Faculty Bibliography

High-mobility group A2 (HMGA2) is commonly overexpressed in large leiomyomas. HMGA2 is an important regulator of cell growth, differentiation, apoptosis, and transformation. As a predicted target of Let-7 microRNAs (Let-7s), HMGA2 can be repressed by Let-7s in vitro. MicroRNA profiling analysis revealed that Let-7s were significantly dysregulated in uterine leiomyomas: high in small leiomyomas and lower in large leiomyomas. To evaluate whether Let-7 repression of HMGA2 plays a major role in leiomyomas, we analyzed the molecular relationship of HMGA2 and Let-7s, both in vitro and in vivo. We first characterized that exogenous Let-7 microRNAs could directly repress the dominant transcript of HMGA2, HMGA2a. This repression was also identified for two cryptic HMGA2 transcripts in primary leiomyoma cultures. Second, we found that the endogenous Let-7s were biologically active and played a major role in the regulation of HMGA2. Then, we illustrated that Let-7 repression of HMGA2 inhibited cellular proliferation. Finally, we examined the expression levels of Let-7c and HMGA2 in a large cohort of leiomyomas (n = 120), and we found high levels of Let-7 and low levels of HMGA2 in small leiomyomas, and low levels of Let-7 and high levels of HMGA2 in large leiomyomas. Our findings suggest that the Let-7-mediated repression of HMGA2 mechanism can be an important molecular event in leiomyoma growth. (Mol Cancer Res 2008;6(4):663-73)

ARA70 is a coactivator of androgen receptor (AR), a ligand-dependent transcription factor that plays an important role in prostate cancer. There are 2 variants of ARA70, the full length 70 kd ARA70alpha isoform and the internally spliced 35 kd ARA70beta isoform. Recent studies have suggested different expression and roles of the 2 isoforms in several endocrine malignancies, including prostate, breast, and ovarian cancers. To study the roles of these isoforms in cancers, we produced isoform-specific polyclonal antibodies. The anti-ARA70alpha antibody was raised in rabbits against 326 amino acid peptide corresponding to the internal deletion missing from ARA70beta (ARA70id), whereas the anti-ARA70beta antibody was raised against 18 amino acid polypeptide spanning the splice junction, with Gln-Gln motif unique to ARA70beta. The antisera were affinity purified on CNBr-activated sepharose 4B, and their specificity tested against bacterially expressed, Ni-column-purified ARA70alpha, ARA70beta, and ARA70id. The anti-ARA70alpha antibody recognized ARA70alpha and ARA70id, but not ARA70beta. The anti-ARA70beta antibody was specific to ARA70beta and did not cross-react with ARA70alpha or ARA70id. We then used these antibodies to detect ARA70 isoforms in crude extracts made of prostate cancer cell lines and performed immunohistochemical localization of these proteins in prostate tissues. ARA70beta localized to the cytosol, whereas ARA70alpha was found in the nucleus, supporting the notion of their dissimilar functions

ARA70 was first identified as a gene fused to the ret oncogene in thyroid carcinoma and subsequently as a co-activator for androgen receptor (AR). Two isoforms of ARA70 have been identified: a 70-kDa version called ARA70 alpha and an internally spliced 35-kDa variant termed ARA70 beta. We have previously reported that ARA70 alpha expression is reduced in prostate cancer, and its overexpression inhibits proliferation of LNCaP prostate cancer cells. However, the function of the ARA70 beta isoform in prostate cancer is not understood. In this report we examined the effects of ARA70 beta on AR transcriptional regulation as well as prostate cancer cellular proliferation and invasion. Although both ARA70 alpha and ARA70 beta functioned as transcriptional co-activators of AR in cell-based reporter assays, ARA70 beta overexpression, in contrast to ARA70 alpha, promoted prostate cancer cellular proliferation and invasion through Matrigel. Interestingly, genome-wide expression profiling of cells expressing ARA70 beta revealed an increase in the expression of genes involved in the control of cell division and adhesion, compatible with a role for ARA70 beta in proliferation and invasion. Consistent with its function in promoting cell growth and invasion, ARA70 beta expression was increased in prostate cancer. Our findings implicate ARA70 beta as a regulator of tumor cell growth and metastasis by affecting gene expression

Human uterine leiomyomas (ULMs) are the most common neoplasms of women. Many genes are dysregulated in ULMs and some of this dysregulation may be due to abnormal expression of micro-RNAs (miRNAs). In this study, 55 ULMs and matched myometrium were collected from 41 patients for microarray-based global miRNA expression analysis. Of 206 miRNAs examined, 45 miRNAs were significantly up- or down-regulated in ULMs in comparison to the matched myometrium (P < 0.001). The top five dysregulated miRNAs in ULMs are the let-7 family, miR-21, miR-23b, miR-29b, and miR-197. Four polycistronic clusters of miRNAs were either up- or down-regulated, but not in a mixed pattern, indicative of coordinated regulation of these miRNAs. Significance analysis revealed that subsets of miRNAs were strongly associated with tumor sizes and race. By prediction analysis we identified some important tumorigenic genes previously identified in ULMs that may be targeted by the dysregulated miRNAs. HMGA2 was identified as one of target genes of the let-7 family of miRNAs and has been found to be suppressed by let-7 in vitro. (This article contains Supplementary material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.) (c) 2007 Wiley-Liss, Inc

The determination of tissue thickness in paraffin blocks in the histology laboratory has been largely based on visual estimates. More accurate methods are required for the construction of tissue microarrays (TMAs) to assure a greater yield of cores in sections through the TMA block. We describe an accurate radiographic method to determine tissue thickness in donor paraffin blocks and have validated its application to TMA construction. Individual radiographic analysis was performed on paraffin donor blocks used for the construction of TMAs for determination of donor block tissue thickness. Consecutive numbered slide sections through the TMA block were then examined for the presence or loss of cores in the 150th TMA slide (from the final third of the TMA block) and correlated with the thickness of the individual donor blocks determined radiographically. At the 150th TMA slide, 202 of 1340 cores (15.1%) were depleted. Radiographic measurement showed a greater thickness of the donor paraffin block tissue (2.02 mm) corresponding to the retained cores as compared with the donor tissue (1.54 mm) of the depleted cores (P < 0.001). With progressive slide sections through a TMA block, the retention of tissue cores shows a significant correlation with donor block tissue thickness. Radiographic determination of tissue thickness in donor paraffin blocks can be used in TMA construction. Prior knowledge of tissue thickness in TMA construction can prompt compensatory steps that can enhance the yield of valuable samples and assure sufficient numbers of adequate cores for statistical analysis in biomarker evaluations