Cellular models for the study of the pharmacology and signaling of melanin-concentrating hormone receptors.

Cellular models for the study of the neuropeptide melanin-concentrating hormone (MCH) have become indispensable tools for pharmacological profiling and signaling analysis of MCH and its synthetic analogues. Although expression of MCH receptors is most abundant in the brain, MCH-R(1) is also found in different peripheral tissues. Therefore, not only cell lines derived from nervous tissue but also from peripheral tissues that naturally express MCH receptors have been used to study receptor signaling and regulation. For screening of novel compounds, however, heterologous expression of MCH-R(1) or MCH-R(2) genes in HEK293, Chinese hamster ovary, COS-7, or 3T3-L1 cells, or amplified MCH-R(1) expression/signaling in IRM23 cells transfected with the G(q) protein gene are the preferred tools because of more distinct pharmacological effects induced by MCH, which include inhibition of cAMP formation, stimulation of inositol triphosphate production, increase in intracellular free Ca(2+) and/or activation of mitogen-activated protein kinases. Most of the published data originate from this type of model system, whereas data based on studies with cell lines endogenously expressing MCH receptors are more limited. This review presents an update on the different cellular models currently used for the analysis of MCH receptor interaction and signaling.

Receptor-mediated tumor targeting with radiopeptides. Part 1. General principles and methods.

Radiolabeled peptides have become important tools for preclinical cancer research, and in nuclear oncology they serve as diagnostic and more recently also as therapeutic agents. In the latter application, radiolabeled peptides represent a distinct sector of the molecular targeting approach, which in many areas of therapy implements the old "magic bullet" concept by specifically directing the therapeutic agent to the site of action. Although in the past few years the development of receptor-mediated targeting for therapy has been confined to some radiolabeled antibodies and to somatostatin/SRIF, research into an increasing number of radiolabeled peptides and their receptors expressed by different tumors will soon lead to a wider use of peptide radiopharmaceuticals. In a consecutive series of six reviews we present a comprehensive overview of the literature on receptor-mediated tumor targeting with the different radiopeptides currently studied. Part 1 summarizes the concepts and methods of radiopeptide targeting, the selection of radioisotopes, chelators, the criteria of peptide ligand development and some general aspects of diagnostic and therapeutic application of peptide radiopharmaceuticals.

Prognostic and predictive relevance of DNAM-1, SOCS6 and CADH-7 genes on chromosome 18q in colorectal cancer.

PURPOSE: Chromosome 18q deletion has been described as a negative prognostic factor in colorectal cancer (CRC). The relationship between its supposed negative prognostic influence and the inactivation of candidate tumor suppressors deleted in colorectal cancer, Smad2 and Smad4 has not been definitively established. The aim of the present study was to evaluate the genetic status of three novel putative tumor suppressors, Cadh-7, DNAX accessory molecule-1 (Dnam-1) and suppressor of cytokine signaling (Socs6) on chromosome 18q and to correlate molecular results with patient survival and benefit from adjuvant chemotherapy. EXPERIMENTAL DESIGN: One hundred and ninety representative patient samples from a randomized multicenter study of the Swiss Group for Clinical Cancer Research of 5-fluorouracil (5-FU)- based adjuvant chemotherapy were screened for the gene copy status of Cadh-7, Socs6 and Dnam-1 using real-time quantitative PCR assay, and the molecular results were correlated with clinical outcome. RESULTS: Loss of gene copy number was found in 26.8, 37.9 and 54.2% for Cadh-7, Dnam-1 and Socs6, respectively. Only Dnam-1 deletion was an independent negative prognostic factor for the 5-year overall survival (OS) in the untreated group of patients (hazard ratio = 2.44; p = 0.01). On the contrary, loss of Cadh-7 gene copy number was a favourable prognostic factor for disease-free survival (hazard ratio = 0.43; p = 0.03) and OS (hazard ratio = 0.29; p = 0.01) in the untreated control population. Furthermore and most importantly, patients with Dnam-1 deletion who received adjuvant chemotherapy had a significantly lower risk of death compared to untreated patients with Dnam-1 deletion (hazard ratio = 0.51; p = 0.05), whereas those with Dnam-1 retention did not derive any benefit from 5-FU-based treatment (hazard ratio = 1.68; p = 0.16). CONCLUSIONS: Loss of Dnam-1 gene copy number and retention of Cadh-7 might be indicators of worse prognosis, and Dnam-1 deletion might predict for a beneficial response to adjuvant 5-FU-based chemotherapy in patients with CRC. The confirmation of our findings in large independent randomized studies is needed.

Prognostic and predictive relevance of microsatellite instability in colorectal cancer.

Microsatellite instability (MSI) is the phenotypic hallmark of a deficient DNA mismatch-repair system, observed in 10-20% of sporadic colorectal cancers (CRC). Since the prognostic and predictive value of this genetic alteration has been assessed mainly in non-randomised, uncontrolled studies, we investigated the potential of MSI to predict patient survival and response to adjuvant chemotherapy in tumour specimens from a randomised trial of the Swiss Group for Clinical Cancer Research (SAKK) that tested the value of 5-fluorouracil/mitomycin adjuvant chemotherapy. MSI status was determined in matched normal and tumour tissue samples from 160 patients using a panel of 9 microsatellite markers. There was no correlation between high frequency MSI (MSI-H) and overall (OS) or disease-free survival (DFS) in the untreated control group of patients (HR=1.13, p=0.80; and HR=0.89, p=0.81, respectively). Furthermore, MSI-H phenotype did not predict for a larger benefit of adjuvant chemotherapy on OS or DFS (HR=0.49, p=0.41; HR=0.49, p=0.41, respectively), making a potential value of this molecular marker as a predictive factor in CRC unlikely. Our data do not confirm the prognostic relevance of MSI-H status in colorectal cancer patients found in some other studies. In addition, microsatellite instability did not correlate with the extent of chemotherapy benefit, although we observed a statistically non-significant favourable impact of 5-FU-based treatment in the MSI-H group compared to MSI-L/MSS patients. Larger prospective randomised trials are required to conclusively establish a potential clinical significance of MSI in colorectal cancer.

Expression and characterization of melanin-concentrating hormone receptors on mammalian cell lines.

The neuropeptide melanin-concentrating hormone (MCH) is expressed in central and peripheral tissues where it participates in the complex network regulating energy homeostasis as well as in other physiologically important functions. Two MCH receptor subtypes, MCH-R1 and MCH-R2, have been cloned which signal through activation of Gi/o/q proteins and hence regulate different intracellular signals, such as inhibition of cAMP formation, stimulation of IP3 production, increase in intracellular free Ca2+ and/or activation of MAP kinases. Most of the data were obtained with cell systems heterologously expressing either of the MCH receptors. Fewer reports exist on studies with cell lines which endogenously express MCH receptors. Here, we describe human and other mammalian cell lines with which MCH receptor activation can be studied under "natural" conditions and we summarize the characteristics and signaling pathways of the MCH receptors in the different cell systems.

Receptor-mediated tumor targeting with radiopeptides. Part 1. General concepts and methods: applications to somatostatin receptor-expressing tumors.

Radiolabeled peptides have become important tools in nuclear oncology, both as diagnostics and more recently also as therapeutics. They represent a distinct sector of the molecular targeting approach, which in many areas of therapy will implement the old "magic bullet" concept by specifically directing the therapeutic agent to the site of action. In this three-part review, we present a comprehensive overview of the literature on receptor-mediated tumor targeting with the different radiopeptides currently studied. Part I summarizes the general concepts and methods of targeting, the selection of radioisotopes, chelators, and the criteria of peptide ligand development. Then, the >400 studies on the application to somatostatin/somatostatin-release inhibiting factor receptor-mediated tumor localization and treatment will be reviewed, demonstrating that peptide radiopharmaceuticals have gained an important position in clinical medicine.

SMAD7 is a prognostic marker in patients with colorectal cancer.

Chromosomal region 18q21 is frequently deleted in colorectal cancer (CRC) and is associated with poor prognosis. Potential tumor suppressor mechanisms altered by 18q21 deletion include mediation of TGFbeta signaling by SMADs. Following the definition of SMAD4 deletion as a negative predictive marker for chemotherapy benefit in patients with CRC, we aimed to evaluate the clinical relevance of the deletion of other SMAD genes clustered in this region: SMAD2 and SMAD7 in 264 CRC biopsies from a previous clinical study. In contrast to SMAD2 deletion, for which no clinical relevance was observed, hazard ratios (HR) in a multivariate analysis associated with SMAD7 deletion [overall survival (OS): HR = 0.43, p = 0.0012; disease-free survival (DFS): HR = 0.50, p = 0.0033] indicated a favorable outcome for these patients. In addition, SMAD7 duplication had a hazardous effect on survival [OS: HR = 2.10, p = 0.020; DFS: HR = 2.06, p = 0.015]. Moreover, the HRs associated with one additional copy of SMAD7 were 1.76, p = 0.00024 [OS] and 1.64, p = 0.00048 [DFS] respectively, showing a graded effect of SMAD7 on patient outcome depending on gene copy number that suggests a dose-and-effect basis. Since SMAD7 blocks TGFbeta signaling, these data are consistent with the loss of SMAD7 rendering carcinoma cells more sensitive to cell growth arrest/apoptotic effect of TGFbeta, whereas gain of SMAD7 function might result in TGFbeta resistance, thereby emphasizing the role of TGFbeta in tumor suppression.

DCR3 locus is a predictive marker for 5-fluorouracil-based adjuvant chemotherapy in colorectal cancer.

Adjuvant chemotherapy reduces the incidence of distant metastasis and increases survival of patients with colorectal cancer. However, predictive markers are needed to define subsets of patients with stage II and III disease that may benefit from adjuvant treatment. A secreted member of the TNF receptor superfamily, the decoy receptor 3 (DcR3), was reported to be amplified in colorectal cancer as a negative regulator of Fas-mediated apoptosis. We analyzed DcR3 gene copy number and protein expression in a large series of tumors from a randomized multicenter trial of 5-fluorouracil/mitomycin C (FU/MMC) adjuvant chemotherapy of the Swiss Group for Clinical Cancer Research (SAKK 40/81), using real-time quantitative PCR and immunohistochemistry on tumor microarrays. Results of gene status and protein expression of DcR3 were correlated with disease-free and overall survival of patients. We observed amplification of the DcR3 gene in 185/294 (63%) and overexpression of the DcR3 protein in 163/223 (73%) of colorectal tumors. Multivariate analysis showed no prognostic effect of DcR3 gene amplification and protein overexpression. However, adjuvant chemotherapy was significantly more beneficial in patients with normal DcR3 gene copy number than in patients with amplification (DFS: HR 2.84, 95% CI 1.16-6.98, p = 0.02; OS: HR 3.15, 95% CI 1.19-8.32, p = 0.02), whereas DcR3 protein overexpression did not influence the effect of adjuvant chemotherapy (DFS: HR 1.02, 95% CI 0.65-1.60, p = 0.95; OS: HR 0.95, 95% CI 0.61-1.49, p = 0.83). We conclude that amplification of the 20q13 locus is a predictive marker for adjuvant chemotherapy in colorectal cancer.