Copy-number analysis of topoisomerase and thymidylate synthase genes in frozen and FFPE DNAs of colorectal cancers.

BACKGROUND: Archived formalin-fixed, paraffin-embedded specimens represent an important resource for pharmacogenomic analysis in retrospective clinical studies but the quality of results from formalin-fixed, paraffin-embedded samples is of concern due to the fact of the degradation of DNAs and RNAs from formalin-fixed, paraffin-embedded tissues. METHODS: In the present study, we used DNA from fresh frozen as well as formalin-fixed, paraffin-embedded tumor to detect copy-number changes in colorectal cancer, and our data shows that formalin-fixed, paraffin-embedded DNAs were able to deliver reliable copy-number data, and that quantitative PCR had the ability to detect copy-number changes from deletion to amplification, with high concordance of copy-number calls among formalin-fixed, paraffin-embedded and frozen DNAs. RESULTS: The amplification of topoisomerase I and deletion of thymidylate synthase were found in 23% (12/52) and 27% (14/52) of colorectal cancers, but EGF receptor amplification was not common (5/52, <10%). Among 52 colorectal cancers, 31 tumors were both topoisomerase I and thymidylate synthase diploid, which may have a worse outcome for tumor chemotherapy; and there were five tumors with favorable genomics (topoisomerase I amplification and thymidylate synthase deletion). Furthermore, topoisomerase I-amplified tumors had a two-times higher RNA level and a nearly twofold higher protein expression level than did the diploid tumors (p < 0.001 and 0.01, respectively), but there were no correlations between copy-number status and RNA or protein level for thymidylate synthase. CONCLUSIONS: Our study suggests a potential pharmacogenomic influence of topoisomerase I copy-number alteration on its RNA/protein expressions, which could be reflected on tumor response to chemotherapy in human colorectal cancer.

Image-based evaluation of the molecular events underlying HC11 mammary epithelial cell differentiation.

We have developed an image-based technique for signal pathway analysis, target validation, and compound screening related to mammary epithelial cell differentiation. This technique used the advantages of optical imaging and the HC11-Lux model system. The HC11-Lux cell line is a subclone of HC11 mammary epithelial cells transfected stably with a luciferase construct of the beta-casein gene promoter (p-344/-1betac-Lux). The promoter activity was imaged optically in real time following lactogenic induction. The imaging signal intensity was closely correlated with that measured using a luminometer following protein extraction (R=0.99, P<0.0001) and consistent with the messenger RNA (mRNA) level of the endogenous beta -casein gene. Using this technique, we examined the roles of JAK2/Stat5A, Raf-1/MEK/MAKP, and PI3K/Akt signal pathways with respect to differentiation. The imaging studies showed that treatment of the cells with epidermal growth factor (EGF), AG490 (JAK2-specific inhibitor), and LY294002 (PI3K-specific inhibitor) blocked lactogenic differentiation in a dose-dependent manner. PD98059 (MEK-specific inhibitor) could reverse EGF-mediated differentiation arrest. These results indicate that these pathways are essential in cell differentiation. This simple, sensitive, and reproducible technique permits visualization and real-time evaluation of the molecular events related to milk protein production. It can be adopted for high-throughput screening of small molecules for their effects on mammary epithelial cell growth, differentiation, and carcinogenesis.

MAGED2: a novel p53-dissociator.

The tumor suppressor protein p53 is a transcription factor that is frequently mutated in human cancers. In response to DNA damage, unmutated or wild-type (wt) p53 protein is stabilized and activated by post-transcriptional modifications that enable it to induce either apoptosis or cell cycle arrest. Using a yeast p53-dissociator assay, we identified MAGED2 as a potential negative regulator of wt p53 activity. Subsequently, using co-immunoprecipitation and reporter gene assays in human cultured cells that are often adopted for functional analysis of p53 we demonstrated that MAGED2 interacted physically with p53 and modified its activity. Finally, we were able to illustrate expression of both p53 and MAGED2 within the same subcellular compartment, i.e. either nucleus or cytoplasm, in 2,682 human cancer tissue specimens using a common cancer tissue microarray and antibodies against MAGED2 and p53. The present results implicate MAGED2, a novel protein, as a p53-dissociator.

DNA repair pathway profiling and microsatellite instability in colorectal cancer.

BACKGROUND: The ability to maintain DNA integrity is a critical cellular function. DNA repair is conducted by distinct pathways of genes, many of which are thought to be altered in colorectal cancer. However, there has been little characterization of these pathways in colorectal cancer. METHOD: By using the TaqMan real-time quantitative PCR, RNA expression profiling of 20 DNA repair pathway genes was done in matched tumor and normal tissues from 52 patients with Dukes' C colorectal cancer. RESULTS: The relative mRNA expression level across the 20 DNA repair pathway genes varied considerably, and the individual variability was also quite large, with an 85.4 median fold change in the tumor tissue genes and a 127.2 median fold change in the normal tissue genes. Tumor-normal differential expression was found in 13 of 20 DNA repair pathway genes (only XPA had a lower RNA level in the tumor samples; the other 12 genes had significantly higher tumor levels, all P<0.01). Coordinated expression of ERCC6, HMG1, MSH2, and POLB (RS>or=0.60) was observed in the tumor tissues (all P<0.001). Apoptosis index was not correlated with expression of the 20 DNA repair pathway genes. MLH1 and XRCC1 RNA expression was correlated with microsatellite instability status (P=0.045 and 0.020, respectively). An inverse correlation was found between tumor MLH1 RNA expression and MLH1 DNA methylation (P=0.003). CONCLUSION: Our study provides an initial characterization of the DNA repair pathways for understanding the cellular DNA damage/repair system in human colorectal cancer.

Differential expression of the AP-1 transcription factor family members in human colorectal epithelial and neuroendocrine neoplasms.

We immunohistochemically examined 75 human colorectal neoplasms (adenoma, 27; adenocarcinoma, 24; neuroendocrine carcinoma, 24) for the expression of activator protein (AP)-1 family proteins. Nuclear and cytoplasmic expression levels of c-Jun and Fra-1 proteins were markedly elevated in adenomas, adenocarcinomas and neuroendocrine carcinomas compared with nonneoplastic colorectal epithelial cells. JunB also was overexpressed in these tumors but with a predominantly cytoplasmic staining pattern. Overexpression of Fra-2 was evident in carcinomas but less frequent in adenomas. Expression levels of JunD and c-Fos were high in nonneoplastic colorectal epithelial cells and remained so in neoplasms. FosB was undetectable in nonneoplastic and neoplastic colorectal tissues. Neuroendocrine carcinomas exhibited an AP-1 expression profile similar to adenocarcinomas except for infrequent overexpression of c-Jun in poorly differentiated variants. Hierarchical clustering separated the majority of malignant from benign tumors based on AP-1 expression patterns. AP-1 transcription factor family members are expressed differentially in nonneoplastic and neoplastic colorectal tissues. Up-regulation of c-Jun and Fra-1 is an early event in human colorectal tumorigenesis. Overexpression of Fra-2 may participate in tumor progression.

Altered expression of TFF-1 and CES-2 in Barrett's Esophagus and associated adenocarcinomas.

Identification of biomarkers to recognize individuals with Barrett's esophagus (BE) predisposed to develop malignancy is currently a pressing issue. We utilized gene expression profiling to compare molecular signatures of normal esophagus and stomach, BE, and adenocarcinoma (AC) to identify such potential biomarkers. Over 22,000 genes were analyzed by oligonucleotide microarrays on 38 unique RNA Unsupervised and supervised clusterings were performed on a subset of 2849 genes that varied most significantly across the specimens. Immunohistochemistry (IHC) for two of the significantly differentially expressed gene products was performed on tissue microarrays. Unsupervised clustering identified two discernable molecular BE profiles, one of which was similar to normal gastric tissue ("BE1"), and another that was shared by several of the AC specimens ("BE2"). The BE1 profile included expression of several genes that have been described as tumor-suppressor genes, most notably trefoil factor 1 (TFF-1). The BE2 profile included expression of genes previously found overexpressed in cancers, such as carboxylesterase-2 (CES-2). IHC demonstrated the loss of TFF-1 late in the progression of BE to AC. It also revealed CES-2 as being upregulated in AC documented to have arisen in the presence of BE. These potential biomarkers, as well as the relative expression of genes from BE1 versus those from BE2, may be validated in the future to aid in risk stratification and guide treatment protocols in patients with BE and associated AC.

A mouse model for developing treatment for secondary liver tumors.

Metastatic colorectal cancer is the second most common cause of cancer mortality. The liver is a common site of metastasis and only a minority of patients with liver metastases are candidates for potentially curative surgical resection. The treatment of patients with unresectable liver tumors is a major clinical problem and survival remains low. Many animal models of hepatic metastasis do not result in disease which resembles the advanced cancer setting. The purpose of this study was to establish a murine model for use in the evaluation of therapy for secondary liver cancer. Human colon cancer cells were injected directly into the portal vein of nude mice. Magnetic resonance imaging (MRI), performed weekly, was used to follow the time-course and characteristics of tumor growth. As expected, tumor size increased proportionately with time, following inoculation, and the MRI images correlated well with gross pathology findings at necropsy with respect to both tumor size and location. The tumors retained important morphology and biological characteristics of human colon cancer. Mice bearing liver metastasis were treated with irinotecan or drug vehicle. MRI evaluation pre/post-therapy gave an objective measure of therapeutic response. Irinotecan therapy was able to double the survival (median 76-93 days) compared to vehicle alone (median 43-46 days). This murine model is reproducible, rapid, inexpensive and has an excellent success rate for the development of liver metastasis (100%). When used in conjunction with small animal MRI, this model allows the efficient evaluation of the therapeutics of liver metastasis without the use of repeated laparotomy or splenectomy and without requiring large numbers of animals undergoing terminal experiments.

Analysis of variation in mouse TPMT genotype, expression and activity.

Although the mouse has great potential for pharmacogenomic discovery, little is known about variation in drug response or genetic variation in pharmacologically relevant genes between inbred mouse strains. We therefore assessed variation in gene sequence, mRNA expression and protein activity of thiopurine methyltransferase (TPMT) in multiple inbred mouse strains. TPMT activity was measured by high-performance liquid chromatography detection of 6-MMP produced by incubation of liver homogenates with 6-MP. Genetic variation was assessed by resequencing and single nucleotide polymorphism (SNP) genotyping using pyrosequencing technology. mRNA expression was measured by real-time polymerase chain reaction. We observed an almost five-fold variation in TPMT activity, with strains falling into distinct low and high activity groups. This pattern of TPMT activity was highly correlated with expression of TPMT mRNA among strains, and high TPMT expression is dominant in F1 hybrids. To correlate genotype with phenotype, 29 SNPs and one insertion/deletion were genotyped throughout the TPMT gene and upstream 10 kb. Only two haplotypes were observed across all 30 polymorphisms, corresponding to the low and high activity groups. These results suggest that differential mouse TPMT activity is due to variation in mRNA expression. In addition, the identified pattern of low haplotype diversity suggests that the mouse is likely to be useful for pharmacogenomic discovery by associating haplotype blocks with drug response phenotypes among inbred strains.

Pharmacogenomic profiling of the PI3K/PTEN-AKT-mTOR pathway in common human tumors.

The protein synthetic machinery is activated by a variety of genetic alterations during tumor progression and represents an attractive target for cancer therapy. The mammalian target of rapamycin (mTOR) plays an important role in regulating protein translation through phosphorylation of p70 S6 kinase 1 (S6K1), a protein involved in ribosome biogenesis, and 4E-BP1 (eIF-4E binding protein), a translation repressor. It has been shown that mTOR has a direct linkage to the phosphatidylinositol-3'-kinase (PI3K)/PTEN-AKT survival pathway. Recent studies have demonstrated that mTOR inhibition by rapamycin or its analogues have remarkable activity against a wide range of human cancers in vitro and in human tumor xenograft models. Phase I clinical evaluations also suggested an anti-tumor effect of rapamycin analogue such as CCI-779. The clinical challenge for the application of this class of anticancer drug is the ability to prospectively identify which tumors will be sensitive to mTOR inhibition. Recent studies have identified cellular markers that are associated with the in vitro activity of rapamycin or CCI-779. However, there have been no reports on how these cellular markers are expressed together in human tumor specimen. In this study, multiple components of the PI3K/PTEN-AKT-mTOR pathway were evaluated by immunohistochemistry in tissue arrays containing 124 tumors from 8 common tumor types. The results show variable expression of all the signaling proteins. For example, mTOR expression was low in brain tumors, but high in the rest of tumors. High levels of 4E-BP1 were seen in colonic adenocarcinoma and low levels in lymphoma. Phospho-AKT (p-AKT) and phospho-S6K1 (p-S6K1) were the only proteins that had significantly correlated protein expression (rs=0.51, p<0.001). Since low PTEN, high p-AKT and high p-S6K1 expression render tumors sensitive to mTOR inhibition in vitro, these criteria were used to model tumor sensitivity. Overall, 26% of tumors (32/124) are predicted to be sensitive to mTOR inhibition, with variable rates for different tumors (melanoma 0% vs ovarian 41%). This is the first report on the PI3K/PTEN-AKT-mTOR pathway in common human tumors and evaluation of the coordinated expression of different signaling proteins. This study should provide a useful tool for selecting future targeted phase II and III clinical trials in the development of this exciting class of agents.

Therapeutic synergy between irinotecan and 5-fluorouracil against human tumor xenografts.

PURPOSE: Although the combination of irinotecan and 5-Fluorouracil is clinically active, it is associated with significant toxicity and resistance. Studies were carried out to define the optimal dosage, sequence, and timing for the combination in mice bearing xenografted human tumors. EXPERIMENTAL DESIGN: The maximum tolerated dose of irinotecan and 5-Fluorouracil in combination was determined in nude mice. Therapeutic efficacy against established human colon carcinoma xenografts, HCT-8 and HT-29, and human head and neck squamous cell carcinoma xenografts, FaDu and A253, was determined using the rugs individually, simultaneously, and in sequence with various intervals in between. Treatments were i.v. weekly x 4. Immunohistochemical and reverse transcription-PCR measurements of relevant drug-metabolizing enzymes, apoptosis-related proteins, cell cycle distribution, cyclin A, and S phase fraction expression were carried out and compared with the therapeutic outcome. RESULTS: The maximum tolerated dose of irinotecan resulted in cure rates of 30% or less in all xenografts. No cures were achieved with FUra alone. Concurrent administration of irinotecan and FUra, or of FUra 24 h before irinotecan, resulted in cure rates of <20%, except for FaDu (60%). Administration of irinotecan 24 h before FUra resulted in the highest cure rates, 80% in HCT-8, 0% in HT-29, 100% in FaDu, and 10% in A253. CONCLUSIONS: The optimal therapeutic synergy was achieved when irinotecan was administered 24 h before 5-Flurouracil. Sensitivity to this combination was associated with poor differentiation status, higher cyclin A index, recruitment of cells into S phase, and induction of Bax expression and apoptosis.

Human carboxylesterase 2 is commonly expressed in tumor tissue and is correlated with activation of irinotecan.

The prodrug irinotecan is an active agent for the treatment of advanced colorectal cancer and a number of other solid tumors. Irinotecan is converted in vivo to SN-38 (7-ethyl-10-hydroxy-camptothecin), the active metabolite that causes cell death, by human liver carboxylesterases. Previous studies suggest that human carboxylesterase 2 (CES2) is the key activating isoform. Although conversion of irinotecan to SN-38 by liver carboxylesterase is an inefficient process, clinical data indicate that irinotecan has significant antitumor activity. This scenario raises the possibility that local conversion of irinotecan to SN-38 by CES2 in tumor tissues might occur. The expression profile of CES2 protein in human tumor tissues was evaluated in a tissue array of 18 different types of human cancer and in a panel of normal human liver samples by immunohistochemistry and Western blot, respectively. Cytosolic CES2 expression was observed in 101 of 154 tumors (66%) and 55 of 60 normal tissues (92%). Among the 18 types of tumors analyzed, 2 types (gallbladder tumor and lymphoma) did not express CES2, 5 types expressed weak CES2, and 11 types expressed moderate to intense CES2. In functional studies, CES2 protein was highly variable among liver samples, with a 15-fold range in cytosol and a 3-fold range in microsome fractions. Liver microsomal CES2 protein expression was significantly correlated with irinotecan activation to SN-38 (R(s) = 0.70; P = 0.007). This study confirms that CES2 is a key enzyme for irinotecan activation. Tumor CES2 expression may contribute to variable response to irinotecan chemotherapy for solid tumors.

Comprehensive evaluation of carboxylesterase-2 expression in normal human tissues using tissue array analysis.

Carboxylesterases play an important role in the hydrolytic biotransformation of a number of structurally diverse endogenous compounds and medications. Several distinct carboxylesterase isoforms have been described in human liver, brain, and placenta. Carboxylesterase-2 has been identified as the key enzyme in the metabolic activation of the irinotecan, a topoisomerase I inhibitor commonly used in the treatment of many solid tumors. The tissue distribution and intensity of protein expression of carboxylesterase-2 have not been defined in any organ or tissue. This study used a carboxylesterase-2-specific antibody and tissue array analysis to detect carboxylesterase-2 expression in human normal tissues by immunohistochemistry. Carboxylesterase-2 is present in a wide variety of organs and tissues. The highest carboxylesterase-2 expression occurs in hepatocyte, small intestine mucosa, kidney proximal convoluted tubule, and adrenal cortex cells. The results suggest that liver and gastrointestinal tract with carboxylesterase-2 are likely the most important sites of conversion of irinotecan to the active metabolite SN-38, but carboxylesterase-2 within the other tissues may be contributive to this process. In the central nervous system, carboxylesterase-2 expression was confined to capillary endothelial cells, consistent with the enzyme having a role to protect the central nervous system from toxic esters and perhaps being a component of a blood-brain barrier system.

Three-dimensional image of hepatocellular carcinoma under confocal laser scanning microscope.

AIM:To investigate the application of confocal laser scanning microscopy (CLSM) in tumor pathology and three-dimensional (3-D) reconstruction by CLSM in pathologic specimens of hepatocellular carcinoma (HCC).METHODS:The 30&mgr;m thick sections were cut from the paraffin embedded tissues of HCC, hyperplasia and normal liver,stained with DNA fluorescent probe YOYO-1 iodide and examined by CLSM to collect optical sections of nuclei and 3-D images reconstructed.RESULTS:HCC displayed chaotic arrangement of carcinoma cell nuclei, marked pleomorphism, indented and irregular nuclear surface, and irregular and coarse chromatin texture.CONCLUSION:The serial optical tomograms of CLSM can be used to create 3-D reconstruction of cancer cell nuclei. Such 3-D impressions might be helpful or even essential in making an accurate diagnosis.