PTCH-1 and MDM2 expression in ameloblastoma from a West African sub-population: implication for chemotherapeutics

Introduction Ameloblastoma is a slow growing, painless odontogenic swelling which can attain sizes that result in severe deformities of the craniofacial complex. It is the most commonly encountered odontogenic tumor in Nigeria. Surgical intervention is currently the method of treatment; however identification of altered molecular pathways may inform chemotherapeutic potential. The Protein Patched homolog 1 (PTCH-1) is overexpressed in ameloblastoma. Also, mutation in the MDM2 gene can reduce the tumor suppressor function of p53 and promote ameloblastoma growth. No study however has characterized the molecular profile of African cases of ameloblastoma with a view to developing chemotherapeutic alternatives. The objective was to characterize the PTCH-1 genetic profile of Ameloblastoma in Nigerian patients as a first step in investigating its potential for chemotherapeutic intervention. Methods Twenty-eight FFPE blocks of ameloblastoma cases from Nigerian patients were prepared for antibody processing to PTCH-1 (Polyclonal Anti-PTCH antibody ab39266) and MDM2 (Monoclonal Anti-MDM2 antibody (2A10) ab16895). Cytoplasmic brown staining was considered as positive for PTCH while nuclear staining was positive for MDM2. Results Moderate and strong expressions for PTCH in ameloblast and stellate reticulum were 78.6% and 60.7% respectively. Only 3 (10.7%) cases expressed MDM2. Conclusion The importance of our study is that it supports, in theory, anti-PTCH/SHH chemotherapeutics for Nigerian ameloblastoma cases and also infers the possible additional use of anti-p53 agents.


Introduction
Ameloblastoma is a slow growing, painless odontogenic neoplasm which causes expansion of the buccal and lingual jaw plates and sometimes infiltration of soft tissue. Although it is slow growing, the tumor can attain sizes that result in severe deformities of the craniofacial complex [1]. Ameloblastoma of the jaws is the most commonly encountered odontogenic tumor in Africa [2][3][4]. A Nigerian study reported that ameloblastoma was the commonest odontogenic tumor and constituted 63% of odontogenic tumors [4].
Surgical intervention is currently the main method of treatment and although chemotherapy has been proposed, it is not yet a conventional method of treating ameloblastoma [1]. The identification of altered molecular signaling pathways [5,6] however, may inform potential nonsurgical approaches of management.
The Sonic Hedgehog (SHH) pathway has members that play a critical role in tooth development, are involved in odontogenic tumorigenesis and induce or promote carcinogenesis in organs [7][8][9]. Protein Patched homolog 1 (PTCH-1), a tumor suppressor protein, is one of such members. DeVilliers et al [10] identified the overexpression of PTCH-1 in all ameloblastoma cases they studied.
The high expression of PTCH is important because certain therapeutic compounds have proven effective as antagonists of the PTCH in SHH pathway. Cyclopamine for example, acts at the level of SHH signaling and is effective in reducing the viability of cancer cells by blocking activation of the SHH response pathway and abnormal cell growth. Studies have shown the effectiveness of cyclopamine on cells of breast cancer, gastric cancer, medulloblastoma and oral squamous cell carcinoma [11,12]. No study however has characterized the molecular profile of African cases of ameloblastoma with a view to developing therapeutic alternatives to surgery. The p53-MDM2 paradigm is the best-studied relationship between a tumor suppressor gene and an oncogene. These two genes form an auto-regulatory feedback loop in which p53 positively regulates MDM2 levels and MDM2 in-turn negatively regulates p53 by inhibiting its functions as a transcription factor and also inducing intracellular p53 degradation [13]. Therefore a mutation in the MDM2 gene promoter can enhance MDM2 expression and consequently reduce the tumor suppressor function of p53 [14].
MDM2 overexpression has been reported in ameloblastoma and hence linked to tumor progression [15]. We aim to describe PTCH-1 and MDM2 expression in ameloblastoma from Nigerian patients to characterize the possible direct or implied chemotherapeutic potential presented by PTCH-1 and p53 in this neoplasm. Cytoplasmic brown staining was considered as positive for PTCH while nuclear staining was positive for MDM2. The Sinicrope [16] scoring method was used to evaluate both the intensity of the

Discussion
Our immunohistochemical based study found moderate to strong expression of PTCH in ameloblastoma. A study by DeVilliers et al [10] that determined the microgenomic profile of ameloblastoma via several molecular methods reported that most members of the Sonic Hedgehog pathway were under-expressed except for PTCH that was over-expressed by two-folds. They additionally validated protein expression in their study by immunohistochemistry.
Heikinheimo et al [5] however documented an under-expression of PTCH and other Sonic Hedgehog pathway members via several molecular methods. DeVilliers et al [10] had earlier suggested that the over-expression of PTCH could be used for chemotherapeutics and from our findings we support this proposition in Nigerian ameloblastoma cases. Shiori et al [17] recently reported that the proliferation of the ameloblastoma AM-1 cell line was significantly inhibited in the presence of SHH neutralizing antibody and cyclopamine. Keratocystic odontogenic tumour, which is in the same odontogenic tumor group as ameloblastoma, and also has PTCH dysfunction implicated in its pathogenesis has been shown to respond significantly to cyclopamine in a dose-dependent manner by targeting the SHH pathway [18]. Strong expression for PTCH in ameloblast-like region was noted in 60% of cystic ameloblastoma. This is comparable to the expression in other neoplastic odontogenic cysts [19] and may also support the report of Rosenstein et al [20] who documented that the percentage of cells in the cell cycle was higher in cystic ameloblastoma compared to the solid variant. Thus, although the cystic ameloblastoma may appear innocuous, it actually has higher biologic behavior at the ameloblast cellular level and may thus have a higher expression of PTCH which is a gene related to proliferation. Hemangiomatous ameloblastoma is a rare variant and although there was moderate to strong PTCH expression in the ameloblast and stellate reticulum-like regions of these cases, they are not sufficient for prognostic or therapeutic prediction.
MDM2 negatively inhibits p53 and hence the over-expression of MDM2 will greatly suppress the tumor suppressor activity of p53 [21]. MDM2 expression in this study was very poor and this may suggest that in Nigerian patients with ameloblastoma, MDM2 does not play a significant role in p53 inhibition. Other studies in non-Nigerian patients have shown that p53 in ameloblastoma are mostly suppressed by MDM2 and not mutated. Hence, if MDM2 is detached from them, they can resume their tumor suppressor action.
However, our study raises the question of primary mutation in the p53 tumor suppressor protein in Nigerian ameloblastoma cases and not p53 inhibition by MDM2. Further studies are needed to establish the status of the p53 gene in Nigerian ameloblastoma cases. A study from Malaysia reported a frame-shift type mutation of p53 in ameloblastoma at exon 4 and 7 [21]. They then suggested that this mutation could serve for potential therapeutics. Anti-p53 agents Page number not for citation purposes 4 could reduce the size of large and or unresectable tumors close to vital structures [21].

Conclusion
The significance of our study therefore is that it supports, in theory, anti-PTCH/SHH chemotherapeutics for Nigerian ameloblastoma cases and also infers the possible additional use of anti-p53 agents.