Introduction
The low-density lipoprotein receptor-related protein 1 (LRP1, ID 6692 in the Human Gene Nomenclature database) is a member of the low-density lipoprotein receptor (LDLR) family [1]. Receptors of this family mediate the internalization of a variety of extracellular macromolecules and macromolecular complexes, including lipoproteins, proteinases, proteinase–inhibitor complexes and extracellular matrix proteins [2]. Recent studies have demonstrated that several receptors of this family are also involved in ligand-mediated signal transduction [3].
LRP1 is a large endocytic receptor that recognizes more than 30 different ligands. Proteinases and molecules associated with the regulation of proteolytic activity are the largest group of LRP1 ligands. Certain proteinases bind directly to LRP1, whereas several other proteinases only bind once complexed with their specific inhibitors [2]. LRP1 is involved in the tight regulation of serine proteinases of the plasminogen/plasmin system and also in the regulation of certain matrix metalloproteinase family members [1, 4, 5]. Through the regulation of extracellular proteolytic activity, LRP1 has an important role in the regulation of cellular growth, cell migration, tissue repair and remodeling, and tumor progression and invasion [1, 3, 6].
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In addition, it has been shown that LRP1 is a signaling receptor involved in inositol and Wnt signaling, Ras activation and the activation of Src, mitogen-activated protein kinases and protein kinase A. Although the function of LRP1 in these processes is less well understood, several authors have suggested that LRP1 might modulate mitogenic signaling, cell adhesion, cell proliferation and apoptosis [3, 7, 8].
Besides the role of LRP1 in protease regulation and cell signaling, Binder and colleagues [9] also suggested that it acts, through the binding of the heat shock protein gp96 on the surface of antigen-presenting cells, as a sensor for necrotic cell death leading to proinflammatory immune responses.
Because of its potential role in processes mentioned above, the relationship between LRP1 expression and cancer establishment, progression and invasion has been studied. Increased expression of LRP1 has been shown in human malignant astrocytes and the glioblastoma cell line U87 [10, 11]. In addition, Li and colleagues [6] observed that LRP1 promotes invasiveness of breast cancer cells in vitro. However, previous studies also showed that LRP1 expression decreases with aggressiveness in several human tumor cell lines (lung carcinoma, osteosarcoma) compared with nontumor cell lines [12, 13].
In addition, LRP1 gene amplification has been observed in some astrocytomas [14], and Grimsley and colleagues [15] reported that the concentration of LRP1 protein increases with the passage number of cultured Hep G2 hepatoma cells, possibly owing to the selection of cells with LRP1 gene amplification. Another interesting finding has recently been observed [16, 17]: in these papers it was suggested that LRP1B, a member of the LDLR family with striking homology to LRP1, represents a tumor suppressor gene in non-small cell lung cancer cell lines [16] and urothelial cancer cells [17]. In their studies the authors reported frequent alteration of the LRP1B gene in non-small cell lung cancer cell lines and urothelial cancer cells, respectively.
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The common silent C766T polymorphism in exon 3 of the LRP1 gene has been associated with Alzheimer's disease [18, 19], suggesting that this polymorphism might be of functional importance or might be a marker of other functional changes located in this gene. Our study was undertaken to investigate an association between the C766T LRP1 polymorphism and the occurrence of breast cancer in a sample of Czech women of Caucasian origin.
Materials and methods
Subjects
The populations tested comprised 164 females diagnosed with breast cancer (mean age 57 years, range 35–80 years), as a control population, and 183 age-compatible female volunteers (mean age 57 years, range 40–94 years) with no history of cancer of any type. The cancer patients were recruited in collaboration with several oncologists and surgeons in the city of Brno, Czech Republic. All cancers were confirmed histologically. The control subjects were also recruited from several districts of the city of Brno by general practitioners from their clientele. Both the patients and the controls were unrelated Caucasians of Czech nationality. The study was approved by the Committee for Ethics of Medical Experiments on Human Subjects, Medical Faculty, Masaryk University, Brno.
Methods
Genomic DNA was extracted from peripheral blood leukocytes. The LRP1 C766T genotypes were determined by polymerase chain reaction (PCR) and a subsequent restriction analysis with RsaI restriction endonuclease as described by Hollenbach and colleagues [19]. PCR was performed in a total volume of 25 μl containing 50 ng of genomic DNA, 10 pmol of each primer (5'-GGGGTCCAGGACTGCATGTA-3' and 5'-AAGTCCG-TACCTCGGCAGTG-3'), 1 × reaction buffer (MBI Fermentas, Vilnius, Lithuania), 200 μM deoxyribonucleotides (Roche Diagnostics, Mannheim, Germany), 3.0 mM Mg2+ and 0.2 units of Taq polymerase (Fermentas). The amplification conditions were as follows: initial denaturation at 95°C for 3 min, followed by 34 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, elongation at 72°C for 20 s and final elongation at 72°C for 2 min. PCR products were digested with 5 units of RsaI (Fermentas). Digested fragments were separated on 4.0% MetaPhor agarose gels and detected by ethidium bromide staining. The presence of the T allele was characterized by fragments of 51 and 8 base pairs, the presence of the C allele by fragments of 32, 19 and 8 base pairs.
Statistical analysis
Differences in genotype distributions from those expected for Hardy–Weinberg equilibrium were tested by the χ2 test. The significances of differences in allele frequencies among groups were tested by Fisher's exact test. Odds ratios and 95% confidence intervals were calculated. P < 0.05 was considered statistically significant. For all statistical analyses, the data analysis software system STATISTICA, version 6 (StatSoft, Inc; http://www.statsoft.com was used.
Results
Allele frequencies and genotype distribution of the C766T LRP1 polymorphism in the breast cancer and the control groups are shown in Table 1. The distribution of genotypes in the breast cancer and the control groups did not differ from Hardy–Weinberg equilibrium. The distribution of alleles in the control group is identical with that previously reported in control subjects from the same Caucasian population [20]. The LRP1 T allele frequency was significantly higher in women with breast cancer than in the control subjects (0.21 versus 0.15; P = 0.01963). The combined number of subjects with the T allele (CT heterozygotes and TT homozygotes) was significantly higher in the breast cancer subjects than in the controls (odds ratio 1.743, 95% confidence interval 1.112–2.732).
Table 1
Distribution of genotypes and allele frequencies of the LRP1 C766T polymorphism in both breast cancer and control groups
Genotypes | Allele frequencies | ||||||
|---|---|---|---|---|---|---|---|
Group |
n
|
CC
|
CT
|
TT
|
C
|
T
|
P
|
Controls | 183 | 132 | 47 | 4 | 0.85 | 0.15 | |
Breast cancer | 164 | 98 | 62 | 4 | 0.79 | 0.21 | 0.01963 |
Discussion
Tumorigenesis is a multistep process that involves cell transformation, invasive growth, angiogenesis and spread of the tumor into distant sites [21]. It has previously been suggested that LRP1 is involved in the regulation of some of these processes.
The breakdown of the basement membrane and extracellular matrix facilitates cancer cell invasion and migration into the surrounding normal tissues [22]. This critical step in cancer progression is mediated by serine proteinases and metalloproteinases [23]. It has been demonstrated that LRP1 modulates the extracellular levels of several matrix metalloproteinases [4, 5] as well as the extracellular level of urokinase plasminogen activator (uPA), a serine proteinase that activates plasminogen [2]. Extracellular proteolytic activity is an important factor in cancer establishment and progression [24‐26]. However, recent studies indicate that proteinases participate not only in cancer progression and metastasis but that they are important in all stages of tumorigenesis including tumor establishment, growth and neovascularization [25]. Degradation of the extracellular matrix mediated by these proteinase systems perturbs cellular signaling, interferes with the adhesiveness of cells and activates several growth factors, all of which processes are important in tumorigenesis [25‐27].
Lutz and colleagues [7] have recently shown that LRP1 modulates the mitogen-induced c-Jun amino-terminal kinase (JNK) signaling pathway and therefore the transcriptional activity of the transcription factor activator protein 1 (AP-1), which has been implicated in a variety of biological processes including cell proliferation, apoptosis and oncogenic transformation [28]. Moreover, Lutz and colleagues found that the expression of a fusion protein bearing the intracellular carboxy-terminal tail of human LRP1 and the transmembrane region plus the extracellular IgG-Fc domain selectively reduced DNA-damage-induced apoptosis in neuronal cells. The authors suggested that LRP1 is involved, by modulating the JNK signaling transduction pathway, in apoptotic and stress responses [7].
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JNK lies downstream in the Ras signaling pathway. Ras proteins are key transducers of growth signals regulated by receptors on the cell surface [29]. Recent studies have shown that the phosphorylated cytoplasmic tail of LRP1 associates both in vivo and in vitro with the cellular docking protein Shc [30]. It has been suggested that this association is important for the phosphorylation of Shc. Phosphorylated Shc proteins are important mediators of Ras activation. Phosphorylation of tyrosine residues of LRP1 is thus believed to be involved in signal transduction. The role of LRP1 in other types of cell signaling has been also suggested [3, 8].
Our study was designed to investigate the role of C766T LRP1 polymorphism in breast cancer development. We observed that the LRP1 T allele frequency was significantly higher in patients with breast cancer than in the age and sex-compatible control subjects. These findings imply that female bearers of the LRP1 T allele are more susceptible to breast cancer development than women with the LRP1 CC genotype. However, large studies will be needed to confirm this finding.
We are aware that association studies have several limitations. The heterogeneity between patients and control subjects in terms of age and ethnicity could explain false positive results. Moreover, small population-based samples might not be representative in terms of the allele and genotype frequencies of whole population. However, it seems that this is not true of this study. Patient and control subjects were age-compatible unrelated Caucasians recruited from a homogenous Czech population. Moreover, the LRP1 T allele frequency of the control group from this study is almost identical with that previously observed in a large sample of subjects from the same Caucasian population [20]. The fact that allele and genotype frequencies in all samples studied did not differ from Hardy–Weinberg equilibrium also suggests that the observed allele and genotype frequencies are representative of the Czech population.
Previous reports suggested that LRP1 promotes invasiveness of breast cancer cells in vitro [6] and that the expression of LRP1 in human breast cancer cells is induced by hypoxia [31], estrogen and cell density [32]. In addition, Chazaud and colleagues [33] reported that uPA and LRP1 are localized at filopodia of invasive breast cancer cells, and that the formation/internalization of a complex of LRP1 with uPA, uPA receptor and plasminogen activator inhibitor-1 is required for the attachment and migration of cancer cells in vitro.
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So far, little is known about the functional importance of the silent C766T LRP1 polymorphism. Numerous studies have associated this polymorphism with an increased risk of Alzheimer's disease [18, 19] suggesting that this polymorphism is probably a marker of other functional changes located in this gene. However, although the C766T polymorphism causes neither an amino acid change nor an alteration of a consensus splicing site, we cannot discount the C766T variant as a neutral polymorphism. A functional LRP1 polymorphism could (1) influence the expression of the LRP1 gene and thus the number of LRP1 receptors on the cell surface, (2) influence the affinity of LRP1 for its ligands and thus the uptake of several proteases and their inhibitors, and (3) alter the signaling activity of LRP1. The exact role of the C766T polymorphism in LRP1 remains to be resolved.
Conclusion
The results of this study indicate that the T allele of the C766T polymorphism in the LRP1 gene is associated with an increased risk of breast cancer development in female Caucasian subjects. Further studies are needed to establish an influence of this and other LRP1 polymorphisms on LRP1 gene expression and/or protein function. The role of LRP1 polymorphisms in other types of cancer also remains to be solved.
Acknowledgements
Recruitment of patients and control subjects was made in co-operation with Dr D Macková, Dr I Kocáková and Dr O Bednarík. Excellent technical assistance from Ms Tereza Vávrová, Ms Andrea Stejskalová and Ms Anna Pekárková is gratefully acknowledged. This study was supported by grant CEZ J07/98:141100002 from the Ministry of Education, Youth and Physical Training of the Czech Republic.
Competing interests
None declared.