Abstract Immortalized models of the mouse B lymphocyte lineage have proven to be valuable resources for a wide variety of B cell neoplasms and for the study of normal B cell differentiation. Whether these models have been engineered in mice (in vivo) or in cell culture (in vitro), there are distinct advantages and disadvantages to both. Homogeneity and ease of accessibility can be considered a benefit of working with cultured cells, whereas the contribution from accessory or stromal cells can be an important element not readily available in the in vitro model system. We originally used immortalized Burkitt's Lymphoma (BL) cells to discover a cluster of microRNAs, miR-1204∼1208, that map within the noncoding PVT1 locus in the region of human 8q24 region down-stream of MYC. The BL cell lines allowed us to isolate a large amount of RNA from a homogeneous resource that enhanced discovery of low-level transcripts. Using probes for mouse miR-1204∼1208 to examine expression in a panel of mouse cell lines representing different stages of B cell development, we were able to show that expression of miR-1204∼1208 appeared to arise at the small B cell stage and that these higher levels of expression continued through to the mature plasma cell. This suggested that the pre-B cell or naïve small B cell stages may be most illuminating in assigning targets and function to miR-1204∼1208 or PVT1. To determine if over-expression of one of these miRNAs, miR-1204, influences the latency and/or type of B cell malignancy we used two mouse transgenic (TG) models of B cell malignancy, H2-Ld-hu-IL6 and iMyc, lentiviral expression of miR-1204 reduced the latency of tumor development in both models and in the case of iMyc-TG, there was also a shift in tumor type from late stage plasmacytoma to the earlier stage of large B cell lymphoma. However, further interpretation of these results was confounded by heterogeneity of lentiviral integration and expression among tumors. Thus, we turned to several human and mouse in vitro models of B cell development, representing pro-B, pre-B, small B, mature B and plasma cell stages to address the effects of modulating the expression of miR-1204 and its host noncoding transcript, PVT1. Over-expression of miR-1204 has been achieved through the use of lentiviruses or synthetic mimics and suppressed expression has been achieved through application of synthetic inhibitors. We also over-expressed PVT1 using a lentiviral vector and suppressed PVT1 expression through the use of siRNA corresponding to various exons of PVT1. Resultant changes in growth and morphology of these cell lines hint that microarray expression analyses will reveal functional targets of miR-1204 in normal and malignant lymphoid development. It will also be of interest to examine whether over-expression or inhibition of miR-1204 plays an additional role in maturation of the normal B cell. Citation Format: Konrad Huppi, Oliver L. Ou, Jason J. Pitt, Brady Wahlberg, Tamara L. Jones, Vishala Neppalli, Siegfried Janz, Natasha J. Caplen. Noncoding RNAs of the 8q24 locus: Consequences of the over-expression or suppression of miR-1204 and PVT1 in developing B cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1834. doi:10.1158/1538-7445.AM2013-1834

Understanding the functional effects of the wide-range of aberrant genetic characteristics associated with the human chromosome 8q24 region in cancer remains daunting due to the complexity of the locus. The most logical target for study remains the MYC protooncogene, a prominent resident of 8q24 that was first identified more than a quarter of a century ago. However, many of the amplifications, translocation breakpoints and viral integration sites associated with 8q24 are often found throughout large regions surrounding the MYC locus and often include other loci. In addition, Chr. 8q24 is host to a number of SNPs associated with cancer risk. Yet, the lack of a direct correlation between cancer risk alleles and MYC expression has also raised the specter of doubt that MYC is the sole target of these genetic associations. The 8q24 region has been described as a gene desert because of the paucity of functionally annotated genes located within this region. In this review, we examine the current evidence for the involvement of other loci within the 8q24 region, most of which are non-coding transcripts, either in concert with MYC or independent of MYC, as possible candidate gene targets in malignancy.

The 8q24 locus has been found to be involved in many types of cancers as a consequence of somatic changes associated with chromosome instability including amplification, translocation or deletion or frequent viral integration (HPV). A number of SNPs in Genome Wide Association (GWA) studies have also implicated the 8q24 locus as a region of susceptibility for many types of cancer. The most likely 8q24 candidate target may be the MYC proto-oncogene that is a well characterized transcription factor. However, the assumed correlation between MYC expression and disease is lacking suggesting a connection between 8q24 involvement and disease is much more complicated than simply targeting MYC. While other transcription units also reside within the 8q24 locus (PRNCR1, POU5F1P1, PVT1 and the miRNA cluster miR-1204∼1208), they are remarkable in that no coding potential has been readily associated with any of these genes. Thus, the region has been referred to as the “8q24 Gene Desert”. With the renewed realization that many noncoding RNAs do have a functional role, the location of the miR-1204∼1208 cluster of miRNAs within the PVT1 lincRNA transcriptional unit actually suggests an “Oasis of transcription” that could be the additional or alternative target to MYC. We have now compared expression of transcripts of the miR-1204∼1208 cluster with MYC and PVT1 in multiple cancer cell lines and we have found them to be uniformly up-regulated in expression, particularly in those cell lines with amplified or translocated 8q24. These results confirm a pilot study of microdissected primary prostate tumors that also show correlated high expression in MYC, miR-1204∼1208 and PVT1. To further analyze the functional role of the 8q24 transcripts, we have now introduced mimics or inhibitors of each miRNA and siRNAs corresponding to MYC and PVT1 into prostate or colon cell lines (with and without amplified 8q24). In amplified prostate cell lines, mimics of miR-1204, miR-1206 and miR-1208 appear to be as effective as MYC or PVT1 siRNAs in arresting cell growth and inducing apoptosis. This result is not seen in normal prostate cells. Predictably, inhibitors of the miR-1204, miR-1206 and miR-1208 seem to have no effect or seem to enhance cellular proliferation. In colon cell lines with amplified 8q24, miR-1204, miR-1206, miR-1207 and miR-1208 mimics all appear to be as effective in arresting growth as silencing MYC or PVT1. What is particularly striking is that down-regulation or silencing of PVT1 produces the same effect as over-expression (mimic) of the embedded cluster of miRNAs suggesting opposing actions of miRNAs and host transcript. Experiments to determine whether synergy or promoter competition of the PVT1/miR-1204∼1208 cluster is specifically active in amplified 8q24 will be presented. Nevertheless, we can already assign viable functional roles to transcripts besides MYC that could represent alternative molecular targets within the 8q24 locus for cancer susceptibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 199. doi:1538-7445.AM2012-199

Understanding the functional effects of the wide-range of aberrant genetic characteristics associated with the human chromosome 8q24 region in cancer remains daunting due to the complexity of the locus. The most logical target for study remains the MYC proto-oncogene, a prominent resident of 8q24 that was first identified more than a quarter of a century ago. However, many of the amplifications, translocation breakpoints, and viral integration sites associated with 8q24 are often found throughout regions surrounding large expanses of the MYC locus that include other transcripts. In addition, chr.8q24 is host to a number of single nucleotide polymorphisms associated with cancer risk. Yet, the lack of a direct correlation between cancer risk alleles and MYC expression has also raised the possibility that MYC is not always the target of these genetic associations. The 8q24 region has been described as a "gene desert" because of the paucity of functionally annotated genes located within this region. Here we review the evidence for the role of other loci within the 8q24 region, most of which are non-coding transcripts, either in concert with MYC or independent of MYC, as possible candidate gene targets in malignancy.

Tumor progression is the continual selection of variant subpopulations of malignant cells that have acquired increasing levels of genetic instability (Nowell Science 1976, 194, 23–28). This instability is manifested as chromosomal aneuploidy or translocations, viral integration or somatic mutations that typically affect the expression of a gene (oncogene) that is especially damaging to the proper function of a cell. With the recent discovery of non-coding RNAs such as microRNAs (miRNAs), the concept that a target of genetic instability must be a protein-encoding gene is no longer tenable. Over the years, we have conducted several studies comparing the location of miRNA genes to positions of genetic instability, principally retroviral integration sites and chromosomal translocations in the mouse as a means of identifying miRNAs of importance in carcinogenesis. In this current study, we have used the most recent annotation of the mouse miRome (miRBase, release 16.0), and several datasets reporting the sites of integration of different retroviral vectors in a variety of mouse strains and mouse models of cancer, including for the first time a model that shows a propensity to form solid tumors, as a means to further identify or define, candidate oncogenic miRNAs. Several miRNA genes and miRNA gene clusters stand out as interesting new candidate oncogenes due to their close proximity to common retroviral integration sites including miR-29a/b/c and miR106a~363. We also discussed some recently identified miRNAs including miR-1965, miR-1900, miR-1945, miR-1931, miR-1894, and miR-1936 that are close to common retroviral integration sites and are therefore likely to have some role in cell homeostasis.

Abstract Several SNPs in the area surrounding the PVT1 noncoding gene region on human chromosome 8q24 have recently been found by Genome Wide Association (GWA) to be associated with susceptibility to a number of malignancies including Hodgkin's Lymphoma and other diseases including Type II Diabetes and End Stage Renal Disease in Type I Diabetes. This genetically unstable region is one of the most frequent targets of retroviral integration of the Human Papilloma Virus Type 18 (HPV18 in Cervical Carcinoma) and is the frequent target of chromosomal translocation in lymphoid malignancies such as Non-Hodgkin's Lymphoma [incl. Burkitt's Lymphoma (BL) and Diffuse Large B Cell Lymphoma (DLBL)]. Gene amplification in carcinomas such as breast, prostate, ovarian, colon, pancreatic, esophageal, as well as osteogenic sarcoma usually encompasses the proximal region of PVT1 along with its well-known neighboring transcript, MYC, whereas chromosomal deletion (in hematodermic NK lymphoma) affects the distal segment of PVT1. In a search for functionality to PVT1, many laboratories (including our own) have cloned and sequenced transcripts from the PVT1 region from many species in addition to human, with the conclusion that PVT1 is a noncoding or lincRNA with many alternatively spliced isoforms. We have systematically documented these isoforms with a particular focus on promoter and regulatory elements that are found in the region. Our laboratory has also identified a cluster of at least 6 miRNAs (miR-1204∼1208) that span the PVT1 region adding yet another level of complexity to the locus. To begin to assign some function to PVT1 based transcripts in these disease phenotypes, we have made 4th generation (CMV promoter with WPE enhancer) over-expressing lentiviral constructs containing either a large segment of PVT1 (Exons 1b-10) or the most proximal and abundant miRNA, miR-1204. These constructs follow earlier studies with a 3rd generation lentivirus (CMV promoter) containing miR-1204 that rapidly generated DLBL in cooperation with MYC or IL6 transgenes (∼16 days or 82 days respectively). While these results pointed to over-expression of miR-1204 playing a specific role in the development of B cell malignancy, additional studies with the LentiCMV-miR-1204 construct in other tumor cell lines (prostate and breast) also revealed potential phenotypic changes in cell proliferation and migration. With the new 4th generation constructs, experiments are now underway in cell lines and mouse models to determine if over-expression of miR-1204 or PVT1 alone is sufficient to induce phenotypic changes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1181. doi:10.1158/1538-7445.AM2011-1181

Abstract SNPs in the area surrounding MYC on human 8q24 have recently been found by Genome Wide Association (GWA) to be associated with susceptibility to a number of malignancies including prostate, breast, colorectal and bladder carcinomas (CA). This genetically unstable region is also a frequent target of chromosomal translocation (Tx), amplification or retroviral integration in a number of CAs such as breast, prostate, ovarian, colon, pancreatic, and cervical. In one example, essentially 100% of patients with Burkitt's lymphoma exhibit one of three characteristic non-random chromosomal Txs that places MYC or the immediate surrounding region in close proximity to enhancers of the immunoglobulin (Ig) heavy chain or light chain loci. Although de-regulated MYC expression could be assumed to be the target of the genomic instability or GWA-based susceptibility, no clear correlation between MYC expression and disease has been established. A possible alternative target has been identified in a series of transcripts cloned from the PVT1 region downstream of MYC. However, the extent of alternative splicing coupled with the lack of a coding region has made it difficult to assign a specific role for any PVT1-derived transcripts. Recently, we have identified a cluster of small RNAs exhibiting the hairpin formation, sequence conservation and expression characteristics of miRNAs (miR-1204∼1208) within the transcriptional domain of PVT1. Increased expression of several of these miRNAs in tumors harboring amplified MYC/PVT1 or Burkitt lymphomas with the 8q24 Tx suggests a possible role for these miRNAs in tumorigenesis, especially for miR-1204 which is found 60 kb downstream of MYC, flanking exon 1b of PVT1. An increased expression of miR-1204 in pre-B cells compared to pro-B cells also suggested a lymphoid specific developmental pattern of expression. Lentiviral constructs of miR-1204 under control of a CMV promoter (LentiCMV-miR-1204) revealed a possible effect on MYC, but in a pre-B (not pro-B) specific environment. Introduction of LentiCMV-miR-1204 into mice harboring either a MYC (C.iMYC) or IL6 (C.IL6) transgene resulted in high frequency and rapid onset of large B cell lymphomas (∼16 or 82 days vs. 91 or 117 days for controls, respectively). While these results point to over-expression of miR-1204 playing a specific role in the development of B cell malignancy, additional studies with the LentiCMV-miR-1204 construct in other tumor cell lines (prostate and breast) also reveal phenotypic changes in cell proliferation and migration. Further studies using lentiviruses with alternative promoters for miR-1204 and the other 8q24 associated miRNAs are underway to identify downstream targets and pathways not only in lymphoid malignancy but also in the wide range of malignancies associated with GWA-susceptibility and genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1952.

Genomic instability has been observed in many different types of cancers. While genetic alterations often cover a large spectrum of genomic events including gross rearrangement or amplification or deletion of chromosomal regions, some alterations are minimally detectable including small microsatellite or indel mutations and retroviral integration. In fact, the study of events associated with genomic instability has led to the discovery of many new and important genes associated with critical regulatory pathways. More recently, small regulatory RNAs or microRNAs have been found to reside throughout the genome and it has been suggested that such genetic alterations could also target microRNAs. In this chapter we will examine genomic instability that may alter human or mouse microRNA expression and function. Using both computational and experimental approaches, we outline possible associations between regions of genomic instability and novel microRNA candidates or already established and known microRNAs.