By Debananda Pati, PhD and Pulivarthi Rao, PhD

Introduction

Most cancers are highly aneuploid; i.e., they contain an aberrant number of chromosomes, and in most cases these chromosomes display major structural abnormalities including amplifications, deletions and translocations. This type of chromosomal instability (CIN) can contribute to tumor initiation and progression by altering the expression of proto-oncogene or tumor suppressor gene.

To date, the mechanism of aneuploidy remains an unresolved problem in cancer biology. The proposed study was to understand the mechanism of aneuploidy in osteosarcoma (OS). OS provides a prototype to study the chromosomal instability because these tumors often present with complex karyotypes including translocations and a high number of chromosomal amplifications and deletions, suggesting that genomic instability is linked to the development of this tumor.^1-7 Exposure to ionizing radiation, for example during radiotherapy of other cancers, is a high-risk factor for development of osteosarcoma at the irradiated site, further suggesting that DNA breakage and genomic instability contribute to the development of OS.⁸ We recently identified cell cycle regulatory gene – CDC5L from 6p amplicon, which is amplified and overexpressed in OS.⁹

Recent Results: CDC5L in Osteosarcoma

The basis for this study stems from our previous work on the identification of a cell cycle regulator gene – CDC5L from 6p12p21 amplicon, which is amplified and overexpressed in OS. Further, we showed an oncogenic nature of CDC5L using an in vivo assay. The proposed study was designed to correlate the CDC5L expression with genomic instability in primary tumors and cell lines and functional significance of CDC5L by overexpressing CDC5L cDNA and down-regulating by SiRNA.

1. CDC5L Overexpression Correlates with High Genomic Instability in Osteosarcoma

We correlated CDC5L expression with chromosomal instability in primary tumors and osteosarcoma cell lines (Figure 1). The genomic instability index score was calculated by combining total losses and gains (numerical genomic instability) were identified by chromosomal CGH and correlated with the expression in patient samples. In case of cell lines, we calculated structural genomic instability score identified by spectral karyotyping (SKY) and correlated with the CDC5L expression. To determine whether the genomic instability is independent of CDC5L overexpression, we screened for TP53 mutations in a panel of OS patients.

Interestingly, the genomic instability observed in CDC5L overexpressed tumors was negative for TP53 mutations. Therefore, the genomic instability observed in osteosarcoma was correlated with high expression of CDC5L. In addition, we observed several mitotic defects in primary tumors and cell lines with high expression of CDC5L (Figure 1).

2. Functional significance of CDC5L in OS

2.1. Creation of direct osteoblast-specific CDC5L transgenic mice

We have recently generated direct osteoblast-specific transgenic mice that express CDC5L under the control of the 2.3-kb alpha (1) collagen chain gene promoter (Tg Col1a1-Cdc5L) in a coat color vector containing tyrogenase minigene (9; kindly provided by Dr. Brandon Lee at BCM) and the WPRE posttranscriptional sequences.

Transgenic founders were generated by pronuclear injection at the BCM mouse transgenic core on a C57/BL6 albino background. In April, 2010, four founders (two males and two females) out of 20 pups born to two surrogate moms were identified at birth by eye color change and confirmed by PCR by using specific primer pairs shown in Figure 2. We are currently breeding these mice with C57BL6 WT mice for further characterization.

Preliminary immunohistochemical analysis is underway on paraffin sections of newborn hind limbs with rabbit-anti-rabbit Cdc5L antibody to detect Cdc5L overexpression compared to control mice. We are carefully monitoring these mice for any osteogenic phenotype.

2.2 Functional significance of down-regulation of CDC5L

Knockdown of CDC5L in U2OS and HeLa cell lines compromised their proliferative capacity but to a varying degree (Figure 3).

The molecular basis of the underlying proliferative defect induced by the deficiency of CDC5L was further interrogated by gene expression pattern in these cell lines. Table 1 summarizes the top 10 differentially expressed genes.

Of these genes, GTF2H1 and Caspase 7 are previously implicated in cell cycle progression and cancer, respectively. Previous study on general transcription factor (GTF2H1) confirmed CDK-activating kinase complex as a distinct component of this gene, suggesting a link, by the phosphorylation of the cell division cycle 2 or CDK2, between GTF2H1 and the processes of transcription, DNA repair, and cell cycle progression. Caspase 7 is one of the apoptosis-related genes whose inactivation can lead to loss of its apoptotic function and contribute to the pathogenesis of several human cancers. We analyzed 334 differentially expressed genes to identify any relevant pathways and functionally associated genes in CDC5L deficient cell lines by Ingenuity Pathways Analysis (Ingenuity Systems, Inc., Redwood City, CA). Several interesting pathways namely hypoxia signaling and some metabolic pathways associate with knockdown of CDC5L (Figure 4). Of the several functionally associated genes, cell cycle and cancer genes seems to be significant (Figure 4).

Summary of our work on CDC5L and Osteosarcoma

Combined array CGH, Q RT-PCR, Immunohistochemistry and Western blot analysis suggest the amplification and overexpression of CDC5L (positive regulator of cell cycle G2/M progression) as the target for 6p12-p21 amplicon found in osteosarcoma.

In vivo assay demonstrated the Oncogeneic nature of CDC5L.

Tumors with high expression of CDC5L displayed high levels of genomic instability.

Overexpression of CDC5L through genomic gain/amplification and independent of TP53 mutation is likely to contribute to high genomic instability in Osteosarcoma.

Generated direct osteoblast-specific transgenic mice that express CDC5L under the control of the 2.3-kb alpha (1) collagen chain gene promoter. We are currently breeding these mice with C57BL6 WT mice for further characterization.

Future Directions

Future work will focus on the following three specific goals:

1. Identify genes regulated by CDC5L. This will shed light on the precise mechanism by which CDC5L-like proteins regulate cell cycle progression, specifically the G2/M transition, and it is also likely to provide vital insight into the molecular mechanisms of the development of malignancy.
2. Investigate the formation of osteosarcoma induced by CDC5L overexpression in relation to p53 and Rb using osteoblast specific transgenic Cre mice.
3. Design and screen inhibitors of CDC5L function that might prove useful for arresting cell proliferation in osteosarcoma.

V7N4 ESUN Copyright © 2010 Liddy Shriver Sarcoma Initiative.

By Xin-Yan Lu, MD; Yi-Jue Zhao, MD; Debananda Pati, PhD and Pulivarthi Rao, PhD

Abstract

Osteosarcoma (OS) is the most frequent bone neoplasm in children and often present with a high number of chromosomal amplifications and deletions, suggesting that genomic instability is linked to the tumor development. Recently, we identified CDC5L, a cell cycle regulator gene as target for 6p12-p21 amplicon found in OS. Our recent in vivo studies, suggest that CDC5L was a potential oncogene. Overexpression of CDC5L through genomic gain/amplification is likely to lead to aberrant cell cycle control and may contribute to the malignant phenotype in osteosarcoma. The role of CDC5L in the development of genomic instability and progression of OS needs to be addressed.

An Introduction to Osteosarcoma and Genomic Instability

Osteosarcoma (OS) is the most frequent primary bone tumor of children and young adults accounting for about 5.3% of all pediatric cancers. The tumors typically arise in the metaphyseal regions of long bones, including the distal femur, proximal tibia, and proximal humerus. Several types of OS are recognized based mainly on the location of the lesion, associated bone, or related disease entity. Currently, only the histological response (degree of necrosis) to therapy represent gold standard for predicting the outcome in a patient with non-metastatic osteosarcoma at the time of definitive surgery.¹ Patients with lower degree of necrosis have a higher risk of relapse and poor outcome even after chemotherapy and complete resection of the primary tumor. Therefore, a better understanding of the underlying molecular genetic events leading to tumor initiation and progression could result in the identification of potential diagnostic and therapeutic targets. OS is often present with complex chromosomal rearrangements, deletions and amplifications, suggesting that genomic instability is linked to the development of this tumor (Figure 1).^2-8 Exposure to ionizing radiation, for example during radiotherapy of other cancers, is a high-risk factor for development of OS at the irradiated site, further suggesting that DNA breakage and genomic instability contribute to the development of this tumor.⁹

Although genomic instability in tumors has been recognized for over a century, it’s molecular and biochemical causes are not well understood. The molecular mechanisms generating genomic instability are thought diverse, but all involve deregulation of some aspect of chromosomal replication and segregation.^10-13 Genomic instability can arise when there are defects in the mechanisms that ensure faithful sister chromatid segregation during cell division and in cell cycle checkpoints that guard mitotic fidelity. The checkpoints are sensor mechanisms within the cell, which monitor the cellular environment and determine whether appropriate conditions have been fulfilled before it may progress further through a cell division cycle. Aberration in cell cycle checkpoints can lead to inappropriate proliferation – the hallmark of cancer. Therefore, to understand the links between cell cycle checkpoints and cancer, we must first understand the molecular machinery, which drives cell cycle progression and chromosomal segregation.

Recently, others and we have identified several chromosomal regions that are recurrently amplified in OS by chromosomal comparative genomic hybridization (cCGH) and array-based CGH.^2-8 Of these, amplification of 6p12-p21 and 17p11.2 appeared to be markers of an early genetic change in OS (Figsures 2 and 3). The 6p12-p21 genomic region is particularly interesting because of its association with poor prognosis in patients with osteosarcoma. We recently performed array CGH-based copy number analysis on primary osteosarcoma tumors and identified a 9.4 Mb amplicon at 6p12-p21. Our preliminary work has identified CDC5L – a cell division cycle gene that is essential for the progression of the G2 phase and entry into mitosis as probable candidate for genomic instability from 6p12-p21 amplicon in OS.¹⁴ CDC5L has also been suggested to play a role in transcription because of sequence similarities in certain domains with the proto-oncogenic transcription factor MYB.^15-16 Because of its functional role in cell division and transcription, CDC5L is most likely, play an important role in genomic instability. To our knowledge until now, no data is available to implicate CDC5L in genomic instability and genesis of OS. A complete molecular understanding of CDC5L and its regulation has therefore important implications and applications for cancer biology.

Frequent amplification and overexpression of CDC5L

Amplification of 6p12-p21 is frequent and poor prognostic marker in osteosarcoma. Based on our region-specific array CGH analysis on primary osteosarcoma tumors, we refined the amplicon to 7.9Mb on 6p and identified 10 highly amplified clones.¹⁴

The candidate genes consisting of MAPK14, MAPK1, CDKN1A, PIM1, MDGA1, BTB9, DNAH8, CCND3, PTK7, CDC5L and RUNX2 were identified within the amplified BAC clones from 6p12-p21 using the gene information available from the University of California at Santa Cruz Biotechnology.

To assess whether the observed DNA copy number increase in above genes corresponds to increased transcript level, qRT-PCR analysis of 11 genes encoded in 6p12-p21 amplicon was performed on 13 primary tumors and 7 osteosarcoma cell lines. Only three of eleven genes, i.e. CCND3, CDC5L and RUNX2 showed consistent overexpression in all OS patient samples. To assess if increased CDC5L, RUNX2 and CCND3 transcripts result in increased protein level, Western blot analysis was performed on seven osteosarcoma cell lines.¹⁴ In general, CDC5L protein was overexpressed compared to CCND3 and RUNX2. The protein expression level of CDC5L, RUNX2 and CCND3 was also analyzed by IHC on sections from six individual cases and 52 osteosarcoma-specific tissue microarray. Based on the intensity scores, we classified tumors into low, medium and high expressing groups. Consistent with Western blot analysis, high expression of CDC5L was noted in >23% of tumors compared to RUNX2 (~8%) and CCND3.

Oncogenic activity of CDC5L

We used an in vivo assay to demonstrate that CDC5L as a potential oncogene. In this assay, we utilized ectopically expressed mouse CDC5L in NIH3T3 cells to form malignant tumors in SCID mice when compared to the controls (non-transfected NIH3T3 cells).¹⁴ NIH3T3 cells were stably transfected with mouse CDC5L cDNA in a myc epitope tagged vector (pCS2MT). After initial screening, we selected two clones based on their varying level of CDC5L expression. Tumorigenicity was assayed by inoculating SCID mice with different cell doses from clone in supraclavicular region. After 2-3 weeks of inoculation, both clones developed tumors at supraclavicular region. These tumors exhibited high-level expression of CDC5L and complex chromosomal rearrangements, assayed by Western blot and spectral karyotypic (SKY) analysis, respectively (Figure 4).

Amplification and Overexpression of CDC5L Correlates with High Levels of Genomic Instability

To determine the overexpression of CDC5L correlated with the genomic instability in osteosarcoma patients and cell lines we generated genomic instability scores. We found a positive correlation between overexpression of CDC5L and high-levels of genomic/chromosomal instability in primary tumors and osteosarcoma cell lines (Figures 5 & 6). The genomic instability index score was calculated by combining total losses and gains (numerical genomic instability) were identified by chromosomal CGH and correlated with the expression in patient samples. In case of cell lines we calculated structural genomic instability score identified by multicolor spectral karyotyping (SKY) and correlated with the CDC5L expression. In addition, we established that the genomic instability was independent of TP53 status of the tumors. These results demonstrate that the overexpression of CDC5L through genomic gain/amplification and independent of TP53 mutation is likely to lead to aberrant cell cycle control and may contribute genomic instability in osteosarcoma.

Functional Significance of CDC5L Overexpression

We propose to study the functional significance of overexpressed CDC5L in OS by stably expressing CDC5L cDNA and vector controls into normal osteoblast (NOS) cells and then examine its potential to develop gross chromosomal abnormalities (a hall mark of osteosarcoma) and development of bone tumors in the nude mice xenograft models. For high-level stable gene expression, we will utilize the lentiviral expression system. Mouse CDC5L cDNA will be cloned into a CMV promoter-driven lentiviral expression vector (pLenti6). Gene activity can be detected within the first 24 hours, or Blasticidin can be used to select for stably integrated target cells long after transduction. Western blot analysis will be used to document the CDC5L overexpression in the lentiviral-transduced cells. Transduced mCDC5L cells will be followed in vitro in primary cultures for genomic instability by measuring the loss or gain of chromosomes using conventional G-banding and SKY. As a comparison, we will mix the lentivirus with the NOS cell pellet and put them into collagen gel cultures for one to two rounds before injection into nude mice. Lines that show normal karyotypes after one or two passages will be selected and injected into the nude mouse xenografts.

We will use CDC5L transduced-NOS and non-transduced control for the xenograft experiments to analyze a statistically significant result. Over a period, two to six month mice will be evaluated for tumor formation using various imaging techniques (Computer Tomography, X-ray). These studies include observation of the animals for the development of any tumor progression using CT and/or X-ray imaging in one-month intervals for a period of six months. The functional significance of down-regulation of CDC5L will be studied using RNAi sequence and we will test cells after treatment for CDC5L protein by Western blot and genomic instability by FACS and cytogenetic analysis.

In summary, we identified a cell cycle regulatory gene-CDC5L as a potential oncogene from 6p12-p21 amplification found in osteosarcoma. Overexpression of CDC5L through genomic gain/amplification is likely to lead to aberrant cell cycle control and may contribute to the malignant phenotype in osteosarcoma. The identification of genes regulated by CDC5L will shed light not only on the precise mechanism by which CDC5L-like proteins regulate cell cycle progression, specifically the G2/M transition, but is also likely to provide vital insight into the molecular mechanisms of the development of malignancy. Critically, inhibitors of CDC5L function might prove useful for arresting cell proliferation in osteosarcoma.

V6N3 ESUN Copyright © 2009 Liddy Shriver Sarcoma Initiative.

Grant Funding

The Liddy Shriver Sarcoma Initiative funded this $50,000 grant in June 2009, and it was covered in press release by Texas Children's Hospital. The study was made possible, in part, by generous donations made in memory of Sean Keane (Irish Media Ball), Frank Shafer (Sarcoma Walk), and Brandon Gordon (Golf for Gordie), all of whom lost their lives to osteosarcoma; and by generous donations made in honor of Logan Brasic (Soccer ‘Round the Clock), Matthew Siegle (Fishin’ for the Cure), Emma Koertzen (Stewart’s Stampede), and Lauren Chelenza (Pearl S. Buck Elementary School Walk-a-Thon), who are all still fighting this disease.