The Drosha-DGCR8 complex initiates microRNA maturation by precise cleavage of the stem loops that are embedded in primary transcripts (pri-miRNAs). Here we propose a model for this process that is based upon evidence from both computational and biochemical analyses. A typical metazoan pri-miRNA consists of a stem of approximately 33 bp, with a terminal loop and flanking segments. The terminal loop is unessential, whereas the flanking ssRNA segments are critical for processing. The cleavage site is determined mainly by the distance (approximately 11 bp) from the stem-ssRNA junction. Purified DGCR8, but not Drosha, interacts with pri-miRNAs both directly and specifically, and the flanking ssRNA segments are vital for this binding to occur. Thus, DGCR8 may function as the molecular anchor that measures the distance from the dsRNA-ssRNA junction. Our current study thus facilitates the prediction of novel microRNAs and will assist in the rational design of small hairpin RNAs for RNA interference.

Purpose: The intratumoral heterogeneity (ITH) and the evolution of genomic architectures associated with the development of distant metastases are not well understood in colorectal cancers.Experimental Design: We performed multiregion biopsies of primary and liver metastatic regions from five colorectal cancers with whole-exome sequencing and copy number profiling.Results: In addition to a substantial level of genetic ITH, multiregion genetic profiling identifies the subclonal mutational architecture, leading to the region-based or spatial categorization of somatic mutations and the inference of intratumoral evolutionary history of cancers. The universal mutations (those observed in all the regional biopsies) are enriched in known cancer genes such as APC and TP53 with distinct mutational spectra compared with biopsy-or region-specific mutations, suggesting that major operative mutational mechanisms and their selective pressures are not constant across the metastatic progression. The phylogenies inferred from genomic data show branching evolutionary patterns where some primary biopsies are often segregated with metastastic lesions. Our analyses also revealed that copy number changes such as the chromosomal gains of c-MYC and chromothripsis can be region specific and the potential source of genetic ITH.Conclusions: Our data show that the genetic ITH is prevalent in colorectal cancer serving as a potential driving force to generate metastasis-initiating clones and also as a means to infer the intratumoral evolutionary history of cancers. The paucity of recurrent metastasis-clonal events suggests that colorectal cancer distant metastases may not follow a uniform course of genomic evolution, which should be considered in the genetic diagnosis and the selection of therapeutic targets for the advanced colorectal cancer.

Caveolin-1 (Cav1) is an integral membrane, scaffolding protein found in plasma membrane invaginations (caveolae). Cav1 regulates multiple cancer-associated processes. In breast cancer, a tumor suppressive role for Cav1 has been suggested; however, Cav1 is frequently overexpressed in aggressive breast cancer subtypes, suggesting an oncogenic function in advanced-stage disease. To further delineate Cav1 function in breast cancer progression, we evaluated its expression levels among a panel of cell lines representing a spectrum of breast cancer phenotypes. In basal-like (the most aggressive BC subtype) breast cancer cells, Cav1 was consistently upregulated, and positively correlated with increased cell proliferation, anchorage-independent growth, and migration and invasion. To identify mechanisms of Cav1 gene regulation, we compared DNA methylation levels within promoter ‘CpG islands' (CGIs) with ‘CGI shores', recently described regions that flank CGIs with less CG-density. Integration of genome-wide DNA methylation profiles (‘methylomes') with Cav1 expression in 30 breast cancer cell lines showed that differential methylation of CGI shores, but not CGIs, significantly regulated Cav1 expression. In breast cancer cell lines having low Cav1 expression (despite promoter CGI hypomethylation), we found that treatment with a DNA methyltransferase inhibitor induced Cav1 expression via CGI shore demethylation. In addition, further methylome assessments revealed that breast cancer aggressiveness associated with Cav1 CGI shore methylation levels, with shore hypermethylation in minimally aggressive, luminal breast cancer cells and shore hypomethylation in highly aggressive, basal-like cells. Cav1 CGI shore methylation was also observed in human breast tumors, and overall survival rates of breast cancer patients lacking estrogen receptor α (ERα) negatively correlated with Cav1 expression. Based on this first study of Cav1 (a potential oncogene) CGI shore methylation, we suggest this phenomenon may represent a new prognostic marker for ERα-negative, basal-like breast cancer.

Surgical archives of tumor specimens are often impure. The presence of RNA transcripts from nontumor cells, such as immune and stromal cells, can impede analyses of cancer expression profiles. To systematically analyze the impact of tumor purity, the gene expression profiles and tumor purities were obtained for 7,794 tumor specimens across 21 tumor types (available in The Cancer Genome Atlas consortium). First, we observed that genes with roles in immunity and oxidative phosphorylation were significantly inversely correlated and correlated with the tumor purity, respectively. The expression of genes implicated in immunotherapy and specific immune cell genes, along with the abundance of immune cell infiltrates, was substantially inversely correlated with tumor purity. This relationship may explain the correlation between immune gene expression and mutation burden, highlighting the need to account for tumor purity in the evaluation of expression markers obtained from bulk tumor transcriptome data. Second, examination of cluster membership of gene pairs, with or without controlling for tumor purity, revealed that tumor purity may have a substantial impact on gene clustering across tumor types. Third, feature genes for molecular taxonomy were analyzed for correlation with tumor purity, and for some tumor types, feature genes representing the mesenchymal and classical subtypes were inversely correlated and correlated with tumor purity, respectively. Our findings indicate that tumor purity is an important confounder in evaluating the correlation between gene expression and clinicopathologic features such as mutation burden, as well as gene clustering and molecular taxonomy. .

Background: MicroRNAs (miRNAs) are small noncoding RNAs, which play significant roles as posttranscriptional regulators. The functions of animal miRNAs are generally based on complementarity for their 5' components. Although several computational miRNA target-gene prediction methods have been proposed, they still have limitations in revealing actual target genes.