Loss of Cdk2 and cyclin A2 impairs cell proliferation and tumorigenesis.

Cell-cycle inhibition has yet to offer a generally effective approach to cancer treatment, but a full evaluation of different combinations of cell-cycle inhibitors has not been evaluated. Cyclin A2, a core component of the cell cycle, is often aberrantly expressed in cancer where it may impact cell proliferation. In this study, we investigated the role of cyclin A2 in tumorigenesis using a conditional genetic knockout mouse model. Cyclin A2 deletion in oncogene-transformed mouse embryonic fibroblasts (MEF) suppressed tumor formation in immunocompromised mice. These findings were confirmed in mice with cyclin A2-deficient hepatocytes, where a delay in liver tumor formation was observed. Because cyclin A2 acts in complex with Cdk2 in the cell cycle, we explored a hypothesized role for Cdk2 dysregulation in this effect through conditional deletions of both genes. In oncogene-transformed MEFs lacking both genes, tumor formation was strongly suppressed in a manner associated with decreased proliferation, premature senescence, and error-prone recovery from serum deprivation after immortalization. Whereas loss of cyclin A2 led to a compensatory increase in Cdk1 activity, this did not occur with loss of both Cdk2 and cyclin A2. Our work offers a rationale to explore combinations of Cdk1 and Cdk2 inhibitors as a general approach in cancer therapy.

CLIC4 regulates TGF-ß-dependent myofibroblast differentiation to produce a cancer stroma.

Cancer stroma has a profound influence on tumor development and progression. The conversion of fibroblasts to activated myofibroblasts is a hallmark of reactive tumor stroma. Among a number of factors involved in this conversion, transforming growth factor (TGF)-ß has emerged as a major regulator. CLIC4, an integral protein in TGF-ß signaling, is highly upregulated in stroma of multiple human cancers, and overexpression of CLIC4 in stromal cells enhances the growth of cancer xenografts. In this study, we show that conditioned media from tumor cell lines induces expression of both CLIC4 and the myofibroblast marker alpha smooth muscle actin (α-SMA) in stromal fibroblasts via TGF-ß signaling. Genetic ablation of CLIC4 in primary fibroblasts prevents or reduces constitutive or TGF-ß-induced expression of α-SMA and extracellular matrix components that are markers of myofibroblasts. CLIC4 is required for the activation of p38 map kinase by TGF-ß, a pathway that signals myofibroblast conversion in stromal cells. This requirement involves the interaction of CLIC4 with PPM1a, the selective phosphatase of activated p38. Conditioned media from fibroblasts overexpressing CLIC4 increases tumor cell migration and invasion in a TGF-ß-dependent manner and promotes epithelial to mesenchymal transition indicating that high stromal CLIC4 serves to enhance tumor invasiveness and progression. Thus, CLIC4 is significantly involved in the development of a nurturing tumor microenvironment by enhancing TGF-ß signaling in a positive feedback loop. Targeting CLIC4 in tumor stroma should be considered as a strategy to mitigate some of the tumor enhancing effects of the cancer stroma.

Spontaneous skin erosions and reduced skin and corneal wound healing characterize CLIC4(NULL) mice.

Cutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-ß (TGF-ß) signaling, and mice lacking components of the TGF-ß signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-ß pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4(NULL) mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4(NULL) mice, resolution of inflammation was delayed, and expression of ß4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4(NULL) skin. The induced level of phosphorylated Smad2 in response to TGF-ß was reduced in cultured CLIC4(NULL) keratinocytes relative to in wild-type cells, and CLIC4(NULL) keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-ß. CLIC4(NULL) keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-ß pathway.

Inducible NOS-induced chloride intracellular channel 4 (CLIC4) nuclear translocation regulates macrophage deactivation.

Nuclear translocation of cytosolic CLIC4 is an essential feature of its proapoptotic and prodifferentiation functions. Here we demonstrate that CLIC4 is induced concurrently with inducible nitric oxide synthase (iNOS) and S-nitrosylated in proinflammatory peritoneal macrophages. Chemical inhibition or genetic ablation of iNOS inhibits S-nitrosylation and nuclear translocation of CLIC4. In macrophages, iNOS-induced nuclear CLIC4 coincides with the pro- to anti-inflammatory transition of the cells because IL-1ß and CXCL1 mRNA remain elevated in CLIC4 and iNOS knockout macrophages at late time points, whereas TNFα mRNA is elevated only in the iNOS knockout macrophages. Active IL-1ß remains elevated in CLIC4 knockout macrophages and in macrophages in which CLIC4 nuclear translocation is prevented by the NOS inhibitor l-NAME. Moreover, overexpression of nuclear-targeted CLIC4 down-regulates IL-1ß in stimulated macrophages. In mice, genetically null for CLIC4, the number of phagocytosing macrophages stimulated by LPS is reduced. Thus, iNOS-induced nuclear CLIC4 is an essential part of the macrophage deactivation program.

Cyclin-dependent kinase 1 (Cdk1) is essential for cell division and suppression of DNA re-replication but not for liver regeneration.

Cyclin-dependent kinase 1 (Cdk1) is an archetypical kinase and a central regulator that drives cells through G2 phase and mitosis. Knockouts of Cdk2, Cdk3, Cdk4, or Cdk6 have resulted in viable mice, but the in vivo functions of Cdk1 have not been fully explored in mammals. Here we have generated a conditional-knockout mouse model to study the functions of Cdk1 in vivo. Ablation of Cdk1 leads to arrest of embryonic development around the blastocyst stage. Interestingly, liver-specific deletion of Cdk1 is well tolerated, and liver regeneration after partial hepatectomy is not impaired, indicating that regeneration can be driven by cell growth without cell division. The loss of Cdk1 does not affect S phase progression but results in DNA re-replication because of an increase in Cdk2/cyclin A2 activity. Unlike other Cdks, loss of Cdk1 in the liver confers complete resistance against tumorigenesis induced by activated Ras and silencing of p53.

Cdk2 and Cdk4 activities are dispensable for tumorigenesis caused by the loss of p53.

The loss of p53 induces spontaneous tumors in mice, and p53 mutations are found in approximately 50% of human tumors. These tumors are generally caused by a number of events, including genomic instability, checkpoint defects, mitotic defects, deregulation of transcriptional targets, impaired apoptosis, and G(1) deregulation or a combination of these effects. In order to determine the role of proteins involved in G(1) control in tumorigenesis, we focused on Cdk2 and Cdk4, two cyclin-dependent kinases that in association with cyclin E and cyclin D promote the G(1)/S phase transition. We analyzed the consequence of loss of Cdk2 in p53-null animals by generating Cdk2(-/-) p53(-/-) mice. These mice are viable and developed spontaneous tumors, predominantly lymphoblastic lymphomas, similar to p53(-/-) mice. In contrast, the genotypes Cdk4(-/-) p53(-/-) were mostly lethal, with few exceptions, and Cdk2(-/-) Cdk4(-/-) p53(-/-) mice die during embryogenesis at embryonic day 13.5. To study the oncogenic potential, we generated mouse embryonic fibroblasts (MEFs) and found that p53(-/-), Cdk2(-/-) p53(-/-), Cdk4(-/-) p53(-/-), and Cdk2(-/-) Cdk4(-/-) p53(-/-) MEFs grew at similar rates without entering senescence. Ras-transformed MEFs of these genotypes were able to form colonies in vitro and induce tumors in nude mice. Our results suggest that tumorigenicity mediated by p53 loss does not require either Cdk2 or Cdk4, which necessitates considering the use of broad-spectrum cell cycle inhibitors as a means of effective anti-Cdk cancer therapy.

Nesprin-2 Giant (NUANCE) maintains nuclear envelope architecture and composition in skin.

Giant isoforms, encoded by Nesprin-1 (Syne1) and Nesprin-2 (Syne2), are multifunctional actin-binding and nuclear-envelope-associated proteins belonging to the spectrin superfamily. Here, we investigate the function of Nesprin-2 Giant (NUANCE) in skin by generating mice lacking the actin-binding domain of Nesprin-2 (Nesprin-2DeltaABD). This loss results in a slight but significant thickening of the epidermis, which is a consequence of the increased epithelial nuclear size. Nonetheless, epidermal proliferation and differentiation appear normal in the knockout epidermis. Surprisingly, Nesprin-2 C-terminal-isoform expression and nuclear envelope localization were affected in certain tissues. Nuclei of primary dermal knockout fibroblasts and keratinocytes were heavily misshapen, displaying a striking similarity to nuclear deformations characteristic of laminopathies. Furthermore, emerin, the protein involved in the X-linked form of Emery-Dreifuss muscular dystrophy (EDMD), was unevenly distributed along the nuclear envelope in mutant fibroblasts, often forming aggregates in the deformed nuclear envelope areas. Thus, Nesprin-2 is an important scaffold protein implicated in the maintenance of nuclear envelope architecture. Aged knockout fibroblasts readily generated, by alternative splicing and alternative translation initiation, aberrant Nesprin-2 Giant isoforms that lacked an ABD but that were sufficient to restore nuclear shape and emerin localization; this suggests that other regions of Nesprin-2 Giant, potentially including its spectrin repeats, are crucial for these functions.

Lamin A/C-dependent localization of Nesprin-2, a giant scaffolder at the nuclear envelope.

The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.