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國久 智成医学部附属病院 乳腺内分泌外科講師
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■ 論文- BACKGROUND/AIM: Triple-negative breast cancer (TNBC) is a recalcitrant disease. The present study examined the efficacy of methionine restriction and the poly ADP-ribose polymerase (PARP)-inhibitor olaparib on BRCA1/2 wild-type and BRCA1 mutated TNBC cell lines. MATERIALS AND METHODS: The human BRCA1/2 wild-type cell line MDA-MB-231, and BRCA1-mutant cell lines MDA-MB-436 and HCC1937 were used to examine sensitivity to recombinant methioninase (rMETase) or a methionine-free medium or to olaparib or the combination of a methionine-free medium and olaparib. Cell viability was examined using the WST-8 reagent as well as by direct cell counting after Hoechst 33342 staining. RESULTS: MDA-MB-231 was sensitive to a methionine-free medium and rMETase and resistant to olaparib. The combination of olaparib and a methionine-free medium was not synergistic on MDA-MB-231 cells. MDA-MB-436 cells were not sensitive to a methionine-free medium but were sensitive to olaparib and rMETase. The combination of olaparib and a methionine medium was not synergistic in MDA-MB-436 cells. HCC1937 cells were sensitive to a methionine-free medium, partially sensitive to rMETase, partially resistant to olaparib, and sensitive to the combination of a methionine-free medium and olaparib. All three cell lines were sensitive to rMETase, with MDA-MB-436 being the most sensitive. CONCLUSION: Methionine restriction and olaparib showed synergistic efficacy on the BRCA1-mutant TNBC cell line HCC1937. The BRCA1-mutant cell line MDA-MB-436 was most sensitive to rMETase. The BRCA1/2 wild-type TNBC cell line MDA-MB-231 was sensitive to a methionine-free medium but resistant to olaparib. Therefore, methionine restriction has clinical potential for BRCA1/2 wild-type and BRCA1-mutant olaparib-resistant and -sensitive TNBC.2025年04月, Anticancer research, 45(4) (4), 1367 - 1372, 英語, 国際誌研究論文（学術雑誌）
- BACKGROUND/AIM: Exosomes are extracellular vesicles produced by both normal and cancer cells. Previous research has demonstrated that circulating exosomes derived from cancer cells may create a niche for future metastasis, distant from the primary tumor. In the present report, circulating exosomes were captured and quantified based on exosome-surface proteins in pre- and post-operative serum of breast cancer patients, focusing on the exosome markers CD9 and CD63, as well as HER2, a therapeutic target for breast cancer. MATERIALS AND METHODS: Eight breast cancer patients were recruited, and their pre- and post-operative serum samples were analyzed for CD63 and CD9; or CD9 and human epidermal growth factor receptor-2 (HER2), double-positive exosomes. An ExoCounter with antibody-conjugated beads was used to capture serum-derived exosomes. Sera from patients with tumors larger than 10 mm were used for analysis. The resected breast cancer was also histopathologically analyzed for the presence of HER2. RESULTS: CD63 and CD9 double-positive serum exosomes and CD9 and HER2 double-positive serum exosomes decreased after surgery in breast-cancer patients whose tumors expressed HER2, as determined by histopathological analysis. CONCLUSION: Serum exosomes expressing CD9, CD63 and HER2 are candidate biomarkers of tumor burden in HER2-positive breast-cancer patients.2024年, Cancer genomics & proteomics, 21(6) (6), 580 - 584, 英語, 国際誌研究論文（学術雑誌）
- BACKGROUND/AIM: BRCA1/2 mutations in breast cancer cells impair homologous recombination and promote alternative end joining (Alt-EJ) for DNA-damage repair. DNA polymerase theta, encoded by POLQ, plays a crucial role in Alt-EJ, making it a potential therapeutic target, particularly in BRCA1/2-mutant cancers. Methionine restriction is a promising approach to target cancer cells due to their addiction to this amino acid. The present study investigated the expression of POLQ in BRCA1/2 wild-type and BRCA1-mutant breast cancer cells under methionine restriction. MATERIALS AND METHODS: POLQ mRNA expression was measured using qRT-PCR in BRCA1/2 wild-type (MDA-MB-231) and BRCA1- mutant (HCC1937 and MDA-MB-436) breast-cancer cells under normal, or serum-restricted, or serum- and methionine-restricted conditions. RESULTS: Compared to BRCA1/2 wild-type cells, BRCA1-mutant cells displayed significantly higher basal POLQ expression in normal medium. Methionine restriction further increased POLQ expression in the BRCA1-mutant cells but decreased it in the BRCA1/2 wild-type cells. CONCLUSION: The present findings suggest that methionine restriction showed differential effects on POLQ expression, potentially impacting Alt-EJ activity, in BRCA1/2 wild-type and BRCA1-mutant breast-cancer cells. Further investigation is needed to explore the potential of combining methionine restriction with DNA-repair inhibitors, such as PARP inhibitors, to overcome drug resistance in BRCA1/2 mutant cancers.2024年, Cancer genomics & proteomics, 21(4) (4), 399 - 404, 英語, 国際誌研究論文（学術雑誌）
- BACKGROUND/AIM: Breast cancer that is estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and human epidermal growth factor receptor-2 (HER2)-negative is termed triple-negative breast cancer (TNBC). Cytotoxic chemotherapy remains the first choice of treatment against TNBC due to lack of specific therapeutic targets. TNBC is not classified based on therapeutic targets, but recently, the development of targeted therapies - including immune checkpoint inhibitors and poly (adenosine diphosphate-ribose) polymerase inhibitors - has gained attention. This study aimed to examine a novel target-oriented TNBC classification to further facilitate targeted therapy by classifying TNBC based on the breast cancer 1 (BRCA1)-like as well as the protein expression of HER2, programmed death ligand 1 (PD-L1), androgen receptor (AR), cytokeratin 5/6, and epidermal growth factor receptor (EGFR). PATIENTS AND METHODS: We enrolled 17 patients with primary TNBC who did not receive preoperative chemotherapy and underwent surgery at the Kobe University Hospital, Japan, between January 1, 2018, and July 31, 2019. Immunohistochemical staining was performed on tumor specimens, while a BRCAness test was performed using multiplex ligation-dependent probe amplification (MLPA) analysis. A BRCAness score 0.5 or higher was considered BRCA1-like. RESULTS: Tumors were classified as HER2-low (immunohistochemistry score 1+ or 2+ and FISH negative), PD-L1 positive, AR positive, or BRCA1-like. HER2-low, PD-L1 positive, AR positive, and BRCA1-like were detected in 11 (64.7%), 4 (23.5%), 6 (35.3%), and 6 (35.3%) samples. The tumor of only one patient could not be classified into any of these categories. CONCLUSION: Almost all TNBC cases can be classified according to treatable targets.2023年11月, Anticancer research, 43(11) (11), 5067 - 5072, 英語, 国際誌研究論文（学術雑誌）
- Metal nanoparticles (NPs), such as gold NPs (AuNPs), are particularly sensitive to X-rays, and thus specific accumulation of AuNPs in a tumor would allow radiotherapy with low energy X-rays and reduced side effects. AuNPs can be generated using HAuCl4 and the natural polyphenol epigallocatechin-3-gallate (EGCG) in the presence of citrate. Here, we generated EGCG-AuNPs in the presence of several additives and examined the accumulation of these NPs in mouse tumors following intravenous administration. EGCG-AuNPs 15 nm in diameter in the presence of sodium alginate accumulated more in tumors compared to 40-nm-diameter EGCG-AuNPs. Furthermore, the results of in vitro cellular uptake and serum protein absorption studies suggest that adsorption of 15–16 kDa serum proteins to EGCG-AuNPs suppresses accumulation in tumors. Thus, tendency to adsorb specific proteins on EGCG-AuNPs surface should be tailored for enhancing their accumulation in tumors.2023年04月, JCIS Open, 9研究論文（学術雑誌）
- BACKGROUND/AIM: Methionine addiction is the elevated requirement for exogenous methionine for growth and survival of cancer cells, termed the Hoffman effect. Methionine-addicted cancer cells synthesize normal or excess amounts of methionine but still need an external source of methionine. Methionine restriction (MR) by either a methionine-free medium or in vivo by a low-methionine diet or by methioninase, selectively arrests cancer cells in the late S/G2 cell cycle phase, but not normal cells. The present study focuses on the comparison of production and secretion of exosomes by cancer cells under MR and normal conditions. MATERIALS AND METHODS: MDA-MB-231 cells (triple-negative breast cancer), containing exosomes labeled with CD63-GFP (CD63-GFP exosomes), were visualized by fluorescence microscopy. MDA-MB-231 cells expressing exosome-specific CD63-GFP were cultured in methionine-containing (MET+) or in methionine-free (MET-) DMEM conditions. Exosomes were isolated from conditioned medium of cultured MDA-MD-231 cells by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA) and Western blotting. RESULTS: When MDA-MB-231-CD63-GFP cells were cultured under MR conditions, they arrested their growth and CD63-GFP-expressing exosomes were strongly increased in the cells. MR resulted in approximately a 2-fold increase in exosome production and secretion per cell, even though cell growth was arrested. Methionine restriction thus resulted in elevated exosome production and secretion per surviving cell. CONCLUSION: Exosome production and secretion in the cancer cells increased under MR, suggesting a relation between MR and exosome production and secretion.2023年, Cancer genomics & proteomics, 20(5) (5), 412 - 416, 英語, 国際誌研究論文（学術雑誌）
- S. Karger {AG}, 2022年03月, Case Reports in Oncology, 351 - 355研究論文（学術雑誌）
- BACKGROUND/AIM: Aldehyde dehydrogenase (ALDH) 1A1 is a well-known marker for cancer stem cells (CSCs), characterized by self-renewal capacity and multidrug resistance in breast cancer. We developed a near-infrared turn-on fluorescence probe for ALDH1A1, C5S-A, which is suitable for observing and analyzing viable cells. Here, we demonstrated the utility of C5S-A in CSC research using breast cancer cell lines. MATERIALS AND METHODS: To evaluate concordance between C5S-A and conventional stem cell markers, breast cancer cells sorted for ALDEFLUOR-positive cells and for CD44+/CD24- cell populations were stained with C5S-A. Tumorigenicity of C5S-A-positive cells was examined by mammosphere formation assay and subcutaneous transplantation to immunodeficient mice. Additionally, to determine how long fluorescence from a single staining remained observable, we cultured breast cancer cells for 5 days after C5S-A staining. We then evaluated whether C5S-A-positive cells possessed resistance to cytotoxic drugs by chronological imaging. RESULTS: C5S-A staining showed good concordance with conventional breast CSC markers, and good utility for research into CSC characteristics in breast cancer cell lines, including tumorigenesis. Additionally, C5S-A was observable for more than 3 days with a single staining. Using this property, we then confirmed that C5S-A-positive cells possessed resistance to cytotoxic drugs, which is one of the characteristics of CSCs. CONCLUSION: We showed that C5S-A is suitable for CSC research using breast cancer cell lines, and confirmed its utility in observing cells over time.2022年03月, Anticancer research, 42(3) (3), 1199 - 1205, 英語, 国際誌研究論文（学術雑誌）
- The high incidence and mortality of cancer make it a global health issue. However, conventional cancer therapies have several disadvantages, especially serious side effects due to low selective toxicity to cancer cells. Gold nanoparticles (AuNPs) are an excellent drug carrier, enhance drug delivery efficiency, and hold promise for photothermal and radiation therapies. (-)-Epigallocatechin-3-gallate (EGCG) is the major polyphenolic antioxidant constituent of green tea, has a potent antitumor effect, and binds specifically to the 67 kDa laminin receptor, which is overexpressed on the surface of several cancer cell lines such as HeLa and MDA-MB-231 cells. We synthesized EGCG-modified AuNPs (EGCG-AuNPs) using ratios (nEGCG/ngold) from 1:2 to 10:1 and evaluated their size, morphology, stability, antioxidant ability, cytotoxicity, cellular uptake, and uptake mechanisms in vitro in comparison with the conventional AuNPs prepared by using citrate as the reducing agent (citrate-AuNPs). In HeLa cells, EGCG-AuNPs (10:1) (135 nm diameter, sea-urchin-like shape) exhibited the highest cellular uptake. Conversely, EGCG-AuNPs (1:2) (39 nm diameter, spherical shape) were preferentially taken up by MDA-MB-231 cells. Cellular uptake of EGCG-AuNPs toward normal cells (NIH3T3 cells) was found to be in a nonspecific manner, and the amount of uptake was suppressed. X-ray irradiation after cellular uptake of EGCG-AuNPs (1:2) in MDA-MB-231 cells significantly enhanced irradiation-induced cell death. These findings suggest enhanced cellular uptake of EGCG-AuNPs with a 39 nm diameter and their potential use in combinatorial therapeutics of EGCG-AuNPs for breast cancer.2022年01月, ACS Applied Bio Materials, 5(1) (1), 355 - 365[査読有り]研究論文（学術雑誌）
- [A Case of Metastatic Recurrent Breast Cancer Successfully with Olaparib in Late Line Therapy].A 44-years-old woman who underwent bilateral mastectomy was treated with chemotherapy after axillary lymph nodes and liver metastases recurrence. She was referred to our hospital for BRCA1/2 germline test and the test revealed BRCA2 pathogenic mutation. Before the administration of olaparib as the fourth-line therapy, liver dysfunction, caused by extensive liver metastasis, was observed. The liver damage improved, and tumor markers decreased immediately as shown in the blood test and CT examination results after 2 months; indicating marked reduction of liver metastasis. In the OlympiAD trial, the patients received olaparib as either the first-, second- or third-line treatment; however, few data on the efficacy of olaparib in the patients, as a late line treatment, were reported. In this article, we report a case of a woman in whom olaparib was used as the fourth-line treatment for metastatic recurrent breast cancer. A high therapeutic effect was obtained and the quality of life has been maintained in her for the past 1 year.2021年09月, Gan to kagaku ryoho. Cancer & chemotherapy, 48(9) (9), 1153 - 1155, 日本語, 国内誌研究論文（学術雑誌）
- Anticancer Research USA Inc., 2020年06月, Anticancer Research, 40(6) (6), 3091 - 3096[査読有り]研究論文（学術雑誌）
- Oncogenic miRNAs Identified in Tear Exosomes From Metastatic Breast Cancer Patients (vol 40, pg 3091, 2020)3091INT INST ANTICANCER RESEARCH, 2020年08月, ANTICANCER RESEARCH, 40(8) (8), 4805 - 4805, 英語その他
- 日本学術振興会, 科学研究費助成事業, 基盤研究(B), 神戸大学, 2024年04月01日 - 2027年03月31日乳癌の早期診断のための涙液バイオマーカーの探索
- 日本学術振興会, 科学研究費助成事業, 基盤研究(A), 神戸大学, 2022年04月01日 - 2026年03月31日細胞外小胞を用いたリキッドバイオプシーによる癌に対する術前化学療法の効果予測
- 日本学術振興会, 科学研究費助成事業, 基盤研究(C), 神戸大学, 2021年04月01日 - 2024年03月31日涙液を用いた新しい乳癌早期診断法の確立乳癌は女性のがんの中では一番罹患者数が多く、また他のがんに比べて比較的若くに発症することなどの特徴を持つ疾患である。一方乳癌は検診などによる早期発見によって、早期治療介入につながり、予後が改善することがわかっている。一般的に乳癌の早期発見のために、マンモグラフィを用いた検診が行われている。研究者らはマンモグラフィよりも簡便、低侵襲、高感度な検査方法を模索するうちに、涙液中に乳癌由来のマイクロRNAが含まれていることを発見した。次に涙液中の乳癌由来物質の中で、より特異性の高い物質として細胞外小胞に着目するようになった。リキッドバイオプシーの手法としてエクソソームなどの細胞外小胞を超高感度に検出する方法として竹内らの開発したTearExo法を用いることとし、共同研究として乳癌患者の涙液を測定し、その結果を報告した。TearExo法は、細胞外小胞の表面に発現しているタンパク質に対する抗体と蛍光レポーター分子を導入したナノ空孔をもつセンサチップを用いて、細胞外小胞を超高感度に自動分析する方法である。新たな乳癌早期診断法として、細胞外小胞を腫瘍マーカーとした超高感度で選択性の高い乳癌のリキッドバイオプシーを確立することを本研究の目的としている。 今後TearExo法の臨床応用に向けて、より多くの検体を用いた精度の検証が必要であると考える。そのため目標症例数を100例とし、涙液のサンプリングと計測を予定している。今年度は研究計画書を作成し、神戸大学の倫理委員会の承認を得ることができた。これまではTearExo法の測定は少数の検体で行ってきたが、今後は安定した基板の大量生産が必要となったので、そのシステム構築を行っている。
- 日本学術振興会, 科学研究費助成事業, 基盤研究(C), 神戸大学, 2021年04月01日 - 2024年03月31日がん早期診断における涙液エクソソームの有用性の検討乳がんの死亡率は上昇の一途をたどっており、現在では日本人女性の11人に1人が乳がんにかかるといわれ、早期発見により適切な治療が行われれば良好な経過が期待できるといわれているものの実に年間約13.000人の女性が乳がんでなくなっており、その対策は急務である。一方で乳癌の発症延齢は40歳代と 60歳代後半と2つのピークがあり、40代～50代女性のがん死亡原因の第1位である。そのため40歳から健診が勧められているが、乳がんの検診率は約40%と先進国の中で最も低い。仕事や育児、介護などに追われている40代～50代女性には検診に行くこと自体のハードルの高さが問題となっている。近年はさまざまな体液を用いたがんの早期診断研究がなされており、その社会実現性も高いと考えるが、血液はやはり侵襲性が高く、尿は女性にとって心理的侵襲性も少なくないといえる。特に乳がんや子宮頸癌などは20-30代女性で急増しており、20代からのがんの早期診断という点で、より気軽に採取が可能である体液サンプルを利用した早期診断方法が必要であると考え、涙に着目した。申請者らは乳がん転移患者から涙液由来エクソソームを採取し、oncogenic miRNAが高発現していることを明らかにした。しかし、この涙液由来エクソソーム内にがん細胞から放出されたエクソソームが含まれているのかなど涙液の有効性について疑念は拭えない状態であった。そのため、涙液にがん細胞由来エクソソームが含まれており、涙液エクソソームががんの早期診断に有用であることを明らかにする必要があると考え本研究の着手した。 21年度は倫理委員会からの承認を得て、前向きに35例の患者の涙サンプルの採取を実施し現在順次解析に取り組んでいる。
- 日本学術振興会, 科学研究費助成事業, 基盤研究(B), 神戸大学, 2021年04月01日 - 2024年03月31日涙を用いた卵巣がんの早期発見のための非浸襲・迅速・超高感度細胞外小胞検出法の開発