To study the multistep process of cervical cancer development, we analyzed 128 frozen cervical samples spanning normalcy, increasingly severe cervical intraepithelial neoplasia (CIN1-CIN3), and cervical cancer (CxCa) from multiple perspectives, revealing a cascade of progressive changes. Compared with normal tissue, expression of many DNA replication/repair and cell proliferation genes was increased in CIN1/ CIN2 lesions and further sustained in CIN3, consistent with high-risk human papillomavirus (HPV)-induced tumor suppressor inactivation. The CIN3-to-CxCa transition showed metabolic shifts, including decreased expression of mitochondrial electron transport complex components and ribosomal protein genes. Significantly, despite clinical, epidemiological, and animal model results linking estrogen and estrogen receptor alpha (ERα) to CxCa, ERα expression declined >15-fold from normalcy to cancer, showing the strongest inverse correlation of any gene with the increasing expression of p16, a marker for HPV-linked cancers. This drop in ERα in CIN and tumor cells was confirmed at the protein level. However, ERα expression in stromal cells continued throughout CxCa development. Our further studies localized stromal ERα to FSP1+, CD34+, SMA− precursor fibrocytes adjacent to normal and precancerous CIN epithelium, and FSP1−, CD34−, SMA+ activated fibroblasts in CxCas. Moreover, rank correlations with ERα mRNA identified IL-8, CXCL12, CXCL14, their receptors, and other angiogenesis and immune cell infiltration and inflammatory factors as candidates for ERα-induced stroma-tumor signaling pathways. The results indicate that estrogen signaling in cervical cancer has dramatic differences from ERα+ breast cancers, and imply that estrogen signaling increasingly proceeds indirectly through ERα in tumor-associated stromal fibroblasts.cervical cancer | HPV | estrogen | tumor microenvironment | stroma G lobally, cervical cancer (CxCa) is the second most common cancer in women, with >500,000 new cases each year, half of which are ultimately fatal (1). In the developed world, routine CxCa screening for abnormal cervical cytology, human papillomavirus (HPV), or both (2) has strongly reduced CxCa incidence, demonstrating the value of recognizing and removing early neoplasms (3). Such screening programs present a rare opportunity to study the sequential molecular changes in the development of a human cancer (4).CxCa development is related to infection with high-risk oncogenic HPVs, most prominently HPV16 and HPV18 (5-7). HPV oncoproteins E6 and E7 are best known for blocking tumor suppressor functions of p53 and Rb, respectively, but have numerous additional interaction partners (8-13). Most cervical HPV infections are cleared (14), but in a fraction of cases persistent infections lead to increasingly severe grades of dysplasia (cervical intraepithelial neoplasia grades 1, 2, and 3; CIN1, CIN2, CIN3) and ultimately to invasive cancer. Although the key steps of the carcinogenic process, HPV infection, progression to precancer, and invas...

BackgroundHigh throughput sequencing technology provides us unprecedented opportunities to study transcriptome dynamics. Compared to microarray-based gene expression profiling, RNA-Seq has many advantages, such as high resolution, low background, and ability to identify novel transcripts. Moreover, for genes with multiple isoforms, expression of each isoform may be estimated from RNA-Seq data. Despite these advantages, recent work revealed that base level read counts from RNA-Seq data may not be randomly distributed and can be affected by local nucleotide composition. It was not clear though how the base level read count bias may affect gene level expression estimates.ResultsIn this paper, by using five published RNA-Seq data sets from different biological sources and with different data preprocessing schemes, we showed that commonly used estimates of gene expression levels from RNA-Seq data, such as reads per kilobase of gene length per million reads (RPKM), are biased in terms of gene length, GC content and dinucleotide frequencies. We directly examined the biases at the gene-level, and proposed a simple generalized-additive-model based approach to correct different sources of biases simultaneously. Compared to previously proposed base level correction methods, our method reduces bias in gene-level expression estimates more effectively.ConclusionsOur method identifies and corrects different sources of biases in gene-level expression measures from RNA-Seq data, and provides more accurate estimates of gene expression levels from RNA-Seq. This method should prove useful in meta-analysis of gene expression levels using different platforms or experimental protocols.

Background: The Zap1 transcription factor is a central player in the response of yeast to changes in zinc status. We previously used transcriptome profiling with DNA microarrays to identify 46 potential Zap1 target genes in the yeast genome. In this new study, we used complementary methods to identify additional Zap1 target genes.

Successful addiction treatment depends on maintaining long-term abstinence, making relapse prevention an essential therapeutic goal. However, exposure to environmental cues associated with drug use often thwarts abstinence efforts by triggering drug using memories that drive craving and relapse. We sought to develop a dual approach for weakening cocaine memories through phosphoproteomic identification of targets regulated in opposite directions by memory extinction compared with reconsolidation in male Sprague-Dawley rats that had been trained to self-administer cocaine paired with an audiovisual cue. We discovered a novel, inversely regulated, memorydependent phosphorylation event on calcium-calmodulin-dependent kinase II ␣ (CaMKII␣) at serine (S)331. Correspondingly, extinction-associated S331 phosphorylation inhibited CaMKII␣ activity. Intra-basolateral amygdala inhibition of CaMKII promoted memory extinction and disrupted reconsolidation, leading to a reduction in subsequent cue-induced reinstatement. CaMKII inhibition had no effect if the memory was neither retrieved nor extinguished. Therefore, inhibition of CaMKII represents a novel mechanism for memory-based addiction treatment that leverages both extinction enhancement and reconsolidation disruption to reduce relapse-like behavior.

Conflicting reports are available with regard to the effects of childhood abuse and neglect on hippocampal function in children. While earlier imaging studies and some animal work have suggested that the effects of early-life stress (ELS) manifest only in adulthood, more recent studies have documented impaired hippocampal function in maltreated children and adolescents. Additional work using animal modes is needed to clarify the effects of ELS on hippocampal development. In this regard, genomic, proteomic, and molecular tools uniquely available in the mouse make it a particularly attractive model system to study this issue. However, very little work has been done so far to characterize the effects of ELS on hippocampal development in the mouse. To address this issue, we examined the effects of brief daily separation (BDS), a mouse model of ELS that impairs hippocampal-dependent memory in adulthood, on hippocampal development in 28-day-old juvenile mice. This age was chosen because it corresponds to the developmental period in which human imaging studies have revealed abnormal hippocampal development in maltreated children. Exposure to BDS caused a significant decrease in the total protein content of synaptosomes harvested from the hippocampus of 28-day-old male and female mice, suggesting that BDS impairs normal synaptic development in the juvenile hippocampus. Using a novel liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM) assay, we found decreased expression of many synaptic proteins, as well as proteins involved in axonal growth, myelination, and mitochondrial activity. Golgi staining in 28-day-old BDS mice showed an increase in the number of immature and abnormally shaped spines and a decrease in the number of mature spines in CA1 neurons, consistent with defects in synaptic maturation and synaptic pruning at this age. In 14-day-old pups, BDS deceased the expression of proteins involved in axonal growth and myelination, but did not affect the total protein content of synaptosomes harvested from the hippocampus, or protein levels of other synaptic markers. These results add two important findings to previous work in the field. First, our findings demonstrate that in 28-day-old juvenile mice, BDS impairs synaptic maturation and reduces the expression of proteins that are necessary for axonal growth, myelination, and mitochondrial function. Second, the results suggest a sequential model in which BDS impairs normal axonal growth and myelination before it disrupts synaptic maturation in the juvenile hippocampus.