Calaluce et al. BMC Cancer 2010, 10:126 http://www.biomedcentral.com/1471-2407/10/126 This discordance has been attributed to posttranscrip- tional control mechanisms affecting mRNA stability and translation. Steady-state mRNA levels of genes, con- trolled partially or totally at this level, may be mislead- ing. Gygi and colleagues have shown that correlation between mRNA and protein levels could not be pre- dicted from only mRNA steady-state levels [4]. They observed that some genes had the same mRNA levels but protein levels varied more than 20 fold. Conversely, some proteins were of equal expression but their respec- tive mRNA level varied by more than 30-fold. They con- cluded that “transcript levels provide little predictive value with respect to the extent of protein expression” [4]. Additionally, Idekar and colleagues have described similar results for the galactose gene [5]. Although our understanding of transcriptional gene regulation is advanced, posttranscriptional gene regula- tion remains largely unexplored. It is becoming clear, however, that this is an important mode of gene regula- tion, especially for proinflammatory genes. These genes appear to be posttranscriptionally regulated by RNA binding proteins (RBPs) which interact with AU-rich elements (AREs) in the 3’ untranslated region (UTR) of mRNAs. Approximately 3,000 human genes contain AREs, representing 8% of the human genome [6]. Many of these genes which possess AREs are in areas of tran- sient biological responses, including cell growth and dif- ferentiation, immune responses, signal transduction, transcriptional and translational control, hematopoiesis, apoptosis, nutrient transport, and metabolism [6,7]. New methodologies have provided global identifica- tion of in vivo mRNA targets of different RBPs. One of these, termed the ribonomic approach, involves the immunoprecipitation of ribonucleoprotein complexes (RNPs) with antibodies against different RBPs, extrac- tion of mRNA, and hybridization to microarrays [8-10]. This approach, also referred to as RIP-Chip, enables investigators to identify groups of posttranscriptionally regulated mRNAs coordinately controlled by RBPs dur- ing various biological processes. A new paradigm, the posttranscriptional operon hypothesis, has been devel- oped which states that RBPs coordinately regulate the expression of biologically related molecules [11,12]. This paradigm is being confirmed by the work of many dif- ferent laboratories as our understanding of posttran- scriptional regulation broadens and putative operons are described [8,13-17]. HuR is an RBP that binds to AREs of many proto-oncogenes and labile mRNAs. It has emerged as a key regulatory factor which stabilizes and translationally enhances its targets mRNAs, and affects their transport from the nucleus to the cytoplasm [18-20]. HuR belongs to the ELAV (embryonic lethal abnormal vision) family found in mammalian cells con- taining four members: HuR, HuB, HuC, and HuD. HuR Page 2 of 15 is the only ubiquitously expressed member. The other family members are found primarily in the central ner- vous system and gonadal tissue [18]. Many HuR targets are cytokines, chemokines, and other early-response genes [21,22]. Of the hallmarks of cancer originally described by Hanahan and Weinberg, HuR has been demonstrated to control expression of genes in multiple areas of malig- nant transformation [23]. Consequently, HuR has been suggested to function as a tumor maintenance gene, per- missive for malignant transformation, tumor growth, and perhaps metastasis [24]. HuR has been described in the literature as controlling the expression of many cancer- relevant genes, including those that encode these pro- teins: Prothymosin-a, Bcl-2, Mcl-1, SirT1, TGF-b, MMP- 9, MTC-1, uPA, VEGF-a, HIF1-a and cyclins A1 (CCN A1), B1 and D1 [25-35]. Increased levels of HuR have been associated with a more aggressive breast cancer and a worse prognosis [36-38]. Of significance, HuR has been described as posttranscriptionally regulating the expres- sion of many breast cancer relevant genes including those that encode Glut-1, ERa, COX-2, IL-8, Cyclin E1, and most recently BRCA-1 [36,39-44]. HuR RIP-Chip analysis has recently identified Thrombospondin 1 as a key HuR target in the MCT-1 transformed estrogen receptor positive (ER+) cell line MCF-7 [45]. Its interac- tions, however, are complex and, at times, HuR may interact with miRNAs such as Let-7 to translationally suppress the expression of C-MYC mRNA [46]. Since HuR has been described as regulating the expression of many cancer relevant genes, we asked whether it may coordinately regulate breast cancer genes in ER+ and ER- breast cancer. We performed a HuR RIP-Chip analysis on MDA-MB-231 (ER-) and MCF-7 (ER+) cell lines to identify cancer-relevant genes, not known to be regulated by HuR, and potential novel breast cancer targets. Our studies indicated that HuR was associated with unique subsets of mRNAs in each cell line as well as a subset of HuR associated mRNA targets common to both. We chose two cancer-asso- ciated genes, CD9 and CALMODULIN 2 (CALM2), highly expressed in both cell lines, and functionally vali- dated the role of HuR in regulating their expression. Unexpectedly, HuR differentially regulated the same tar- get, CD9, in both cell lines in an opposite manner. Moreover, we found presumptive differential regulation of CALM2 by HuR, as HuR interacted only with CALM2 mRNA, but not with family members CALM1 and CALM3 mRNAs. We discovered that HuR interacts with many breast cancer-relevant genes not previously known to be controlled by HuR, and target genes which have not been shown to be cancer related. This latter category may indeed represent novel cancer genes dis- covered by HuR RIP-Chip analysis.