Abstract Oral cancer has a severe impact on the quality of life (QOL) of patients. Treatment for the advanced cases is given priority for reducing deaths due to oral cancer and keeping QOL after the treatment. Several reports have demonstrated that inactivation of a single critical oncogene in cancer cells can induce the terminal differentiation or apoptosis. In this study, we have attempted to identify a critical oncogene for oral cancer progression. Using Applied Biosystems Human Genome Survey Arrays, we determined the gene expression profiles in 10 oral squamous cell carcinoma (OSCC) primary tissues and 10 human OSCC cell lines. This array contains 32,878 60-mer oligonucleotides probes representing a set of 29,098 individual human genes. We identified Akt1 mRNA that was commonly expressed in all OSCC tissues and cultured cells but not in non-neoplastic tissues and cells. Subsequently, immunohistochemical examination and Western blot analysis showed the overexpression of Akt1 protein in 59 of 63 OSCC primary tissues (94%) but not in oral normal epithelium. Overexpression of Akt1 and phosphorylated Akt1 proteins was also detected in all human OSCC cultured cells by sandwich ELISA. Akt is frequently activated in many types of human cancer, and has been implicated in tumorigenesis and malignancy. To clarify the oncogenic function of Akt1 in human OSCC cells, we used RNA interfering (RNAi) method. We designed and synthesized 8 small interfering RNAs for specific Akt1 (siAkt1), and then tested their RNAi effects. Akt in mammals comprises three highly homologous members known as Akt1, Akt2 and Akt3. Transfection with siAkt1 at the concentration of 1 nM markedly suppressed the expression of Akt1 protein but not Akt2 and Akt3, and significantly reduced the growth rate of human OSCC cells on the plastic surface and in collagen gels. Furthermore, atelocollagen-mediated systemic siAkt1 administration markedly inhibited the growth of human OSCC tumors which had been subcutaneously xenografted in athymic nude mice without interferon responses. These results suggest that Akt1 functions as a critical oncogene in human OSCC cells and targeting Akt1 appears a novel approach for treatment with oral cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4505.
It is widely known that human malignancies are caused by genomic and epigenomic alterations. In oral squamous cell carcinoma(OSCC), numerous gene alterations have also been reported. However, in clinical practice, these findings are not utilized at all. Therefore, we have attempted to identify gene alterations that would be useful in the diagnosis and treatment of OSCC. First, we extracted genomic DNA and total RNA from the primary tumor and adjacent normal tissues, respectively. Subsequently, targeted ultradeep sequencing by a next-generation sequencer with the use of a custom panel specialized for OSCC was performed to detect gene mutations. Gene mutations detected only in tumor derived genomic DNA were defined as tumor-specific gene mutations. Furthermore, comprehensive gene expression analysis by microarray was conducted, and the gene expression profiles from tumor and normal tissues were compared for detecting gene expression alterations. In the mutation analysis using 48 OSCC cases, tumor-specific gene mutations were observed in TP53 90%, NOTCH1 38%, CDKN2A 29%, PIK3CA 13%, and HRAS 8% of cases. Some genetic mutation was detected in all except for one case. On the other hand, in the expression analysis using 150 OSCC cases, 143 genes showing significant expression changes of 5-fold or more in tumor tissues were observed. Subsequently, when we examined the relationship between these gene alterations and cervical lymph node metastasis, we found 29 genes that showed a significant correlation with lymph node metastasis. These results revealed the somatic mutations and aberrant expression of specific genes in OSCC. Detection of these gene alterations is useful for prediction of latent cervical lymph node metastasis and detection of minimal residual disease by liquid biopsy.
Recently, numerous tumor-suppressive microRNAs (TS-miRs) have been identified in human malignancies. Here, we attempted to identify novel TS-miRs in oral squamous cell carcinoma (OSCC). First, we transfected human OSCC cells individually with 968 synthetic miRs mimicking human mature miRs individually, and the growth of these cells was evaluated using the WST-8 assay. Five miR mimics significantly reduced the cell growth rate by less than 30%, and the miR-1289 mimic had the most potent growth inhibitory effect among these miRs. Subsequently, we assessed the in vivo growth-inhibitory effects of miR-1289 using a mouse model. The administration of the miR-1289 mimic-atelocollagen complex significantly reduced the size of subcutaneously xenografted human OSCC tumors. Next, we investigated the expression of miR-1289 in OSCC tissues using reverse transcription-quantitative PCR. The expression level of miR-1289 was significantly lower in OSCC tissues than in the adjacent normal oral mucosa. Furthermore, 15 genes were identified as target genes of miR-1289 via microarray and Ingenuity Pathway Analysis (IPA) microRNA target filtering. Among these genes, the knockdown of magnesium transporter 1 (MAGT1) resulted in the most remarkable cell growth inhibition in human OSCC cells. These results suggested that miR-1289 functions as a novel TS-miR in OSCC and may be a useful therapeutic tool for patients with OSCC.
6028 Background: Lymph node stage is an important prognostic factor in squamous cell carcinoma of the head and neck (SCCHN). We previously reported the clinical usefulness of sentinel lymph node (SLN) biopsy diagnosed by concurrently performing histological examination using semiserial sections and genetic analysis by quantitative RT-PCR. However, these methods took about 3 hours. In this study, we have attempted to develop a more efficient method for intraoperative genetic detection of lymph node metastasis in SCCHN. Methods: A total of 291 lymph nodes (59 patients) resected on SLN biopsy for cN0 SCCHN or neck dissection for cN1/2 SCCHN were diagnosed by one-step nucleic acid amplification (OSNA) method using GD-100. The primary site was tongue, gingiva, oral floor, buccal mucosa, and pharynx in 44% (26), 37% (22), 10% (6), 5% (3), and 3% (2), respectively. OSNA consists of a short homogenization step followed by amplification of cytokeratin 19 (CK19) mRNA directly from the lysate. It is characterized by the use of 4 different primers specifically designed to recognize 6 distinct regions, so the CK19 primers do not amplify the known CK19 pseudogenes. The reaction process proceeds at a constant temperature (65°C) during strand displacement reaction. Amplification and detection of CK19 mRNA can be completed in a single step. Each lymph node was divided into two halves to diagnose metastasis. An alternative half was used for the OSNA assay with cytokeratin 19 (CK19) mRNA, and the remaining block was subjected to semiserial sectioning, sliced at 200-μm intervals and then examined by H&E and cytokeratin AE1/AE3 immunohistochemical staining. Results: Fifty-four of 291 lymph nodes were pathologically metastasis-positive. The optimal cut-off for the copy number of CK19 mRNA in assessing lymph node metastasis was 300 copies/μl, which had the highest diagnostic accuracy. The sensitivity and specificity of OSNA assay with CK19 mRNA was 92.6% (50/54) and 97% (230/237), respectively. An overall concordance rate between the OSNA assay and histopathology was 96.2%. The OSNA assay could be completed within 30 minutes. Conclusions: The OSNA assay showing high sensitivity and specificity can be used as a novel genetic detection tool of lymph node metastasis in SCCHN patients. No significant financial relationships to disclose.
Abstract This study investigates the role of interleukin (IL)-6 in oral squamous cell carcinoma (OSCC), evaluating its potential as a prognostic marker and therapeutic target. This study involved 95 untreated OSCC patients, with pre-surgical serum IL-6 levels measured by Electro chemiluminescence immunoassay (ECLIA). Patients were categorized into high (78 patients) and low (17 patients) IL-6 groups based on a cutoff value of 8 pg/mL.A strong correlation was observed between higher IL-6 levels and lower overall survival (P<0.0001) and disease-free survival (P=0.0283). Serum IL-6 levels also showed a strong positive correlation with certain blood test markers such as neutrophil and lymphocyte fractions and C-reactive protein (CRP), and a negative correlation with albumin levels. These findings suggest a link between systemic inflammation, malnutrition, and the Inflammation-based prognostic score (IBPS).Further analysis through microarray assessed the gene expression in primary tumor tissues, revealing 513 genes with more than a two-fold change in the high IL-6 group compared to controls. Gene Ontology analysis indicated increased metabolic activity in the high IL-6 group and suppressed inflammation and immune-related molecules in the low IL-6 group.Immunohistochemical staining for IL-6 in tumor tissues showed its presence mainly in the stroma, particularly in cancer-associated fibroblasts (CAFs) identified by vimentin and α-SMA positivity. Both OSCC cell lines and CAFs established from OSCC tissues demonstrated high IL-6 secretion, especially in cell lines derived from lymph node metastases. Furthermore, cell lines from primary, lymph node, and lung metastases exhibited high IL-6 secretion, with HRAS mutations also identified. Knockdown of HRAS using siRNA reduced IL-6 secretion.Finally, in vivo experiments using an anti-IL-6 receptor antibody showed that blocking IL-6 signaling reduced the proliferation of OSCC cell lines.The study concludes that IL-6 is a useful prognostic and therapeutic target in OSCC. The wide applicability of IL-6 testing and the clinical use of anti-human IL-6R antibody drugs, combined with aggressive nutritional intervention, suggest a potential paradigm shift in OSCC treatment. Citation Format: Hiroyuki Goda, Tomoko Adachi, Koh-ichi Nakashiro, Nobuyuki Kuribayashi, Daisuke Uchida. Interleukin6 as a Potential Prognostic Marker and Therapeutic Target in Oral Squamous Cell Carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB329.
Abstract Purpose: Dendritic cells (DCs) are professional antigen-presenting cells that play a central role in initiating adaptive and innate immune responses. DC-based vaccination is widely tested as a promising therapeutic cancer vaccine in multiple clinical trials. Programed cell death 1(PD-1)/PD-1 Ligands (PD-Ls) pathway are involved in the negative regulation of T cell-functions. PD-1 is expressed on T cells, and PD-Ls are expressed on DCs as well as some cancer cells. More recent studies have indicated that certain chemotherapeutic agents might enhance the cancer vaccine-mediated immune responses. In the current study, we conducted the in vitro experiments to examine whether or not chemotherapeutic drugs which are used for patients with head and neck cancer, may enhance T cell-stimulating ability of DCs via regulation of the expression of PD-Ls. Methods: Healthy volunteer-derived human peripheral blood monocyte were cultured with IL-4 and GM-CSF for 5 days to generate immature DCs, and then the DCs were matured by the stimulation with OK-432 which is a streptococcal immune adjuvant, for 24 hours, followed by the treatment with each chemotherapeutic drug such as 5-FU, CDDP, docetaxel or gemcitabine for 24 hours. Viability of DCs was assessed by WST-8. IL-10 and IL-12 in the supernatants from the DC cultures were measured by ELISA. Expression of mRNAs and proteins for PD-Ls was analyzed by quantitative real-time PCR and flow cytometry, respectively. DCs were co-cultured with allogeneic T cells (E/T ratio=1/20, 1/200 or 1/2000), then IFN-γ in the supernatants were measured by ELISA to evaluate T cell-stimulating ability of the DCs. Results: IL-12 production by DCs was significantly enhanced by OK-432 stimulation. Although CDDP, docetaxel and gemcitabine inhibited the OK-432-induced IL-12 production on the DCs, 5-FU did not. 5-FU and gemcitabine significantly inhibited the IL-10 production by OK-432-stimulated DCs. Expression of PD-L1 and PD-L2 genes as well as of their proteins on the DCs which was increased by OK-432 stimulation, was significantly suppressed by the treated with 5-FU or docetaxel. OK-432-stimulated DCs-treated with 5-FU strongly enhanced both proliferation and IFN-γ production of allogeneic T cells as compared with DCs stimulated only with OK-432. Conclusions: The findings obtained from the current study suggested the possibility that 5-FU may enhance the T cell-stimulating ability of dendritic cells via regulation of the expression of PD-L1 and PD-L2, and that the combination therapy of DC-based cancer vaccine with 5-FU may elicit better anti-tumor efficacy than only vaccination. Citation Format: Tomoyuki Tano, Masato Okamoto, Hiroyuki Goda, Yohei Fujita, Koh-ichi Nakashiro, Hiroyuki Hamakawa. Enhancement of the T cell-stimulating ability of dendritic cells by 5-FU via regulation of the expression of ligands for programed cell death 1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4722. doi:10.1158/1538-7445.AM2013-4722
