Transcription factor E4F1 is essential for epidermal stem cell maintenance and skin homeostasis.

A growing body of evidence suggests that the multifunctional protein E4F1 is involved in signaling pathways that play essential roles during normal development and tumorigenesis. We generated E4F1 conditional knockout mice to address E4F1 functions in vivo in newborn and adult skin. E4F1 inactivation in the entire skin or in the basal compartment of the epidermis induces skin homeostasis defects, as evidenced by transient hyperplasia in the interfollicular epithelium and alteration of keratinocyte differentiation, followed by loss of cellularity in the epidermis and severe skin ulcerations. E4F1 depletion alters clonogenic activity of epidermal stem cells (ESCs) ex vivo and ends in exhaustion of the ESC pool in vivo, indicating that the lesions observed in the E4F1 mutant skin result, at least in part, from cell-autonomous alterations in ESC maintenance. The clonogenic potential of E4F1 KO ESCs is rescued by Bmi1 overexpression or by Ink4a/Arf or p53 depletion. Skin phenotype of E4F1 KO mice is also delayed in animals with Ink4a/Arf and E4F1 compound gene deficiencies. Our data identify a regulatory axis essential for ESC-dependent skin homeostasis implicating E4F1 and the Bmi1-Arf-p53 pathway.

The Hsp90 cochaperone TTT promotes cotranslational maturation of PIKKs prior to complex assembly.

Phosphatidylinositol 3-kinase-related kinases (PIKKs) are a family of kinases that control fundamental processes, including cell growth, DNA damage repair, and gene expression. Although their regulation and activities are well characterized, little is known about how PIKKs fold and assemble into active complexes. Previous work has identified a heat shock protein 90 (Hsp90) cochaperone, the TTT complex, that specifically stabilizes PIKKs. Here, we describe a mechanism by which TTT promotes their de novo maturation in fission yeast. We show that TTT recognizes newly synthesized PIKKs during translation. Although PIKKs form multimeric complexes, we find that they do not engage in cotranslational assembly with their partners. Rather, our findings suggest a model by which TTT protects nascent PIKK polypeptides from misfolding and degradation because PIKKs acquire their native state after translation is terminated. Thus, PIKK maturation and assembly are temporally segregated, suggesting that the biogenesis of large complexes requires both dedicated chaperones and cotranslational interactions between subunits.

Heterogeneity in sarcoma cell lines reveals enhanced motility of tetraploid versus diploid cells.

Soft tissue sarcomas with complex genomics are very heterogeneous tumors lacking simple prognosis markers or targeted therapies. Overexpression of a subset of mitotic genes from a signature called CINSARC is of bad prognosis, but the significance of this signature remains elusive. Here we precisely measure the cell cycle and mitosis duration of sarcoma cell lines and we found that the mitotic gene products overexpression does not reflect variation in the time spent during mitosis or G2/M. We also found that the CINSARC cell lines, we studied, are composed of a mixture of aneuploid, diploid, and tetraploid cells that are highly motile in vitro. After sorting diploid and tetraploid cells, we showed that the tetraploid cell clones do not possess a proliferative advantage, but are strikingly more motile and invasive than their diploid counterparts. This is correlated with higher levels of mitotic proteins overexpression. Owing that mitotic proteins are almost systematically degraded at the end of mitosis, we propose that it is the abnormal activity of the mitotic proteins during interphase that boosts the sarcoma cells migratory properties by affecting their cytoskeleton. To test this hypothesis, we designed a screen for mitotic or cytoskeleton protein inhibitors affecting the sarcoma cell migration potential independently of cytotoxic activities. We found that inhibition of several mitotic kinases drastically impairs the CINSARC cell invasive and migratory properties. This finding could provide a handle by which to selectively inhibit the most invasive cells.

Whole-genome duplication increases tumor cell sensitivity to MPS1 inhibition.

Several lines of evidence indicate that whole-genome duplication resulting in tetraploidy facilitates carcinogenesis by providing an intermediate and metastable state more prone to generate oncogenic aneuploidy. Here, we report a novel strategy to preferentially kill tetraploid cells based on the abrogation of the spindle assembly checkpoint (SAC) via the targeting of TTK protein kinase (better known as monopolar spindle 1, MPS1). The pharmacological inhibition as well as the knockdown of MPS1 kills more efficiently tetraploid cells than their diploid counterparts. By using time-lapse videomicroscopy, we show that tetraploid cells do not survive the aborted mitosis due to SAC abrogation upon MPS1 depletion. On the contrary diploid cells are able to survive up to at least two more cell cycles upon the same treatment. This effect might reflect the enhanced difficulty of cells with whole-genome doubling to tolerate a further increase in ploidy and/or an elevated level of chromosome instability in the absence of SAC functions. We further show that MPS1-inhibited tetraploid cells promote mitotic catastrophe executed by the intrinsic pathway of apoptosis, as indicated by the loss of mitochondrial potential, the release of the pro-apoptotic cytochrome c from mitochondria, and the activation of caspases. Altogether, our results suggest that MPS1 inhibition could be used as a therapeutic strategy for targeting tetraploid cancer cells.

Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5.

BACKGROUND: The two myogenic regulatory factors Myf5 and MyoD are basic helix-loop-helix muscle transcription factors undergoing differential cell cycle dependent proteolysis in proliferating myoblasts. This regulated degradation results in the striking expression of these two factors at distinct phases of the cell cycle, and suggests that their precise and alternated disappearance is an important feature of myoblasts, maybe connected to the maintenance of the proliferative status and/or commitment to the myogenic lineage of these cells. One way to understand the biological function(s) of the cyclic expression of these proteins is to specifically alter their degradation, and to analyze the effects of their stabilization on cells. To this aim, we undertook the biochemical analysis of the mechanisms governing Myf5 mitotic degradation, using heterologous systems. RESULTS: We show here that mitotic degradation of Myf5 is conserved in non-myogenic cells, and is thus strictly under the control of the cell cycle apparatus. Using Xenopus egg extracts as an in vitro system to dissect the main steps of Myf5 mitotic proteolysis, we show that (1) Myf5 stability is regulated by a complex interplay of phosphorylation/dephosphorylation, probably involving various kinases and phosphatases, (2) Myf5 is ubiquitylated in mitotic extracts, and this is a prerequisite to its degradation by the proteasome and (3) at least in the Xenopus system, the E3 responsible for its mitotic degradation is not the APC/C (the major E3 during mitosis). CONCLUSION: Altogether, our data strongly suggest that the mitotic degradation of Myf5 by the ubiquitin-proteasome system is precisely controlled by multiple phosphorylation of the protein, and that the APC/C is not involved in this process.

E4F1 deficiency results in oxidative stress-mediated cell death of leukemic cells.

The multifunctional E4F1 protein was originally discovered as a target of the E1A viral oncoprotein. Growing evidence indicates that E4F1 is involved in key signaling pathways commonly deregulated during cell transformation. In this study, we investigate the influence of E4F1 on tumorigenesis. Wild-type mice injected with fetal liver cells from mice lacking CDKN2A, the gene encoding Ink4a/Arf, developed histiocytic sarcomas (HSs), a tumor originating from the monocytic/macrophagic lineage. Cre-mediated deletion of E4F1 resulted in the death of HS cells and tumor regression in vivo and extended the lifespan of recipient animals. In murine and human HS cell lines, E4F1 inactivation resulted in mitochondrial defects and increased production of reactive oxygen species (ROS) that triggered massive cell death. Notably, these defects of E4F1 depletion were observed in HS cells but not healthy primary macrophages. Short hairpin RNA-mediated depletion of E4F1 induced mitochondrial defects and ROS-mediated death in several human myeloid leukemia cell lines. E4F1 protein is overexpressed in a large subset of human acute myeloid leukemia samples. Together, these data reveal a role for E4F1 in the survival of myeloid leukemic cells and support the notion that targeting E4F1 activities might have therapeutic interest.