Ryuichi Murase

Pathways mediating the effects of cannabidiol on the reduction of breast cancer cell proliferation, invasion, and metastasis

Invasion and metastasis of aggressive breast cancer cells are the final and fatal steps during cancer progression. Clinically, there are still limited therapeutic interventions for aggressive and metastatic breast cancers available. Therefore, effective, targeted, and non-toxic therapies are urgently required. Id-1, an inhibitor of basic helix-loop-helix transcription factors, has recently been shown to be a key regulator of the metastatic potential of breast and additional cancers. We previously reported that cannabidiol (CBD), a cannabinoid with a low toxicity profile, down-regulated Id-1 gene expression in aggressive human breast cancer cells in culture. Using cell proliferation and invasion assays, cell flow cytometry to examine cell cycle and the formation of reactive oxygen species, and Western analysis, we determined pathways leading to the down-regulation of Id-1 expression by CBD and consequently to the inhibition of the proliferative and invasive phenotype of human breast cancer cells. Then, using the mouse 4T1 mammary tumor cell line and the ranksum test, two different syngeneic models of tumor metastasis to the lungs were chosen to determine whether treatment with CBD would reduce metastasis in vivo. We show that CBD inhibits human breast cancer cell proliferation and invasion through differential modulation of the extracellular signal-regulated kinase (ERK) and reactive oxygen species (ROS) pathways, and that both pathways lead to down-regulation of Id-1 expression. Moreover, we demonstrate that CBD up-regulates the pro-differentiation factor, Id-2. Using immune competent mice, we then show that treatment with CBD significantly reduces primary tumor mass as well as the size and number of lung metastatic foci in two models of metastasis. Our data demonstrate the efficacy of CBD in pre-clinical models of breast cancer. The results have the potential to lead to the development of novel non-toxic compounds for the treatment of breast cancer metastasis, and the information gained from these experiments broaden our knowledge of both Id-1 and cannabinoid biology as it pertains to cancer progression.

Serum autoantibody to sideroflexin 3 as a novel tumor marker for oral squamous cell carcinoma

The purpose of this study is to establish a tumor marker that can be applied for the early detection and follow‐up of oral cancer patients. Employing the proteomic approach using MALDI TOF‐MS, 2‐DE, patients sera and culturing cell lines, the serum autoantibodies (autoAbs) were screened and the serum levels were estimated by ELISA. Targeting the tumor cell invasion into the surrounding stromal tissues, MRC‐5 human fibroblasts were employed as the target cells and a mitochondrial membrane protein, sideroflexin 3 (SFXN3), was identified. The serum anti‐SFXN3‐autoAb levels elevated in patients with the oral squamous cell carcinoma significantly: with 77% sensitivity and 89% specificity against control samples. The serum anti‐SFXN3‐autoAb levels were mildly correlated with the primary tumor sizes, however, the levels were slightly highly elevated in T1 early cancer. An immunohistochemical analysis revealed that the SFXN3 protein is expressed in the stromal fibroblasts between the caner nests and also in the basal layer of the squamous epithelium. Changes in the serum anti‐SFXN3‐autoAb levels after therapy correlated with the clinical tumor burden. These findings demonstrated that the serum anti‐SFXN3‐autoAb is worthy of clinical evaluation as a potentially of the novel tumor maker for the early detection of oral squamous cell carcinoma.

Targeting Id1 reduces proliferation and invasion in aggressive human salivary gland cancer cells

BackgroundSalivary gland cancer (SGC) is one of the common malignancies of the head and neck area. It develops in the minor and major salivary glands and sometimes metastasizes to other organs, particularly to the lungs. Inhibitors of differentiation (Id) proteins are negative regulators of basic helix-loop-helix transcription factors that control malignant cell behavior and tumor aggressiveness in many tissues. In this study, our goal was to determine the potential role of Id proteins, particularly Id1, during human SGC cell progression.MethodsWe first determined the expression levels of Id1 and Id2 in four SGC cell lines: two adenocarcinoma of the salivary gland (HSG and HSY) and two adenoid cystic carcinoma (ACC2 and ACCM) cell lines. We then used constructs that expressed antisense cDNAs to Id1 or Id2 to knockdown the expression of these proteins in cell lines where they were highly expressed, and determined the effects of the knockdown on cell proliferation, migration and invasion.ResultsId1 mRNA and protein were detectable in all cell lines, and expression of Id2 was variable, from absent to high. The ACC2 and ACCM cell lines expressed both Id1 and Id2, but Id1 was expressed at a higher level in the more aggressive ACCM cell line in comparison toACC2 cells as confirmed by Id1 promoter-reporter assays. We therefore focused on the ACCM cells for the remainder of the study. We found that proliferation and invasiveness of ACCM cells were strongly reduced after Id1 knockdown whereas Id2 suppression had only a slight effect. Results of scratch and colony formation assays also confirmed that ACCM cell aggressiveness was significantly reduced upon Id1 knockdown. Finally, this knockdown resulted in reduced c-myc and enhanced cyclin-dependent kinase inhibitor p21 expression.ConclusionsThese results demonstrate that Id1 plays an important role in the control of human SGC cell aggressiveness and suggest a potential role as a marker of diagnosis, prognosis and progression of SGCs. Id1 suppression could represent a novel and effective approach for the treatment of salivary gland cancer.

Novel Therapeutic Strategies for Malignant Salivary Gland Tumors: Lessons Learned from Breast Cancer

Malignant salivary gland tumors (MSGTs) account for 2–6% of all head and neck cancers. Despite the rarity, MSGTs have been of great interest due to a wide variety of pathological features and high metastasis rates resulting in poor prognosis. Surgical resection followed by radiation therapy represents the main treatment of this malignancy. Adjuvant therapy is reserved for the management of local recurrence, no longer amenable to additional local therapy, and for metastasis. Based on the studies from other types of tumors, particularly breast cancer, the expression and function of sex steroid hormone receptors in cancer have been extensively studied and applied to diagnosis and treatment. Although a number of studies in MSGTs have been published, the rationale for hormone therapy is still controversial due to the disparate results and insufficient number of cases. However, some recent reports have demonstrated that certain salivary gland neoplasms are similar to breast cancer, not only in terms of the pathological features, but also at the molecular level. Here, we shed light on the biological similarity between MSGTs and certain types of breast cancer, and describe the potential use of hormone and additional therapies for MSGTs.

Double sentinel lymph node mapping with indocyanine green and 99m-technetium–tin colloid in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) frequently metastasizes to cervical lymph nodes, which is the most known prognostic factor. Screening methods to identify sentinel lymph nodes (SLNs) are therefore of great interest for the management of potential neck metastasis. The purpose of this study was to evaluate the clinical benefit of double SLN mapping with indocyanine green (ICG) and 99m-technetium-tin colloid ((99m)Tc-tin colloid) for sentinel node navigation surgery (SNNS). Between 2007 and 2010, 16 patients diagnosed with OSCC were investigated by SLN biopsy using the double mapping method. (99m)Tc-tin colloid was injected into the peri-tumoural region on the preoperative day, and ICG was administered intraoperatively in the same position to assist in detecting nodes during surgery. Based on the gamma-ray signal and near-infrared (NIR) fluorescence of ICG, SLNs were identified and thereafter assessed pathologically and genetically for cancer involvement. Radio-guided detection was successful for all patients. ICG mapping identified a relatively larger number of nodes, suggesting that several non-SLNs were potentially involved. The double mapping method assisted surgeons to explore SLNs. Since the ICG fluorescence was shielded by the subcutaneous fatty tissue and the muscle layer including platysma and sternocleidomastoid, it was necessary to retract the tissue away from nodes.

Objective validity of an implant-retained overdenture with a ball attachment system after marginal mandibulectomy

Implant-retained overdentures are known to improve oral function, but the clinical impact on patients who have had mandibular resections is still debatable. We have treated 16 patients who had such resections for oral cancer and consequent loss of the alveolar ridge, with overdentures supported by osseointegrated implants and ball attachments. To quantify their functional improvement, we evaluated their maximum bite force and masticatory performance. Their function improved significantly, (from 77.5N - 365N, 371% increase in maximum bite force, p<0.001) and masticatory performance increased (from 2.5 - 7.7, 208%, p<0.0001) after the overdentures had been inserted. While individual changes in maximum bite force showed no significant correlation, those in masticatory performance correlated significantly, which suggests that the subjects with poor masticatory function are likely to benefit from retention of an implant. These results indicate that implant-retained overdentures are an effective way to rehabilitate patients after marginal mandibular resection.

A Case of Impacted Supernumerary Fourth Molar in the Bilateral Mandibular Ramus

Abstract The occurrence of multiple supernumerary teeth in individuals without any associated syndrome is rare. In this report, a rare case of a 48-year-old woman who had an impacted supernumerary fourth molar in the bilateral mandibular ramus is described. She presented with a swelling in the left cheek region. Radiographic examination revealed an impacted supernumerary tooth in the left mandibular ramus with pericoronal resorption of the bone, suggesting peripheral inflammation. She also had an impacted supernumerary tooth on the right side. After administering an antibiotic and antiinflammatory drug, tooth extraction was performed under general anesthesia.

Prognostic impact of preoperative serum interleukin‑6 levels in patients with early‑stage oral squamous cell carcinoma, defined by sentinel node biopsy

Failure to detect recurrence and lymph node metastasis early represents a fundamental barrier to the improvement of survival rate in early stage oral squamous cell carcinoma (OSCC). The present study evaluated the association between serum interleukin-6 (IL-6) level and clinical outcomes in patients with early stage OSCC patients defined by sentinel node biopsy (SNB). A total of 53 patients with clinical stage I/II OSCC who underwent SNB were enrolled. SNB was determined by a radioisotope method, and was evaluated by histopathological examination and genetic analysis. Preoperative sera were measured for IL-6 by ELISA. In the clinical stage I/II patients, disease-free survival (DFS) was demonstrated to be higher in patients with negative SNB compared with patients with positive SNB. In total, 13 patients were demonstrated to exhibit lymph node metastasis by SNB or were reclassified to pathological stage T4 subsequent to analysis of the surgically resected specimens. Thus, 40 patients were diagnosed with early stage OSCC. Of these 40 patients, DFS of the patients with low serum IL-6 was significantly higher compared with the patients with high serum IL-6 (P=0.012). In 19 patients with negative SNB and low serum IL-6, the disease-free rate was 100%. These findings suggested that SNB staging and serum IL-6 level have a high prognostic value in patients with early stage OSCC. Additional investigation and longer follow-up times are warranted to improve understanding of the group of patients that may benefit from this procedure.

Abstract 1017: Id-1 gene and protein as therapeutic target for head and neck squamous cell carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Squamous cell carcinoma is one of the most common malignancies in head and neck region. Primary tumors, especially derived from undifferentiated cells, tend to have aggressive phenotypes, which are mainly characterized by local recurrence and distant metastasis. In this study we found that Id-1, a negative regulator of tissue specific transcription factors, is highly expressed in head and neck squamous carcinoma cells (HNSCC), and that the expression is correlated with the malignant phenotype, i.e., cell proliferation and invasion. Id-1 knockdown dramatically reduces squamous cell invasion that is accompanied by profound morphologic changes and robust reduction in expression levels of mesenchymal markers.In addition, reduction in tumor growth and neoangiogenesis were observed in vivo, indicating that knock-down of Id-1 drastically suppresses VEGFA expression. Our results also suggested that VEGFA-autocrine loop activates tylosine kinase pathways including Srk and FAK, and therefore manifests invasive phenotype. With clinical materials obtained from HNSCC patients, significant correlation was observed between Id-1 and VEGFA. Furthermore, we demonstrated that a non toxic compound, cannabidiol, significantly downregulates Id-1 gene expression and associated squamous cell proliferation and invasiveness. Taken together, these results suggest that Id-1 controls cancer cell phenotype in culture and in vivo, and that drugs targeting Id-1 represent a novel and promising strategy for improving the therapy and outcome of patients with HNSCC. Citation Format: Ryuichi Murase, Sean McAllister, Yohei Fujita, Tomoki Sumida, Koichi Nakashiro, Pierre Desprez, Hiroyuki Hamakawa. Id-1 gene and protein as therapeutic target for head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1017. doi:10.1158/1538-7445.AM2014-1017