Research Article Whole-Genome Profiling in Liposarcomas Reveals Genetic Alterations Common to Specific Telomere Maintenance Mechanisms Jay E. Johnson,1 Edward J. Gettings,2 Jaclyn Schwalm,2 Jianming Pei,2 Joseph R. Testa,2 Samuel Litwin,2 Margaret von Mehren,2 and Dominique Broccoli1 1Department of Laboratory Oncology Research, Curtis and Elizabeth Anderson Cancer Institute, Memorial Health University Medical Center, Savannah, Georgia and 2Department of Medical Oncology, Population Science Division and Human Genetics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania Abstract tional telomere that activates DNA damage checkpoints (2, 3), Telomere attrition ultimately leads to the activation of initiating protective cellular responses, such as apoptosis (4) or protective cellular responses, such as apoptosis or senescence. senescence (5). Although such responses typically limit the like- Impairment of such mechanisms can allow continued prolif- lihood that an aberrantly proliferating cell will cause a tumor, eration despite the presence of dysfunctional telomeres. Under impairment of pathways responsible for the maintenance of such conditions, high levels of genome instability are often genome integrity can allow continued proliferation despite the engendered. Data from both mouse and human model systems presence of dysfunctional telomeres. Under these conditions, high indicate that a period of genome instability might facilitate levels of genome instability are often engendered (6). Interestingly, tumorigenesis. Here, we use a liposarcoma model system to data from both mouse and human model systems indicate that assay telomere maintenance mechanism (TMM)–specific a transient period of genome instability might actually promote genetic alterations. A multiassay approach was used to assess cancer formation (6, 7). the TMMs active in tumors. Genomic DNA from these sam- Activation of a telomere maintenance mechanism (TMM) has ples was then analyzed by high-resolution DNA mapping array been found to stabilize telomeres, facilitate evasion of cell cycle to identify genetic alterations. Our data reveal a higher level checkpoints, and allow the unlimited cellular proliferation that of genome instability in alternative lengthening of telomere is a hallmark of cancer (8, 9). Maintenance of telomeric DNA is (ALT)–positive tumors compared with telomerase-positive usually accomplished through the action of telomerase, a large tumors, whereas tumors lacking both mechanisms have multisubunit complex that adds polynucleotide repeats to pre- relatively low levels of genome instability. The bulk of the existing telomeres (10). In humans, the core telomerase holoen- genetic changes are amplifications, regardless of the mode zyme consists of a catalytic protein subunit (hTERT) and a of telomere maintenance used. We also identified genetic template RNA (hTER). Whereas most tumors use telomerase to changes specific to the ALT mechanism (e.g., deletion of chro- maintain telomeric arrays, sarcomas often use a telomerase- mosome 1q32.2-q44)as well as changes that are underrepre- independent mechanism called alternative lengthening of telo- sented among ALT-positive tumors, such as amplification of meres (ALT; refs. 11, 12). chromosome 12q14.3-q21.2. Taken together, these studies pro- There are several differences between cells that use ALT for vide insight into the molecular pathways involved in the regu- telomere maintenance and those that use telomerase. Whereas lation of ALT and reveal several loci that might be exploited telomerase-positive cells contain relatively short telomeres, telo- either as prognostic markers or targets of chemotherapeutic meric tracts in ALT-positive cells are heterogeneously sized and intervention. [Cancer Res 2007;67(19):9221–8] have a greater mean length than their telomerase-positive coun- terparts (11). Another characteristic of ALT-positive cells is the Introduction presence of extrachromosomal DNA circles made up of telomeric sequence. ALT-positive cells also contain cell cycle–regulated Telomeres are specialized structures found at the ends of linear colocalizations of telomeric DNA, the telomere binding proteins DNA molecules and are required for the maintenance of eukaryotic TRF1 and TRF2, and the promyelocytic leukemia (PML) nuclear chromosomes. These nucleoprotein structures guard against chro- body in structures called ALT-associated PML bodies (APBs; refs. mosome end-to-end fusions and prevent the natural ends of chro- 13–15). Interestingly, several lines of evidence implicate telomeric mosomes from being recognized as dsDNA breaks (1). Telomeres recombination as playing a role in ALT. For example, telomere suffer sequence attrition each time a cell replicates its genome dynamics in ALT-positive cells are consistent with a recombina- due, in part, to the incomplete nature of semiconservative DNA tion-based mechanism of elongation (16). Furthermore, a tag from replication. Processing events necessary for the maintenance of a single marked telomere is readily transferred to other telo- telomere structure might also contribute to sequence attrition. meres in ALT cells (17). Moreover, several groups have shown Eventually, unchecked loss of telomeric DNA produces a dysfunc- that there is increased telomeric sister chromatid exchange in ALT cells (17–19). Finally, several studies have shown that survivors of telomerase deficiency in yeast maintain their telomeres by a Requests for reprints: Dominique Broccoli, Department of Laboratory Oncology Research, Curtis and Elizabeth Anderson Cancer Institute, Memorial Health University mechanism that is likely functionally similar to ALT in humans and Medical Center, 4700 Waters Avenue, Savannah, GA 31404. Phone: 912-350-0957; that this mechanism proceeds by DNA recombination (20–22). Fax: 912-350-1269; E-mail: [email protected]. I2007 American Association for Cancer Research. Dysfunctional telomeres cause genome instability, whereas both doi:10.1158/0008-5472.CAN-07-1133 ALT and telomerase rescue telomere dysfunction. Several studies www.aacrjournals.org 9221 Cancer Res 2007; 67: (19). October 1, 2007 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. Cancer Research have explored the relationship between genome instability and and ALT-negative tumors, we used Fisher’s exact two-sided test. This test telomerase activation, including recent work using a human breast was applied at each SNP locus where the fraction of ALT-positive tumors cancer model system (23). The effect of ALT activation on the levels with a CN change was compared with the fraction of ALT-negative tumors of genome instability in human cancer has not yet been deter- also showing a change. This method was used to analyze amplifications of SNPs within chromosome 12q and deletion of SNPs within chromosome 1q. mined. Here, we use DNA mapping array technology to assay For chromosome 12q, 360 adjacent SNPs were compared. Eight hundred genomic imbalances in a set of liposarcomas, allowing for direct seventy-seven SNPs were compared for chromosome 1q. comparison of the levels of genome instability associated with the We used the Jonckheere-Terpstra test of the null hypothesis that there is various TMMs. This approach has the added benefit of facilitating no relation of the TMM with the levels of either genome or telomere the identification of TMM-specific genetic alterations, analysis instability versus the alternative that instability levels vary with the mode of of which should contribute to our understanding of the ALT and telomere maintenance; specifically, that the instability observed in unknown telomerase mechanisms. tumors is less than that of telomerase-positive tumors, which in turn is less than that of ALT-positive tumors. PCR sequencing of TP53. At least 150 ng of genomic DNA were Materials and Methods subjected to PCR amplification using primer pairs to amplify the regions Tumor specimens. Our tumor sample set consisted of 37 liposarcomas containing exons 2 to 4 (5¶-CCAGGTGACCCAGGGTTGGAAG-3¶ and 5¶-GA- collected between 1992 and 2006 and made available for analysis from AGCCAAAGGGTGAAGAGGAAT-3¶)or5to9(5¶-TTCACTTGTGCCCTGA- the Fox Chase Cancer Center (FCCC) Tumor Bank. Of these samples, 34 CTT-3¶ and 5¶-CTGGAAACTTTCCACTTGAT-3¶). For amplification of exons were described previously with respect to both morphologic characteristics 2 to 4, reactions were done in a buffer containing 16.6 mmol/L ammonium and TMMs (24). In the intervening time, three new samples were obtained acetate, 67 mmol/L Tris-OH(pH8.8), 6.7 Amol/L EDTA (pH8.0), 1% DMSO, and tested for the presence of TMMs. Tumors were obtained from males and 10 mmol/L h-mercaptoethanol, and 3.5 mmol/L magnesium chloride. females with equal frequency, and the median age of the patients was Reactions were incubated at 94jC for 3 min and subjected to two rounds 65 years. Our collection contained mostly grade 1 tumors (n = 27), several each of the following cycles: 94jC for 1 min, 62jC for 1 min, and 72jC for grade 2 tumors (n = 7), and the remainder were grade 3 (n = 3). Eighteen 1.5 min; 94jC for 1 min, 61jC for 1 min, and 72jC for 1.5 min. Next, samples were primary tumors, with the remainder (n = 19) representing reactions were subjected to an additional 33 cycles of amplification using recurrences. Two tumors thought previously to be related were shown by the following conditions: 94jC for 1 min, 60jC for 1 min, and 72jCfor genotyping to actually be two genetically