ΔNp63α is an oncogene that targets chromatin remodeler Lsh to drive skin stem cell proliferation and tumorigenesis.

The p53 homolog p63 is essential for development, yet its role in cancer is not clear. We discovered that p63 deficiency evokes the tumor-suppressive mechanism of cellular senescence, causing a striking absence of stratified epithelia such as the skin. Here we identify the predominant p63 isoform, ΔNp63α, as a protein that bypasses oncogene-induced senescence to drive tumorigenesis in vivo. Interestingly, bypass of senescence promotes stem-like proliferation and maintains survival of the keratin 15-positive stem cell population. Furthermore, we identify the chromatin-remodeling protein Lsh as a new target of ΔNp63α that is an essential mediator of senescence bypass. These findings indicate that ΔNp63α is an oncogene that cooperates with Ras to promote tumor-initiating stem-like proliferation and suggest that Lsh-mediated chromatin-remodeling events are critical to this process.

TAp63 induces senescence and suppresses tumorigenesis in vivo.

p63 is distinct from its homologue p53 in that its role as a tumour suppressor is controversial, an issue complicated by the existence of two classes of p63 isoforms. Here we show that TAp63 isoforms are robust mediators of senescence that inhibit tumorigenesis in vivo. Whereas gain of TAp63 induces senescence, loss of p63 enhances sarcoma development in mice lacking p53. Using a new TAp63-specific conditional mouse model, we demonstrate that TAp63 isoforms are essential for Ras-induced senescence, and that TAp63 deficiency increases proliferation and enhances Ras-mediated oncogenesis in the context of p53 deficiency in vivo. TAp63 induces senescence independently of p53, p19(Arf) and p16(Ink4a), but requires p21(Waf/Cip1) and Rb. TAp63-mediated senescence overrides Ras-driven transformation of p53-deficient cells, preventing tumour initiation, and doxycycline-regulated expression of TAp63 activates p21(Waf/Cip1), induces senescence and inhibits progression of established tumours in vivo. Our findings demonstrate that TAp63 isoforms function as tumour suppressors by regulating senescence through p53-independent pathways. The ability of TAp63 to trigger senescence and halt tumorigenesis irrespective of p53 status identifies TAp63 as a potential target of anti-cancer therapy for human malignancies with compromised p53.

p63, cellular senescence and tumor development.

Deficiency of p63, a p53-related protein, causes severe defects in epithelial morphogenesis. Studies of p63-compromised mouse models reveal that p63 deficiency induces cellular senescence both in cultured cells and in vivo, through regulation p19(Arf)/p53 and p16(Ink4a)/Rb pathways. An extensive tumor study of p63-compromised mice demonstrated that p63 deficiency does not predispose to, but rather protects from, tumor development. These findings further implicate p63 as a negative regulator of the tumor suppressive mechanism of cellular senescence.

p63 heterozygous mutant mice are not prone to spontaneous or chemically induced tumors.

Homology between p63 and p53 has suggested that these proteins might function similarly. However, the majority of data from human tumors have not supported a similar role for p63 in tumor suppression. To investigate this issue, we studied spontaneous tumorigenesis in p63+/- mice in both WT and p53-compromised backgrounds. We found that p63+/- mice were not tumor prone and mice heterozygous for both p63 and p53 had fewer tumors than p53+/- mice. The rare tumors that developed in mice with compromised p63 were also distinct from those of p53+/- mice. Furthermore, p63+/- mice were not prone to chemically induced tumorigenesis, and p63 expression was maintained in carcinomas. These findings demonstrate that, in agreement with data from human tumors, p63 plays a markedly different biological role in cancer than p53.

p63 deficiency activates a program of cellular senescence and leads to accelerated aging.

The p53 tumor suppressor plays a key role in organismal aging. A cellular mechanism postulated to drive the aging process is cellular senescence, mediated in part by p53. Although senescent cells accumulate in elderly individuals, most studies have relied on correlating in vitro senescence assays with in vivo phenotypes of aging. Here, using two different mouse models in which the p53-related protein p63 is compromised, we demonstrate that cellular senescence and organismal aging are intimately linked and that these processes are mediated by p63 loss. We found that p63(+/-) mice have a shortened life span and display features of accelerated aging. Both germline and somatically induced p63 deficiency activates widespread cellular senescence with enhanced expression of senescent markers SA-beta-gal, PML, and p16(INK4a). Using an inducible tissue-specific p63 conditional model, we further show that p63 deficiency induces cellular senescence and causes accelerated aging phenotypes in the adult. Our results thus suggest a causative link between cellular senescence and aging in vivo, and demonstrate that p63 deficiency accelerates this process.

Expression of a constitutively active mutant of M-Ras in normal bone marrow is sufficient for induction of a malignant mastocytosis/mast cell leukemia, distinct from the histiocytosis/monocytic leukemia induced by expression of activated H-Ras.

Expression of constitutively activated M-Ras in normal murine bone-marrow cells was sufficient to induce the factor-independent, in vitro growth and differentiation of colonies of macrophages and neutrophils, and the generation of immortal lines of factor-independent mast cells, and, upon in vivo injection of the transduced cells, a fatal mastocytosis/mast-cell leukemia. In contrast, expression of constitutively activated H-Ras in bone-marrow cells resulted in the in vitro growth, in the absence of exogenous factors, of colonies that contained only macrophages and of lines of cells resembling dendritic cells, and, upon in vivo injection of the transduced cells, a fatal histiocytosis/monocytic leukemia. Macrophages generated by bone-marrow cells expressing activated M-Ras or activated H-Ras differed morphologically, the latter appearing more activated, a difference abrogated by an inhibitor of Erk activation. Inhibition of either Erk or PI3 kinase blocked the capacity of both activated M-Ras and activated H-Ras to support proliferation and viability. However, inhibition of p38 MAPK activity suppressed proliferation of bone-marrow cells expressing activated H-Ras, but enhanced that of bone-marrow cells expressing activated M-Ras. Thus, expression of either activated M-Ras or H-Ras in normal hematopoietic cells was sufficient for transformation but each resulted in the generation of distinct lineages of cells.

Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry.

The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions.

Defining the involvement of p38alpha MAPK in the production of anti- and proinflammatory cytokines using an SB 203580-resistant form of the kinase.

Despite its lack of specificity, the inhibitor SB 203580 has been widely used to implicate p38 mitogen-activated protein kinase (MAPK) in the synthesis of many cytokines. Here we show unequivocally that the production of interleukin (IL)-1beta, IL-6, IL-10, and tumor necrosis factor alpha (TNFalpha) requires p38 MAPK activity by demonstrating that the inhibitory effects of SB 203580 were reversed by expression of an SB 203580-resistant form of p38alpha (SBR-p38alpha) that fails to bind to SB 203580. This strategy established the requirement for p38 activity for the lipopolysaccharide-stimulated production of IL-10, IL-1beta, and IL-6 by the monocytic cell WEHI 274 and the production of IL-6 and TNFalpha stimulated by ligation of the Fc-gamma receptor of the mast cell MC/9. Expression of SBR-p38alpha in primary macrophages abrogated the ability of SB 203580 to inhibit the lipopolysaccharide-stimulated production of TNFalpha but not of IL-10. Expression of SBR-p38alpha in primary T lymphocytes abrogated the ability of SB 203580 to inhibit the production of interferon-gamma induced by co-ligation of CD3 and CD28 but not the production of interferon-gamma or IL-10 induced by IL-12. These results suggest that the levels of p38 MAPK activity required for maximal cytokine production vary with different cytokines and stimuli.

Ras and relatives--job sharing and networking keep an old family together.

Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cells, with roles in growth, survival, differentiation, cytokine production, chemotaxis, vesicle-trafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B are also frequently mutated in human cancer and leukemia. Besides the four p21 Ras proteins, the Ras subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (R-Ras3), Rap1A, Rap1B, Rap2A, Rap2B, RalA, and RalB. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors through which they transmit signals and of GTPase activating proteins that downregulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members, although the presence of well-conserved orthologs in Caenorhabditis elegans suggests that their individual roles are both specific and vital. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and functions previously attributed to one family member may be shared with other members of the Ras family. Here we discuss similarities and differences in activation, effector usage, and functions of different members of the Ras subfamily. We also review the possibility that the differential localization of Ras proteins in different parts of the cell membrane may govern their responses to activation of cell surface receptors.

Activation of small GTPases of the Ras and Rho family by growth factors active on mast cells.

The small GTPases of the Ras and Rho families are activated by the key growth factors for mast cell development and survival, SLF and IL-3. While there are many clues that activation of Ras and Rho proteins play critical roles in growth, survival and differentiation, as well as in functions, such as migration and degranulation, limitations in the specificity of experimental tools still obscure their precise functions. There is increasing evidence that differences in subcellular localization of closely related GTPases determines important differences in their function. However, other data also point to differences in sensitivity to activation by GEF and in the effectors they engage.