Adenocarcinomas of uncertain origin are a frequent problem for surgical pathologists. To determine the utility of immunostaining for cytokeratin 7 and cytokeratin 20 in the separation of pulmonary adenocarcinomas from colonic adenocarcinomas, we studied routinely processed, formalin-fixed tissue from 151 of these tumors using commercially available monoclonal antibodies and an avidin-biotin immunohistochemical technique. Used alone, neither cytokeratin 7 immunostaining or cytokeratin 20 immunostaining reliably separated these tumors. However, the immunophenotype of cytokeratin 7 positive/cytokeratin 20 negative was seen in 86% of the pulmonary adenocarcinomas, and in 0% of the colonic adenocarcinomas. Conversely, the cytokeratin 7-negative/cytokeratin 20-positive immunophenotype was seen in 77% of the colonic carcinomas, and in 0% of the pulmonary tumors. In conclusion, cytokeratin 7/cytokeratin 20 immunostaining patterns may be helpful in separating pulmonary adenocarcinomas from colonic adenocarcinomas.

IL-17 is a proinflammatory cytokine produced by activated Th17 cells and other immune cells. IL-17–producing Th17 cells are major contributors to chronic inflammatory and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Although the transcriptional regulation of Th17 cells is well understood, the posttranscriptional regulation of IL-17 gene expression remains unknown. The RNA-binding protein HuR positively regulates the stability of many target mRNAs via binding the AU-rich elements present in the 3′ untranslated region of many inflammatory cytokines including IL-4, IL-13, and TNF-α. However, the regulation of IL-17 expression by HuR has not been established. CD4+ Th17 cells from HuR knockout mice had decreased IL-17 steady-state mRNA and protein levels compared with wild-type Th17 cells, as well as decreases in frequency of IL-17+ cells. Moreover, we demonstrated that HuR directly binds to the IL-17 mRNA 3′ untranslated region by using RNA immunoprecipitation and biotin pulldown assays. In addition, the knockout of HuR decreased cellular proliferation of CD4+ T cells. Mice with adoptively transferred HuR KO Th17 cells had delayed initiation and reduced disease severity in the onset of experimental autoimmune encephalomyelitis compared with wild-type Th17 cells. Our results reveal a HuR-induced posttranscriptional regulatory mechanism of Th17 differentiation that influences IL-17 expression. These findings may provide novel therapeutic targets for the treatment of Th17-mediated autoimmune neuroinflammation.

BackgroundThe discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs).MethodsThe ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established.ResultsWe used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, CD9 and CALM2 mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis.ConclusionThis is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.

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