NUMB controls p53 tumour suppressor activity.

NUMB is a cell fate determinant, which, by asymmetrically partitioning at mitosis, controls cell fate choices by antagonising the activity of the plasma membrane receptor of the NOTCH family. NUMB is also an endocytic protein, and the NOTCH-NUMB counteraction has been linked to this function. There might be, however, additional functions of NUMB, as witnessed by its proposed role as a tumour suppressor in breast cancer. Here we describe a previously unknown function for human NUMB as a regulator of tumour protein p53 (also known as TP53). NUMB enters in a tricomplex with p53 and the E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing ubiquitination and degradation of p53. This results in increased p53 protein levels and activity, and in regulation of p53-dependent phenotypes. In breast cancers there is frequent loss of NUMB expression. We show that, in primary breast tumour cells, this event causes decreased p53 levels and increased chemoresistance. In breast cancers, loss of NUMB expression causes increased activity of the receptor NOTCH. Thus, in these cancers, a single event-loss of NUMB expression-determines activation of an oncogene (NOTCH) and attenuation of the p53 tumour suppressor pathway. Biologically, this results in an aggressive tumour phenotype, as witnessed by findings that NUMB-defective breast tumours display poor prognosis. Our results uncover a previously unknown tumour suppressor circuitry.

Alterations of the Notch pathway in lung cancer.

Notch signaling regulates cell specification and homeostasis of stem cell compartments, and it is counteracted by the cell fate determinant Numb. Both Numb and Notch have been implicated in human tumors. Here, we show that Notch signaling is altered in approximately one third of non-small-cell lung carcinomas (NSCLCs), which are the leading cause of cancer-related deaths: in approximately 30% of NSCLCs, loss of Numb expression leads to increased Notch activity, while in a smaller fraction of cases (around 10%), gain-of-function mutations of the NOTCH-1 gene are present. Activation of Notch correlates with poor clinical outcomes in NSCLC patients without TP53 mutations. Finally, primary epithelial cell cultures, derived from NSCLC harboring constitutive activation of the Notch pathway, are selectively killed by inhibitors of Notch (gamma-secretase inhibitors), showing that the proliferative advantage of these tumors is dependent upon Notch signaling. Our results show that the deregulation of the Notch pathway is a relatively frequent event in NSCLCs and suggest that it might represent a possible target for molecular therapies in these tumors.

Rac3-induced neuritogenesis requires binding to Neurabin I.

Rac3, a neuronal GTP-binding protein of the Rho family, induces neuritogenesis in primary neurons. Using yeast two-hybrid analysis, we show that Neurabin I, the neuronal F-actin binding protein, is a direct Rac3-interacting molecule. Biochemical and light microscopy studies indicate that Neurabin I copartitions and colocalizes with Rac3 at the growth cones of neurites, inducing Neurabin I association to the cytoskeleton. Moreover, Neurabin I antisense oligonucleotides abolish Rac3-induced neuritogenesis, which in turn is rescued by exogenous Neurabin I but not by Neurabin I mutant lacking the Rac3-binding domain. These results show that Neurabin I mediates Rac3-induced neuritogenesis, possibly by anchoring Rac3 to growth cone F-actin.

Modular structure of the human lamin B2 replicator.

The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation.

Early mitotic degradation of the homeoprotein HOXC10 is potentially linked to cell cycle progression.

Hox proteins are transcription factors involved in controlling axial patterning, leukaemias and hereditary malformations. Here, we show that HOXC10 oscillates in abundance during the cell cycle, being targeted for degradation early in mitosis by the ubiquitin-dependent proteasome pathway. Among abdominal-B subfamily members, the mitotic proteolysis of HOXC10 appears unique, since the levels of the paralogous HOXD10 and the related homeoprotein HOXC13 are constant throughout the cell cycle. When two destruction box motifs (D-box) are mutated, HOXC10 is stabilized and cells accumulate in metaphase. HOXC10 appears to be a new prometaphase target of the anaphase-promoting complex (APC), since its degradation coincides with cyclin A destruction and is suppressed by expression of a dominant-negative form of UbcH10, an APC-associated ubiquitin-conjugating enzyme. Moreover, HOXC10 co-immunoprecipitates the APC subunit CDC27, and its in vitro degradation is reduced in APC-depleted extracts or by competition with the APC substrate cyclin A. These data imply that HOXC10 is a homeoprotein with the potential to influence mitotic progression, and might provide a link between developmental regulation and cell cycle control.