Grant Funds Research on Chromosomal Instability in Osteosarcoma

November 2014: The Liddy Shriver Sarcoma Initiative has awarded a $50,000 grant for promising osteosarcoma research at Massachusetts General Hospital. In this study, Amity Lynn Manning, PhD aims to reveal vulnerabilities within osteosarcoma cells that can be exploited to render the disease less aggressive and more sensitive to traditional therapies.

Osteosarcoma is a rare cancer that usually affects children, adolescents and young adults. The disease is treated with a combination of aggressive therapies, but a significant number of patients deal with disease relapse and progression.

Osteosarcoma cells are known to display a high degree of chromosomal instability (CIN). The link between CIN and poor prognosis has been observed for some time, but the mechanisms that contribute to CIN are not well understood. Thus, the effect of suppressing CIN in osteosarcoma has yet to be tested.

What is Chromosomal Instability (CIN)?

Normal cells divide to produce two new cells, each with an identical number of chromosomes. Osteosarcoma cells, however, frequently experience errors in this process that result in each of the two new cells receiving different numbers of chromosomes. When such defects in chromosome segregation happen frequently, as they do in osteosarcoma, it is known as chromosome instability and has important implications in cancer. Chromosome instability can promote tumor cell evolution, and contribute to drug resistance and relapse.

Manning's recent work has shown that loss of the retinoblastoma tumor suppressor protein (RB), which is common in osteosarcoma, leads to defects in cell division that impact chromosomal stability. Manning and her team have begun to explore the the manipulation of CIN, and in this new study they plan to suppress CIN in osteosarcoma to test its effects. The researchers hope to reveal weaknesses in chromosomally unstable cancer cells that can be exploited to enhance therapeutic response.

This study's findings have the potential to be relevant to a number of cancers that exhibit CIN. However, by performing this work on osteosarcoma, new therapeutic targets that are specific to the disease may be identified, which could ultimately lead to improved outcomes for osteosarcoma patients.

Grant Funding

This study is made possible by generous donations made in the memory of Hallie Brown. The Initiative is indebted to her family and friends. Together, we are making a difference.

From the Investigator

Amity Lynn Manning, PhDAmity Lynn Manning, PhD: My research interests lie in understanding how cancers cells are able to evolve and adapt in ways that enable them to evade current therapeutic approaches. I am thrilled to be working with the Liddy Shriver Sarcoma Initiative towards a better understanding of osteosarcoma biology and the identification of new and clinically relevant approaches to target osteosarcoma cells.

There is no better way to ensure that the most important questions are asked and answered in a manner that has a direct and powerful impact on patient treatment than by seeking feedback from expert clinicians and researchers in the field. The Initiative helps to accomplish this twofold, first with the peer-review process and again with the publication of the experimental plan in the ESUN. This grant application process has proven to be quite collaborative and has already facilitated my development of new relationships with sarcoma clinicians and researchers.

Suppression of CIN in Osteosarcoma to Enhance Therapeutic Response

Figure 1: CIN impacts drug response .

Figure 1: CIN impacts drug response

Osteosarcomas are often quite aggressive, with treatment including pre-operative chemotherapy, followed by surgery and post-operative chemotherapy. However, many patients have a bad histological response to pre-operative treatment, predictive of a worse long-term prognosis. In addition, chemotherapeutic treatment for those with recurring or metastatic disease is often ineffective. Despite decades of study, the identification of novel therapeutic approaches has been slow and overall survival rates for osteosarcoma remains at 60-70%. Unlike many sarcomas that can be characterized by specific chromosome translocations and gene mutations, osteosarcomas exhibit complex rearrangements, including structural and numerical aberrations and wide variability between cells.1 As a result, few conserved genetic changes that may indicate effective therapeutic targets have been identified. Indeed, this type of genomic instability, termed chromosome instability (CIN), results in the generation of aneuploid cells and has important implications in cancer. For example, it has been demonstrated that 'shuffling' of genomic content by CIN can facilitate loss of tumor suppressors and increased copy number of oncogenes (Baker et al, 2009).2 Furthermore, the genomic diversity generated by CIN promotes the development of cancer cells that are metastatic, resistant to chemotherapeutics and are more prone to tumor relapse (Figure 1).2-9 Consequently, CIN correlates with poor patient prognosis.3 Unfortunately, the exploitation of chromosome instability as a therapeutic target has been limited because key factors and mechanisms that contribute to this defect are unclear. Thus, defining the underlying molecular mechanism(s) that govern generation and tolerance of CIN in osteosarcomas is critical, not only to enhance our overall understanding of how such solid tumors arise, but also to provide insight into how these tumors may be efficiently and specifically targeted by novel therapeutics.

Tumor Suppressors and Oncogenes

Tumor Suppressors are genes which function in normal cells to limit cell proliferation. These genes can respond to cues, including the presence of DNA damage and nutrient limitations, to put a 'brake' on progression of the cell cycle. The corruption of one or more tumor suppressor pathways allows cancer cells to continue to grow when normal cells would not.

Oncogenes are derived from proto-oncogenes that function to promote cellular proliferation, serving as cellular 'accelerators' and setting the pace for normal cell growth. In cancer cells oncogenes exhibit increased activity and/or expression, allowing for avoidance of programmed cell death and increased cell growth and proliferation.

Like a car with no brakes and the accelerator pressed down: cancer cells in which oncogenes (cellular accelerator) are activated, and tumor suppressor pathways (cellular brake) are corrupted, progress forward in the cell cycle with little regulation. Such unregulated proliferation is a hallmark of cancer.

In addition to CIN, another feature common in osteosarcoma is the loss of the RB1 tumor suppressor gene. Sporadic osteosarcomas show alteration of the RB1 gene in 70% of cases, with loss of a single copy of RB1 linked with poor prognosis. The RB1 tumor suppressor protein (pRB) is well known for its ability to repress transcription and to prevent cell proliferation. Functional inactivation of pRB compromises the ability of cells to respond to signals that normally suppress cell proliferation and results in the mis-expression of genes that drive cell division. Lesions leading to the inactivation of RB are thought to occur in most cancer cells, creating a cellular environment that is permissive for inappropriate cell proliferation.

Unexpectedly, our recent work has demonstrated that pRB additionally functions in the maintenance of genomic stability such that inactivation of RB family proteins promotes CIN and aneuploidy.10 Given that RB1 loss predisposes afflicted patients to osteosarcomas and that frequency of RB-pathway defects in sporadic osteosarcomas is high, these studies raise the tantalizing idea that mutational events that promote tumor cell proliferation may also contribute much of the aneuploidy seen in this cancer.

Purpose of Investigation

Osteosarcomas have been particularly evasive to traditional chemotherapeutics, a feature thought to be due to the high degree of CIN in these cancers. However, the prevailing model that suppression of CIN may be of therapeutic benefit has yet to be tested. We will begin to address this question by first delineating the mechanism(s) by which CIN occurs in osteosarcoma and identifying novel suppressors and enhancers of CIN. This new knowledge will be exploited, together with what is already known of RB's role in maintaining genome stability, to examine the influence suppression of CIN will have on survival, growth and drug response in osteosarcoma cells. This study will lead to a better understanding of osteosarcoma cell biology and reveal vulnerabilities that can be exploited to render osteosarcoma less aggressive and more sensitive to traditional therapeutics.

Aim #1. Characterize defects that contribute to CIN in Osteosarcoma cells.

Figure 2. pRB loss causes defects in cell division.

Figure 2. pRB loss causes defects in cell division.

We have previously shown that pRB-loss in non-transformed cells leads to several mitotic defects including changes in chromosome cohesion and deformation of centromeric chromatin, thereby compromising mitotic fidelity and promoting aneuploidy.11 In addition, this work provided an enticing link between loss of pRB function, which is found in most osteosarcomas, and merotely, a specific type of chromosome mal-attachment, which is recognized as one of the most common mechanisms for chromosome mis-segregation in cancer cells (Figure 2).12,13 We have since shown that in cells where pRB loss promotes segregation errors, the CIN phenotype is limited by the presence of p53.14 Osteosarcomas frequently lose or mutate RB1 and TP53, or components of their respective regulatory pathways, and are well characterized as having high incidence of CIN. In preliminary work examining mitotic progression, we have observed that osteosarcoma cell lines similarly exhibit mitotic defects and lagging chromosomes during anaphase, consistent with CIN. However, it is not yet know if such defects in osteosarcomas can similarly be attributed to changes in chromatin structure, and RB1 and/or p53 loss.

Approach 1-1. To characterize the role of pRB loss on mitotic defects and CIN in a panel of osteosarcoma cell lines.

Variation in chromosome copy number within a population is a consequence of frequent chromosome segregation errors characteristic of CIN. To identify such heterogeneity, we will employ a Fluorescent in situ hybridization (FISH)-based assay to score chromosome copy number variations in a panel of osteosarcoma cell lines that have been obtained through MGH's Center for Molecular Therapeutics (CMT). Measures of numerical heterogeneity (NH) will be used as an indicator of CIN status. Knowledge of the CIN status of this collection of cell lines provides a unique opportunity to explore the role of pRB in regulating mitotic fidelity and CIN. To this end, mitotic defects will be characterized in a selection of osteosarcomas that harbor RB1 loss or mutation, or in which pRB function has been compromised via misregulation of upstream CDK activity. Observed mitotic defects and measures of CIN will be compared to that seen in osteosarcoma lines lacking obvious pRB pathway lesions or in which pRB has been experimentally re-expressed or reactivated. Preliminary work utilized the SAOS2 osteosarcoma cell line engineered to express pRB from an inducible construct. Using this cell line, we have shown that re-introduction of pRB is sufficient to suppress some defects, including changes in mitotic sister chromatid cohesion (Figure 3). Additional osteosarcoma lines lacking RB1 will be similarly engineered with inducible constructs. Lines in which pRB is present but functionally inactivated by increased CDK activity, will be treated with CDK inhibitors to render pRB functional once again. Immunofluorescence and FISH-based assays will be used to examine the effects of re-expression or re-activation of pRB on mitotic progression and CIN respectively.

Approach 1-2. To determine the mechanism(s) by which sister chromatid cohesion is compromised in osteosarcoma cells.

Our work suggests changes in the regulation of mitotic chromatid cohesion are involved in forming anaphase mal-attachments, yet it is unclear how such changes occur following pRB loss and what implications this may have for the cell. Part of the puzzle arises from the fact that pRB is thought to be functionally inactivated as cells progress towards S-phase, long before mitosis. If this conventional view of pRB regulation is correct, a link between pRB and mitotic fidelity must be due to changes earlier in the cell cycle.

The mechanism(s) by which sister chromatid cohesion is compromised in osteosarcoma cells will initially be investigated using quantitative PCR and western blot techniques to measure changes in mRNA and protein levels of cohesin complex components and functional regulators of cohesion both before and after induction of pRB expression.

To compliment the expression analysis described above, and explain chromosome cohesion defects in pRB-deficient osteosarcoma cells, functional changes in cohesin chromatin association will be assessed. A cell synchronization and fractionation approach will be used to determine specific changes in cohesin regulation that result following reintroduction/reactivation of pRB in a panel of osteosarcoma cell lines. In parallel, Chromatin Immuno-Precipitation (ChIP) assays will be used to identify specific genomic sites where cohesion is disrupted following pRB loss. Together these experiments will define the temporal and genomic context in which pRB loss compromises chromosome structure.

Understanding Cellular Changes and Sensitivities in the Context of CIN

Complimentary and future approaches will be taken to identify changes, in addition to RB1 loss, that may correlate with CIN and represent therapeutic targets. First, genomic and expression profiles, as well as drug sensitivities of osteosarcoma lines will be compared to measurements of CIN. This will be accomplished through a series of collaborations with clinical researchers at Massachusetts General Hospital's Cancer Center. Dr Cyril Benes runs the Center for Molecular Therapeutics (CMT) at MGH and has acquired a massive database of genomic and expression data, as well as sensitivity to over 300 drugs across more than a thousand tumor cell lines. This collection includes the panel of Osteosarcoma cell lines to be used in these experiments. Much of this data is publically available through the Wellcome trust Sanger Institute's Genomics of Drug Sensitivity (GDS) Project. Knowledge gained from Aim 1 of this proposal will enable the CMT data to be examined in new and exciting ways to identify genomic or expression changes that may serve as biomarkers of CIN, or indicate novel drug targets. Analyzing known drug sensitivities to newly characterized CIN in cell lines used in this study has the potential to uncover existing drugs or drug combinations to which CIN osteosarcomas are exquisitely sensitive.

Aim #2. Identify suppressors & enhancers of mitotic defects induced by pRB loss.

Figure 3: Defects in osteosarcoma are sensitive to RB function.

Figure 3: Defects in osteosarcoma are sensitive to RB function.

Previous work has suggested that cohesion defects play an integral role in the loss of mitotic fidelity in cells lacking pRB. Preliminary data shows that re-expressing pRB is sufficient to suppress some mitotic defects, effectively enhancing sister chromatid cohesion (Figure 3). In addition, we have shown that cohesion changes in pRB-depleted cells can be moderated by co-depletion of Wapl, a regulator of cohesion stability. This shows it is possible to suppress specific defects and opens the possibility to examine how suppression of defects influences CIN. Manipulation of these, or other players that promote cohesion and/or suppress segregation errors and the resulting CIN, may stabilize the cancer genome, suppressing a tumor cell's potential for evolution and rendering it more sensitive to existing therapeutics. Alternatively, defects in chromosome structure may represent a weakness that can be exploited to sensitize high CIN osteosarcoma cells to death. Both approaches will be tested.

To examine these possibilities, siRNAs or hairpin constructs will be utilized to target regulators of chromatin structure and chromosome segregation in a subset of osteosarcoma cell lines. By measuring changes in chromatin structure, levels of cohesin loading, cell viability, and rates of chromosome mis-segregation, it will be determined whether the depletion of these targets suppresses mitotic defects in osteosarcoma cells or sensitizes them to cell death. Defects in cohesion and centromere function lead to a specific type of mal attachment known as merotely, which is strongly linked to chromosome mis-segregation. We will test whether manipulation of these candidates allow for the correction of merotelic attachments and suppression of segregation errors.

Numerous regulators have been described to contribute to chromosome structure and mitotic fidelity and numerous candidates are likely to influence pRB-loss phenotypes in osteosarcoma cells. Analysis of both conditions that enhance and suppress mitotic defects will help to identify the mechanism(s) by which pRB loss contributes to CIN. It is unclear that all candidates will be similarly effective or function as drug-able targets in a tumor setting. Therefore, to enhance the likelihood of identifying relevant drug targets, numerous regulators involved in multiple pathways that contribute to CIN (i.e. regulation of chromatin structure, kinetochore function and error correction) will be identified and tested.

Tumor Relevance

While cultured cell lines are an invaluable tool to understand tumor cell biology and assess dynamic processes like cell division, their usefulness is ultimately limited by their ability to recapitulate what is happening in vivo. Therefore, with the help of Dr Edwin Choy, Dr Gregory Cote, and Dr Miguel Rivera at MGH, changes found in cell lines that correlate with CIN can be compared to those seen in patient samples. Together, this data will compliment the experimental approaches described above to enhance our understanding of changes that cause CIN in Osteosarcoma. The combined collaborative advisement of basic and clinical researchers will enhance efforts to identify new and clinically relevant approaches to target osteosarcoma.

Aim 3. Assess the tumor relevance of pRB's regulation of chromosome stability and test the influence of suppression of CIN on tumor cell growth.

Although CIN has been shown in mouse models to be sufficient for tumorigenesis, the tumors that form are infrequent, and occur later in life.15,16 A more likely role for CIN has been proposed in the acquired resistance to cancer therapy and tumor relapse.2,17 Osteosarcoma is one context where pRB's contribution to genome stability may be particularly relevant as this tumor type exhibits frequent lesions in the RB pathway and a high degree of CIN. However, the effect of suppressing CIN in established tumor cells has not been tested.

To address this question, cell growth and survival will be scored in a panel of osteosarcoma lines following suppression of CIN. CIN will be suppressed by depleting Wapl, or other candidates identified in Aim 2, and cells will be assessed for viability and proliferation. Effects on CIN will be scored using FISH-based measures of NH, and mechanism(s) of suppression will be analyzed as described above (Aims 1 & 2). This aim will identify conditions capable of suppressing CIN in tumor cell lines and reveal if CIN-suppressing mechanisms are pRB-specific.

To test the contribution of CIN to anchorage independent growth and acquired drug resistance in vitro, cell lines stably expressing inducible hairpin constructs (such as shWapl) will be generated and cell growth in liquid and soft agar conditions will be measured and compared between parental and depleted cells in the presence or absence of drug treatment. Assays to score acquired drug resistance are well established18 and likely to reveal changes that impact drug response. These findings will be ultimately assessed further with in vivo mouse xenograft models.

Impact Statement and Future Directions

Published work shows that CIN contributes to drug resistance,5 suggesting that manipulation of CIN will alter drug response. However, this hypothesis remains untested and it is unknown if suppression of CIN in osteosarcoma cells will be of therapeutic value. The identification of the specific mitotic defects that result from the loss of pRB and that may drive chromosome mis-segregation will enable this hypothesis to be tested. This experimental proposal will examine the underlying defects that contribute to CIN in osteosarcoma and reveal ways to correct or exploit these changes as a point of weakness. CIN is thought to be a major driving force in tumor development, evolution, and drug resistance. The identification of mechanisms by which to manipulate CIN opens a new therapeutic strategy that may render osteosarcomas more sensitive to existing chemotherapeutics and improve patient outcome.

By Amity Lynn Manning, PhD
Instructor in Medicine at Massachusetts General Hospital


1. Bayani, J., et al. (2007) Genomic mechanisms and measurement of structural and numerical instability in cancer cells. Semin Cancer Biol 17, 5-18.

2. Baker, D.J., et al. (2009) Whole chromosome instability caused by Bub1 insufficiency drives tumorigenesis through tumor suppressor gene loss of heterozygosity. Cancer Cell 16, 475-486.

3. Choi, C.M., et al. (2009) Chromosomal instability is a risk factor for poor prognosis of adenocarcinoma of the lung: Fluorescence in situ hybridization analysis of paraffin-embedded tissue from Korean patients. Lung Cancer 64, 66-70.

4. Kuukasjarvi, T., et al. (1997) Genetic heterogeneity and clonal evolution underlying development of asynchronous metastasis in human breast cancer. Cancer Res 57, 1597-1604.

5. Lee, A.J., et al. (2011) Chromosomal instability confers intrinsic multidrug resistance. Cancer research 71, 1858-1870.

6. McClelland, S.E., et al. (2009) Chromosomal instability: a composite phenotype that influences sensitivity to chemotherapy. Cell Cycle 8, 3262-3266.

7. Nowell, P.C. (1976) The clonal evolution of tumor cell populations. Science 194, 23-28.

8. Rajagopalan, H. and Lengauer, C. (2004) Aneuploidy and cancer. Nature 432, 338-341.

9. Swanton, C., et al. (2009) Chromosomal instability determines taxane response. Proc Natl Acad Sci U S A 106, 8671-8676.

10. Manning, A.L. and Dyson, N.J. (2011) pRB, a tumor suppressor with a stabilizing presence. Trends Cell Biol 21, 433-441.

11. Manning, A.L., et al. (2010) Loss of pRB causes centromere dysfunction and chromosomal instability. Genes Dev 24, 1364-1376.

12. Ganem, N.J., et al. (2009) A mechanism linking extra centrosomes to chromosomal instability. Nature 460, 278-282.

13. Thompson, S.L. and Compton, D.A. (2008) Examining the link between chromosomal instability and aneuploidy in human cells. J Cell Biol 180, 665-672.

14. Manning, A.L., et al. (2013) Whole chromosome instability resulting from the synergistic effects of pRB and p53 inactivation. Oncogene.

15. Iwanaga, Y., et al. (2007) Heterozygous deletion of mitotic arrest-deficient protein 1 (MAD1) increases the incidence of tumors in mice. Cancer Res 67, 160-166.

16. Michel, L.S., et al. (2001) MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature 409, 355-359.

17. Sotillo et al.

18. Qi, J., et al. (2011) Multiple mutations and bypass mechanisms can contribute to development of acquired resistance to MET inhibitors. Cancer research 71, 1081-1091.

19. Wendt, K.S., et al. (2008) Cohesin mediates transcriptional insulation by CCCTC-binding factor. Nature 451, 796-801.

20. Parelho, V., et al. (2008) Cohesins functionally associate with CTCF on mammalian chromosome arms. Cell 132, 422-433.

21. Kline-Smith, S.L., et al. (2004) Depletion of centromeric MCAK leads to chromosome congression and segregation defects due to improper kinetochore attachments. Mol Biol Cell 15, 1146-1159.

22. Maney, T., et al. (1998) Mitotic centromere-associated kinesin is important for anaphase chromosome segregation. J Cell Biol 142, 787-801.

23. Bakhoum, S.F., et al. (2009) Genome stability is ensured by temporal control of kinetochore-microtubule dynamics. Nat Cell Biol 11, 27-35.

  • Figure 1: CIN impacts drug response.
    Chromosomally unstable (CIN) cells experience a high rate of whole chromosome segregations errors during cell division. The subsequent gain or loss of entire whole chromosomes, and the genes contained therein, result in aneuploidy. The ongoing ‘shuffling’ of the genome through this process of chromosome instability throughout chemotherapeutic treatment can promote the acquisition of drug resistant cells and tumor relapse.
  • Figure 2. pRB loss causes defects in cell division.
    Depletion of pRB promotes merotelic attachments, which are visualized as lagging chromosomes in anaphase (arrow head), and high rates of chromosome segregation errors.
  • Figure 3: Defects in osteosarcoma are sensitive to RB function.
    Re-expression of pRb with a tet-inducible promoter is sufficient to promote sister chromatid cohesion, thereby decreasing the gap between sister kinetochores.