Masato Enari

KIF5B-RET fusions in lung adenocarcinoma

We identified in-frame fusion transcripts of KIF5B (the kinesin family 5B gene) and the RET oncogene, which are present in 1–2% of lung adenocarcinomas (LADCs) from people from Japan and the United States, using whole-transcriptome sequencing. The KIF5B-RET fusion leads to aberrant activation of RET kinase and is considered to be a new driver mutation of LADC because it segregates from mutations or fusions in EGFR, KRAS, HER2 and ALK, and a RET tyrosine kinase inhibitor, vandetanib, suppresses the fusion-induced anchorage-independent growth activity of NIH3T3 cells.

Druggable Oncogene Fusions in Invasive Mucinous Lung Adenocarcinoma

Purpose: To identify druggable oncogenic fusions in invasive mucinous adenocarcinoma (IMA) of the lung, a malignant type of lung adenocarcinoma in which KRAS mutations frequently occur. Experimental Design: From an IMA cohort of 90 cases, consisting of 56 cases (62%) with KRAS mutations and 34 cases without (38%), we conducted whole-transcriptome sequencing of 32 IMAs, including 27 cases without KRAS mutations. We used the sequencing data to identify gene fusions, and then performed functional analyses of the fusion gene products. Results: We identified oncogenic fusions that occurred mutually exclusively with KRAS mutations: CD74-NRG1, SLC3A2-NRG1, EZR-ERBB4, TRIM24-BRAF, and KIAA1468-RET. NRG1 fusions were present in 17.6% (6/34) of KRAS-negative IMAs. The CD74-NRG1 fusion activated HER2:HER3 signaling, whereas the EZR-ERBB4 and TRIM24-BRAF fusions constitutively activated the ERBB4 and BRAF kinases, respectively. Signaling pathway activation and fusion-induced anchorage-independent growth/tumorigenicity of NIH3T3 cells expressing these fusions were suppressed by tyrosine kinase inhibitors approved for clinical use. Conclusions: Oncogenic fusions act as driver mutations in IMAs without KRAS mutations, and thus represent promising therapeutic targets for the treatment of such IMAs. Clin Cancer Res; 20(12); 3087–93. ©2014 AACR.

Fas-induced DNA fragmentation and proteolysis of nuclear proteins.

Fas is a member of the tumour necrosis factor (TNF) receptor family. Activation of Fas by its ligand or an agonistic anti‐Fas antibody causes apoptosis in Fas‐bearing cells, by activating various members of the caspase family.

Requirement of ATM for Rapid p53 Phosphorylation at Ser46 without Ser/Thr-Gln Sequences

ABSTRACT p53 phosphorylation at Ser46 following DNA damage is important for preferential transactivation of proapoptotic genes. Here, we report that ataxia-telangiectasia mutated (ATM) kinase is responsible for Ser46 phosphorylation of p53 during early-phase response to DNA damage. To elucidate the direct phosphorylation of p53 at Ser46 by ATM, an ATM mutant (ATM-AS) sensitive to ATP analogues was engineered. In vitro kinase assays revealed that p53 was phosphorylated at Ser46 by ATM-AS, even when ATP analogues were used as phosphate donors, although this phosphorylation site is not in an SQ motif, a consensus ATM site. Furthermore, Ser46 phosphorylation by ATM was dependent on the N- and C-terminal domains of p53, unlike Ser15 phosphorylation. Immunofluorescence analyses showed that Ser46-phosphorylated p53 was observed as foci in response to DNA damage and colocalized with γ-H2AX or Ser1981-phosphorylated ATM. These results suggest that ATM phosphorylates a noncanonical serine residue on p53 by mechanisms different from those for the phosphorylation of Ser15.

Regulation of clathrin‐mediated endocytosis by p53

The p53 gene encodes a multi‐functional protein to prevent tumorigenesis. Although there have been many reports of the nuclear functions of p53, little is known about the cytosolic functions of p53. Here, we found that p53 is present in cytosol as well as nuclei under unstressed conditions and binds to clathrin heavy chain (CHC). CHC is known to play a role in receptor‐mediated endocytosis. Based on our findings, we examined the effect of p53 on clathrin‐mediated endocytosis of epidermal growth factor receptor (EGFR). Surprisingly, p53 co‐localized with CHC at the plasma membrane in response to EGF stimulation. In cells with ablated p53 expression by RNAi, EGFR internalization was delayed and intracellular signaling from EGFR was altered. Thus, our findings provide evidence that cytosolic p53 may participate in the regulation of clathrin‐mediated endocytosis to control the correct signaling from EGFR.

TSPAN12 is a critical factor for cancer–fibroblast cell contact-mediated cancer invasion

Significance Cancer-associated fibroblasts (CAFs) are abundant and promote cancer proliferation, invasion, and metastasis. Mutations in the p53 gene and decreased p53 expression are often detected in CAFs, and a dysfunction in p53 in CAFs contributes to cancer progression. However, how host-derived p53 influences cancer cells remains unclear. We herein established coculture systems to monitor enhancements in invasiveness and proliferation elicited by p53-depleted fibroblasts and demonstrated that tetraspanin 12 (TSPAN12), identified as a p53-regulated gene, was required for these processes through the contact of cancer cells with stromal fibroblasts and β-catenin–mediated CXC chemokine ligand 6 (CXCL6) secretion. These results suggest that antibodies against TSPAN12 and CXCL6 may be effective therapeutic agents for cancer. Communication between cancer cells and their microenvironment controls cancer progression. Although the tumor suppressor p53 functions in a cell-autonomous manner, it has also recently been shown to function in a non–cell-autonomous fashion. Although functional defects have been reported in p53 in stromal cells surrounding cancer, including mutations in the p53 gene and decreased p53 expression, the role of p53 in stromal cells during cancer progression remains unclear. We herein show that the expression of α-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts (CAFs), was increased by the ablation of p53 in lung fibroblasts. CAFs enhanced the invasion and proliferation of lung cancer cells when cocultured with p53-depleted fibroblasts and required contact between cancer and stromal cells. A comprehensive analysis using a DNA chip revealed that tetraspanin 12 (TSPAN12), which belongs to the tetraspanin protein family, was derepressed by p53 knockdown. TSPAN12 knockdown in p53-depleted fibroblasts inhibited cancer cell proliferation and invasion elicited by coculturing with p53-depleted fibroblasts in vitro, and inhibited tumor growth in vivo. It also decreased CXC chemokine ligand 6 (CXCL6) secretion through the β-catenin signaling pathway, suggesting that cancer cell contact with TSPAN12 in fibroblasts transduced β-catenin signaling into fibroblasts, leading to the secretion of CXCL6 to efficiently promote invasion. These results suggest that stroma-derived p53 plays a pivotal role in epithelial cancer progression and that TSPAN12 and CXCL6 are potential targets for lung cancer therapy.

Overexpression of the DNA sensor proteins, absent in melanoma 2 and interferon-inducible 16, contributes to tumorigenesis of oral squamous cell carcinoma with p53 inactivation

The development of oral squamous cell carcinoma (OSCC) is a multistep process that requires the accumulation of genetic alterations. To identify genes responsible for OSCC development, we performed high‐density single nucleotide polymorphism array analysis and genome‐wide gene expression profiling on OSCC tumors. These analyses indicated that the absent in melanoma 2 (AIM2) gene and the interferon‐inducible gene 16 (IFI16) mapped to the hematopoietic interferon‐inducible nuclear proteins. The 200‐amino‐acid repeat gene cluster in the amplified region of chromosome 1q23 is overexpressed in OSCC. Both AIM2 and IFI16 are cytoplasmic double‐stranded DNA sensors for innate immunity and act as tumor suppressors in several human cancers. Knockdown of AIM2 or IFI16 in OSCC cells results in the suppression of cell growth and apoptosis, accompanied by the downregulation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells activation. Because all OSCC cell lines have reduced p53 activity, wild‐type p53 was introduced in p53‐deficient OSCC cells. The expression of wild‐type p53 suppressed cell growth and induced apoptosis via suppression of nuclear factor kappa‐light‐chain‐enhancer of activated B cells activity. Finally, the co‐expression of AIM2 and IFI16 significantly enhanced cell growth in p53‐deficient cells; in contrast, the expression of AIM2 and/or IFI16 in cells bearing wild‐type p53 suppressed cell growth. Moreover, AIM2 and IFI16 synergistically enhanced nuclear factor kappa‐light‐chain‐enhancer of activated B cells signaling in p53‐deficient cells. Thus, expression of AIM2 and IFI16 may have oncogenic activities in the OSCC cells that have inactivated the p53 system. (Cancer Sci 2012; 103: 782–790)

Prevalence of human papillomavirus 16/18/33 infection and p53 mutation in lung adenocarcinoma

Human papillomavirus (HPV) infection is a causative event for the development of uterine cervical carcinoma. Human papillomavirus (HPV) 16, 18, and 33 DNA has been also detected frequently in lung adenocarcinomas (AdCs) in East Asian countries; however, its prevalence in Japan remains unclear. We therefore screened for HPV 16/18/33 DNA in 297 lung AdCs in a Japanese population by multiplex PCR with type‐specific primers. As reported previously, HPV 16 DNA was detected in two cervical cancer cell lines, CaSki and SiHa, while HPV 18 DNA was detected in HeLa cells, and 0.1–1.0 copies of HPV‐DNA per cell were detectable by this method. However, with this method, none of the 297 lung AdCs showed positive signals for HPV 16/18/33 DNA, indicating that HPV‐DNA is not or is very rarely integrated in lung AdC genomes in the Japanese. Furthermore, none of the lung AdCs showed positive signals by nested PCR with HPV 16/18 type‐specific primers. Therefore, we further attempted to detect HPV 16/18/33 DNA in 91 lung cancer cell lines, including 40 AdC cell lines. Among them, 30 have been established in Japan and the remaining 61 in the USA. No HPV signals were obtained in any of the 91 cell lines by either multiplex or nested PCR, while the p53 gene was mutated in 81 of them including 35 of the 40 AdC cell lines. These results indicate that HPV 16/18/33 infection does not play a major role in the development of lung AdC in Japan nor in the USA. (Cancer Sci 2010)

correction: A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD

This corrects the article DOI: 34112

NuMA Is Required for the Selective Induction of p53-Target Genes

ABSTRACT The p53 tumor suppressor protein is a transcription factor controlling various outcomes, such as growth arrest and apoptosis, through the regulation of different sets of target genes. The nuclear mitotic apparatus protein (NuMA) plays important roles in spindle pole organization during mitosis and in chromatin regulation in the nucleus during interphase. Although NuMA has been shown to colocalize with several nuclear proteins, including high-mobility-group proteins I and Y and GAS41, the role of NuMA during interphase remains unclear. Here we report that NuMA binds to p53 to modulate p53-mediated transcription. Acute and partial ablation of NuMA attenuates the induction of the proarrested p21 gene following DNA damage, subsequently causing impaired cell cycle arrest. Interestingly, NuMA knockdown had little effect on the induction of the p53-dependent proapoptotic PUMA gene. Furthermore, NuMA is required for the recruitment of cyclin-dependent kinase 8 (Cdk8), a component of the Mediator complex and a promoter of p53-mediated p21 gene function. These data demonstrate that NuMA is critical for the target selectivity of p53-mediated transcription.