Cross-species hybridization of microarrays for studying tumor transcriptome of brain metastasis.

Although the importance of the cellular microenvironment (soil) during invasion and metastasis of cancer cells (seed) has been well-recognized, technical challenges have limited the ability to assess the influence of the microenvironment on cancer cells at the molecular level. Here, we show that an experimental strategy, competitive cross-species hybridization of microarray experiments, can characterize the influence of different microenvironments on cancer cells by independently extracting gene expression data of cancer and host cells when human cancer cells were xenografted into different organ sites of immunocompromised mice. Surprisingly, the analysis of gene expression data showed that the brain microenvironment induces complete reprogramming of metastasized cancer cells, resulting in a gain of neuronal cell characteristics and mimicking neurogenesis during development. We also show that epigenetic changes coincide with transcriptional reprogramming in cancer cells. These observations provide proof of principle for competitive cross-species hybridization of microarray experiments to characterize the effect of the microenvironment on tumor cell behavior.

Short rare hTERT-VNTR2-2nd alleles are associated with prostate cancer susceptibility and influence gene expression.

BACKGROUND: The hTERT (human telomerase reverse transcriptase) gene contains five variable number tandem repeats (VNTR) and previous studies have described polymorphisms for hTERT-VNTR2-2nd. We investigated how allelic variation in hTERT-VNTR2-2nd may affect susceptibility to prostate cancer. METHODS: A case-control study was performed using DNA from 421 cancer-free male controls and 329 patients with prostate cancer. In addition, to determine whether the VNTR polymorphisms have a functional consequence, we examined the transcriptional levels of a reporter gene linked to these VNTRs and driven by the hTERT promoter in cell lines. RESULTS: Three new rare alleles were detected from this study, two of which were identified only in cancer subjects. A statistically significant association between rare hTERT-VNTR2-2nd alleles and risk of prostate cancer was observed [OR, 5.17; 95% confidence interval (CI), 1.09-24.43; P = 0.021]. Furthermore, the results indicated that these VNTRs inserted in the enhancer region could influence the expression of hTERT in prostate cancer cell lines. CONCLUSIONS: This is the first study to report that rare hTERT VNTRs are associated with prostate cancer predisposition and that the VNTRs can induce enhanced levels of hTERT promoter activity in prostate cancer cell lines. Thus, the hTERT-VNTR2-2nd locus may function as a modifier of prostate cancer risk by affecting gene expression.

The hTERT-VNTR2-2nd alleles are involved in genomic stability in gastrointestinal cancer.

BACKGROUND: hTERT contains a high density of minisatellites, of which rare alleles of hTERT-VNTR2-2nd have been reported to be associated with prostate cancer. This shows an association between VNTR and cancer, but this repeat sequence is likely to be associated with genomic instability. Therefore, we investigated the effects of hTERT-VNTR2-2nd on gastrointestinal cancer and the relationship between repeated sequence and chromosome instability. METHODS: A case-control study was performed using DNA from 818 cancer-free controls, 539 cases with gastric cancer, 275 cases with colon cancer and 274 cases with rectal cancer. To determine whether minisatellites affect gene expression, expression levels were examined using TERT-reporter vectors in cell lines. In addition, the length of the hTERT-VNTR2-2nd alleles were determined in blood and cancer tissues from 107 gastric cancers, 112 colon cancers and 76 rectal cancers patients to determine whether the repeat sequence was associated with genomic instability during cancer development. RESULTS: No statistically significant association between hTERT-VNTR2-2nd and risk of gastrointestinal cancer was detected. However, it has been shown that VNTRs inserted into the enhancer region can regulate the expression of TERT in gastrointestinal cancer cells. Moreover, hTERT-VNTR2-2nd was analyzed in matched blood and cancer tissue from patients with gastrointestinal cancer and in seven among 294 subjects, and hTERT-VNTR2-2nd was found to be rearranged. CONCLUSIONS: We suggest that minisatellites are associated with genomic instability in cancer and that the hTERT-VNTRs region may increase hTERT expression in gastrointestinal cancer cells.

A polymorphic minisatellite region of BORIS regulates gene expression and its rare variants correlate with lung cancer susceptibility.

Aberrant expression of BORIS/CTCFL (Brother of the Regulator of Imprinted Sites/CTCF-like protein) is reported in different malignancies. In this study, we characterized the entire promoter region of BORIS/CTCFL, including the CpG islands, to assess the relationship between BORIS expression and lung cancer. To simplify the construction of luciferase reporter cassettes with various-sized portions of the upstream region, genomic copies of BORIS were isolated using TAR cloning technology. We analyzed three promoter blocks: the GATA/CCAAT box, the CpG islands and the minisatellite region BORIS-MS2. Polymorphic minisatellite sequences were isolated from genomic DNA prepared from the blood of controls and cases. Of the three promoter blocks, the GATA/CCAAT box was determined to be a critical element of the core promoter, while the CpG islands and the BORIS-MS2 minisatellite region were found to act as regulators. Interestingly, the polymorphic minisatellite region BORIS-MS2 was identified as a negative regulator that repressed the expression levels of luciferase reporter cassettes less effectively in cancer cells compared with normal cells. We also examined the association between the size of BORIS-MS2 and lung cancer in a case-control study with 590 controls and 206 lung cancer cases. Rare alleles of BORIS-MS2 were associated with a statistically significantly increased risk of lung cancer (odds ratio, 2.04; 95% confidence interval, 1.02-4.08; and P=0.039). To conclude, our data provide information on the organization of the BORIS promoter region and gene regulation in normal and cancer cells. In addition, we propose that specific alleles of the BORIS-MS2 region could be used to identify the risk for lung cancer.

Analysis of promoter methylation and polymorphic minisatellites of BORIS and lack of association with gastric cancer.

BORIS is a member of the cancer-testis gene family that comprises genes normally expressed only in testis but abnormally activated in different malignancies. In this study, we examined the relation between BORIS expression and gastric cancer, which is the most common cancer in Korea. Abnormal BORIS expression in the patient's gastric cancer tissues was observed. We checked the methylation status of the gene in gastric cancer tissue, because the regulation by methylation in its CpG islands is well known for BORIS. However, there was no correlation between the methylation status and gene expression. Then, we focused on the minisatellites (variable number of tandem repeats) of BORIS as another possible regulator for this abnormal expression. Previously, we reported the characterization of BORIS-MS2 and determined the frequency of alleles in cancer patients. A case-control study was performed using DNA from 774 controls and 496 patients with gastric cancer. There was no significant difference observed in the overall distribution of minisatellite alleles. These results suggest that additional different regulators for the abnormal BORIS expression in gastric cancer may exist. Additionally, we performed a segregation analysis of BORIS-MS2 with genomic DNA obtained from two generations of five families and from three generations of two families. BORIS-MS2 alleles were transmitted through meiosis following Mendelian inheritance, which suggests that this polymorphic minisatellite could be a useful marker for paternity mapping and DNA fingerprinting.

Gene expression signature-based prognostic risk score in gastric cancer.

PURPOSE: Despite continual efforts to develop a prognostic model of gastric cancer by using clinical and pathologic parameters, a clinical test that can discriminate patients with good outcomes from those with poor outcomes after gastric cancer surgery has not been established. We aim to develop practical biomarker-based risk score that can predict relapse of gastric cancer after surgical treatment. EXPERIMENTAL DESIGN: Microarray technologies were used to generate and analyze gene expression profiling data from 65 gastric cancer patients to identify biomarker genes associated with relapse. The association of expression patterns of identified genes with relapse and overall survival was validated in independent gastric cancer patients. RESULTS: We uncovered two subgroups of gastric cancer that were strongly associated with the prognosis. For the easy translation of our findings into practice, we developed a scoring system based on the expression of six genes that predicted the likelihood of relapse after curative resection. In multivariate analysis, the risk score was an independent predictor of relapse in a cohort of 96 patients. We were able to validate the robustness of the six-gene signature in an additional independent cohort. CONCLUSIONS: The risk score derived from the six-gene set successfully prognosticated the relapse of gastric cancer patients after gastrectomy.

Susceptibility for breast cancer in young patients with short rare minisatellite alleles of BORIS.

In this study, we characterized two blocks of minisatellites in the 5' upstream region of the BORIS gene (BORIS-MS1, -MS2). BORIS-MS2 was found to be polymorphic; therefore, this locus could be useful as a marker for DNA fingerprinting. We assessed the association between BORIS-MS2 and breast cancer by a case-control study with 428 controls and 793 breast cancers cases. Rare alleles in the younger group (age, <40) were associated with a statistically significant increased risk of breast cancer (odds ratio, 4.84; 95% confidence interval, 1.06-22.22; and P = 0.026). A statistically significant association between the short rare alleles and cancer was identified in the younger group (8.02; 1.01-63.83; P = 0.021). Kaplan-Meier estimates showed that poor prognosis was associated with patients who contained the rare alleles. Our data suggest that the short rare alleles of BORIS-MS2 could be used to identify the risk for breast cancer in young patients.