Small proline-rich protein-1B is overexpressed in human oral squamous cell cancer stem-like cells and is related to their growth through activation of MAP kinase signal.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are considered to be essential for tumor maintenance, recurrence and metastasis. Therefore, eradication of CSCs/CICs is essential to cure cancers. However, the molecular mechanisms of CSCs/CICs are still elusive. In this study, we investigated the molecular mechanism of the cell growth of oral CSCs/CICs. Oral CSCs/CICs were isolated as aldehyde dehydrogenase 1 bright (ALDH1(br)) cells by the ALDEFLUOR assay. Small proline-rich protein-1B (SPRR1B) gene was shown to be overexpressed in ALDH1(br) cells by a cDNA microarray and RT-PCR. SPRR1B was shown to have a role in cell growth and maintenance of ALDH1(br) cells by SPRR1B overexpression and knockdown experiments. To elucidate the molecular mechanism by which SPRR1B regulates cell growth, further cDNA microarray analysis was performed using SPRR1B-overexpressed cells and cells with SPRR1B knocked down by siRNA. Expression of the tumor suppressor gene Ras association domain family member 4 (RASSF4) was found to be suppressed in SPRR1B-overexpressed cells. On the other hand, the expression of RASSF4 was enhanced in cells in which SPRR1B expression was knocked down by SPRR1B-specific siRNA. RASSF4 has an RA (Ras association) domain, and we thus hypothesized that RASSF4 modulates the MAP kinase signal downstream of the Ras signal. MAP kinase signal was activated in SPRR1B-overexpressed cells, whereas the signal was suppressed in SPRR1B knocked down cells. Taken together, the results indicate that the expression of SPRR1B is upregulated in oral CSCs/CICs and that SPRR1B has a role in cell growth by suppression of RASSF4.

High expression of ALDH1 and SOX2 diffuse staining pattern of oral squamous cell carcinomas correlates to lymph node metastasis.

One of the major factors involved in the prognosis of oral squamous cell carcinoma (OSCC) patients is metastasis. Recent progress in cancer stem-like cell/cancer-initiating cell (CSC/CIC) research indicates that CSCs are related to metastasis. Aldehyde dehydrogenase 1 - (ALDH1) and SRY-related HMG-box gene 2 (SOX2) have recently been shown to be putative CSC markers for several human malignancies. The aim of this study was to determine the association of ALDH1 and SOX2 expression in oral squamous cell carcinoma (OSCC) with lymph node metastasis. Immunohistochemical staining of ALDH1, SOX2 and Ki67 was performed in 80 OSCC tissues. High expression rates of ALDH1 (2%-40%) were found to be related to lymph node metastasis (P = 0.0017). Interestingly, we found that SOX2 staining could be classified into two patterns: (i) peripheral staining pattern; and (ii) diffuse staining pattern. The diffuse staining pattern showed a significant correlation with lymph node metastasis (P < 0.001). No correlation was found between Ki67 staining and lymph node metastasis (P = 0.4724). The ALDH1 positive staining rates in metastatic lymph nodes were higher than that in corresponding primary OSCC tissues. These results indicate that high expression rates of ALDH1 and SOX2 diffuse staining patterns might be novel prediction markers for OSCC lymph node metastasis.

SOX2 is overexpressed in stem-like cells of human lung adenocarcinoma and augments the tumorigenicity.

Recently, the SOX2 gene has been reported to be amplified in human lung squamous cell carcinomas. However, its roles in human lung adenocarcinomas are still elusive. In this study, we analyzed the functions of SOX2 in cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) derived from human lung adenocarcinoma. Human lung CSCs/CICs were isolated as higher tumorigenic side population (SP) cells using Hoechst 33342 dye from several lung cancer cell lines. Four of nine lung cancer cell lines were positive for SP cells (LHK2, 1-87, A549, Lc817). The ratios of SP cells ranged from 0.4% for Lc817 to 2.8% for LHK2. To analyze the molecular aspects of SP cells, we performed microarray screening and RT-PCR analysis, and isolated SOX2 as one of a SP cell-specific gene. SOX2 was expressed predominantly in LHK2 and 1-87 SP cells, and was also expressed in several other cancer cell lines. The expression of SOX2 protein in primary human lung cancer tissues were also confirmed by immunohistochemical staining, and SOX2 was detected in more than 80% of primary lung cancer tissues. To address SOX2 molecular functions, we established a SOX2-overexpressed LHK2 and A549 cell line (LHK2-SOX2 and A549-SOX2). LHK2-SOX2 cells showed higher rates of SP cells and higher expression of POU5F1 compared with control cells. LHK2-SOX2 and A549-SOX2 cells showed relatively higher tumorigenicity than control cells. On the other hand, SOX2 mRNA knockdown of LHK2 SP cells by gene-specific siRNA completely abrogated tumorigenicity in vivo. These observations indicate that SOX2 has a role in maintenance of stemness and tumorigenicity of human lung adenocarcinoma CSCs/CICs and is a potential target for treatment.

Phase I clinical trial of survivin-derived peptide vaccine therapy for patients with advanced or recurrent oral cancer.

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is abundantly expressed in most malignancies, but is hardly detectable in normal adult tissues. Previously we have identified a human leukocyte antigen (HLA)-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), recognized by CD8(+) cytotoxic T lymphocytes (CTL). Survivin-2B80-88-specific CTL were induced efficiently from peripheral blood mononuclear cells (PBMC) of oral cancer patients after stimulation with the peptide in vitro. We conducted a phase I clinical study to evaluate the safety and the efficacy of survivin-2B80-88 peptide vaccination in HLA-A24-positive patients with advanced or recurrent oral cancer. The vaccines were given subcutaneously or intratumorally six times at 14-day intervals. Eleven patients were enrolled and 10 patients completed the vaccination protocol. No adverse events were observed in any patients. In two patients, the levels of serum squamous cell carcinoma (SCC) antigen decreased transiently during the period of vaccination. Tumor regression that was compatible with a partial response (PR) was noted in one patient. The remaining nine patients experienced progressive disease (PD). Immunologically, an increase of the peptide-specific CTL frequency was detected in six of the eight patients evaluated by HLA-A24/peptide tetramer analysis. The present clinical trial revealed that survivin-2B peptide vaccination was safe and had therapeutic potential for oral cancer patients. However, subsequent clinical trials in combination with various adjuvant drugs will be required to improve the immunological and therapeutic efficacy. This trial was registered with University Hospital Medical Information Network (UMIN) number UMIN000000976.

Clonal diversity of cytotoxic T lymphocytes that recognize autologous oral squamous cell carcinoma.

Cytotoxic T lymphocytes (CTLs) play an essential role in immunologic responses for tumor rejection. In the past decade, various melanoma tumor-associated antigens (TAAs) have been identified, and several clinical trials of vaccination immunotherapy and adoptive immunotherapy using such antigens with or without adjuvants have had fascinating results. However, this has not been the case with oral squamous cell carcinoma (OSCC) because of the difficulty of establishing oral cancer cell lines and CTLs against autologous oral cancer cells. Therefore, few oral cancer antigens have been identified with such CTLs. We herein present the successful establishment of an oral squamous cell carcinoma cell line, POT-1, and an HLA-A24-restricted CTL line (TcPOT-1) from a patient's autologous peripheral blood lymphocytes. TcPOT-1 recognized autologous POT-1 cells in an HLA-A24-restricted manner, and also allogeneic HLA-A24 (+) OSCC cell lines OSC-70 and HSC-2. We also succeeded in isolating two distinct CTL clones from TcPOT-1, HLA-A24-restricted CTL clone 4F11 and HLA-A33-restricted clone 4A11. Both of these clones recognized autologous POT-1 but not allogeneic OSSC cell lines. These data imply that the TcPOT-1 CTL line may include several CTL subpopulations with distinct antigen specificities, such as an HLA-A24-restricted POT-1-specific clone, HLA-A33-restricted POT-1-specific clone, and HLA-A24-restricted allogeneic OSCC-recognizing clone. Therefore, precise analysis of TcPOT-1-recognizing antigens may provide us with important information on as-yet-unknown tumor rejection antigens in OSCC.

Clinical assessment of the relationship between the third molar and the inferior alveolar canal using panoramic images and computed tomography.

PURPOSE: To predict the relationship between lower third molars and the inferior alveolar canal (IAC) from panoramic radiographs, and to establish criteria for using computed tomography (CT). MATERIALS AND METHODS: A retrospective cohort study was performed involving 443 patients (695 teeth). Predictor variables were the distance between the third molar and the IAC, and findings according to the Rood's criteria. Outcome variables were the absence of cortication between the third molar and the IAC on the CT image, and injury of the inferior alveolar nerve (IAN). Statistical analysis was performed to assess the relationship between predictor and outcome variables. RESULTS: All patients had preoperative panoramic radiographs, and 71 patients (119 teeth) also had CT images. On CT examination, 48 teeth (40.3%) showed absence of cortication. Injury of the IAN was reported in 7 cases (1.0%), 5 of which exhibited absence of cortication; the remaining 2 did not have CT scans. Five of the 48 cases showing absence of cortication exhibited IAN injury, and none of the cases with cortication exhibited IAN injury. On the panoramic images, the following signs were strongly correlated with absence of cortication: a superimposed relationship between the third molar and the IAC; darkness of the root; and diversion and narrowing of the IAC. CONCLUSION: Presence of Rood's criteria was a predictor for a contact relationship between the third molar and the IAC, and an indication for CT examination. However, a superimposed relationship and the absence of Rood's criteria did not necessarily signify a separate relationship between third molar and the IAC.