Thomas K. Weber

Erlotinib, an effective epidermal growth factor receptor tyrosine kinase inhibitor, induces p27KIP1 up-regulation and nuclear translocation in association with cell growth inhibition and G1/S phase arrest in human non-small-cell lung cancer cell lines.

Erlotinib, a small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has been shown to have potent antitumor effects against human non-small-cell lung cancer (NSCLC) cell growth; however, the mechanism of such an effect is not elucidated. Here, we demonstrate that erlotinib-induced cell growth inhibition in EGFR high-expressing human H322 NSCLC cells was accompanied by G1/S phase arrest, which was largely caused by a decrease in expression of G1/S-related cyclins, suppression of activities of cyclin-dependent kinase (CDK) 2 and CDK4, induction of CDK inhibitor p27KIP1, and retinoblastoma hypophosphorylation. To further understand the role of p27KIP1 in G1/S arrest and cell growth inhibition by erlotinib, we determined its effect on the expression of p27KIP1 at transcriptional and posttranscriptional levels. Studies using real-time reverse transcription-polymerase chain reaction analysis and p27 promoter-driven luciferase reporter showed that erlotinib treatment resulted in the promotion of p27 gene transcription. In addition, erlotinib treatment led to an increase in p27KIP1 half-life by inhibiting p27KIP1 phosphorylation at Thr187 and by down-regulating Skp2 expression. Furthermore, immunofluorescence staining and cell fractionation showed that erlotinib treatment led to p27KIP1 translocation to the nucleus. Knockdown of p27KIP1 expression with p27KIP1 small interfering RNA significantly abrogated erlotinib-induced G1 phase arrest and cell growth inhibition, suggesting that induction of p27KIP1 is required for G1 arrest and cell growth inhibition by erlotinib. It is noteworthy that we found that G1 arrest and p27KIP1 up-regulation by erlotinib occurred in the tested sensitive cell lines but to a lesser extent in the resistant cell lines. Taken together, these results suggest that erlotinib inhibits human NSCLC cell growth predominantly by inducing p27KIP1 expression and by suppressing cell-cycle events involved in the G1/S transition.

Staging Intra-Abdominal Desmoid Tumors in Familial Adenomatous Polyposis: A Search for a Uniform Approach to a Troubling Disease

INTRODUCTIONDesmoid tumors are a clinical problem in 12 to 15 percent of patients with familial adenomatous polyposis. There is no predictably effective treatment for intra-abdominal desmoid tumors, which sometimes cause significant complications by their effects on the ureters or bowel. The relative rarity and the clinical heterogeneity of intra-abdominal desmoid tumors make randomized studies difficult to do. In this article a staging system is proposed to make multi-institutional studies easier.METHODSIntra-abdominal desmoid tumors can be staged according to their size, clinical presentation and growth pattern.CONCLUSIONA way of staging intra-abdominal desmoid tumors is proposed to facilitate stratification by disease severity during collaborative studies of various treatments.

Increased screening colonoscopy rates and reduced racial disparities in the New York citywide campaign: An urban model

OBJECTIVES:In 2003, in response to low colonoscopy screening rates and significant sociodemographic disparities in colonoscopy screening in New York City (NYC), the NYC Department of Health and Mental Hygiene, together with the Citywide Colon Cancer Control Coalition, launched a multifaceted campaign to increase screening. We evaluated colonoscopy trends among adult New Yorkers aged 50 years and older between 2003 and 2007, the first five years of this campaign.METHODS:Data were analyzed from the NYC Community Health Survey, an annual, population-based surveillance of New Yorkers. Annual prevalence estimates of adults who reported a timely colonoscopy, one within the past 10 years, were calculated. Multivariate models were used to analyze changes over time in associations between colonoscopy screening and sociodemographic characteristics.RESULTS:Overall, from 2003 to 2007 the proportion of New Yorkers aged 50 years and older who reported timely colonoscopy screening increased from 41.7% to 61.7%. Racial/ethnic and sex disparities observed in 2003 were eliminated by 2007: prevalence of timely colonoscopy was similar among non-Hispanic whites, non-Hispanic blacks, Hispanics, men, and women. However, Asians, the uninsured, and those with lower education and income continued to lag in receipt of timely colonoscopies.CONCLUSIONS:The increased screening colonoscopy rate and reduction of racial/ethnic disparities observed in NYC suggest that multifaceted, coordinated urban campaigns can improve low utilization of clinical preventive health services and reduce public-health disparities.

Colon Cancer Screening Practices in New York City, 2003 Results of a Large Random-Digit Dialed Telephone Survey

New York City (NYC) has one of the highest concentrations of gastroenterologists in the country, yet only 33% of colorectal cancers in NYC are diagnosed early, and approximately 1500 New Yorkers die from colorectal cancer each year.

Differential expression of DOC-1 in microsatellite-unstable human colorectal cancer

The precise genetic mechanism of malignant transformation in DNA mismatch repair deficient, microsatellite-unstable colorectal cancer (CRC) has yet to be elucidated. We employed cDNA microarray to identify patterns of gene expression among CRC cell lines and to compare directly lines with and without microsatellite instability. This study was undertaken to test the hypothesis that microsatellite-unstable CRC cell lines demonstrate specific patterns of gene expression that differ significantly from those observed among microsatellite-stable CRC. Multiple differential expression patterns were identified. Genes demonstrating differential expression included deleted-in-oral-cancer-1 (DOC-1), a highly conserved growth suppressor. DOC-1 expression correlated with microsatellite status, with significantly decreased expression in microsatellite-unstable cell lines and constitutive expression in microsatellite-stable cell lines. We also observed alterations in the biologic behavior of p12DOC-1-deficient cell lines, with increased Su2009phase and decreased apoptosis compared to microsatellite-stable (DOC-1+) cell lines. Transfection of p12DOC-1 into SW48, which lacks p12DOC-1 expression, resulted in cell cycle and apoptosis profiles similar to other p12DOC-1+ cell lines. These results support the hypothesis that microsatellite-unstable CRC is characterized by novel patterns of gene expression different from those associated with microsatellite-stable CRC, and demonstrate that p12DOC-1 has tumor suppressor potential in colon epithelial cells.

An A13 Repeat within the 3′-Untranslated Region of Epidermal Growth Factor Receptor (EGFR) Is Frequently Mutated in Microsatellite Instability Colon Cancers and Is Associated with Increased EGFR Expression

Colorectal cancers (CRC) with microsatellite instability (MSI) have clinical, pathologic, genetic, and epigenetic features distinct from microsatellite-stable CRC. Examination of epidermal growth factor receptor (EGFR) mRNA and protein expression levels in a panel of colon cancer cell lines identified strong expression of EGFR in multiple cell lines with MSI. Although no relationship between EGFR overexpression and the length of a CA dinucleotide repeat in intron 1 was observed, a variant A13/A14 repeat sequence within the 3-untranslated region (3-UTR) of the EGFR gene was identified, which was mutated by either mononucleotide or dinucleotide adenosine deletions in 64% of MSI cell lines and 69% of MSI colon tumors. Using a Tet-Off system, we show that this mutation increases EGFR mRNA stability in colon cancer cells, providing a mechanistic basis for EGFR overexpression in MSI colon cancer cell lines. To determine whether this mutation is a driver or a bystander event in MSI colon cancer, we examined the effect of pharmacologic and molecular inhibition of EGFR in EGFR 3-UTR mutant MSI cell lines. Cell lines with an EGFR 3-UTR mutation and that were wild-type (WT) for downstream signaling mediators in the Ras/BRAF and PIK3CA/PTEN pathways were sensitive to EGFR inhibition, whereas those harboring mutations in these signaling mediators were not. Furthermore, in cell lines WT for downstream signaling mediators, those with EGFR 3-UTR mutations were more sensitive to EGFR inhibition than EGFR 3-UTR WT cells, suggesting that this mutation provides a growth advantage to this subset of MSI colon tumors.

The intestinal epithelial cell differentiation marker intestinal alkaline phosphatase (ALPi) is selectively induced by histone deacetylase inhibitors (HDACi) in colon cancer cells in a Kruppel-like factor 5 (KLF5)-dependent manner.

Background: Differentiation induction represents a potential cancer treatment strategy. Results: Colon cancer cell lines respond differentially to HDACi-mediated induction of the differentiation marker ALPi. HDACi induction of ALPi is KLF5-dependent. Conclusion: HDACi induce ALPi in a subset of colon cancer cell lines in a KLF5-dependent manner. Significance: Colon cancer cell lines are differentially responsive to HDACi-induced differentiation. The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect.

Immortalization and neoplastic transformation of normal rat colon epithelium: an in vitro model of colonic neoplastic progression.

BACKGROUNDnBecause of the refractory nature of colon epithelium to growth and maintenance in vitro, the cell lines currently available for study are derived from tumors. Unlike most epithelial model systems, there exist no preneoplastic, nontumorigenic colon cell lines for manipulation and study.nnnMETHODSnIntact fetal rat colon was cultured in the presence of a feeder layer of cells producing a retrovirus that harbors the SV40 LT gene resulting in the establishment of immortalized colon cell lines.nnnRESULTSnThe epithelial and intestinal origin of cell lines was established from the constitutive expression of keratin and villin, respectively. All cell lines displayed an absence of anchorage independent growth and failed to produce tumors in vivo. Neoplastic transformation of immortalized rat colon epithelial cell lines was achieved following introduction of individual oncogenic ras gene members or the v-src oncogene. Probing of cell lysates with phosphotyrosine antibodies revealed altered phosphotyrosyl protein profiles associated with different stages of colonic neoplastic progression.nnnCONCLUSIONSnThe establishment of immortalized nontumorigenic colon epithelial cell lines facilitates the biochemical analysis of events associated with different stages of colonic neoplastic progression. In addition, this simple culture technique lends itself to studies involving alternative genetic elements implicated in the genesis of colon tumors.

Deciphering the Colon Cancer Genes-Report of the InSiGHT-Human Variome Project Workshop, UNESCO, Paris 2010

The Human Variome Project (HVP) has established a pilot program with the International Society for Gastrointestinal Hereditary Tumours (InSiGHT) to compile all inherited variation affecting colon cancer susceptibility genes. An HVP‐InSiGHT Workshop was held on May 10, 2010, prior to the HVP Integration and Implementation Meeting at UNESCO in Paris, to review the progress of this pilot program. A wide range of topics were covered, including issues relating to genotype–phenotype data submission to the InSiGHT Colon Cancer Gene Variant Databases (chromium.liacs.nl/LOVD2/colon_cancer/home.php). The meeting also canvassed the recent exciting developments in models to evaluate the pathogenicity of unclassified variants using in silico data, tumor pathology information, and functional assays, and made further plans for the future progress and sustainability of the pilot program. Hum Mutat 32:1–4, 2011.

Modulation of CDK2-AP1 (p12DOC-1) expression in human colorectal cancer

We have previously demonstrated an association between microsatellite instability and decreased CDK2-AP1 (p12DOC−1) expression in human colorectal cancer (CRC) cell lines. In those same studies, induction of CDK2-AP1 expression promoted both cell cycle arrest and apoptosis. The goals of our present study were to better understand the mechanisms leading to reduced CDK2-AP1 expression in microsatellite unstable (MSI) CRC and to study further the effect of CDK2-AP1 modulation on cell proliferation and apoptosis utilizing RNA interference (RNAi) techniques. We used direct sequencing to screen for mutations of the poly (T)8 microsatellite-like region in the 3′ end of the CDK2-AP1 gene in 24 CRC cell lines. We then utilized an in vitro human mismatch repair (MMR) recombinant system to assess for correction of the mutation and changes in CDK2-AP1 expression secondary to hMLH1 transfection. We also investigated the effect of CDK2-AP1 modulation in four settings: (1) native CDK2-AP1 absence, (2) endogenous CDK2-AP1 expression, (3) RNAi-induced CDK2-AP1 inhibition and (4) induced CDK2-AP1 over expression. The mutation – del T poly (T)8 – at the 3′ end of the CDK2-AP1 gene was found in 3/12 (25%) of MSI CRC cell lines, but in none of the microsatellite stable samples (0/12). Interestingly, when wild-type MMR protein – MLH1 – was induced in an in vitro human recombinant system, the del T poly (T)8 mutation was reversed and CDK2-AP1 expression increased. RNAi-mediated CDK2-AP1 inhibition was associated with decreased apoptosis and increased cell proliferation in CDK2-AP1-non deficient CRC cell lines. We conclude that mutations in the microsatellite-like sequence of the CDK2-AP1 gene in MSI CRC are associated with decreased CDK2-AP1 expression. In addition, modulation of CDK2-AP1 expression in human CRC alters cell proliferation and apoptosis.