Impact Objectives • Investigate systematically the physiological role of placenta-specific kinase, NRK

• Clarify the cellular and molecular mechanism of hormone-dependent breast cancer to develop new therapies

NRK’s role in breast cancer Consulting cellular and molecular biologist Assistant Professor Kimitoshi Denda, Associate Professor Nobuhiro Hayashi and Assistant Professor Toshiaki Fukushima talk about their work on understanding the role of NRK in breast cancer, which was conceived in partnership with Professor Masayuki Komada

Can you talk about to the further studies in cell fate; quiescence, your work on the proliferation, apoptosis, differentiation and phenotype analysis senescence in breast tissue. I sincerely hope of NRK KO mice that our findings on the NRK-action will via histological lead to a more profound understanding of and histochemical the mechanisms of breast cancer evaluation? suppression and thus advance our knowledge Assistant Professor Associate Professor Assistant Professor on high-throughput diagnosis and therapy Kimitoshi Denda Nobuhiro Hayashi Toshiaki Fukushima TF: We analysed also from the perspective of preventive the expression medicine. Why is research on the elucidation of cell levels of several marker used for proliferation suppression mechanisms of the classification applied to the type of What are your research plans for value? human breast cancer, which is essential for the coming year? therapeutic strategy. Our results indicated that KD: Dysregulation of cell proliferation the NRK-deficient mice often develop breast TF: We are trying to elucidate molecular mechanisms are key factors in the cancer similar to human luminal B-type. This mechanisms by which NRK regulates cell development of cancer. Investigations into type of cancer cells is hormone-sensitive proliferation in placenta. Our preliminary tumour suppressor products that (oestrogen-receptor positive) and highly data indicate NRK affects the specific signal control these mechanisms are indispensable proliferative (Ki-67 positive). Importantly, pathway activated by growth factors. Although for better realising how cancer initiates and hormone-sensitive breast cancer accounts NRK KO mice frequently develop breast progresses as well as to develop targeted for more than 70 per cent of the total breast cancers similar to human luminal B-type, our molecular drugs for effective therapy. cancer patients. The dysfunction of NRK previous study was focused on the early stage may be involved in the pathogenesis of a of the cancers. Subsequent study is needed to What is the importance of the physiological substantial fraction of human breast cancers. understand the progression of the cancer, and roles of NRK? In particular, the NRK KO mice show signs of how the cancer cells evolve under untreated breast tumours after repetitive pregnancy and conditions or by the treatment with anti- NH: NRK (Nik-related kinase) is a serine/ parturition without exception. We speculate oestrogen therapy in NRK KO mice. threonine protein kinase that is found that the tumour development was influenced inducibly expressed in the placenta. In the by the exposure of mammary epithelial cells to KD: Together with another outstanding NRK knockout (KO) animal models, placental oestrogen during pregnancy. research topic on parturition, we would like overgrowth occurs, suggesting its crucial to perfectly clarify the mechanisms of cell fate role in the regulation of cell proliferation How will your findings contribute to diagnosis control including carcinogenesis. What we and development in placental tissues. In and treatment? should not overlook is that hormone-sensitive addition, the NRK KO foetuses exhibit cells are restricted not only in reproductive delayed delivery in contrast to the case with NH: The established notion that ever- organs but in liver, lung, smooth muscle, the wild-type foetuses, which may highlight increasing expression of oncogenes and adipose tissue and so on. I want to also its role in a currently-undiscovered delivery the signals manifested in their protein elucidate how and why some tumours often induction signalling mechanism. On the other products would result in cancer progression alter their nature to hormone sensitivity, as hand, surprisingly, we observed a frequent by early studies has been revised to include prostate cancer exacerbated by testosterone. occurrence of breast cancer in NRK KO mice. that oncoprotein signalling would bear I also desire to conduct a preparation of It made us to recognise NRK as a tumour counteracting response in cells toward oestrogen-independent breast cancer model suppressor protein. proliferation by more recent researches. Our mice by breeding these with other genetically exploration has the potential to contribute modified mice. l

76 www.impact.pub Understanding NRK: a step towards new therapies Consulting cellular and molecular biologist Dr Kimitoshi Denda and his team at the Tokyo Institute of Technology are furthering our understanding of the role of NRK to develop knowledge about, and perhaps a cure for breast cancer

Breast cancer is the most common in the NRK mutant female mice, suggesting neoplastic growth of mammary epithelial malignant tumour affecting women in many that it is a potent tumour-suppressor cells. countries. It is a typical heterogeneous gene, as expected from its structure in tumour which is alleged to cause resistance to advance, involved in the regulation of Proceeding with Dr Hayashi, Associate medical treatment. The sex steroid hormone cell proliferation and development of the Professor of the same institute, Denda is oestrogen is believed to influence the onset mammary gland tissues.’ seeking to clarify the molecular mechanisms or progression of more than 70 per cent of lying behind the NRK-null phenotypes by breast cancer, and an increase in oestrogen Oestrogen plays a critical role in the analysing the protein expression profiles has been linked with cancer development in development of most breast cancers. of key regulators in the NRK-KO tissues. In postmenopausal women. Unfortunately, as Denda and his team focused on the NRK the proteome assay, by using the wild-type Dr Kimitoshi Denda, Assistant Professor at encoded on the X . They (WT) placental tissues as a control, a pair the Tokyo Institute of Technology, explains the recognised that the NRK-mutant mice that of 2D gel images were compared to identify pathogenesis of breast cancer is still poorly experienced repetitive pregnancies were and localise around 1800 protein spots, understood. This is why Denda and his team more likely to develop breast tumours. The corresponding to 18 distinct proteins. ‘It are conducting research into the NRK gene, in type of breast cancer in those mice was is tempting to speculate that dysfunction the hopes of discovering its physiological roles tentatively classified as non-invasive tubular of NRK may play relevant roles in cellular in healthy individuals as well as in tumour adenocarcinomas by hisotological analysis. proliferation, cell cycle progression and pathogenesis. ‘The breast tumours found in the NRK-null resistance to apoptosis and oncogenesis is mice were positive for oestrogen receptor its consequence,’ he observes. THE ROLE OF NRK (ER+) and cell proliferation marker Ki67 and ‘NRK encoded on the negative for receptor tyrosine kinase HER2/ One of the substantially decreased proteins is a physiologically-mysterious protein ErbB2, suggesting the same characteristics is annexin A5 (ANXA5) in NRK-KO placentas kinase that belongs to the germinal centre as the human luminal B breast cancer,’ says (submitted for publication). ANXA5, well- kinase IV family,’ outlines Denda. ‘NRK is Denda. ‘Together with the dense clumps known as an expedient diagnostic tool for suggested to be involved in the induction of ER+ found within the NRK-deficient detecting apoptotic cells, exhibits strong of actin cytoskeletal reorganisation mammary gland tissues in late pregnancy, Ca2+-dependent binding affinity towards through cofilin phosphorylation during our results lead to the hypothesis that phosphatidylserine in the outer leaflet of late embryogenesis.’ He points out that excessive blood oestrogen and a faulty the plasma membrane. It is believed to be NRK is highly-expressed, especially in the proliferation of mammary cells could be the involved in coagulation regulation locally placenta, and its gene KO causes placental cause of mammary tumorigenesis in NRK- and the gene knockout of ANXA5 shows overgrowth, ‘indicating that NRK is a mutant mice,’ Denda observes. a significant decrease in the number of crucial modulator of cell proliferation and foetuses during pregnancy compared to WT, development in placental tissues’. Evidence THE CHALLENGES OF NRK and a decrease in neonatal body weight. also suggests that NRK plays a role in NRK is supposed to be a tumour ANXA5 is also thought to be deeply involved delivery-inducing signalling dispatched from suppressor gene product. ‘It is a gatekeeper in various pathogenesis during pregnancy. the placenta. that impacts cellular proliferation directly, Denda believed that this might cause excess rather than a caretaker or landscaper,’ says proliferation of specified tissues, as is the Surprisingly, Denda also observed that there Denda. However, when mutated, NRK has case in placentomegaly. is a frequent occurrence of breast tumours been shown to exert some influence on the

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FUNDING Ministry of Education, Culture, Sports, Science, and Technology of Japan • Takeda Denda’s research progressed further thanks Denda hopes to present their research Science Foundation to recent developments in advanced mass for wider international audiences to share spectrometry methods, which enabled knowledge that contributes towards PARTNERS AND TEAM MEMBERS him and his team to monitor numerous conquering cancer. Associate Professor Yoh-ichi Tagawa phosphorylation sites in proteins. By (TIT; Tokyo Institute of Technology) • Dr furthering the profiling analysis of the gene As Dr Hayashi stated previously, the Kanako Wakabayashi-Nakao (International product changes in the NRK-gene mutant conventional conception that oncogene University of Health and Welfare) • Professor Shinya Yamanaka (Kyoto placental tissues, he hopes to be able to signalling would result in cancer cell University) • Professor Emeritus Naomi elucidate the functional mechanism of proliferation has been overwritten in Kitamura (TIT) • Professor Keiji Miyazawa placental development and differentiation that excessively elevated signalling by (University of Yamanashi) • Professor during pregnancy, thereby determining oncoproteins such as RAS, MYC, and RAF Masataka Tanno (Kawasaki Saiwai targets of current chemotherapeutic can provoke counteracting responses Hospital) • Associate Professor Junji treatments for perinatal medicine. from cells, specifically induction of Hirota (TIT) • Dr Isao Kii (RIKEN cell senescence/apoptosis by more Cluster for Science) • Associate Professor Mitsuru Futakuchi (Nagasaki University) This research has resulted in a number recent research. In the upcoming years, • Assistant Professor Jun Nakayama of challenges. For example, how and why Denda seeks to learn more about the (Waseda University) • Dr Akinori Endo does NRK dominantly expressed contribute mechanisms of cell fate control including (TIT) • Dr Takayo Yanagawa (Tokai to suppress the onset of cancer in normal cell carcinogenesis and how these can University School of Medicine) • Assistant mammary tissues? Denda points out that alternate hormone-dependence using NRK- Professor Toshiaki Tanaka (TIT), and • occurrence of breast cancer is only restricted KO animal models. Cancer is recognised Associate Professor Akira Kato (TIT) in the multiparous NRK KO female mice, as a disease of clonal evolution within the CONTACT and thereby oestrogen probably triggers body, and individuality of cancer cells such Dr Kimitoshi Denda carcinogenesis. ‘We hypothesise that there as hormone sensitivity can be degenerated Project Coordinator is a possibility of breast tumour progression with time. The research team at the Tokyo due to malfunction of intercommunication Institute of Technology hope to develop a E: [email protected] among the (feto-)placenta and the major reliable line oestrogen-independent breast W: https://kaken.nii.ac.jp/en/grant/ oestrogen producing ovary and mammary cancer mouse models through selective KAKENHI-PROJECT-15K14378/ gland tissues.’ breeding, thus further contributing towards W: https://www.titech.ac.jp/english/ news/2016/036094.html

BIOS These results lead to the hypothesis that excessive Dr Kimitoshi Denda received his PhD from the Tokyo Institute of Technology. He currently serves as an Assistant Professor blood oestrogen and a faulty proliferation of at the Tokyo Institute of Technology. Denda’s research interests include deterrent mammary cells may be the cause of mammary mechanisms of cell proliferation by a novel X-linked tumour suppressor and tumorigenesis in NRK-mutant mice understanding the molecular basis of labour induction for consequent drug discovery.

LOOKING TO THE FUTURE understanding and finding a treatment Dr Nobuhiro Hayashi received a PhD The team have demonstrated one of the for triple-negative breast cancer, a distinct from Tokyo Institute of Technology, and female-specific critical roles for NRK subtype that is difficult to manage. subsequently he expanded his research on protein science as Assistant Professor with through generation and phenotypic Professor Koiti Titani at Fujita Health analysis of mice deficient for the protein This research supports the idea that University. He is now a researcher in the field kinase gene. ‘We offer evidence that NRK have the NRK gene product of PROTEOMICS as Associate Professor at is involved in suppressing the pathological corresponding to the orthologue enzyme the School of Life Science and Technology, development of breast tumour associated in mouse cells. As previously mentioned, Tokyo Institute of Technology. with the mammary gland hyperplasia during as human luminal B type breast cancer pregnancy by phenotype analysis of the estimated immunohistologically found in NRK-KO mutant mice,’ Denda observes. breast cancer in NRK-mutant mice, this research offers hope that scientists such Denda believes in the importance of as Denda and his team will unveil more developing skills, such as next-generation about the mechanisms of breast cancer genome editing, in future generations suppression. This should lead them to of researchers. He contributes towards develop diagnosis techniques and curative teaching applied techniques in studies therapies against tumours other than with genetically modified mice to young surgical ablation or chemotherapy. This is scientists to discover and unravel the why Denda continues with his research and, developmental, physiological and critically, educate new scientists to further pathophysiological cues. In the future, our understanding of breast cancer. l

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