Due to the poor correlation between steady state mRNA levels and protein products, traditional microarray analysis may miss many genes which are regulated primarily at the level of mRNA stability and translation. Posttranscriptional gene regulation is becoming increasingly recognized as an important form of cellular control. The importance of microRNAs and RNA binding proteins (RBPs) is now beginning to be better appreciated. We study the elav (embryonic lethal abnormal vision) family of RBPs, which are paraneoplastic antigens, over-expressed in a variety of malignancies, including breast cancer. Antibodies against elav family members are believed to be cancer-protective. The elav family of RBPs binds to the AU-rich elements (AREs) found in the 3\#8217; untranslated regions (UTRs) of many early-response genes, including proto-oncogenes and cell cycle regulators. HuR, the ubiquitously expressed family member, has been described to play a role in cancer progression. HuR stabilizes and translationally up regulates the expression of its target mRNAs. Elevated levels of cytoplasmic HuR directly correlate with increased invasiveness of malignancy and poor prognosis for many cancers, including those of the breast. Recently, HuR has been implicated in tamoxifen resistance in breast cancer. HuR has been described to control the expression of genes in all six different areas of transformation originally described by Hanahan and Weinberg. Hence, it has been suggested that HuR may serve as a tumor maintenance gene which allows for cancers, once they are established, to proliferate. Therefore, it is of interest to discover in vivo HuR targets, as these genes may play vital roles in transformed cells. We have developed methods which enable us to identify en masse, in vivo targets of RBPs such as HuR from cell lines and now for the first time, solid tissues as well. We call this ribonomic analysis or RNA immunoprecipitations applied to microarrays, RIP-on-Chip. This technique, in which no cross linking is used, allows one to en masse identify different in vivo mRNA targets which different RBPs interact with. We have used RIP-on-Chip to identify distinct subsets of HuR associated mRNAs in MDA MB231 and MCF-7 breast cancer cell lines. Whereas some gene targets are well appreciated cancer genes, a number do not have clear cut associations with cancer and may well represent novel targets. To further investigate the role of HuR in triple negative breast cancer, we have over expressed HuR in MB231 cells, which results in accelerated growth. Therefore, we conclude that HuR pull downs can identify not only known but also novel cancer targets and RIP-on-Chip analysis identifies distinct subsets of cancer relevant genes.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3808.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO