Aberrant expression of microRNA (miRNA) in human cancers is a common phenomenon. miRNAs regulate many tumor suppressor genes and oncogenes, therefore acting as oncogenes or tumor suppressor genes themselves to directly regulate cancer cell survival and proliferation.
In tumor cells, inhibiting miRNAs that are overexpressed, and restoring intracellular levels of those that are lost or otherwise underexpressed, are attractive approaches for cancer treatment. Additionally, the role miRNAs play in modulating cancer cell response to chemotherapeutic agents suggests that they may be a target for improving drug response in cancer therapy.
These naturally occurring 19–23 nucleotide long, single-stranded noncoding RNAs, which regulate gene expression largely by decreasing levels of target messenger RNAs, were highlighted at the recent Select Biosciences “Genomics Research” conference.
“As we start to look at the full genomic pattern of a tumor tissue as opposed to a normal tissue we see lots of changes over the course of tumor development. miRNA likely has hundreds of targets in the cell. The goal is to identify specific mutations and pair those with drugs that inhibit or activate pathways,” explained Anthony Saleh, Ph.D., IRTA scientist, National Institute on Deafness and Other Communication Disorders at NIH.
To determine the targets, researchers use genomic, transcriptomic, or proteomic approaches. The difficulty with omics-type experiments is the scope. Myriad miRNAs are overexpressed in cancer, although most are not very highly overexpressed. Targets are painstakingly narrowed down by expression level, consistent expression levels across samples, and biological function.
“We study miRNA expression in head and neck squamous cell carcinoma (HNSCC) lines grown from patients’ tumors and have identified a number of overexpressed miRNAs that are potentially oncogenic, in particular the miR-30 family,” continued Dr. Saleh.
“Other studies showed NF-kappaB was activating expression. NF-kappaB is, in general, an oncogene, promoting tumor formation. We also identified TP53 and Notch1, which are important tumor suppressors for HNSCC, as miR-30 targets. So in HNSCC miR-30 is being stimulated by an oncogene and suppressing two important tumor suppressor pathways.”
TP53, the most common tumor suppressor pathway, primarily functions to direct cell fate decisions. In 90% of HNSCC cases, TP53 is nonfunctional, deleted, or mutated.
Notch1 has different roles in different cancers and highlights the difficulty in studying cancer. In HNSCC it is a tumor cell suppressor. In other cancers it is an oncogene.
“Now we are determining if miR-30 might be a potential target for sensitizing tumors. If we decrease the levels of miR-30 through inhibitors, we may be able to slow down the cancer growth and, perhaps, kill it in combination with other chemotherapeutic drugs or radiation,” concluded Dr. Saleh.
Dx, Px, and Tx Tools
“If you consider the critical role of miRNAs in cancer coupled with their unique mechanism of action, it is clear that miRNAs represent a new class of diagnostic, prognostic, and therapeutic tools,” commented Alexander Pertsemlidis, Ph.D., assistant professor, Greehey Children’s Cancer Research Institute at the University of Texas Health Science Center at San Antonio.
“The idea of screening libraries of miRNA mimics, which increase intracellular levels of miRNAs, and inhibitors, which decrease those levels, has been around for a while, but is represented more in the patent literature than in the biomedical literature. We use high-throughput screening (HTS) to identify miRNA mimics and inhibitors that specifically sensitize cancer cells to widely used anticancer drugs.”
For example, miR-337-3p appears to be lost in lung cancer cells, with replacement selectively sensitizing cells to paclitaxel by enhancing taxane-induced G2/M arrest through downregulation of its targets. Increasing levels of miR-337-3p may provide a novel therapeutic tool for the treatment of non-small-cell lung cancer in conjunction with taxanes.
Conversely, miR-139-5p is present at aberrantly high levels in neuroendocrine lung cancer cells, and when removed with an inhibitor, selectively decreases tumor cell viability. Inhibiting miR-139-5p may therefore be a tool for treating neuroendocrine lung tumors.
Dr. Pertsemlidis’ investigations contribute to the understanding of miRNA roles in, and beyond, lung cancer pathogenesis. A number of miRNAs are aberrantly expressed in several different cancer types. Mimics or inhibitors that are selectively cytotoxic in lung cancer may also have therapeutic applications in breast cancer, and miRNA targets important to disease progression in neuroendocrine lung cancers may also play critical roles in other neuroendocrine cancers, such as neuroblastoma.
Standardizing RNAi Screens
Cellecta designs and constructs pooled libraries expressing short hairpin RNAs (shRNA) and performs genetic screens using these libraries to identify genes required for any cellular response with a strong selectable phenotype, such as genes required for growth and proliferation of cancer cells.
shRNA consists of a sense strand, short loop sequence, and antisense strand that, when expressed in cells, interferes with gene expression similar to synthetic siRNA. In addition to single shRNA screening, Cellecta has developed an approach to combinatorially screen shRNA pairs to discover synthetically lethal and synergistically lethal gene combinations.
The company’s shRNA libraries for pooled screening have optimized structures to increase stability, protocols for uniform en masse cloning of large heterogeneous pools of shRNA sequences into the lentiviral library vector, and specially designed shRNA-specific barcode sequences incorporated for HT sequencing to enable accurate assessment of the libraries’ shRNAs.
“It is not trivial to create a complex yet representative library that expresses multiple shRNAs to different targets, with each construct containing a unique sequenceable barcode that identifies the specific shRNA it contains,” commented Alex Chenchik, Ph.D., president and CSO.
With grant funding from the NIH National Center for Research Resources and National Human Genome Institute, Cellecta established The Decipher Project to help standardize the process of RNAi-based screens. “Cellecta’s goal with Decipher is to provide researchers investigating disease progression, cell-signaling pathways, or genes involved in a range of biological processes, the basic tools to conduct genome-scale genetic screens,” said Dr. Chenchik.
Through Decipher, Cellecta offers academic labs no-cost access to three plasmid human shRNA libraries that target 15,000 genes, and two for mouse that target 10,000 genes. In exchange, researchers agree to publicly share all genetic screen data generated after publication. In the last year, over 100 researchers accessed the Decipher libraries, according to Dr. Chenchik.