RASSF1A is part of a complex similar to the Drosophila Hippo/Salvador/Lats tumor-suppressor network

The Ras Association Domain Family 1A (RASSF1A) gene is one of the most frequently silenced genes in human cancer. RASSF1A has been shown to interact with the proapoptotic kinase MST1. Recent work in Drosophila has led to the discovery of a new tumor-suppressor pathway involving the Drosophila MST1 and MST2 ortholog, Hippo, as well as the Lats/Warts serine/threonine kinase and a protein named Salvador (Sav). Little is known about this pathway in mammalian cells. We report that complexes consisting of RASSF1A, MST2, WW45 (the human ortholog of Sav), and LATS1 exist in human cells. MST2 enhances the RASSF1A-WW45 interaction, which requires the C-terminal SARAH domain of both proteins. Components of this complex are localized at centrosomes and spindle poles from interphase to telophase and at the midbody during cytokinesis. Both RASSF1A and WW45 activate MST2 by promoting MST2 autophosphorylation and LATS1 phosphorylation. Mitosis is delayed in Rassf1a(-/-) mouse embryo fibroblasts and frequently results in cytokinesis failure, similar to what has been observed for LATS1-deficient cells. RASSF1A, MST2, or WW45 can rescue this defect. The complex of RASSF1A, MST2, WW45, and LATS1 consists of several tumor suppressors, is conserved in mammalian cells, and appears to be involved in controlling mitotic exit.     

Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence

The p16(INK4a) cyclin-dependent kinase inhibitor has a key role in establishing stable G1 cell-cycle arrest through activating the retinoblastoma (Rb) tumour suppressor protein pRb in cellular senescence. Here, we show that the p16(INK4a) /Rb-pathway also cooperates with mitogenic signals to induce elevated intracellular levels of reactive oxygen species (ROS), thereby activating protein kinase Cdelta (PKCdelta) in human senescent cells. Importantly, once activated by ROS, PKCdelta promotes further generation of ROS, thus establishing a positive feedback loop to sustain ROS-PKCdelta signalling. Sustained activation of ROS-PKCdelta signalling irreversibly blocks cytokinesis, at least partly through reducing the level of WARTS (also known as LATS1), a mitotic exit network (MEN) kinase required for cytokinesis, in human senescent cells. This irreversible cytokinetic block is likely to act as a second barrier to cellular immortalization ensuring stable cell-cycle arrest in human senescent cells. These results uncover an unexpected role for the p16(INK4a)-Rb pathway and provide a new insight into how senescent cell-cycle arrest is enforced in human cells.     

The hedgehog-PKA pathway regulates two distinct steps of the differentiation of retinal ganglion cells: the cell-cycle exit of retinoblasts and their neuronal maturation

In the developing zebrafish retina, neurogenesis is initiated in cells adjacent to the optic stalk and progresses to the entire neural retina. It has been reported that hedgehog (Hh) signalling mediates the progression of the differentiation of retinal ganglion cells (RGCs) in zebrafish. However, the progression of neurogenesis seems to be only mildly delayed by genetic or chemical blockade of the Hh signalling pathway. Here, we show that cAMP-dependent protein kinase (PKA) effectively inhibits the progression of retinal neurogenesis in zebrafish. Almost all retinal cells continue to proliferate when PKA is activated, suggesting that PKA inhibits the cell-cycle exit of retinoblasts. A cyclin-dependent kinase (cdk) inhibitor p27 inhibits the PKA-induced proliferation, suggesting that PKA functions upstream of cyclins and cdk inhibitors. Activation of the Wnt signalling pathway induces the hyperproliferation of retinal cells in zebrafish. The blockade of Wnt signalling inhibits the PKA-induced proliferation, but the activation of Wnt signalling promotes proliferation even in the absence of PKA activity. These observations suggest that PKA inhibits exit from the Wnt-mediated cell cycle rather than stimulates Wnt-mediated cell-cycle progression. PKA is an inhibitor of Hh signalling, and Hh signalling molecule morphants show severe defects in cell-cycle exit of retinoblasts. Together, these data suggest that Hh acts as a short-range signal to induce the cell-cycle exit of retinoblasts. The pulse inhibition of Hh signalling revealed that Hh signalling regulates at least two distinct steps of RGC differentiation: the cell-cycle exit of retinoblasts and RGC maturation. This dual requirement of Hh signalling in RGC differentiation implies that the regulation of a neurogenic wave is more complex in the zebrafish retina than in the Drosophila eye.     

Transient deactivation of ERK signalling is sufficient for stable entry into G0 in primary avian fibroblasts

Re-entry into the cell cycle from quiescence requires the activation of mitogen-activated protein (MAP) kinases of the extracellular-signal-regulated kinase (ERK) family [1,2]. The relationship between ERK and cell-cycle control is, however, complex, as ERK activation can also lead to terminal differentiation [3] or a senescence-like growth arrest [4]. Here, we report that reversible cell-cycle exit induced by serum withdrawal in primary avian fibroblasts is associated with rapid deactivation of ERK, but ERK activity is subsequently regenerated and sustained at high levels in fully quiescent (G0) cells. As in proliferating cells, ERK activation during G0 required the MAPkinase kinase MEK and was partially dependent on cell adhesion. Active, phosphorylated ERK was concentrated in the nucleus in cycling cells, but was largely confined to the cytoplasm during G0. This was unexpected, as activatory phosphorylation mediated by MEK is thought to play an important role in promoting nuclear translocation [5,6]. These results indicate that transient deactivation of ERK signalling can be sufficient for stable cell-cycle exit, and that MEK-mediated phosphorylation is not sufficient for nuclear translocation of active ERK in G0. Cytoplasmic sequestration may prevent active ERK from accessing critical nuclear cell-cycle targets, thus allowing quiescent or post-mitotic cells to retain ERK activity for other physiological functions.     

Interaction of LATS1 with SMAC links the MST2/Hippo pathway with apoptosis in an IAP-dependent manner

Metastatic malignant melanoma is the deadliest skin cancer, and it is characterised by its high resistance to apoptosis. The main melanoma driving mutations are part of ERK pathway, with BRAF mutations being the most frequent ones, followed by NRAS, NF1 and MEK mutations. Increasing evidence shows that the MST2/Hippo pathway is also deregulated in melanoma. While mutations are rare, MST2/Hippo pathway core proteins expression levels are often dysregulated in melanoma. The expression of the tumour suppressor RASSF1A, a bona fide activator of the MST2 pathway, is silenced by promoter methylation in over half of melanomas and correlates with poor prognosis. Here, using mass spectrometry-based interaction proteomics we identified the Second Mitochondria-derived Activator of Caspases (SMAC) as a novel LATS1 interactor. We show that RASSF1A-dependent activation of the MST2 pathway promotes LATS1-SMAC interaction and negatively regulates the antiapoptotic signal mediated by the members of the IAP family. Moreover, proteomic experiments identified a common cluster of apoptotic regulators that bind to SMAC and LATS1. Mechanistic analysis shows that the LATS1-SMAC complex promotes XIAP ubiquitination and its subsequent degradation which ultimately results in apoptosis. Importantly, we show that the oncogenic BRAF^V600E mutant prevents the proapoptotic signal mediated by the LATS1-SMAC complex while treatment of melanoma cell lines with BRAF inhibitors promotes the formation of this complex, indicating that inhibition of the LATS1-SMAC might be necessary for BRAF^V600E-driven melanoma. Finally, we show that LATS1-SMAC interaction is regulated by the SMAC mimetic Birinapant, which requires C-IAP1 inhibition and the degradation of XIAP, suggesting that the MST2 pathway is part of the mechanism of action of Birinapant. Overall, the current work shows that SMAC-dependent apoptosis is regulated by the LATS1 tumour suppressor and supports the idea that LATS1 is a signalling hub that regulates the crosstalk between the MST2 pathway, the apoptotic network and the ERK pathway.Subject terms: Protein-protein interaction networks, Extracellular signalling molecules     

Sonic hedgehog opposes epithelial cell cycle arrest.

Stratified epithelium displays an equilibrium between proliferation and cell cycle arrest, a balance that is disrupted in basal cell carcinoma (BCC). Sonic hedgehog (Shh) pathway activation appears sufficient to induce BCC, however, the way it does so is unknown. Shh-induced epidermal hyperplasia is accompanied by continued cell proliferation in normally growth arrested suprabasal cells in vivo. Shh-expressing cells fail to exit S and G2/M phases in response to calcium-induced differentiation and also resist exhaustion of replicative growth capacity. In addition, Shh blocks p21(CIP1/WAF1)-induced growth arrest. These data indicate that Shh promotes neoplasia by opposing normal stimuli for epithelial cell cycle arrest.     

salvador Promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines

The number of cells in an organism is determined by regulating both cell proliferation and cell death. Relatively few mechanisms have been identified that can modulate both of these processes. In a screen for Drosophila mutations that result in tissue overgrowth, we identified salvador (sav), a gene that promotes both cell cycle exit and cell death. Elevated Cyclin E and DIAP1 levels are found in mutant cells, resulting in delayed cell cycle exit and impaired apoptosis. Salvador contains two WW domains and binds to the Warts (or LATS) protein kinase. The human ortholog of salvador (hWW45) is mutated in three cancer cell lines. Thus, salvador restricts cell numbers in vivo by functioning as a dual regulator of cell proliferation and apoptosis.     

Id-1 modulates senescence and TGF-beta1 sensitivity in prostate epithelial cells

BACKGROUND INFORMATION: Loss of sensitivity to TGF-beta1 (transforming growth factor beta1)-induced growth arrest is an important step towards malignant transformation in human epithelial cells, and Id-1 (inhibitor of differentiation or DNA binding-1) has been associated with cell proliferation and cell-cycle progression. Here, we investigated the role of Id-1 in cellular sensitivity to TGF-beta1. RESULTS: Using an immortalized prostate epithelial cell line, NPTX cells, we suppressed Id-1 expression through antisense strategy. We found that inhibition of Id-1 expression suppressed cell proliferation and at the same time induced cellular senescence and G2/M cell-cycle arrest. In addition, inactivation of Id-1 made cells more vulnerable to TGF-beta1-induced growth arrest. The sensitization effect on TGF-beta1 was associated with up-regulation of two downstream effectors of the TGF-beta1 pathway, p21WAF1/Cip1 and p27KIP1. CONCLUSION: Our results indicate that endogenous Id-1 levels might be a crucial factor in the development of resistance to TGF-beta1-induced growth suppression in human prostate epithelial cells.     

Notch1 activation reduces proliferation in the multipotent hematopoietic progenitor cell line FDCP-mix through a p53-dependent pathway but Notch1 effects on myeloid and erythroid differentiation are independent of p53

Signaling mediated by activation of the transmembrane receptor Notch influences cell-fate decisions, differentiation, proliferation, and cell survival. Activated Notch reduces proliferation by altering cell-cycle kinetics and promotes differentiation in hematopoietic progenitor cells. Here, we investigated if the G(1) arrest and differentiation induced by activated mNotch1 are dependent on tumor suppressor p53, a critical mediator of cellular growth arrest. Multipotent wild-type p53-expressing (p53(wt)) and p53-deficient (p53(null)) hematopoietic progenitor cell lines (FDCP-mix) carrying an inducible mNotch1 system were used to investigate the effects of proliferation and differentiation upon mNotch1 signaling. While activated Notch reduced proliferation of p53(wt)-cells, no change was observed in p53(null)-cells. Activated Notch upregulated the p53 target p21(cip/waf) in p53(wt)-cells, but not in p53(null)-cells. Induction of the p21(cip/waf) gene by activated Notch was mediated by increased binding of p53 to p53-binding sites in the p21(cip/waf) promoter and was independent of the canonical RBP-J binding site. Re-expression of p53(wt) in p53(null) cells restored the inhibition of proliferation by activated Notch. Thus, activated Notch inhibits proliferation of multipotent hematopoietic progenitor cells via a p53-dependent pathway. In contrast, myeloid and erythroid differentiation was similarly induced in p53(wt) and p53(null) cells. These data suggest that Notch signaling triggers two distinct pathways, a p53-dependent one leading to a block in proliferation and a p53-independent one promoting differentiation.     

Histone deacetylase 1 regulates retinal neurogenesis in zebrafish by suppressing Wnt and Notch signaling pathways

In the developing vertebrate retina, progenitor cells initially proliferate but begin to produce postmitotic neurons when neuronal differentiation occurs. However, the mechanism that determines whether retinal progenitor cells continue to proliferate or exit from the cell cycle and differentiate is largely unknown. Here, we report that histone deacetylase 1 (Hdac1) is required for the switch from proliferation to differentiation in the zebrafish retina. We isolated a zebrafish mutant, ascending and descending (add), in which retinal cells fail to differentiate into neurons and glial cells but instead continue to proliferate. The cloning of the add gene revealed that it encodes Hdac1. Furthermore, the ratio of the number of differentiating cells to that of proliferating cells increases in proportion to Hdac activity, suggesting that Hdac proteins regulate a crucial step of retinal neurogenesis in zebrafish. Canonical Wnt signaling promotes the proliferation of retinal cells in zebrafish, and Notch signaling inhibits neuronal differentiation through the activation of a neurogenic inhibitor, Hairy/Enhancer-of-split (Hes). We found that both the Wnt and Notch/Hes pathways are activated in the add mutant retina. The cell-cycle progression and the upregulation of Hes expression in the add mutant retina can be inhibited by the blockade of Wnt and Notch signaling, respectively. These data suggest that Hdac1 antagonizes these pathways to promote cell-cycle exit and the subsequent neurogenesis in zebrafish retina. Taken together, these data suggest that Hdac1 functions as a dual switch that suppresses both cell-cycle progression and inhibition of neurogenesis in the zebrafish retina.     

Ectopic expression of p27Kip1 in oligodendrocyte progenitor cells results in cell-cycle growth arrest

Oligodendrocyte differentiation is a complex process believed to be controlled by an intrinsic mechanism associated with cell-cycle arrest. Recently, the cell-cycle inhibitor protein p27^Kip1 has been proposed as a key element in causing growth arrest of oligodendrocyte precursor cells. To investigate the effects of p27 upon oligodendrocyte cell development, we have introduced the p27 cDNA in oligodendrocyte progenitor cells using an adenovirus vector. Progenitor cells normally express low levels of p27. After adenoviral infection and p27 overexpression, progenitor cells were able to undergo cell-cycle arrest, even in the presence of strong mitogens. The effects of p27 were shown to be directly upon cyclin-dependent kinase-2 (CDK2), the protein kinase complex responsible for G1/S transition, as immunodepletion of oligodendrocyte extracts of p27 protein resulted in the activation of CDK2 activity. However, cells that became growth arrested owing to infection with p27 adenovirus did not display conventional oligodendrocyte differentiation markers, such as O4 or O1. Taken together, these data provide mechanistic evidence indicating that p27 is primarily involved in oligodendroglial progenitor proliferation by inhibiting CDK2 activity and inducing oligodendrocyte cell-cycle arrest. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 431–440, 1998     

Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro

Diamond-Blackfan anemia (DBA) is caused by aberrant ribosomal biogenesis due to ribosomal protein (RP) gene mutations. To develop mechanistic understanding of DBA pathogenesis, we studied CD34⁺ cells from peripheral blood of DBA patients carrying RPL11 and RPS19 ribosomal gene mutations and determined their ability to undergo erythroid differentiation in vitro. RPS19 mutations induced a decrease in proliferation of progenitor cells, but the terminal erythroid differentiation was normal with little or no apoptosis. This phenotype was related to a G₀/G₁ cell cycle arrest associated with activation of the p53 pathway. In marked contrast, RPL11 mutations led to a dramatic decrease in progenitor cell proliferation and a delayed erythroid differentiation with a marked increase in apoptosis and G₀/G₁ cell cycle arrest with activation of p53. Infection of cord blood CD34⁺ cells with specific short hairpin (sh) RNAs against RPS19 or RPL11 recapitulated the two distinct phenotypes in concordance with findings from primary cells. In both cases, the phenotype has been reverted by shRNA p53 knockdown. These results show that p53 pathway activation has an important role in pathogenesis of DBA and can be independent of the RPL11 pathway. These findings shed new insights into the pathogenesis of DBA.