100K GeneChip microarray data
In the present study, we performed 100K GeneChip microarray analyses of 26 primary MCL and six MCL cell lines. The use of short synthetic oligonucleotides as arrayed elements enabled the detection of genomic imbalances with high resolution (approximately 24 kb) and genotyping was performed simultaneously to identify regions of pUPD. Overall, the identified genomic imbalance pattern was consistent with those previously described by cytogenetics or arrayCGH studies in MCL (Kohlhammer et al, 2004; de Leeuw et al, 2004; Rubio-Moscardo et al, 2005b; Tagawa et al, 2005; Mestre-Escorihuela et al, 2007; Pinyol et al, 2007).
Using the GeneChip mapping technique, we not only delineated previously reported alterations but also identified novel regions of recurrent genomic imbalance in MCL. Taking advantage of the high resolution of the used SNP arrays, we focused on detecting high-level amplifications and small regions of homozygous loss. In this way, novel regions harbouring potential oncogenes or candidate TSG were identified that might be involved in tumourigenesis. Moreover, we introduced two useful bioinformatic approaches for identifying homozygous losses in 100K GeneChip data and point out advantages and disadvantages.
In line with a recent study of five MCL cell lines (Nielaender et al, 2006), LOH analysis of primary MCL tumor tissue demonstrated that pUPD is a recurrent genetic mechanism in MCL tumourigenesis. Genomic distribution of detected pUPD in the analysed primary MCL showed that recurrently affected regions, such as 11q and 13q, are commonly targeted by deletions in MCL. Furthermore, we explicitly demonstrated TSG inactivation by pUPD targeting the TP53 locus in 17p13.1. A homozygous missense mutation affecting a DNA binding domain was detected. This mutation is frequent in lymphomas (28%) and has been reported to be deleterious for TP53-DNA interaction (http://www-p53.iarc.fr). TP53 inactivation by chromosomal deletion is a common chromosomal event in MCL and is associated with poor prognosis (Rubio-Moscardo et al, 2005b). The present study showed that pUPD is an alternative mechanism to chromosomal deletion leading to homozygosity of a TSG inactivating mutation. Thus, pUPD seems to be a critical genetic event in MCL pathogenesis.
Genes encoding microtubule-associated proteins as targets of chromosomal aberrations
Interestingly, analysis of the 100K GeneChip data identified different genes encoding microtubule-associated proteins (MAPs) to be involved in chromosomal alterations in MCL. MAPs are cellular proteins that are associated with microtubules and alter their dynamics. Microtubule dynamic property is crucial for the assembly of the mitotic spindle and the attachment and movement of chromosomes along the spindle (Zhai et al, 1996). Microtubule-targeting drugs suppressing microtubule dynamics are widely used as cancer chemotherapeutic agents (Jordan & Wilson, 2004). In addition to their direct involvement in the physical process of mitosis, microtubules also serve as scaffolds for signalling molecules (Mollinedo & Gajate, 2003). The family of MAPs includes products of oncogenes, tumour suppressors and apoptosis regulators, suggesting that alteration of microtubule dynamics and changes in the scaffolding properties of microtubules may be critical events in tumourigenesis and tumour progression (Bhat & Setaluri, 2007). Until now, alterations in microtubule organisation have not been reported in MCL (Jares et al, 2007).
In this study, a homozygous deletion of the MAP2 locus was identified in the MCL cell line UPN-1. Real time RT-PCR revealed absence of MAP2 expression in UPN-1 and REC-1. REC-1 harbours a heterozygous deletion of part of the long arm of chromosome 2 and epigenetic studies showed complete DNA methylation of the CpG island of the remaining MAP2 allele. Moreover, the DNAs of the MCL cell lines JEKO-1 and HBL-2 were also partially hypermethylated. MSP analysis demonstrated partial hypermethylation in 90% of 20 investigated primary MCL. In one of these cases showing partial hypermethylation, a point mutation affecting the coding sequence of MAP2 was identified. These findings suggest that DNA hypermethylation is a frequent mechanism leading to MAP2 gene inactivation in MCL. MAP2 protein participates in the stabilisation of microtubules and is predominantly expressed in neurons, where it is essential for the regulation of organelle transport within axon and dendrites (Sanchez et al, 2000). Moreover, MAP2 was the first protein shown to copurify and interact directly with the regulatory subunit of the protein kinase A (PKA), also known as cAMP-dependent protein kinase (cAPK) (Vallee et al, 1981; Theurkauf & Vallee, 1982). Among other cellular effects, PKA-catalysed phosphorylation modulates cell growth, cell division and actin cytoskeleton rearrangements. MAP2 protein operates as an A-kinase anchoring protein (AKAP) and targets the PKA to microtubules. Attachment to microtubules occurs through its tubulin-binding domain (Serrano et al, 1984; Hirokawa, 1994). MAP2 also harbours a conserved binding site for phosphatase PP2A, although direct binding of the phosphatase has yet to be reported. PP2A represents a family of heterotrimeric serine/threonine phosphatases implicated in the regulation of a plethora of cellular processes such as apoptosis, transcription, translation, DNA replication, signal transduction, protection against tumourigenesis and cell division (Janssens et al, 2005). Soltani et al (2005) reported that MAP2 expression is associated with prognosis in melanoma. A five-year clinical follow-up study showed longer disease-free survival of patients whose primary tumors express abundant MAP2 as compared with patients with weak or no MAP2 expression. Moreover, exogenous expression by adenovirus leads to cell cycle arrest, growth inhibition and apoptosis in metastatic melanoma cells (Fang et al, 2001; Soltani et al, 2005). Thus, lack of MAP2 expression might be also associated with MCL pathogenesis.
The p53 protein is also associated with microtubules in vitro and in vivo (Giannakakou et al, 2000) and has been reported to regulate other microtubule-associated proteins (Murphy et al, 1999; Johnsen et al, 2000; Mirza et al, 2002). TP53 inactivation by chromosomal deletions or by mutations is a common genetic alteration in MCL. Galmarini et al (2003) demonstrated that microtubule protein composition was altered in TP53 mutants (mut-p53) and dynamic instability of microtubules was significantly increased. Mutation analyses in this report identified seven primary MCL cases to harbour homozygous mutations in the coding sequence of TP53. In all these cases, the second allele got lost by chromosomal deletion or pUPD. Similarly, TP53 was shown to be homozygously mutated in four of six investigated MCL cell lines (Amin et al, 2003; M’Kacher et al, 2003; Camps et al, 2006; Zamo et al, 2006). Galmarini et al (2003) also reported that the MAP6 (also called STOP) protein and its corresponding mRNA-expression were increased in the mut-p53 cells, than in the wt-p53 cells suggesting negative transcriptional regulation of MAP6 by p53 protein. Interestingly, our MCL GeneChip data showed high-level amplification of MAP6 in the three MCL cell lines MAVER-1, JEKO-1 and HBL-2. Real-time RT-PCR detected higher expression of MAP6 in JEKO-1, GRANTA-519 and REC-1 compared with non-tumours tonsil tissue. The apparent lack of correlation between MAP6 dosage and expression in MAVER-1 and HBL-2 might be caused by putative non-neuronal transcripts variants that cannot be detected with the primers used. According to this, the murine homologue shows non-neuronal transcript variants, which lacks whole exons (Bosc et al, 2003). So far, no human non-neuronal transcript variant has been identified. High-level amplification of MAP6 was frequently detected in MCL cell lines but in none of the investigated primary MCL. As cell lines are frequently derived from cases with advanced disease, this finding might indicate that MAP6 amplification is associated with disease progression. In line with this hypothesis, a chromosomal rearrangement of another MAP gene, i.e. MAP4, was recently identified as secondary alteration in a large B-cell lymphoma (DLBCL), which was present at relapse but not at initial diagnosis (Murga Penas et al, 2006). According to our GeneChip data, it is widely assumed that cell lines generally harbour an increased number of chromosomal aberrations compared to the primary tumour cells.
Our study provides evidence that alterations of microtubule dynamics might be critical in MCL tumourigenesis and tumour progression. Nineteen of the 26 primary MCL from the SNP array panel and five of the six MCL cell lines harboured a genetic or epigenetic defect in at least one of the three microtubule-associated genes MAP2, MAP6 and TP53. Six primary cases were not analysed by methylation-specific methods. Only one case and one MCL cell line, in which all three gene loci were analysed, did not show any of the investigated alterations.
The complexity of the mitotic spindle requires fine-tuning of the dynamics of all microtubules for proper function. MAPs can either stabilise or destabilise microtubules. Changes in levels of expression have been reported to correlate with aggressiveness of cancer cells or their sensitivity to microtubule-targeting agents. Although plenty of studies exist regarding microtubule-associated proteins in neurons, where they play a critical role in neurite outgrowth and dendrite development, their mechanisms of operation in mitotic spindle regulation are rather unclear. Mitotic spindle organisation is a fine-tuning process and investigation of the involved proteins is difficult due to low dosage. In contrast to abundant gene expression in brain tissue, expression of MAP2 and MAP6 is hardly detectable in other kinds of tissues. In our study, genomic alterations, such as MAP6 amplification or MAP2 and TP53 inactivation, provide evidence for the involvement of microtubule-associated genes in MCL tumourigenesis. Alterations in microtubule dynamics and mitotic spindle organisation might contribute to the karyotype complexity and chromosomal instability that are characteristic features of MCL. Supporting this hypothesis, a recent study underlined the high expression level of centrosome-associated gene products in blastoid MCL (Neben et al, 2007). MCL has one of the worst prognoses amongst all lymphomas. There is no therapy that can be considered as standard. Resistance against microtubule-targeting chemotherapeutic agents may be the consequence of changes in microtubule dynamics.
In conclusion, our study demonstrated that 100K GeneChip microarray analyses is a useful strategy to analyse MCL genomes with regard to genomic imbalances, particularly homozygous deletions, as well as pUPD. Moreover, we identified novel candidate TSG and oncogene loci that might harbour genes involved in MCL pathogenesis. Interestingly, different genes encoding microtubule-associated proteins could be identified as targets of chromosomal aberrations in MCL. Our findings suggest that alteration of microtubule dynamics is a critical genetic event in MCL.