GLI2 and P53 Cooperate to Regulate IGFBP-3 Mediated Chondrocyte Apoptosis in the Progression From Benign to Malignant Cartilage Tumours Today I would introduce you to some work that has been going on in our lab. Specifically, the genetic events that are involved in the progression of enchondroma to chondrosarcoma Louisa Ho, Aneta Stojanovski, Heather Whetstone, Qingxia Wei, Elaine Mau, Benjamin Alman, Jay Wunder The Hospital for Sick Children, Toronto, ON Canada CTOS -November 14, 2008

Endochondral Ossification Articular chondrocytes Growth plate chondrocytes epiphysis Before I get into a discussion of cartilage tumors, I would like to provide a brief overview of how mammalian long bones develop normally. This process is called endochondral ossification, whereby a cartilage template of the long bone is formed, followed by its replacement by bone. Longitudinal growth of the bone occurs throughout the ossification process, which involves 2 opposite growth plates shown in this diagram by the dark blue lines. Within these blue lines or growth plates, chondrocytes undergo controlled phases of proliferation, differentiation and apoptosis Following chondrocyte apoptosis, blood vessel invasion ensues, leading to the replacement of the cartilaginous matrix with trabecular bone that becomes filled with bone marrow. Secondary ossification centers also arise at the epiphyses or ends of the long bones, so that ossification is occurring at either side of the growth plate. Eventually, chondrocyte proliferation slows down, resulting in the closure of the growth plate. Next, imagine that we zoomed into the one of these blue lines…

Growth Plate Chondrocyte Differentiation Periarticular Perichondrium PTHrP Resting Proliferation Within the growth plate, as I mentioned before, chondrocytes undergo distinct phases of differentiation. From the resting zone chondrocytes enter the proliferation stage by undergoing successive mitotic divisions to form a columns of proliferative cells. Chondrocytes that stop proliferating then differentiate to prehypertrophic and then hypertrophic chondrocytes, that are characterized by an increase in cell size, and eventually undergo programmed cell death or apoptosis. The progression of chondrocytes through these phases are tightly regulated by Indian hedgehog and Parathyroid hormone related protein (PTHrP) by a negative feedback mechanism. From the prehypertrophic chondrocytes, Ihh is secreted to either directly or indirectly induce PTHrP production, which is believed to be expressed from the periarticular perichondrium that surrounds the cartilage template at the epiphyses. PTHrP then diffuses to its receptor, expressed by proliferative and prehypertrophic chondrocytes. As a result, the rate of differentiation of proliferative chondrocytes into prehypertrophic chondrocytes slows down, limiting the source of Ihh production, namely the number of prehypertrophic chondrocytes, and allowing for continued chondrocyte proliferation. As well, Ihh has been shown to act by PTHrP-independent mechanisms to directly stimulate chondrocyte proliferation and promote the differentiation of reserve chondrocytes into proliferative chondrocytes. Other factors regulating chondrocyte fate include the insulin growth factor (or IGF) pathway, but I will come back to explain this pathway in greater detail later on in my talk. First I would like to return my focus to the Ihh and describe in further detail the hedgehog signaling pathway. Ihh Prehypertrophy Hypertrophy Apoptosis

Hedgehog Signalling Pathway Sufu Gli GliR PKA, CI, GSK3β Smo Ptc Hedgehog GliAct Ptc, Gli1, IGF2 No ligand With Hh ligand In the absence of the hedgehog ligand, its receptor Patched inhibits the pathway resulting in the proteolytic processing of its downstream effectors, called the Gli transcription factors to a repressor form or for degradation. Conversely, when the hedgehog ligand is able to bind to its receptor and activate the pathway, Gli is released from the inhibitory complex and is processed to an activated form to regulate expression of its target genes. There are 3 Gli transcription factors in vertebrates. All of them possess a C-terminal activation domain shown in red, whereas only Gli2 and Gli3 have an N terminal repression domain. However, recent studies have shown that Gli1 and Gli2 function primarily as activators, whereas Gli3 functions mainly as a repressor. My project focuses on Gli2 and its transcriptional activities in regulating growth plate development.

Cartilage Tumours Enchondromas are common benign cartilaginous tumors of bone Arise on the metaphyseal side of the growth plate Solitary or multifocal disease May cause pain, skeletal deformity, bony weakness leading to pathologic fracture Enchondromatosis syndromes show a higher risk (32%) of malignant transformation to chondrosarcoma Treatment is usually limited to surgical excision, given its resistance to chemotherapy Mice in which Gli2 is overexpressed within the growth plate develop ECA-like lesions (Hopyan, 2002) More specifically, I am interested in determining the genetic events involved in the formation of benign cartilaginous tumors, such as enchondromas and how they progress to malignant forms of chondrosarcoma. Enchondromas are common benign cartilaginous tumors of the bone that arise on the metaphyseal side of the growth plate. They may occur as solitary lesions, as shown by the lobular shape in this X-ray, or as a multifocal disease called enchondromatosis, in which multiple lesions are present within the bone. Symptoms of ECAs include pain, skeletal deformity, bony weakness leading to pathological fracture or often show no symptoms at all. While malignant progression of solitary ECAs are believed to be rare, the multifocal enchondromatosis syndromes show a much higher risk of transformation to CSA. In humans, CSAs are the second most common primary bone cancer, with osteosarcoma being the first. Since CSAs are particularly resistant to chemotherapy, available treatment options are inadequate and are usually limited surgical excision or amputation. With this in mind, previous studies in our lab aimed to determine the genetic factors involved in the development of these cartilage tumors. Transgenic mice were generated in which the Gli2 transcription factor, which I mentioned earlier to be one of the downstream effectors of the hedgehog signalling pathway, was expressed under the Col2 promoter, resulting in targeted overexpression in the growth plate. These mice developed cartilage lesions below the growth plate, similar to the ECAs, but did not progress to malignant forms of cartilage tumors.

? Hypothesis: Research Questions Multiple genetic events are involved in the progression of enchondroma to chondrosarcoma Research Questions What are the mechanisms by which benign tumors become more aggressive? Therefore, we hypothesized that multiple genetic events are involved in the progression of enchondromas to chondrosarcoma. To test this hypothesis we asked the following questions: What are the mechanisms by which benign tumors become more aggressive and does increased Hh signalling lead to more aggressive lesions? In this talk I will largely focus on the first question and only briefly touch upon the second question in my future directions. ? ECA CSA

Additional genetic events: P53 tumour suppressor Tg(Gli2;ColIIA1) p53 +/- To introduce additional genetic events in our Gli2 mouse model, we crossed them with p53 deficient mice to generate double transgenic mice. The p53 tumor suppressor was an obvious candidate as it is the most commonly mutated gene found in human cancers, including chondrosarcoma, which can be attributed to its diverse roles in regulating cell cycle arrest, apoptosis and senescence in response to cellular stress. When we examined the histological appearance of their cartilage lesions, as previously mentioned we observed thatGli2 overexpressing mice develop small ECA-like lesions, composed of cells with occasional binucleate lacunae in a cartilage matrix (A,C,E). However, Gli2 OE mice that were also deficient in p53 developed larger cartilage lesions with increased cellularity, variability in cytological appearance, pleiomorphic nuclei in many of the cells, and a similar appearance to that of a low grade CSA (B,D,F). As well lesions that have a histological appearance consistent with an undifferentiated sarcoma developed a small number of the mice, and were associated with loss of the wild type p53 allele (G). Interestingly, it was also observed that the frequency of these larger cartilage lesions in Tg(Gli2;ColIIAI);p53+/- mice increased with age (H).

Changes in the embryonic growth plate Gli2;P53-/- Gli2OE WT P53-/- Phenotypic differences more apparent during embryonic development Proliferation Differentiation Apoptosis Ossification To determine which stages of chondrocyte development is altered in these transgenic mice, phenotypic differences were analyzed in the embryonic growth plate. Such differences were shown to be more apparent at embryonic stages, as shown by the decreased length of the developing bone of transgenic mice. Specifically we assessed whether differences in proliferation, differentiation, apoptosis and ossification could be observed.

Changes in the embryonic growth plate Differentiation Apoptosis E C D A To assess changes in chondrocyte differentiation, we stained embryonic growth plates with ColX, which is a marker specifically expressed by hypertrophic chondrocytes. The lengths of the ColX region was then measured. As shown in Figure A, it was observed that overexpression of Gli2 resulted in a decreased length of the ColX expressing zone, whereas the p53 deficient mice had no apparent effect. However, surprisingly, the double transgenic mice showed an increase in length of the ColX expressing zone, which is opposite to that observed by a Gli2 overexpression alone. While the length of ColX expressing cells is frequently used as a measure of differentiation, or in other words, the amount of post-proliferative chondrocytes that become hypertrophic chondrocytes, the length of the ColX expressing zone can also be affected by the amount of hypertrophic cells undergoing apoptosis. Therefore, using the TUNEL assay or staining for active-caspase3 it was observed that a significant decrease in apoptosis in the double transgenic mice, but not in mice overexpressing Gli2 or with a p53 deficiency alone. This suggest that the combined effect of the Gli2OE and p53 deficiency resulted in a significant decrease in apoptosis, and a possible interaction between the pathways. It is also important to note that increases in proliferation was observed in the Gli2 transgenic and p53 deficient mice, with a more dramatic effect in the double transgenic. Therefore, it is likely that the development of the larger cartilage lesions observed in the Gli2;p53 mice is due to greater increases in proliferation and a substantial decrease in apoptosis occurring in the developing growth plate. B

Gli transcription factors directly regulate IGFBP-3 expression Previous work in our lab found in a microarray analysis that C2C12 and C3H10T1/2 cell lines with increased Hh signaling revealed IGFBP-3 as a differentially expressed gene compared to WT cells (A). Since IGFBP-3 is known to be a p53 target gene that is involved in the induction of apoptosis, we wanted to determine whether IGFBP3 was downregulated in our mouse models. Indeed this was the case, in which decreased igfbp3 was observed in Gli2OE and p53 deficient mice and showed a further reduction in the double transgenic. Next we asked whether Gli could directly regulate IGFBP3 expression. A search of the promoter and the 5’ region upstream of the igfbp3 gene showed the presence of a Gli consensus binding site, in which Gli was confirmed to bind to this site using a ChIP assay. D C

IGFBP-3 promoter assay Gli consensus binding site Mutated binding site 5`-GACCACCAG-3` Mutated binding site 5`-GACGAGGAG-3` Next we transfected 2 cell lines with a luciferase reporter consisting of the IGFBP3 promoter and 5’ region, as well as a construct in which the Gli binding site was mutated. As expected, activation of the Hh pathway resulted in a decrease in IGFBP3 expression but was not observed in the cells transfected with the mutant construct. Here treatment with IGF was used as a positive control, which has previously been shown to increase IGFBP3 expression.

Limb Explants – Changes in Apoptosis WT p53-/- IGFBP-3 SHH IGFBP-3+SHH While the data suggests thus far that p53 and Gli2 can regulate the expression of IGFBP3, to determine whether IGFBP3 can induce apoptosis and essentially rescue the deficiency in apoptosis observed in our mouse models we treated limb explants with IGFBP3, Hh ligand and a combination of both treatments. Therefore, what we would expect to find than would be an increase in apoptosis in limbs treated with IGFBP3, a decrease in limbs treated with the Hh ligand and no effect with combined treatment. Gli2;p53-/- No Tx + Shh IGFBP-3 +IGFBP-3 +Shh

Human Chondrosarcoma and IGFBP-3 Fold change While we have shown that reduced IGFBP3 expression is involved in cartilage tumor progression in our mouse models, it is also important to show that same effect can be observed in human CSAs. Decreased IGFBP-3 expression detected by real-time PCR is correlated with increasing grade of chondrosarcoma (A). Reduced IGFBP-3 staining in CSA (B) compared to growth plate cartilage (C). Furthermore, treatment of human CSA cells in culture with IGFBP-3 results in an increase in Caspase 3 and 7 activity, indicative of the induction of apoptosis. IGFBP-3

Model under investigation Periarticular Perichondrium PTHrP Resting p53 Proliferation Ihh Prehypertrophy IGFBP-3 Hypertrophy Overall our data suggests an interaction between the Hh/Gli and p53 signaling pathways in growth plate development and maintenance, in which its combined deregulation inhibits apoptosis through decreased expression of IGFBP3, which contributes to the transformation of benign cartilage neoplasia. Apoptosis Gli Overall our data suggests an interaction between the Hh/Gli and p53 signaling pathways in growth plate development and maintenance, in which its combined deregulation contributes to the transformation of benign cartilage neoplasia.

Possible Treatment targets P53 deficiency X X IGF signaling Hh signaling No Tumor Benign Malignant

Acknowledgements Dr. Jay Wunder Alman lab members Dr. Cohick Committee members Dr. Eldad Zachsenhaus Dr. Rita Kandel Alman lab members Aneta Stojanovski Heather Whetstone Elaine Mau Qing Xia Wei