Publications by Author: Frank J Slack

Many diseases, especially cancer, are caused by the abnormal expression of non-coding microRNAs (miRNAs), which regulate gene expression, leading to the development of miRNA-based therapeutics. Synthetic miRNA inhibitors have shown promising efficacy in blocking the activity of aberrant miRNAs that are upregulated in disease-specific pathologies. On the other hand, miRNAs that aid in preventing certain diseases and are reduced in expression in the disease state need different strategies. To tackle this, miRNA mimics, which mimic the activity of endogenous miRNAs, can be delivered for those miRNAs downregulated in different disease states. However, the delivery of miRNA mimics remains a challenge. Here, we report a cationic polylactic-co-glycolic acid (PLGA)-poly-L-histidine delivery system to deliver miRNA mimics. We chose miR-34a mimics as a proof of concept for miRNA delivery. miR-34a-loaded PLGA-poly-L-histidine nanoparticles (NPs) were formulated and biophysically characterized to analyze the structural properties of miRNA mimic-loaded NPs. In vitro efficacy was determined by investigating miR-34a and downstream target levels and performing cell viability and apoptosis assays. We confirmed in vivo efficacy through prolonged survival of miR-34a NP-treated A549-derived xenograft mice treated intratumorally. The results of these studies establish PLGA-poly-L-histidine NPs as an effective delivery system for miRNA mimics for treating diseases characterized by downregulated miRNAs.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal of human cancers. Clinical trials of various chemotherapy, radiotherapy, targeted agents and combination strategies have generally failed to provide meaningful improvement in survival for patients with unresectable disease. Photodynamic therapy (PDT) is a photochemistry-based approach that enables selective cell killing using tumor-localizing agents activated by visible or near-infrared light. In recent years, clinical studies have demonstrated the technical feasibility of PDT for patients with locally advanced PDAC while a growing body of preclinical literature has shown that PDT can overcome drug resistance and target problematic and aggressive disease. Emerging evidence also suggests the ability of PDT to target PDAC stroma, which is known to act as both a barrier to drug delivery and a tumor-promoting signaling partner. Here, we review the literature which indicates an emergent role of PDT in clinical management of PDAC, including the potential for combination with other targeted agents and RNA medicine.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal of human cancers. The dismal response of PDAC to virtually all therapeutics is associated, in part, with a characteristically dense fibrotic stroma. This stroma not only acts as a barrier to drug perfusion, but also promotes tumor survival through paracrine crosstalk and biophysical interactions. Photodynamic therapy (PDT) is being explored for PDAC treatment, though the impact of tumor-promoting stromal crosstalk on PDT response in PDAC is not well-characterized. The current study assesses the effect of tumor-stroma interactions on response to PDT or chemotherapy in heterocellular 3D cocultures using PDAC cells and two different fibroblastic cell types (pancreatic stellate cells, PSCs, and a normal human fibroblast cell line, MRC5) embedded in extracellular matrix (ECM). While stromal fibroblasts promote resistance to chemotherapy as expected, PDAC 3D nodules in coculture with fibroblasts exhibit increased response to PDT relative to homotypic cultures. These results point to the potential for PDT to overcome tumor-promoting stromal interactions associated with poor therapeutic response in PDAC.

Multiple RNA-binding proteins and non-coding RNAs, such as microRNAs (miRNAs), are involved in post-transcriptional gene regulation through recognition motifs in the 3' untranslated region (UTR) of their target genes. The KRAS gene encodes a key signaling protein, and its messenger RNA (mRNA) contains an exceptionally long 3' UTR; this suggests that it may be subject to a highly complex set of regulatory processes. However, 3' UTR-dependent regulation of KRAS expression has not been explored in detail. Using extensive deletion and mutational analyses combined with luciferase reporter assays, we have identified inhibitory and stabilizing cis-acting regions within the KRAS 3' UTR that may interact with miRNAs and RNA-binding proteins, such as HuR. Particularly, we have identified an AU-rich 49-nt fragment in the KRAS 3' UTR that is required for KRAS 3' UTR reporter repression. This element contains a miR-185 complementary element, and we show that overexpression of miR-185 represses endogenous KRAS mRNA and protein in vitro. In addition, we have identified another 49-nt fragment that is required to promote KRAS 3' UTR reporter expression. These findings indicate that multiple cis-regulatory motifs in the 3' UTR of KRAS finely modulate its expression, and sequence alterations within a binding motif may disrupt the precise functions of trans-regulatory factors, potentially leading to aberrant KRAS expression.

While microRNAs (miRNAs) and the KRAS oncogene are known to be dysregulated in various cancers, little is known about the role of miRNAs in the regulation of KRAS in cancer. Here we review a selection of studies published in 2014 that have contributed to our understanding of the molecular mechanisms of KRAS regulation by miRNAs and the clinical relevance of sequence variants that may interfere with functional miRNA-mediated KRAS regulation.

While cancer is a serious health issue, there are very few genetic biomarkers that predict predisposition, prognosis, diagnosis, and treatment response. Recently, sequence variations that disrupt microRNA (miRNA)-mediated regulation of genes have been shown to be associated with many human diseases, including cancer. In an early example, a variant at one particular single nucleotide polymorphism (SNP) in a let-7 miRNA complementary site in the 3' untranslated region (3' UTR) of the KRAS gene was associated with risk and outcome of various cancers. The KRAS oncogene is an important regulator of cellular proliferation, and is frequently mutated in cancers. To discover additional sequence variants in the 3' UTR of KRAS with the potential as genetic biomarkers, we resequenced the complete region of the 3' UTR of KRAS in multiple non-small cell lung cancer and epithelial ovarian cancer cases either by Sanger sequencing or capture enrichment followed by high-throughput sequencing. Here we report a comprehensive list of sequence variations identified in cases, with some potentially dysregulating expression of KRAS by altering putative miRNA complementary sites. Notably, rs712, rs9266, and one novel variant may have a functional role in regulation of KRAS by disrupting complementary sites of various miRNAs, including let-7 and miR-181.

Targeted therapeutic approaches have seen tremendous advances in the last decade, for good reason. Specifically intervening with a disease-causing gene can revert the deleterious phenotype while eliminating the toxicity often associated with broad-spectrum agents. Unfortunately, because these selective agents hit one target in a single location, acquired resistance is often high. An arguably better treatment approach includes coupling multiple targeted agents or using an agent that hits an individual target in several independent locations and/or alters multiple relevant targets in the disease-causing pathway(s), precisely the approach taken by Nishimura and colleagues in their recent report aimed at identifying a better treatment option for ovarian cancer.

Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One approach involves utilizing cells derived from the model under evaluation. The approach to generate such cells varies based on the cell of origin and often the genetics of the cell. Here we describe the steps involved in generating epithelial cells from the lungs of KrasLSL-G12D/+; p53LSL-R172/+ mice (Kasinski and Slack, 2012). These mice develop aggressive lung adenocarcinoma following cre-recombinase dependent removal of a stop cassette in the transgenes and subsequent expression of Kra-G12D and p53R172 . While this protocol may be useful for the generation of epithelial lines from other genetic backgrounds, it should be noted that the Kras; p53 cell line generated here is capable of proliferating in culture without any additional genetic manipulation that is often needed for less aggressive backgrounds.

MicroRNAs, a class of small, non-coding RNAs, are now widely known for their importance in many aspects of biology. These small regulatory RNAs have critical functions in diverse biological events, including development and disease. Recent findings show that microRNAs are essential for lifespan determination in the model organisms, Caenorhabditis elegans and Drosophila, suggesting that microRNAs are also involved in the complex process of ageing. Further, short RNA fragments derived from longer parental RNAs, such as transfer RNA cleavage fragments, have now emerged as a novel class of regulatory RNAs that inhibit translation in response to stress. In addition, the RNA editing pathway is likely to act in the double-stranded RNA-mediated silencing machinery to suppress unfavorable RNA interference activity in the ageing process. These multiple, redundant layers in gene regulatory networks may make it possible to both stably and flexibly regulate genetic pathways in ensuring robustness of developmental and ageing processes.

Lung cancer is the leading cause of cancer deaths worldwide, and current therapies fail to treat this disease in the vast majority of cases. The RAS and p53 pathways are two of the most frequently altered pathways in lung cancers, with such alterations resulting in loss of responsiveness to current therapies and decreased patient survival. The microRNA-34 (mir-34) gene family members are downstream transcriptional targets of p53, and miR-34 expression is reduced in p53 mutant tumors; thus, we hypothesized that treating mutant Kras;p53 tumors with miR-34 would represent a powerful new therapeutic to suppress lung tumorigenesis. To this end we examined the therapeutically resistant Kras(LSL-G12D)(/+);Trp53(LSL-R172H)(/+) mouse lung cancer model. We characterized tumor progression in these mice following lung-specific transgene activation and found tumors as early as 10 weeks postactivation, and severe lung inflammation by 22 weeks. Tumors harvested from these lungs have elevated levels of oncogenic miRNAs, miR-21 and miR-155; are deficient for p53-regulated miRNAs; and have heightened expression of miR-34 target genes, such as Met and Bcl-2. In the presence of exogenous miR-34, epithelial cells derived from these tumors show reduced proliferation and invasion. In vivo treatment with miR-34a prevented tumor formation and progression in Kras(LSL-G12D)(/+);Trp53(LSL-R172H)(/+) mice. Animals infected with mir-34a-expressing lentivirus at the same time as transgene activation had little to no evidence of tumorigenesis, and lentivirus-induced miR-34a also prevented further progression of preformed tumors. These data support the use of miR-34 as a lung tumor-preventative and tumor-static agent.