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Haploinsufficiency of the c-myc transcriptional repressor FIR, as a dominant negative-alternative splicing model, promoted p53-dependent T-cell acute lymphoblastic leukemia progression by activating Notch1

Kazuyuki Matsushita _, Kouichi Kitamura, Bahityar Rahmutulla, Nobuko Tanaka, Takayuki Ishige, Mamoru Satoh, Tyuji Hoshino, Satoru Miyagi, Takeshi Mori, Sakae Itoga, Hideaki Shimada, Takeshi Tomonaga, Minoru Kito, Yaeko Nakajima-Takagi, Shuji Kubo, Chiaki Nakaseko, Masahiko Hatano, Takashi Miki, Masafumi Matsuo, Masaki Fukuyo, Atsushi Kaneda, Atsushi Iwama and Fumio Nomura

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Oncotarget. 2015; 6:5102-5117. https://doi.org/10.18632/oncotarget.3244

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Abstract

Kazuyuki Matsushita1,2,*, Kouichi Kitamura1,2,*, Bahityar Rahmutulla1, Nobuko Tanaka1, Takayuki Ishige1,2, Mamoru Satoh1, Tyuji Hoshino3, Satoru Miyagi4, Takeshi Mori5, Sakae Itoga2, Hideaki Shimada6, Takeshi Tomonaga7, Minoru Kito8, Yaeko Nakajima-Takagi4, Shuji Kubo9, Chiaki Nakaseko10, Masahiko Hatano11, Takashi Miki12, Masafumi Matsuo5,13, Masaki Fukuyo14, Atsushi Kaneda14, Atsushi Iwama4 and Fumio Nomura1,2

1 Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Inohana, Chiba, Japan

2 Division of Laboratory Medicine, Chiba University Hospital, Inohana, Chiba, Japan

3 Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana, Chiba, Japan

4 Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Inohana, Chuo-ku, Chiba, Japan

5 Department of Pediatrics, Graduate School of Medicine, Kobe University, Kusunoki-cho, Kobe, Japan

6 Department of Surgery, School of Medicine, Toho University, Omori-nishi, Ota-ku, Tokyo, Japan

7 Laboratory of Proteome Research, National Institute of Biomedical Innovation, Saito-Asagi, Ibaraki, Osaka, Japan

8 Oriental Yeast Co., Ltd. Azusawa, Itabashi-ku, Tokyo, Japan

9 Department of Genetics, Hyogo College of Medicine, Mukogawa-cho, Nishinomiya, Hyogo Prefecture, Japan

10 Department of Haematology, Chiba University Hospital, Inohana, Chiba, Japan

11 Department of Biomedical Science, Graduate School of Medicine, Chiba University, Inohana, Chiba, Japan

12 Department of Medical Physiology, Graduate School of Medicine, Chiba University, Inohana, Chiba, Japan

13 Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, Arise, Ikawadani, Nishi, Kobe, Japan

14 Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Inohana, Chiba, Japan

* These authors contributed equally to this work

Correspondence:

Kazuyuki Matsushita, email:

Keywords: FBP interacting repressor (FIR), splicing variant, haplo-insufficiency, leukemia, P53, T-ALL

Received: November 27, 2014 Accepted: December 27, 2014 Published: December 31, 2014

Abstract

FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR+/−) C57BL6 mice were generated. FIR complete knockout (FIR−/−) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR+/− mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR+/-TP53-/- generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR+/−TP53−/− compared with that in FIR+/+TP53−/− mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion, the alternative splicing of FIR, which generates FIRΔexon2, may contribute to both colorectal carcinogenesis and leukemogenesis.


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