Integrating analysis of proteome profile and drug screening identifies therapeutic potential of MET pathway for the treatment of malignant peripheral nerve sheath tumor.

BACKGROUND: Malignant peripheral nerve sheath tumor (MPNST) is an aggressive sarcoma with a poor prognosis that requires novel therapeutic agents. Proteome information is useful for identifying new therapeutic candidates because it directly reflects the biological phenotype. Additionally, in vitro drug screening is an effective tool to identify candidate drugs for common cancers. Hence, we attempted to identify novel therapeutic candidates for MPNST by integrating proteomic analysis and drug screening. METHODS: We performed comprehensive proteomic analysis on 23 MPNST tumor samples using liquid chromatography - tandem mass spectrometry to identify therapeutic targets. We also conducted drug screening of six MPNST cell lines using 214 drugs. RESULTS: Proteomic analysis revealed that the MET and IGF pathways were significantly enriched in the local recurrence/distant metastasis group of MPNST, whereas drug screening revealed that 24 drugs showed remarkable antitumor effects on the MPNST cell lines. By integrating the results of these two approaches, MET inhibitors, crizotinib and foretinib, were identified as novel therapeutic candidates for the treatment of MPNST. CONCLUSIONS: We successfully identified novel therapeutic candidates for the treatment of MPNST, namely crizotinib and foretinib, which target the MET pathway. We hope that these candidate drugs will contribute to the treatment of MPNST.

Artesunate inhibits intestinal tumorigenesis through inhibiting wnt signaling.

Artesunate (ART) is a clinically approved antimalarial drug and was revealed as a candidate of colorectal cancer chemopreventive agents in our drug screening system. Here, we aimed to understand the suppressive effects of ART on intestinal tumorigenesis. In vitro, ART reduced T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter transcriptional activity. In vivo, ART inhibited intestinal polyp development. We found that ART reduces TCF1/TCF7 nuclear translocation by binding the Ras-related nuclear protein (RAN), suggesting that ART inhibits TCF/LEF transcriptional factor nuclear translocation by binding to RAN, thereby inhibiting Wnt signaling. Our results provide a novel mechanism through which artesunate inhibits intestinal tumorigenesis.

Drug screening with a novel tumor-derived cell line identified alternative therapeutic options for patients with atypical teratoid/rhabdoid tumor.

Atypical teratoid/rhabdoid tumor (AT/RT) is a rare intracranial tumor occurring predominantly in young children. The prognosis is poor, and no effective treatment is currently available. To develop novel effective therapies, there is a need for experimental models for AT/RT. In this research, we established a cell line from a patient's AT/RT tissue (designated ATRT_OCGH) and performed drug screening using 164 FDA-approved anti-cancer agents, to identify candidates for therapeutic options. We found that bortezomib, a proteasome inhibitor, was among the agents for which the cell line showed high sensitivity, along with tyrosine kinase inhibitors, topoisomerase inhibitors, and histone deacetylase inhibitors, which are known to exert anti-AT/RT effects. Concomitant use of panobinostat potentiated the inhibitory effect of bortezomib on AT/RT cell proliferation. Our findings may provide a rationale for considering combination therapy of panobinostat and bortezomib for treatment of AT/RT.

Deferasirox Might Be Effective for Microcytic Anemia and Neurological Symptoms Associated with Aceruloplasminemia: A Case Report and Review of the Literature.

The patient was a 64-year-old man presented with difficulty in walking, articulation, and swallowing, as well as cognitive impairment. He had refractory microcytic anemia and diabetes mellitus. His serum levels of iron, copper, and ceruloplasmin were low. Magnetic resonance imaging suggested iron deposition in the basal ganglia, thalami, cerebellar dentate nuclei, and cerebral and cerebellar cortices. He was diagnosed with aceruloplasminemia after a ceruloplasmin gene analysis. Iron chelation therapy with deferasirox improved his anemia and cerebellar symptoms, which included dysarthria and limb ataxia. The present study and previous reports indicate that cerebellar symptoms with aceruloplasminemia might respond to deferasirox in less than one year.

Establishment of novel patient-derived models of dermatofibrosarcoma protuberans: two cell lines, NCC-DFSP1-C1 and NCC-DFSP2-C1.

Dermatofibrosarcoma protuberans (DFSP) is a common type of dermal sarcoma, characterized by the presence of the unique collagen type I alpha 1 chain (COL1A1)-PDGFB translocation, which causes constitutive activation of the platelet-derived growth factor ß (PDGFB) signaling pathway. Patients with DFSP exhibit frequent local recurrence, and novel therapeutic approaches are required to achieve better clinical outcomes. Patient-derived cancer cell lines are essential in the preclinical research. Here, we established novel patient-derived DFSP cell lines from two patients with DFSP and designated these cell lines NCC-DFSP1-C1 and NCC-DFSP2-C1. Tumors of the two patients with DFSP had COL1A1-PDGFB translocations with distinct COL1A1 breakpoints, e.g., in exons 33 and 15, and the translocations were preserved in the established cell lines. NCC-DFSP1-C1 and NCC-DFSP2-C1 cells exhibited similar morphology and limited capability of proliferation in vitro, forming spheroids when seeded on low-attachment tissue culture plates. In contrast, NCC-DFSP1-C1 cells had considerably higher invasive capability than NCC-DFSP2-C1 cells. Overall proteome contents were similar between NCC-DFSP1-C1 and NCC-DFSP2-C1 cells. Notably, in vitro screening studies identified anticancer drugs that showed antiproliferative effects at considerably low concentrations in the DFSP cell lines. Bortezomib, mitoxantrone, ponatinib, and romidepsin were more cytotoxic to NCC-DFSP1-C1 cells than to NCC-DFSP2-C1 cells. These cell lines will be useful tools for developing novel therapeutic strategies to treat DFSP.

Establishment of a novel patient-derived Ewing's sarcoma cell line, NCC-ES1-C1.

Ewing's sarcoma is an aggressive mesenchymal tumor characterized by the presence of a unique EWSR1-FLI1 translocation. Ewing's sarcoma primarily occurs in the bone and soft tissues. Cell lines enable researchers to investigate the molecular backgrounds of disease and the significance of genetic alterations in relevant cellular contexts. Here, we report the establishment and characterization of a novel Ewing's sarcoma cell line following primary Ewing's sarcoma tumor tissue culture. The established cell line was authenticated by DNA microsatellite short tandem repeat analysis, characterized by in vitro assays, and named NCC-ES1-C1. The NCC-ES1-C1 cell line grew well for 15 mo and was subcultured more than 50 times during this period. Characterization of the cells revealed that they were not adherent and showed floating features. In conclusion, we successfully established a novel Ewing's sarcoma cell line, NCC-ES1-C1, from primary tumor tissue. The cell line has the characteristic EWSR1-FLI1 gene fusion and exhibits aggressive growth in vitro. Thus, the NCC-ES1-C1 cell line will be a useful tool for investigating the mechanisms of disease and the biological role of the EWSR1-FLI1 fusion gene.

Complementary Critical Functions of Zfy1 and Zfy2 in Mouse Spermatogenesis and Reproduction.

The mammalian Y chromosome plays a critical role in spermatogenesis. However, the exact functions of each gene in the Y chromosome have not been completely elucidated, partly owing to difficulties in gene targeting analysis of the Y chromosome. Zfy was first proposed to be a sex determination factor, but its function in spermatogenesis has been recently elucidated. Nevertheless, Zfy gene targeting analysis has not been performed thus far. Here, we adopted the highly efficient CRISPR/Cas9 system to generate individual Zfy1 or Zfy2 knockout (KO) mice and Zfy1 and Zfy2 double knockout (Zfy1/2-DKO) mice. While individual Zfy1 or Zfy2-KO mice did not show any significant phenotypic alterations in fertility, Zfy1/2-DKO mice were infertile and displayed abnormal sperm morphology, fertilization failure, and early embryonic development failure. Mass spectrometric screening, followed by confirmation with western blot analysis, showed that PLCZ1, PLCD4, PRSS21, and HTT protein expression were significantly deceased in spermatozoa of Zfy1/2-DKO mice compared with those of wild-type mice. These results are consistent with the phenotypic changes seen in the double-mutant mice. Collectively, our strategy and findings revealed that Zfy1 and Zfy2 have redundant functions in spermatogenesis, facilitating a better understanding of fertilization failure and early embryonic development failure.

Proteomic approach toward personalized sarcoma treatment: lessons from prognostic biomarker discovery in gastrointestinal stromal tumor.

Sarcomas range from curable tumors to those causing death via metastasis and recurrence. Thus, there is an urgent need for biomarker identification in order to assess the degree of malignancy, predict prognosis, and evaluate possible therapies. Various proteomic approaches and different clinical materials have been used to this end, and candidate biomarkers have been reported for the different types of sarcomas. However, the sample size used in these biomarker studies was generally insufficient, and thus far, no biomarker has been proved useful in clinics. Given that sarcomas are rare, biomarker validation in this setting is more challenging than in other malignancies. In gastrointestinal stromal tumor, adjuvant therapy has proven to be effective. However, only 40% patients experience metastasis after curative surgery alone, and the rest of the patients may not need adjuvant therapy. Using a proteomic approach, we identified pfetin (potassium channel tetramerization domain containing 12, KCTD 12) as a novel prognostic biomarker for sarcoma, and immunohistochemically confirmed its clinical usefulness by a multiinstitutional validation study. Here, we describe our experience and discuss the critical points in the discovery of this biomarker.

Molecular background of alpha-fetoprotein in liver cancer cells as revealed by global RNA expression analysis.

alpha-Fetoprotein (AFP) is considered to be a diagnostic and prognostic biomarker in hepatocellular carcinoma (HCC). However, the role of AFP in the development of HCC is presently obscure. We hypothesized that a certain set of genes is expressed in a manner coordinate with AFP, and that these genes essentially contribute to the malignant characteristics of AFP-producing HCC. To address this hypothesis, we carried out global mRNA expression analysis of 21 liver cancer cell lines that produce varying levels of AFP. We identified 213 genes whose mRNA expression levels were significantly correlated with that of AFP (P < 0.0001). These included liver-specific transcription factors for AFP and other albumin family genes. Eighteen HCC-associated genes and 11 genes associated with malignancies other than HCC showed significant correlations with AFP production levels. Genes involved in lipid catabolism, blood coagulation, iron metabolism, angiogenesis, and the Wnt and mitogen-activated protein kinase pathways were also identified. Text data mining revealed that participation in the transcription factor network could explain the connection between 78 of the identified genes. Glypican 3, which is a component of the Wnt pathway and contributes to HCC development, had the fifth highest correlation coefficient with AFP. Reactivity to specific antibodies confirmed the significant correlation between AFP and glypican 3 expression in HCC tissues. These observations suggest that AFP-producing liver cancer cells may have a unique molecular background consisting of cancer-associated genes. From this genome-wide association study, novel aspects of the molecular background of AFP were revealed, and thus may lead to the identification of novel biomarker candidates.