Yoshimi Arima

Abstract Background: The NF1 tumor suppressor gene encodes neurofibromin and is a functional Ras GTPase-activating protein (RasGAP) that negatively regulates the Ras signaling pathway by accelerating the conversion of activated Ras-GTP to inactive Ras-GDP. NF1 gene germline mutations cause various Neurofibromatosis type 1 (NF1) symptoms, including neurofibroma development. We are developing in vitro models that recapitulate the pathological and clinical properties of neurofibromas with the aim of developing therapeutic strategies to treat patients with NF1 gene-deficient tumors. Methods: Neurofibroma cells and dedifferentiated fat (DFAT) cells were established from NF1 patient tumors. Tissue samples were obtained during tumor resection at our hospital from patients who met NIH clinical diagnostic criteria for NF1. Whole-blood specimens were also obtained for gene analysis. All patients provided written informed consent. The institutional review board at our university approved this aspect of our study. Tumor tissues were dissociated in DMEM containing collagenase. The neurofibroma cells at the bottom of the tube and the floating stromal adipocytes were collected separately after centrifugation. To establish DFAT cells, the stromal adipocytes were placed in a culture flask filled with 20% FBS-DMEM, and then the flask was inverted and incubated at 37 °C in a humidified atmosphere of 5% CO2. The stromal adipocytes floated up through the medium and adhered to the ceiling of the flask. After 1 week, the cells were firmly attached to the ceiling and had dedifferentiated. The DFAT cells as well as the neurofibroma cells can be passaged. The DFAT cells exhibited multipotent differentiation abilities into a variety of cell types. Results: We established neurofibroma cells and DFAT cells from NF1-associated neurofibromas. We performed flow cytometry analysis and found that the cells derived from NF1 patients expressed SOX10, S100, and CD90, all of which are expressed in Schwann cells. We identified the NF1 mutations in patients by next-generation sequencing. Peripheral blood specimens from patients 1 and 2 were positive for c.1466A>G, p.Tyr489Cys and c.3213_3214delAA, p.Ser1072Hisfs*16 mutations of NF1, respectively. We also identified NF1 mutations in the cells that we had established from tumors. In the tumor specimen of patient 1, we identified an additional somatic mutation, c.6772C>T, p.Arg2258X of NF1 gene. Conclusions: We established NF1 gene-deficient neurofibroma cells and NF1 gene-deficient DFAT cells from the tumor tissues from NF1 patients with NF1 gene mutations. These cells may well be useful in studying the pathophysiology of NF1 gene-deficient tumors as well as cell-based drug screening to facilitate the development of new treatments. Note: This abstract was not presented at the meeting. Citation Format: Yoshimi Arima, Hiroyuki Nobusue, Shigeki Sakai, Kazuo Kishi, Toshiki Takenouchi, Kenjiro Kosaki, Hideyuki Saya. Establishment of neurofibroma cells and dedifferentiated fat (DFAT) cells from tumor tissues from patients diagnosed with NF1 (Neurofibromatosis type 1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5311.

Abstract Biliary tract cancers (BTCs) are relatively rare in the Western world, but the rates of BTCs have risen worldwide in the past few decades. BTCs, which arise from epithelial cells in bile ducts, include intrahepatic and extrahepatic cholangiocarcinomas, gallbladder carcinomas, and ampullary carcinomas. The differences between these subtypes are not fully understood because of the limited number of genetically engineered mouse models for BTCs. Genetic mutations in the KRAS gene and the epigenetic changes in Ink4a/Arf genes have been frequently detected in BTCs, and it has been considered that those alterations are associated with the development and maintenance of human BTCs. To clarify the biological features of intrahepatic cholangiocarcinomas and gallbladder carcinomas, we developed syngeneic mouse models based on a combination of organoid 3D cultures and the transplantation of oncogene-expressed syngeneic bile duct epithelial cells. We isolated the epithelial cell adhesion molecule (EpCAM)-positive intrahepatic bile duct epithelial cells and the gallbladder epithelial cells from the Ink4a/Arf knockout C57BL/6 mice, and the cells were maintained as organoids under 3D culture conditions. Those cells were then infected with retroviruses expressing KRASG12V. Lethal metastatic adenocarcinomas having differentiated components formed in our mice when we transplanted the KRASG12V-expressing cells into the livers of wild-type C57BL/6 mice. The tumors resembled human desmoplastic intrahepatic cholangiocarcinomas and gallbladder carcinomas. Our mouse models can be useful for investigating pathogenesis and therapeutic strategies for human BTCs. Citation Format: Akiyoshi Kasuga, Takashi Semba, Hiromasa Takaishi, Takanori Kanai, Hideyuki Saya, Yoshimi Arima. Intrahepatic cholangiocarcinoma and gallbladder carcinoma mouse model based on transplantation of syngeneic tumor-initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4083.

Abstract Background: Clarifying the cells of origin in human lung adenocarcinoma will contribute to a better understanding of oncogene-induced lung adenocarcinomas and to the development of new treatments. Alveolar type II cells and club cells (Clara cells) have been shown to be the cells of origin by lineage-tracing approaches and conditional transgenic mice. Additionally, bronchioalveolar stem cells (BASCs), which are adult murine distal lung epithelial stem cells, are considered to be the cells of origin in lung adenocarcinomas because BASCs have both self-renewal abilities and differentiation properties. We attempted to enrich BASCs from mouse lungs and determine whether or not BASCs have the potential to form tumors driven by the oncogene. Methods: The epithelial cell adhesion molecule (EpCAM)+/CD45-/CD31- lung cells were isolated from the Ink4a/Arf-/- C57BL/6 mice by using a fluorescence activated cell sorting (FACS). The cells were then subjected to 3D culture on Matrigel with B27, EGF, KGF, ROCK inhibitor in serum-free DMEM/F-12. For detection of BASCs, we investigated the expressions of EpCAM and Sca-1 by flow cytometric analyses. We also investigated the expressions of SP-C and CC-10 by immunofluorescence analyses. The cells were then infected with bicistronic retroviruses having green fluorescent protein (GFP) and either KRASG12V or EML4-ALK genes. The GFP-positive oncogene-expressing cells were sorted by FACS and expanded under 3D culture conditions. Cells were then transplanted into the recipient C57BL/6 mice so that we could investigate their tumorigenic activities. Results: Flow cytometric analyses revealed that the EpCAM+/Sca-1+ cells, which are putative BASCs, were enriched in the 3D culture of the isolated EpCAM+/CD45-/CD31- lung cells from Ink4a/Arf-/- mice. The percentage of EpCAM+/Sca-1+ cell fraction was 36.97%, 92.63%, 99.98%, at primary EpCAM+/CD45-/CD31- lung cells and after the first and second passage, respectively. We also detected a small population of SP-C+/CC-10+ cells in colonies formed under our 3D culture conditions. The oncogene-expressing cells maintained the population of EpCAM+/Sca-1+ cells; 88.64-99.89% and 72.72-99.12%, KRASG12V and EML4-ALK, respectively. These cells formed differentiated tumors which were similar to human lung adenocarcinomas, having papillary, acinar, and solid components. Conclusions: The EpCAM+/Sca-1+ lung cells, putative BASCs, from Ink4a/Arf-/- mice were enriched in 3D culture. These cells formed histologically different types of lung adenocarcinomas after the transduction of KRASG12V or EML4-ALK, suggesting that we had established the source of the lung adenocarcinoma cells. Citation Format: Takashi Semba, Ryo Sato, Akiyoshi Kasuga, Makoto Suzuki, Hideyuki Saya, Yoshimi Arima. Enriching EpCAM+/Sca-1+ mouse bronchioalveolar stem cells to generate lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1983.

Abstract The NF1 tumor suppressor gene encodes neurofibromin and is a functional Ras GTPase-activating protein (RasGAP) involved in negatively regulating the Ras signal by accelerating the conversion of activated Ras-GTP to inactive Ras-GDP. NF1 gene germline mutations cause Neurofibromatosis type 1 (NF1, von Recklinghausen disease). We hypothesized that additional genetic alterations promote the malignancy of NF1-associated tumors. To test our hypothesis, we inoculated a GFP-labeled human NF1-deficient cell line, sNF96.2-GFP, which has a frame-shift mutation (c.3683delC, p.Asn1229MetfsTer11) in the NF1 gene, into the renal sub-capsules of immunodeficient mice. A subclonal cell line, the A-1 cell, was established from the developed tumor. We found that A-1 cells show much higher tumorigenic activity and phosphorylation status of MEK and Akt than the parental sNF96.2-GFP cells. We analyzed the genomic DNA of both the sNF96.2 and the A-1 cells by using the next-generation sequencing and our medical exome panel of 4813 genes, which are known to be responsible for most human genetic disorders. We identified 18 heterozygous variants within coding regions of 17 genes that were present in the A-1 cells, but not in the original sNF96.2 cells. We found a single base substitution (c.1508G>T, p.Gly503Val) in the PTPN11 gene, which encodes the tyrosine phosphatese SHP-2, and is associated with the regulation of the Ras signaling pathway. It is critical to note that constitutional gain-of-function mutations in the PTPN11 gene cause Noonan Syndrome in humans due to activation of the Ras pathway. To determine the role of PTPN11 mutation in NF1-associated tumors, we established a cell line overexpressing PTPN11mut in sNF96.2-GFP cells. We inoculated parental cells and PTPN11mut cells into the subcutaneous of nude mice, and we found that PTPN11mut cells show much higher tumorigenic activity than the parental sNF96.2-GFP cells. Our data suggests that this additional gene mutation in PTPN11 promotes the malignant characteristics of NF1-associated tumors. Citation Format: Yoshimi Arima, Ritsuko Harigai, Ryo Sato, Toshiki Takenouchi, Kenjiro Kosaki, Hideyuki Saya. Additional mutation in PTPN11 gene promotes tumorigenesis of the NF1 gene mutated cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3418. doi:10.1158/1538-7445.AM2017-3418

Abstract Background: LATS1 participates in the Hippo pathway, which is best characterized in Drosophila melanogaster. Hippo pathway regulates cell proliferation and cell death to ensure appropriate organ size is maintained. LATS1 knockout mice develop ovarian tumor and soft tissue sarcoma, indicating that LATS1 functions as a tumor suppressor. Indeed, LATS1 expression is reduced in breast cancers due to hypermethylation of its promoter region, which is well correlated with poor prognosis of the patients. However in budding yeast, Dbf2 kinase which is the yeast counterpart of LATS1 phosphorylates and activates Cdc14 phosphatase to faithfully execute mitotic exit. This signaling pathway is known as mitotic exit network (MEN). Since there has been reported no phosphatase substrate of LATS1, we pursued the novel target of LATS1 to elucidate the significance of MEN in mammals and clarified a novel role of LATS1. Methods: We screened LATS1 substrates using phosphopeptide enrichment techniques coupled with high-accuracy mass spectrometry. We verified that the identified peptides are substrates of LATS1 both in vitro and in vivo. After determining the phosphorylation sites of the novel substrate, we analyzed functional significance of this phosphorylation-mediated signaling pathway. Results: Phosphoproteomic screening identified myosin phosphatase-targeting subunit1 (MYPT1) as a new substrate for LATS1. LATS1 directly and preferentially phosphorylated serine 445 (S445) of MYPT1. An MYPT1 mutant (S445A) failed to dephosphorylate threonine 210 of polo-like kinase1 (PLK1), thereby activating PLK1. This suggests that LATS1 promotes MYPT1 to antagonize PLK1 activity. Consistent with this, LATS1-depleted HeLa cells or fibroblasts from LATS1 knock-out mice showed increased PLK1 activity. We also found that DNA damage-induced LATS1 activation caused PLK1 suppression via the phosphorylation of MYPT1 S445. Furthermore, LATS1 knock-down caused impairment of G2 checkpoint arrest after DNA damage. Conclusions: These results indicate that LATS1 phosphorylates a phosphatase as does the yeast Dbf2 and demonstrate a novel link between two mitotic kinases, LATS1 and PLK1. PLK1 is overexpressed in many cancers and its expression levels often correlate with poor prognosis of the patients. The oncogenic properties of PLK1, such as inducing genomic instability by incomplete G2 checkpoint, would be enhanced under the condition of reduced LATS1 expression. Thus, our data raise the intriguing possibility that cancer patients with reduced LATS1 expression in their tumors are potential candidates for treatment with PLK inhibitors. Our data also suggests that mammalian MEN signaling pathway plays an important role for the G2 DNA damage checkpoint response other than mitotic exit. Citation Format: Tatsuyuki Chiyoda, Shinji Kuninaka, Kenta Masuda, Takatsune Shimizu, Yoshimi Arima, Daisuke Aoki, Hideyuki Saya. The Hippo pathway component LATS1 phosphorylates MYPT1 to counteract PLK1 and regulate G2 DNA damage checkpoint. [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 562. doi:10.1158/1538-7445.AM2013-562

Abstract Triple-negative breast cancer (TNBC) is classified as a heterogeneous group, because TNBCs are defined as breast cancer negative for the estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2 (HER2). We show that TNBC cell lines can be divided into those which express the repressor of transcription Zinc finger E-box binding homeobox 1 (ZEB1) and the tight junction protein claudin-1. ZEB1 has been identified as a key transcription factor eliciting epithelial-mesenchymal transition (EMT) and cancer progression. We found that knockdowns of ZEB1 in TNBC cells induce an epithelial-like morphology and induce the mRNA expression of claudin-1.> The claudin family is the principal constituent of tight junctions in epithelial cells. We also found that ZEB1 binds to a claudin-1 promoter sequence by DNA-mediated pull-down assay. Furthermore, in breast cancer specimens, we observed a reciprocal expression of ZEB1 and claudin-1. These findings suggest that ZEB1 represses claudin-1 expressions in TNBCs. We then analyzed the association of clinical outcomes with ZEB1 and claudin-1 expressions by using a publicly available gene expression database of breast cancer patients. The overall survival rates in the category which tested positive for ZEB1 and claudin-1 is poor. To further characterize the effects of ZEB1-positive breast cancer cells on this phenotype of cancer, we investigated tumor formations following the co-inoculation of ZEB1-expressing MDA-MB-231 cells and claudin-1-expressing HCC1937 cells into nude mice. Although HCC1937 cells alone didn't form tumors in our orthotopic xenograft models, tumor formations were observed when the cells were mixed with MDA-MB-231 cells. These data suggest that the ZEB1-expressing breast cancer cells do promote tumorigenicity of claudin-1-positive cancer cells. We hypothesized that the heterogeneity on gene expressions within the tumor tissue is associated with malignancy and therapeutic resistance of breast cancers. Citation Format: Yoshimi Arima, Mari Hosonaga, Hideyuki Saya. The epithelial-mesenchymal transition-related transcription factor ZEB suppresses Claudin expression in breast cancer cells. [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 522. doi:10.1158/1538-7445.AM2013-522