Nidogen 1 and 2 gene promoters are aberrantly methylated in human gastrointestinal cancer
© Ulazzi et al. 2007
Received: 30 January 2007
Accepted: 28 February 2007
Published: 28 February 2007
Nidogens are highly conserved proteins of basement membranes. Two nidogen proteins, nidogen 1 and nidogen 2, are known in mammals.
We show that CpG islands of both NID1 and NID2 genes are aberrantly methylated in human cancer samples and cancer cell lines. For both genes, methylation was correlated with loss of gene transcription in human cell lines. Furthermore, demethylation of the NID1 and NID2 promoters restored gene transcription, demonstrating that methylation was responsible for silencing nidogen genes. In primary tumors, we detected NID1 promoter methylation in 67% of colon cancer samples and in 90% of gastric cancers. NID2 promoter was methylated in 29% of colon and 95% of gastric cancers. Immuno-staining for nidogen-2 confirmed the correlation between aberrant methylation and loss of nidogen expression also in primary tumors, implying that aberrant methylation was a mechanism for inhibiting nidogens expression in human gastrointestinal tumors.
These results suggest that loss of nidogens expression has a potential pathogenetic role in colon and stomach tumorigenesis. Nidogens are believed to connect laminin and collagen IV networks, hence stabilizing the basement membrane structure. Nidogens are also important for cell adhesion, as they establish contacts with various cellular integrins. Loss of nidogen expression may favor invasion and metastasis of cancer cells by loosening cell interaction with basal membrane and by weakening the strength of the basement membrane itself, first barrier from the connective vascularized matrix.
Basement membrane is a thin layer of specialized extracellular matrix. Its main components include collagen IV, laminins, heparan sulfate proteoglycan (perlecan) and nidogens. It can be found under sheets of epithelial and endothelial cells and surrounding muscle, fat and muscle cells. It is essential for tissue compartmentalization and maintenance of cell phenotypes; it also supplies stimuli for tissue development and remodeling [1, 2].
Among its components, nidogen binds and forms ternary complex with collagen IV and laminin, connecting the two networks and stabilizing the tri-dimensional structure of the basement membrane . Nidogen thus serves the important role of establishing and maintaining basement membrane and tissue architecture.
Nidogen also interacts with cell receptor molecules and controls cell polarization, migration and invasion. Nidogen-1, in particular, interacts with β1 family of integrin receptors and with αvβ3 integrin [4–7]. Through interactions with the leukocyte response integrin, nidogen favors neutrophil chemotaxis during the inflammation process. The interactions between cells and basement membranes regulate various cellular processes, including differentiation, proliferation and apoptosis.
In human, two nidogen proteins, nidogen-1 (150 kDa) and nidogen-2 (200 kDa), have been identified. The two proteins share a 46% primary sequence identity and a very similar three dimensional structure, consisting of three globular domains connected by a flexible link and a rod [8, 9].
Both nidogens are co-expressed in various tissues and interact with collagens I and IV and perlecan at comparable level. Differently, in vitro binding of nidogen-2 to laminin γ1 is weaker than for nidogen-1. Also, nidogen-2, differently from nidogen-1, does not interact with fibulins. Both nidogens are also cell-adhesive. The above features indicate that the two proteins might fulfill similar if not identical functions, may be interchangeable in many, but not all the cases and may compensate each other deficiency [10, 11]. This suggestion is confirmed by the findings that both nidogen-1 and nidogen-2 are ubiquitous components of basement membranes underneath epithelia of most of the major organ systems . Furthermore, while deficient mice for either nidogen-1 or nidogen-2 [12, 13] have no overt phenotypic abnormalities and basement membranes appear to be normal, preliminary reports of double mutants indicate a perinatal lethal phenotype and altered basement membrane ultrastructure . At least in the case of nidogen-1 knock-out, nidogen 2 compensation was demonstrated. An increase in nidogen-2 expression and unexpected high affinity of nidogen-2 for laminin γ1 was discovered, thus explaining the lack of phenotype of the NID1-null mice .
In spite of the fact that it is established that abnormal stromal microenvironment contributes to tumor formation and progression and nidogens play a key role in the maintenance of the structural integrity of basement membranes, which establish a barrier for cell movement, migration and invasion, nidogen defects have not been linked to human cancer. Here, we report that methylation of nidogen 1 and 2 promoters is responsible for loss of their gene expression and is frequent in human gastrointestinal tumors.
Materials and methods
The cell lines used in this study were MCF7, MDA-MB-231 and BT-20 from breast carcinoma, SW48 and RKO from colon carcinoma, HEP3B from hepatocellular carcinoma, RT4 from bladder carcinoma, SiHa and HeLa from cervical carcinoma, SK-LU-1 from lung carcinoma, HEY from ovary carcinoma, G401 from a renal rhabdoid tumor, U937 from a histiocytic lymphoma, K562 from a blast crisis of a chronic myeloid leukemia, G361 from a melanoma, RD from a rhabdomyosarcoma. These cell lines were obtained from the American Type Culture Collection (ATCC) and grown in Iscove Medium (Sigma-Alldrich) supplemented with 10% Fetal Bovine Serum (Sigma-Alldrich) at 37°C in 5% CO2 atmosphere.
Treatment with zebularine and trichostatin
Treatment of cells with Zebularine (synthesized at the Department of Pharmaceutical Sciences, University of Ferrara) was carried out for 72 hours at 500 μM concentration. Trichostatin (TSA) (Sigma-Alldrich) treatment was for 24 hours at 500 nM. When combined with zebularine, TSA treatment was added during the last 24 hours. Immediately after treatments, cells were harvested for DNA and RNA purification.
Tumor and normal primary samples
Samples from primary tumors and non-neoplastic specimens were from colorectal, gastric tissues, and from peripheral blood leukocytes. Unselected gastric carcinomas were collected from 20 patients. Colorectal carcinomas (CRC) were collected from 49 patients. Additional files 1 and 2 describe the characteristics of tumor samples. Normal controls included biopsies of stomach mucosa from 13 patients with gastric ulcers and colon mucosa from 24 patients with diverticulosis. All patients included in the study had a histological confirmed diagnosis. Informed consent was obtained from each subject included in the study.
DNA purification and methylation specific PCR
NID1 and NID2 oligonucleotides for MSP and RT-PCR analyses
T (°C) annealing
RNA purification and RT-PCR
Total RNA was prepared using the TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA) in accordance to manufacturer's instructions. RNA was reverse transcribed using Superscript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) in the presence of oligo-dT and random primers. The primers used for NID1 and NID2 cDNA amplification are shown in Table 1. Detection of the GAPDH gene expression was used for cDNA quality control and normalization. Oligonucleotides for GAPDH cDNA amplification were: GAPDH-RNA1107F (5'-CTA TAA ATT GAG CCC GCA GCC-3') and GAPDH-RNA1257R (5'-CCC AAT ACG ACC AAA TCC GT-3'). All PCR reactions were performed with Hot Star Taq polymerase (Qiagen) in a total volume of 12 μl, with the following condition: 35 cycles (25 for GAPDH) of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec. Nucleotide sequence of PCR product was determined to confirm specificity of RT-PCR products.
Nidogen immunohistochemical analysis
Immunohistochemical analysis of nidogen-2 expression was performed on formalin-fixed paraffin-embedded tumor tissue sections using the ultraView Universal DAB detection system and the BenchMark XT automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA). Rabbitt antisera against nidogen-2 was produced by one of the co-author (TS).
Human cancer cell lines are methylated at the NID1 and NID2 promoters
We detected NID1 as a gene reactivated by treatment with the demethylating agent zebularine in the human cancer cell line MDA-MB-231 (data not shown). To confirm that reactivation was induced by promoter demethylation, we analyzed the methylation status of the NID1 promoter in the MDA-MB-231 cell line and in several other human cell lines. In addition, since two nidogen homologous genes exist in the human genome, we also analyzed the methylation status of the promoter of the NID2 gene.
NID1 and NID2 promoter methylation in human primary gastrointestinal neoplasms
NID1 and NID2 promoters DNA methylation
NID1 methylation (%)
NID2 methylation (%)
Stomach, normal mucosa
Colon, normal mucosa
Removal of basement membrane is essential for cell invasion , a regulated process that occurs in trophoblast implantation, organogenesis and angiogenesis . In tumorigenesis, invasion through the basement membrane is a feature of malignancy . Disruption of the integrity of the basement membrane creates an invasion-permissive environment, which may promote cancer cell proliferation and invasion . Basement membrane abnormalities may cause an increase in tumor susceptibility  by bypassing the need for degradation of the basement membrane and by triggering pro-proliferative effects on tumor cells via interaction with stromal fibroblast growth factors, cytokines, and/or matrix proteases .
Examples of abnormalities of basement membranes in human cancer have been described. Malignant melanomas lacked basement membranes when they migrated into the dermis, a process accompanied by a drastically reduced staining for collagen type IV and nidogen . Wilms' tumors are coated with a layer of amorphous material that does not represent a basement membrane, since it contains polysialic acid but not laminin, laminin-nidogen complex, or low density proteoglycan . The role of basement membrane in normalization of tumor cells was functionally proven, for example, by experiments with pre-malignant breast epithelial cells, which undergo growth arrest and differentiate in the presence of a reconstituted physiological basement membrane . Overall, these observations suggest that major alterations in the composition of basement membranes are important in tumor establishment or progression.
Here, we report for the first time that loss of nidogen expression, a common basement membrane protein, is frequent in human gastrointestinal tumors. The mechanism underlying nidogen loss is aberrant promoter methylation of both NID1 and NID2 genes. Our results indicate that aberrant methylation at nidogen genes is an epigenetic defect that is absent in non-neoplastic mucosa and muscolaris, and potentially imparts a selective advantage to tumor cells. The lack of expression of nidogen proteins, which are both present in the basement membranes of the normal adult intestine , impairs the possibility of compensation for constitution of basement membrane. It has also been reported that nidogen can protect laminin-1 against proteolysis , suggesting that nidogen absence may cause additional basement membrane proteins degradation and remodeling. Nidogen loss will certainly affect basement membrane structural integrity. Potentially, this mechanism may facilitate the route to invasion for genetically altered epithelial cells and favor metastasis by promoting angiogenesis.
We wish to thank Augusto Bevilacqua, Pietro Zucchini, Annalisa Peverati and Iva Pivanti for the excellent technical support. This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC), Ministero dell'Istruzione, Università e Ricerca, Ministero della Salute and by Progetto CAN2006-Comitato dei Sostenitori Unife.
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