Biomedical subjects

William C Hahn

Publications and source records attributed to William C Hahn.

At least 19 recordsLinked to original sources

PubMed2006

Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis.

DNA-responsive checkpoints prevent cell-cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. Ser287 phosphorylation is a major locus of G2/M checkpoint control, although several checkpoint-independent kinases can phosphorylate this site. We reported previously that mitotic entry requires 14-3-3 removal and Ser287 dephosphorylation. We show here that DNA-responsive checkpoints also activate PP2A/B56delta phosphatase complexes to dephosphorylate Cdc25 at a site distinct from Ser287 (T138), the phosphorylation of which is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. Our data suggest that creation of a 14-3-3 "sink," consisting of phosphorylated 14-3-3 binding intermediate filament proteins, including keratins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56delta as a central checkpoint effector and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis.

14-3-3 Proteins↗

PubMed2006

Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators.

Although androgen receptor (AR)-mediated signaling is central to prostate cancer, the ability to modulate AR signaling states is limited. Here we establish a chemical genomic approach for discovery and target prediction of modulators of cancer phenotypes, as exemplified by AR signaling. We first identify AR activation inhibitors, including a group of structurally related compounds comprising celastrol, gedunin, and derivatives. To develop an in silico approach for target pathway identification, we apply a gene expression-based analysis that classifies HSP90 inhibitors as having similar activity to celastrol and gedunin. Validating this prediction, we demonstrate that celastrol and gedunin inhibit HSP90 activity and HSP90 clients, including AR. Broadly, this work identifies new modes of HSP90 modulation through a gene expression-based strategy.

Antibiotics, Antineoplastic↗

PubMed2006

Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9.

Leukaemias and other cancers possess a rare population of cells capable of the limitless self-renewal necessary for cancer initiation and maintenance. Eradication of these cancer stem cells is probably a critical part of any successful anti-cancer therapy, and may explain why conventional cancer therapies are often effective in reducing tumour burden, but are only rarely curative. Given that both normal and cancer stem cells are capable of self-renewal, the extent to which cancer stem cells resemble normal tissue stem cells is a critical issue if targeted therapies are to be developed. However, it remains unclear whether cancer stem cells must be phenotypically similar to normal tissue stem cells or whether they can retain the identity of committed progenitors. Here we show that leukaemia stem cells (LSC) can maintain the global identity of the progenitor from which they arose while activating a limited stem-cell- or self-renewal-associated programme. We isolated LSC from leukaemias initiated in committed granulocyte macrophage progenitors through introduction of the MLL-AF9 fusion protein encoded by the t(9;11)(p22;q23). The LSC were capable of transferring leukaemia to secondary recipient mice when only four cells were transferred, and possessed an immunophenotype and global gene expression profile very similar to that of normal granulocyte macrophage progenitors. However, a subset of genes highly expressed in normal haematopoietic stem cells was re-activated in LSC. LSC can thus be generated from committed progenitors without widespread reprogramming of gene expression, and a leukaemia self-renewal-associated signature is activated in the process. Our findings define progression from normal progenitor to cancer stem cell, and suggest that targeting a self-renewal programme expressed in an abnormal context may be possible.

Animals↗

PubMed2006

Comparative oncogenomics identifies NEDD9 as a melanoma metastasis gene.

Genomes of human cancer cells are characterized by numerous chromosomal aberrations of uncertain pathogenetic significance. Here, in an inducible mouse model of melanoma, we characterized metastatic variants with an acquired focal chromosomal amplification that corresponds to a much larger amplification in human metastatic melanomas. Further analyses identified Nedd9, an adaptor protein related to p130CAS, as the only gene within the minimal common region that exhibited amplification-associated overexpression. A series of functional, biochemical, and clinical studies established NEDD9 as a bona fide melanoma metastasis gene. NEDD9 enhanced invasion in vitro and metastasis in vivo of both normal and transformed melanocytes, functionally interacted with focal adhesion kinase and modulated focal contact formation, and exhibited frequent robust overexpression in human metastatic melanoma relative to primary melanoma. Thus, comparative oncogenomics has enabled the identification and facilitated the validation of a highly relevant cancer gene governing metastatic potential in human melanoma.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Androgen-dependent regulation of Her-2/neu in prostate cancer cells.

The mechanisms underlying the progression of prostate cancer to a state of resistance to hormone ablation remain poorly understood. Here, we have investigated the relationship between androgen receptor (AR) and Her-2/neu in prostate cancer cells. Overexpression of Her-2/neu (c-ErbB2) activates the AR pathway and confers a survival and growth advantage to prostate cancer cells in an androgen-deficient milieu. In vitro, the absence of androgens or AR blockade induced Her-2/neu protein expression and phosphorylation. In contrast, upon readministration of androgens, Her-2/neu mRNA, protein, and phosphorylation levels decreased linearly with increasing concentrations of dihydrotestosterone as LNCaP cells reentered the cell cycle. In vivo, induction of Her-2/neu by castration in orthotopically injected LNCaP cells resulted in a progressive increase in prostate-specific antigen secretion into the mouse serum, indicating that Her-2/neu-mediated, AR-dependent transcription occurs following castration and results in tumor cell growth. Finally, selection of LNCaP cells stably transfected with short hairpin RNA specific for AR resulted in Her-2/neu overexpression. Similarly, knockdown of Her-2/neu led to induction of AR. However, when Her-2/neu and AR were simultaneously targeted, we observed cell death, whereas surviving cells retained low level expression of Her-2/neu. Thus, induction and activation of Her-2/neu occurs in an androgen-depleted environment or as a result of AR inactivation, promoting ablation-resistant survival of prostate cancer cells. These data provide the biochemical rationale to target Her-2/neu in hormone-refractory prostate cancer.

Androgen Receptor Antagonists↗

PubMed2006

PP2A regulates BCL-2 phosphorylation and proteasome-mediated degradation at the endoplasmic reticulum.

Anti-apoptotic activity of BCL-2 is mediated by phosphorylation at the endoplasmic reticulum (ER), but how this phosphorylation is regulated and the mechanism(s) by which it regulates apoptosis are unknown. We purified macromolecular complexes containing BCL-2 from ER membranes and found that BCL-2 co-purified with the main two subunits of the serine/threonine phosphatase, PP2A. The association of endogenous PP2A and BCL-2 at the ER was verified by co-immunoprecipitation and microcystin affinity purification. Knock down or pharmacological inhibition of PP2A caused degradation of phosphorylated BCL-2 and led to an overall reduction in BCL-2 levels. We found that this degradation was due to the action of the proteasome acting selectively at the ER. Conversely, overexpression of PP2A caused elevation in endogenous BCL-2. Most importantly, we found that PP2A knock down sensitized cells to several classes of death stimuli (including ER stress), but this effect was abolished in a genetic background featuring knock in of a non-phosphorylatable BCL-2 allele. These studies support the hypothesis that PP2A-mediated dephosphorylation of BCL-2 is required to protect BCL-2 from proteasome-dependent degradation, affecting resistance to ER stress.

Amino Acid Sequence↗

PubMed2006

A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen.

To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.

Animals↗

PubMed2006

Roots and stems: stem cells in cancer.

Cancer develops from normal tissues through the accumulation of genetic alterations that act in concert to confer malignant phenotypes. Although we have now identified some of the genes that when mutated initiate tumor formation and drive cancer progression, the identity of the cell population(s) susceptible to such transforming events remains undefined for the majority of human cancers. Recent work indicates that a small population of cells endowed with unique self-renewal properties and tumorigenic potential is present in some, and perhaps all, tumors. Although our understanding of the biology of these putative cancer stem cells remains rudimentary, the existence of such cells has implications for current conceptualizations of malignant transformation and therapeutic approaches to cancer.

Cell Transformation, Neoplastic↗

PubMed2006

Minimizing the risk of reporting false positives in large-scale RNAi screens.

Large-scale RNA interference (RNAi)-based analyses, very much as other 'omic' approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens.

Databases, Genetic↗

PubMed2006

Genome-scale loss-of-function screening with a lentiviral RNAi library.

The discovery that RNA interference (RNAi) is functional in mammalian cells led us to form The RNAi Consortium (TRC) with the goal of enabling large-scale loss-of-function screens through the development of genome-scale RNAi libraries and methodologies for their use. These resources form the basis of a loss-of-function screening platform created at the Broad Institute. Our human and mouse libraries currently contain >135,000 lentiviral clones targeting 27,000 genes. Initial screening efforts have demonstrated that these libraries and methods are practical and powerful tools for high-throughput lentivirus RNAi screens.

Animals↗

PubMed2005

Involvement of PP2A in viral and cellular transformation.

Although the small DNA tumor virus SV40 (simian virus 40) fails to replicate in human cells, understanding how SV40 transforms human and murine cells has and continues to provide important insights into cancer initiation and maintenance. The early region of SV40 encodes two oncoproteins: the large T (LT) and small t (ST) antigens. SV40 LT contributes to murine and human cell transformation in part by inactivating the p53 and retinoblastoma protein tumor suppressor proteins. SV40 ST inhibits the activity of the protein phosphatase 2A (PP2A) family of serine-threonine phosphatases, and this interaction is required for SV40-mediated transformation of human cells. PP2A regulates multiple signaling pathways, suggesting many possible targets important for viral replication and cell transformation. Genetic manipulation of particular PP2A subunits has confirmed a role for specific complexes in transformation, and recent work implicates the perturbation of the phosphatidylinositol 3-kinase/Akt pathway and c-Myc stability in transformation by ST and PP2A. Mutations in PP2A subunits occur at low frequency in human tumors, suggesting that alterations of PP2A signaling play a role in both experimentally induced and spontaneously arising cancers. Unraveling the complexity of PP2A signaling will not only provide further insights into cancer development but may identify novel targets with promise for therapeutic manipulation.

Cell Transformation, Neoplastic↗

PubMed2005

Creating oral squamous cancer cells: a cellular model of oral-esophageal carcinogenesis.

Immortalization and malignant transformation are important steps in tumor development. The ability to induce these processes from normal human epithelial cells with genetic alterations frequently found in the corresponding human cancer would significantly enhance our understanding of tumor development. Alterations in several key intracellular regulatory pathways (the pRB, p53, and mitogenic signaling pathways and the telomere maintenance system) appear to be sufficient for the neoplastic transformation of normal human cells. Nevertheless, in vitro transformation models to date depend on viral oncogenes, most prominently the simian virus 40 early region, to induce immortalization and malignant transformation of normal human epithelial cells. Here, we demonstrate a transformation model creating oral-esophageal cancer cells by using a limited set of genetic alterations frequently observed in the corresponding human cancer. In a stepwise model, cyclin D1 overexpression and p53 inactivation led to immortalization of oral keratinocytes. Additional ectopic epithelial growth factor receptor overexpression followed by c-myc overexpression as well as consecutive reactivation of telomerase induced by epithelial growth factor receptor sufficed to transform oral epithelial cells, truly recapitulating the development of the corresponding human disease.

Carcinoma, Squamous Cell↗

PubMed2005

Oncogenic transformation by inhibitor-sensitive and -resistant EGFR mutants.

BACKGROUND: Somatic mutations in the kinase domain of the epidermal growth factor receptor tyrosine kinase gene EGFR are common in lung adenocarcinoma. The presence of mutations correlates with tumor sensitivity to the EGFR inhibitors erlotinib and gefitinib, but the transforming potential of specific mutations and their relationship to drug sensitivity have not been described. METHODS AND FINDINGS: Here, we demonstrate that EGFR active site mutants are oncogenic. Mutant EGFR can transform both fibroblasts and lung epithelial cells in the absence of exogenous epidermal growth factor, as evidenced by anchorage-independent growth, focus formation, and tumor formation in immunocompromised mice. Transformation is associated with constitutive autophosphorylation of EGFR, Shc phosphorylation, and STAT pathway activation. Whereas transformation by most EGFR mutants confers on cells sensitivity to erlotinib and gefitinib, transformation by an exon 20 insertion makes cells resistant to these inhibitors but more sensitive to the irreversible inhibitor CL-387,785. CONCLUSION: Oncogenic transformation of cells by different EGFR mutants causes differential sensitivity to gefitinib and erlotinib. Treatment of lung cancers harboring EGFR exon 20 insertions may therefore require the development of alternative kinase inhibition strategies.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Cancer-associated PP2A Aalpha subunits induce functional haploinsufficiency and tumorigenicity.

The introduction of SV40 small t antigen or the suppression of PP2A B56gamma subunit expression contributes to the experimental transformation of human cells. To investigate the role of cancer-associated PP2A Aalpha subunit mutants in transformation, we introduced several PP2A Aalpha mutants into immortalized but nontumorigenic human cells. These PP2A Aalpha mutants exhibited defects in binding to other PP2A subunits and impaired phosphatase activity. Although overexpression of these mutants failed to render immortalized cells tumorigenic, partial suppression of endogenous PP2A Aalpha expression activated the AKT pathway and permitted cells to form tumors in immunodeficient mice. These findings suggest that cancer-associated Aalpha mutations contribute to cancer development by inducing functional haploinsufficiency, disturbing PP2A holoenzyme composition, and altering the enzymatic activity of PP2A.

Amino Acid Sequence↗

PubMed2005

The telomerase reverse transcriptase regulates chromatin state and DNA damage responses.

Constitutive expression of telomerase prevents senescence and crisis by maintaining telomere homeostasis. However, recent evidence suggests that telomerase is dynamically regulated in normal cells and also contributes to transformation independently of net telomere elongation. Here, we show that suppression of the telomerase catalytic subunit [human telomerase reverse transcriptase (hTERT)] expression abrogates the cellular response to DNA double strand breaks. Loss of hTERT does not alter short-term telomere integrity but instead affects the overall configuration of chromatin. Cells lacking hTERT exhibit increased radiosensitivity, diminished capacity for DNA repair, and fragmented chromosomes, demonstrating that loss of hTERT impairs the DNA damage response.

Cell Line↗

PubMed2005

Analysis of telomeric single-strand overhang length in human endometrial cancers.

The 3' single-strand telomeric overhang (3'-OH) is a key component of telomere structure. Although telomere length has been well analyzed in a variety of human cancers, no information is available on the 3'-OH length in cancers. In the present study, we examined the 3'-OH length in normal and malignant endometria using telomere-oligonucleotide ligation assay. Although 3'-OH lengths varied among patients, 3'-OH length observed in endometrial cancers was significantly shorter than that found in samples derived from normal endometria (P < 0.001: Student's t-test), suggesting that erosion of 3'-OH length induces impaired telomeric integrity and genomic instability, leading to carcinogenesis. Interestingly, we found that the most aggressive subtypes of endometrial cancers harbored significantly longer 3'-OH length than those with non-aggressive subtypes (P < 0.001: Sheffe's test), suggesting that cancer cells with long 3'-OH length have growth advantage due to their stabilized telomere ends. In contrast, we failed to observe an association between overall telomere length and any clinicopathological characteristics of endometrial cancers. These findings suggest that erosion of 3'-OH length, rather than overall telomere length, play roles in endometrial carcinogenesis. Furthermore, long 3'-OH may serve as a molecular marker for aggressive phenotype of tumors.

Blotting, Southern↗

PubMed2005

Telomerase: regulation, function and transformation.

Work from several laboratories over the past decade indicates that the acquisition of constitutive telomerase expression is a critical step during the malignant transformation of human cells. Normal human cells transiently express low levels of telomerase, the ribonucleoprotein responsible for extending and maintaining telomeres, and exhibit telomere shortening after extended passage, whereas most cancers exhibit constitutive telomerase expression and maintain telomeres at stable lengths. These observations establish a direct connection between immortalization and stabilization of telomere structure. However, recent work suggests that telomerase also contributes to cancer development beyond its role in maintaining stable telomere lengths. In this review, we summarize recent observations that support the concept that telomerase plays multiple roles in facilitating human cell transformation.

Cell Transformation, Neoplastic↗

PubMed2005

The Cdk1 complex plays a prime role in regulating N-myc phosphorylation and turnover in neural precursors.

Myc family transcription factors are destabilized by phosphorylation of a conserved amino-terminal GSK-3beta motif. In proliferating cerebellar granule neuron precursors (CGNPs), Sonic hedgehog signaling induces N-myc expression, and N-myc protein is stabilized by insulin-like growth factor-mediated suppression of GSK-3beta. N-myc phosphorylation-mediated degradation is a prerequisite for CGNP growth arrest and differentiation. We investigated whether N-myc phosphorylation and turnover are thus linked to cell cycle exit in primary mouse CGNP cultures and the developing cerebellum. We report that phosphorylation-induced turnover of endogenous N-myc protein in CGNPs increases during mitosis, due to increased priming phosphorylation of N-myc for GSK-3beta. The priming phosphorylation requires the Cdk1 complex, whose cyclin subunits are indirect Sonic hedgehog targets. These findings provide a mechanism for promoting growth arrest in the final cycle of neural precursor proliferation competency, or for resetting the cell cycle in the G1 phase, by destabilizing N-myc in mitosis.

Animals↗