The major focus of my lab is on investigating roles of microRNA regulation in lung cancer pathogenesis. There are three ongoing projects: (1) microRNA regulation of drug sensitivity in non-small cell lung cancer, (2) microRNA repression of tumor suppressor gene FUS1, and (3) variation in microRNAs and microRNA target sites in human populations. These investigations are based on (a) microRNA isolation from cell lines and both frozen and FFPE tissues, (b) expression profiling using several different platforms, including Agilent, Illumina and Exiqon microarrays and TaqMan qRT-PCR assays, (c) data analysis including defining miRNA and mRNA expression signatures and calculating over-representation of microRNA target sites in sets of differentially expressed genes, (d) microRNA manipulation using precursor mimics and chemically stabilized inhibitors, either individually or by high-throughput screen, (e) microRNA target prediction, using both publicly available methods and those that we have developed in-house, and (f) in vivo validation, using orthotopic and non-orthotopic mouse models of human lung cancer.
microRNA regulation of drug sensitivity in non-small cell lung cancer. We are using a combination of in vitro and in silico approaches to address the questions of whether microRNA expression profiles correlate with chemosensitivity/resistance in lung cancers, whether microRNAs play a functional role in chemosensitivity/resistance of lung cancer cells, and whether microRNA expression levels can be manipulated to increase drug sensitivity in cultured lung cancer cells. To address these questions, we are using a panel of lung cancer and immortalized normal lung epithelial lines, on which in vitro sensitivity profiles to drugs commonly used in the treatment of lung cancer (which can show variations of >1,000 fold) and mRNA expression profiles (providing gene expression signatures for sensitivity/resistance) have been measured, and a library of chemically synthesized inhibitors for all known human miRNAs in conjunction with a high-throughput screening platform. We are pursuing this study along three avenues: (1) correlating microRNA expression in NSCLC cell lines with response of the cell lines to paclitaxel; (2) identifying microRNAs that modulate cellular viability and sensitivity to paclitaxel; and (3) defining molecular signatures of microRNA expression changes in response to short-term and long-term paclitaxel treatment.
microRNA regulation of tumor suppressor gene FUS1. We are investigating microRNA regulation of FUS1, a tumor suppressor gene. Although loss of one 3p21.3 allele is frequently seen in lung cancer, the FUS1 mRNA transcript can be detected in most lung cancer cells, while Fus1 protein can not be detected on immunoblots, suggesting that miRNAs may play a role in repression of Fus1 expression. The FUS1 gene contains a highly conserved 3’UTR, and we predicted that the 3’UTR of FUS1 contains target sites for multiple miRNAs. We have identified three microRNAs – miR-93, miR-98 and miR-197 – that translationally repress tumor suppressor gene FUS1. We showed that exogenous over-expression of these miRNAs inhibits Fus1 protein expression. We confirmed that the three miRNAs target the 3’UTR region of the FUS1 transcript, and that individual deletion of the three miRNA target sites in the FUS1 3’UTR restores the expression level of Fus1 protein. We further found that miR-93 and miR-98 are expressed at higher levels in small cell lung cancer cell lines (SCLC) than in non-small cell lung cancer cell lines (NSCLC) and immortalized human bronchial epithelial cells (HBECs). Finally, we found that elevated miR-93 and miR-197 expression is correlated with reduced Fus1 expression in NSCLC tumor specimens compared to normal lung. These results suggest that the three miRNAs are negative regulators of Fus1 expression in lung cancers. Inhibiting one or more of these miRNAs is potentially an effective way to abrogate the endogenous repression of Fus1, such that the combination of FUS1-based vector and miRNA inhibitor delivery would more effectively restore the tumor suppressor function of FUS1 than traditional vector-based FUS1 gene therapy.
Variation in miRNA loci and target sites. We are also interested in tying microRNA regulation to study of variation in human populations and the assessment of disease risk. We sequenced nine loci containing miRNAs predicted to target genes related to lipid and sterol homeostasis in 1,917 human subjects. These subjects were drawn from the Dallas Heart Study (DHS), a single-site, multi-ethnic, population-based probability sample. We sequenced a total of 7.2 kb of genomic DNA, covering 17 mature miRNAs and 13 miRNA passenger strands. We identified 115 single nucleotide polymorphisms (SNPs) and 8 insertion/deletion polymorphisms (indels); the majority (89 of 123) of these polymorphisms were extremely rare, with minor allele frequencies of less than 0.01 across all ethnicities. The density of SNPs within mature microRNAs and passenger strands (~3.5 SNPs per kb) is much lower than the density within other regions of the miRNA precursors and the flanking regions (~7 to 12 SNPs per kb, depending on ethnicity), as is π, the average per nucleotide heterozygosity (on the order of 10-5 for mature miRNAs and passenger strands and 10-4 for other precursor and flanking regions). Sequencing of approximately 100 other miRNA loci across a smaller sample of 32 African American subjects from the DHS confirmed the rarity of SNPs in mature miRNAs and their passenger strands. Several of the SNPs typed in this study showed associations with measured levels of plasma lipids, suggesting a role for miRNAs in lipid and sterol homeostasis; these associations match observations made in other published genome-wide association studies (GWAS). Together, these data provide evidence of selective pressure imposing sequence constraints on miRNAs and their precursors, as variation may affect overall miRNA expression, thereby altering the regulation of specific target genes.