AKT-induced tamoxifen resistance is overturned by RRM2 inhibition.

UNLABELLED: Acquired tamoxifen resistance develops in the majority of hormone-responsive breast cancers and frequently involves overexpression of the PI3K/AKT axis. Here, breast cancer cells with elevated endogenous AKT or overexpression of activated AKT exhibited tamoxifen-stimulated cell proliferation and enhanced cell motility. To gain mechanistic insight on AKT-induced endocrine resistance, gene expression profiling was performed to determine the transcripts that are differentially expressed post-tamoxifen therapy under conditions of AKT overexpression. Consistent with the biologic outcome, many of these transcripts function in cell proliferation and cell motility networks and were quantitatively validated in a larger panel of breast cancer cells. Moreover, ribonucleotide reductase M2 (RRM2) was revealed as a key contributor to AKT-induced tamoxifen resistance. Inhibition of RRM2 by RNA interference (RNAi)-mediated approaches significantly reversed the tamoxifen-resistant cell growth, inhibited cell motility, and activated DNA damage and proapoptotic pathways. In addition, treatment of tamoxifen-resistant breast cancer cells with the small molecule RRM inhibitor didox significantly reduced in vitro and in vivo growth. Thus, AKT-expressing breast cancer cells upregulate RRM2 expression, leading to increased DNA repair and protection from tamoxifen-induced apoptosis. IMPLICATIONS: These findings identify RRM2 as an AKT-regulated gene, which plays a role in tamoxifen resistance and may prove to be a novel target for effective diagnostic and preventative strategies.

Regional hypermethylation and global hypomethylation are associated with altered chromatin conformation and histone acetylation in colorectal cancer.

Regional DNA hypermethylation and global DNA hypomethylation are 2 epigenetic alterations associated with colorectal cancers. However, their correlation with microsatellite instability (MSI) and chromosomal instability (CIN) in colorectal cancer, and their relationship with chromatin conformation and histone modification are not clear. In this study, we analyzed regional and global methylation in 16 cell lines and 64 primary colorectal cancers. We found that MSI and CIN are 2 alternative events in most cell lines and tumors. Furthermore, regional hypermethylation and global hypomethylation are also alternative events in most cases. We also observed a strong correlation between MSI and regional hypermethylation and between CIN and global hypomethylation. We further analyzed chromatin conformation and histone acetylation in cell lines with CIN or MSI. CIN cancers had open chromatin conformation and enriched histone acetylation in repetitive as well as in gene-specific regions. MSI cancers, on the other hand, had closed chromatin conformation and low levels of histone acetylation. After a MSI cell line was treated with 5-aza-2'-deoxycytidine or trichostatin A, the closed chromatin conformation became open, and histone acetylation was enriched. These observations support our hypothesis that in colorectal cancer, regional hypermethylation and global hypomethylation are associated with altered chromatin conformation and histone acetylation, which might have a causal correlation with MSI and CIN, respectively.

Fractional genomic alteration detected by array-based comparative genomic hybridization independently predicts survival after hepatic resection for metastatic colorectal cancer.

PURPOSE: Although liver resection is the primary curative therapy for patients with colorectal hepatic metastases, most patients have a recurrence. Identification of molecular markers that predict patients at highest risk for recurrence may help to target further therapy. EXPERIMENTAL DESIGN: Array-based comparative genomic hybridization was used to investigate the association of DNA copy number alterations with outcome in patients with colorectal liver metastasis resected with curative intent. DNA from 50 liver metastases was labeled and hybridized onto an array consisting of 2,463 bacterial artificial chromosome clones covering the entire genome. The total fraction of genome altered (FGA) in the metastases and the patient's clinical risk score (CRS) were calculated to identify independent prognostic factors for survival. RESULTS: An average of 30 +/- 14% of the genome was altered in the liver metastases (14% gained and 16% lost). As expected, a lower CRS was an independent predictor of overall survival (P = 0.03). In addition, a high FGA also was an independent predictor of survival (P = 0.01). The median survival time in patients with a low CRS (score 0-2) and a high (> or =20%) FGA was 38 months compared with 18 months in patients with a low CRS and a low FGA. Supervised analyses, using Prediction Analysis of Microarrays and Significance Analysis of Microarrays, identified a set of clones, predominantly located on chromosomes 7 and 20, which best predicted survival. CONCLUSIONS: Both FGA and CRS are independent predictors of survival in patients with resected hepatic colorectal cancer metastases. The greater the FGA, the more likely the patient is to survive.

High-resolution analysis of DNA copy number alterations in colorectal cancer by array-based comparative genomic hybridization.

Array-based comparative genomic hybridization (CGH) allows for the simultaneous examination of thousands of genomic loci at 1-2 Mb resolution. Copy number alterations detected by array-based CGH can aid in the identification and localization of cancer causing genes. Here we report the results of array-based CGH in a set of 125 primary colorectal tumors hybridized onto an array consisting of 2463 bacterial artificial chromosome clones. On average, 17.3% of the entire genome was altered in our samples (8.5 +/- 6.7% gained and 8.8 +/- 7.3% lost). Losses involving 8p, 17p, 18p or 18q occurred in 37, 46, 49 and 60% of cases, respectively. Gains involving 8q or 20q were observed 42 and 65% of the time, respectively. A transition from loss to gain occurred on chromosome 8 between 41 and 48 Mb, with 25% of cases demonstrating a gain of 8p11 (45-53 Mb). Chromosome 8 also contained four distinct loci demonstrating high-level amplifications, centering at 44.9, 60, 92.7 and 144.7 Mb. On 20q multiple high-level amplifications were observed, centering at 32.3, 37.8, 45.4, 54.7, 59.4 and 65 Mb. Few differences in DNA copy number alterations were associated with tumor stage, location, age and sex of the patient. Microsatellite stable and unstable (MSI-H) tumors differed significantly with respect to the frequency of alterations (20 versus 5%, respectively, P < 0.01). Interestingly, MSI-H tumors were also observed to have DNA copy number alterations, most commonly involving 8q. This high-resolution analysis of DNA copy number alterations in colorectal cancer by array-based CGH allowed for the identification of many small, previously uncharacterized, genomic regions, such as on chromosomes 8 and 20. Array-based CGH was also able to identify DNA copy number changes in MSI-H tumors.

A pituitary-derived MEG3 isoform functions as a growth suppressor in tumor cells.

Human pituitary adenomas are the most common intracranial neoplasm. Typically monoclonal in origin, a somatic mutation is a prerequisite event in tumor development. To identify underlying pathogenetic mechanisms in tumor formation, we compared the difference in gene expression between normal human pituitary tissue and clinically nonfunctioning pituitary adenomas by cDNA-representational difference analysis. We cloned a cDNA, the expression of which was absent in these tumors, that represents a novel transcript from the previously described MEG3, a maternal imprinting gene with unknown function. It was expressed in normal human gonadotrophs, from which clinically nonfunctioning pituitary adenomas are derived. Additional investigation by Northern blot and RT-PCR demonstrated that this gene was also not expressed in functioning pituitary tumors as well as many human cancer cell lines. Moreover, ectopic expression of this gene inhibits growth in human cancer cells including HeLa, MCF-7, and H4. Genomic analysis revealed that MEG3 is located on chromosome 14q32.3, a site that has been predicted to contain a tumor suppressor gene involved in the pathogenesis of meningiomas. Taken together, our data suggest that MEG3 may represent a novel growth suppressor, which may play an important role in the development of human pituitary adenomas.

DNA damage-induced inhibition of securin expression is mediated by p53.

Tumor suppressor p53 induces the cellular response to DNA damage mainly by regulating expression of its downstream target genes. The human securin is an anaphase inhibitor, preventing premature chromosome separation through inhibition of separase activity. It is also known as the product of the human pituitary tumor-transforming gene, pttg, a proto-oncogene. Here we report that the expression of human securin is suppressed in cells treated with the DNA-damaging drugs doxorubicin and bleomycin. This suppression requires functional p53. Analysis of the human securin promoter reveals that DNA-binding sites for Sp1 and NF-Y are both required for activation of securin expression; however, only the NF-Y site is essential for the suppression by p53. Our study indicates that securin is a p53 target gene and may play a role in p53-mediated cellular response to DNA damage.