User Publications
Published works from users of human isogenic cell-line pairs (X-MAN lines) engineered using adeno-associated viral (AAV) vectors (GENESIS).
Genetic inactivation of AKT1, AKT2, and PDPK1 in human colorectal cancer cells clarifies their roles in tumor growth regulation
Proc Natl Acad Sci U S A. 2010 Feb 9;107(6):2598-603. Epub 2010 Jan 20.
Phosphotidylinositol-3-kinase (PI3K) signaling is altered in the majority of human cancers. To gain insight into the roles of members of this pathway in growth regulation, we inactivated AKT1, AKT2, or PDPK1 genes by targeted homologous recombination in human colon cancer cell lines. Knockout of either AKT1 or AKT2 had minimum effects on cell growth or downstream signaling. In contrast, knockout of both AKT1 and AKT2 resulted in markedly reduced proliferation in vitro when growth factors were limiting and severely affected experimental metastasis in mice. Unexpectedly, AKT1 and AKT2 appeared to regulate growth through FOXO proteins, but not through either GSK3beta or mTOR. In contrast, inactivation of PDPK1 affected GSK3beta and mTOR activation. These findings show that the PI3K signaling pathway is wired differently in human cancer cells than in other cell types or organisms, which has important implications for the design and testing of drugs that target this pathway.
Knock in of the AKT1 E17K mutation in human breast epithelial cells does not recapitulate oncogenic PIK3CA mutations
Oncogene. 2010 Jan 25. epub/doi:10.1038/onc.2009.516.
An oncogenic mutation (G49A:E17K) in the AKT1 gene has been described recently in human breast, colon, and ovarian cancers. The low frequency of this mutation and perhaps other selective pressures have prevented the isolation of human cancer cell lines that harbor this mutation thereby limiting functional analysis. Here, we create a physiologic in vitro model to study the effects of this mutation by using somatic cell gene targeting using the nontumorigenic human breast epithelial cell line, MCF10A. Surprisingly, knock in of E17K into the AKT1 gene had minimal phenotypic consequences and importantly, did not recapitulate the biochemical and growth characteristics seen with somatic cell knock in of PIK3CA hotspot mutations. These results suggest that mutations in critical genes within the PI3-kinase (PI3K) pathway are not functionally equivalent, and that other cooperative genetic events may be necessary to achieve oncogenic PI3K pathway activation in cancers that contain the AKT1 E17K mutation.
RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth
Nature 2010 Published Online, doi:10.1038/nature08833
Activating mutations in KRAS and BRAF are found in more than 30% of all human tumours and 40% of melanoma, respectively, thus targeting this pathway could have broad therapeutic effects1. Small molecule ATP-competitive RAF kinase inhibitors have potent antitumour effects on mutant BRAF(V600E) tumours but, in contrast to mitogen-activated protein kinase kinase (MEK) inhibitors, are not potent against RAS mutant tumour models, despite RAF functioning as a key effector downstream of RAS and upstreamofMEK2,3. Here we showthat ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on the cellular context. In BRAF(V600E) tumours, RAF inhibitors effectively block the mitogen-activated protein kinase (MAPK) signalling pathway and decrease tumour growth. Notably, in KRAS mutant and RAS/RAF wild-type tumours, RAF inhibitors activate the RAF–MEK–ERK pathway in a RAS-dependent manner, thus enhancing tumour growth in some xenograft models. Inhibitor binding activates wild-type RAF isoforms by inducing dimerization, membrane localization and interaction with RAS–GTP. These events occur independently of kinase inhibition and are, instead, linked to direct conformational effects of inhibitors on the RAF kinase domain. On the basis of these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signalling pathways, depending on the cellular context. Furthermore, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors.
Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors
EMBO Mol Med 2009; 1, 315–322
The tumour suppressor gene, phosphatase and tensin homolog (PTEN), is one of the most commonly mutated genes in human cancers. Recent evidence suggests that PTEN is important for the maintenance of genome stability. Here, we show that PTEN deficiency causes a homologous recombination (HR) defect in human tumour cells. The HR deficiency caused by PTEN deficiency, sensitizes tumour cells to potent inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), both in vitro and in vivo. PARP inhibitors are now showing considerable promise in the clinic, specifically in patients with mutations in either of the breast cancer susceptibility genes BRCA1 or BRCA2. The data we present here now suggests that the clinical assessment of PARP inhibitors should be extended beyond those with BRCA mutations to a larger group of patients with PTEN mutant tumours.
Deletion of PTEN Promotes Tumorigenic Signaling, Resistance to Anoikis, and Altered Response to Chemotherapeutic Agents in Human Mammary Epithelial Cells
Cancer Res 2009; 69: (21). November 1, 2009
Many cancers, including breast cancer, harbor loss-of-function mutations in the catalytic domain of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or have reduced PTEN expression through loss of heterozygosity and/or epigenetic silencing mechanisms. However, specific phenotypic effects of PTEN inactivation in human cancer cells remain poorly defined without a direct causal connection between the loss of PTEN function and the development or progression of cancer. To evaluate the biological and clinical relevance of reduced or deleted PTEN expression, a novel in vitro model system was generated using human somatic cell knockout technologies. Targeted homologous ecombination allowed for a single and double allelic deletion, which resulted in reduced and deleted PTEN expression, respectively. We determined that heterozygous loss of PTEN in the nontumorigenic human mammary epithelial cell line MCF-10A was sufficient for activation of the phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase pathways, whereas the homozygous absence of PTEN expression led to a further increased activation of both pathways. The deletion of PTEN was able to confer growth factor–independent proliferation, which was confirmed by the resistance of the PTEN−/− MCF-10A cells to small-molecule inhibitors of the epidermal growth factor receptor. However, neither heterozygous nor homozygous loss of PTEN expression was sufficient to promote anchorage-independent growth, but the loss of PTEN did confer apoptotic resistance to cell rounding and matrixdetachment. Finally, MCF-10A cells with the reduction or loss of PTEN showed increased susceptibility to the chemotherapeutic drug doxorubicin but not paclitaxel.
Glucose deprivation contributes to the development of KRAS pathway mutations in tumor cells
Published Online August 6, 2009 /Science/ DOI: 10.1126/science.1174229
Tumor progression is driven by genetic mutations, but little is known about the environmental conditions that select for these mutations. Studying the transcriptomes of paired colorectal cancer cell lines that differed only in the mutational status of their KRAS or BRAF genes, we found that GLUT1, encoding glucose transporter-1, was one of three genes consistently upregulated in cells with KRAS or BRAF mutations. The mutant cells exhibited enhanced glucose uptake and glycolysis and survived in low glucose conditions, phenotypes that all required GLUT1 expression. In contrast, when cells with wild-type KRAS alleles were subjected to a low glucose environment, very few cells survived. Most surviving cells expressed high levels of GLUT1, and 4% of these survivors had acquired new KRAS mutations. The glycolysis inhibitor 3-bromopyruvate preferentially suppressed the growth of cells with KRAS or BRAF mutations. Together, these data suggest that glucose deprivation can drive the acquisition of KRAS pathway mutations in human tumors.
Tumours with PI3K activation are resistant to dietary restriction
Published Online April 2009 / Nature / Vol 458 / doi:10.1038/nature07782
Dietary restriction delays the incidence and decreases the growth of various types of tumours, but the mechanisms underlying the sensitivity of tumours to food restriction remain unknown. Here we show that certain human cancer cell lines, when grown as tumour xenografts in mice, are highly sensitive to the anti-growth effects of dietary restriction, whereas
others are resistant. Cancer cells that form dietary-restriction-resistant tumours carry mutations that cause constitutive activation of the phosphatidylinositol-3-kinase (PI3K) pathway and in culture proliferate in the absence of insulin or insulin-like growth factor 1. Substitution of an activated mutant allele of PI3K with wild-type PI3K in otherwise isogenic cancer cells, or the restoration of PTEN expression in a PTEN-null cancer cell line, is sufficient to convert a dietary-restriction-resistant tumour into one that is dietary-restriction-sensitive. Dietary restriction does not affect a PTEN-null mouse model of prostate cancer, but it significantly decreases tumour burden in a mouse model of lung cancer
lacking constitutive PI3K signalling. Thus, the PI3K pathway is an important determinant of the sensitivity of tumours to dietary restriction, and activating mutations in the pathway may influence the response of cancers to dietary restriction-mimetic therapies.
A syngeneic variance library for functional annotation of human variation: application to BRCA2
Cancer Res., 2008, 68(13), Pages 5023-5030.
The enormous scope of natural human genetic variation is now becoming defined. To accurately annotate these variants, and to identify those with clinical importance, is often difficult to assess through functional assays. We explored systematic annotation by using homologous recombination to modify a native gene in hemizygous (wt/ exon) human cancer cells, generating a novel syngeneic variance library (SyVaL). We created a SyVaL of BRCA2 variants: nondeleterious, proposed deleterious, deleterious, and of uncertain significance. We found that the null states BRCA2 ex11/ ex11 and BRCA2 ex11/Y3308X were deleterious as assessed by a loss of RAD51 focus formation on genotoxic damage and by acquisition of toxic hypersensitivity to mitomycin C and etoposide, whereas BRCA2 ex11/Y3308Y, BRCA2 ex11/P3292L, and BRCA2 ex11/P3280H had wild-type function. A proposed phosphorylation site at codon 3291 affecting function was confirmed by substitution of an acidic residue (glutamate, BRCA2 ex11/S3291E) for the native serine, but in contrast to a prior report, phosphorylation was dispensable (alanine, BRCA2 ex11/S3291A) for BRCA2-governed cellular phenotypes. These results show that SyVaLs offer a means to comprehensively annotate gene function, facilitating numerical and unambiguous readouts. SyVaLs may be especially useful for genes in which functional assays using exogenous expression are toxic or otherwise unreliable. They also offer a stable, distributable cellular resource for further research.
Survivin depletion preferentially reduces the survival of activated K-Ras-transformed cells
Mol Cancer Ther., 2007, 6(1), Pages 269-276.
To identify cancer-specific targets, we have conducted a synthetic lethal screen using a small interfering RNA (siRNA) library targeting approximately 4,000 individual genes for enhanced killing in the DLD-1 colon carcinoma cell line that expresses an activated copy of the K-Ras oncogene. We found that siRNAs targeting baculoviral inhibitor of apoptosis repeat-containing 5 (survivin) significantly reduced the survival of activated K-Ras-transformed cells compared with its normal isogenic counterpart in which the mutant K-Ras gene had been disrupted (DKS-8). In addition, survivin siRNA induced a transient G(2)-M arrest and marked polyploidy that was associated with increased caspase-3 activation in the activated K-Ras cells. These results indicate that tumors expressing the activated K-Ras oncogene may be particularly sensitive to inhibitors of the survivin protein.
Epitope tagging of endogenous genes in diverse human cell lines
Nucleic Acids Research 2008 36(19):e127; doi:10.1093/nar/gkn566
Epitope tagging is a powerful and commonly used approach for studying the physical properties of proteins and their functions and localization in eukaryotic cells. In the case of Saccharomyces cerevisiae, it has been possible to exploit the high efficiency of homologous recombination to tag proteins by modifying their endogenous genes, making it possible to tag virtually every endogenous gene and perform genome-wide proteomics experiments. However, due to the relative inefficiency of homologous recombination in cultured human cells, epitope-tagging approaches have been limited to ectopically expressed transgenes, with the attendant limitations of their nonphysiological transcriptional regulation and levels of expression. To overcome this limitation, a modification and extension of adeno-associated virus-mediated human somatic cell gene targeting technology is described that makes it possible to simply and easily create an endogenous epitope tag in the same way that it is possible to knock out a gene. Using this approach, we have created and validated human cell lines with epitope-tagged alleles of two cancer-related genes in a variety of untransformed and transformed human cell lines. This straightforward approach makes it possible to study the physical and biological properties of endogenous proteins in human cells without the need for specialized antibodies for individual proteins of interest.
Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control
PNAS., 2008, 105(52), Pages 20752-20757.
Chk1 is widely known as a DNA damage checkpoint signaling protein. Unlike many other checkpoint proteins, Chk1 also plays an essential but poorly defined role in the proliferation of unperturbed cells. Activation of Chk1 after DNA damage is known to require the phosphorylation of several C-terminal residues, including the highly conserved S317 and S345 sites. To evaluate the respective roles of these individual sites and assess their contribution to the functions of Chk1, we used a gene targeting approach to introduce point mutations into the endogenous human CHK1 locus. We report that the essential and nonessential functions of Chk1 are regulated through distinct phosphorylation events and can be genetically uncoupled. The DNA damage response function of Chk1 was nonessential. Targeted mutation of S317 abrogated G2/M checkpoint activation, prevented subsequent phosphorylation of Chk1, impaired efficient progression of DNA replication forks, and increased fork stalling, but did not impact viability. Thus, the nonessential DNA damage response function of Chk1 could be unambiguously linked to its role in DNA replication control. In contrast, a CHK1 allele with mutated S345 did not support viability, indicating an essential role for this residue during the unperturbed cell cycle. A distinct, physiologic mode of S345 phosphorylation, initiated at the centrosome during unperturbed mitosis was independent of codon 317 status and mechanistically distinct from the ordered and sequential phosphorylation of serine residues on Chk1 induced by DNA damage. Our findings suggest an essential regulatory role for Chk1 phosphorylation during mitotic progression.
Chromatid cohesion defects may underlie chromosome instability in human colorectal cancers
PNAS, 2008, 105(9): Pages 3443-3348
Although the majority of colorectal cancers exhibit chromosome instability (CIN), only a few genes that might cause this phenotype have been identified and no general mechanism underlying their function has emerged. To systematically identify somatic mutations in potential CIN genes in colorectal cancers, we determined the sequence of 102 human homologues of 96 yeast CIN genes known to function in various aspects of chromosome transmission fidelity. We identified 11 somatic mutations distributed among five genes in a panel that included 132 colorectal cancers. Remarkably, all but one of these 11 mutations were in the homologs of yeast genes that regulate sister chromatid cohesion. We then demonstrated that down-regulation of such homologs resulted in chromosomal instability and chromatid cohesion defects in human cells. Finally, we showed that down-regulation or genetic disruption of the two major candidate CIN genes identified in previous studies (MRE11A and CDC4) also resulted in abnormal sister chromatid cohesion in human cells. These results suggest that defective sister chromatid cohesion as a result of somatic mutations may represent a major cause of chromosome instability in human cancers.
Identification and characterization of a diamine exporter in colon epithelial cells
J Biol Chem., 2008, 283(39), Pages 26428-26435.
SLC3A2, a member of the solute carrier family, was identified by proteomic methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine, compared to transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using shRNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared to its isogenic clone Hkh2 cells, which lacks an activated K-RAS allele. Spermidine/spermine N1-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody, as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.
Chk1 Instability Is Coupled to Mitotic Cell Death of p53-deficient Cells in Response to Virus-induced DNA Damage Signaling
Journal of Molecular Biology, Volume 372, Issue 2, 14 September 2007, Pages 397-406
Adeno-associated virus (AAV) DNA, by mimicking a stalled replication fork, provokes a DNA damage response that can arrest cells in the G2/M phase of the cell-cycle. This response depends strictly on DNA damage signaling kinases ATR and Chk1. Here, we used AAV to study long-term effects of DNA damage signaling in cells with altered p53 status. In HCT116 cells, in response to damage signaling, p53 represses transcription of the genes encoding mitotic regulators Cdc25C, cyclin B1, and Plk1 to establish a firm G2 arrest. Isogenic cells lacking p53 maintain these three proteins at constant levels yet can still arrest initially in G2 because Chk1 signaling inhibits their enzymatic activities. Unexpectedly, the levels of Chk1 fall abruptly in a proteasome-dependent manner after two days of arrest in G2. In p53-deficient cells, this Chk1 instability is coupled to recovery of the phosphatase activity of Cdc25C and in the kinase activities of Plk1 and Cdk1/cyclin B1. Consequently, the p53-deficient cells enter lethal mitosis. Thus, the Chk1-mediated arrest is transient: it initially causes cells to accumulate in G2 until p53-dependent transcriptional repression of mitotic proteins takes over. p53-deficient cells cannot maintain the DNA damage signaling-induced G2 arrest after Chk1 has disappeared, and continue into catastrophic mitosis. Restoring Chk1 prevents the cells from entering such mitosis. These results reveal a mechanism based on Chk1 stability that regulates mitotic entry after DNA damage and elucidate the controversial phenomenon of p53-promoted cell survival in the face of damage signaling.
High-throughput screening identifies novel agents eliciting hypersensitivity in Fanconi pathway-deficient cancer cells
Cancer Res., 2007, 67(5), Pages 2169-2177.
Inactivation of the Fanconi anemia (FA) pathway occurs in diverse human tumors among the general population and renders those tumors hypersensitive to DNA interstrand-cross-linking (ICL) agents. The identification of novel agents to which FA pathway–deficient cells were hypersensitive could provide new therapeutic opportunities and improve our molecular understanding of the FA genes. Using high-throughput screening, we assessed the growth of isogenic human cancer cells that differed only in the presence or absence of single FA genes upon treatment with 880 active drugs and 40,000 diverse compounds. We identified several compounds to which FA pathway–deficient cells were more sensitive than FA pathway–proficient cells, including two groups of structurally related compounds. We further investigated the compound eliciting the strongest effect, termed 80136342. Its mechanism of action was distinct from that of ICL agents; 80136342 did not cause increased chromosomal aberrations, enhanced FANCD2 monoubiquitination, H2AX phosphorylation, p53 activation, or ICL induction. Similar to ICL agents, however, 80136342 caused a pronounced G2 arrest in FA pathway–deficient cells. When applied in combination with ICL agents, 80136342 had at least additive toxic effects, excluding interferences on ICL-induced toxicity and facilitating a combinational application. Finally, we identified one particular methyl group necessary for the effects of 80136342 on FA–deficient cells. In conclusion, using high-throughput screening in an isogenic human FA cancer model, we explored a novel approach to identify agents eliciting hypersensitivity in FA pathway–deficient cells. We discovered several attractive candidates to serve as lead compounds for evaluating structure-activity relationships and developing therapeutics selectively targeting FA pathway–deficient tumors.
Human cancer cells require ATR for cell cycle progression following exposure to ionizing radiation
Oncogene., 2007, 26(18), Pages 2535-2542.
The vast majority of cancer cells have defective checkpoints that permit the cell cycle to progress in the presence of double-strand DNA breaks (DSBs) caused by ionizing radiation (IR) and radiomimetic drugs. ATR (ataxia telangiectasia-mutated and Rad3-related) has recently been shown to be activated by DSBs, although the consequences of this activity are largely unknown. In this report, we use advanced gene targeting methods to generate biallelic hypomorphic ATR mutations in human colorectal cancer cells and demonstrate that progression of the cancer cell cycle after IR treatment requires ATR. Cells with mutant ATR accumulated at a defined point at the beginning of the S phase after IR treatment and were unable to progress beyond that point, whereas cells at later stages of the S phase during the time of irradiation progressed and completed DNA replication. The prolonged arrest of ATR mutant cancer cells did not involve the ataxia telangiectasia mutated-dependent S-phase checkpoint, but rather closely resembled a previously characterized form of cell cycle arrest termed S-phase stasis. As ATR strongly contributed to clonogenic survival after IR treatment, these data suggest that blocking ATR activity might be a useful strategy for inducing S-phase stasis and promoting the radiosensitization of checkpoint-deficient cancer cells.
Gene-specific selection against experimental fanconi anemia gene inactivation in human cancer
Cancer Biol Ther., 2007, 6(5), Pages 654-660.
The Fanconi anemia (FA) gene family comprises at least 12 genes interacting in a common pathway involved in DNA repair. To gain insight into the role of FA gene inactivation occurring in tumors among the general population, we endogenously targeted in cancer cells four FA genes that act at different stages of the FA pathway. After successful mono-allelic deletion of all genes, the sequential homozygous deletion was achieved only for FANCC and FANCG, acting upstream, but not for BRCA2 or FANCD2, acting downstream in the FA pathway. Targeting of the second allele in in BRCA2 and FANCD2 heterozygote clones resulted in redeletion exclusively of the already defective allele in multiple instances (13x concerning BRCA2, 25x concerning FANCD2), strongly suggesting a detrimental phenotype. Unlike complete FANCD2 disruption, the mere reduction of FANCD2 protein levels had no discernible effect. In addition, we confirmed that human cancer cells harboring the Seckel ATR mutation display impaired FANCD2 monoubiquitination and FANCD2 nuclear focus formation, as well as an increased sensitivity to DNA interstrand-crosslinking agents. Nevertheless, these cells were viable, indicating an ATR-independent function of FANCD2, distinct from its major known functions, to be responsible for the detrimental effects of FANCD2 loss. In conclusion, we established the downstream FA genes FANCD2 and BRCA2 to represent particularly vulnerable parts of the FA pathway, providing direct evidence for the paradoxical assumption that their inactivation could be predominantly selected against in cancer cells. This would explain why certain FA gene defects, despite an apparent selection for FA pathway inactivation in cancer, are rarely observed in tumors among the general population.
Loss of ataxia telangiectasia mutated- and Rad3-related function potentiates the effects of chemotherapeutic drugs on cancer cell survival
Mol Cancer Ther., 2007, 6(4), Pages 1406-1413.
The diverse responses of human cells to various forms of DNA damage are controlled by a complex network of signaling proteins. There has been considerable interest in the components of this signaling apparatus as potential targets for new forms of anticancer therapy. In this report, we examine the contributions of an upstream signaling molecule, the ataxia telangiectasia mutated– and Rad3-related (ATR) protein kinase, to the resistance of cancer cells to DNA-damaging agents that are commonly used as anticancer therapeutics. Loss of ATR function in knock-in cancer cells strikingly enhanced the effects of several of the most commonly used therapeutic compounds, impeding the progression of the cell cycle and reducing long-term cancer cell survival. Loss of ATR function potentiated the toxicity of alkylating agents most strikingly, antimetabolites moderately, and double-strand break–inducing agents to a lesser extent. These results suggest that specific inhibition of ATR activity will be a valid strategy to increase the effectiveness of currently used modes of therapy.
P21 gene knock down does not identify genetic effectors seen with gene knock out
Cancer Biol Ther., 2007, 6(7), Pages 1025-1030.
RNA interference (RNAi) has become a popular tool for analyzing gene function in cancer research. The feasibility of using RNAi in cellular and animal models as an alternative to conventional gene knock out approaches has been demonstrated. Although these studies show that RNAi can recapitulate phenotypes seen in knock out animals and their derived cell lines, a systematic study rigorously comparing downstream effector genes between RNAi and gene knock out has not been performed. Here we present data contrasting the phenotypic and genotypic changes that occur with either stable knock down via RNAi of the cyclin dependent kinase inhibitor p21 versus its somatic cell knock out counterpart in the human mammary epithelial cell line MCF-10A. Our results demonstrate that p21 knock down clones display a growth proliferative response upon exposure to Transforming Growth Factor-Beta Type 1 (TGFbeta) similar to p21 knock out clones. However, gene expression profiles were significantly different in p21 knock down cells versus p21 knock out clones. Importantly p21 knock down clones did not display increased gene expression of interleukin-1alpha (IL-1alpha), a critical effector of this growth response previously validated in p21 knock out cells. We conclude that gene knock out can yield additional vital information that may be missed with gene knock down strategies.
Activation of p53-Dependent Growth Suppression in Human Cells by Mutations in PTEN or PIK3CA
Molecular and Cellular Biology, January 2007, p. 662-677, Vol. 27, No. 2
In an effort to identify genes whose expression is regulated by activated phosphatidylinositol 3-kinase (PI3K) signaling, we performed microarray analysis and subsequent quantitative reverse transcription-PCR on an isogenic set of PTEN gene-targeted human cancer cells. Numerous p53 effectors were upregulated following PTEN deletion, including p21, GDF15, PIG3, NOXA, and PLK2. Stable depletion of p53 led to reversion of the gene expression program. Western blots revealed that p53 was stabilized in HCT116 PTEN–/– cells via an Akt1-dependent and p14ARF-independent mechanism. Stable depletion of PTEN in untransformed human fibroblasts and epithelial cells also led to upregulation of p53 and senescence-like growth arrest. Simultaneous depletion of p53 rescued this phenotype, enabling PTEN-depleted cells to continue proliferating. Next, we tested whether oncogenic PIK3CA, like inactivated PTEN, could activate p53. Retroviral expression of oncogenic human PIK3CA in MCF10A cells led to activation of p53 and upregulation of p53-regulated genes. Stable depletion of p53 reversed these PIK3CA-induced expression changes and synergized with oncogenic PIK3CA in inducing anchorage-independent growth. Finally, targeted deletion of an endogenous allele of oncogenic, but not wild-type, PIK3CA in a human cancer cell line led to a reduction in p53 levels and a decrease in the expression of p53-regulated genes. These studies demonstrate that activation of PI3K signaling by mutations in PTEN or PIK3CA can lead to activation of p53-mediated growth suppression in human cells, indicating that p53 can function as a brake on phosphatidylinositol (3,4,5)-triphosphate-induced mitogenesis during human cancer pathogenesis.
Targeted Disruption of FANCC and FANCG in Human Cancer Provides a Preclinical Model for Specific Therapeutic Options
Gastroenterology, Volume 130, Issue 7, Pages 2145-2154
How specifically to treat pancreatic and other cancers harboring Fanconi anemia gene mutations has raised great interest recently, yet preclinical studies have been hampered by the lack of well-controlled human cancer models. Methods: We endogenously disrupted FANCC and FANCG in a human adenocarcinoma cell line and determined the impact of these genes on drug sensitivity, irradiation sensitivity, and genome maintenance. Results: FANCC and FANCG disruption abrogated FANCD2 monoubiquitination, confirming an impaired Fanconi anemia pathway function. On treatment with DNA interstrand–cross-linking agents, FANCC and FANCG disruption caused increased clastogenic damage, G2/M arrest, and decreased proliferation. The extent of hypersensitivity varied among agents, with ratios of inhibitory concentration 50% ranging from 2-fold for oxaliplatin to 14-fold for melphalan, a drug infrequently used in solid tumors. No hypersensitivity was observed on gemcitabine, etoposide, 3-aminobenzamide, NU1025, or hydrogen peroxide. FANCC and FANCG disruption also resulted in increased clastogenic damage on irradiation, but only FANCG disruption caused a subsequent decrease in relative survival. Finally, FANCC and FANCG disruption increased spontaneous chromosomal breakage, supporting the role of these genes in genome maintenance and likely explaining why they are mutated in sporadic cancer. Conclusions: Our human cancer cell model provides optimal controls to elucidate fundamental biologic features of individual Fanconi anemia gene defects and facilitates preclinical studies of therapeutic options. The impact of Fanconi gene defects on drug and irradiation sensitivity renders these genes promising targets for a specific, genotype-based therapy for individual cancer patients, providing a strong rationale for clinical trials.
The colorectal microRNAome
PNAS, 2006, 103(10), Pages 3687-3692
MicroRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms. The public miRNA database contains 321 human miRNA sequences, 234 of which have been experimentally verified. To explore the possibility that additional miRNAs are present in the human genome, we have developed an experimental approach called miRNA serial analysis of gene expression (miRAGE) and used it to perform the largest experimental analysis of human miRNAs to date. Sequence analysis of 273,966 small RNA tags from human colorectal cells allowed us to identify 200 known mature miRNAs, 133 novel miRNA candidates, and 112 previously uncharacterized miRNA* forms. To aid in the evaluation of candidate miRNAs, we disrupted the Dicer locus in three human colorectal cancer cell lines and examined known and novel miRNAs in these cells. These studies suggest that the human genome contains many more miRNAs than currently identified and provide an approach for the large-scale experimental cloning of novel human miRNAs in human tissues.
Hypoxia-inducible factor-1alpha promotes nonhypoxia-mediated proliferation in colon cancer cells and xenografts
Cancer Res., 2006, 66(3), Pages1684-1693.
Hypoxia-inducible factor-1 (HIF-1 ) is a transcription factor that directly transactivates genes important for the growth and metabolism of solid tumors. HIF-1 is overexpressed in cancer, and its level of expression is correlated with patient mortality. Increased synthesis or stability of HIF-1 can be induced by hypoxia-dependent or hypoxia-independent factors. Thus, HIF-1 is expressed in both nonhypoxic and hypoxic cancer cells. The role of HIF-1 in nonhypoxia-mediated cancer cell proliferation remains speculative. We have disrupted HIF-1 by targeted homologous recombination in HCT116 and RKO human colon cancer cells. Loss of HIF-1 significantly reduced nonhypoxia-mediated cell proliferation in vitro and in vivo. Paradoxically, loss of HIF-1 expression did not grossly affect the hypoxic compartments within tumor xenografts in vivo, although HIF-1 promoted cell proliferation and survival under hypoxia in vitro. To further test the role of HIF-1 within tumor compartments, we generated cells with combined disruptions of both HIF-1 and vascular endothelial growth factor (VEGF). In all xenografts, disruption of VEGF led to marked expansion of the hypoxic compartments and growth delay. Nonetheless, the presence or absence of HIF-1 did not grossly affect these expanded hypoxic compartments. These data provide compelling evidence that, in a subset of colon cancers, (a) HIF-1 is a positive factor for nonhypoxia-mediated cell proliferation in vitro and in vivo and (b) HIF-1 is a positive factor for cell proliferation and survival under hypoxic conditions in vitro, but does not grossly contribute to the tumor hypoxic compartments in vivo.
Targeted deletion of MKK4 in cancer cells: a detrimental phenotype manifests as decreased experimental metastasis and suggests a counterweight to the evolution of tumor-suppressor loss
Cancer Res., 2006, 66(11), Pages 5560-5564.
Tumor-suppressors have commanded attention due to the selection for their inactivating mutations in human tumors. However, relatively little is understood about the inverse, namely, that tumors do not select for a large proportion of seemingly favorable mutations in tumor-suppressor genes. This could be explained by a detrimental phenotype accruing in a cell type-specific manner to most cells experiencing a biallelic loss. For example, MKK4, a tumor suppressor gene distinguished by a remarkably consistent mutational rate across diverse tumor types and an unusually high rate of loss of heterozygosity, has the surprisingly low rate of genetic inactivation of only approximately 5%. To explore this incongruity, we engineered a somatic gene knockout of MKK4 in human cancer cells. Although the null cells resembled the wild-type cells regarding in vitro viability and proliferation in plastic dishes, there was a marked difference in a more relevant in vivo model of experimental metastasis and tumorigenesis. MKK4(-/-) clones injected i.v. produced fewer lung metastases than syngeneic MKK4-competent cells (P = 0.0034). These findings show how cell type-specific detrimental phenotypes can offer a paradoxical and yet key counterweight to the selective advantage attained by cells as they experiment with genetic null states during tumorigenesis, the resultant balance then determining the observed biallelic mutation rate for a given tumor-suppressor gene.
Ischemia Induced K-ras Mutations in Human Colorectal Cancer Cells: Role of Microenvironmental Regulation of MSH2 Expression
Cancer Research 63, 8134-8141, September 15, 2005
Mutation of the K-ras gene is one of the most common genetic alterations in solid tumors, including colorectal cancer. The relatively late emergence of K-ras mutations in colorectal cancer is particularly striking in the class of mismatch repair–deficient tumors associated with early-onset microsatellite instability. We, therefore, tested the hypothesis that the microsatellite instability phenotype itself does not efficiently trigger K-ras mutations in colorectal cancer cells, but rather that tumor-associated microenvironmental conditions (e.g., hypoxia and hypoglycemia) contribute to this event by modulating genetic instability. We examined K-rasG13D mutation using PCR-RFLP analysis in two different microsatellite instability colorectal cancer cell lines (HCT116 and DLD-1) and their variants in which the mutant (but not the wild-type) K-ras allele has been genetically disrupted (Hkh-2 and Dks-8). We found K-rasG13D mutation to occur at far greater incidence in cells derived from xenografted tumors or exposed to conditions of combined hypoxia and hypoglycemia in vitro. Interestingly, this mutagenesis was neither enhanced by induced oxidative damage nor prevented by the antioxidant vitamin E. Moreover, the accumulation of K-ras mutations was paralleled by down-regulation of the key mismatch repair protein MSH2 in xenografted tumors, particularly in hypoperfused areas and under hypoglycemic conditions (in vitro). In contrast, the microsatellite stable colorectal cancer cell line Caco-2 neither accumulated K-ras mutations nor showed down-regulation of MSH2 under these conditions. Thus, our study suggests that ischemia may not simply select for, but can actually trigger, increased mutation rate in crucial colorectal cancer oncoproteins. This finding establishes a novel linkage between genetic instability, tumor ischemia, and genetic tumor progression and carries important implications for applying anticancer therapies involving tumor hypoxia (e.g., antiangiogenesis) in microsatellite instability cancers.
Mutant PIK3CA promotes cell growth and invasion of human cancer cells
Cancer Cell, 2005, Volume 7, Issue 6, Pages 561-573
PIK3CA is mutated in diverse human cancers, but the functional effects of these mutations have not been defined. To evaluate the consequences of PIK3CA alterations, the two most common mutations were inactivated by gene targeting in colorectal cancer (CRC) cells. Biochemical analyses of these cells showed that mutant PIK3CA selectively regulated the phosphorylation of AKT and the forkhead transcription factors FKHR and FKHRL1. PIK3CA mutations had little effect on growth under standard conditions, but reduced cellular dependence on growth factors. PIK3CA mutations resulted in attenuation of apoptosis and facilitated tumor invasion. Treatment with the PI3K inhibitor LY294002 abrogated PIK3CA signaling and preferentially inhibited growth of PIK3CA mutant cells. These data have important implications for therapy of cancers harboring PIK3CA alterations.
SMAC/Diablo-dependent apoptosis induced by nonsteroidal antiinflammatory drugs (NSAIDs) in colon cancer cells
PNAS, 2004, 101(48), Pages 16897-16902
Nonsteroidal antiinflammatory drugs (NSAIDs) form a paradigm for the chemoprevention of cancer, preventing colonic tumor progression in both experimental animals and humans. However, the mechanisms underlying the antineoplastic effects of NSAIDs are currently unclear. We found that the mitochondrial second mitochondrial-derived activator of caspase (SMAC)/direct inhibitor of apoptosis protein-binding protein with low pI (Diablo) protein translocates into the cytosol during NSAID-induced apoptosis in colon cancer cells. When SMAC/Diablo is disrupted by homologous recombination and RNA interference in these cells, the NSAID-induced apoptosis is abrogated. Biochemical markers of apoptosis, such as caspase activation, cytosolic release of cytochrome c and apoptosis-inducing factor, and mitochondrial membrane potential change, are accordingly decreased. These results establish that SMAC/Diablo is essential for the apoptosis induced by NSAIDs in colon cancer cells.
SMAC/X-linked inhibitor of apoptosis protein (XIAP) is a nonredundant modulator of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human cancer cells
Cancer Res. 2004, 1;64(9), Pages 3006-3008
Although the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to play an important role in the immunosurveillance of neoplasia, apoptotic factors that modulate the sensitivity of cancer cells to TRAIL are poorly understood. The inhibitor of apoptosis proteins (IAPs) have generated considerable interest as potential targets for cancer therapy, but the lack of a phenotype in X-linked IAP (XIAP) knockout mice has generated speculation that IAP function may be redundant. Using gene targeting technology, we show that disruption of the gene encoding XIAP in human cancer cells did not interfere with basal proliferation, but caused a remarkable sensitivity to TRAIL. These results demonstrate that XIAP is a nonredundant modulator of TRAIL-mediated apoptosis and provide a rationale for XIAP as a therapeutic target.
Increase in frequency of mutant K-ras after xenograft implantation of colorectal carcinoma cells
Proc. Amer. Assoc. Cancer Tes, Volume 45, 2004
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Mutations in cancer are able to transform the K-ras gene from a proto-oncogene to an oncogene. All known mutations limit the ability of the protein to break down GTP to GDP leading to continuous growth signaling in affected cells. Mutations in codon 12 or 13 of K-ras gene occur in approximately half of all colorectal cancers, 75-100% of pancreatic cancer and almost 50% of lung adenocarcinomas. The prevalence of this mutation in solid tumors suggests that it arises early in cancer development and tumor progression. Therefore, we addressed the issue that environmental conditions might influence, or select for mutagenicity at this locus. To identify the association between phenotype and genotype and the influence of environment on the likelihood of K-ras mutation, we examined the growth of the paired isogenic cell lines HCT 116 (K-rasWT/K-rasG13D) and Hkh2 (K-rasWT/Null) in mice. The HCT 116 formed tumors in all mice within 10 days after subcutaneous injection of one or two million cells. Injection of one million Hkh-2 cells did not lead to any tumor formation. Surprisingly, eight of the nine mice injected with two million cells grew tumors from these apparently non-tumorogenic Hkh-2 cells, but with an average latency of 21 days. Tumor growth was monitored and xenografts were collected for analysis once tumors reached a size of at least 300 mm3. The cell lines and the tumors were tested for K-ras mutations using a nested polymerase chain reaction (PCR) and restriction enzyme digestions. For HCT 116, 100% of the cell line showed at least one mutant allele at codon 13, whereas the Hkh-2 cell line showed less than 5% mutation at this locus. All HCT 116 tumors showed an enrichment for mutation at codon 13. About 50% mutation was detected for each tumor formed by the Hkh-2 cells. Thus, the exposure of the cells to tumor conditions (e.g. hypoxia, three-dimensional growth) has revealed a possible connection between these environmental conditions and the prevalence of mutations in the K-ras gene. Presumably, this occurs by inducing point mutations in wild type K-ras (HCT 116 and Hkh-2), and/or positive selection for those few cells originally harboring the K-ras mutation (Hkh-2). This study suggests possible mechanisms for inducing K-ras mutation early in tumorogenesis. (Supported by Canadian Institutes of Health Research (CIHR)).
Tumour suppression: disruption of HAUSP gene stabilizes p53
Nature, 2004, 428(6982), Page 416
Ubiquitination of p53 is the principal mechanism through which p53 concentrations in the cell are regulated1, 2 in order to maintain its effects on tumori-genesis and normal cell growth. The protein HAUSP (also known as USP7) is a ubiquitin-specific protease (deubiquitinase)3, 4 that has been shown by Li et al. to bind to p53 (ref. 5); in overexpression experiments, Li et al. showed that p53 could be stabilized as a result of deubiquitination by HAUSP and suggested that HAUSP may thereby act as a tumour suppressor5, 6. Here we use a different approach to investigate the relationship between HAUSP and p53 stability, in which we disrupt the HAUSP gene in human cells by targeted homologous recombination. Instead of the expected increase in ubiquitinated p53 and destabilization of p53, we find that disruption of HAUSP results in the opposite phenotype, leading to stabilization and functional activation of p53 in our system. It may be that HAUSP can deubiquitinate other proteins such as MDM2, another regulator of p53, and that the balance between the deubiquitination of the different targets of HAUSP determines the steady-state level of p53.
Inactivation of hCDC4 can cause chromosomal instability
Nature, 2004, 428(6978), Pages 77-81.
Aneuploidy, an abnormal chromosome number, has been recognized as a hallmark of human cancer for nearly a century; however, the mechanisms responsible for this abnormality have remained elusive. Here we report the identification of mutations in hCDC4 (also known as Fbw7 or Archipelago) in both human colorectal cancers and their precursor lesions. We show that genetic inactivation of hCDC4, by means of targeted disruption of the gene in karyotypically stable colorectal cancer cells, results in a striking phenotype associated with micronuclei and chromosomal instability. This phenotype can be traced to a defect in the execution of metaphase and subsequent transmission of chromosomes, and is dependent on cyclin E–a protein that is regulated by hCDC4 (refs 2-4). Our data suggest that chromosomal instability is caused by specific genetic alterations in a large fraction of human cancers and can occur before malignant conversion.
B-Raf is dispensable for K-Ras-mediated oncogenesis in human cancer cells
Cancer Res., 2004, 64(6), Pages 1932-1937.
Oncogenic mutations in B-Raf and Kirsten-Ras (K-Ras) are mutually exclusive during human cancer pathogenesis. In an effort to study the biological basis of this epistasis, gene targeting was used to create isogenic sets of human cancer cells differing only in presence or absence of endogenous oncogenic K-Ras or wild-type B-Raf. Whereas cells lacking the K-Ras oncogene were unable to efficiently form xenograft tumors, isogenic cells retaining activated K-Ras but deleted for B-Raf remained highly tumorigenic. Deletion of oncogenic K-Ras failed to reduce the activation state of B-Raf or ERK1/2, despite the requirement of oncogenic K-Ras for tumorigenesis. Genechip analysis revealed numerous genes in which the regulation by oncogenic K-Ras did not require B-Raf. These studies suggest that despite the mutual exclusivity of K-Ras and B-Raf mutations in human cancer and the well-described role for Raf proteins as Ras effectors, B-Raf is dispensable for K-Ras-mediated oncogenesis in a human cancer cell line. Additional studies are required to demonstrate the generalizability of these unexpected findings.
Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses
Nucleic Acids Res. 2004, 2;32(1) Page e3
Emerging evidence suggests that recombinant adeno-associated viral (rAAV) vectors can be used for specific gene targeting in human somatic cells. We have developed an rAAV vector construction procedure employing fusion PCR and a single cloning step that considerably simplifies the knockout process. We demonstrate its utility by disrupting genes at specific positions within human colon cancer cells as well as within immortalized normal epithelial cells. This technology should be broadly applicable to in vitro studies that require the manipulation of the human genome.
PTEN gene targeting reveals a radiation-induced size checkpoint in human cancer cells
Cancer Res., 2004, 64(19), Pages 6906-6914.
Following DNA damage, human cells arrest primarily in the G1 and G2 phases of the cell cycle. Here, we show that after irradiation, human cancer cells with targeted deletion of PTEN or naturally occurring PTEN mutations can exert G1 and G2 arrests but are unable to arrest in size. Pharmacological inhibition of phosphoinositol-3-kinase or mTOR in PTEN–/– cells restored the size arrest, whereas siRNA-mediated depletion of TSC2 in PTEN+/+ cells attenuated the size arrest. Radiation treatment potentiated Akt activation in PTEN–/– but not PTEN+/+ cells. Finally, abrogation of the size arrest via PTEN deletion conferred radiosensitivity both in vitro and in vivo. These results identify a new tumor suppressor gene-regulated, DNA damage-inducible arrest that occurs simultaneously with the G1 and G2 arrests but is genetically separable from them. We suggest that aberrant regulation of cell size during cell cycle arrest may be important in human cancer pathogenesis.
The Chk2 tumor suppressor is not required for p53 responses in human cancer cells
J Biol Chem, 2003, 278(23), Pages 20475-204579
Ionizing radiation damages chromosomal DNA and activates p53-dependent transcription in mammalian cells. The Chk2 protein kinase has been hypothesized to be the primary mediator of this response. We have rigorously tested this hypothesis in human cells by disrupting the CHK2 gene through homologous recombination. We found that the p53 response was unexpectedly robust in such cells. Phosphorylation of p53 at serine 20, accumulation of p53 protein, transcriptional activation of p53 target genes, and cell cycle arrest and apoptotic death phenotypes were completely intact regardless of CHK2 status. Our results indicate that Chk2 kinase is not required for p53 activation in human cells and explain why CHK2 and TP53 mutations can jointly occur in human tumors.
PUMA mediates the apoptotic response to p53 in colorectal cancer cells
PNAS, 2003, 18;100(4), Pages 1931-1936
Although several genes that might mediate p53-induced apoptosis have been proposed, none have previously been shown to play an essential role in this process through a rigorous gene disruption approach. We used a gene-targeting approach to evaluate p53-mediated death in human colorectal cancer cells. Expression of p53 in these cells induces growth arrest through transcriptional activation of the cyclin-dependent kinase inhibitor p21. If p21 is disrupted via gene targeting, the cells die through apoptosis. If the PUMA gene is also disrupted in such cells, apoptosis is prevented. The effects of PUMA on apoptosis were observed after exogenous overexpression of p53 as well as after exposure to hypoxia, a physiologic activator of p53, and DNA damage. The PUMA protein interacts with Bcl-X(L) and promotes mitochondrial translocation and multimerization of Bax. Accordingly, genetic disruption of BAX makes cells resistant to the apoptosis resulting from PUMA expression. These results suggest that the balance between PUMA and p21 is pivotal in determining the responses to p53 activation and provide a model for understanding the basis of p53 mutations in human cancer.
Haploinsufficiency of hTERT leads to telomere dysfunction and radiosensitivity in human cancer cells
Cancer Biol Ther., 2003, 2(6), Pages 679-684.
One of the most consistent differences between cancer cells and normal somatic cells is the continuous expression of telomerase, an enzyme that is important for maintenance of chromosome ends, or telomeres. It is believed that telomerase expression allows cancer cells to maintain their telomeres after many cell divisions and thereby avoid replicative senescence. We have tested this hypothesis by targeting the gene encoding the catalytic subunit of the telomerase holoenzyme, hTERT, in a human cancer cell line. Heterozygous disruption of hTERT led to a reduction in telomerase activity, telomere shortening, activation of DNA damage signaling and the appearance of a subpopulation of cells that displayed features of senescence. Targeted cells were radiosensitive, as compared with parental controls that had two intact hTERT alleles, and expressed a classical marker of senescence after irradiation. These results suggest that telomerase inhibitors might be useful in the sensitization of cancer cells to DNA damaging agents.
Human somatic cell knockouts reveal determinants of sensitivity and resistance to proteasome inhibitor PS-341
Cancer Biol Ther., 2003,2(6):Pages 700-701.
No abstract available.
Targeted inactivation of CTNNB1 reveals unexpected effects of beta-catenin mutation
PNAS 2002, 11;99(12), Pages 8265-8270
Inactivating mutations of the adenomatous polyposis coli gene (APC) or activating mutations of the beta-catenin gene (CTNNB1) initiate colorectal neoplasia. To address the biochemical and physiologic effects of mutant beta-catenin, we disrupted either the mutant or wild-type CTNNB1 allele in a human colorectal cancer cell line. Cells with only wild-type beta-catenin had decreased colony-forming ability when plated at low density, although their growth was similar to that of parental cells when passaged under routine conditions. Immunohistochemistry and cell-fractionation studies suggested that mutant beta-catenin activity was distinguished primarily by cellular localization and not by protein degradation. Surprisingly, we found mutant beta-catenin bound less well to E-cadherin than did wild-type beta-catenin, and the membranous localization of wild-type and mutant beta-catenin was accordingly distinct. These findings pose several challenges to current models of APC/beta-catenin function.
DNMT1 and DNMT3b cooperate to silence genes in human cancer cells
Nature, 2002, 416(6880), Pages 552-556
Inactivation of tumour suppressor genes is central to the development of all common forms of human cancer. This inactivation often results from epigenetic silencing associated with hypermethylation rather than intragenic mutations. In human cells, the mechanisms underlying locus-specific or global methylation patterns remain unclear. The prototypic DNA methyltransferase, Dnmt1, accounts for most methylation in mouse cells, but human cancer cells lacking DNMT1 retain significant genomic methylation and associated gene silencing12. We disrupted the human DNMT3b gene in a colorectal cancer cell line. This deletion reduced global DNA methylation by less than 3%. Surprisingly, however, genetic disruption of both DNMT1 and DNMT3b nearly eliminated methyltransferase activity, and reduced genomic DNA methylation by greater than 95%. These marked changes resulted in demethylation of repeated sequences, loss of insulin-like growth factor II (IGF2) imprinting, abrogation of silencing of the tumour suppressor gene p16 INK4a, and growth suppression. Here we demonstrate that two enzymes cooperatively maintain DNA methylation and gene silencing in human cancer cells, and provide compelling evidence that such methylation is essential for optimal neoplastic proliferation.
DEC1 is a downstream target of TGF-beta with sequence-specific transcriptional repressor activities
PNAS, 2002, 99(5), Pages 2848-2853
To identify genes that mediate transforming growth factor-beta (TGF-beta) signaling, a colorectal cancer cell line that was sensitive to the growth inhibitory effects of this cytokine was created. We then determined the global gene expression profiles of these cells, and those of HaCaT human keratinocytes, in the presence and absence of TGF-beta. Of the several genes identified in this screen, DEC1 was of particular note in light of the rapidity and consistency of its induction and its potential biochemical activities. We identified a consensus DNA-binding site for DEC1 and showed that DEC1 could repress the transcription of a reporter containing this binding site in its promoter. Finally, both alleles of the DEC1 locus in HaCaT cells were inactivated through targeted homologous recombination. This approach revealed that DEC1 induction was not required for the growth inhibition mediated by TGF-beta in this line. However, DEC1 may function in concert with other signaling components to mediate certain biologic effects of TGF-beta.
Oncogenic beta-catenin is required for bone morphogenetic protein 4 expression in human cancer cells
Cancer Res., 2002, 15;62(10), Pages 2744-2748.
Somatic cell gene targeting was used to create an isogenic set of human colon cancer cells that differs only in the presence or absence of their endogenous activated beta-catenin oncogene. Affymetrix Genechip expression profiling of parental cells and gene-targeted derivatives identified numerous novel genes whose expression was dependent on the presence of oncogenic beta-catenin. The transforming growth factor-beta family member bone morphogenetic protein 4 (BMP4), whose receptor is mutated in a rare inherited gastrointestinal cancer predisposition syndrome, was the most highly differentially expressed gene. Additional experiments revealed that the oncogenic allele of beta-catenin specifically is absolutely required for BMP4 expression and secretion by human cancer cells and that BMP4 is overexpressed and secreted by human colon cancer cells with mutant adenomatous polyposis coli genes. These data identify the presence of regulatory interactions between the Wnt and BMP signaling pathways in cancer pathogenesis, providing an intriguing connection between the sporadic and inherited forms of a common human malignancy.
Targeted inactivation of p53 in human cells does not result in aneuploidy
Cancer Res., 2002, 62(4), Pages 1129-33
Because p53 mutation and aneuploidy usually coexist, it has been suggested that p53 inactivation leads to aneuploidy. We have rigorously tested this hypothesis in diploid human cell lines in which p53 was experimentally inactivated by targeted homologous recombination. Cells completely deficient in p53 did not become aneuploid, although a slight tendency toward tetraploidization was observed. No increased rates of numerical or structural chromosomal instabilities were observed in the p53-deficient cells. Rates of sister chromatid exchange and homologous recombination were also unaffected by p53 status. These results show that inactivation of p53 does not, in and of itself, lead to the development of aneuploidy.
Proof-of-principle: oncogenic beta-catenin is a valid molecular target for the development of pharmacological inhibitors
Mol Cancer Ther., 2002,1(14), Pages 1355-1359.
Activation of beta-catenin is a critical step in the pathogenesis of many common human cancers and is the initiating event in adenocarcinoma of the colon. Because activation of beta-catenin provides a gain-of-function, it is tempting to speculate that specific pharmacological inhibition of activated beta-catenin might reverse the tumorigenic properties of human cancer cells and therefore form the basis of an effective anticancer strategy. In an effort to provide proof-of-principle for such a strategy, we used a novel clonal growth assay based on human somatic cell gene targeting to determine whether activated beta-catenin remains a necessary oncogenic stimulus in advanced human cancer cells. Using this approach, we demonstrate that beta-catenin is a necessary oncogene in human SW48 and DLD1 colon cancer cells but not in HCT116 cells. These data indicate that activated beta-catenin can remain a critical oncogenic stimulus throughout the progression of human colon cancer and suggest that the small molecule inhibitors of activated beta-catenin currently under development will be effective anticancer therapeutics in a subset of malignant colon cancers.
Securin is required for chromosomal stability in human cells
Cell. 2001, 105(4), Pages 445-57
Abnormalities of chromosome number are the most common genetic aberrations in cancer. The mechanisms regulating the fidelity of mitotic chromosome transmission in mammalian cells are therefore of great interest. Here we show that human cells without an hSecurin gene lose chromosomes at a high frequency. This loss was linked to abnormal anaphases during which cells underwent repetitive unsuccessful attempts to segregate their chromosomes. The abnormal mitoses were associated with biochemical defects in the activation of separin, the sister-separating protease, rendering it unable to cleave the cohesin subunit Scc1 efficiently. These results illuminate the function of mammalian securin and show that it is essential for the maintenance of euploidy.
Genetic disruption of PPARdelta decreases the tumorigenicity of human colon cancer cells
PNAS., 2001, 98(5), Pages 2598-2603.
Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that have been implicated in a variety of biologic processes. The PPARdelta isotype was recently proposed as a downstream target of the adenomatous polyposis coli (APC)/beta-catenin pathway in colorectal carcinogenesis. To evaluate its role in tumorigenesis, a PPARdelta null cell line was created by targeted homologous recombination. When inoculated as xenografts in nude mice, PPARdelta -/- cells exhibited a decreased ability to form tumors compared with PPARdelta +/- and wild-type controls. These data suggest that suppression of PPARdelta expression contributes to the growth-inhibitory effects of the APC tumor suppressor.
High-throughput drug screening of the DPC4 tumor-suppressor pathway in human pancreatic cancer cells
Ann Surg., 2001, 233(5), Pages 696-703.
Various tumor-suppressor genes are mutated in all human cancers. Specifically, DPC4 (deleted in pancreatic carcinoma, locus 4 or MADH4/SMAD4) is a tumor-suppressor gene mutated in approximately 50% of human pancreatic adenocarcinomas. DPC4 plays an important role in the well-studied transforming growth factor-beta (TGFbeta) signaling pathway. It would be useful to identify therapies that augment or restore the downstream functions of this critical signal transduction pathway, in hopes that such therapy would have a rational role in anticancer therapy. METHODS: Using a commercially available plasmid vector with a luciferase reporter gene already incorporated, a DPC4-specific reporter construct was genetically engineered. This was done by inserting six copies of the palindromic Smad binding element (6SBE), which is a DNA binding element specific for DPC4, in front of the minimal promoter in the plasmid. This construct was then stably integrated into the genome of a human pancreatic cancer cell line (PANC-1) that has wild-type DPC4. Several stably transfected clones were tested for basal luciferase expression and inducibility with TGFbeta, which is known to activate the DPC4 signal transduction pathway. A single transfected clone was chosen for the drug screen based on basal luciferase (reporter) expression and TGFbeta inducibility. A systematic screen of the chemical library was then performed, using luciferase activity to detect DPC4 activity and induction of the signaling pathway. RESULTS: A high-throughput system based on this stably integrated reporter system was used to screen a library of 16,320 random compounds to identify agents that conferred robust augmentation of the DPC4 signal transduction pathway. Of the 16,320 compounds screened, 11 were associated with a 2- to 5-fold induction of luciferase activity, and one with a 12-fold activation. The latter compound was shown to be a novel histone deacetylase inhibitor and was further characterized. These results confirm the feasibility of a specific high-throughput reporter system to screen a large compound library in human cells efficiently. The screening identified several compounds capable of augmenting DPC4-specific luciferase reporter activity, and a specific mechanism for one compound was identified. The discovery of such agents will aid our understanding of complex tumor-suppressive signaling pathways and may identify other potential therapeutic targets within this critical signaling pathway. In addition, random drug screening provides an unbiased method for identifying drugs or lead compounds for potential therapeutic use.
Role of BAX in the apoptotic response to anticancer agents
Science. 2000, 290(5493), Pages 989-992.
To assess the role of BAX in drug-induced apoptosis in human colorectal cancer cells, we generated cells that lack functional BAX genes. Such cells were partially resistant to the apoptotic effects of the chemotherapeutic agent 5-fluorouracil, but apoptosis was not abolished. In contrast, the absence of BAX completely abolished the apoptotic response to the chemopreventive agent sulindac and other nonsteroidal anti-inflammatory drugs (NSAIDs). NSAIDs inhibited the expression of the antiapoptotic protein Bcl-XL, resulting in an altered ratio of BAX to Bcl-XL and subsequent mitochondria-mediated cell death. These results establish an unambiguous role for BAX in apoptotic processes in human epithelial cancers and may have implications for cancer chemoprevention strategies.
Cooperative effects of genes controlling the G(2)/M checkpoint
Genes Dev. 2000 Jul 1;14(13):1584-8.
It is believed that multiple effectors independently control the checkpoints permitting transitions between cell cycle phases. However, this has not been rigorously demonstrated in mammalian cells. The p53-induced genes p21 and 14-3-3ς are each required for the G2 arrest and allow a specific test of this fundamental tenet. We generated human cells deficient in both p21 and 14-3-3ς and determined whether the double knockout was more sensitive to DNA damage than either single knockout. p21−/− 14-3-3ς−/− cells were significantly more sensitive to DNA damage or to the exogenous expression of p53 than cells lacking only p21 or only 14-3-3ς. Thus, p21 and 14-3-3ς play distinct but complementary roles in the G2/M checkpoint, and help explain why genes at the nodal points of growth arrest pathways, like p53, are the targets of mutation in cancer cells.
14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage
Nature., 1999, 401(6753), Pages 616-20.
14-3-3Sigma is a member of a family of proteins that regulate cellular activity by binding and sequestering phosphorylated proteins. It has been suggested that 14-3-3sigma promotes pre-mitotic cell-cycle arrest following DNA damage, and that its expression can be controlled by the p53 tumour suppressor gene. Here we describe an improved approach to the generation of human somatic-cell knockouts, which we have used to generate human colorectal cancer cells in which both 14-3-3sigma alleles are inactivated. After DNA damage, these cells initially arrested in the G2 phase of the cell cycle, but, unlike cells containing 14-3-3sigma, the 14-3-3sigma-/- cells were unable to maintain cell-cycle arrest. The 14-3-3sigma-/- cells died (‘mitotic catastrophe’) as they entered mitosis. This process was associated with a failure of the 14-3-3sigma-deficient cells to sequester the proteins (cyclin B1 and cdc2) that initiate mitosis and prevent them from entering the nucleus. These results may indicate a mechanism for maintaining the G2 checkpoint and preventing mitotic death.
Disruption of p53 in human cancer cells alters the responses to therapeutic agents
J Clin Invest., 1999, 104(3), Pages 263-269.
We have examined the effects of commonly used chemotherapeutic agents on human colon cancer cell lines in which the p53 pathway has been specifically disrupted by targeted homologous recombination. We found that p53 had profound effects on drug responses, and these effects varied dramatically depending on the drug. The p53-deficient cells were sensitized to the effects of DNA-damaging agents as a result of the failure to induce expression of the cyclin-dependent kinase inhibitor p21. In contrast, p53 disruption rendered cells strikingly resistant to the effects of the antimetabolite 5-fluorouracil (5-FU), the mainstay of adjuvant therapy for colorectal cancer. The effects on 5-FU sensitivity were observed both in vitro and in vivo, were independent of p21, and appeared to be the result of perturbations in RNA, rather than DNA, metabolism. These results have significant implications for future efforts to maximize therapeutic efficacy in patients with defined genetic alterations.
Requirement for p53 and p21 to sustain G2 arrest after DNA damage
Science., 1998, 282(5393), Pages 1497-1501.
After DNA damage, many cells appear to enter a sustained arrest in the G2 phase of the cell cycle. It is shown here that this arrest could be sustained only when p53 was present in the cell and capable of transcriptionally activating the cyclin-dependent kinase inhibitor p21. After disruption of either the p53 or the p21 gene, radiated cells progressed into mitosis and exhibited a G2 DNA content only because of a failure of cytokinesis. Thus, p53 and p21 appear to be essential for maintaining the G2 checkpoint in human cells.
Targeted deletion of Smad4 shows it is required for transforming growth factor beta and activin signaling in colorectal cancer cells
PNAS., 1998, 3;95(5), Pages 2412-2416.
Smad4 (DPC4) is a candidate tumor suppressor gene that has been hypothesized to be critical for transmitting signals from transforming growth factor (TGF) beta and related ligands. To directly test this hypothesis, the Smad4 gene was deleted through homologous recombination in human colorectal cancer cells. This deletion abrogated signaling from TGF-beta, as well as from the TGF-beta family member activin. These results provide unequivocal evidence that mutational inactivation of Smad4 causes TGF-beta unresponsiveness and provide a basis for understanding the physiologic role of this gene in tumorigenesis.
Repair Defect in p21 WAF1/CIP1 -/- human cancer cells
Cancer Res., 1996, 15;56(10), Pages 2250-2255.
p53 induction and cell cycle arrest occur following DNA damage, possibly to allow repair prior to replication. p21WAF1/CIP1, a cyclin-cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen-interacting protein, is induced by p53 and mediates the cell cycle arrest. To investigate a role for p21 in DNA repair in vivo, we studied the expression of in vitro damaged reporter DNA transfected into p21 +/+ or -/- HCT116 human colon cancer cells. Introduction of UV-damaged or cis-platinum-damaged cytomegalovirus-driven ß-galactosidase reporter DNA into tumor cells revealed a significant decrease (2–5-fold) in reporter expression in p21 -/- versus +/+ cells. In the absence of DNA damage, there was a significant increase (2–3-fold) in the number of 6-TG-resistant colonies derived from p21 -/- versus +/+ cells. Reintroduction of wild-type p21, but not a p21 C-terminal truncation mutant which lacks the proliferating cell nuclear antigen interaction domain, stimulated (2–3-fold) the repair capacity of the p21-deficient cells. We conclude that p21 deficiency is associated with a defect in DNA repair, which could lead to an increased sensitivity of tumor cells to DNA damage.
p21 is necessary for the p53-mediated G1 arrest in human cancer cells
Cancer Res., 1995, 55(22), Pages 5187-5190.
DNA-damaging agents induce a p53-dependent G1 arrest that may be critical for p53-mediated tumor suppression. It has been suggested that p21WAF1/CIP1, a cdk inhibitory protein transcriptionally regulated by p53, is an effector of this arrest. To test this hypothesis, an isogenic set of human colon adenocarcinoma cell lines differing only in their p21 status was created. The parental cell line underwent the expected cell cycle changes upon induction of p53 expression by DNA damage, but the G1 arrest was completely abrogated in p21-deficient cells. These results unambiguously establish p21 as a critical mediator of one well-documented p53 function and have important implications for understanding cell cycle checkpoints and the mechanism(s) through which p53 inhibits human neoplasia.
Altered growth of human colon cancer cell lines disrupted at activated Ki-ras
Science., 1993, 260(5104), Pages 85-88.
Point mutations that activate the Ki-ras proto-oncogene are presented in about 50 percent of human colorectal tumors. To study the functional significance of these mutations, the activated Ki-ras genes in two human colon carcinoma cell lines, DLD-1 and HCT 116, were disrupted by homologous recombination. Compared with parental cells, cells disrupted at the activated Ki-ras gene were morphologically altered, lost the capacity for anchorage-independent growth, grew more slowly both in vitro and in nude mice, and showed reduced expression of c-myc. Thus, the activated Ki-ras gene plays a key role in colorectal tumorigenesis through altered cell differentiation and cell growth.