Liposarcoma

Telomere maintenance mechanisms in liposarcomas

Dominique Broccoli, Ph.D.

Department of Laboratory Oncology Research

Curtis and Elizabeth Anderson Cancer Institute

Memorial University Medical Center

Savannah, Georgia

There are a number of characteristics that distinguish cancer cells from normal cells. One such characteristic of tumor cells is their infinite ability to grow and divide. These cells are in essence immortal. Indeed, many cell lines used in research laboratories today were originally derived from human tumors decades ago.

Liposarcoma

Liposarcoma is a tumor derived from primitive cells that undergo adipose differentiation. It is largely a disease of adults, its incidence peaking between the ages of 40 and 60 years, and it shows a slight predominance toward men.

Dr. Mark Gebhardt and Dr. Peter Buecker have written an in-depth review article, Liposarcoma, which appeared in ESUN, Vol. 1, N0. 5, 2004. Their article gives an extensive set of references to articles dealing with this disease. Click here to access it.

How do cells acquire the ability to divide and multiply indefinitely? The answer lies in the behavior of telomeres, specialized structures at the ends of eukaryotic chromosomes. The genetic material is organized as a group of discrete linear DNA molecules, each containing many genes, which are packaged into structures called chromosomes. Humans have 46 chromosomes. To correctly pass on all the genes each time a cell divides the DNA on every chromosome must first be copied and then separated. Structures on the chromosome help to achieve this. The centromere (see Figure 1) is the region of the chromosome that governs separation. Telomeres are structures at the end of the chromosome that act to cap each end of the linear DNA molecule, much as the plastic tips on shoelaces keep them from unraveling.

Figure 1: Cartoon of chromosome

DNA and RNA

An introduction to DNA, RNA and proteins can be found on the Nobel website. After clicking on the above hyperlink, make sure to read the section “Learn how to navigate in the document” to take full advantage of this tutorial.

Telomeres were originally defined based on observations in flies and corn that chromosome ends behave differently from double-stranded DNA breaks induced by radiation or chemicals. Double-stranded DNA breaks are highly unstable genetic structures that activate the cellular DNA damage response and may result in chromosome rearrangements. In contrast, naturally occurring chromosome ends, or telomeres, do not activate these cellular pathways or induce chromosome rearrangements despite being the end of a linear DNA molecule. This is achieved by packaging the chromosome end with specific proteins to form a cap-like structure, thereby sequestering the end of the DNA molecule.

Figure 2: Telomeres are packaged into lariat structures called T-loops.

The Protective Function of Telomeres

The protective function of telomeres is due to the formation of a nucleoprotein complex, comprised of 6 protein subunits (TRF1, TRF2, RAP1, POT1, TIN2 and TPP1) called shelterin (1), in complex with the telomeric 5’(TTAGGG)3’ hexanucleotide repeats. The shelterin complex sequesters the end of the DNA molecule and prevents it from activating DNA damage pathways (1, 2). This is thought to be achieved by organizing telomeres into lariat structures, called telomere loops or t-loops, formed by invasion of the 3’ single-stranded overhang into the duplex telomeric repeats, thereby sequestering the chromosome end and providing a chromosome "cap" (3).

The term “cellular pathway” refers to the sequence of events that result in a final cellular event. The cellular "DNA damage response pathway" is one such cascade of discrete events that is triggered by alterations to the structure of DNA. In this pathway, proteins in the cell first sense the damage. These sensing molecules then transmit damage information to other proteins, which prevent the cell from dividing, and lead to recruitment of proteins that will repair the DNA damage.

What do telomeres have to do with the infinite cellular division potential of cancer cells? It has become clear over the past two decades that telomeres limit the total number of times many cells can divide. In many cells of the body, telomeres become shorter with each cell division because DNA sequences are lost as a result of DNA replication. This sequence loss is a consequence of the way DNA is replicated. Eventually, insufficient telomeric DNA remains at the chromosome ends to form functional telomeric complexes. The uncapped chromosome end resembles a double-stranded DNA break which activates the cellular DNA damage response and halts further cell division. Thus, proliferation-associated telomere sequence loss acts as a tumor suppressor mechanism by limiting the total number of divisions any given cell can undergo. In other words, normal changes in telomeres that occur as a result of cellular aging protect against cancer.

Figure 3. Telomere dynamics during tumorigenesis.

Hampering Chromosomal Replicaion

Complete replication of chromosomal termini is hampered by the unidirectional nature and primer requirements of conventional DNA polymerases. Due to these features, a region of unreplicated DNA will remain on the parental DNA strand acting as the template for lagging strand synthesis following removal of the most terminal primer. This has become known as the end replication problem (4).

Enzymes are proteins in cells that perform biochemical reactions. Most of the DNA on the chromosome is copied by enzymes called DNA polymerases. DNA polymerases make a new linear piece of DNA (a daughter strand) using the original DNA (the parental strand) as a template and can only move in one direction. These enzymes require a primer, a small piece of RNA, in order to start adding new bases (the building blocks of DNA). The RNA is removed leaving a piece of parental DNA at the end of the chromosome that has not been copied. This is why sequences are lost each time the DNA is replicated. This inability of DNA polymerases to copy the end of the DNA molecule is known as the end replication problem.

As discussed above, telomeres also act to confer stability on chromosome ends. Mutations in DNA damage response genes, such as the tumor suppressor p53, may prevent a cell from responding appropriately to shortened telomeres that are seen by the cell as DNA damage. In this scenario the cell is unable to sense or repair the damage. As a consequence, continued cellular divisions occur without chromosome caps. This, in turn, leads to elevated chromosome instability resulting in chromosome rearrangements (mutations). This high level of instability, driven by critically short telomeres, has been observed in human tumors and is thought to actually contribute to, and even promote, tumor formation by increasing the rate at which tumor promoting mutations are acquired (5-7).

Articles Discussing Telomere Loss of Funcation

as a Tumor Promoting Event

L. Chin, S.E. Artandi, Q. Shen, A. Tam, S.L. Lee, G.J. Gottlieb, C.W. Greider and R.A. DePinho p53 deficiency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis, Cell 97 (1999) 527-538.

R.A. DePinho and K. Polyak Cancer chromosomes in crisis, Nat Genet 36 (2004) 932-934.

Chin, K., de Solorzano, C. O., Knowles, D., Jones, A., Chou, W., Rodriguez, E. G., Kuo, W. L., Ljung, B. M., Chew, K., Myambo, K., Miranda, M., Krig, S., Garbe, J., Stampfer, M., Yaswen, P., Gray, J. W., and Lockett, S. J. (2004) Nat Genet 36(9), 984-988.

From the discussion above it is clear that telomeres play a central role in cancer formation. Ultimately, for a tumor to continue to grow the cell(s) must circumvent the telomere length-dependent limitation on continual cellular division and the ensuing rampant chromosome instability. In most carcinomas, this is achieved through the action of an enzyme called telomerase (8). Telomerase is composed of a catalytic reverse transcriptase subunit and an RNA template molecule (9). Telomerase is able to add sequences directly on to the end of the DNA molecule causing the telomeres to become longer. Telomerase is normally active in the germline (egg and sperm) thereby ensuring that each generation starts life with telomeres of the appropriate length. But during development and early life telomerase is turned off in many cells. This leads to the replication associated loss of DNA from chromosome ends and contributes to aging. Experiments have demonstrated that forced expression of telomerase is sufficient to confer replicative immortality on human cells. Importantly, these cells are not tumorigenic, but they are capable of unlimited cell division. Because telomerase is activated in many carcinomas, significant effort has been expended to determine the molecular mechanisms that regulate telomerase activity and to validate telomerase inhibition as an anticancer strategy (10).

Telomerase is the enzyme that helps to copy the telomeres at the very ends of the chromosomes. It does this by adding DNA on the end of the parental strand, in the opposite direction from that of DNA polymerases. Because there is no parental molecule to copy telomerase uses an RNA molecule that is part of the enzyme as the template. The end result is that the parental molecule is extended thereby balancing sequence lost due to the end-replication problem.

The DNA sequences that comprise genes code for specific proteins. This DNA is first copied into RNA in a process called transcription. The RNA is then copied into a protein in a process called translation. Because DNA is being made from RNA, rather than the more usual DNA to RNA to protein pathway, the polymerase that makes the DNA is called a reverse trascriptase.

Telomerase activity in germline ensures that every generation starts life with the right size chromosomes. Tumor formation requires mutation in a number of cellular pathways. Some of these mutations lead to activation of growth promoting pathways while others result in the loss of growth inhibiting pathways. By turning off telomerase and limiting the number of divisions most cells can undergo a strong barrier to tumor formation is put in place because this is another pathway that must be activated.

A telomerase-independent mechanism to maintain telomeres, called Alternative Lengthening of Telomeres (ALT) has also been described (11). Various lines of evidence suggest that ALT occurs by recombination-based mechanism(s) (12, 13). For example, experimental evidence supports a mechanism wherein a short telomere may invade a longer telomere and uses it as the template to extend the shorter chromosome end (13). Telomerase-independent mechanisms for telomere maintenance, such as ALT, provide an alternative route whereby tumor cells may overcome the growth limitation imposed by critically short telomeres. Additionally, tumors using ALT for telomere maintenance will be refractory to treatment with telomerase inhibitors. It is therefore crucial to determine the pathways that regulate these mechanisms if we are to develop strategies that can inhibit telomere maintenance in all tumors. Until recently, we have not been able to study these pathways in human tumors due to the rare activation of telomerase-independent mechanisms in carcinomas.

Alternative Lengthening of Telomeres (ALT)

Formally, ALT describes all telomerase-independent telomere maintenance mechanisms. However, to date all but one of the identified human cell lines that use ALT for telomere maintenance have several distinctive characteristics in common. Telomere length in these cell lines is highly heterogeneous, with repeats ranging in size from less than 4 kb to greater than 25 kb (13). ALT-positive cells also have elevated levels of extrachromosomal circular DNA molecules that are composed, at least in part, of telomeric repeats (14). In addition, the frequency of telomeric recombination, measured as telomere-specific sister chromatid exchange, is elevated in cells that use ALT relative to primary or telomerase-positive cells (15). Finally, a subset of cells in ALT-positive cell lines contain large multiprotein complexes, termed ALT-associated PML nuclear bodies (APBs), in which telomere binding proteins, such as TRF1 and TRF2, and telomeric DNA co-localize with the promyelocytic leukemia (PML) nuclear body (16). APBs are tightly correlated with ALT, appearing concomitantly with activation of this telomere maintenance pathway. We, and others, have demonstrated that the formation of APBs is coordinately regulated with the cell cycle (17, 18).

Within the last 5 years several groups, including ours, have demonstrated that in certain subsets of tumors, most notably sarcomas, ALT and telomerase are used in an equivalent frequency of tumors (14-18). Surprisingly, a significant proportion of sarcomas, up to 50% in liposarcomas, do not have characteristics of either telomere maintenance mechanism. Because a high frequency of sarcomas utilize non-telomerase based mechanism(s) of telomere maintenance, these tumors are the only system that can reveal differences in the behavior (aggressiveness, drug sensitivity, etc) of tumors using the different pathways. Activation of ALT as the only means of telomere maintenance within the tumor is correlated with a better prognosis in glioblastoma multiforme (14). In contrast, in liposarcomas, activation of ALT is associated with slightly worse prognosis than activation of telomerase (19) and, the best prognosis is correlated with the absence of characteristics indicative of activation of either telomere maintenance mechanism, which is also the case in osteosarcomas (18).

We hypothesized that the differences in survival for patients whose tumors differed in the telomere maintenance pathway that had been activated resulted as a consequence of fundamental differences in the biology of these cancers. In recently published work (20), we used a technique called whole genome profiling to compare the genetic composition of a panel of liposarcomas that we had previously characterized for which telomere maintenance mechanism was active. This allowed us to identify regions that were commonly lost or amplified in these tumors. It also allowed us to compare the total number of changes across the tumor subtypes. The results indicate that: (1) ALT-positive liposarcomas have more changes than telomerase-positive tumors; and (2) tumors with an active telomere maintenance mechanism have more changes than tumors with no evidence of an active telomere maintenance mechanism. In addition, this whole genome profiling revealed two sizable genetic alterations distinguishing ALT-positive and ALT-negative lesions. Specifically, ALT-positive liposarcomas frequently lose part of chromosome 1, a genetic change not previously reported for liposarcomas. In contrast, the amplification on chromosome 12 that is characteristic of liposarcomas is rarely observed in ALT-positive tumors. Together these data suggest that ALT-positive and ALT-negative tumors are fundamentally different biological entities. These genetic differences are likely responsible for differences in human tumors using different telomere pathways.

Treatment of soft tissue sarcomas has been hampered by chemotherapy with minimal activity as well as significant toxicity. Selecting patients that would derive benefit from therapy in the form of tumor response would save patients from enduring toxicity and identify a group of patients that would not only benefit from response to therapy, but potentially have prolonged survival. The results discussed above have implications for treatment of liposarocmas. First, evidence suggests that sensitivity to DNA damaging chemotherapy drugs is correlated with genomic instability (21, 22). Our observation that ALT-positive tumors have increased instability relative to non-ALT tumors (17, 23) raise the possibility that these tumors might be, in general, more sensitive to chemotherapy than tumors with lower levels of instability. Secondly, the genes contained in the regions exhibiting telomere maintenance mechanism associated changes in DNA content will be good candidates for new drugs targeting specific tumors based upon the telomere maintenance pathway that is active. These studies will provide additional depth to our understanding of factors that predict tumor response to chemotherapy and will provide new insight into the development and evolution of soft tissue sarcomas.

In our new study, entitled “Expression profiles of liposarcomas that have activated different telomere maintenance mechanisms,” which is supported by the Liddy Shriver Sarcoma Initiative, we plan to continue our evaluation of telomere maintenance mechanism associated differences in liposarcomas. We hypothesize that the fundamental genetic differences we have already identified in liposarcomas will be paralleled by differences in the constellation of genes that are expressed. Gene expression profiles for a number of soft tissue sarcomas, including liposarcomas, have been reported previously (24, 25). There is a clear difference between liposarcomas in the expression profile based upon histologic subtype. For example, well-differentiated liposarcomas express genes also found to be expressed in normal adipose tissue (26). However, these studies do not stratify tumors based upon telomere maintenance mechanism. Given our recent data suggesting that liposarcomas utilizing different telomere maintenance mechanisms have specific genetic alterations and different prognosis, it is important to carry out these experiments not only controlling for histologic subtype but also for telomere maintenance mechanism. In our current project, we will complete this expression analysis utilizing a panel of liposarcomas that have been characterized with respect to which telomere maintenance mechanism is active. The genes identified through such studies have the potential to be useful diagnostic and/or prognostic markers, may predict response to treatment, and will be tested as potential candidates for new chemotherapy drugs.

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