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Expression of the epidermal growth factor ligands amphiregulin (AREG) and epiregulin (EREG) is positively correlated with a response to EGFR-targeted therapies in colorectal cancer. Gene-body methylation sites, which show a strong inverse correlation with AREG and EREG gene expression, were identified in cell lines using targeted 454 FLX-bisulfite sequencing and SIRPH analyses for AREG/EREG promoters and intragenic CpGs. Upon treatment of colorectal cancer cells with 5-aza-2'-desoxycytidine, methylation decreases at specific intragenic CpGs accompanied by upregulation of AREG and EREG gene expression. The same AREG gene-body methylation was also found in human colorectal cancer samples and is independent of KRAS and NRAS mutations. Methylation is specifically decreased in the tumor epithelial compartment as compared to stromal tissue and normal epithelium. Investigation of a promoter/enhancer function of the AREG exon 2 region revealed a potential promoter function in reverse orientation. Retrospective comparison of the predictive power of AREG gene-body methylation versus AREG gene expression using samples from colorectal cancer patients treated with anti-EGFR inhibitors with complete clinical follow-up revealed that AREG expression is superior to AREG gene methylation. AREG and EREG genes undergo a complex regulation involving both intragenic methylation and promoter-dependent control.

Lung cancer is a highly aggressive neoplasm with 85% mortality. To identify new tumor-associated genes, we compared the expression profile of a primary metastasizing adenocarcinoma with normal airway epithelial cells. Two cDNA libraries of up- and downregulated genes were generated, comprising 253 and 299 clones, respectively. The sequence analysis revealed 205 different known genes and 314 cDNA fragments of unknown functions. Northern-blot analysis of 167 clones confirmed differential expression in 58%, and indicated a similar expression pattern in additional lung-cancer cell lines for selected clones, strengthening the value of this model for the identification of new candidate genes in lung carcinogenesis.

Despite the steadily increasing number of patients suffering from squamous-cell carcinomas of the oropharyngeal region, little is known about the molecular steps involved in the induction of these neoplasms. We investigated oropharyngeal cancers from 38 patients for mutations in the p53 tumour-suppressor gene. The majority of patients (74%) had a history of tobacco and alcohol abuse. Five had lymph-node metastases, 3 had multiple primary carcinomas and 2 presented with multiple primary tumours and lymph-node metastases. Exons 5 through 8 of the p53 gene were screened by single-strand conformation polymorphism analysis followed by direct DNA sequencing. A total of 16 tumours (42%) contained point mutations which were scattered throughout exons 5 to 8. Most mutations (56%) were transitions, predominantly G-->A. Among the transversions, G-->T mutations prevailed; these have also been found in smoking-related lung cancer. One carcinoma of the soft palate showed a mutation which was retained in a lymph-node metastasis. In another patient, 2 primary carcinomas had different mutations, indicating that they had developed independently. Similar results were obtained in a case with a p53 mutation in the third of 3 primary tongue carcinomas which developed over a period of 23 years. One lymph-node metastasis had a 12-bp deletion which was not detected in any of the primary malignancies. The frequent occurrence of p53 mutations in oropharyngeal carcinomas supports the view that they play a role in the initiation or progression of the malignant phenotype.

Most malignant features of cancer cells are triggered by activated oncogenes and the loss of tumor suppressors due to mutation or epigenetic inactivation. It is still unclear, to what extend the escape of emerging cancer cells from recognition and elimination by the immune system is determined by similar mechanisms. We compared the transcriptomes of HCT116 colorectal cancer cells deficient in DNA methyltransferases (DNMTs) and of cells, in which the RAS pathway as the major growth-promoting signaling system is blocked by inhibition of MAPK. We identified the MHC Class I genes HLA-A1/A2 and the ULBP2 gene encoding 1 of the 8 known ligands of the activating NK receptor NKG2D among a cluster of immune genes up-regulated under the conditions of both DNMT-deficiency and MEK-inhibition. Bisulphite sequencing analyses of HCT116 with DNMT deficiency or after MEK-inhibition showed that de-methylation of the ULPB2 promoter correlated with its enhanced surface expression. The HLA-A promoters were not methylated indicating that components of the HLA assembly machinery were also suppressed in DNMT-deficient and MEK-inhibited cells. Increased HLA-A2 surface expression was correlated with enhanced recognition and lysis by A2-specific CTL. On the contrary, elevated ULBP2 expression was not reflected by enhanced recognition and lysis by NK cells. Cosuppression of HLA Class I and NKG2D ligands and genes encoding peptide transporters or proteasomal genes mediates a strong functional link between RAS activation, DNMT activity and disruption of the antigen presenting system controlling immune recognition in colorectal cancer cells.

Ovarian cancer is one of the most lethal gynecological malignancies and the small success rate of routine therapeutic methods justifies efforts to develop new approaches. Evaluation of targets for effective inhibition of ovarian cancer cell growth should precipitate clinical application of gene silencing therapy. In our previous work, we showed upregulation of HMGA2 gene expression as a result of Ras-induced rat ovarian surface epithelial cell transformation. This gene codes the HMGA2 protein, a member of the high-mobility group AT-hook (HMGA) family of nonhistone chromatin proteins. Genome-wide studies revealed upregulation of the HMGA2 gene in human ovarian carcinomas. Herein we have evaluated over-expression of the HMGA2 gene, relevant to ovarian cancer, in subsets of human specimens and cell lines by in situ RNA hybridization and RT-PCR. Transient silencing of HMGA2 gene by means of siRNA inhibited proliferation of those ovarian cancer cells, which over-express this gene initially. Growth suppression was mediated by cell-cycle arrest. Stable silencing of highly expressed HMGA2 gene by shRNAi in A27/80, Ovcar-3 and OAW-42 ovarian cancer cell lines resulted in growth inhibition because of G1 arrest and increase of apoptosis as well. The tumor growth inhibition effect of HMGA2 silencing for Ovcar-3 cells was validated in vivo. Our findings revealed that the HMGA2 gene represents a promising target for gene silencing therapy in ovarian cancer.

Reactive oxygen species (ROS) are known to be involved in different pro- and anticarcinogenic mechanisms. However, their influence on the maintenance of the transformed phenotype has not been studied so far. Here we show that the anchorage-independent growth of transformed murine fibroblasts is inhibited by antioxidants and radical scavengers in a concentration-dependent and reversible manner. These agents also reduce TGF-beta-dependent stimulation of colony formation in soft agar, pointing to their specific interference with TGF-beta-triggered signal chains involved in the maintenance of the transformed state.

Cancer patients with tumors of similar grading, staging and histogenesis can have markedly different treatment responses to different chemotherapy agents. So far, individual markers have failed to correctly predict resistance against anticancer agents. We tested 30 cancer cell lines for sensitivity to 5-fluorouracil, cisplatin, cyclophosphamide, doxorubicin, etoposide, methotrexate, mitomycin C, mitoxantrone, paclitaxel, topotecan and vinblastine at drug concentrations that can be systemically achieved in patients. The resistance index was determined to designate the cell lines as sensitive or resistant, and then, the subset of resistant vs. sensitive cell lines for each drug was compared. Gene expression signatures for all cell lines were obtained by interrogating Affymetrix U133A arrays. Prediction Analysis of Microarrays was applied for feature selection. An individual prediction profile for the resistance against each chemotherapy agent was constructed, containing 42–297 genes. The overall accuracy of the predictions in a leave-one-out cross validation was 86%. A list of the top 67 multidrug resistance candidate genes that were associated with the resistance against at least 4 anticancer agents was identified. Moreover, the differential expressions of 46 selected genes were also measured by quantitative RT-PCR using a TaqMan micro fluidic card system. As a single gene can be correlated with resistance against several agents, associations with resistance were detected all together for 76 genes and resistance phenotypes, respectively. This study focuses on the resistance at the in vivo concentrations, making future clinical cancer response prediction feasible. The TaqMan-validated gene expression patterns provide new gene candidates for multidrug resistance. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat. © 2005 Wiley-Liss, Inc.

The TIG3 gene is a retinoic acid inducible class II tumor suppressor gene downregulated in several human tumors and malignant cell lines. Diminished TIG3 expression correlates with decreased differentiation whereas forced expression of TIG3 suppresses oncogenic signaling pathways and subsequently induces differentiation or apoptosis in tumor cells. Analysis of TIG3 mRNA expression in a large set of cDNA pools derived from matched tumor and normal human tissues showed a significant downregulation of TIG3 in 29% of the cDNA samples obtained from ovarian carcinomas. Using in situ hybridization, we demonstrated expression of TIG3 in the epithelial lining of 7 normal ovaries but loss of TIG3 expression in 15/19 of human ovarian carcinoma tissues. In SKOV-3, CAOV-3 and ES-2 ovarian carcinoma cell lines, downregulation of TIG3 mRNA was reversible and dependent on an activated MEK-ERK signaling pathway. Re-expression of TIG3 mRNA in these cells upon specific interference with the MEK-pathway was correlated with growth inhibition of the cells. In OVCAR-3 and A27/80 ovarian carcinoma cells, TIG3 suppression is MEK-ERK independent, but expression could be reconstituted upon interferon gamma (IFNγ) induction. Overexpression of TIG3 in A27/80 ovarian carcinoma cells significantly impaired cell growth and despite increased mRNA levels, TIG3 protein was hardly detectable. These results suggest that TIG3 is negatively regulated by an activated MEK-ERK signaling pathway. Further mechanisms must interfere with TIG3 expression that are independent of MEK and partially include interferon-responsive components. © 2005 Wiley-Liss, Inc.

Rat-1 cells which had been transformed with the activated Ha-ras-1 gene from human EJ bladder carcinoma cells were fused with diploid embryonic rat fibroblasts. Four selected cell hybrids expressed the human transforming gene product p21 at levels of 10 to 30% compared to 100% in the transformed parental cells. The hybrid cells, however, exhibited normal morphology, anchorage requirement for proliferation, and largely extended latency periods of tumorigenicity in newborn rats. Tumorigenic hybrid derivatives contained lower numbers of chromosomes than the tetraploid parental hybrids. DNA of the non-tumorigenic cell hybrids transformed Rat-1 cells to anchorage-independent proliferation as expected for the transforming human Ha-ras gene present in the donor DNA. We conclude that the transforming properties of the activated Ha-ras gene in Rat-1 cells can be suppressed at the post-translational level by the presence of the genome from diploid embryonic rat fibroblasts but additional controls of expression of the transforming gene are likely to exist. Normal cells contain suppressor gene(s) which safeguard these cells against transformation by the product of the transforming Ha-ras-1 oncogene.