Highly Expressed Genes in Rapidly Proliferating Tumor Cells as New Targets for Colorectal Cancer Treatment.

PURPOSE: The clinical management of colorectal cancer patients has significantly improved because of the identification of novel therapeutic targets such as EGFR and VEGF. Because rapid tumor proliferation is associated with poor patient prognosis, here we characterized the transcriptional signature of rapidly proliferating colorectal cancer cells in an attempt to identify novel candidate therapeutic targets. EXPERIMENTAL DESIGN: The doubling time of 52 colorectal cancer cell lines was determined and genome-wide expression profiling of a subset of these lines was assessed by microarray analysis. We then investigated the potential of genes highly expressed in cancer cells with faster growth as new therapeutic targets. RESULTS: Faster proliferation rates were associated with microsatellite instability and poorly differentiated histology. The expression of 1,290 genes was significantly correlated with the growth rates of colorectal cancer cells. These included genes involved in cell cycle, RNA processing/splicing, and protein transport. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and protoporphyrinogen oxidase (PPOX) were shown to have higher expression in faster growing cell lines and primary tumors. Pharmacologic or siRNA-based inhibition of GAPDH or PPOX reduced the growth of colon cancer cells in vitro. Moreover, using a mouse xenograft model, we show that treatment with the specific PPOX inhibitor acifluorfen significantly reduced the growth of three of the seven (42.8%) colon cancer lines investigated. CONCLUSIONS: We have characterized at the transcriptomic level the differences between colorectal cancer cells that vary in their growth rates, and identified novel candidate chemotherapeutic targets for the treatment of colorectal cancer.

Brush border myosin Ia inactivation in gastric but not endometrial tumors.

Brush border Myosin Ia (MYO1A) has been shown to be frequently mutated in colorectal tumors with microsatellite instability (MSI) and to have tumor suppressor activity in intestinal tumors. Here, we investigated the frequency of frameshift mutations in the A8 microsatellite in exon 28 of MYO1A in MSI gastric and endometrial tumors and found a high mutation rate in gastric (22/47; 46.8%) but not endometrial (3/48; 6.2%) tumors. Using a regression model, we show that MYO1A mutations are likely to confer a growth advantage to gastric, but not endometrial tumors. The mutant MYO1A(7A) protein was shown to lose its membrane localization in gastric cancer cells and a cycloheximide-chase assay demonstrated that the mutant MYO1A(7A) protein has reduced stability compared to the wild type MYO1A. Frequent MYO1A promoter hypermethylation was also found in gastric tumors. Promoter methylation negatively correlates with MYO1A mRNA expression in a series of 58 non-MSI gastric primary tumors (Pearson's r = -0.46; p = 0.0003) but not in a cohort of 54 non-MSI endometrial tumors and treatment of gastric cancer cells showing high MYO1A promoter methylation with the demethylating agent 5-aza-2'-deoxycytidine, resulted in a significant increase of MYO1A mRNA levels. We found that normal gastric epithelial cells, but not normal endometrial cells, express high levels of MYO1A. Therefore, when considered together, our findings suggest that MYO1A has tumor suppressor activity in the normal gastric epithelium but not in the normal endometrium and inactivation of MYO1A either genetically or epigenetically may confer gastric epithelial cells a growth advantage.

Brush border myosin Ia has tumor suppressor activity in the intestine.

The loss of the epithelial architecture and cell polarity/differentiation is known to be important during the tumorigenic process. Here we demonstrate that the brush border protein Myosin Ia (MYO1A) is important for polarization and differentiation of colon cancer cells and is frequently inactivated in colorectal tumors by genetic and epigenetic mechanisms. MYO1A frame-shift mutations were observed in 32% (37 of 116) of the colorectal tumors with microsatellite instability analyzed, and evidence of promoter methylation was observed in a significant proportion of colon cancer cell lines and primary colorectal tumors. The loss of polarization/differentiation resulting from MYO1A inactivation is associated with higher tumor growth in soft agar and in a xenograft model. In addition, the progression of genetically and carcinogen-initiated intestinal tumors was significantly accelerated in Myo1a knockout mice compared with Myo1a wild-type animals. Moreover, MYO1A tumor expression was found to be an independent prognostic factor for colorectal cancer patients. Patients with low MYO1A tumor protein levels had significantly shorter disease-free and overall survival compared with patients with high tumoral MYO1A (logrank test P = 0.004 and P = 0.009, respectively). The median time-to-disease recurrence in patients with low MYO1A was 1 y, compared with >9 y in the group of patients with high MYO1A. These results identify MYO1A as a unique tumor-suppressor gene in colorectal cancer and demonstrate that the loss of structural brush border proteins involved in cell polarity are important for tumor development.

ERM/ETV5 up-regulation plays a role during myometrial infiltration through matrix metalloproteinase-2 activation in endometrial cancer.

We have described recently the Ets family transcription factor, ERM/ETV5, specifically up-regulated in endometrioid endometrial carcinoma (EEC) and associated with myometrial infiltration. Ets family members have been correlated to tumor progression by up-regulating the expression of matrix-degrading proteases. In the present study, we investigated the possibility that in EEC, ERM/ETV5 may act by inducing the expression of genes involved in extracellular matrix remodeling. Unraveling the molecular events associated with the initiation of tumor invasion would represent an obvious improvement for EEC patients. The overexpression of ERM/ETV5 induced scattering in the endometrial cancer cell line Hec-1A, correlating to increased matrix metalloproteinase-2 (MMP-2) gelatinase activity. Both chromatin immunoprecipitation and reversion experiments with RNA interference and specific MMP-2 inhibitor showed a functional link between ERM/ETV5 overexpression and MMP-2 activation. The increased MMP-2 activity associated with overexpressed ERM/ETV5 in a mouse model conferred invasive capacity to endometrial tumors. Orthotopically implanted overexpressing ERM/ETV5 tumors presented a more aggressive and infiltrative pattern of myometrial invasion. Finally, the specific localization of ERM/ETV5 and MMP-2 at the invasive front of myometrial infiltrating human endometrial carcinomas further reinforced the hypothesis of a role for ERM/ETV5 in the early steps of endometrial dissemination. Taken together, these results lead us to propose that in EEC, ERM/ETV5 acts through MMP-2 gelatinolytic activity to confer invasive capabilities, associated with an initial switch to myometrial infiltration. They also postulate ERM/ETV5 as a valuable marker for patient stratification and a transcription pathway that should be evaluated for therapies specifically targeting the initial steps of EEC dissemination.

Tumour selection advantage of non-dominant negative P53 mutations in homozygotic MDM2-SNP309 colorectal cancer cells.

BACKGROUND: Mdm2 is a natural inhibitor of p53 function and its overexpression impairs p53 transcriptional activity. T-->G single-nucleotide polymorphism at position 309 (SNP309) of mdm2 induces overexpression of mdm2, but inhibits p53. OBJECTIVES: To determine whether SNP309 is a risk-modifier polymorphism in colorectal cancer (CRC) and whether tumour selection of P53 mutations are influenced by SNP309. METHODS: Single-stranded conformation polymorphism and automatic sequencing were performed. RESULTS: SNP309 is not associated with the risk of CRC or recurrence of tumours. These data do not over-ride the tumour-selection capabilities of P53 mutations in CRC. However, a significant association with non-dominant-negative P53 mutations (p = 0.02) was found. CONCLUSIONS: MDM2-SNP309 favours tumour selection of non-dominant negative P53 mutations in CRC, which also show an earlier age of tumour onset.

Mutational dynamics in human tumors confirm the neutral intrinsic instability of the mitochondrial D-loop poly-cytidine repeat.

Somatic mutations at a mitochondrial noncoding polycytidine (C)(n) repeat (polyC) have been associated with tumor progression. We analyzed whether these alterations are due to the inherent mutability of repeated sequences. Insertion and deletion mutations were found in colon (n = 114), stomach (n = 105), endometrium (n = 53), breast (n = 45), lung (n = 35), and prostate (n = 20) tumors. The mutation frequency in colon, gastric, and endometrial tumors was 23, 17, and 11%, respectively, which paralleled the relative extent of microsatellite instability in long mononucleotide repeats observed in tumors with mismatch repair deficiency (colon > stomach > endometrium, relative ratio 10:8:4). Colon tumors with mutations of more than one nucleotide were more advanced in tumor progression. Further, two tumors showing a T > C mutation that restored the homopolymeric repeat, harbored sequential deletion mutations of up to 4 and 6 nucleotides. These results illustrate that the increased mutability of repeated mitochondrial sequences is dependent on the repetitive structure of the DNA molecule and suggest that mutations in the (C)(n) repeat, whether homoplasmic or not, and by extrapolation, mitochondrial mutations in general, are not the result of selective pressure during tumorigenesis. We also suggest that the (C)(n) repeat may be used as an universal molecular clock to estimate the relative mitotic history of tumors.

Mechanisms of inactivation of the receptor tyrosine kinase EPHB2 in colorectal tumors.

The receptor tyrosine kinase EPHB2 has recently been shown to be a direct transcriptional target of TCF/beta-catenin. Premalignant lesions of the colon express high levels of EPHB2 but the expression of this kinase is reduced or lost in most colorectal carcinomas. In addition, inactivation of EPHB2 has been shown to accelerate tumorigenesis initiated by APC mutation in the colon and rectum. In this study, we investigated the molecular mechanisms responsible for the inactivation of EPHB2 in colorectal tumors. We show here the presence of mutations in repetitive sequences in exon 17 of EPHB2 in 6 of 29 adenomas with microsatellite instability (MSI), and 101 of 246 MSI carcinomas (21% and 41%, respectively). Moreover, we found EPHB2 promoter hypermethylation in 54 of the 101 colorectal tumors studied (53%). Importantly, EPHB2 expression was restored after treatment of EPHB2-methylated colon cancer cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. In conclusion, in this study, we elucidate the molecular mechanisms of inactivation of EPHB2 and show for the first time the high incidence of frameshift mutations in MSI colorectal tumors and aberrant methylation of the regulatory sequences of this important tumor suppressor gene.

SMAD4 levels and response to 5-fluorouracil in colorectal cancer.

We have recently reported that low tumor levels of SMAD4, a key mediator of transforming growth factor-beta superfamily signaling, can predict the probability of recurrence in patients with Dukes C colorectal cancer who had surgery as the only form of treatment. However, standard treatment for Dukes C colorectal cancer patients currently involves the administration of 5-fluorouracil (5-FU)-based adjuvant chemotherapy after surgery. Approximately 30% to 40% of these patients present with recurrence and die within 5 years, and there is great need for markers capable of predicting poor prognosis after the combined surgery/adjuvant treatment. In this study, we evaluate the prognostic value of SMAD4 in patients treated with surgery and 5-FU-based adjuvant therapy. We used immunohistochemistry and quantitative real-time reverse transcription-PCR to measure the levels of SMAD4 protein and mRNA expression in the primary tumors and a number of lymph node metastases from a series of 75 Dukes C colorectal cancer patients with at least 6 years of follow-up. Patients with tumors expressing low levels of SMAD4 protein or mRNA showed significantly shorted disease-free and overall survival than patients with high tumor levels of SMAD4. The median survival of patients with low SMAD4 protein or mRNA tumor levels was 1.4 and 1.2 years, respectively, whereas patients with high SMAD4 tumor level had a median survival of >9.3 years. In addition, the protein and mRNA levels of SMAD4 in lymph node metastases was significantly lower than in primary tumors (P = 0.006). In contrast, allelic imbalance in chromosome 18q21 was of no prognostic significance in these patients. In conclusion, low SMAD4 tumor levels identified a subset of patients with poor prognosis following surgery and 5-FU-based adjuvant therapy; therefore, these patients could be good candidates to receive combined treatment with additional chemotherapeutic agents such as CPT-11 and/or oxaliplatin.

Gene-expression profiling predicts recurrence in Dukes' C colorectal cancer.

BACKGROUND & AIMS: Although approximately 50% of Dukes' C colorectal cancer patients are surgically cured, it is currently not possible to distinguish these patients from those at high risk of recurrence. The recent advent of routine adjuvant chemotherapy for these patients has greatly complicated the identification of new markers predicting the response to surgery, which is now reliant on archived materials. Microarray analysis allows fine tumor classification but cannot be used with paraffin-embedded archival samples. METHODS: We used microarray analysis of a unique set of fresh-frozen tumor samples from Dukes' C patients who had surgery as the only form of treatment to identify molecular signatures that characterize tumors from patients with good and bad prognosis. RESULTS: Unsupervised hierarchical clustering and a K-nearest neighbors-based classifier identified groups of patients with significantly different survival (P = .019 and P = .0001). Expression profiling outperformed previously reported genetic markers of prognosis such as TP53 and K-RAS mutational status and allelic imbalance in chromosome 18q, which were of limited prognostic power in this study. Functional categories significantly enriched in gene-expression differences included protein transport and folding. The prognostic potential of the RAS homologue RHOA, one of the most differentially expressed genes, was further investigated using immunohistochemistry and a tissue microarray containing 137 independent Dukes' C tumor samples. Reduced RHOA expression was associated with significantly shorter survival (P = .01). CONCLUSIONS: This study shows that gene-expression profiling of surgical tumor samples can predict recurrence in Dukes' C patients. Therefore, this approach could be used to guide decisions concerning the clinical management of these patients.

SMAD4 as a prognostic marker in colorectal cancer.

More than 50% of patients with Dukes C colorectal cancer have disease recurrence and die within 5 years after surgical removal of their primary tumor. It is currently not possible to distinguish patients with good and bad prognosis. SMAD4 is an important tumor suppressor gene that mediates transforming growth factor-beta superfamily signaling and is located in chromosome 18q21, a region with frequent genetic losses in these tumors. Allelic imbalance in 18q has been linked to poor prognosis in a subset of colorectal cancer patients. Therefore, we generated a tissue microarray containing triplicate tumor samples from 86 Dukes C patients and used immunohistochemistry to assess the relative expression level of SMAD4 and its value as a prognostic marker. In addition, SMAD4 was screened for mutations and two polymorphic microsatellite markers were used to assess the presence of allelic imbalance in these tumors. Patients with tumors expressing high SMAD4 levels had significantly better overall (P < 0.025) and disease-free (P < 0.013) survival than patients with low levels. This identifies SMAD4 as a prognostic marker for Dukes C colorectal cancer. Although all tumors with absent SMAD4 staining showed allelic imbalance in 18q21, tumors with 18q21 allelic imbalance as a group showed no difference in SMAD4 levels compared with tumors without allelic imbalance, suggesting that additional mechanisms of SMAD4 down-regulation exist. In addition, although SMAD4 mutations were found in five tumors, they were not associated with shorter survival. In conclusion, the level of expression of SMAD4 was found to be a more sensitive marker than 18q21 allelic imbalance and SMAD4 mutations, which were of no prognostic significance for these patients.

Low levels of microsatellite instability characterize MLH1 and MSH2 HNPCC carriers before tumor diagnosis.

Microsatellite instability (MSI) characterizes tumors arising in patients with hereditary non-polyposis colorectal cancer (HNPCC) syndrome. HNPCC is a hereditary autosomal dominant disease caused by germline mutations in genes from the DNA (MMR) mismatch repair system. In these tumors, the loss of MMR compromises the genome integrity, allowing the progressive accumulation of mutations and the establishment of a mutator phenotype in a recessive manner. It is not clear, however, whether MSI can be detected in HNPCC carriers before tumor diagnosis. The aim of this study was to evaluate the presence of genetic instability in MMR gene carriers in peripheral blood lymphocytes of carriers and non-carriers members of two HNPCC families harboring a germline MLH1 and MSH2 mutation, respectively. An extensive analysis of the allelic distribution of single molecules of the polyA tract bat26 was performed using a highly sensitive PCR-cloning approach. In non-carriers, the allelic distribution of single bat26 molecules followed a gaussian distribution with no bat26 alleles shorter than (A)21. All mutation carriers showed unstable alleles [(A)20 or shorter] with an overall frequency of 5.6% (102/1814). We therefore suggest that low levels of genomic instability characterize MMR mutation carriers. These observations suggest that somatic mutations accumulate well before tumor diagnosis. Even though it is not clear whether this is due to the presence of a small percentage of cells with lost MMR or due to MMR haploinsufficiency, detection of these short unstable alleles might help in the identification of asymptomatic carriers belonging to families with no detectable MMR gene mutations.

Molecular patterns of nuclear and mitochondrial microsatellite alterations in breast tumors.

The widespread distribution of microsatellite sequences within the human genome has allowed researchers to identify alternative patterns of microsatellite alterations in cancer cells. Among them, aneuploid patterns of nuclear microsatellites, pseudo-diploid microsatellite instability patterns, and also patterns of microsatellite instability within the mitochondrial genome. In this context, while aneuploid patterns of multiple genomic gains and losses had already been described in breast tumors, data on nuclear microsatellite instability still remain controversial and limited data on mitochondrial instability are available. In order to clarify this, we undertook an extensive analysis of nuclear and mitochondrial microsatellite alterations in breast ductal adenocarcinomas, stratified by grades. No instability was detected in any of the 40 dinucleotide microsatellites analysed nor in bat26 and APDelta3 mononucleotide repeats, clearly concluding that microsatellite instability is not a feature of ductal breast tumorigenesis. Instead, microsatellites defined a clear pattern of aneuploid genomic gains and losses among which, losses of BRCA1 at D17S855 and gains of plakoglobin at D17S846 significantly associated to grade III tumors and poor prognosis. On the other hand, mitochondrial instability at the transcription control region was also detected in 10.8% of cases. None of the new mitochondrial variants was found in the normal tissue counterparts, confirming that these new variants arise as sporadic somatic mutations in the tumor cells. Also, no association was found between heteroplasmy in the normal tissue and mitochondrial instability in the tumors. We therefore suggest that these new variants arise in tumors as a consequence of the progressive accumulation of slippage somatic mutations and the intrinsic instability of these microsatellite sequences. Finally, our results also confirm that mitochondrial instability does not associate with nuclear MSI.