Enrichment of SOX2-Positive Cells in BRAF V600E Mutated and Recurrent Ameloblastoma

Abstract

Ameloblastoma is the most common benign odontogenic neoplasm, but with an aggressive behavior and a high recurrence rate. Nowadays wide surgical resection is the current recommended treatment, which can cause further loss of function and esthetics. Recent studies point to the stem/progenitor cells as both initiators and propagators of the tumors. Elucidation of the cellular and molecular mechanisms underlying the tumor stem cells is of broad interest for understanding tumorigenesis and for developing effective targeted therapies. SRY related HMG box gene 2 (SOX2) is a transcription factor that plays important roles in development, stem cell renewal, and cancer formation. Few studies have revealed increased SOX2 expression in atypical ameloblastoma and ameloblastic carcinoma. For the development of personalized medicine for ameloblastoma, biomarkers that provide prognostic or predictive information regarding a tumor's nature or its response to treatment are essential. Thus, in this study, we aimed to study if SOX2-positive cells exist in ameloblastomas and their correlation with the clinicopathologic parameters. Our data suggested BRAF(V600E) mutation might contribute to the expansion of SOX2-positive cells. The identification of BRAF(V600E) mutation and the amplification of SOX2-positive cells in ameloblastomas imply the possible benefit of applying BRAF and SOX2 inhibitors in recurrent and un-resectable ameloblastomas.

Keywords: BRAF V600E; SOX2; ameloblastoma; recurrence; stem cells.

Figure 1

Different types of ameloblastoma with hematoxylin and eosin (H&E) stain and immunohistochemical stains (IHC). Histopathological subtypes were as followed: follicular (A1–3), plexiform (B1–3), and unicystic (C1–3,D1–3). The (D) series are the intraluminal portion of a unicystic ameloblastoma. The (E1–3) are normal oral squamous epithelium including in the specimen, serving as an internal positive control. (A2,B2,C2,D2,E2: 1:100 dilution (Cell Signaling); A3,C3,D3,E3: 1:1000 dilution (Millipore); B3: 1:500 dilution (Millipore). Original magnification ×200 in A–D; ×100 in E).

Figure 2

Remnants of odontogenic epithelium in dental follicle containing SOX2+ cells. (Immunohistochemical staining showing SOX2+ cells in dental follicular tissue with (A) few remnants and (B) more remnants.) (Original magnification ×200).

Figure 3

Comparison of the location of positive cells under SOX2 and Ki-67 immunostain. The cells show exclusively positive to one of these two antibodies. (A) follicular type; (B) plexiform type; (C) unicystic type, mural subtype. (Original magnification ×200).

Figure 4

Immunofluorescence for SOX2 and Ki-67. Confirming SOX2+ and Ki-67+ cells were in different population of tumor cells. (A) Follicular type; (B,C) plexiform type; (D) follicular type with soft tissue invasion; (E) plexiform type with heavy inflammation; (F) oral squamous epithelium. (Original magnification ×200; C series: ×400).

Figure 5

SOX2 and Ki-67 IHC labeling index in three paired cases of primary and recurrent lesions in three patients. The representative photographs were taken from three patients’ primary and recurrent lesions. (A1: recurrent and A2: primary lesions of patient 1; B1: recurrent and B2: primary lesions of patient 2; C1: recurrent and C2: primary lesions of patient 3).

Figure 6

SOX2 knockdown in ameloblastoma cell lines reduces the cell viability. (A1–3,B1–3) The cell viability of AM1 and AM3 cells at 2 days, 4 days, and 6 days post SOX2 suppression. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 7

Representative wild type BRAF and BRAF(V600E) mutant sequence chromatograms of BRAF exon 15 (portion) were shown. * The BRAF c.1799 T > A mutation resulting in a heterogeneous thymine-to-adenine transversion at nucleotide position 1799, which results in a valine-to-glutamate substitution at residue 600 (BRAF V600E mutation rs113488022) was confirmed to be present in ameloblastomas.

Figure 8

SOX2-positive rate distribution among BRAF(V600E) wild type (WT) and mutant cases. * p < 0.05.

Figure 9

Western blot analysis of AM1 and its resistant clones to BRAF inhibitors. The AM1 cells express BRAF(V600E), while it was downregulated in the treated groups. BRAF inhibition induces upregulation of stemness marker SOX2 in AM1.