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Mutations in the BRCA2 tumor suppressor gene have been associated with an increased risk of developing prostate cancer. One of the paradoxes concerning BRCA2 is the fact that its inactivation affects genetic stability and is deleterious for cellular and organismal survival, while BRCA2-mutated cancer cells adapt to this detriment and malignantly proliferate. Therapeutic strategies for tumors arising from BRCA2 mutations may be discovered by understanding these adaptive mechanisms. In this study, we conducted forward genetic synthetic viability screenings in Caenorhabditis elegans brc-2 (Cebrc-2) mutants and found that Ceubxn-2 inactivation rescued the viability of Cebrc-2 mutants. Moreover, loss of NSFL1C, the mammalian ortholog of CeUBXN-2, suppressed the spindle assembly checkpoint (SAC) activation and promoted the survival of BRCA2- deficient cells. Mechanistically, NSFL1C recruited USP9X to inhibit the polyubiquitination of AURKB and reduce the removal of AURKB from the centromeres by VCP, which is essential for SAC activation. SAC inactivation is common in BRCA2-deficient prostate cancer patients, but PP2A inhibitors could reactivate the SAC and achieve BRCA2- deficient prostate tumor synthetic lethality. Our research reveals the survival adaptation mechanism of BRCA2-deficient prostate tumor cells and provides different angles for exploring synthetic lethal inhibitors in addition to targeting DNA damage repair pathways., Introduction Germline mutations of BRCA2 predispose humans to prostate, breast, ovarian, and pancreatic cancers (1, 2). As a tumor suppressor protein, BRCA2 is essential for stabilizing stalled replication forks (2, [...]

Oncogenic mutations drive anabolic metabolism, creating a dependency on nutrient influx through transporters, receptors, and macropinocytosis. While sphingolipids suppress tumor growth by downregulating nutrient transporters, macropinocytosis and autophagy still provide cancer cells with fuel. Therapeutics that simultaneously disrupt these parallel nutrient access pathways have potential as powerful starvation agents. Here, we describe a water-soluble, orally bioavailable synthetic sphingolipid, SH-BC-893, that triggers nutrient transporter internalization and also blocks lysosome-dependent nutrient generation pathways. SH-BC-893 activated protein phosphatase 2A (PP2A), leading to mislocalization of the lipid kinase PIKfyve. The concomitant mislocalization of the PIKfyve product PI(3,5)P2 triggered cytosolic vacuolation and blocked lysosomal fusion reactions essential for LDL, autophagosome, and macropinosome degradation. By simultaneously limiting access to both extracellular and intracellular nutrients, SH-BC-893 selectively killed cells expressing an activated form of the anabolic oncogene Ras in vitro and in vivo. However, slower-growing, autochthonous PTEN-deficient prostate tumors that did not exhibit a classic Warburg phenotype were equally sensitive. Remarkably, normal proliferative tissues were unaffected by doses of SH-BC-893 that profoundly inhibited tumor growth. These studies demonstrate that simultaneously blocking parallel nutrient access pathways with sphingolipid-based drugs is broadly effective and cancer selective, suggesting a potential strategy for overcoming the resistance conferred by tumor heterogeneity., Introduction To meet the anabolic demands of cell division, oncogenic mutations drive glucose and glutamine transporter gene expression (1-4). The LDL receptor is similarly upregulated in cancer cells to provide [...]

It has long been assumed that p53 suppresses tumor development through induction of apoptosis, possibly with contributions by cell cycle arrest and cell senescence.sup.1,2. However, combined deficiency in these three processes does not result in spontaneous tumor formation as observed upon loss of p53, suggesting the existence of additional mechanisms that are critical mediators of p53-dependent tumor suppression function.sup.3-5. To define such mechanisms, we performed in vivo shRNA screens targeting p53-regulated genes in sensitized genetic backgrounds. We found that knockdown of Zmat3, Ctsf and Cav1, promoted lymphoma/leukemia development only when PUMA and p21, the critical effectors of p53-driven apoptosis, cell cycle arrest and senescence, were also absent. Notably, loss of the DNA repair gene Mlh1 caused lymphoma in a wild-type background, and its enforced expression was able to delay tumor development driven by loss of p53. Further examination of direct p53 target genes implicated in DNA repair showed that knockdown of Mlh1, Msh2, Rnf144b, Cav1 and Ddit4 accelerated MYC-driven lymphoma development to a similar extent as knockdown of p53. Collectively, these findings demonstrate that extensive functional overlap of several p53-regulated processes safeguards against cancer and that coordination of DNA repair appears to be an important process by which p53 suppresses tumor development. In vivo shRNA screens in sensitized genetic backgrounds identify p53-activated target genes involved in DNA repair that enable its tumor suppressor function., Author(s): Ana Janic [sup.1] [sup.2] , Liz J. Valente [sup.1] [sup.2] [sup.3] , Matthew J. Wakefield [sup.1] [sup.4] , Leon Di Stefano [sup.1] , Liz Milla [sup.1] [sup.2] , Stephen [...]

Tumours often become resistant to treatment, but how this occurs is poorly understood. An analysis of how the protein Rb affects tumour growth and the response to therapy might cast light on the problem. Assessing how the protein Rb affects cancer reveals possible drug targets., Author(s): Seth M. Rubin, Julien Sage Author Affiliations: Manipulating the tumour-suppressor protein Rb in lung cancer reveals possible drug targets The development of cancer invariably involves cellular proteins that have [...]

The tumour-suppressor protein PTEN is mostly found in the cell cytoplasm, tethered to endosome vesicles. This localization regulates the enzyme's activity towards specific lipids and influences its control of cell [...]

The Drosophila eyes are used as model system to find out that elevated Notch signaling in combination with activation of components of the Polycomb complex of transcriptional repressors lead to metastatic growth of tumors through epigenetic silencing of the Rbf gene. Rbf is the Drosophila homologue of the retinoblastoma tumorosuppressor gene (Rb) and it represents a novel link between Notch signaling, tumor growth and metastasis.

The p53 tumor suppressor protein plays a central role in cell cycle regulation, DNA repair, and apoptosis. Recent studies indicate that DNA damage and somatic mutations in the p53 gene can occur because of genotoxic stress in many tissues, including the skin, colon, and synovium. Although somatic mutations in the p53 gene have been demonstrated in rheumatoid arthritis (RA) synovial tissue and synoviocytes, no information is available on the location or extent of p53 mutations. Using microdissected RA synovial tissue sections, we observed abundant p53 transition mutations, which are characteristic DNA damage caused by oxidative stress, p53 mutations, as well as p53 mRNA expression, were located mainly in the synovial intimal lining rather than the sublining (P < 0.01). Clusters of p53 mutant subclones were observed in some microdissected regions, suggesting oligoclonal expansion. Because IL-6 gene expression is regulated by wild-type p53, IL-6 mRNA expression in microdissected tissues was quantified by using real-time PCR. The regions with high rates of p53 mutations contained significantly greater amounts of IL-6 mRNA compared with the low mutation samples (P < 0.02). The microdissection findings suggest that p53 mutations are induced in RA synovial tissues by inflammatory oxidative stress. This process, as in sun-exposed skin and inflamed colonic epithelium, provides some of the mutant clones with a selective growth advantage. A relatively low percentage of cells containing p53 mutations can potentially affect neighboring cells and enhance inflammation through the elaboration of proinflammatory cytokines.

A key tumor suppressor mechanism that is disrupted frequently in human cancer involves the ARF and p53 genes. In mouse fibroblasts, the Arf gene product responds to abnormal mitogenic signals to activate p53 and trigger either cell cycle arrest or apoptosis. Recent evidence indicates that Arf also has p53-independent functions that may contribute to its tumor suppressor activity. Using [Arf.sup.-/-] and [p53.sup.-/-] mice, we have discovered a p53-independent requirement for Arf in the developmental regression of the hyaloid vascular system (HVS) in the mouse eye. Arf is expressed in the vitreous of the eye and is induced before HVS regression in the first postnatal week. In the absence of Arf, failed HVS regression causes a pathological process that resembles persistent hyperplastic primary vitreous, a developmental human eye disease thought to have a genetic basis. These findings demonstrate an essential and unexpected role for Arf during mouse eye development, provide insights into the potential genetic basis for persistent hyperplastic primary vitreous, and indicate that Arf regulates vascular regression in a p53-independent manner. The latter finding raises the possibility that Arf may function as a tumor suppressor at least in part by regulating tumor angiogenesis.

The functional role of p53 in nitric oxide (NO)-mediated vascular smooth muscle cell (VSMC) apoptosis remains unknown. In this study, VSMC from [p53.sup.-/-] and [p53.sup.+/+] murine aortas were exposed to exogenous or endogenous sources of NO. Unexpectedly, [p53.sup.-/-] VSMC were much more sensitive to the proapoptotic effects of NO than were [p53.sup.+/+] VSMC. Furthermore, this paradox appeared to be specific to NO, because other proapoptotic agents did not demonstrate this differential effect on [p53.sup.-/-] cells. NO-induced apoptosis in [p53.sup.-/-] VSMC occurred independently of cGMP generation. However, mitogen-activated protein kinase (MAPK) pathways appeared to play a significant role. Treatment of the [p53.sup.-/-] VSMC with S-nitroso-N-acetylpenicillamine resulted in a marked activation of p38 MAPK and, to a lesser extent, of c-Jun N[H.sub.2]-terminal kinase, mitogen-activated protein kinase kinase (MEK) 1/2, and p42/44 (extracellular signal-regulated kinase, ERK). Furthermore, basal activity of the MEK-p42/44 (ERK) pathway was increased in the [p53.sup.+/+] VSMC. Inhibition of p38 MAPK with SB-203580 or of MEK1/2 with PD-98059 blocked NO-induced apoptosis. Therefore, p53 may protect VSMC against NO-mediated apoptosis, in part, through differential regulation of MAPK pathways. mitogen-activated protein kinase; guanosine 3',5'-cyclic monophosphate, p38; c-Jun N[H.sub.2]-terminal kinase; p42/44

Results demonstrate that Drosophila phosphate and tensin homolog (DPTEN) protein controls cell number and size and mediates subcellular organization of the actin cytoskeleton. Data further indicate that DPTEN modulates tissue mass by inhibiting a protein kinase and the insulin receptor substrate homolog Chico.

Research was conducted to test whether the tumor suppressor gene product , p19(super Arf) hinders oncogenic transformation in primary cells and whether its role is abrogated when p53 is neutralized by viral oncoproteins and dominant-negative mutants. Cell cultures were monitored and maintained by calcium phosphate using the same amounts of DNA as in p53 degradation. Results showed high levels of genetic instability in p53 and that potent tumor suppressor activity resulted from the encoding of unrelated antioncogenic proteins with cooperating modes of action.

The efficacy of chemotherapeutic agents may be determined by a number of different factors, including the genotype of the tumor cell. The p53 tumor suppressor gene frequently is mutated in human tumors, and this may contribute to chemotherapeutic resistance. We tested the requirement for wild-type p53 in the response of tumor cells to treatment with paclitaxel (trade name Taxol), an antineoplastic agent that stabilizes cellular microtubules. Although paclitaxel is broadly effective against human tumor xenografts in mice, including some known to carry p53 mutations, we found that p53-containing mouse tumor cells were significantly more sensitive to direct treatment with this drug than were p53-deficient tumor cells. In an attempt to reconcile this apparent discrepancy, we examined the requirement for p53 in the cytotoxic effects of tumor necrosis factor [Alpha] (TNF-[Alpha]), a cytokine released from murine macrophages upon paclitaxel treatment. Conditioned medium from paclitaxel-treated macrophages was capable of inducing p53-independent apoptosis when applied to transformed mouse embryonic fibroblasts and was inhibitable by antibodies against TNF-[Alpha]. Furthermore, in response to direct treatment with TNF-[Alpha], both wild-type and p53-deficient tumor cells underwent apoptosis to similar extents and with similar kinetics. Our results suggest that the efficacy of paclitaxel in vivo may be due not only to its microtubule-stabilizing activity, but its ability to activate local release of an apoptosis-inducing cytokine.

Protein tyrosine phosphatases (PTPs) have long been thought to play a role in tumor suppression due to their ability to antagonize the growth promoting protein tyrosine kinases. Recently, a candidate tumor suppressor from 10q23, termed P-TEN, was isolated, and sequence homology was demonstrated with members of the PTP family, as well as the cytoskeletal protein tensin. Here we show that recombinant P-TEN dephosphorylated protein and peptide substrates phosphorylated on serine, threonine, and tyrosine residues, indicating that P-TEN is a dual-specificity phosphatase. In addition, P-TEN exhibited a high degree of substrate specificity, showing selectivity for extremely acidic substrates in vitro. Furthermore, we demonstrate that mutations in P-TEN, identified from primary tumors, tumor cells lines, and a patient with Bannayan-Zonana syndrome, resulted in the ablation of phosphatase activity, demonstrating that enzymatic activity of P-TEN is necessary for its ability to function as a tumor suppressor.

The mouse p53 protein generated by alternative splicing (p53as) has amino acid substitutions at its C terminus that result in constitutively active sequence-specific DNA binding (active form), whereas p53 protein itself binds inefficiently (latent form) unless activated by C-terminal modification. Exogenous p53as expression activated transcription of reporter plasmids containing p53 binding sequences and inhibited growth of mouse and human cells lacking functional endogenous p53. Inducible p53as in stably transfected p53 null fibroblasts increased [p21.sup.WAF1/Cip-1/Sdi] and decreased bcl-2 protein steady-state levels. Endogenous p53as and p53 proteins differed in response to cellular DNA damage. p53 protein was induced transiently in normal keratinocytes and fibroblasts whereas p53as protein accumulation was sustained in parallel with induction of [p21.sup.WAF1/Cip-1/Sdi] protein and mRNA, in support of p53as transcriptional activity. Endogenous p53 and p53as proteins in epidermal tumor cells responded to DNA damage with different kinetics of nuclear accumulation and efficiencies of binding to a p53 consensus DNA sequence. A model is proposed in which C-terminally distinct p53 protein forms specialize in functions, with latent p53 forms primarily for rapid non.sequence-specific binding to sites of DNA damage and active p53 forms for sustained regulation of transcription and growth.

Replication protein A (RPA) is required for both DNA replication and nucleotide excision repair. Previous studies have shown that RPA interacts with the tumor sup-pressor p53. Herein, we have mapped a 20-amino acid region in the N-terminal part of p53 that is essential for its binding to RPA. This region is distinct from the minimal activation domain of p53 previously identified. We also demonstrate that UV radiation of cells greatly reduces the ability of RPA to bind to p53. Interestingly, damage-induced hyperphosphorylated RPA does not associate with p53. Furthermore, down-regulation of the RPA/p53 interaction is dependent upon the capability of cells to perform global genome repair. On the basis of these data, we propose that RPA may participate in the coordination of DNA repair with the p53-dependent checkpoint control by sensing UV damage and releasing p53 to activate its downstream targets.