Publications by Author: Frank J Slack

MicroRNAs (miRNAs) are emerging as potential cancer therapeutics, but effective delivery mechanisms to tumor sites are a roadblock to utility. Here we show that systemically delivered, synthetic miRNA mimics in complex with a novel neutral lipid emulsion are preferentially targeted to lung tumors and show therapeutic benefit in mouse models of lung cancer. Therapeutic delivery was demonstrated using mimics of the tumor suppressors, microRNA-34a (miR-34a) and let-7, both of which are often down regulated or lost in lung cancer. Systemic treatment of a Kras-activated autochthonous mouse model of non-small cell lung cancer (NSCLC) led to a significant decrease in tumor burden. Specifically, mice treated with miR-34a displayed a 60% reduction in tumor area compared to mice treated with a miRNA control. Similar results were obtained with the let-7 mimic. These findings provide direct evidence that synthetic miRNA mimics can be systemically delivered to the mammalian lung and support the promise of miRNAs as a future targeted therapy for lung cancer.

Transcriptional regulation of microRNA (miRNA) expression is one of the least understood aspects of miRNA biogenesis. In C. elegans the list of miRNAs whose transcriptional control has been described in some detail is currently limited to four: let-7, lin-4, lsy-6, and mir-61. Each of these genes has been shown experimentally to be transcriptionaly regulated by cis- and/or trans-acting factors that either promote or inhibit expression. Additionally, computational methods based on conservation among miRNA genes have yielded predicted regulatory sequences in C. elegans that may function to regulate miRNA expression on a genome-wide scale.

Interactions between tumor and stromal cells are well known to play a prominent roles in progression of pancreatic ductal adenocarcinoma (PDAC). As knowledge of stromal crosstalk in PDAC has evolved, it has become clear that cancer associated fibroblasts can play both tumor promoting and tumor suppressive roles through a combination of paracrine crosstalk and juxtacrine interactions involving direct physical contact. Another major contributor to dismal survival statistics for PDAC is development of resistance to chemotherapy drugs. Though less is known about how the acquisition of chemoresistance impacts upon tumor-stromal crosstalk. Here, we use 3D co-culture geometries to recapitulate juxtacrine interactions between epithelial and stromal cells. In particular, extracellular matrix (ECM) overlay cultures in which stromal cells (pancreatic stellate cells, or normal human fibroblasts) are placed adjacent to PDAC cells (PANC1), result in direct heterotypic cell adhesions accompanied by dramatic fibroblast contractility which leads to highly condensed macroscopic multicellular aggregates as detected using particle image velocimetry (PIV) analysis to quantify cell velocities over the course of time lapse movie sequences. To investigate how drug resistance impacts these juxtacrine interactions we contrast cultures in which PANC1 are substituted with a drug resistant subline (PANC1-OR) previously established in our lab. We find that heterotypic cell-cell interactions are highly suppressed in drug-resistant cells relative to the parental PANC1 cells. To investigate further we conduct RNA-seq and bioinformatics analysis to identify differential gene expression in PANC1 and PANC1-OR, which shows that negative regulation of cell adhesion molecules, consistent with increased epithelial mesenchymal transition (EMT), is also consistent with loss of hetrotypic cell-cell contact necessary for the contractile behavior observed in drug naïve cultures. Overall these findings elucidate the role of drug-resistance in inhibiting an avenue of stromal crosstalk which is associated with tumor suppression and also help to establish cell culture conditions useful for further mechanistic investigation.

Interactions between tumor and stromal cells are well known to play prominent roles in progression of pancreatic ductal adenocarcinoma (PDAC). As knowledge of stromal crosstalk in PDAC has evolved, it has become clear that cancer associated fibroblasts can play both tumor promoting and tumor suppressive roles through a combination of paracrine crosstalk and juxtacrine interactions involving direct physical contact. Another major contributor to dismal survival statistics for PDAC is development of resistance to chemotherapy drugs, though less is known about how the acquisition of chemoresistance impacts upon tumor-stromal crosstalk. Here, we use time lapse imaging and image analysis to study how co-culture geometry impacts interactions between epithelial and stromal cells. We show that extracellular matrix (ECM) overlay cultures in which stromal cells (pancreatic stellate cells, or normal human fibroblasts) are placed adjacent to PDAC cells (PANC1) result in direct heterotypic cell adhesions accompanied by dramatic fibroblast contractility. We analyze these interactions in co-cultures using particle image velocimetry (PIV) analysis to quantify cell velocities over the course of time lapse movie sequences. We further contrast co-cultures of PANC1 with those containing a drug resistant subline (PANC1-OR) previously established in our lab and find that heterotypic cell-cell interactions are suppressed in the latter relative to the parental line. We use RNA-seq and bioinformatics analysis to identify differential gene expression in PANC1 and PANC1-OR, which shows that negative regulation of cell adhesion molecules, consistent with increased epithelial mesenchymal transition (EMT), is also correlated with reduction in the hetrotypic cell-cell contact necessary for the contractile behavior observed in drug naïve cultures. Overall these findings elucidate the role of drug-resistance in inhibiting an avenue of stromal crosstalk which is associated with tumor suppression and also help to establish cell culture conditions useful for further mechanistic investigation.

Neuroendocrine (NE) prostate cancer (NEPC) is a lethal subtype of castration-resistant prostate cancer (PCa) arising either de novo or from transdifferentiated prostate adenocarcinoma following androgen deprivation therapy (ADT). Extensive computational analysis has identified a high degree of association between the long noncoding RNA (lncRNA) H19 and NEPC, with the longest isoform highly expressed in NEPC. H19 regulates PCa lineage plasticity by driving a bidirectional cell identity of NE phenotype (H19 overexpression) or luminal phenotype (H19 knockdown). It contributes to treatment resistance, with the knockdown of H19 re-sensitizing PCa to ADT. It is also essential for the proliferation and invasion of NEPC. H19 levels are negatively regulated by androgen signaling via androgen receptor (AR). When androgen is absent SOX2 levels increase, driving H19 transcription and facilitating transdifferentiation. H19 facilitates the PRC2 complex in regulating methylation changes at H3K27me3/H3K4me3 histone sites of AR-driven and NEPC-related genes. Additionally, this lncRNA induces alterations in genome-wide DNA methylation on CpG sites, further regulating genes associated with the NEPC phenotype. Our clinical data identify H19 as a candidate diagnostic marker and predictive marker of NEPC with elevated H19 levels associated with an increased probability of biochemical recurrence and metastatic disease in patients receiving ADT. Here we report H19 as an early upstream regulator of cell fate, plasticity, and treatment resistance in NEPC that can reverse/transform cells to a treatable form of PCa once therapeutically deactivated.

MicroRNAs (miRNAs) have increasingly been shown to be involved in human cancer, and interest has grown about the potential use of miRNAs for cancer therapy. miRNA levels are known to be altered in cancer cells, including in non-small cell lung cancer (NSCLC), a subtype of lung cancer that is the most prevalent form of cancer worldwide and that lacks effective therapies. The let-7 miRNA is involved in the regulation of oncogene expression in cells and directly represses cancer growth in the lung. let-7 is therefore a potential molecular target for tumor therapy. However, applications of RNA interference for cancer research have been limited by a lack of simple and efficient methods to deliver oligonucleotides (ONs) to cancer cells. In this study, we have used in vitro and in vivo approaches to show that HCC827 cells internalize hydrophobically modified let-7b miRNAs (hmiRNAs) added directly to the culture medium without the need for lipid formulation. We identified functional let-7b hmiRNAs targeting the HMGA2 mRNA, one of the let-7 target genes upregulated in NSCLC, and show that direct uptake in HCC827 cells induced potent and specific gene silencing in vitro and in vivo. Thus, hmiRNAs constitute a novel class of ONs that enable functional studies of genes involved in cancer biology and are potentially therapeutic molecules.

For decades, research into cancer biology focused on the involvement of protein-coding genes. Only recently was it discovered that an entire class of molecules, termed non-coding RNA (ncRNA), plays key regulatory roles in shaping cellular activity. An explosion of studies into ncRNA biology has since shown that they represent a diverse and prevalent group of RNAs, including both oncogenic molecules and those that work in a tumor suppressive manner. As a result, hundreds of cancer-focused clinical trials involving ncRNAs as novel biomarkers or therapies have begun and these are likely just the beginning.