Genetic and Epidemiologic Trends of Norovirus Outbreaks in the United States from 2013 to 2016 Demonstrated Emergence of Novel GII.4 Recombinant Viruses.

Noroviruses are the most frequent cause of epidemic acute gastroenteritis in the United States. Between September 2013 and August 2016, 2,715 genotyped norovirus outbreaks were submitted to CaliciNet. GII.4 Sydney viruses caused 58% of the outbreaks during these years. A GII.4 Sydney virus with a novel GII.P16 polymerase emerged in November 2015, causing 60% of all GII.4 outbreaks in the 2015-2016 season. Several genotypes detected were associated with more than one polymerase type, including GI.3, GII.2, GII.3, GII.4 Sydney, GII.13, and GII.17, four of which harbored GII.P16 polymerases. GII.P16 polymerase sequences associated with GII.2 and GII.4 Sydney viruses were nearly identical, suggesting common ancestry. Other common genotypes, each causing 5 to 17% of outbreaks in a season, included GI.3, GI.5, GII.2, GII.3, GII.6, GII.13, and GII.17 Kawasaki 308. Acquisition of alternative RNA polymerases by recombination is an important mechanism for norovirus evolution and a phenomenon that was shown to occur more frequently than previously recognized in the United States. Continued molecular surveillance of noroviruses, including typing of both polymerase and capsid genes, is important for monitoring emerging strains in our continued efforts to reduce the overall burden of norovirus disease.

Subcellular localization of activated AKT in estrogen receptor- and progesterone receptor-expressing breast cancers: potential clinical implications.

Activated v-AKT murine thymoma viral oncogene homolog 1 (AKT)/protein kinase B (PKB) kinase (pAKT) is localized to the plasma membrane, cytoplasm, and/or nucleus in 50% of cancers. The clinical importance of pAKT localization and the mechanism(s) controlling this compartmentalization are unknown. In this study, we examined nuclear and cytoplasmic phospho-AKT (pAKT) expression by immunohistochemistry in a breast cancer tissue microarray (n = 377) with approximately 15 years follow-up and integrated these data with the expression of estrogen receptor (ER)alpha, progesterone receptor (PR), and FOXA1. Nuclear localization of pAKT (nuclear-pAKT) was associated with long-term survival (P = 0.004). Within the ERalpha+/PR+ subgroup, patients with nuclear-pAKT positivity had better survival than nuclear-pAKT-negative patients (P < or = 0.05). The association of nuclear-pAKT with the ERalpha+/PR+ subgroup was validated in an independent cohort (n = 145). TCL1 family proteins regulate nuclear transport and/or activation of AKT. TCL1B is overexpressed in ERalpha-positive compared with ERalpha-negative breast cancers and in lung metastasis-free breast cancers. Therefore, we examined the possible control of TCL1 family member(s) expression by the estrogen:ERalpha pathway. Estradiol increased TCL1B expression and increased nuclear-pAKT levels in breast cancer cells; short- interfering RNA against TCL1B reduced nuclear-pAKT. Overexpression of nuclear-targeted AKT1 in MCF-7 cells increased cell proliferation without compromising sensitivity to the anti-estrogen, tamoxifen. These results suggest that subcellular localization of activated AKT plays a significant role in determining its function in breast cancer, which in part is dependent on TCL1B expression.

Estradiol-regulated microRNAs control estradiol response in breast cancer cells.

Estradiol (E2) regulates gene expression at the transcriptional level by functioning as a ligand for estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). E2-inducible proteins c-Myc and E2Fs are required for optimal ERalpha activity and secondary estrogen responses, respectively. We show that E2 induces 21 microRNAs and represses seven microRNAs in MCF-7 breast cancer cells; these microRNAs have the potential to control 420 E2-regulated and 757 non-E2-regulated mRNAs at the post-transcriptional level. The serine/threonine kinase, AKT, alters E2-regulated expression of microRNAs. E2 induced the expression of eight Let-7 family members, miR-98 and miR-21 microRNAs; these microRNAs reduced the levels of c-Myc and E2F2 proteins. Dicer, a ribonuclease III enzyme required for microRNA processing, is also an E2-inducible gene. Several E2-regulated microRNA genes are associated with ERalpha-binding sites or located in the intragenic region of estrogen-regulated genes. We propose that the clinical course of ERalpha-positive breast cancers is dependent on the balance between E2-regulated tumor-suppressor microRNAs and oncogenic microRNAs. Additionally, our studies reveal a negative-regulatory loop controlling E2 response through microRNAs as well as differences in E2-induced transcriptome and proteome.

Interplay between estrogen receptor and AKT in estradiol-induced alternative splicing.

BACKGROUND: Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. METHODS: MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. RESULTS: We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF. CONCLUSION: E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.