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Yaoyu Chen,
Jinyun Chen,
Alice Loo,
Savina Jaeger,
Linda Bagdasarian,
Jianjun Yu,
Franklin Chung,
Joshua Korn,
David Ruddy,
Ribo Guo,
Margaret E McLaughlin,
Fei Feng,
Ping Zhu, Frank Stegmeier,
Raymond Pagliarini,
Dale Porter,
Wenlai Zhou
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ABSTRACT: The molecular chaperone heat shock protein 90 (HSP90) facilitates the appropriate folding of various oncogenic proteins and is necessary for the survival of some cancer cells. HSP90 is therefore an attractive drug target, but the efficacy of HSP90 inhibitor may be limited by HSP90 inhibition induced feedback mechanisms. Through pooled RNA interference screens, we identified that heat shock factor 1(HSF1) is a sensitizer of HSP90 inhibitor. A striking combinational effect was observed when HSF1 knockdown plus with HSP90 inhibitors treatment in various cancer cell lines and tumor mouse models. Interestingly, HSF1 is highly expressed in hepatocellular carcinoma (HCC) patient samples and HCC is sensitive to combinational treatment, indicating a potential indication for the combinational treatment. To understand the mechanism of the combinational effect, we identified that a HSF1-target gene DEDD2 is involved in attenuating the effect of HSP90 inhibitors. Thus, the transcriptional activities of HSF1 induced by HSP90 inhibitors provide a feedback mechanism of limiting the HSP90 inhibitor's activity, and targeting HSF1 may provide a new avenue to enhance HSP90 inhibitors activity in human cancers.
Oncotarget 04/2013; · 4.78 Impact Factor
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Alix Scholer-Dahirel,
Michael R Schlabach,
Alice Loo,
Linda Bagdasarian,
Ronald Meyer,
Ribo Guo,
Steve Woolfenden,
Kristine K Yu,
Judit Markovits,
Karen Killary,
Dmitry Sonkin,
Yung-Mae Yao,
Markus Warmuth,
William R Sellers,
Robert Schlegel, Frank Stegmeier,
Rebecca E Mosher,
Margaret E McLaughlin
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ABSTRACT: Persistent expression of certain oncogenes is required for tumor maintenance. This phenotype is referred to as oncogene addiction and has been clinically validated by anticancer therapies that specifically inhibit oncoproteins such as BCR-ABL, c-Kit, HER2, PDGFR, and EGFR. Identifying additional genes that are required for tumor maintenance may lead to new targets for anticancer drugs. Although the role of aberrant Wnt pathway activation in the initiation of colorectal cancer has been clearly established, it remains unclear whether sustained Wnt pathway activation is required for colorectal tumor maintenance. To address this question, we used inducible β-catenin shRNAs to temporally control Wnt pathway activation in vivo. Here, we show that active Wnt/β-catenin signaling is required for maintenance of colorectal tumor xenografts harboring APC mutations. Reduced tumor growth upon β-catenin inhibition was due to cell cycle arrest and differentiation. Upon reactivation of the Wnt/β-catenin pathway colorectal cancer cells resumed proliferation and reacquired a crypt progenitor phenotype. In human colonic adenocarcinomas, high levels of nuclear β-catenin correlated with crypt progenitor but not differentiation markers, suggesting that the Wnt/β-catenin pathway may also control colorectal tumor cell fate during the maintenance phase of tumors in patients. These results support efforts to treat human colorectal cancer by pharmacological inhibition of the Wnt/β-catenin pathway.
Proceedings of the National Academy of Sciences 09/2011; 108(41):17135-40. · 9.68 Impact Factor
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Tara L Naylor,
Huaping Tang,
Boris A Ratsch,
Andreas Enns,
Alice Loo,
Liqing Chen,
Peter Lenz,
Nigel J Waters,
Walter Schuler,
Bernd Dörken,
Yung-Mae Yao,
Markus Warmuth,
Georg Lenz, Frank Stegmeier
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ABSTRACT: The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis. The ABC subtype of DLBCL is associated with constitutive activation of the NF-κB pathway, and oncogenic lesions have been identified in its regulators, including CARD11/CARMA1 (caspase recruitment domain-containing protein 11), A20/TNFAIP3, and CD79A/B. In this study, we offer evidence of therapeutic potential for the selective PKC (protein kinase C) inhibitor sotrastaurin (STN) in preclinical models of DLBCL. A significant fraction of ABC DLBCL cell lines exhibited strong sensitivity to STN, and we found that the molecular nature of NF-κB pathway lesions predicted responsiveness. CD79A/B mutations correlated with STN sensitivity, whereas CARD11 mutations rendered ABC DLBCL cell lines insensitive. Growth inhibitory effects of PKC inhibition correlated with NF-κB pathway inhibition and were mediated by induction of G₁-phase cell-cycle arrest and/or cell death. We found that STN produced significant antitumor effects in a mouse xenograft model of CD79A/B-mutated DLBCL. Collectively, our findings offer a strong rationale for the clinical evaluation of STN in ABC DLBCL patients who harbor CD79 mutations also illustrating the necessity to stratify DLBCL patients according to their genetic abnormalities.
Cancer Research 02/2011; 71(7):2643-53. · 7.86 Impact Factor
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ABSTRACT: The spliceosome, a dynamic assembly of proteins and RNAs, catalyzes the excision of intron sequences from nascent mRNAs. Recent work has suggested that the activity and composition of the spliceosome are regulated by ubiquitination, but the underlying mechanisms have not been elucidated. Here, we report that the spliceosomal Prp19 complex modifies Prp3, a component of the U4 snRNP, with nonproteolytic K63-linked ubiquitin chains. The K63-linked chains increase the affinity of Prp3 for the U5 snRNP component Prp8, thereby allowing for the stabilization of the U4/U6.U5 snRNP. Prp3 is deubiquitinated by Usp4 and its substrate targeting factor, the U4/U6 recycling protein Sart3, which likely facilitates ejection of U4 proteins from the spliceosome during maturation of its active site. Loss of Usp4 in cells interferes with the accumulation of correctly spliced mRNAs, including those for alpha-tubulin and Bub1, and impairs cell cycle progression. We propose that the reversible ubiquitination of spliceosomal proteins, such as Prp3, guides rearrangements in the composition of the spliceosome at distinct steps of the splicing reaction.
Genes & development 07/2010; 24(13):1434-47. · 12.08 Impact Factor
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Shih-Min A Huang,
Yuji M Mishina,
Shanming Liu,
Atwood Cheung, Frank Stegmeier,
Gregory A Michaud,
Olga Charlat,
Elizabeth Wiellette,
Yue Zhang,
Stephanie Wiessner, [......],
Chris Lu,
Daniel Curtis,
Marc W Kirschner,
Christoph Lengauer,
Peter M Finan,
John A Tallarico,
Tewis Bouwmeester,
Jeffery A Porter,
Andreas Bauer,
Feng Cong
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ABSTRACT: The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.
Nature 09/2009; 461(7264):614-20. · 36.28 Impact Factor
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ABSTRACT: The RAS pathway is one of the most frequently deregulated pathways in cancer. RAS signals through multiple effector pathways, including the RAF/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAPK and phosphatidylinositol 3-kinase (PI3K)-AKT signaling cascades. The oncogenic potential of these effector pathways is illustrated by the frequent occurrence of activating mutations in BRAF and PIK3CA as well as loss-of-function mutations in the tumor suppressor PTEN, a negative regulator of PI3K. Previous studies have found that whereas BRAF mutant cancers are highly sensitive to MEK inhibition, RAS mutant cancers exhibit a more variable response. The molecular mechanisms responsible for this heterogeneous response remain unclear. In this study, we show that PI3K pathway activation strongly influences the sensitivity of RAS mutant cells to MEK inhibitors. Activating mutations in PIK3CA reduce the sensitivity to MEK inhibition, whereas PTEN mutations seem to cause complete resistance. We further show that down-regulation of PIK3CA resensitizes cells with co-occurring KRAS and PIK3CA mutations to MEK inhibition. At the molecular level, the dual inhibition of both pathways seems to be required for complete inhibition of the downstream mammalian target of rapamycin effector pathway and results in the induction of cell death. Finally, we show that whereas inactivation of either the MEK or PI3K pathway leads to partial tumor growth inhibition, targeted inhibition of both pathways is required to achieve tumor stasis. Our study provides molecular insights that help explain the heterogeneous response of KRAS mutant cancers to MEK pathway inhibition and presents a strong rationale for the clinical testing of combination MEK and PI3K targeted therapies.
Cancer Research 05/2009; 69(10):4286-93. · 7.86 Impact Factor
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ABSTRACT: Deregulation of the PI3K signaling pathway is observed in many human cancers and occurs most frequently through loss of PTEN phosphatase tumor suppressor function or through somatic activating mutations in the Class IA PI3K, PIK3CA. Tumors harboring activated p110alpha, the protein product of PIK3CA, require p110alpha activity for growth and survival and hence are expected to be responsive to inhibitors of its lipid kinase activity. Whether PTEN-deficient cancers similarly depend on p110alpha activity to sustain activation of the PI3K pathway has been unclear. In this study, we used a single-vector lentiviral inducible shRNA system to selectively inactivate the three Class IA PI3Ks, PIK3CA, PIK3CB, and PIK3CD, to determine which PI3K isoforms are responsible for driving the abnormal proliferation of PTEN-deficient cancers. Down-regulation of PIK3CA in colorectal cancer cells harboring mutations in PIK3CA inhibited downstream PI3K signaling and cell growth. Surprisingly, PIK3CA depletion affected neither PI3K signaling nor cell growth in 3 PTEN-deficient cancer cell lines. In contrast, down-regulation of the PIK3CB isoform, which encodes p110beta, resulted in pathway inactivation and subsequent inhibition of growth in both cell-based and in vivo settings. This essential function of PIK3CB in PTEN-deficient cancer cells required its lipid kinase activity. Our findings demonstrate that although p110alpha activation is required to sustain the proliferation of established PIK3CA-mutant tumors, PTEN-deficient tumors are dependent instead on p110beta signaling. This unexpected finding demonstrates the need to tailor therapeutic approaches to the genetic basis of PI3K pathway activation to achieve optimal treatment response.
Proceedings of the National Academy of Sciences 10/2008; 105(35):13057-62. · 9.68 Impact Factor
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ABSTRACT: Defects in chromosome-microtubule attachment trigger spindle-checkpoint activation and delay mitotic progression. How microtubule attachment is sensed and integrated into the steps of checkpoint-signal amplification is poorly understood. In a functional genomic screen targeting human kinases and phosphatases, we identified a microtubule affinity-regulating kinase kinase, TAO1 (also known as MARKK) as an important regulator of mitotic progression, required for both chromosome congression and checkpoint-induced anaphase delay. TAO1 interacts with the checkpoint kinase BubR1 and promotes enrichment of the checkpoint protein Mad2 at sites of defective attachment, providing evidence for a regulatory step that precedes the proposed Mad2-Mad1 dependent checkpoint-signal amplification step. We propose that the dual functions of TAO1 in regulating microtubule dynamics and checkpoint signalling may help to coordinate the establishment and monitoring of correct congression of chromosomes, thereby protecting genomic stability in human cells.
Nature Cell Biology 06/2007; 9(5):556-64. · 19.49 Impact Factor
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ABSTRACT: Mutations in the cylindromatosis (CYLD) gene cause benign tumors of skin appendages, referred to as cylindromas. The CYLD gene encodes a deubiquitinating enzyme that removes Lys-63-linked ubiquitin chains from I kappa B kinase signaling components and thereby inhibits NF-kappaB pathway activation. The dysregulation of NF-kappaB activity has been proposed to promote cell transformation in part by increasing apoptosis resistance, but it is not clear whether this is CYLD's only or predominant tumor-suppressing function. Here, we show that CYLD is also required for timely entry into mitosis. Consistent with a cell-cycle regulatory function, CYLD localizes to microtubules in interphase and the midbody during telophase, and its protein levels decrease as cells exit from mitosis. We identified the protein kinase Plk1 as a potential target of CYLD in the regulation of mitotic entry, based on their physical interaction and similar loss-of-function and overexpression phenotypes. Our findings raise the possibility that, as with other genes regulating tumorigenesis, CYLD has not only tumor-suppressing (apoptosis regulation) but also tumor-promoting activities (enhancer of mitotic entry). We propose that this additional function of CYLD could provide an explanation for the benign nature of most cylindroma lesions.
Proceedings of the National Academy of Sciences 06/2007; 104(21):8869-74. · 9.68 Impact Factor
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Frank Stegmeier,
Michael Rape,
Viji M. Draviam,
Grzegorz Nalepa,
Mathew E. Sowa,
Xiaolu L. Ang,
E. Robert McDonald III,
Mamie Z. Li,
Gregory J. Hannon,
Peter K. Sorger,
Marc W. Kirschner,
J. Wade Harper,
Stephen J. Elledge
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ABSTRACT: The spindle checkpoint prevents chromosome mis-segregation by delaying sister chromatid separation until all chromosomes have achieved bipolar attachment to the mitotic spindle. Its operation is essential for accurate chromosome segregation, whereas its dysregulation can contribute to birth defects and tumorigenesis. The target of the spindle checkpoint is the anaphase-promoting complex (APC), a ubiquitin ligase that promotes sister chromatid separation and progression to anaphase. Using a short hairpin RNA screen targeting components of the ubiquitin-proteasome pathway in human cells, we identified the deubiquitinating enzyme USP44 (ubiquitin-specific protease 44) as a critical regulator of the spindle checkpoint. USP44 is not required for the initial recognition of unattached kinetochores and the subsequent recruitment of checkpoint components. Instead, it prevents the premature activation of the APC by stabilizing the APC-inhibitory Mad2–Cdc20 complex. USP44 deubiquitinates the APC coactivator Cdc20 both in vitro and in vivo, and thereby directly counteracts the APC-driven disassembly of Mad2–Cdc20 complexes (discussed in an accompanying paper). Our findings suggest that a dynamic balance of ubiquitination by the APC and deubiquitination by USP44 contributes to the generation of the switch-like transition controlling anaphase entry, analogous to the way that phosphorylation and dephosphorylation of Cdk1 by Wee1 and Cdc25 controls entry into mitosis.
Nature 04/2007; 446(7138):876-881. · 36.28 Impact Factor
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ABSTRACT: The synthetic multivulva (synMuv) genes negatively regulate Ras-mediated vulval induction in the nematode Caenorhabditis elegans. The synMuv genes define three classes, A, B, and C, such that double mutants carrying mutations in genes of any two classes are multivulva. The class B synMuv genes include lin-35, a homolog of the retinoblastoma (Rb) tumor suppressor gene, as well as homologs of genes that function with Rb in transcriptional regulation. We screened for additional synMuv mutations using a strategy different from that of previous synMuv genetic screens. Some of the mutations we recovered affect new synMuv genes. We present criteria for assigning synMuv mutations into different genetic classes. We also describe the molecular characterization of the class B synMuv gene lin-65.
Genetics 07/2006; 173(2):709-26. · 4.01 Impact Factor
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ABSTRACT: The advent of RNA interference has led to the ability to interfere with gene expression and greatly expanded our ability to perform genetic screens in mammalian cells. The expression of short hairpin RNA (shRNA) from polymerase III promoters can be encoded in transgenes and used to produce small interfering RNAs that down-regulate specific genes. In this study, we show that polymerase II-transcribed shRNAs display very efficient knockdown of gene expression when the shRNA is embedded in a microRNA context. Importantly, our shRNA expression system [called PRIME (potent RNA interference using microRNA expression) vectors] allows for the multicistronic cotranscription of a reporter gene, thereby facilitating the tracking of shRNA production in individual cells. Based on this system, we developed a series of lentiviral vectors that display tetracycline-responsive knockdown of gene expression at single copy. The high penetrance of these vectors will facilitate genomewide loss-of-function screens and is an important step toward using bar-coding strategies to follow loss of specific sequences in complex populations.
Proceedings of the National Academy of Sciences 10/2005; 102(37):13212-7. · 9.68 Impact Factor
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ABSTRACT: Chromosome segregation is triggered by the cleavage of cohesins by separase. Here we show that in budding yeast separation of the ribosomal DNA (rDNA) and telomeres also requires Cdc14, a protein phosphatase known for its role in mitotic exit. Cdc14 shares this role with the FEAR network, which activates Cdc14 during early anaphase, but not the mitotic exit network, which promotes Cdc14 activity during late anaphase. We further show that CDC14 is necessary and sufficient to promote condensin enrichment at the rDNA locus and to trigger rDNA segregation in a condensin-dependent manner. We propose that Cdc14 released by the FEAR network mediates the partitioning of rDNA by facilitating the localization of condensin thereto. This dual role of the FEAR network in initiating mitotic exit and promoting chromosome segregation ensures that exit from mitosis is coupled to the completion of chromosome segregation.
Cell 06/2004; 117(4):455-69. · 32.40 Impact Factor
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ABSTRACT: The protein phosphatase Cdc14 is a key regulator of exit from mitosis in budding yeast. Its activation during anaphase is characterized by dissociation from its inhibitor Cfi1/Net1 in the nucleolus and is controlled by two regulatory networks. The Cdc14 early anaphase release (FEAR) network promotes activation of the phosphatase during early anaphase, whereas the mitotic exit network (MEN) activates Cdc14 during late stages of anaphase.
Here we investigate how the FEAR network component Spo12 regulates Cdc14 activation. We identify the replication fork block protein Fob1 as a Spo12-interacting factor. Inactivation of FOB1 leads to premature release of Cdc14 from the nucleolus in metaphase-arrested cells. Conversely, high levels of FOB1 delay the release of Cdc14 from the nucleolus. Fob1 associates with Cfi1/Net1, and consistent with this observation, we find that the bulk of Cdc14 localizes to the Fob1 binding region within the rDNA repeats. Finally, we show that Spo12 phosphorylation is cell cycle regulated and affects its binding to Fob1.
Fob1 functions as a negative regulator of the FEAR network. We propose that Fob1 helps to prevent the dissociation of Cdc14 from Cfi1/Net1 prior to anaphase and that Spo12 activation during early anaphase promotes the release of Cdc14 from its inhibitor by antagonizing Fob1 function.
Current Biology 04/2004; 14(6):467-80. · 9.65 Impact Factor
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ABSTRACT: Completion of the cell cycle requires the temporal and spatial coordination of chromosome segregation with mitotic spindle disassembly and cytokinesis. In budding yeast, the protein phosphatase Cdc14 is a key regulator of these late mitotic events. Here, we review the functions of Cdc14 and how this phosphatase is regulated to accomplish the coupling of mitotic processes. We also discuss the function and regulation of Cdc14 in other eukaryotes, emphasizing conserved features.
Annual Review of Genetics 02/2004; 38:203-32. · 22.23 Impact Factor
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ABSTRACT: In budding yeast, the protein phosphatase Cdc14 controls exit from mitosis. Its activity is regulated by a competitive inhibitor Cfi1/Net1, which binds to and sequesters Cdc14 in the nucleolus. During anaphase, Cdc14 is released from its inhibitor by the action of two regulatory networks. The Cdc Fourteen Early Anaphase Release (FEAR) network initiates Cdc14 release from Cfi1/Net1 during early anaphase, and the Mitotic Exit Network (MEN) promotes Cdc14 release during late anaphase. Here, we investigate the relationship among FEAR network components and propose an order in which they function to promote Cdc14 release from the nucleolus. Furthermore, we examine the role of the protein kinase Cdc5, which is a component of both the FEAR network and the MEN, in Cdc14 release from the nucleolus. We find that overexpression of CDC5 led to Cdc14 release from the nucleolus in S phase-arrested cells, which correlated with the appearance of phosphorylated forms of Cdc14 and Cfi1/Net1. Cdc5 promotes Cdc14 phosphorylation and, by stimulating the MEN, Cfi1/Net1 phosphorylation. Furthermore, we suggest that Cdc14 release from the nucleolus only occurs when Cdc14 and Cfi1/Net1 are both phosphorylated.
Molecular Biology of the Cell 12/2003; 14(11):4486-98. · 4.94 Impact Factor
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ABSTRACT: In budding yeast, the phosphatase Cdc14, a key regulator of exit from mitosis, is released from its inhibitor Cfi1/Net1 in the nucleolus during anaphase. A signaling cascade, known as the mitotic exit network (MEN), controls this release. We have identified a regulatory network, the FEAR (Cdc fourteen early anaphase release) network that promotes Cdc14 release from the nucleolus during early anaphase. The FEAR network is comprised of the polo kinase Cdc5, the separase Esp1, the kinetochore-associated protein Slk19, and Spo12. We also show that the FEAR network initiates Cdc14 release from Cfi1/Net1 during early anaphase, and MEN maintains Cdc14 in the released state during late anaphase. We propose that one function of Cdc14 released by the FEAR network is to stimulate MEN activity.
Cell 02/2002; 108(2):207-20. · 32.40 Impact Factor
