-Case Studies of profiling T-UCR expression in Cancer

Recent genome-wide expression studies show that a subset of ultraconserved regions (UCRs), known as transcribed ultraconserved regions (T-UCRs), are abnormally expressed in a number of human cancers, such as leukemia, colorectal carcinoma, and hepatocellular carcinoma (Braconi, et al., 2011; Calin, et al., 2007; Lujambio, et al., 2010). In addition, the expression profile of T-UCRs appears to be well-correlated with clinical prognosis in patients with neuroblastoma (Mestdagh, et al., 2010, Scaruffi, et al. 2010). These new discoveries offer great promise for the use of T-UCR expression patterns in the diagnosis and prognosis of specific human cancers.

Case Study 1: T-UCR Expression is altered in Human Leukemia and Carcinomas

By comparing the expression patterns of T-UCRs between normal and tumor tissues of the same origin, Calin, et al. (2007) demonstrated that out of 962 possible T-UCRs, 88 T-UCRs are differentially expressed in a variety of tumor types (Table 1), including leukemia, colorectal carcinoma (CRC), and hepatocellular carcinoma (HCC; Calin, et al., 2007).

UCR Name	Type and Location	Significance	Upstream, Host, and Downstream Genes	CAGR Location and Host Gene Cancer Connection
uc.29	nonexonic	high CRC versus normal	LMO4, \N AF118089
uc.73	possibly exonic	low CLL versus CD5; high CRC versus normal	AK126774, BC017741 ZFHX1B
uc.111	possibly exonic	high CRC versus normal	AK128398, \N AB051544	yes
uc.112	nonexonic	high CRC versus normal	TBC1D5, \N SATB1
uc.134	possibly exonic	high CRC versus normal	AF257098, MGC12197, MLF1
uc.135	exonic	low CLL versus CD5	GOLPH4, EVI1 ARPM1	yes in antisense with EVI-1 oncogene overexpressed by t(3;21)(q26;q22)
uc.206	nonexonic	high CRC versus normal	SP8, \N SP4
uc.230	possibly exonic	high CRC versus normal	AK096400, \N TFEC
uc.233	exonic	low CLL versus CD5	C7orf21, CENTG3 ASB10	in sense with CENTG3
uc.291	possibly exonic	low CLL versus CD5	AK024492, C10orf11 KCNMA1
uc.292	exonic	high CRC versus normal	AF338191, MLR2 C10orf12	in sense with MLR2
uc.339	possibly exonic	high CRC versus normal	ATP5G2, \N KIAA1536	yes
uc.341	exonic	high CRC versus normal	HOXC11, HOXC10 HOXC9	yes in sense with HOXC10
uc.388	nonexonic	high CRC versus normal	BX641000, TCF12 FLJ14957
uc.399	nonexonic	high CRC versus normal	CYLD, \N SALL1
uc.420	exonic	high CRC versus normal	POLG2, DDX5 LOC90799	in sense with DDX5, downregulated in colon

Table 1. Most Differentially Expressed UCRs in Leukemias and Carcinomas. Adapted from Calin, et al. (2007).

Case Study 2: Genome-wide expression profiling identified T-UCRs associated with Hepatocellular carcinoma

Using a custom microarray containing probes for all 481 UCRs, Braconi, et al. (2011) demonstrated that the expression of 56 T-UCRs is either up- or downregulated in HepG2 hepatocellular carcinoma cells compared with normal liver (Fig. 1). No bias in the distribution of the various subclasses of T-UCRs was observed in the malignant cells (Fig. 2; Braconi, et al., 2011). The authors further showed that one T-UCR in particular, uc.338, is highly overexpressed in HepG2 cells, and that uc.338 depletion by siRNA leads to a reduction in the number of actively dividing cells as well as decreased growth in soft agar assays. These results indicate that at least one T-UCR can, either directly or indirectly, contribute to malignancy.

Figure 1. UCR expression is altered in hepatocellular carcinoma. Genome-wide expression profiling in hepatocellular carcinoma and normal liver cells identified 56 UCRS that are inappropriately expressed in malignant hepatocytes. 19 T-UCRs are at least 2-fold up- or downregulated, while 6 (labeled) are more than 3-fold differentially expressed. From Braconi,et al. (2011).

Figure 2. Distribution of aberrantly expressed UCRs in hepatocellular carcinoma. Green bars: All UCRs expressed in the hepatocellular carcinoma cell line HepG2 are distributed according to their subtypes (Exonic, Possibly exonic, Non exonic). Red bars: No dramatic change in the distribution of UCRs according to subtypes is observed for those UCRs that are differentially expressed in HepG2 relative to normal liver. Adapted from Braconi, et al. (2011).

Case study 3: An integrative genomics screen uncovers a correlation of some T-UCRs with clinical prognosis factors in neuroblastoma

Genome-wide expression profiling revealed correlations between specific T-UCR expression levels and important clinicogenetic parameters such as MYCN amplification status in neuroblastoma. Mestdagh, et al. (2010) describe a "signature" of seven T-UCRs that are upregulated in tumors in which MYCN is amplified compared with the less aggressive, MYCN-non-amplified tumors (Figure 3).

Figure 3. T-UCR expression as a function of MYCN amplification in neuroblastoma. Among 49 patients, the transcriptional levels of seven "signature" T-UCRs are elevated in MYCN-amplified (MNA; left) compared with non-MYCN (MNN) tumors. Ordinate: Pathway score. Adapted from Mestdagh, et al. (2010).

Case Study 4: T-UCR expression as a predictor of survival in neuroblastoma

By comparing 8 short-versus 12 long-term survivors, Scaruffi, et al. (2009) found that the expression levels of 54 T-UCRs are higher in neuroblastoma patients exhibiting long-term (5 years or greater) survival than in short-term survivors, suggesting that T-UCR expression can be used to gauge the prognosis of individuals with this disease. Further, these authors reported that the presence of greater than or fewer than 15 differentially regulated T-UCRs correlated positively with either long- or short-term survival, respectively. While it is not yet clear if the differential expression of any one T-UCR is a causative agent, rather than an effect, of neuroblastoma, this is another example demonstrating that T-UCRs could possibly serve as effective indicators of cancer occurrence, the stage of disease progression, and the chances of a patient's long-term outlook (Scaruffi, et al., 2009).

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References:

Braconi, C., Valeri, N., Kogure, T., Gasparini, P., Huang, N., Nuovo, G.J., Terracciano, L., Croce, C.M., and Patel, T. (2011). Expression and functional role of a transcribed noncoding RNA with an ultraconserved element in hepatocellular carcinoma. Proc Natl Acad Sci U S A 108, 786-791.
Calin, G.A., Liu, C.G., Ferracin, M., Hyslop, T., Spizzo, R., Sevignani, C., Fabbri, M., Cimmino, A., Lee, E.J., Wojcik, S.E., et al. (2007). Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas. Cancer Cell 12, 215-229.
Lujambio, A., Portela, A., Liz, J., Melo, S.A., Rossi, S., Spizzo, R., Croce, C.M., Calin, G.A., and Esteller, M. (2010). CpG island hypermethylation-associated silencing of non-coding RNAs transcribed from ultraconserved regions in human cancer. Oncogene 29, 6390-6401.
Mestdagh, P., Fredlund, E., Pattyn, F., Rihani, A., Van Maerken, T., Vermeulen, J., Kumps, C., Menten, B., De Preter, K., Schramm, A., et al. (2010). An integrative genomics screen uncovers ncRNA T-UCR functions in neuroblastoma tumours. Oncogene 29, 3583-3592.
Scaruffi, P., Stigliani, S., Moretti, S., Coco, S., De Vecchi, C., Valdora, F., Garaventa, A., Bonassi, S., and Tonini, G.P. (2009). Transcribed-Ultra Conserved Region expression is associated with outcome in high-risk neuroblastoma. BMC Cancer 9, 441.