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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.

Accumulation of the COMMD1 protein as a druggable pharmacology event to target cancer cells has not been evaluated so far in cancer animal models. We have previously demonstrated that a second-generation peptide, with cell-penetrating capacity, termed CIGB-552, was able to induce apoptosis mediated by stabilization of COMMD1. Here, we explore the antitumor effect by subcutaneous administration of CIGB-552 in a therapeutic schedule. Outstandingly, a significant delay of tumor growth was observed at 0.2 and 0.7 mg/kg (p

The para-methoxybenzyl-substituted vanadocene dichloride (Vanadocene Y) (1) and its diselenocyanate (Selenocyanato-Vanadocene Y) (2) were tested in vitro in an anti-angiogenesis assay against human umbilical vein endothelial cells (HUVEC) delivering IC50 values of 0.92 ± 0.03 μM (1) and 37 ± 11 μM (2). In a cytotoxicity assay against the human renal cancer cells, CAKI-1, the compounds demonstrated IC50 values of 0.55 ± 0.09 μM (1) and 0.25 ± 0.03 μM (2). Then both compounds were given at their maximum tolerable dose, MTD, of 20 mg/kg/d (1) or 40 mg/kg/d (2) on four consecutive days or at 50% of the MTD on five consecutive days per week for three weeks to overall four cohorts of eight CAKI-1 tumor-bearing female NMRI:nu/nu mice each, while a further cohort was treated with solvent only. Both MTD-treated mouse cohorts showed a statistically significant tumor growth reduction with respect to the solvent-treated control group with an optimal T/C value of 47% on day 39 of the experiment. Immunohistological analysis revealed that the expression of the proliferation marker Ki-67 was reduced due to long-term treatment with 2.

Accumulation of the COMMD1 protein as a druggable pharmacology event to target cancer cells has not been evaluated so far in cancer animal models. We have previously demonstrated that a second-generation peptide, with cell-penetrating capacity, termed CIGB-552, was able to induce apoptosis mediated by stabilization of COMMD1. Here, we explore the antitumor effect by subcutaneous administration of CIGB-552 in a therapeutic schedule. Outstandingly, a significant delay of tumor growth was observed at 0.2 and 0.7 mg/kg (p 0.01) or 1.4 mg/kg (p 0.001) after CIGB-552 administration in both syngeneic murine tumors and patient-derived xenograft models. Furthermore, we evidenced that (131)I-CIGB-552 peptide was actually accumulated in the tumors after administration by subcutaneous route. A typical serine-proteases degradation pattern for CIGB-552 in BALB/c mice serum was identified. Further, biological characterization of the main metabolites of the peptide CIGB-552 suggests that the cell-penetrating capacity plays an important role in the cytotoxic activity. This report is the first in describing the antitumor effect induced by systemic administration of a peptide that targets COMMD1 for stabilization. Moreover, our data reinforce the perspectives of CIGB-552 for cancer targeted therapy.

The use of next generation sequencing has expanded our view on whole mammalian methylome patterns. In particular, it provides a genome-wide insight of local DNA methylation diversity at single nucleotide level and enables the examination of single chromosome sequence sections at a sufficient statistical power. We describe a bisulfite-based sequence profiling pipeline, Bi-PROF, which is based on the 454 GS-FLX Titanium technology that allows to obtain up to one million sequence stretches at single base pair resolution without laborious subcloning. To illustrate the performance of the experimental workflow connected to a bioinformatics program pipeline (BiQ Analyzer HT) we present a test analysis set of 68 different epigenetic marker regions (amplicons) in five individual patient-derived xenograft tissue samples of colorectal cancer and one healthy colon epithelium sample as a control. After the 454 GS-FLX Titanium run, sequence read processing and sample decoding, the obtained alignments are quality controlled and statistically evaluated. Comprehensive methylation pattern interpretation (profiling) assessed by analyzing 10 (2)-10 (4) sequence reads per amplicon allows an unprecedented deep view on pattern formation and methylation marker heterogeneity in tissues concerned by complex diseases like cancer.

Azacytidine is an established nucleoside drug that is well known for its ability to modulate epigenetic gene regulation by inhibition of DNA methylation. Despite recent advances in the clinical development of azacytidine, the use of the drug is limited by its low bioavailability and dependency on variably expressed nucleoside transporters for cellular uptake. We show here that CP-4200, an elaidic acid derivative of azacytidine, has strong epigenetic modulatory potency in human cancer cell lines, as evidenced by efficient depletion of DNA methyltransferase protein, genome-wide DNA demethylation, and robust reactivation of epigenetically silenced tumor suppressor genes. Importantly, however, the cellular uptake of CP-4200 was substantially less dependent on the nucleoside transporters that are known to be involved in azacytidine uptake. In agreement with this notion, CP-4200 showed a significantly higher antitumoral activity than azacytidine in an orthotopic mouse tumor model for acute lymphocytic leukemia. Together, these data represent a detailed characterization of the CP-4200 mode of action and suggest that elaidic acid modification improves the therapeutic efficacy of azacytidine. Mol Cancer Ther; 9(5); 1256–64. ©2010 AACR.

Improved methods for the preclinical characterization of anti-cancer drugs in larger panels of primary lung cancer xenograft models have recently described (Fichtner et al. Clin Cancer Res. 2008;14(20):6456-68). These models consider more thoroughly the heterogeneity and genetic instability of tumors in a way ultimately enabeling treatment decisions on an individualized basis. The approach is based on large scale screening for activity in new in vivo models combined with extensive characterization of these models (gene expression and mutations). Bioinformatic methods are then used to further evaluate the mechanism of action of new drugs, to investigate possible resistance mechanisms and provide a rationale for combination therapies. Here we describe the development of a panel of primary patient derived colorectal cancer models. Primary colon carcinoma tissue samples were collected using a standardised procedure. Tumour pieces were transplanted onto immunodeficient mice immediately after surgery. A panel of 28 stably passagable colon cancer xenografts could be established as permanent tumour models. The results obtained so far, show that these patient-derived colon cancer models feature a high coincidence with the original tumour regarding histology and gene expression profiling. Response analysis according to clinical trial criteria revealed that the standard drug Oxaliplatin induce a overall response in 8 of the 28 xenograft models. Gene expression analyses were performed to identify predictive profiles for Oxaliplatin sensitivity. As one example, the effectiveness of Oxaliplatin seems to be related to the differential expression of genes which products are involved in the wnt pathway. The analysis of drug sensitivity in this set of models together with bioinformatics coorelations will provide hypotheses for further studies to identify patients that will more likely benefit from a drug as well as provide a rationale for combinations to be tested in clinical trials. These new approaches may help to better integrate research results in clinical development and may increase the success of new anti-cancer drugs in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1584. doi:10.1158/1538-7445.AM2011-1584

Azacytidine is an established nucleoside drug that is well known for its ability to modulate epigenetic gene regulation by inhibition of DNA methylation. Despite recent advances in the clinical development of azacytidine, the use of the drug is limited by its low bioavailability and dependency on variably expressed nucleoside transporters for cellular uptake. To investigate the function of CP-4200, an elaidic acid ester of azacytidine, we performed DNA methyltransferase depletion assays, DNA methylation analysis using capillary electrophoresis and Infinium methylation arrays, transport studies and mouse experiments. We show here that CP-4200 has strong epigenetic modulatory potency in human cancer cell lines, as evidenced by efficient depletion of DNA methyltransferase protein, genome-wide DNA demethylation and robust reactivation of epigenetically silenced tumor suppressor genes. Importantly, new results show that the uptake mechanisms of colon and leukemic cancer cell lines for CP-4200 and its parent drug azacytidine are fundamentally different, with CP-4200 being substantially less dependent on nucleoside transporters. Correspondingly, CP-4200 showed a significantly higher antitumoral activity than azacytidine in an orthotopic mouse tumor model for acute lymphocytic leukemia. These results establish a fundamentally novel approach for the delivery of azacytidine to human cancer cells which might have important therapeutic implications. Reference: Brueckner, B., et al. (2010). Mol. Cancer Ther. 9: 1256-1264. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2013. doi:10.1158/1538-7445.AM2011-2013