduced cells than in control cells as determined by Gimsa staining. The values shown are the mean ± SE of three separate samples. (D) SIM2s inhibits breast cancer cell proliferation. Equal numbers of control and SIM2s-expressing cells were plated in six-well plates and counted daily over a 7-day period. Significant differences in cell proliferation due to re-introduction of SIM2s were apparent by Day 5 (*P < 0.001) and continued through Day 7 (**P < 0.0001). Data represent results from at least two independent experiments. The values shown are the mean ± SE of triplicate samples. (E) SIM2s inhibits MDA-MB-435 cell growth on soft agar. The left portion of the figures shows representative images of colonies that grew on soft agar in control (top) and SIM2s (bottom) MDA-MB-435 cells. Following 14 days of growth, the number of colonies > 200 µm in size were counted (right portion of figure). Re-introduction of SIM2s into MDA-MB-435 cells significantly reduced colony formation on soft agar (*P < 0.005). Values are the mean number of colonies ± SE of three plates per group.

 
The ability to grow in soft agar is a hallmark of the transformed phenotype. To assess if re-introduction of SIM2s reduces anchorage-independent growth of breast cancer cells, vector control- and SIM2s-infected MDA-MB-435 cells were plated in soft agar and assayed for colony formation over 2 weeks. Gross observation revealed that SIM2s-infected cells do not grow as well in soft agar as the control cells (Figure 3E). After 14 days, the number of colonies > 200 µm was significantly lower in the SIM2s-infected cells (Figure 3E).

SIM2s inhibits invasion and downregulates MMP3 expression and activity
Since we found that SIM2s expression is inversely related to cellular invasiveness (Figure 1), we next wanted to determine if SIM2s affected the invasive potential of MDA-MB-435 cells. Control- and SIM2s-transduced cells were grown in a Matrigel-coated modified Boyden chamber, which mimics the three-step process of invasion: adhesion, proteolytic dissolution of the extracellular matrix (ECM) and migration. Consistent with our hypothesis that SIM2s is a tumor suppressor, forced expression of SIM2s significantly blocked invasion over 80% in comparison with the control cell line (Figure 4A).


Figure 4
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  		Fig. 4 SIM2s-dependent inhibition of invasion potential, MMP3 expression and enzymatic activity in MDA-MB-435 cells. (A) Cellular invasion assay. MDA-MB-435 cells (1 x 105 cells per sample) were plated in serum-free media in the upper compartment of a modified Boyden chamber coated with Matrigel. The lower compartment contained 10% FBS to serve as the chemo-attractant. After 24 h, the number of cells that had migrated to the bottom surface of the membrane were stained with Diff-Quick and counted. Significantly less SIM2s-expressing cells were able to invade and migrate to the lower chamber (*P < 0.005). Values are the average number of cells per five fields per membrane of three separate plates, and are expressed as percent of control (mean ± SE). (B) SIM2s inhibits basal and PMA-induced MMP3 expression in MDA-MB-435 cells. Quantification of MMP3 mRNA levels in control and SIM2s- transfected MDA-MB-435 cells by real-time RT–PCR. The values shown are the mean ± SE of three separate samples, *P < 0.005. (C) Casein zymography and western blot analysis of MMP3 activity and protein, respectively, in control and SIM2s-infected cells.

 
The ability of SIM2s to decrease invasiveness of MDA-MB-435 cells suggested that SIM2s affects one or more genes responsible for ECM remodeling. RT–PCR analyses of control- and SIM2s-transduced cells for expression of several MMP genes was performed (data not shown). Of the MMP genes analyzed in these preliminary experiments, MMP3 was most significantly repressed by SIM2s. This finding was noteworthy as MMP3 is known to play important roles in cell migration, breast cancer progression and epithelial to mesenchymal transitions (EMTs) (24,25). To better characterize the effects of SIM2s on MMP3 expression, control- and SIM2s-transduced MDA-MB-435 cells were analyzed for MMP3 mRNA by real-time RT–PCR. SIM2s significantly repressed both basal and PMA-induced MMP3 gene expression (Figure 4B), which correlated with MMP3 protein and enzymatic activity as determined by western blot and casein gel zymography (Figure 4C).

SIM2s decreases MMP3 expression and binds the MMP3 promoter
To determine if the effects of SIM2s on MMP3 expression are mediated at the transcriptional level, a human MMP3 promoter-controlled luciferase reporter gene was analyzed for SIM2s responsiveness. MDA-MB-435 cells were transfected with a luciferase reporter gene under the control of the human MMP3 promoter in the presence or absence of SIM2s. SIM2s significantly decreased basal and PMA-induced luciferase activity in a dose-dependent manner (Figure 5A). Consistent with SIM2s functioning as a transcriptional repressor, we determined by ChIP analyses that SIM2s bound the MMP3-Luc reporter in the presence of transfected SIM2s (Figure 5B) and also interacted with the endogenous MMP3 promoter in SIM2s-transduced MDA-MB-435 cells (Figure 5C). No SIM2s binding was observed on the E-cadherin promoter, which was used as a negative control (Figure 5C).


Figure 5
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  		Fig. 5 SIM2s represses MMP3 transcription by binding the MMP3 promoter. (A) Effects of SIM2s on basal and PMA-induced expression from human MMP3 reporter constructs. MDA-MB-435 cells containing –2264 to +37 of the human MMP3 promoter were tested for basal (open bars) and PMA-induced (black bars) luciferase expression in the presence of increasing amounts of SIM2s expression construct. Plasmids [0.45 µg MMP3-luc construct, various amounts of pcDNA3-SIM2sHA (0.0, 0.4, 1.0 or 2.0 µg) plus pcDNA3 (to equal 2.0 µg total expression construct) and 0.2 µg ß-Gal expression vector] were introduced into MDA-MB-435 cells by transfection. Following a 24 h recovery period, fresh medium containing DMSO or PMA (50 ng/ml) was added and luciferase and ß-galactosidase activities were measured 24 h later. The values shown are the means ± SE for three separate plates. (B) Transfected SIM2s binds the MMP3-Luc reporter plasmid. 293t cells were co-transfected with the –2264 to +37 MMP3-Luc reporter and either a control (pcDNA3) or SIM2s expression vector. Following formalin cross-linking, chromatin was sonicated and immunoprecipitated with anti-SIM2 (Chemicon) or IgG (negative control) antibodies and analyzed by PCR using primers that target the reporter vector. (C) SIM2s binds the endogenous MMP3 promoter in SIM2s-transduced MDA-MB-435 cells. Chromatin immunoprecipitation analysis of the MMP3 and E-cadherin (negative control) promoters in control and MDA-MB-435-SIM2s cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
SIM2 was initially identified by positional cloning around the DS critical region of chromosome 21 and is believed to contribute to many of the physiological abnormalities associated with DS (13,14). In these studies, we present evidence that a splice variant of SIM2, SIM2s, has tumor suppressor activity when re-expressed in invasive breast cancer cells. We found that forced expression of SIM2s in MDA-MB-435 cells significantly inhibited proliferation, anchorage-independent growth and invasion potential. The decrease in invasion appears to be due, in part, to SIM2s' ability to repress MMP3 expression through direct inhibition of the MMP3 promoter. These results are consistent with the observation that SIM2 is a transcriptional repressor and imply that SIM2s has tumor suppressor activity.

MMP play a major role in a number of biological processes, including morphogenesis, polarity determination, apoptosis and angiogenesis (26–29). These processes occur at some stages in normal development, and during pathological conditions such as tumor progression and metastasis. Invasion requires active degradation of the ECM and basement membrane, which requires the actions of MMPs. Not surprisingly, over-expression of MMP3 in the mouse mammary gland causes increased lateral branching, precocious alveolar development, hyperplasia, EMT and cancer (30,31). MMP3 gene expression is primarily regulated at the promoter level and is induced by growth factors, cytokines, ECM components and EMT-dependent transcription factors like SLUG and SNAIL (32–34). We have found that re-expression of SIM2s decreases MMP3 mRNA levels by transcriptional repression of the MMP3 promoter. This is consistent with other tumor suppressor genes such as the tumor metastasis suppressor nm23-H1, which inhibits invasion and metastasis by competing for and blocking activator binding to the YB1 response element on the MMP2 promoter (35). Similarly, expression of the ETS transcription factor TEL abrogates RAS transformed NIH 3T3 cell growth and tumorigenicity by suppressing MMP3 mRNA and promoter activity (36).

Our results are surprising since others have proposed that SIM2s has oncogenic activity (17–19,37); however, the observation that SIM2s may have contradictory roles in cancer progression is not unique. For example, in breast, ovarian and lung cancers, reduced caveolin-1 (CAV1) expression has been found to be associated with increased invasiveness and histological grade (38). This is supported by in vivo studies, which showed that mammary glands from Cav1–/– mice undergo precocious alveolar development, hyperplasia and increased incidence of tumor formation (39). In contrast, CAV1 is upregulated in metastatic bladder, thyroid and prostate carcinomas (40,41). It has been proposed that CAV1 is transiently downregulated in migrating cells and re-established after intravasation, suggesting a biphasic expression pattern during cancer progression. Alternatively, deletion of, or mutations in, downstream targets of CAV1 signaling could account for these differences.

Tissue and cell context also play a role in determining the oncogenic and tumor suppressive activity of a specific gene. Ras and Notch are signaling molecules utilized throughout development to control and regulate multiple biological processes by enhancing or antagonizing common pathways (42). This is best observed in cancers where interactions between Ras and Notch signaling can be an important determinant of transformation potential. Activation of ras signaling is frequently observed during cancer progression; however, this phenomenon is tissue-dependent as ras has also been shown to act as a tumor suppressor (43–45). Similarly, over-expression of Notch induces cancer in salivary and mammary tissues, but inhibits cell growth and induces keratinocyte differentiation in skin (46–48). These observations suggest that the tumorigenic effects of Notch and Ras signaling are dependent upon their cellular environment. Tissue-specific differences in cross-talk between Ras and Notch signaling pathways, and the outcome of these interactions, adds another level of complexity. Thus, it is possible that discrepancies in SIM2s tumor suppressive and proposed oncogenic activities may be due in part to similar, as yet uncharacterized phenomena.

The unique profile of cancer risk in DS patients suggests that more than one gene may be responsible for the observed differences in cancer susceptibilities. Most likely, these effects are the result of complex interactions between gene products encoded by chromosome 21 and downstream targets of these genes on other chromosomes. Chromosome 21 contains ~250 genes, of which many have been implicated as playing a role in the DS-associated cancer promotion or protection effects. A recent report linked the increased incidence of DS acute megakaryoblastic leukemia to the transcription factor GATA1 (49,50). Another putative oncogene located on chromosome 21, ETS2, has been shown to be upregulated in testicular germ cell tumors from DS subjects (51). Interestingly, despite evidence suggesting that ETS2 plays a role in breast cancer progression (52,53), over-expression of ETS2 in DS patients is not associated with increased breast cancer incidence. The RIP140 gene, which is also located on chromosome 21, interacts with many members of the nuclear steroid receptor family including the estrogen receptor where it functions as a co-repressor (54).

The tumor suppressive activity of trisomy 21 may be the result of changes in ECM and angiogenic regulators. Several genes regulating ECM composition have been linked to chromosome 21 including collagen 6a (COL6A), superoxide dismutase 2 (SOD2), ß2 integrin (ITGB2) and endostatin (COL18A1) (8,55). In addition, it has recently been shown that the DS critical region-1 protein is induced by VEGF and inhibits thrombin and VEGF signaling, ultimately blocking endothelial proliferation and angiogenesis (56). Our results suggest that SIM2s abrogates breast cancer cell invasion and MMP3 gene expression, implying that SIM2s may also function as a metastasis inhibitor. Studies are under way to determine the role SIM2s plays in normal development and further characterize its effects on tumorigenesis.


   Acknowledgments
 
This work was supported by awards from the National Cancer Institute RO1CA111551 (W.W.P.), RO1CA85944 (P.S.), National Institutes of Environmental Health Center Award Pilot Project P30ES09106 and Howard Hughes Pre-doctoral Fellowship (T.G).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 1, 2006; revised June 26, 2006; accepted June 30, 2006.


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