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Multiple sequential genetic and epigenetic alterations underlie cancer development and progression. Overcoming cellular senescence is an early step in cancer pathogenesis. Here, we demonstrate that a noncoding regulatory RNA, microRNA-16 (miR-16), has the potential to induce cellular senescence. First, we examined the expression of miR-16 in primary cutaneous T-cell lymphoma (CTCL) and other non-Hodgkin T/natural killer (NK)-cell lymphomas and found that miR-16 was downregulated than that in the corresponding normal cells. Notably, miR-16 expression was reduced as the primary CTCL progressed from the early stage to the advanced stage. Next, we transduced CTCL cells with miR-16 to examine whether this miRNA exhibited tumor-suppressive effects in CTCL cells. In CTCL cells expressing wild-type p53, forced expression of miR-16 enhanced p21 expression via downregulation of the polycomb group protein Bmi1, thereby inducing cellular senescence. Alternatively, in CTCL cells lacking functional p53, miR-16 induced compensatory apoptosis. The miR-16 transfection significantly decreased senescent cells and increased apoptotic cells in p21-knockdown CTCL cells expressing wild-type p53, suggesting that the presence or absence of p21 may be the most important condition in the senescence-apoptosis switch in CTCL lymphomagenesis. Furthermore, we found that the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) restored the expression of miR-16 and its essential targets, induced senescence in CTCL cells expressing wild-type p53 and promoted apoptosis in cells with nonfunctional p53. Moreover, we found that other T/NK-cell lymphoma cell lines showed similar tumor-suppressive effects in response to miR-16 and SAHA and that these effects were dependent on p53 status. These results suggested that epigenetic silencing of miR-16 may be a key step during lymphoma development. Elucidation of the essential targets of miR-16 and SAHA provides a basis for the clinical application of SAHA in the treatment of CTCL and other non-Hodgkin T/NK-cell lymphomas.

Deletions of the 3p arm have been detected in various solid tumors, but no study to date has investigated this deletion in diffuse large B-cell lymphoma (DLBCL). Recently, we demonstrated that 3p14.2 was deleted in approximately 30% of DLBCL cases by use of a genome-wide array-comparative genomic hybridization (CGH). For a more detailed examination of the genomic losses at 3p14.2, here we made use of contig BAC array for 3p14.2, and found that 12 DLBCL samples displayed losses. All of the deleted regions were located within the fragile histidine triad (FHIT) gene, and the most frequent region of loss was mapped to 0.4 Mbp of the region encompassing the introns 4 and 5 and exon 5 of the FHIT gene. Concomitant analysis of transcripts showed that the FHIT gene was aberrantly transcribed in 31% of the DLBCL samples examined and that the lost exons of the aberrant transcripts were correlated with genomic deletions. These findings indicate that (1) loss of genomic material at 3q14.2 is responsible for exon losses of the FHIT gene, and (2) genomic loss of the FHIT gene is one of the causes of the generation of aberrant transcripts.

The proto-oncogene BMI1 and its product, Bmi1, is overexpressed in various types of tumors, particularly in aggressive tumors and tumors resistant to conventional chemotherapy. BMI1/Bmi1 is also crucially involved in cancer-initiating cell maintenance, and is recurrently upregulated in mantle cell lymphoma (MCL), especially aggressive variants. Recently, side population (SP) cells were shown to exhibit tumor-initiating characteristics in various types of tumors. In this study, we show that recurrent MCL cases significantly exhibit upregulation of BMI1/Bmi1. We further demonstrate that clonogenic MCL SP shows such tumor-initiating characteristics as high tumorigenicity and self-renewal capability, and that BMI1 was upregulated in the SP from recurrent MCL cases and MCL cell lines. On screening for upstream regulators of BMI1, we found that expression of microRNA-16 (miR-16) was downregulated in MCL SP cells by regulating Bmi1 in the SPs, leading to reductions in tumor size following lymphoma xenografts. Moreover, to investigate downstream targets of BMI1 in MCL, we performed cross-linking/chromatin immunoprecipitation assay against MCL cell lines and demonstrated that Bmi1 directly regulated pro-apoptotic genes such as BCL2L11/Bim and PMAIP1/Noxa, leading to enhance anti-apoptotic potential of MCL. Finally, we found that a proteasome inhibitor bortezomib, which has been recently used for relapsed MCL, effectively induced apoptosis among MCL cells while reducing expression of Bmi1 and increasing miR-16 in MCL SP. These results suggest that upregulation of BMI1 and downregulation of miR-16 in MCL SP has a key role in the disease’s progression by reducing MCL cell apoptosis. Our results provide important new insight into the pathogenesis of MCL and strongly suggest that targeting BMI1/Bmi1 might be an effective approach to treating MCL, particularly refractory and recurrent cases.

Mantle cell lymphoma (MCL) is characterized by 11q13 chromosomal translocation and CCND1 overexpression, but additional genomic changes are also important for lymphomagenesis. To identify the genomic aberrations of MCL at higher resolutions, we analysed 29 patient samples and seven cell lines using array-based comparative genomic hybridization (array CGH) consisting of 2348 artificial chromosome clones, which cover the whole genome at a 1.3 mega base resolution. The incidence of identified genomic aberrations was generally higher than that determined with chromosomal CGH. The most frequent imbalances detected by array CGH were gains of chromosomes 3q26 (48%), 7p21 (34%), 6p25 (24%), 8q24 (24%), 10p12 (21%) and 17q23 (17%), and losses of chromosomes 2p11 (83%), 11q22 (59%), 13q21 (55%), 1p21-p22 (52%), 13q34 (52%), 9q22 (45%), 17p13 (45%), 9p21 (41%), 9p24 (41%), 6q23-q24 (38%), 1p36 (31%), 8p23 (34%), 10p14 (31%), 19p13 (28%), 5q21 (21%), 22q12 (21%), 1q42 (17%) and 2q13 (17%). Our analyses also detected several novel recurrent regions of loss located at 1p36, 1q42.2-q43, 2p11.2, 2q13, 17p13.3 and 19p13.2-p13.3, as well as recurrent regions of homozygous loss such as 2p11 (Ig(kappa)), 2q13 and 9p21.3-p24.1 (INK4a/ARF). Of the latter, we investigated the 2q13 loss, which led to identification of homozygous deletions of the proapoptotic gene BIM. The high-resolution array CGH technology allowed for the precise identification of genomic aberrations and identification of BIM as a novel candidate tumor suppressor gene in MCL.

Genetic amplification at chromosome 8p23.1 has been reported in some solid tumors. Translocation of 8p23.1 has also been reported in hematological malignancies and head and neck squamous cell cancer. In an attempt to clarify whether this translocation is implicated in lymphomagenesis, we performed FISH analysis of the immunoblastic B-cell lymphoma cell line OCI-LY8, which has chromosome translocation at 8p23.1, with various BAC clones. We found split signals on BAC, RP11-18L2 where the MASL1 gene is located. This translocation was found to produce a chimeric transcript of MASL1 exon 1 with a cryptic exon from the genome region at 14q21. Our study indicates that MASL1 is not only a target gene for genomic amplification but also for chromosomal translocation. Since tumorigenic activity of the MASL1 has not been proven, its in vitro transforming activity was studied and in vivo nude mice assay were performed. Although no in vitro transforming activity was detected by focus formation, the in vivo tumorigenesis assay with nude mice showed that both MASL1 and chimeric MASL1 possess tumorigenic activity. This suggests that MASL1 is an important oncogene not only for solid tumors but also for hematologic malignancies.