SMAD4 is inactivated in the majority of pancreatic ductal adenocarcinomas (PDAC) with concurrent mutational inactivation of the INK4A/ARF tumor suppressor locus and activation of the KRAS oncogene. Here, using genetically engineered mice, we determined the impact of SMAD4 deficiency on the development of the pancreas and on the initiation and/or progression of PDAC-alone or in combination with PDAC-relevant mutations. Selective SMAD4 deletion in the pancreatic epithelium had no discernable impact on pancreatic development or physiology. However, when combined with the activated KRAS G12D allele, SMAD4 deficiency enabled rapid progression of KRAS G12D -initiated neoplasms. While KRAS G12D alone elicited premalignant pancreatic intraepithelial neoplasia (PanIN) that progressed slowly to carcinoma, the combination of KRAS G12D and SMAD4 deficiency resulted in the rapid development of tumors resembling intraductal papillary mucinous neoplasia (IPMN), a precursor to PDAC in humans. SMAD4 deficiency also accelerated PDAC development of KRAS G12D INK4A/ARF heterozygous mice and altered the tumor phenotype; while tumors with intact SMAD4 frequently exhibited epithelial-to-mesenchymal transition (EMT), PDAC null for SMAD4 retained a differentiated histopathology with increased expression of epithelial markers. SMAD4 status in PDAC cell lines was associated with differential responses to transforming growth factor-␤ (TGF-␤) in vitro with a subset of SMAD4 wild-type lines showing prominent TGF-␤-induced proliferation and migration. These results provide genetic confirmation that SMAD4 is a PDAC tumor suppressor, functioning to block the progression of KRAS G12D -initiated neoplasms, whereas in a subset of advanced tumors, intact SMAD4 facilitates EMT and TGF-␤-dependent growth.[Keywords: Smad4; pancreatic cancer; epithelial-to-mesenchymal transition mouse models; TGF-␤] Supplemental material is available at http://www.genesdev.org. PDAC (pancreatic ductal adenocarcinoma) ranks as the fourth leading cause of cancer mortality in the United States and carries a median survival of <6 mo (Li et al. 2004). Hallmarks of this disease include the infiltration of the tumor with a proliferative stroma (desmoplasia), early invasion and metastasis, and pronounced genomic instability (Solcia et al. 1995). PDAC is characterized by a highly recurrent pattern of genetic lesions consisting of activating mutations of KRAS and inactivation of INK4A (via mutation, deletion, or promoter methylation) in virtually all cases, inactivation of the p53-ARF pathway in ∼87% of cases (including tumors with deletions of the INK4A/ARF locus), and SMAD4 inactivation in ∼53% (Hansel et al. 2003). Hence, SMAD4 status can be considered as a distinguishing molecular feature of two major classes of PDAC. Significant ongoing efforts are being directed toward the elucidation of how specific signature mutations contribute to the genesis and progression of PDAC and influence its tumor biological features.

Activating KRAS mutations and p16 Ink4a inactivation are near universal events in human pancreatic ductal adenocarcinoma (PDAC). In mouse models, Kras G12D initiates formation of premalignant pancreatic ductal lesions, and loss of either Ink4a͞Arf (p16 Ink4a ͞p19 Arf ) or p53 enables their malignant progression. As recent mouse modeling studies have suggested a less prominent role for p16 Ink4a in constraining malignant progression, we sought to assess the pathological and genomic impact of inactivation of p16 Ink4a , p19 Arf , and͞or p53 in the Kras G12D model. Rapidly progressive PDAC was observed in the setting of homozygous deletion of either p53 or p16 Ink4a , the latter with intact germ-line p53 and p19 Arf sequences. Additionally, Kras G12D in the context of heterozygosity either for p53 plus p16 Ink4a or for p16 Ink4a ͞p19 Arf produced PDAC with longer latency and greater propensity for distant metastases relative to mice with homozygous deletion of p53 or p16 Ink4a ͞p19 Arf . Tumors from the double-heterozygous cohorts showed frequent p16 Ink4a inactivation and loss of either p53 or p19 Arf . Different genotypes were associated with specific histopathologic characteristics, most notably a trend toward less differentiated features in the homozygous p16 Ink4a ͞p19 Arf mutant model. High-resolution genomic analysis revealed that the tumor suppressor genotype influenced the specific genomic patterns of these tumors and showed overlap in regional chromosomal alterations between murine and human PDAC. Collectively, our results establish that disruptions of p16 Ink4a and the p19 ARF -p53 circuit play critical and cooperative roles in PDAC progression, with specific tumor suppressor genotypes provocatively influencing the tumor biological phenotypes and genomic profiles of the resultant tumors.array comparative genomic hybridization ͉ mouse models ͉ pancreatic cancer ͉ KRAS ͉ tumor suppressor P ancreatic ductal adenocarcinoma (PDAC) ranks as the fourth leading cause of cancer mortality in the United States and causes Ͼ200,000 deaths worldwide annually (1, 2). Histopathological analyses have identified precursor lesions, pancreatic intraepithelial neoplasias (PanIN), which appear to progress through increasingly severe stages of cellular atypia leading to invasive PDAC (3). These lesions show multistep molecular progression that includes early activating KRAS mutations and telomere attrition, and subsequent inactivation of p16 Ink4a , p14 ARF , p53, and͞or SMAD4 tumor suppressors in a high percentage of cases (4-6).The Ink4a͞Arf locus (hereafter denoted p16 Ink4a ͞p19 Arf ) encodes tumor suppressors p16 INK4A and p14 ARF (p19 Arf in the mouse). p16 INK4A is a G 1 cyclin-dependent kinase (CDK) inhibitor that binds to CDK4 and CDK6 and prevents their association with D-type cyclins (7), thereby facilitating CDK4͞6-cyclin D-mediated phosphorylation and inactivation of retinoblastoma protein (RB) and S-phase entry. p16 INK4A -mediated tumor suppression may relate to its induction by activated oncogenes and consequen...

Chemoresistance to platinum therapy is a major obstacle that needs to be overcome in the treatment of ovarian cancer patients. The high rates and patterns of therapeutic failure seen in patients are consistent with a steady accumulation of drug-resistant cancer stem cells (CSCs). This study demonstrates that the Notch signaling pathway and Notch3 in particular are critical for the regulation of CSCs and tumor resistance to platinum. We show that Notch3 overexpression in tumor cells results in expansion of CSCs and increased platinum chemoresistance. In contrast, γ-secretase inhibitor (GSI), a Notch pathway inhibitor, depletes CSCs and increases tumor sensitivity to platinum. Similarly, a Notch3 siRNA knockdown increases the response to platinum therapy, further demonstrating that modulation of tumor chemosensitivity by GSI is Notch specific. Most importantly, the cisplatin/GSI combination is the only treatment that effectively eliminates both CSCs and the bulk of tumor cells, indicating that a dual combination targeting both populations is needed for tumor eradication. In addition, we found that the cisplatin/GSI combination therapy has a synergistic cytotoxic effect in Notch-dependent tumor cells by enhancing the DNA-damage response, G 2 /M cell-cycle arrest, and apoptosis. Based on these results, we conclude that targeting the Notch pathway could significantly increase tumor sensitivity to platinum therapy. Our study suggests important clinical applications for targeting Notch as part of novel treatment strategies upon diagnosis of ovarian cancer and at recurrence. Both platinum-resistant and platinum-sensitive relapses may benefit from such an approach as clinical data suggest that all relapses after platinum therapy are increasingly platinum resistant.

To determine the role of the phosphatidylinositol 3-kinase (PI3-K) pathway in pancreas development, we generated a pancreas-specific knockout of Pten, a negative regulator of PI3-K signaling. Knockout mice display progressive replacement of the acinar pancreas with highly proliferative ductal structures that contain abundant mucins and express Pdx1 and Hes1, two markers of pancreatic progenitor cells. Moreover, a fraction of these mice develop ductal malignancy. We provide evidence that ductal metaplasia results from the expansion of centroacinar cells rather than transdifferentiation of acinar cells. These results indicate that Pten actively maintains the balance between different cell types in the adult pancreas and that misregulation of the PI3-K pathway in centroacinar cells may contribute to the initiation of pancreatic carcinoma in vivo.

Promoter DNA methylation status of six genes in samples derived from 27 bronchial epithelial cells and matching blood samples from 22 former/current smokers and five nonsmokers as well as 49 primary non^small cell lung cancer samples with corresponding blood controls was determined using methylation-specific PCR (MSP). Lung tumor tissues showed a significantly higher frequency of promoter DNA methylation in p16, MGMT, and DAPK (P < 0.05; Fisher's exact test). p16 promoter DNA methylation in tumors was observed at consistently higher levels when compared with all the other samples analyzed (P = 0.001; Fisher's exact test). ECAD and DAPK exhibited statistically insignificant differences in their levels of DNA methylation among the tumors and bronchial epithelial cells from the smokers. Interestingly, similar levels of methylation were observed in bronchial epithelial cells and corresponding blood from smokers for all four genes (ECAD, p16, MGMT, and DAPK) that showed smoking/lung cancer^associated methylation changes. In summary, our data suggest that targeted DNA methylation silencing of ECAD and DAPK occurs in the early stages and that of p16 and MGMT in the later stages of lung cancer progression. We also provide preliminary evidence that peripheral lymphocytes could potentially be used as a surrogate for bronchial epithelial cells to detect altered DNA methylation in smokers.Lung cancer is the leading cause of cancer deaths in the United States, accounting for 28% of all cancer deaths and f157,200 deaths every year (1). Unfortunately, 36% of nonsmall cell lung cancer cases are detected at an advanced stage often after micrometastasis has developed, leading to an alarmingly low 5-year survival rate of only 14.9% (1). Because nearly 87% of lung cancer cases are due to smoking, yet only 10% of smokers develop lung cancer, it may be worthwhile to screen for susceptible individuals to administer effective preventive measures (1, 2). Thus, the establishment of a combination of early diagnostic markers that could be analyzed in clinical samples obtained using relatively noninvasive procedures could become an asset to efficient detection of changes in preneoplastic tissue before tumor formation and metastasis can occur.Differential DNA methylation at CpG islands has been associated with regulation of gene expression and is essential for normal development, X-chromosome inactivation, imprinting, suppression of parasitic DNA sequences, and cancer (3 -5). Aberrant differential methylation of CpG islands in the promoter region of genes that are implicated in different roles including carcinogen activation or detoxification (CYP1A1 and GSTP1), tumor suppression (p14, p15, p16, p73, APC, and BRCA1), DNA repair (hMLH1 and MGMT), and metastasis and invasion (CDH1, ECAD, TIMP1, and DAPK) occurs in several cancers, including lung cancer (3 -10). Thus, the DNA methylation status of critical genes is not only ideal for use as diagnostic markers but also as therapeutic targets for lung cancer. In this study, we chose to a...

Intravenous electrocardiography guidance to position catheters obtains a satisfactory catheter tip placement that is in accordance with transesophageal echocardiography views. The surface landmark technique does not result in reliable placement at the superior vena cava-right atrium junction.

BackgroundThe complexity of the human plasma proteome represents a substantial challenge for biomarker discovery. Proteomic analysis of genetically engineered mouse models of cancer and isolated cancer cells and cell lines provide alternative methods for identification of potential cancer markers that would be detectable in human blood using sensitive assays. The goal of this work is to evaluate the utility of an integrative strategy using these two approaches for biomarker discovery.Methodology/Principal FindingsWe investigated a strategy that combined quantitative plasma proteomics of an ovarian cancer mouse model with analysis of proteins secreted or shed by human ovarian cancer cells. Of 106 plasma proteins identified with increased levels in tumor bearing mice, 58 were also secreted or shed from ovarian cancer cells. The remainder consisted primarily of host-response proteins. Of 25 proteins identified in the study that were assayed, 8 mostly secreted proteins common to mouse plasma and human cancer cells were significantly upregulated in a set of plasmas from ovarian cancer patients. Five of the eight proteins were confirmed to be upregulated in a second independent set of ovarian cancer plasmas, including in early stage disease.Conclusions/SignificanceIntegrated proteomic analysis of cancer mouse models and human cancer cell populations provides an effective approach to identify potential circulating protein biomarkers.

Dilution end point loss of heterozygosity (LOH) analysis, a novel approach for the analysis of LOH, was used to evaluate allelic losses with the use of 21 highly polymorphic microsatellite markers at nine chromosomal sites most frequently affected in smoking-related non-small cell lung cancers. Allelotyping was done for bronchial epithelial cells and matching blood samples from 23 former and current smokers and six nonsmokers as well as in 33 adenocarcinomas and 25 squamous cell carcinomas (SCC) and corresponding matching blood from smokers. Major conclusions from these studies are as follows: (a) LOH at chromosomal sites 8p, 9p, 11q, and 13q (P > > 0.05, Fisher's exact test) are targeted at the early stages, whereas LOH at 1p, 5q, 17p, and 18q (P < < 0.05, Fisher's exact test) occur at the later stages of non-small cell lung cancer progression; (b) LOH at 1p, 3p, 5q, 8p, 9p, 11q, 13q, 17p, and 18q occurs in over 45% of the tobacco smokers with SCC and adenocarcinoma; (c) compared with bronchial epithelial cells from smokers, there is a significantly higher degree of LOH at 1p, 5q, and 18q in adenocarcinoma and at 1p, 3p, and 17p in SCC (P < < 0.05, Fisher's exact test). We propose that lung cancer progression induced by tobacco smoke occurs in a series of target gene inactivations/activations in defined modules of a global network. The gatekeeper module consists of multiple alternate target genes, which is inclusive of but not limited to genes localized to chromosomal loci 8p, 9p, 11q, and 13q.