|Ahead of print publication
Role of phosphatase and tensin homolog in pathogenesis of ameloblastoma: An immunohistochemical study
Bhaskar Narayan, Aadithya B Urs, Jeyaseelan Augustine, Hanspal Singh
Department of Oral Pathology and Microbiology, Maulana Azad Institute of Dental Sciences, New Delhi, India
|Date of Submission||09-Aug-2018|
|Date of Acceptance||11-Sep-2019|
|Date of Web Publication||29-Jan-2020|
Aadithya B Urs,
Department of Oral Pathology and Microbiology, Maulana Azad Institute of Dental Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
Background: Altered molecular signaling pathways in ameloblastoma have been identified to play a pivotal role in the mechanism of oncogenesis, differentiation, and tumor progression. Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathway is one of the signaling pathways that are associated with the pathogenesis of ameloblastoma. Phosphatase and tensin homolog (PTEN) controls cell migration and proliferation. It monitors the level of the Akt and maintains cellular integrity. The present study was aimed to study the immunoexpression of PTEN in ameloblastoma to understand its role in the pathogenesis of ameloblastoma.
Materials and Methods: Twenty cases of ameloblastoma and ten cases of normal tooth germ were subjected to immunohistochemical staining against PTEN.
Results: Strong PTEN immunopositivity was seen in the tooth germs, while weak positivity was seen in the ameloblastoma. The immunoscore for PTEN was calculated by adding the percentage score and the intensity score. Seventeen cases showed the reduced PTEN expression in the epithelial component of ameloblastoma. The unpaired t-test showed a statistically significant difference in the mean PTEN immunoscore in tooth germ and ameloblastoma.
Conclusion: The study showed reduced PTEN immunoreactivity, which plays a role in the pathogenesis of ameloblastoma, through Akt pathway.
Keywords: Ameloblastoma, immunohistochemistry, phosphatase and tensin homolog, tooth germs
|How to cite this URL:|
Narayan B, Urs AB, Augustine J, Singh H. Role of phosphatase and tensin homolog in pathogenesis of ameloblastoma: An immunohistochemical study. J Can Res Ther [Epub ahead of print] [cited 2020 Apr 2]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=277242
| > Introduction|| |
Ameloblastoma is a histologically benign and locally aggressive odontogenic tumor which arises from remnants of odontogenic epithelium, such as rests of the dental lamina. The recurrence rate of ameloblastoma ranges from 8.3% to 21%. The end result is an unavoidable loss of the continuity of the jaws, which necessitate reconstructive techniques.
Pathogenesis of ameloblastoma includes diverse molecular processes which involve bone remodeling, apoptosis, and tooth development. Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is one such signaling pathway responsible for its aggressiveness. PI3K catalyzes the synthesis of the phosphatidylinositol (3, 4, 5)-triphosphate (PIP3), which serves to recruit Akt and to allow its activation by pyruvate dehydrogenase kinase. Activated Akt exerts its effects on several downstream molecules and pathways, including prevention of apoptosis, promoting cell proliferation,,, and activating the mTOR, which regulates cell growth.,,,,
Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene located on human chromosome 10q23.3, which is phosphorylated to a stable state and regulates the PI3K⁄Akt/mTOR pathway by dephosphorylating PIP3 and thus inhibiting the activation of Akt. Inactivation of PTEN gene results in unconditional cell proliferation and a reduction in apoptosis through PI3K⁄Akt/mTOR pathway., The loss of PTEN has been seen in several types of human cancers, including head-and-neck squamous cell carcinoma and in multiple cancer-associated syndromes. Germline PTEN mutations as seen in Cowden syndrome are associated with increased risk for endometrial carcinoma.
Blockade of the PI3K/Akt/mTOR pathway can be a novel therapeutic approach in the treatment of odontogenic neoplasms that holds therapeutic promise and decreases the disfigurement of jaws. Hence, the aim of the present study is to assess the role of PTEN in the pathogenesis of ameloblastoma.
| > Materials and Methods|| |
Formalin-fixed, paraffin-embedded tissue samples of 20 cases of ameloblastoma and ten tissue samples of normal tooth germs from the aborted fetuses were retrieved from the department of oral pathology and microbiology in the tertiary dental health-care hospital after the protocol was approved by the ethical committee constituted by the institution.
The sections were deparaffinized, immersed in decreasing gradient of ethanol and heated in microwave in 1 mM ethylenediaminetetraacetic acid buffer (pH 9.0) for 20 min. The sections were then treated with peroxidase block and incubated with PTEN primary antibody (Mouse Monoclonal Antibody, 6H2.1, Bio SB, Santa Barbara, CA, USA) at 4°C overnight. The standard streptavidin-biotin peroxidase complex method was employed to bind the primary antibody along with anti-immunoglobulin G secondary antibody. The reactions products were visualized after the application of 3,3'-diaminobenzidine reagent (UltraVision Quanto Detection System, HRP DAB, Thermo Scientific Immunohistochemistry Solutions, Germany). Sections were counterstained with hematoxylin. As a negative control, sections were treated with phosphate-buffered saline with the omission of the primary antibody.
The immunostains were reviewed by three independent evaluators. Immunohistochemical reactivity for all stains were graded according to the percentage of positive tumor cells: (0) 0%, (1) <25%, (2) 25%–50%, and (3) >50%, and the intensity of staining: (0) no staining, (1) weak, (2) moderate, or (3) strong, as compared with the negative control tissues. Known positive tissues for PTEN immunopositivity were used as positive controls, while the vascular endothelium, which exhibits a consistently strong, predominantly nuclear, staining for PTEN, served as an internal positive control. The values for the quality (0–3) and quantity (0–3) of staining were added to give a single number for each tumor, which was used in the final evaluation. The value obtained was classified as follows: (0) negative, (<4) weak positive, and (>4) strong positive.
Statistical significance of the difference between test and control for PTEN immunoscore was evaluated by Student's t-test with standard error. A statistically significant difference was considered to be present at P < 0.05.
| > Results|| |
We characterized the quality and quantity of immunostaining for PTEN. Significant statistical difference was seen in the mean total PTEN immunoscore between tooth germs and ameloblastoma (P = 0.00) [Table 1]. The mean total PTEN immunoscore based on the percentage of cells stained and intensity of staining in tooth germ and ameloblastoma was 5.03 and 2.97, respectively [Figure 1]. The results of the present study showed weak positivity (<4) for PTEN in the epithelial components of 85% (17/20) of all ameloblastoma cases. Three cases of ameloblastoma showed strong immunopositivity (>4) for PTEN [Figure 2]. All the cases of tooth germs showed strong PTEN immunopositivity.
|Table 1: Immunohistochemical scores and genetic alterations in ameloblastoma and controls|
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|Figure 1: Mean total phosphatase and tensin homolog immunoscore based on the percentage of cells stained and intensity of staining in tooth germ and ameloblastoma|
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|Figure 2: The microphotograph showing (a) phosphatase and tensin homolog immunoscore (6) in tooth bud (control group) (Inset: Late cap stage [×4]); (b) ameloblastoma exhibiting phosphatase and tensin homolog immunoscore (0); while c, d, e, f showing respective immunoscore of 2, 4, 5, and 6. Inset showing positive control (b, d, and e). Arrowhead showing positive control for a, c, and f|
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| > Discussion|| |
Ameloblastoma is one of the most common odontogenic tumors derived from odontogenic epithelial elements. It is a benign but locally aggressive tumor with a high recurrence rate. Altered molecular signaling pathways in ameloblastoma have also been identified to play a pivotal role in the mechanism of oncogenesis, differentiation, and tumor progression. The PI3K/Akt/mTOR pathway and sonic hedgehog (SHH) pathway are two of the many signaling pathways which are associated with the pathogenesis of ameloblastoma. These pathways converge to control the Gli transcription factor and are responsible for aggressiveness of this tumor.
The PTEN gene located on human chromosome 10q23.3 checks the basal and stimulated PIP3 level to control cell migration and proliferation with other functions, such as genomic integrity, stem cell self-renewal, cellular senescence, and interaction with p53. Under normal physiological conditions, PTEN remains in a phosphorylated form, which is inactive and stable. Once stimulated, it may get dephosphorylated at the C-terminal and bind to the negatively charged plasma membrane (phosphatidylinositol lipid) to perform its function.,,
Over the past decade, a variety of human cancers including head-and-neck squamous cell carcinoma, breast carcinoma, prostate carcinoma, and many cancer-associated syndromes have been reported with the loss of PTEN function., Three rare autosomal dominant cancer syndromes, Cowden syndrome, Lhermitte–Duclos disease, and Bannayan–Zonana syndrome have been reported with germline mutations for PTEN gene, while only three studies have been reported in the past regarding its role in the pathogenesis of the ameloblastoma.,,
Twenty cases of ameloblastoma and ten cases of tooth germs were evaluated for PTEN immunoexpression in the present study. The mean total PTEN immunoscore based on the percentage of cells stained and intensity of staining was higher in tooth germ when compared with ameloblastoma. Kumamoto and Ooya and Scheper et al. also found statistically lower level of PTEN immunoreactivity in the epithelial component of ameloblastoma than in tooth germs. They attributed that decreased PTEN expression might participate in oncogenesis of odontogenic epithelium by activating the Akt signaling pathway. A similar hypothesis could be envisaged in the current study.
Three cases of ameloblastoma showed a relative increase in PTEN immunoexpression despite tumor progression. This observation is quite rare and can be attributed to its unexplored details. Lin Fde et al. in their study suggested that PTEN alone did not control Akt phosphorylation, and other molecular mechanism may be associated with pAkt signaling pathway. Furthermore, the expression of SHH, PTCH, Smoothened (SMO), and Gli1 in ameloblastoma at mRNA and protein levels has been studied by Zhang et al. who suggested the role of these signaling molecules in the growth of ameloblastoma through epithelial–mesenchymal interactions and cell proliferation.
The unavailability of basic regions of PTEN protein may occur as a result of direct interaction with its own highly acidic C-terminus after its phosphorylation. This results in folding of PTEN protein which inhibits its interaction with PDZ domain-containing proteins. Hence, interaction with PIP3 is hampered by the unavailability of basic regions of PTEN protein to bind with the acidic plasma membrane. Hence, dephosphorylation of PIP3 does not take place. Further, signaling will take place through PI3K/Akt/mTOR pathway and lead to tumor progression despite normal PTEN expression.
Our study suggests reduced immunoexpression of PTEN in ameloblastoma, albeit on a small sample size might participate in the oncogenesis of odontogenic epithelium by activating the Akt signaling pathway. Hence, the role of PTEN in monitoring of the PI3K/Akt/mTOR pathway to maintain the cellular and genomic integrity has been reiterated.
To conclude, there are many genes such as PTCH, SHH, PI3K, Akt, ERK, mTOR, Ras, and SMO that are involved in the pathogenesis of ameloblastoma. Due to the scarcity of documentation pertaining to the molecular pathogenesis of ameloblastoma, there is immense scope for further investigation. Targeted molecular therapies could probably alter the way ameloblastoma would be treated in the coming years.
We would like to thank Dr. Puja Sakhuja, Director Professor and Head, Department of Pathology, Govind Ballabh Pant Institute of Postgraduate Medical Education and Research, New Delhi and Dr. Nita Khurana, Professor and Head, Department of Pathology, Maulana Azad Medical College, New Delhi, for their support in standardization of the PTEN immunostaining procedure.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]