SCF(FBW7) regulates cellular apoptosis by targeting MCL1 for ubiquitylation and destruction

The effective use of targeted therapy is highly dependent on the identification of responder patient populations. Loss of FBW7, which encodes a tumour-suppressor protein, is frequently found in various types of human cancer, including breast cancer, colon cancer and T-cell acute lymphoblastic leukaemia (T-ALL). In line with these genomic data, engineered deletion of Fbw7 in mouse T cells results in T-ALL, validating FBW7 as a T-ALL tumour suppressor. Determining the precise molecular mechanisms by which FBW7 exerts antitumour activity is an area of intensive investigation. These mechanisms are thought to relate in part to FBW7-mediated destruction of key proteins relevant to cancer, including Jun, Myc, cyclin E and notch 1 (ref. 9), all of which have oncoprotein activity and are overexpressed in various human cancers, including leukaemia. In addition to accelerating cell growth, overexpression of Jun, Myc or notch 1 can also induce programmed cell death. Thus, considerable uncertainty surrounds how FBW7-deficient cells evade cell death in the setting of upregulated Jun, Myc and/or notch 1. Here we show that the E3 ubiquitin ligase SCF(FBW7) (a SKP1-cullin-1-F-box complex that contains FBW7 as the F-box protein) governs cellular apoptosis by targeting MCL1, a pro-survival BCL2 family member, for ubiquitylation and destruction in a manner that depends on phosphorylation by glycogen synthase kinase 3. Human T-ALL cell lines showed a close relationship between FBW7 loss and MCL1 overexpression. Correspondingly, T-ALL cell lines with defective FBW7 are particularly sensitive to the multi-kinase inhibitor sorafenib but resistant to the BCL2 antagonist ABT-737. On the genetic level, FBW7 reconstitution or MCL1 depletion restores sensitivity to ABT-737, establishing MCL1 as a therapeutically relevant bypass survival mechanism that enables FBW7-deficient cells to evade apoptosis. Therefore, our work provides insight into the molecular mechanism of direct tumour suppression by FBW7 and has implications for the targeted treatment of patients with FBW7-deficient T-ALL.     

Virus-mediated killing of cells that lack p53 activity

A major goal of molecular oncology is to identify means to kill cells lacking p53 function. Most current cancer therapy is based on damaging cellular DNA by irradiation or chemicals. Recent reports support the notion that, in the event of DNA damage, the p53 tumour-suppressor protein is able to prevent cell death by sustaining an arrest of the cell cycle at the G2 phase. We report here that adeno-associated virus (AAV) selectively induces apoptosis in cells that lack active p53. Cells with intact p53 activity are not killed but undergo arrest in the G2 phase of the cell cycle. This arrest is characterized by an increase in p53 activity and p21 levels and by the targeted destruction of CDC25C. Neither cell killing nor arrest depends upon AAV-encoded proteins. Rather, AAV DNA, which is single-stranded with hairpin structures at both ends, elicits in cells a DNA damage response that, in the absence of active p53, leads to cell death. AAV inhibits tumour growth in mice. Thus viruses can be used to deliver DNA of unusual structure into cells to trigger a DNA damage response without damaging cellular DNA and to selectively eliminate those cells lacking p53 activity.     

Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6.

Wild-type p53 protein has many properties consistent with its being the product of a tumour suppressor gene. Although the normal roles of tumour suppressor genes are still largely unknown, it seems that they could be involved in promoting cell differentiation as well as in mediating growth arrest by growth-inhibitory cytokines. Hence, the abrogation of wild-type p53 expression, which is a common feature of many tumours, could eliminate these activities. We have now tested this notion by restoring the expression of p53 in a murine myeloid leukaemic cell line that normally lacks p53. The use of a temperature-sensitive p53 mutant allowed us to analyse cells in which the introduced p53 had either wild-type or mutant properties. Although there seemed to be no effect on differentiation, the introduction of wild-type p53 resulted in rapid loss of cell viability in a way characteristic of apoptosis (programmed cell death). The effect of wild-type p53 was counteracted by interleukin-6. Thus products of tumour suppressor genes could be involved in restricting precursor cell populations by mediating apoptosis.     

Induction of autophagy and inhibition of tumorigenesis by beclin 1

The process of autophagy, or bulk degradation of cellular proteins through an autophagosomic-lysosomal pathway, is important in normal growth control and may be defective in tumour cells. However, little is known about the genetic mediators of autophagy in mammalian cells or their role in tumour development. The mammalian gene encoding Beclin 1, a novel Bcl-2-interacting, coiled-coil protein, has structural similarity to the yeast autophagy gene, apg6/vps30, and is mono-allelically deleted in 40-75% of sporadic human breast cancers and ovarian cancers. Here we show, using gene-transfer techniques, that beclin 1 promotes autophagy in autophagy-defective yeast with a targeted disruption of agp6/vps30, and in human MCF7 breast carcinoma cells. The autophagy-promoting activity of beclin 1 in MCF7 cells is associated with inhibition of MCF7 cellular proliferation, in vitro clonigenicity and tumorigenesis in nude mice. Furthermore, endogenous Beclin 1 protein expression is frequently low in human breast epithelial carcinoma cell lines and tissue, but is expressed ubiquitously at high levels in normal breast epithelia. Thus, beclin 1 is a mammalian autophagy gene that can inhibit tumorigenesis and is expressed at decreased levels in human breast carcinoma. These findings suggest that decreased expression of autophagy proteins may contribute to the development or progression of breast and other human malignancies.     

The pathological response to DNA damage does not contribute to p53-mediated tumour suppression

The p53 protein has a highly evolutionarily conserved role in metazoans as 'guardian of the genome', mediating cell-cycle arrest and apoptosis in response to genotoxic injury. In large, long-lived animals with substantial somatic regenerative capacity, such as vertebrates, p53 is an important tumour suppressor--an attribute thought to stem directly from its induction of death or arrest in mutant cells with damaged or unstable genomes. Chemotherapy and radiation exposure both induce widespread p53-dependent DNA damage. This triggers potentially lethal pathologies that are generally deemed an unfortunate but unavoidable consequence of the role p53 has in tumour suppression. Here we show, using a mouse model in which p53 status can be reversibly switched in vivo between functional and inactive states, that the p53-mediated pathological response to whole-body irradiation, a prototypical genotoxic carcinogen, is irrelevant for suppression of radiation-induced lymphoma. In contrast, delaying the restoration of p53 function until the acute radiation response has subsided abrogates all of the radiation-induced pathology yet preserves much of the protection from lymphoma. Such protection is absolutely dependent on p19(ARF)--a tumour suppressor induced not by DNA damage, but by oncogenic disruption of the cell cycle.     

Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines

During Drosophila development and mammalian embryogenesis, exit from the cell cycle is contingent on tightly controlled downregulation of the activity of Cyclin E-Cdk2 complexes that normally promote the transition from G1 to S phase. Although protein degradation has a crucial role in downregulating levels of Cyclin E, many of the proteins that function in degradation of Cyclin E have not been identified. In a screen for Drosophila mutants that display increased cell proliferation, we identified archipelago, a gene encoding a protein with an F-box and seven tandem WD (tryptophan-aspartic acid) repeats. Here we show that archipelago mutant cells have persistently elevated levels of Cyclin E protein without increased levels of cyclin E RNA. They are under-represented in G1 fractions and continue to proliferate when their wild-type neighbours become quiescent. The Archipelago protein binds directly to Cyclin E and probably targets it for ubiquitin-mediated degradation. A highly conserved human homologue is present and is mutated in four cancer cell lines including three of ten derived from ovarian carcinomas. These findings implicate archipelago in developmentally regulated degradation of Cyclin E and potentially in the pathogenesis of human cancers.     

AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress

Overcoming metabolic stress is a critical step for solid tumour growth. However, the underlying mechanisms of cell death and survival under metabolic stress are not well understood. A key signalling pathway involved in metabolic adaptation is the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Energy stress conditions that decrease intracellular ATP levels below a certain level promote AMPK activation by LKB1. Previous studies showed that LKB1-deficient or AMPK-deficient cells are resistant to oncogenic transformation and tumorigenesis, possibly because of the function of AMPK in metabolic adaptation. However, the mechanisms by which AMPK promotes metabolic adaptation in tumour cells are not fully understood. Here we show that AMPK activation, during energy stress, prolongs cell survival by redox regulation. Under these conditions, NADPH generation by the pentose phosphate pathway is impaired, but AMPK induces alternative routes to maintain NADPH and inhibit cell death. The inhibition of the acetyl-CoA carboxylases ACC1 and ACC2 by AMPK maintains NADPH levels by decreasing NADPH consumption in fatty-acid synthesis and increasing NADPH generation by means of fatty-acid oxidation. Knockdown of either ACC1 or ACC2 compensates for AMPK activation and facilitates anchorage-independent growth and solid tumour formation in vivo, whereas the activation of ACC1 or ACC2 attenuates these processes. Thus AMPK, in addition to its function in ATP homeostasis, has a key function in NADPH maintenance, which is critical for cancer cell survival under energy stress conditions, such as glucose limitations, anchorage-independent growth and solid tumour formation in vivo.     

Abrogation by c-myc of G1 phase arrest induced by RB protein but not by p53.

Inactivating mutations of the retinoblastoma gene (RB) are found in a wide variety of tumour cells. Replacement of wild-type RB can suppress the tumorigenicity of some of these cells, suggesting that the RB protein (Rb) may negatively regulate cell growth. As activation of c-myc expression promotes cell proliferation and blocks differentiation, it may positively regulate cell growth. The c-myc protein is localized in the nucleus and can physically associate with RB protein in vitro, hence c-myc may functionally antagonize RB function. Microinjection of Rb in G1 phase reversibly arrests cell-cycle progression. Here we co-inject RB protein with c-myc, EJ-ras, c-fos or c-jun protein. Co-injection of c-myc, but not EJ-ras, c-fos or c-jun, inhibits the ability of Rb to arrest the cell cycle. The c-myc does not inhibit the activity of another tumour supressor, p53 (ref. 12). Thus, c-myc and RB specifically antagonize one another in the cell.     

Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity

Natural killer (NK) cells attack many tumour cell lines, and are thought to have a critical role in anti-tumour immunity; however, the interaction between NK cells and tumour targets is poorly understood. The stimulatory lectin-like NKG2D receptor is expressed by NK cells, activated CD8+ T cells and by activated macrophages in mice. Several distinct cell-surface ligands that are related to class I major histocompatibility complex molecules have been identified, some of which are expressed at high levels by tumour cells but not by normal cells in adults. However, no direct evidence links the expression of these 'induced self' ligands with tumour cell rejection. Here we demonstrate that ectopic expression of the murine NKG2D ligands Rae1beta or H60 in several tumour cell lines results in potent rejection of the tumour cells by syngeneic mice. Rejection is mediated by NK cells and/or CD8+ T cells. The ligand-expressing tumour cells induce potent priming of cytotoxic T cells and sensitization of NK cells in vivo. Mice that are exposed to live or irradiated tumour cells expressing Rae1 or H60 are specifically immune to subsequent challenge with tumour cells that lack NKG2D ligands, suggesting application of the ligands in the design of tumour vaccines.     

具有激活RB蛋白生物活性小肽的研究

构建了慢病毒载体表达MP1多肽的RFP融合蛋白(RFP-MP1),并研究了它对人肺腺癌细胞株H1299和人骨髓瘤细胞株U2-OS增殖的影响.U2-OS和H1299细胞中RFPMP1的表达导致了RB在蛋白水平上的积累,使细胞生长受到抑制.此外,细胞流式结果发现RFP-MP1使细胞周期阻滞在G1期.进一步研究表明RFP-MP1能够阻滞RB对E2F活性的抑制.这些结果表明,11肽的MP1能够上调肿瘤细胞中RB蛋白的表达水平并且抑制其生长.     

Phosphorylation of Erp1 by p90rsk is required for cytostatic factor arrest in Xenopus laevis eggs

Until fertilization, the meiotic cell cycle of vertebrate eggs is arrested at metaphase of meiosis II by a cytoplasmic activity termed cytostatic factor (CSF), which causes inhibition of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets mitotic cyclins-regulatory proteins of meiosis and mitosis-for degradation. Recent studies indicate that Erp1/Emi2, an inhibitor protein for the APC/C, has an essential role in establishing and maintaining CSF arrest, but its relationship to Mos, a mitogen-activated protein kinase (MAPK) kinase kinase that also has an essential role in establishing CSF arrest through activation of p90 ribosomal S6 kinase (p90rsk), is unclear. Here we report that in Xenopus eggs Erp1 is a substrate of p90rsk, and that Mos-dependent phosphorylation of Erp1 by p90rsk at Thr 336, Ser 342 and Ser 344 is crucial for both stabilizing Erp1 and establishing CSF arrest in meiosis II oocytes. Semi-quantitative analysis with CSF-arrested egg extracts reveals that the Mos-dependent phosphorylation of Erp1 enhances, but does not generate, the activity of Erp1 that maintains metaphase arrest. Our results also suggest that Erp1 inhibits cyclin B degradation by binding the APC/C at its carboxy-terminal destruction box, and this binding is also enhanced by the Mos-dependent phosphorylation. Thus, Mos and Erp1 collaboratively establish and maintain metaphase II arrest in Xenopus eggs. The link between Mos and Erp1 provides a molecular explanation for the integral mechanism of CSF arrest in unfertilized vertebrate eggs.     

Control of plant germline proliferation by SCF(FBL17) degradation of cell cycle inhibitors

Flowering plants possess a unique reproductive strategy, involving double fertilization by twin sperm cells. Unlike animal germ lines, the male germ cell lineage in plants only forms after meiosis and involves asymmetric division of haploid microspores, to produce a large, non-germline vegetative cell and a germ cell that undergoes one further division to produce the twin sperm cells. Although this switch in cell cycle control is critical for sperm cell production and delivery, the underlying molecular mechanisms are unknown. Here we identify a novel F-box protein of Arabidopsis thaliana, designated FBL17 (F-box-like 17), that enables this switch by targeting the degradation of cyclin-dependent kinase A;1 inhibitors specifically in male germ cells. We show that FBL17 is transiently expressed in the male germ line after asymmetric division and forms an SKP1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complex (SCF(FBL17)) that targets the cyclin-dependent kinase inhibitors KRP6 and KRP7 for proteasome-dependent degradation. Accordingly, the loss of FBL17 function leads to the stabilization of KRP6 and inhibition of germ cell cycle progression. Our results identify SCF(FBL17) as an essential male germ cell proliferation complex that promotes twin sperm cell production and double fertilization in flowering plants.     

An inhibitor of Bcl-2 family proteins induces regression of solid tumours

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.     

Efp targets 14-3-3 sigma for proteolysis and promotes breast tumour growth

Oestrogen exerts its influence on target organs through activating oestrogen receptors (ERs) and regulating downstream genes by means of their oestrogen-responsive elements. Efp, a target gene product of ER alpha, is a member of the RING-finger B-box coiled-coil (RBCC) motif family. Efp is predominantly expressed in various female organs as well as in breast cancers, and is thought to be essential for oestrogen-dependent cell proliferation and organ development Efp-disrupted mice display underdeveloped uteri and reduced oestrogen responsiveness. Here we show that Efp is a RING-finger-dependent ubiquitin ligase (E3) that targets proteolysis of 14-3-3 sigma, a negative cell cycle regulator that causes G2 arrest. We demonstrate that tumour growth of breast cancer MCF7 cells implanted in female athymic mice is reduced by treatment with antisense Efp oligonucleotide. Efp-overexpressing MCF7 cells in ovariectomized athymic mice generate tumours in the absence of oestrogen. Loss of Efp function in mouse embryonic fibroblasts results in an accumulation of 14-3-3 sigma, which is responsible for reduced cell growth. These data provide an insight into the cell-cycle machinery and tumorigenesis of breast cancer by identifying 14-3-3 sigma as a target for proteolysis by Efp, leading to cell proliferation.