Koichi Nakashiro

Plasma-membrane-anchored growth factor pro-amphiregulin binds A-type lamin and regulates global transcription.

Amphiregulin (AR), a member of the EGF family, is synthesized as a type I transmembrane protein precursor (proAR) and expressed on the cell surface. Shedding of proAR yields a transmembrane-cytoplasmic fragment (AR-CTF), as well as a soluble AR. Here we demonstrate that the proAR-shedding stimuli trigger endocytosis of both AR-CTF and un-shed proAR. ProAR translocates from the plasma membrane to the inner nuclear membrane, whereas AR-CTF is translocated to the lysosome via retrograde membrane trafficking. Nuclear envelope localization of proAR involves truncation of the C-terminus, which subsequently activates the ER-retrieval signal. The truncated form of proAR interacts with A-type lamin and is retained at the inner nuclear membrane. Heterochromatin formation is then induced and global transcription is transiently suppressed. This study gives new insight into epigenetic chromatin organization in mammalian cells: a plasma-membrane-anchored growth factor is targeted to the inner nuclear membrane where it participates in dynamic chromatin organization and control of transcription.

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.

Abstract 1137: Mutation and function of PIK3CA and HRAS in human oral squamous cell carcinoma cells

Whole-exome sequencing studies showed the mutational landscape of head and neck squamous cell carcinoma including oral cavity. Recent several reports also demonstrated the somatic mutations of oncogenes and tumor suppressor genes in oral squamous cell carcinoma (OSCC), and identified the mutations of HRAS, BRAF, FGFR3, SMAD4, KIT, PTEN, NOTCH1, AKT1, CTNNB1, and PTPN11 to predict disease-free survival of OSCC patients. In this study, we have attempted to elucidate somatic mutations of these genes and their functions in human OSCC cells. We isolated and cultured tumor cells from the resected primary tumor (KT-T), metastatic lymph node (KT-N), and cancerous pleural effusion (KT-M) in the same patient with OSCC. KT-T cells showed epithelial-like shape, whereas both KT-N and KT-M cells indicated the morphological changes to fibroblast-like spindle shape. Genomic DNA was extracted from these cells, and then we performed the mutational analysis by ultra-deep targeted sequencing using Haloplex Cancer Research Panel. All types of KT cells had the mutation of TP53 (R141P, C135S, and E182*) and PIK3CA (H1047R), and an additional active mutation of HRAS (Q61R) was detected in only metastatic KT-N and KT-M cells. Subsequently, we transfected all KT cells with synthetic small interfering RNA (siRNA) specific for PIK3CA (siPIK3CA) and/or HRAS (siHRAS) at the concentration of 10 nM complexed with Lipofectamine RNAiMAX. We confirmed the knockdown effects of these siRNAs in KT cells by quantitative RT-PCR. After transfection of these siRNAs for 72 hours, the growth of KT cells was evaluated by WST-8 assay. Knockdown of PIK3CA or HRAS expression significantly suppressed the growth of KT-T cells only, whereas double knockdown of PIK3CA and HRAS inhibited the growth of KT-N and KT-M cells as well. Next, we investigated the function of mutant HRAS (Q61R) in KT-T cells. Overexpression of mutant HRAS did not change the growth rate and morphology of KT-T cells. Finally, we examined the effects of BEZ-235 (dual PI3K/mTOR inhibitor) and trametinib (MEK1/2 inhibitor) on the growth of KT-T, KT-N, and KT-M cells. BEZ-235 (100 nM) almost completely suppressed the growth of these cells. Trametinib (2 μM) also reduced the growth rate by 16.9% in KT-T, 13.5% in KT-N, and 45.7% in KT-M cells. Furthermore, BEZ-235 combined with trametinib inhibited the cell growth more effectively even at low concentrations. These results suggest that constitutive active mutations of HRAS and PIK3CA support the growth of human OSCC cells and these signaling pathways may be useful therapeutic targets for the patients with OSCC. Citation Format: Hitoshi Akiyama, Koichi Nakashiro, Norihiko Tokuzen, Hiroshi Tanaka, Satoshi Hino, Hiroyuki Hamakawa. Mutation and function of PIK3CA and HRAS in human oral squamous cell carcinoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1137.

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