Calaluce et al. BMC Cancer 2010, 10:126 http://www.biomedcentral.com/1471-2407/10/126 gain insight into putative HuR target sequences [30]. HuR binding was verified by HuR immunoprecipitations, and analyzed by quantitative RT-PCR and biotin pull-downs. Both CD9 and CALM2 mRNAs were enriched in HuR RIPs compared to isotype control IP reactions. Biotin pull- downs verified the binding of HuR protein specifically to the 3’UTR regions of both mRNAs, as had been predicted. CD9 is a tetraspanin molecule which plays important roles in cellular development, activation, growth and motility. It has been implicated in a variety of cancers, including but not limited to gastric cancers and B cell acute leukemia [58-60]. The role of CALM2 in cancer is less well understood but may be linked to cancer since it is involved in con- trolling calcium signaling [61,62]. There are three CAL- MODULIN genes (CALM1, CALM2 and CALM3) highly expressed in both MB-231 and MCF-7 cell lines (Addi- tional File 4, Figure S1). Interestingly, although they are encoded by different genes at different chromosomal locations, all three encode the same open reading frame but differ in the 5’ and 3’ UTRs [[61,63], and [64]]. Only CALM2 mRNA interacts with HuR by RIP analysis. Moreover, previously published reports have indicated the necessity of knocking down all three CALMODULIN mRNAs by siRNA to achieve knock-down of the protein [61]. We conclude that there may be differential HuR associated regulation of these CALMODULIN genes in breast cancer, even though the mechanism needs to be further delineated. Surprisingly, the regulation of both CD9 and CALM2 target genes was dependent upon the cellular milieu. To test the functional consequences of HuR binding to these two transcripts, we prepared cells that stably expressed higher or lower HuR, compared to the parent cells, in both ER+ and ER- breast cancer cell lines. HuR appears to differentially regulate the expression of CD9 in opposite directions in the two different forms of breast cancer. Specifically, HuR over-expression in ER- breast cancer (MB-231) paradoxically decreased CD9 mRNA and protein levels, whereas HuR knock-down increased the CD9 mRNA levels. This is the opposite of what is predicted for most HuR targets, since HuR is thought to stabilize its mRNA targets and often increases their translation. There did not seem to be similar effects upon CALM2 expression. As expected, knock-down of HuR by shRNA decreased expression of CD9 and CALM2 in ER+ breast cancer (MCF-7). Though there are differences in cytoplasmic HuR levels in MB-231 cells as compared with MCF-7, these are modest (10%). This is in keeping, however, with obser- vations that MB-231 cells are more undifferentiated and more aggressive. Moreover, analysis of HuR-associated mRNAs in both ER+ and ER- breast cancer revealed three broad Page 12 of 15 categories of genes. First, there were well known cancer genes, such as PTMA, which are regulated by HuR [27]. Second, there were cancer-related genes, such as CD9 and CALMODULIN, which were not known to be HuR regulated until this report. Third, there were other genes identified by HuR association with unknown can- cer function. These could potentially represent novel cancer targets. Additional proof of HuR involvement with other known cancer genes, such as CD44 and GATA-3, may represent novel insights into the mechan- isms of regulation of these cancer targets (see Additional Files). These results may therefore advance the field by shedding insights into posttranscriptional regulation of known and perhaps unknown cancer target genes. Though the exact mechanisms of HuR differential reg- ulation of CD9 and CALM2 are presently unclear, it may involve microRNA (miRNA) regulation. In a recent report, we described the recruitment by HuR of miRNA let-7 to translationally silence C-MYC expression [46]. It is clear from the findings of laboratories headed by Fili- powicz, Steitz and other investigators, that RBPs and miRNAs are involved in intricate associations to affect downstream translational suppression or activation of target mRNAs to help meet cellular needs [65,66]. Sharp and colleagues proposed that different interactions between RBPs and miRNAs may have evolved as a pro- tective mechanism for the cell against environmental stress [67]. A remaining question is why HuR selectively binds to certain genes containing AREs. Our previous work has demonstrated the role that HuR plays in myogenesis by stabilizing the expression of three critical genes involved in myogenesis: MYOD, MYOGENIN, and p21cip1 [68]. HuR over-expression results in precocious muscle differ- entiation and HuR siRNA knock-down prevents muscle differentiation [69]. It is highly probable that there are more than three HuR targets inside these cells. A speci- fic phenotype potentially arises when HuR levels are altered which may involve interactions with miRNAs, although this theory needs to be fully investigated. Our findings share some similarity to earlier reports of HuR RIP-Chip analysis of MCF-7 cells stably transfected with MCT-1 [45]. These analyses, however, were not gen- ome-wide and employed transfected cells. Nevertheless, thrombospondin, a known important anti-angiogenic fac- tor, was identified as a HuR-regulated target. Combined with earlier reports of the role of HuR in regulating, VEGF-a and HIF1a, HuR may be controlling a “posttran- scriptional mini-operon” involved in angiogenesis [29,32,70]. Further studies are being conducted in our laboratory to investigate the role of HuR in breast cancer angiogenesis using xenograft animal models. It will be par- ticularly important to test the role of HuR upon CD9 and CALM2 expression in breast tumors in vivo.