Why was this research done?
Odontogenic Keratocyts (OKC) is a developmental lesion that arises from odontogenic epithelium, either dental lamina or its remnants, or the basal cells of overlying epithelium. It has a locally aggressive and destructive behavior with significant recurrence rate and the epithelium has a high proliferation rate. Besides the possible local destructive effects on the adjacent teeth, OKCs have been reported to invade salivary glands, skeletal muscle, the maxillary sinus, nasal cavity, mandibular canal, zygoma, orbit and cranium that can assume a locally aggressive and destructive behaviour. OKCs generally have slow but progressively destructive behaviour but atypia of the lining is uncommon, and frank malignant degeneration is rare. For its unique destructive and aggressive features OKC is considered a benign cyst. Although OKCs are considered to have a more aggressive biologic potential than other types of odontogenic cysts, only six cases of carcinoma arising from OKCs have been reported.
However, the regulatory factors involved in cell growth and differentiation in OKC are unknown. The cause of aberrant cellular proliferation could be erroneous programming due to mutation in the regulatory genes. OKC can occur both in isolation as sporadic cysts or as multiple lesions in the feature of inherited Nevoid Basal Cell Carcinoma Syndrome (NBCCS) or Gorlin-Goltz Syndrome. Nearly 80% of NBCCS patients have OKC and cardinal feature.
In the view of the importance of this unique developmental cystic lesion with tumour like behaviour, we intend to study the pathogenesis of OKC. This thesis describes genetic mechanisms involved in the pathogenesis of OKC by demonstrating the expression of different oncogenes, tumour suppressor genes and growth factor receptors in OKC and the impact of these genes in epithelial cell proliferation and differentiation.
Choice of genes:
Genes, such as bcl-2, p53 and c-myc assume central roles, not only as regulator of apoptosis, but also in the process of neoplastic transformation. All are transcribed during embryogenesis. Genes inducing apoptosis include p53 whilst bcl-2 counteracts apoptosis, and c-myc can promote both cell proliferation and apoptosis.
The c-myc proto-oncogene, usually implicated in cell
transformation, differentiation and cell-cycle progression also has a central role in some forms of apoptosis. Bcl-2 mitigates the apoptotic effects of deregulated c-myc expression without affecting its ability to promote continuous cell growth, so providing a novel mechanism of oncogene cooperation and synergy between these two proto-oncogenes Accordingly, a 'two signal' model emerges, in which c-myc can provide the first signal, leading either to apoptosis or to progression, and bcl-2 provides the second signal, which inhibits apoptosis and allows c-myc to drive cells into the cell cycle. Synergy between c-myc and bcl-2 can be rationalised easily if bcl-2 only blocks the apoptotic function of c-myc and leaves its mitogenic activity unaffected.
Because of its role in several neoplastic conditions and p53 is know as "guardian gene" Alteration of the p53 tumour-suppressor gene is probably the most common abnormality in human neoplasia. (Anderson et al, 1992; Hollstein et al, 1991). P53 is commonly mutated in several squamous cell carcinoma of the oral cavity and also in odontogenic tumours. Bcl-2 has been found to cooperate with c-myc in tumour induction or progression (Vaux et al, 1991). Bcl-2 plays a role in protecting cells from p53-induced apoptosis, and c-myc overcomes p53-induced cell cycle arrest. This suggests another model of cooperation in which coordinate regulation of c-myc, bcl-2 and p53 expression through signal-transduction pathways may be involved in the control of cell fate. This has been demonstrated that the c-myc and bcl-2 genes cooperate to inhibit p53 functions and totally overcome p53-induced apoptosis and cell cycle arrest by altering the subcellular trafficking and cells expressing c-myc, bcl-2 and p53 remain viable and proliferate. Because of this synergic and co-operative action amongst these genes and their role in other developmental neoplastic conditions, we included c-myc, bcl-2 and p53 genes in our study to explore their role and expression in OKC.
Because of its aggressive nature, OKC may require additional signalling pathways for cellular proliferation in addition to the presence of bcl-2, c-myc and p53. Non oncogenic hormonal factors, particularly those related to cell proliferation, progression and development seem likely to play a significant role in the pathogenesis of the OKC.
Both EGFr and IGF-1r are known to be involved in various stages of developing tooth germ. During odontogenesis, oral epithelium, outer enamel epithelium and inner enamel epithelium consistently express EGFr in all stages of tooth development and IGF-1r express in both stellate reticulum and stratum intermedium. Both EGF and IGF-1 have diverse roles in odontogenesis; specially play a key role in DNA synthesis with increased mitotic rate result in high proliferation. OKC arise from the epithelial remnants formed at different stages of tooth development, and so that the cyst may reflect the cellular and molecular events occurring during normal odontogenesis. Since OKC epithelium has a high proliferation rate and is derived from tissues expressing these growth factors during odontogenesis, it is therefore likely that there will be an increased expression of EGFr and IGF-1r.
PTCH, a tumour suppressor gene is a candidate gene for NBCCS and OKC is key feature of NBCCS. Mutation of PTCH gene has been identified in a number of familial neoplasia in addition to the tumours of NBCCS. Several sporadic tumours have also shown a PTCH genemutation. PTCH is a receptor for Sonic Hedgehog (Shh) and they form a complex PTCH/HH signal transduction mechanism.
It is likely that OKC follows a coordinate pathway, which includes signals from not only oncogenic (bcl-2, p53, c-myc, PTCH and shh) factors but also other non-oncogenic factors (EGF and IGF-1).
What was done and what was found ?
Accordingly, our experiment shows the immunohistochemical expression of p53, bcl-2, PTCH, Shh and c-myc and also EGFr, IGF-1r in OKC cystic epithelium.
The initial studies undertaken investigated the expression of c-myc, bcl-2 and p53 oncoprotein in OKC. P53 showed moderate positivity in 52% of sporadic and 25% of familial OKC, whereas bcl-2 was positive in 95% of sporadic and 75% of familial OKC. C-myc was not expressed in any of the OKC samples. Our study supports the hypothesis that p53 and bcl-2 are often expressed in an independent manner but with similar effects on tumorigenesis in accordance with previous studies demonstrating a cooperative action of these oncoproteins. OKC epithelial lining cell survival, promoted by bcl-2, increases the risk of epithelial cells becoming exposed to a secondary event such as a p53 mutation causing abnormal cell proliferation. We have also demonstrated that other non-oncogenic factors including Epidermal Growth Factor receptor (EGFr) and Insulin like Growth factor-1 receptor (IGF-1r) are expressed although localisation of the different growth factor receptors varies amongst samples. Both bd-2 and p53 require growth factor receptors and their ligands for cell growth. While cells remain viable due to increased expression of anti-apoptotic bcl-2, p53 often binds with growth factor receptor ligand and antagonises its action. Expression of mutated p53 in OKC indicates that it does not have any control over growth factor receptors, hence OKC epithelium displays increase expression of EGFr and IGF-1r. The cystic epithelium is capable of oncogene and growth factor mediated cell proliferation. Increased expression of cell proliferation marker such as Ki-67 and PCNA indicate that OKC epithelium is highly proliferative in the presence of bcl-2, p53 and growth factor receptors. In addition, differentiation markers, CK13 (90%) and CK14 (70%) also expressed strongly in OKC, however, only 10% showed CK17 expression and no CK10 expression was detected. These findings show that OKC epithelium derives from odontogenic epithelium. In the process of cell maturity and the increased activity of different oncogenes and growth factors, the orientation of CK expression changes, resulting in well-differentiated cystic epithelium with increased differentiation.
Our study showed increased expression of PTCH in 30% of all OKC and Shh in 25%. Only 2 cases showed both PTCH and Shh expression in the same sample. PTCH positive cells do not express Shh, however, they remain in close proximity and often in immediately adjacent cells. Shh signal is diffuse and binds to adjacent upregulated PTCH transmembrane protein to initiate this pathway. Genetic aberrations in either gene cause deregulation of this process resulting in cell proliferation and over-expression of PTCH or Shh. Our study suggests that PTCH and Shh are expressed in an independent manner in neighboring cells to complete the signal transduction mechanism and that of loss of PTCH function may exert an effect in tumorigenesis. In this immunofluroscene study using a polyclonal antibody against PTCH, which may bind with wild type of PTCH, it was considered the necessity to perform subsequent loss of heterozygosity (LOH) study. LOH is an important genetic mechanism, giving rise to neoplasms. Although OKC derived from NBCCS have a germline mutation and a "single' hit is enough to cause OKC, for sporadic OKC, two somatic mutations are required. We have used five of the most common DNA polymorphic markers, which span a large part of the PTCH gene and known to be areas with high, a mutation rate. Our results showed 3 cases of sporadic OKC with substantial LOH for two markers (D9S287 and D9S180). None of the NBCCS related OKC displayed any LOH because germline mutations are mostly point mutations and a single small point mutation in the remaining allele can cause OKC in NBCCS and this cannot be identified by LOH. This observation is in accord with previous studies. Subsequent mutation analysis of these three samples by Direct DNA sequencing with 23 exons of PTCH gene showed: a frameshift mutation (1108delAAGC) in exon 8 (case #10), a large deletion 3509del26 (5"- TGC TTC CCG TGC TTT TGT CTT TCT TT-3') in case #7 and case #16 showed a nonsense mutation in 1149 'G' to 'A' resulting in a ‘TGA’ sequence which creates a stop codon.
What does this all mean ?
An interesting observation of this study is absence of apoptosis in OKC. Although both bcl-2 and p53 have direct role over apoptotic pathway but our result showed no evidence of apoptosis in any sample tested. If cell death is occurring then it is at a very low rate with the emphasis in the cyst lining being proliferating. Bcl-2, which is thought to prevent apoptosis was present in almost 78% of basal cells indicating a possibility of lack of apoptosis due its role in the basal cells. Our results suggest that apoptotic reactions in the lining epithelium of OKCs be suppressed due to high proliferative potentials of OKC lining epithelium, which is confirmed by the expression of Ki-67 and PCNA. At this early stage of OKC formation, increased expression of bcl-2 by basal cells may suggest that thebcl-2 transfection of basal cells prevent their differentiation and extend their viability but does not transform per se. This would allow slow progression of basal cells to the suprabasal layers where the pattern changes with an increased migration and proliferation rate in presence of both p53, IGF-1r and EGFr and consequently loses bcl-2 expression when the cystic lining acquire full proliferation potentials. When basal cells migrate to the upper layers it does not express anti apoptotic bcl-2 any more but apoptosis still not evident due to combined action of p53, EGFr and IGF 1r. This may be due to increased expression of IGF-1, which helps in preventing apoptosis allowing cells to proliferate at a high rate. This observation may have a role in proliferation status of basal cells. When bcl-2 is expressed in the basal layer it only prevents apoptosis, keeping cells viable for further genetic insult. The rate of proliferation is low at this stage, however, these clone of cells give two daughter cells and start migrating towards superficial layers, where the cells encounter further genetic abnormality including p53, EGFr and IGF-1r, which turns these cells into high proliferation status. These daughter cells in presence of bcl-2 retain their capacity to further division. This will facilitate cells to move toward the immediate next layer and awaits to be activated by p53 and growth factors and increase in the number of p53 positive cells (p53 positive cells in the suprabasal layers is 4 times higher than the bcl-2) and migrate more towards the upper suprabasal cells.
Epithelial differentiation known to be influence by several cellular and chemical factors and change of this can be result from the alteration in the epithelial cytoskeletal protein. Recent studies have shown that the pattern of expression of keratin genes is closely linked to the differentiation of epithelium. Therefore keratin represents a molecular tool to examine epithelial differentiation. The current study supports the premise that the OKC may represent a benign cystic neoplasm with no strong trends to malignancy. The patterns of CK expression are consistent with current knowledge and extend our understanding of the OKC.
This finding may explain why the suprabasal cells of OKC, which strongly express CK 13, differentiate at a higher rate than that of neighboring basal cells with CK 14 positivity. However, when CK14 basal cells are proliferating and migrating to upper suprabasal cells, they sacrifice expressing CK 14 and differentiate into CK 13 positive cells.
What was its significance with respect to whv research was done?
In conclusion, pathogenesis of this fast growing benign cystic tumour is yet to be understood fully, however, it is likely that during various stages of tooth development and differentiation, the odontogenic apparatus acquires genetic aberration resulting in OKC. This study does not specifically demonstrate a particular gene or genes involved in the development of OKC (due to the lack of a large sample to predict a particular gene involvement) but it does indicate the involvement of various factors, including several growth factors, oncogenes and tumour suppressor genes. Our study showed that the development of OKC may be due to increased activity of non-oncogenic growth factor receptors (EGFr and IGF- 1) since almost all samples uniformly and homogeneously expressed these factors irrespective of the action of bcl-2 and p53 or PTCH and Shh. However, its growth potentiality can be a result of combined effect of growth factors, oncogenes (bcl-2 and Shh) and tumour suppressor genes (p53 and PTCH) because the cell proliferation index is much higher when growth factors are expressed along with these oncogenic factors. Our results also support the premise that OKC should be considered as a benign cystic neoplasm since LOH is, by definition, a feature of tumorigenic tissue. We also propose that familial and sporadic OKC may follow different pathways in their pathogenesis.