Alcohol promotes breast cancer cell invasion by regulating the Nm23-ITGA5 pathway
© Wong et al; licensee BioMed Central Ltd. 2011
Received: 31 May 2011
Accepted: 12 August 2011
Published: 12 August 2011
Alcohol consumption is an established risk factor for breast cancer metastasis. Yet, the mechanism by which alcohol promotes breast cancer metastases is unknown. The ability of cancer cells to invade through tissue barriers (such as basement membrane and interstitial stroma) is an essential step towards establishing cancer metastasis. In the present study, we identify and examine the roles of two genes, Nm23 and ITGA5, in alcohol-induced breast cancer cell invasion.
Human breast cancer T47D cells were treated with ethanol at various concentrations. Boyden chamber invasion assays were used to measure cellular invasive ability. The mRNA expression level of metastasis suppressor genes including Nm23 was determined by qRT-PCR. ITGA5 was identified using a qRT-PCR array of 84 genes important for cell-cell and cell-extracellular matrix interactions. Nm23 overexpression in addition to Nm23- and ITGA5 knock-down were used to determine the role of the Nm23-ITGA5 pathway on cellular invasive ability of T47D cells. Protein expression levels were verified by Western blot.
Alcohol increased the invasive ability of human breast cancer T47D cells in a dose-dependent manner through the suppression of the Nm23 metastatic suppressor gene. In turn, Nm23 down-regulation increased expression of fibronectin receptor subunit ITGA5, which subsequently led to increased cellular invasion. Moreover, Nm23 overexpression was effective in suppressing the effects of alcohol on cell invasion. In addition, we show that the effects of alcohol on invasion were also inhibited by knock-down of ITGA5.
Our results suggest that the Nm23-ITGA5 pathway plays a critical role in alcohol-induced breast cancer cell invasion. Thus, regulation of this pathway may potentially be used to prevent the establishment of alcohol-promoted metastases in human breast cancers.
List of abbreviations
non metastatic cells 1
integrin alpha 5
KiSS-1 metastasis suppressor
MAP kinase kinase 4
ribonucleotide reductase 1
suppression of tumorigenicity 6
breast cancer metastasis suppressor 1
quantitative reverse-transcriptase polymerase chain reaction
small interfering RNA
Dulbecco's Modified Eagle's Medium
fetal bovine serum
serine/threonine protein kinase
mitogen-activated protein kinase
extracellular signal-regulated kinase
focal adhesion kinase
estrogen receptor alpha.
In 2010, approximately 200,000 women were diagnosed with breast cancer and 40,000 women were expected to die from this disease in the US . Breast cancer is the second leading cause of cancer-related deaths among women in the US, after lung cancer . Often, it is not the primary tumor that leads to the death of cancer patients but, rather, the metastases of the cancerous cells [3, 4]. Breast cancer cells typically spread from the primary tumor site (the breast) to secondary sites (i.e. lungs, liver, bones, etc.) resulting in an increased likelihood of mortality . The invasion of cancer cells into surrounding tissues is an initial step in tumor metastasis and requires the migration of cancer cells and their attachment to the extracellular matrix .
Cell culture and animal studies have previously shown that alcohol consumption increases the risk of developing breast cancer by increasing the ability of breast cancer cells to invade and metastasize [7, 8]. Alcohol consumption increases breast cancer risk in a dose-dependent manner; the risk increases by 10% for each alcoholic drink consumed daily [7–9]. Thus, consumption of two daily alcoholic drinks may lead to a 20% increase in breast cancer risk . A drink is defined as 12 oz of beer or 5 oz of wine . Studies also show that alcohol may increase the risk of breast cancer recurrence in previously diagnosed women, which may affect their survival . Therefore, in order to develop strategies for the prevention and treatment of alcohol-related breast cancers, it is essential to understand the molecular mechanisms by which alcohol promotes the invasive phenotype of the cancer cells. In this study, we show that alcohol promotes the invasive ability of human breast cancer T47D cells in vitro in a dose-dependent manner and show that the Nm23-ITGA5 pathway plays a critical role in the promotion of cancer cell invasion by alcohol.
Metastases suppressing genes encode proteins that hinder the establishment of metastases without blocking the growth of the primary tumor . Two such genes are the human Nm23 genes (Nm23-H1 and Nm23-H2) which have been localized to chromosome 17q21 and encode 17 kDa proteins that use its nucleoside diphosphate (NDP) kinase , histidine kinase , and exonuclease activities  to inhibit multiple metastatic-related processes. Mutants that disrupt the NDP kinase and exonuclease functions of Nm23 still suppress metastasis to varying degrees, suggesting complex and overlapping roles in metastasis regulation . In this report, we focus only on Nm23-H1. Overexpression of Nm23-H1 in tumor cells reduces tumor cell motility and invasion, promotes cellular differentiation, and inhibits anchorage-independent growth and adhesion to fibronectin, laminin, and vascular endothelial cells [16, 17].
While Nm23 works to prevent the spread of breast cancer, ITGA5 produces an integral membrane protein that increases the metastasis of breast cancer cells . ITGA5 is found on chromosome 12q11-q13 and encodes integrin alpha-5, a fibronectin receptor protein . Through binding to fibronectin, an extracellular glycoprotein, ITGA5 facilitates cellular growth and migration [18, 20]. Integrins associate with adaptor proteins, cytoplasmic kinases and transmembrane growth factor receptors to trigger biochemical signaling pathways . Overexpression of ITGA5 leads to increased cellular adhesion and interaction with fibronectin, resulting in promoted tumor metastasis .
In the present study, we report, for the first time, the effects of alcohol on the Nm23-ITGA5 pathway and show that regulation of this pathway is important for in vitro cellular invasion of T47D human breast cancer cells.
Cell culture, transfection, and siRNA
T47D, MCF-7 and MDA-MB-231 breast cancer cells were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured at 37°C, 5% CO2, on 75-cm3 tissue culture flasks (Becton Dickinson Labware, Franklin Lakes, NJ, USA) in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, St Louis, MO, USA). The Nm23 siRNA, ITGA5 siRNA, and negative controls were purchased from Invitrogen (Carlsbad, CA, USA). pcDNA3-Nm23-H1 cDNA and the control vector were kindly provided by Dr. Patricia Steeg (National Cancer Institute, Bethesda, MD, USA). T47D cells were transfected with the above vectors and siRNAs using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Neomycin-resistant clones were isolated by growth in media containing 800 ug/ml G418 (Gibco, St Louis, MO, USA). Alcohol was added to the medium at concentrations of 0.1%, 0.2%, and 0.5% v/v ethanol. RNA and proteins were collected from the cells 48 hours post alcohol treatment.
The in vitro invasion studies were performed using the BD Bio-Coat Matrigel invasion assay system (Becton Dickinson Labware, Franklin Lakes, NJ, USA). To determine the ability of alcohol to affect the invasive ability of breast cancer cells, 2 × 105 T47D cells were suspended in serum-free DMEM medium containing 0.1% bovine serum albumin (BSA) and placed in the upper chamber. The bottom chamber was filled with DMEM containing 10% FBS. The FBS attracted the cancer cells and triggered their migration to the underside of the membrane. Breast cancer cells that have the ability to invade secrete factors which allow them to degrade the Matrigel (e.g., matrix metalloproteinases) and migrate through the 8 μm pores to the lower chamber of the membrane. After 24 hour incubation, the membrane of the upper chamber was cleaned with cotton swabs to remove the Matrigel and the cells that did not migrate. The membrane was fixed and stained using Diff-Quik solutions (Dade-Behring, Newark, DE). Staining of cells allows their visualization and quantification using a light microscope. Five fields of adherent cells were randomly counted in each well with a Nikon Diaphot-TMD (Atlantic Lab Equipment, Salem, MA, USA) inverted microscope at 20× magnification.
Real-time reverse transcription PCR analysis
Primer sequences used for qRT-PCR
F: 5'-ACC TGA AGG ACC GTC CAT TCT TTG C-3'
R: 5'-GGG TGA AAC CAC AAG CCG ATC TCC T-3'
F: 5'-ACC TGC CTC TTC TCA CCA AG-3'
R: 5'-TAG CAG CTG GCT TCC TCT C-3'
F: 5'-GCA ACT TGA AAG CAC TAA ACC-3'
R: 5'-CAT GTA TGG CCT ACA GCC AG-3'
F: 5'-ACT AAG CAC CCT GAC TAT GCT ATC C-3'
R: 5'-CTT CCA TCA CAT CAC TGA ACA CTT T-3'
F: 5'-CAT GAA TCG CCC TGA GGT CAC CTA-3'
R: 5'-GCC TGC ACC TTC TCC ATG CAG CCC-3'
F: 5'-ACT GAG TCA GCT GCG GTT GCG G-3'
R: 5'-AAG ACC TGG AGC TGC CTC TGG CGT GC-3'
F: 5'-CTG TTC AGG GAC AGA ATG TGC T-3'
R: 5'-TCG ATA TGC TTC ACA GTT CTA GGG-3'
F: 5'-TCA CTC CTG AGA TCT GCA AAC AG-3'
R: 5'-TCA CAG TCC GCC AAA TGA AC-3'
F: 5'-CCC TGG AGA CCT GAG AAC CA-3'
R: 5'-CCA CCC GAG TGT AAC CAT AGC-3'
F: 5'-TCC TCT TCT TGA GCT GGA CTC ATT-3'
R: 5'-CGC TCT GCA AAC TGG AGG TC-3'
F: 5'-TGC CTG CGT CCA TCA ACA CT-3'
R: 5'-CAT CAA ACA CCC AAT GCT TGT C-3'
F: 5'-GTC GGG GGC TTC AAC TTA GAC-3'
R: 5'-CCT GGC TGG CTG GTA TTA GC-3'
F: 5'-TAC CTG GTT GAT CCT GCC AG-3'
R: 5'-GAG CTC ACC GGG TTG GTT TTG-3'
Western blot analysis
Cells were lysed using RIPA buffer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 20 mM MgCl2, 1% Nonidet P40 containing protease inhibitors (1 μg/ml PMSF, 1 μg/ml aprotinin and 1 μg/ml pepstatin). Samples were incubated for 1 hour on ice with agitation and centrifuged at 12,000 × g for 20 min. Protein samples were subjected to electrophoresis on 4-12% SDS-polyacrylamide gradient gels and transferred to a PVDF membrane. Membranes were probed with anti-Nm23-H1 (BD Biosciences, San Jose, CA, USA) and anti-actin (Oncogene, Cambridge, MA, USA) antibodies. Protein-antibody complexes were detected with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) followed by enhanced chemiluminescence reaction. Immunoblots were quantified using ImageJ software (NIH website: http://rsbweb.nih.gov/ij/index.html).
Real-time quantitative PCR array of 84 human extracellular matrix and adhesion molecules
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The cDNA was prepared by reverse transcription using the RT2 PCR Array First Strand kit (SA Biosciences, Frederick, MD) as recommended by the manufacturer's instructions. PCR array analysis of 84 genes related to cell-cell and cell-matrix interactions as well as human extracellular matrix and adhesion molecules (RT2 Profiler™ PCR array, PAHS-013A-1, SA Biosciences, Frederick, MD, USA) was performed using the Mastercycler ep Realplex real-time PCR thermocycler (Eppendorf, Wesseling-Berzdorf, Germany). Briefly, 25 μl of PCR mixture, which contained cDNA equivalent to 1 μg RNA in SuperArray RT2 qPCR Master Mix solution, was loaded in each well of the PCR array plate. PCR amplification of cDNA was performed under the following conditions: 10 min at 95°C for one cycle, 15 sec at 95°C, followed by 1 min at 60°C for 40 cycles. All mRNA Ct values for each sample [Ct (sample)] were normalized to glyceraldehyde-3-phosphate dehydrogenase [Ct (GAPDH)] in the same sample. The relative mRNA level was expressed as the value of 2-ΔΔCt (sample).
One-way analysis of variance (ANOVA) was used to test the statistical significance of the qRT-PCR and invasion assay results (SPSS 12.0 student edition, SPSS Inc. Chicago, IL, USA). To detect statistical significance, p value was set at 0.05, and data are presented as the mean ± standard error of the mean (SEM).
Alcohol increases the invasive ability of breast cancer cells in a dose-dependent manner
Alcohol increases breast cancer cell invasiveness by suppressing Nm23 expression
Down-regulation of Nm23 increases ITGA5 expression to promote breast cancer cell invasion
Effects of alcohol and Nm23 overexpression on extracellular matrix and adhesion proteins expression
0.5% EtOH + Nm23-H1
To establish the relationship between alcohol, Nm23, ITGA5 and cell invasion, we knocked down ITGA5 with siRNA in T47D cancer cells and measured the ability of alcohol to affect the invasive ability of these cells. Results show that down-regulating ITGA5 significantly inhibited the ability of T47D breast cancer cells to invade (Figure 5A, p < 0.05). In agreement that decreased ITGA5 expression reduces cell invasive ability, we show that both the Nm23 overexpressing cells and the alcohol-treated Nm23 overexpressing cells have significantly reduced ITGA5 expression (Figure 4A) as well as have an overall lower cell invasive ability (Figure 3A) compared to controls. We also show that alcohol-treated Nm23 overexpressing cells have slightly higher ITGA5 levels compared to non-alcohol-treated Nm23 overexpressing cells (Figure 4A) and this translated to a slightly higher, although not statistically significant, number of invaded cells (Figure 3A). Nm23 and ITGA5 protein expression in T47D cells is shown in Figure 4B. To examine whether the Nm23-ITGA5 effects on invasion were specific to T47D cells, we exposed MCF-7 and MDA-MB-231 cells to various doses of ethanol. We show that alcohol is able to increase Nm23 and decrease ITGA5 in a dose-dependent manner (Figure 4C) and this correlated with increasing cell invasive ability (Figure 1B). Moreover, when ITGA5 was knocked down with siRNA, alcohol was unable to increase the invasion of T47D cancer cells, suggesting that ITGA5 is necessary for alcohol to increase the invasive ability of T47D cancer cells. Furthermore, in ITGA5 knocked-down cells, suppression of Nm23 by siRNA did not rescue their invasive ability (Figure 5A). Results also show that Nm23 knock-down increased ITGA5 expression; however, knockdown of ITGA5 did not affect Nm23 expression (Figure 5B), suggesting that Nm23 is an upstream factor of ITGA5. Depletion of Nm23 and ITGA5 in T47D cells following siRNA transfection is shown in Figure 5C. In summary, the above findings suggest that alcohol increases the invasive ability of breast cancer cells by down-regulating Nm23, which increases ITGA5 expression, and this elevation in ITGA5 increases the ability of breast cancer cells to invade.
We show that alcohol increases the invasive ability of breast cancer cells in a dose-dependent manner. This suggests that alcohol may increase the ability of the cancer to metastasize. In fact, both animal and epidemiological findings suggest that alcohol increase the metastatic ability of breast cancers . Vaeth et al. showed that frequent alcohol drinkers were 1.45-times more likely to be diagnosed with later stage breast cancer than infrequent drinkers . Additionally, animal studies suggest that alcohol consumption increases the incidence of lung metastasis . Thus, it is critical to understand the mechanism by which alcohol promotes the invasive ability of breast cancer cells in order to develop prevention and treatment options for cancer metastasis. Our data suggest that alcohol increases the invasive ability of breast cancer cells via the Nm23 metastasis suppressor gene. More importantly, we show that the invasive ability associated with alcohol can be blocked by regulating Nm23 levels.
The expression of integrins (e.g., ITGA5) in cancer cells is essential as they allow the cells to attach to the endothelium found within the blood vessels of organs such as the lungs (a secondary site for tumor metastasis) . Thus, the levels of integrins such as ITGA5 determine how aggressively the cancer cells may spread to secondary tissues. Our data shows that alcohol exposure increases the expression of the fibronectin receptor subunit ITGA5 in T47D breast cancer cells. Furthermore, overexpression of Nm23 can block the effects of alcohol on ITGA5 expression. Additionally, results show that suppression of Nm23 by siRNA increases the expression of ITGA5 in the cancer cells, thus, indicating that Nm23 regulates ITGA5 expression. Furthermore, we show that down-regulation of ITGA5 is sufficient to block the effects of alcohol on the invasion of T47D cells. Further investigation with other breast cancer cell lines will be necessary before conclusive statements can be made regarding the involvement of the Nm23-ITGA5 pathway in alcohol-induced breast cancer cell invasiveness. Nevertheless, our results indicate that alcohol decreases the expression of Nm23, thereby allowing ITGA5 to be expressed, which in turn allows T47D breast cancer cells to obtain a more invasive phenotype.
Further investigation is also necessary to better understand how alcohol regulates Nm23 expression and how Nm23 regulates ITGA5 expression. It is well accepted that alcohol may promote breast cancer development via the estrogen signaling pathway . As breast cancer cells are able to produce estrogen in vitro, the binding of estrogen to the estrogen receptor α (ERα) may activate downstream PI3K/Akt and MAPK/ERK pathways to promote cell migration [29, 30]. In a recent study, it was reported that estrogen negatively regulates Nm23 expression in vitro . Thus, the modulation of Nm23 expression shown in this study as a result of alcohol exposure may be mediated by estrogen levels. As a NDP kinase, Nm23 may modify cytoskeleton organization and protein trafficking, possibility through ITGA5, to promote cell migration and adhesion to the extracellular matrix (ECM). Previous studies have shown that Nm23 decreases activity of Rac1, a specific nucleotide exchange factor, through binding of Tiam1 [32, 33]. Reduction of Rac1 activation induces the activity of RhoA, a component in the ITGA5-mediated cellular adhesion and migration signalling pathway [34, 33]. Indeed, estrogen has been found to activate RhoA and this activity is necessary for cytoskeletal remodelling and for the enhancement of breast cancer cell migration and invasion . Thus, down-regulation of Nm23 by alcohol may promote RhoA activation through estrogen regulation to favor ITGA5-mediated breast cancer progression.
The ECM and adhesion molecules play a critical role in the invasive phenotype of cancer cells . For example, the binding of integrins to ECM proteins stimulates the phosphorylation of focal adhesion kinase (FAK); this activated FAK can activate signaling pathways such as PI3K, MAPK, and ERK . These pathways have been shown to regulate cell adhesion, motility, invasion, and metastasis . Integrins are heterodimer cell surface receptors composed of α and β subunits. The integrin α5 subunit (ITGA5) dimerizes exclusively with the β1 integrin (ITGB1) to form the classic fibronectin receptor (α5/β1 or ITGA5B1) . The interaction of α5/β1 with fibronectin (FN) plays an important role in the adhesion of cancer cells to the extracellular matrix . Moreover, previous studies have shown that interaction of α5/β1 with FN promotes activation of the ERK and PI3K signaling pathways, which in turn stimulates cells to invade and produce MMPs (e.g., MMP-1 MMP-9) to facilitate invasion . In our studies, we show that the integrin α5 subunit expression is necessary for alcohol to increase the invasive ability of T47D breast cancer cells. It is possible that alcohol stimulates signaling pathways such as ERK and PI3K, via α5/β1, which then increases the invasive phenotype of T47D breast cancer cells. Consequently, activated integrins may facilitate the movement and metastasis of breast cancer cells. In future studies, we will determine if alcohol affects signaling pathways such as FAK, ERK, and PI3K via ITGA5 and elucidate the role of estrogen in alcohol-mediated down-regulation of Nm23.
Our data suggest that alcohol increases breast cancer cell invasion by regulating the Nm23-ITGA5 pathway. Alcohol exposure in human breast cancer T47D cells down-regulated expression of the Nm23 metastasis suppressor gene, leading to increased expression of the ITGA5 fibronectin receptor subunit, and consequently induced cellular invasion in vitro. Results from this work suggest that modulation of the Nm23-ITGA5 pathway may be important for the prevention and treatment of human breast cancers.
This work was supported by American Cancer Society grant ACS RSG CNE-113703 and by grants from the National Institutes of Health: National Cancer Society grant NCI 1K22CA127519-01A1 and National Institute of Environmental Health Sciences Center grants ES09145 and ES007784.
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