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Cadherin-13, a Mediator of Calcium-Dependent Cell-Cell Adhesion, Is Silenced by Methylation in Chronic Myeloid Leukemia and Correlates With Pretreatment Risk Profile and Cytogenetic Response to Interferon Alfa

Jose Roman-Gomez, Juan A. Castillejo, Antonio Jimenez, Francisco Cervantes, Concepcion Boque, Lourdes Hermosin, Angel Leon, Albert Grañena, Dolors Colomer, Anabel Heiniger, Antonio Torres

From the Hematology Department, Reina Sofia Hospital, Cordoba; Hematology Department, Carlos Haya Hospital, Malaga; Hematology Department, Hospital Clinic; and Institut Catala d’Oncologia, Barcelona; and Hematology Department, General Hospital, Jerez, Spain.

Address reprint requests to Jose Roman-Gomez, MD, Hematology Department, Reina Sofia Hospital, Avda, Menendez Pidal s/n, 14004 Cordoba, Spain; email: peperosa{at}teleline.es.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Purpose: Cadherin-13 (CDH13) is a newly characterized cadherin molecule responsible for selective cell recognition and adhesion, the expression of which is decreased by methylation in a variety of human cancers, indicating that the CDH13 gene functions as a tumor suppressor gene. Although defective progenitor-stromal adhesion is a well-recognized feature of chronic myeloid leukemia (CML), the role of CDH13 abnormalities has not been evaluated in this disease.

Patients and Methods: We examined the methylation status of the CDH13 promoter in 179 chronic phase (CP)-CML patients and in 52 advanced-phase samples and correlated it with mRNA expression using methylation-specific polymerase chain reaction (PCR) and reverse transcriptase PCR.

Results: Aberrant de novo methylation of the CDH13 promoter region was observed in 99 (55%) of 179 of CP-CML patients, and 90 of the patients failed to express CDH13 mRNA (P < .0001). Advanced-stage samples (n = 52) showed concordant methylation results with their corresponding CP tumors, indicating that CDH13 methylation was not acquired during the course of the disease. Nevertheless, absence of CDH13 expression was more frequently observed among Sokal high-risk patients (P = .01) and was also independently associated with a shorter median progression-free survival time (P = .03) and poor cytogenetic response to interferon alfa treatment (P = .0001).

Conclusion: Our data indicate that the silencing of CDH13 expression by aberrant promoter methylation occurs at an early stage in CML pathogenesis and probably influences the clinical behavior of the disease.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
CHRONIC MYELOID leukemia (CML) is a neoplastic disease of the hematopoietic stem cell that evolves in three clinical stages: the chronic phase (CP), the accelerated phase (AP), and the blastic crisis (BC). It is characterized by a t(9;22)(q34;q11) reciprocal translocation, which gives rise to a 22q- or Philadelphia (Ph) chromosome and a derivative 9q+. The translocation results in a chimeric BCR-ABL gene on the Ph chromosome, which is expressed as a 210-kd protein.¹ This fusion protein has altered kinase activity, which is the source of its oncogenicity, by affecting signal transduction pathways and gene expression. The following three major mechanisms have been implicated in the malignant transformation by BCR-ABL: altered adhesion to stroma cells and extracellular matrix,² constitutively active mitogenic signaling,³ and reduced apoptosis.⁴

Clinically, an increased expansion of committed myeloid progenitors and precursors, elevated levels of mature granulocytes, and premature release of the progenitor/precursor cells characterize CML. This is postulated to be attributable to defects in the adhesion properties of these cells, perhaps because of inside-to-outside disruption of focal adhesion molecule function and subsequent perturbation of adhesion molecules such as b1 integrin. Although malignant CML progenitors express normal levels of b1 integrin receptors on the cell surface, they demonstrate deficient b1 integrin–mediated adhesion to stroma and a4b1 and a5b1 binding regions of fibronectin, indicating abnormal b1 integrin function.⁵ CML progenitors are also unresponsive to b1 integrin-mediated inhibition of proliferation.⁶ Thus, BCR-ABL–induced abnormalities in integrin function may contribute to both the abnormal circulation and continuous unregulated proliferation of CML hematopoietic progenitors. Alternatively, the role of BCR-ABL protein as a docking protein to recruit downstream molecules to actin, rather than its tyrosine kinase activity, may also contribute to defective adhesion.⁷

Reduced cell-cell adhesiveness is considered to be indispensable for both the early and the late carcinogenic steps. In recent years, there has been increasing interest in a large family of transmembrane glycoproteins, called cadherins, which are the prime mediators of calcium-dependent cell-cell adhesion in normal cells⁸ and are also involved in contact inhibition of cell growth by inducing cell-cycle arrest.⁹ There is increasing evidence that modulation of this complex, which acts as an invasion suppressor and as a major growth and proliferation suppressor, by different mechanisms is an important step in the initiation and progression of human cancers.¹⁰ The cadherin-13 (CDH13, also called H-cadherin and T-cadherin) gene, a newly recognized member of the cadherin superfamily, was recently isolated and has been mapped to 16q24.¹¹ Loss of expression and aberrant methylation of the CDH13 gene have been identified in human cancer cell lines and human primary tumors including lung, breast, gastric, colorectal, and ovarian cancers.^12–14 Although the biologic significance of the alterations of CDH13 in human cancer remains to be determined, there is evidence that loss of CDH13 is associated with an enhanced tumorigenicity of human non–small-cell lung cancer in nude mice, which facilitates the implantation and local growth of these tumors.¹⁵

DNA methylation, or the covalent addition of a methyl group to cytosine within the context of the CpG dinucleotide, has profound effects on the mammalian genome. These effects include transcriptional repression via inhibition of transcription factor binding or the recruitment of methyl-binding proteins and their associated chromatin remodeling factors, X chromosome inactivation, imprinting, and the suppression of parasitic DNA sequences.¹⁶ Normal methylation patterns are frequently disrupted in tumor cells, with global hypomethylation accompanying region-specific hypermethylation. When these hypermethylation events occur within the promoter of a tumor suppressor gene, they will silence the gene and provide the cell with a growth advantage in a manner akin to deletions or mutations.¹⁷ In CML, cancer-linked and transcription-repressing de novo methylation is also found and seems to be gene specific rather than a generalized process. Although most of the key tumor suppressor gene promoters examined for hypermethylation have been found to be normal, hypermethylation of several genes (ie, calcitonin 1 gene, BCR sequences, and ABL Pa promoter) has been observed to track with CML progression.^18–20

Despite the crucial role of the CDH13 gene in cell-cell adhesion and in the contact inhibition of proliferation, the expression and methylation status of this gene have never been evaluated in CML and other hematologic malignancies. In this study, we show that the CDH13 gene is subject to methylation regulation at the transcriptional level and is a target of aberrant methylation in CML cells. Moreover, the methylation status of the CDH13 gene seems to be an important factor in predicting the response to treatment and the clinical outcome of CML.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients
Mononuclear layers of bone marrow cells isolated on a Ficoll-Paque density gradient were obtained from 179 patients with Ph-positive CP-CML, diagnosed between August 1982 and December 2001, in five hematology departments in Spain (Reina Sofia Hospital, Cordoba; Carlos Haya Hospital, Malaga; Hospital Clinic, Barcelona; Institut Catala d’Oncologia, Barcelona; and General Hospital, Jerez, Spain). The study was approved by the investigational review boards in accordance with the policies of the Department of Health and Human Services. All patients gave informed consent for the use of their samples. The patients were unselected for type of therapy (90 patients received chemotherapy alone, and 89 received interferon alfa [IFN]). A diagnostic sample in CP was available for analysis in all patients. Paired samples, for which both a diagnostic sample and an AP/BC sample were available, were analyzed in 52 patients (21 patients in AP, 21 in myeloid BC, and 10 in lymphoid BC). AP and BC were diagnosed according to standard criteria.^21,22 Risk categories according to the Sokal, Kantarjian, and Hasford score systems were determined as previously described.^21,23,24 Hematologic and cytogenetic responses to IFN treatment were evaluated according to the criteria of the Houston group.²⁵ The criterion for complete hematologic response was the normalization of the peripheral WBC count to less than 10 x 10⁹/L with the disappearance of immature circulating cells, the normalization of platelet count (less than 450 x 10⁹/L), and the disappearance of all signs and symptoms of the disease (in particular, splenomegaly).

Cytogenetic response was assessed by analyzing at least 10 metaphases and was defined as good by a complete response (0% Ph-positive metaphases) or partial response (1% to 34% Ph-positive metaphases) and was defined as poor by a minor response (35% to 94% Ph-positive metaphases) or no response (95% to 100% Ph-positive metaphases). Mean time of IFN administration was 30.3 months for good responders and 30.2 months for poor responders.

Methylation-Specific Polymerase Chain Reaction (PCR)
Aberrant promoter methylation of CDH13 gene was determined by the method of methylation-specific PCR (MSP), as reported by Herman et al.²⁶ MSP distinguishes unmethylated alleles of a given gene on the basis of DNA sequence alterations after bisulfite treatment of DNA, which converts unmethylated but not methylated cytosines to uracils. Subsequent PCR using primers specific to sequences corresponding to either methylated or unmethylated DNA sequences was then performed. Primer sequences of CDH13 for the unmethylated reaction were forward (5'-TTGTGGGGTTGTTTTTTGT-3') and reverse (5'-AACTTTTCATTCATACACACA-3'), which amplify a 242–base pair (bp) product. Primer sequences for the methylated reaction were forward (5'-TCGCGGGGTTCGTTTTTCGC-3') and reverse (5'-GACGTTTTCATTCATACACGCG-3'), which amplify a 243-bp product. Briefly, 2 mg of genomic DNA was denatured by treatment with NaOH and modified by sodium bisulfite. DNA samples were purified using Wizard DNA purification resin (Promega Corp, Madison, WI), treated with NaOH, precipitated with ethanol, and resuspended in 20 mL of water. Two milliliters of modified DNA was PCR amplified in a total volume of 50 mL. Hot start PCR was performed for 35 cycles and consisted of denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 1 minute, followed by a final 7-minute extension for all primer sets. DNA samples from bone marrow (n = 20) and peripheral blood (n = 35) from healthy donors age-matched with our CML patients (median age, 50 years; interquartile range, 37 to 70 years) were used as negative controls for methylation-specific assays. Human male genomic DNA universally methylated for all genes (Intergen Co, Purchase, NY) was used as a positive control for methylated alleles. Water blanks were included with each assay. PCR products were visualized on 2% agarose gels stained with ethidium bromide. Results were confirmed by repeating bisulfite treatment and MSP assays for all samples. The sensitivity of this MSP was established by using totally methylated, positive control DNA serially diluted by normal lymphocyte DNA. MSPs with 1:10, 1:100, and 1:1,000 diluted positive control DNA produced detectable methylated bands (data not shown).

Expression of CDH13 Gene
Expression of CDH13 gene was analyzed by the reverse transcriptase PCR (RT-PCR) technique with forward and reverse primers located in exon 2 and exon 3 of the CDH13 gene, respectively. We previously confirmed that genomic DNA was not amplified with these primers. Total RNA was extracted from marrow samples with Ultraspec (Biotecx, Houston, TX) following the manufacturer’s instructions. Reverse transcription was performed on 1 mg total RNA, after the sample was heated at 70°C for 5 minutes, with random hexamers as reaction primer. The reaction was carried out at 42°C for 45 minutes in the presence of 12 U Avian Myeloblastosis virus reverse transcriptase (Boehringer-Mannhein, Germany). Complementary DNA was amplified by means of a primer set that was specific for the CDH13 gene (sense, 5'-TTCAGCAGAAAGTGTTCCATAT-3'; antisense, 5'-GTGCATGGACGAACAGAGT-3'). The PCR reaction was performed as follows: 94°C for 5 minutes, 40 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and incubation at 72°C for 10 minutes. PCR products were resolved on 2% agarose gels. Amplification of RARa gene transcript was performed to assess RNA integrity.²⁷

Statistical Analysis
All calculations were performed with the SPSS statistical package (SPSS, Chicago, IL). Medians and interquartile ranges were calculated for age and clinical and laboratory findings at diagnosis for patients with and without CDH13 expression (Table 1) and tested for any significant differences with the Mann-Whitney U test (for continuous variables) or ² analysis and Fisher’s exact test (for categorical variables). Overall survival (OS) was calculated from time of diagnosis to death from any cause and was censored only for patients known to be alive at last contact. Progression-free survival (PFS) was measured from CML diagnosis to the appearance of AP/BC or death without disease progression, and it was censored only for those patients alive and without evidence of progression at last follow-up. For both OS and PFS calculations, bone marrow transplantation recipients were censored at the time of transplantation. Distributions of OS and PFS curves were estimated by the Kaplan-Meier method, with 95% confidence intervals (CIs) calculated by means of Greenwood’s formula. Comparisons of OS and PFS between groups were based on the log-rank test. Comparisons adjusted for significant prognostic factors were based on Cox regression models and hazards regression models. All progression and survival data were updated on May 31, 2002, and all follow-up data were censored at that point.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

View larger version (46K): [in this window] [in a new window]   Fig 1. (A) Representative methylation-specific polymerase chain reaction in chronic myeloid leukemia samples (lanes 1 to 4) and a methylated control (lane 5). Methylated bands were present in all samples except lane 3. (B) Expression of CDH13 and RARa (as control) in the same specimens. Methylated samples show lack of CDH13 expression.

Of the 179 CP-CML diagnostic samples examined, 99 (55%) demonstrated de novo methylation of the CDH13 promoter region. Ninety (91%) of 99 methylated tumors failed to express detectable mRNA of CDH13, as assessed by RT-PCR (Fig 1). Statistical analysis demonstrated a significant association between methylation of the CDH13 gene and absence of mRNA expression (P < .0001, Table 1). Although methylation of the CDH13 promoter region was significantly associated with lack of CDH13 expression, some discordant values were recorded. Eleven percent of tumors (nine of 79 tumors) that expressed detectable levels of CDH13 also showed methylation of the gene. In an additional 9% (nine of 100 tumors), there was no apparent methylation despite loss of expression, probably indicating other forms of transcriptional silencing in these cases (Table 1).

To determine whether methylation of the CDH13 gene is associated with tumor progression, we examined 52 CML patients in AP or BC and the same tumors in CP. Of these 52 AP/BC samples, 35 (67%) had cells with methylated CDH13 and all of them showed concordant methylation results with their corresponding CP tumors, indicating that methylation of this locus is not detectable with increasing frequency as the CML progresses. Four of the 35 patients with methylated samples were able to express CDH13 at diagnosis and only lacked this expression in more advanced phases of the disease.

CDH13Expression and Pretreatment Clinical Features
The clinical and laboratory characteristics of CML patients with and without CDH13 expression at diagnosis are shown in Table 1. Individual factors such as sex, age, enlarged spleen, peripheral-blood blast cells, platelet count, hemoglobin level, and WBC count were not significantly associated with CDH13 expression. Correlating CDH13 expression with pretreatment risk groups, we detected a significant association between absence of CDH13 expression and high-risk patients assessed by the Sokal scoring system (42% of high-risk patients lacked CDH13 v 20% of low-/intermediate-risk patients; P = .01). A trend toward significance was also observed for the Hasford high-risk group (40% of high-risk patients lacked CDH13 v 21% of low-/intermediate-risk patients; P = .07) and Kantarjian high-risk group (30% of stage III patients lacked CDH13 v 13% of stage I to II patients; P = .08).

CDH13Expression, Response to Treatment, and Clinical Outcome
CML patients in this study were treated with chemotherapy (mainly hydroxyurea, n = 90) or IFN-based regimens (n = 89). Forty-five patients received stem-cell transplantation (14 patients received autologous and 31 patients received allogeneic transplantation). Type of treatment administered and number of patients who received transplantation were similarly distributed between the two CDH13-expressing groups (Table 1). Moreover, mean time of IFN administration was not different between patients with normal CDH13 expression (30 months) and nonexpressing patients (30.2 months). Lack of CDH13 expression correlated with poor response to treatment (Table 1). Thus, complete hematologic response rate was significantly lower among nonexpressing patients than in patients with normal levels of CDH13 (79% v 91%, respectively; P = .05). Moreover, among the 89 patients receiving IFN therapy, a good cytogenetic response was observed in 14 (35%) of 40 patients with normal CDH13 expression but only in two (4%) of 49 nonexpressing patients. This difference was highly significant (P < .0001). A multivariate analysis including the clinical factors described in Table 1 demonstrated that the expression of the CDH13 gene was the only independent factor predicting the cytogenetic response to IFN (Table 2). IFN had no effect on CDH13 expression or methylation in the follow-up of responding and nonresponding patients.

View larger version (30K): [in this window] [in a new window]   Fig 2. Kaplan-Meier analysis of the length of the chronic phase showing prognostic significance of CDH13 expression, Hasford score, Sokal score, and Kantarjian score.

View larger version (29K): [in this window] [in a new window]   Fig 3. Kaplan-Meier analysis of overall survival showing prognostic significance of CDH13 expression, Hasford score, Sokal score, and Kantarjian score.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

Although methylation of the CDH13 promoter region was significantly associated with lack of CDH13 expression, a small number of methylated patients (11%) were able to express detectable levels of CDH13 at diagnosis but lacked this expression as CML progressed. Although the presence of contaminating RNA from normal bone marrow cells cannot be ruled out in these cases, they also may indicate the period of time necessary for methylation to cause stable transcriptional repression. In fact, after methylation of DNA has occurred and before transcriptional inhibition, transcription repressors (ie, methyl CpG-binding proteins) associate with a corepressor complex that includes histone deacetylase. This results in deacetylation of histone, resulting in the formation of a stable repressive complex, tighter nucleosomal packing, and, thus, chromatin compaction and gene silencing.³⁰ Moreover, this study cannot exclude other potential mechanisms of CDH13 silencing because, in a minority of patients (9%), there was no apparent methylation despite loss of CDH13 expression, which may indicate a defect in a certain transcription factor that normally activates CDH13 promoter or an inactivating mutation leading to transcript instability. However, given the prevalence of the methylation, these other mechanisms are not likely to be commonly involved.

Despite a plethora of descriptive studies indicating that de novo methylation in promoter regions is associated with transcriptional silencing of tumor suppressor genes, the mechanistic basis and the initiating signal for ectopic de novo DNA-methyltransferase (DNMT) activity during cancer development remain obscure. CML shows phase-dependent expression of DNMTs.³¹ In the CP, levels of DNMTs are not significantly different from those in normal bone marrow cells,³¹ indicating that overexpression of DNMTs is not involved in the methylation of the CDH13 gene observed in our patients at diagnosis. A more attractive hypothesis is to establish a mechanistic link between genetic (BCR-ABL fusion) and epigenetic changes (CDH13 methylation) during transformation. In fact, it was recently reported that the leukemia-promoting promyelocytic leukemia retinoic acid receptor (PML-RAR) fusion protein induces gene hypermethylation and silencing in acute promyelocytic leukemia.³² This hypothesis suggests a scenario in which oncogenic transcription factors (PML-RAR and perhaps BCR-ABL) recruit DMNTs to target promoters. Newly methylated CpGs (CDH13 promoter in our study) then become docking sites for methyl-binding proteins, which in turn interact with both histone deacetylase complexes and DNMTs. If the initial recruitment step is not prevented, it may eventually lead to the spreading of hypermethylation to the neighboring regions, locking these into a stably silenced chromatin state.³² Whether CDH13 gene is methylated by this mechanism requires further investigation but could explain the recent observation that changes in adhesion induced by BCR-ABL are independent of its tyrosine kinase activity.⁷

What is the functional significance underlying the methylation-mediated transcriptional loss of CDH13 in CML? CDH13 is a unique cadherin cell adhesion molecule that is anchored to the cell-surface membrane through a glycosyl-phosphatidyl-inositol (GPI) moiety.³³ Although CDH13 lacks a typical cytoplasmic domain, which induces reorganization of the actin cytoskeleton,³⁴ CDH13 is still able to mediate calcium-dependent cell-cell adhesion. Moreover, the CDH13 amino acid motif has been well conserved through evolution in vertebrates,³⁵ indicating that CDH13 may have biologic significance in higher animals. In this study, lack of CDH13 expression was found to be correlated with three dismal prognostic features in CML patients: absence of CDH13 expression was more frequently observed among high-risk CML groups and was also associated with shorter PFS time and poor cytogenetic response to IFN. These findings, together with the general expression of CDH13 in normal bone marrow, the emerging role of the CDH13 gene in human cancer,^12–14 the observation that transfection of tumor cells with CDH13 entails a decrease in the proliferative and invasive activities both in vitro and in vivo as a result of challenging the mice with tumorigenic cell lines, and the loss of cancer-cell sensitivity to the action of growth factors^11,36,37 are supportive of CDH13 inactivation contributing directly to the clinical behavior of CML, at least in a subgroup of patients.

IFN, a first-line therapeutic agent in CML, seems to reverse the adhesion defect in this disease by correcting impaired b1 integrin receptor function.³⁸ One could speculate that CDH13 nonexpressing patients have an additional second change affecting adhesion and, therefore, exhibit more difficulties in achieving a good response with IFN. However, it cannot be ruled out that maintenance of the mechanical junctions between cells is not the main function of CDH13 protein. It most probably serves as a signal receptor, a sensor that allows the cell to sense its environment. This hypothesis is corroborated by the data on CDH13 distribution in the membrane. Thus, in the polarized intestinal cells, it is located on the apical part of the cell rather than in the adhesive junctions on the basolateral cell surface.³⁹ It has long been known that many other GPI proteins may activate intracellular signaling.⁴⁰ The absence of the cytoplasmic domain in these proteins implies the presence of a membrane adapter protein. Owing to the interaction with the latter, the signal can be relayed into the cell. It has been reported that, similar to other GPI proteins, CDH13 is located on the cell surface in special plasma membrane domains (caveolae and lipid rafts),⁴¹ which also contain other signal molecules (such as G-proteins, src kinases, ras protein, and transmembrane receptors of growth factors).⁴² It is probable that some of these molecules may serve as messengers during activation of CDH13-dependent signaling and could, therefore, contribute to the CML aggressiveness after CDH13 inhibition observed in our study.

In conclusion, our results strongly indicate that the silencing of CDH13 expression by aberrant promoter methylation occurs at an early stage in the multistage process of CML and plays a role in the clinical behavior of the disease.

	NOTES

 
Supported by grant nos. 01/0662, 99/0103, and 02/1299 from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Spanish Ministry of Health.

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Submitted August 27, 2002; accepted January 7, 2003.

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