On the role of notch, numb and numblike in development and cancer

The development of mutlicellualr organisms relies on a highly conserved signaling network, of which one of the key components is the Notch signaling pathway. The core Notch signaling pathway consists of receptors, ligands and an intracellular mediator. Activation of Notch signaling involves the interaction of the receptor to membrane-bound ligand, leading to the consecutive proteolytic cleavages of the receptor and the release of the Notch intracellular domain (NICD) from the cell membrane. The NICD then translocates to the nucleus and interacts with the DNAbinding transcription factor CSL (CBF-1/Su(H)/Lag-1), resulting in transcriptional activation of E(spl)/Hes family and other target genes.

The work presented in this thesis is focused on two specific aspects of Notch signaling: i) the interplay between Notch and its intrinsic negative regulator Numb/Numblike and the role of Numblike in the early differentiation of embryonic stem (ES) cells; ii) the mechanism on how aberrantly high Notch signaling in T-cell acute lymphoblastic leukemia (T-ALL) translates into deregulated cell cycle control and the transformed cell type.

Numb was originally identified as an inhibitor of Notch signaling pathway in Drosophila melanogaster and acts as a cell-fate determinant during asymmetric cell division. In vertebrates there are two mammalian homologs: Numb and Numblike. In paper I we suggested a model in which Numb/Numblike and Notch had reciprocal antagonistic activities. We found that Numb promoted differentiation in the mouse moyogenic cell line C2C12 at low but not high levels of Notch signaling. In keeping with a differentiation-promoting role, we observed that a C2C12 cell line stably expressing Numblike showed accelerated myogenic differentiation. Unexpectedly we also observed high levels of Notch signaling was accompanied with a reduction of Numb/Numblike at protein levels in C2C12 stable cells. We extended our study and reached the same conclusion in human ovarian carcinoma cell (SKOV-3 cells) and in the developing chick central nervous system. We also found that the Notch mediated reduction of Numblike required the PEST domain in the Numblike protein and was blocked by the proteasome inhibitor MG132. Next, we try to address the role of Numblike in the early differentiation processes as little is known about it. In paper III a mouse ES cell line with inducible expression of Numblike protein was generated. We found that Numblike expression impaired differentiation towards neural and mesodermal differentiation of ES cells. However myocardial differentiation was not affected upon induction of Numblike protein later at Flk-1 positive stage of mesodermal differentiation.

In more than 50% of all T-ALL cases activating mutations of Notch1 have been identified. Thus a better understanding of the effects of Notch signaling strength and timing on the pre-T cell transformation would provide more therapeutic bases. In paper II, we analyzed downstream responses resulting from the high level of Notch1 signaling in T-ALL. Our microarray study revealed that besides Hes1 and Hey1, the canonical downstream targets of Notch signaling, other immediate Notch downstream genes were also activated. Specifically we observed that Notch signaling increased the expression level of the E3 ubiquitin ligase SKP2, thus leading to the reduction of its target protein p27Kip1. Blocking Notch signaling resulted in G0/G1 cell cycle arrest, which could be mimicked by transfection of p27Kip1 or, to a smaller extent, a dominant negative SKP2 allele. In sum our data suggest that the aberrantly high Notch signaling in T-ALL maintains SKP2 at a high level and reduces p27Kip1, leading to more rapid cell cycle progression.

List of scientific papers

I. Chapman G, Liu L, Sahlgren C, Dahlqvist C, Lendahl U (2006). High levels of Notch signaling down-regulate Numb and Numblike. J Cell Biol. 175(4): 535-40
https://pubmed.ncbi.nlm.nih.gov/17116748

II. Dohda T, Maljukova A, Liu L, Heyman M, Grandér D, Brodin D, Sangfelt O, Lendahl U (2007). Notch signaling induces SKP2 expression and promotes reduction of p27Kip1 in T-cell acute lymphoblastic leukemia cell lines. Exp Cell Res. 313(14): 3141-52. Epub 2007 May 5
https://pubmed.ncbi.nlm.nih.gov/17560996

III. Liu L, Lanner F, Lendahl U (2007). Expression of Numblike impairs early mesodermal and neural differentiation in embryonic stem cell. [Manuscript]