Induction of Differentiation by Regulating the Musashi/Numb/Notch Pathway

New insight into cancer therapeutics: Induction of differentiation by regulating the Musashi/Numb/Notch pathway

Yoshinori Nishimoto1, Hideyuki Okano1

1Department of Physiology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan Cell Research (2010) 20:1083-1085. doi:10.1038/cr.2010.122; published online 31 August 2010

The Musashi (Msi) family is a group lular Notch signaling, by binding to cells” which expands to some kinds of of RNA-binding proteins characterized the 3′ untranslated region (UTR) of the solid tumors [10]. Then, Msi1 could by two RNA recognition motifs (RRMs) Numb mRNA [8]. Activation of Notch be a versatile marker of “cancer stem and is evolutionarily conserved [1, 2]. signaling induces the transactivation of cells” especially during an early phase In mammals, two isoforms of this fam- the promoter of the Hes-1 gene (Figure with strong proliferative activities. This ily, Msi1 and Msi2, are co-expressed in 1). The activation of Notch-pathway suggests the possibility that Msi1 might neural precursor cells, including neural is known to regulate the self-renewal have some important roles as a trigger stem cells (NSCs). Msi2 exhibits high of NSCs positively [9]. Previously, of proliferative switching from differ- sequence homology with Msi1, which our laboratory reported that Msi1 and entiated state to cancer production in a is more than 90% at the amino acid Msi2 developmentally controlled self- reverse direction to development. level within the RNA-binding domain. renewal of embryonic cells, as well as Although a previous report showed Msi2 is transcribed over the tissues of adult CNS stem/progenitor cells [6]. that Msi2 gene was rearranged to form ubiquitously [3] in contrast with Msi1 Based on the evidence that tissue stem a Msi2/HoxA9 fusion gene in a chronic enrichment in neural stem cells or pro- cells exist in many adult tissues, includ- myeloid leukemia (CML) case with genitor cells of the peri-ventricular area ing the hematopoietic system, intestine, the 7p15 breakpoint [14], the recent in the embryonic [4] and postnatal [5] mammary gland, testis, skeletal muscle, papers reported by Ito et al. [12] and mammalian brains. When both Msi1 skin, hair follicle and myocardium, oth- Kharas et al. [13] were the first re- and Msi2 genes were knocked-out, er than the CNS and neural crest-derived ports to show clearly the relationship neurosphere formation was markedly tissue, Msi/Numb/Notch signaling is between Msi2-inducing pathway and inhibited; however, single knockout did considered to be associated with many hematopoietic malignancy. These two not bring about such disturbance [6]. adult malignancies [2]. In fact, not only reports suggested a new strategy for the Originally, Msi was identified as a at the normal developmental stages, the therapy of aggressive leukemia. Using required factor for the asymmetric cell Msi-signaling pathway is also reported the in vivo models of CML in a chronic division of the sensory organ precursor to work during tumorigenesis in several phase and in an aggressive blast phase cell (SOP) of the Drosophila adult ex- adult tissues, including glioblastoma [15], it was proposed that Msi2-Numb ternal sensory organ [7]. In mammals, and esophageal, colon, pulmonary, pathway should control the differentia- the function of Msi has been, thus mammary and bladder carcinomas tion of CML cells and that this pathway far, found to activate Notch signaling [2, 10-13]. Intriguingly, in the case of could be the novel target of leukemia through the translational repression of esophageal adenocarcinoma, Msi1 ex- treatment [12]. NUMB, which represses an intracel- pression was the highest in glands dur- Ito et al. first asked whether the levels ing early cancer development. In con- of MSI/NUMB expression and Notch trast, Msi1 level become weaker when signaling altered in the CML model Correspondence: Hideyuki Okano esophageal adenocarcinoma grows into mice [12]. Myeloid blast crisis was Tel: +81-3-5363-3747; Fax: +81-3-3357-5445 E-mail: [email protected], hidokano@ the advanced stage. Such findings have modeled by transplanting hematopoietic a2.keio.jp advocated a concept of “cancer stem stem cells (HSCs)-enriched KLS cells, npg 1084 which was transduced with Bcr-Abl Msi2 gene. Nup98-HoxA9 transduced creased amount of NUMB protein in the and Nup98-HoxA9. HOXA9 regulates with retroviral vector along with Bcr- LAMA-84 cell lines, derived from the expression of many genes that are Abl could overexpress the Msi2 gene blast crisis CML patient, was observed implicated in hematopoiesis and in in KLS cells and upregulation of MSI2 with immunoblot. tumorigenesis. Using the established expression led to downregulation of Then, Ito et al. asked whether hu- mouse model representing chronic NUMB (Figure 1). At the same time, man leukemia in blast crisis showed phase and myeloid blast crisis CML Kharas et al. established doxycycline- aberrant up-regulation of Msi2 as well. (initiated by the Bcr-Abl translocation inducible Msi2 expression systems both Analysis of samples from 30 Korean and progressed by Nup98-HoxA9 ex- in vitro and in vivo, which culminated and British patients showed significant pression) [16], they found Numb was in hematopoietic colony-forming with higher levels of Msi2 gene expression expressed at significantly lower levels more immature myeloid phenotype in in blast crisis CML. As predicted from and Msi2 was particularly elevated in vitro and expansion of hematopoietic the CML mice model, microarray data the blast crisis phase. Notably, Msi2 stem cells (HSCs) and short-term pro- of 90 samples from banks in the United was expressed at higher levels than genitor cells in vivo [13]. They also States showed elevated levels of Msi2, Msi1 in the CML mice model, especially transduced doxycycline-inducible Msi2 HoxA9 and Hes1 and decrement of in hematopoietic stem cell-enriched cells with the Bcr-Abl oncogene and Numb among most of CML patients in populations including KLS cells and transplanted them into mice. Consistent the blast crisis phase. Similarly, Kharas the lineage-negative fraction of blast with the other report, Msi2 induced im- et al. [13] showed the increased level crisis CML. Moreover, they identified mature myeloid leukemia that mimicked of Msi2 and decreased level of Numb a putative HoxA9-binding element CML blastic crisis phase. Conversely, from 33 samples of myeloid blast at the position –5.7 kb in the murine when Msi2 was knocked down, an in- crisis by comparing with 57 samples of chronic phase CML obtained in the United States. It is particularly noteworthy that Ito et al. suggested the apparent clues B Musashi 2 suppressing tumor growth and mortal- HoxA9 –5.7 kb ity rates in the established CML model A NOTCH mice in vivo. They showed that a higher Numb expression made leukemia more MUSASHI2 D AAAAA differentiated and unable to propagate C Numb mRNA disease markedly. In fact, transplanta- (Suppressed translation) Trib2 Hes1 tion of cells expressing Numb decreased the frequency of developing leukemia in the model mice, from 83% to 63%. Chronic phase Blast crisis phase E When the authors transplanted cells Proliferation transfected with Numb, from primary transplanted mice for donor-derived cells, into irradiated recipient second- Differentiation ary mice, the survival rate to evaluate propagation of disease was elevated compared to the control, from 20% Figure 1 Scheme of the CML state in an aggressive blast crisis phase. In to 93%. These results showed that the chronic phase, Numb blocks activation of the Notch signaling (A). During hu- elevated levels of NUMB can inhibit man CML progression, the following changes occurs; Nup98-HoxA9 oncogene disease progression. Additionally, us- binds the putative element at 5.7 kb upstream of transcription start site to trig- ing gene-trap mutants or short hairpin ger the upregulated expression of Musashi2 gene (B). The elevated level of RNA approach, loss of Msi2 impaired MUSASHI2 leads to the downregulation of NUMB, by binding to the 3′ UTR of leukemia growth and increased survival Numb mRNA to inhibit translation (C). Inhibition of Notch signaling is cancelled rates significantlyin vivo, especially in by suppression of NUMB (D), leading to the elevated expression of the targets, blast crisis CML. Similar to NUMB Hes1 gene and Trib2 gene. A series of these activation cause proliferative change from chronic phase to blast crisis phase of CML (E). Red symbols and induction, Msi2-shRNA induced the letters represent increased level of each factor and enhanced activation (or in- differentiation of leukemic cells and inhibition) in blast crisis phase CML. hibited their propagating ability [12].

Cell Research | Vol 20 No 10 | October 2010 npg 1085 Ito et al. also reported the association References Notch pathway molecules are essential of Msi2 with high risk of relapse after for the maintenance, but not the gener- ation, of mammalian neural stem cells. allogeneic transplantation and poorer 1 Okano H, Imai T, Okabe M. Musashi: Genes Dev 2002; 16:846-858. outcomes, indicating the possibility to a translational regulator of cell fates. J 10 Bobryshev YV, Freeman AK, Botelho Cell Sci 2002; 115(Pt 7):1355-1359. make use of MSI2 as an early marker NK, et al. Expression of the putative 2 Okano H, Kawahara H, Toriya M, Na- of advanced CML. On the other hand, stem cell marker Musashi-1 in Barrett's kao K, Shibata S, Imai T. Function of Kharas et al. confirmed that Msi2 ex- esophagus and esophageal adenocarci- RNA-binding protein Musashi-1 in noma. Dis Esophagus 2010 Apr 29. doi: pression had relevance to poor clinical stem cells. Exp Cell Res 2005; 306:349- 10.1111/j.1442-2050.2010.01061.x prognosis among at least congeni- 356. 11 Todaro M, Francipane MG, Medema tally normal AML subjects. Moreover, 3 Sakakibara S, Nakamura Y, Satoh JP, Stassi G. Colon cancer stem cells: among them, they showed that the H, Okano H. Rna-binding protein promise of targeted therapy. Gastroen- Musashi2. Developmentally regulated population, whose gene signatures terology 2010; 138:2151-2162. expression in neural precursor cells and were negatively correlated with genes 12 Ito T, Kwon HY, Zimdahl B, et al. Reg- subpopulations of neurons in mamma- altered by the Msi2-knockdown, was ulation of myeloid leukaemia by the lian CNS. J Neurosci 2001; 21:8091- cell-fate determinant Musashi. Nature associated with poor prognosis. Using 8107. 2010; 466:765-768 multivariate analyses with numbers of 4 Sakakibara S, Imai T, Hamaguchi K, et 13 Kharas MG, Lengner CJ, Al-Shahrour Msi2-associated factors, we might pre- al. Mouse-Musashi-1, a neural RNA- F, et al. Musashi-2 regulates normal dict the prognosis or definitive diagnosis binding protein highly enriched in the hematopoiesis and promotes aggres- more accurately. mammalian CNS stem cell. Dev Biol sive myeloid leukemia. Nat Med 2010; 1996; 176: 230-242. Novel findings about the Msi2/ 16:903-908 5 Sakakibara S, Okano H. Expression Numb/Notch signaling pathway de- 14 Barbouti A, Höglund M, Johansson B, of neural RNA-binding proteins in the scribed above will shed light on a key to et al. A novel gene, MSI2, encoding a postnatal CNS: implications of their putative RNA-binding protein is recur- understanding development of hemato- roles in neuronal and glial cell develop- rently rearranged at disease progression logic malignancies including blast crisis ment. J Neurosci 1997; 17:8300-8312. of chronic myeloid leukemia and forms phase CML. Other recent papers have 6 Sakakibara S, Nakamura Y, Yoshida T, a fusion gene with HOXA9 as a result revealed the relationship between Notch et al. RNA-binding protein Musashi of the cryptic t(7;17)(p15;q23). Cancer family: roles for CNS stem cells and a signaling/Hes1 and CML progression Res 2003; 63:1202-1206. subpopulation of ependymal cells re- [17]. Together with these findings, each 15 Calabretta B, Perrotti D. The biol- vealed by targeted disruption and anti- of Msi family could be a key regulator ogy of CML blast crisis. Blood 2004; sense ablation. Proc Natl Acad Sci USA 103:4010-4022. of several intracellular pathways, decid- 2002; 99:15194-15199. 16 Pear WS, Miller JP, Xu L, et al. Effi- ing self-renewal of the stem cells, tum- 7 Nakamura M, Okano H, Blendy JA, cient and rapid induction of a chronic origenesis and differentiation, which is Montell C. Musashi: a neural RNA- myelogenous leukemia-like myelopro- cell-fate exactly. binding protein required for Drosophi- liferative disease in mice receiving la adult external sensory organ devel- Further studies about relationship P210 bcr/abl-transduced bone marrow. opment. Neuron 1994; 13:67-81. between Msi-signaling pathway and Blood 1998; 92:3780-3792. 8 Imai T, Tokunaga A, Yoshida T, et malignancies will be investigated. Then, 17 Nakahara F, Sakata-Yanagimoto M, al. The neural RNA-binding protein Komeno Y, et al. Hes1 immortalizes differentiation induction into mature Musashi1 translationally regulates committed progenitors and plays a role asymmetric cells by modulating the mammalian numb gene expression by in blast crisis transition in chronic pathway in cancer cells is expected to interacting with its mRNA. Mol Cell myelogenous leukemia. Blood 2010; be the novel therapeutics for advanced Biol 2001; 21:3888-3900. 115:2872-2881. leukemia and other solid carcinomas. 9 Hitoshi S, Alexson T, Tropepe V, et al.