Altered expression of cell proliferation-related and interferon-stimulated genes in colon cancer cells resistant to SN38.

Irinotecan is a topoisomerase I inhibitor widely used as an anticancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance. We have isolated a colon carcinoma cell line, HCT116-SN6, which displays a 6-fold higher resistance to SN38, the active metabolite of irinotecan. In this paper, we studied the molecular mechanisms that cause resistance to SN38 in the HCT116-SN6 cell line. First, we analyzed proliferation, cell cycle distribution, apoptosis, topoisomerase I expression and activity in SN38-resistant (HCT116-SN6) and sensitive (HCT116-s cells). We showed that the SN38-induced apoptosis and the SN38-activated cell cycle checkpoints leading to G(2)/M cell cycle arrest were similar in both cell lines. Topoisomerase I expression and catalytic activity were also unchanged. Then, we compared mRNA expression profiles in the two cell lines using the Affymetrix Human Genome GeneChip arrays U133A and B. Microarray analysis showed that among the genes, which were differentially expressed in HCT116-s and HCT116-SN6 cells, 27% were related to cell proliferation suggesting that proliferation might be the main target in the development of resistance to SN38. This result correlates with the phenotypic observation of a reduced growth rate in HCT116-SN6 resistant cells. Furthermore, 29% of the overexpressed genes were Interferon Stimulated Genes and we demonstrate that their overexpression is, at least partially, due to endogenous activation of the p38 MAP kinase pathway in SN38 resistant cells. In conclusion, a slower cell proliferation rate may be a major cause of acquired resistance to SN38 via a reduction of cell cycle progression through the S phase which is mandatory for the cytotoxic action of SN38. This lower growth rate could be due to the endogenous activation of p38.

Overexpression of phosphoserine aminotransferase PSAT1 stimulates cell growth and increases chemoresistance of colon cancer cells.

BACKGROUND: Colorectal cancer (CRC) is one of the most common causes of cancer death throughout the world. In this work our aim was to study the role of the phosphoserine aminotransferase PSAT1 in colorectal cancer development. RESULTS: We first observed that PSAT1 is overexpressed in colon tumors. In addition, we showed that after drug treatment, PSAT1 expression level in hepatic metastases increased in non responder and decreased in responder patients. In experiments using human cell lines, we showed that ectopic PSAT1 overexpression in colon carcinoma SW480 cell line resulted in an increase in its growth rate and survival. In addition, SW480-PSAT1 cells presented a higher tumorigenic potential than SW480 control cells in xenografted mice. Moreover, the SW480-PSAT1 cell line was more resistant to oxaliplatin treatment than the non-transfected SW480 cell line. This resistance resulted from a decrease in the apoptotic response and in the mitotic catastrophes induced by the drug treatment. CONCLUSION: These results show that an enzyme playing a role in the L-serine biosynthesis could be implicated in colon cancer progression and chemoresistance and indicate that PSAT1 represents a new interesting target for CRC therapy.

Altered memory capacities and response to stress in p300/CBP-associated factor (PCAF) histone acetylase knockout mice.

Chromatin remodeling by posttranslational modification of histones plays an important role in brain plasticity, including memory, response to stress and depression. The importance of H3/4 histones acetylation by CREB-binding protein (CBP) or related histone acetyltransferase, including p300, was specifically demonstrated using knockout (KO) mouse models. The physiological role of a related protein that also acts as a transcriptional coactivator with intrinsic histone acetylase activity, the p300/CBP-associated factor (PCAF), is poorly documented. We analyzed the behavioral phenotype of homozygous male and female PCAF KO mice and report a marked impact of PCAF deletion on memory processes and stress response. PCAF KO animals showed short-term memory deficits at 2 months of age, measured using spontaneous alternation, object recognition, or acquisition of a daily changing platform position in the water maze. Acquisition of a fixed platform location was delayed, but preserved, and no passive avoidance deficit was noted. No gender-related difference was observed. These deficits were associated with hippocampal alterations in pyramidal cell layer organization, basal levels of Fos immunoreactivity, and MAP kinase activation. PCAF KO mice also showed an exaggerated response to acute stress, forced swimming, and conditioned fear, associated with increased plasma corticosterone levels. Moreover, learning and memory impairments worsened at 6 and 12 months of age, when animals failed to acquire the fixed platform location in the water maze and showed passive avoidance deficits. These observations demonstrate that PCAF histone acetylase is involved lifelong in the chromatin remodeling necessary for memory formation and response to stress.

Gene expression signature in advanced colorectal cancer patients select drugs and response for the use of leucovorin, fluorouracil, and irinotecan.

PURPOSE: In patients with advanced colorectal cancer, leucovorin, fluorouracil, and irinotecan (FOLFIRI) is considered as one of the reference first-line treatments. However, only about half of treated patients respond to this regimen, and there is no clinically useful marker that predicts response. A major clinical challenge is to identify the subset of patients who could benefit from this chemotherapy. We aimed to identify a gene expression profile in primary colon cancer tissue that could predict chemotherapy response. PATIENTS AND METHODS: Tumor colon samples from 21 patients with advanced colorectal cancer were analyzed for gene expression profiling using Human Genome GeneChip arrays U133. At the end of the first-line treatment, the best observed response, according to WHO criteria, was used to define the responders and nonresponders. Discriminatory genes were first selected by the significance analysis of microarrays algorithm and the area under the receiver operating characteristic curve. A predictor classifier was then constructed using support vector machines. Finally, leave-one-out cross validation was used to estimate the performance and the accuracy of the output class prediction rule. RESULTS: We determined a set of 14 predictor genes of response to FOLFIRI. Nine of nine responders (100% specificity) and 11 of 12 nonresponders (92% sensitivity) were classified correctly, for an overall accuracy of 95%. CONCLUSION: After validation in an independent cohort of patients, our gene signature could be used as a decision tool to assist oncologists in selecting colorectal cancer patients who could benefit from FOLFIRI chemotherapy, both in the adjuvant and the first-line metastatic setting.

Impact of RNA degradation on gene expression profiles: assessment of different methods to reliably determine RNA quality.

DNA microarray technology enables investigators to measure the expression of several 1000 mRNA species simultaneously in a biological specimen. However, the reliability of the microarray technology to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. Thus, it is of critical importance to standardize sample-handling protocols and to perform a quality assessment of RNA preparations. In this report, 59 human tissue samples were used to evaluate the relationships between RNA quality and gene expression. From Affymetrix GeneChip array data analysis of these samples, we compared the performance of the 28S/18S ratio, two computer methods (RIN and degradometer) and our in-house RNA quality scale (RQS) in assessing RNA quality. The optimal RNA reliability threshold was determined for each method using statistical discrimination measures. We showed that RQS, RIN and degradometer have a similar capacity to detect reliable RNA samples whereas the 28S/18S ratio leads to a misleading categorization. Furthermore, we developed a new approach, based on clustering analyses of full chip expression, to control RNA quality after hybridization experiments. The combination of these methods, allowing monitoring of RNA quality prior to and after the hybridization experiments, ensured reliable and reproducible microarray data.

ABCG2 overexpression in colon cancer cells resistant to SN38 and in irinotecan-treated metastases.

Overcoming drug resistance has become an important issue in cancer chemotherapy. Among all known mechanisms that confer resistance, active efflux of chemotherapeutic agents by proteins from the ATP-binding cassette family has been extensively reported. The aim of the present study was to determine the involvement of ABCG2 in resistance to SN38 (the active metabolite of irinotecan) in colorectal cancer. By progressive exposure to increasing concentrations of SN38, we isolated 2 resistant clones from the human colon carcinoma cell line HCT116. These clones were 6- and 53-fold more resistant to SN38 than the HCT116-derived sensitive clone. Topoisomerase I expression was unchanged in our resistant variants. The highest resistance level correlated with an ABCG2 amplification. This overexpression was associated with a marked decrease in the intracellular accumulation of SN38. The inhibition of ABCG2 function by Ko143 demonstrated that enhanced drug efflux from resistant cells was mediated by the activity of ABCG2 protein and confirmed that ABCG2 is directly involved in acquired resistance to SN38. Furthermore, we show, for the first time in clinical samples, that the ABCG2 mRNA content in hepatic metastases is higher after an irinotecan-based chemotherapy than in irinotecan-naive metastases. In conclusion, this study supports the potential involvement of ABCG2 in the development of irinotecan resistance in vivo.

Drug specific resistance to oxaliplatin is associated with apoptosis defect in a cellular model of colon carcinoma.

To investigate acquired resistance to oxaliplatin, we selected two resistant clones from the HCT116 cell line. We found that the resistant phenotype was associated with resistance to oxaliplatin-induced apoptosis as demonstrated by FACS analysis and by Western blotting of caspase 3 activation. In addition, the resistant phenotype showed a concomitant resistance to lonidamine and arsenic trioxide which are inducers of mitochondrial apoptosis. Furthermore, a complete loss of Bax expression due to a frameshift mutation was observed in the most resistant clone. Taken together, these findings suggest that altered mitochondrial-mediated apoptosis could play a role in oxaliplatin resistance.