The role of epithelial plasticity in prostate cancer dissemination and treatment resistance.

Nearly 30,000 men die annually in the USA of prostate cancer, nearly uniformly from metastatic dissemination. Despite recent advances in hormonal, immunologic, bone-targeted, and cytotoxic chemotherapies, treatment resistance and further dissemination are inevitable in men with metastatic disease. Emerging data suggests that the phenomenon of epithelial plasticity, encompassing both reversible mesenchymal transitions and acquisition of stemness traits, may underlie this lethal biology of dissemination and treatment resistance. Understanding the molecular underpinnings of this cellular plasticity from preclinical models of prostate cancer and from biomarker studies of human metastatic prostate cancer has provided clues to novel therapeutic approaches that may delay or prevent metastatic disease and lethality over time. This review will discuss the preclinical and clinical evidence for epithelial plasticity in this rapidly changing field and relate this to clinical phenotype and resistance in prostate cancer while suggesting novel therapeutic approaches.

Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo.

Alternative splicing generates a vast diversity of protein isoforms from a limited number of protein-coding genes, with many of the isoforms possessing unique, and even contrasting, functions. Fluorescence-based splicing reporters have the potential to facilitate studies of alternative splicing at the single-cell level and can provide valuable information on phenotypic transitions in almost real time. Fibroblast growth factor receptor 2 (FGFR2) pre-mRNA is alternatively spliced to form the epithelial-specific and mesenchymal-specific IIIb and IIIc isoforms, respectively, which are useful markers of epithelial-mesenchymal transitions (EMT). We have used our knowledge of FGFR2 splicing regulation to develop a fluorescence-based reporter system to visualize exon IIIc regulation in vitro and in vivo. Here we show the application of this reporter system to the study of EMT in vitro in cell culture and in vivo in transgenic mice harboring these splicing constructs. In explant studies, the reporters revealed that FGFR2 isoform switching is not required for keratinocyte migration during cutaneous wound closure. Our results demonstrate the value of the splicing reporters as tools to study phenotypic transitions and cell fates at single cell resolution. Moreover, our data suggest that keratinocytes migrate efficiently in the absence of a complete EMT.

The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function.

The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3' to 5' exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.

Different nuclease requirements for exosome-mediated degradation of normal and nonstop mRNAs.

Two general pathways of mRNA decay have been characterized in yeast. In one pathway, the mRNA is degraded by the cytoplasmic form of the exosome. The exosome has both 3' to 5' exoribonuclease and endoribonuclease activity, and the available evidence suggests that the exonuclease activity is required for the degradation of mRNAs. We confirm here that this is true for normal mRNAs, but that aberrant mRNAs that lack a stop codon can be efficiently degraded in the absence of the exonuclease activity of the exosome. Specifically, we show that the endo- and exonuclease activities of the exosome are both capable of rapidly degrading nonstop mRNAs and ribozyme-cleaved mRNAs. Additionally, the endonuclease activity of the exosome is not required for endonucleolytic cleavage in no-go decay. In vitro, the endonuclease domain of the exosome is active only under nonphysiological conditions, but our findings show that the in vivo activity is sufficient for the rapid degradation of nonstop mRNAs. Thus, whereas normal mRNAs are degraded by two exonucleases (Xrn1p and Rrp44p), several endonucleases contribute to the decay of many aberrant mRNAs, including transcripts subject to nonstop and no-go decay. Our findings suggest that the nuclease requirements for general and nonstop mRNA decay are different, and describe a molecular function of the core exosome that is not disrupted by inactivating its exonuclease activity.

Functions of the cytoplasmic exosome.

The exosome consists of a core of ten essential proteins that includes the ribonuclease Rrp44p and is present in both the cytoplasm and nucleus of eukaryotic cells. The cytoplasmic exosome has been extensively characterized in the budding yeast Saccharomyces cerevisiae and some characterization of its metazoan counterpart indicates that most functional aspects are conserved. These studies have implicated the cytoplasmic exosome in the turnover of normal cellular mRNAs, as well as several mRNA surveillance pathways. For this, the exosome needs a set of four proteins that do not partake in nuclear exosome functions. These cofactors presumably direct the exosome to specific cytoplasmic RNA substrates. Here, we review cofactors and functions of the cytoplasmic exosome and provide unanswered questions on the mechanisms of cytoplasmic exosome function.

Functions of the cytoplasmic exosome.

The exosome consists of a core often essential proteins that includes the ribonuclease Rrp44p and is present in both the cytoplasm and nucleus of eukaryotic cells. The cytoplasmic exosome has been extensively characterized in the budding yeast Saccharomyces cerevisiae and some characterization of its metazoan counterpart indicates that most functional aspects are conserved. These studies have implicated the cytoplasmic exosome in the turnover ofnormal cellularmRNAs, as well as several mRNA surveillance pathways. For this, the exosome needs a set of four proteins that do not partake in nuclear exosome functions. These cofactors presumably direct the exosome to specific cytoplasmic RNA substrates. Here, we review cofactors and functions of the cytoplasmic exosome and provide unanswered questions on the mechanisms of cytoplasmic exosome function.

Snail promotes resistance to enzalutamide through regulation of androgen receptor activity in prostate cancer.

Treatment with androgen-targeted therapies can induce upregulation of epithelial plasticity pathways. Epithelial plasticity is known to be important for metastatic dissemination and therapeutic resistance. The goal of this study is to elucidate the functional consequence of induced epithelial plasticity on AR regulation during disease progression to identify factors important for treatment-resistant and metastatic prostate cancer. We pinpoint the epithelial plasticity transcription factor, Snail, at the nexus of enzalutamide resistance and prostate cancer metastasis both in preclinical models of prostate cancer and in patients. In patients, Snail expression is associated with Gleason 9-10 high-risk disease and is strongly overexpressed in metastases as compared to localized prostate cancer. Snail expression is also elevated in enzalutamide-resistant prostate cancer cells compared to enzalutamide-sensitive cells, and downregulation of Snail re-sensitizes enzalutamide-resistant cells to enzalutamide. While activation of Snail increases migration and invasion, it is also capable of promoting enzalutamide resistance in enzalutamide-sensitive cells. This Snail-mediated enzalutamide resistance is a consequence of increased full-length AR and AR-V7 expression and nuclear localization. Downregulation of either full-length AR or AR-V7 re-sensitizes cells to enzalutamide in the presence of Snail, thus connecting Snail-induced enzalutamide resistance directly to AR biology. Finally, we demonstrate that Snail is capable of mediating-resistance through AR even in the absence of AR-V7. These findings imply that increased Snail expression during progression to metastatic disease may prime cells for resistance to AR-targeted therapies by promoting AR activity in prostate cancer.

Cellular migration and invasion uncoupled: increased migration is not an inexorable consequence of epithelial-to-mesenchymal transition.

Metastatic dissemination requires carcinoma cells to detach from the primary tumor and invade through the basement membrane. To acquire these characteristics, epithelial tumor cells undergo epithelial-to-mesenchymal transitions (EMT), whereby cells lose polarity and E-cadherin-mediated cell-cell adhesion. Post-EMT cells have also been shown, or assumed, to be more migratory; however, there have been contradictory reports on an immortalized human mammary epithelial cell line (HMLE) that underwent EMT. In the context of carcinoma-associated EMT, it is not yet clear whether the change in migration and invasion must be positively correlated during EMT or whether enhanced migration is a necessary consequence of having undergone EMT. Here, we report that pre-EMT rat prostate cancer (PC) and HMLE cells are more migratory than their post-EMT counterparts. To determine a mechanism for increased epithelial cell migration, gene expression analysis was performed and revealed an increase in epidermal growth factor receptor (EGFR) expression in pre-EMT cells. Indeed, inhibition of EGFR in PC epithelial cells slowed migration. Importantly, while post-EMT PC and HMLE cell lines are less migratory, both remain invasive in vitro and, for PC cells, in vivo. Our study demonstrates that enhanced migration is not a phenotypic requirement of EMT, and migration and invasion can be uncoupled during carcinoma-associated EMT.

Modulating the RNA processing and decay by the exosome: altering Rrp44/Dis3 activity and end-product.

In eukaryotes, the exosome plays a central role in RNA maturation, turnover, and quality control. In Saccharomyces cerevisiae, the core exosome is composed of nine catalytically inactive subunits constituting a ring structure and the active nuclease Rrp44, also known as Dis3. Rrp44 is a member of the ribonuclease II superfamily of exoribonucleases which include RNase R, Dis3L1 and Dis3L2. In this work we have functionally characterized three residues located in the highly conserved RNB catalytic domain of Rrp44: Y595, Q892 and G895. To address their precise role in Rrp44 activity, we have constructed Rrp44 mutants and compared their activity to the wild-type Rrp44. When we mutated residue Q892 and tested its activity in vitro, the enzyme became slightly more active. We also showed that when we mutated Y595, the final degradation product of Rrp44 changed from 4 to 5 nucleotides. This result confirms that this residue is responsible for the stacking of the RNA substrate in the catalytic cavity, as was predicted from the structure of Rrp44. Furthermore, we also show that a strain with a mutation in this residue has a growth defect and affects RNA processing and degradation. These results lead us to hypothesize that this residue has an important biological role. Molecular dynamics modeling of these Rrp44 mutants and the wild-type enzyme showed changes that extended beyond the mutated residues and helped to explain these results.

The exosome contains domains with specific endoribonuclease, exoribonuclease and cytoplasmic mRNA decay activities.

The eukaryotic exosome is a ten-subunit 3' exoribonucleolytic complex responsible for many RNA-processing and RNA-degradation reactions. How the exosome accomplishes this is unknown. Rrp44 (also known as Dis3), a member of the RNase II family of enzymes, is the catalytic subunit of the exosome. We show that the PIN domain of Rrp44 has endoribonucleolytic activity. The PIN domain is preferentially active toward RNA with a 5' phosphate, suggesting coordination of 5' and 3' processing. We also show that the endonuclease activity is important in vivo. Furthermore, the essential exosome subunit Csl4 does not contain any domains that are required for viability, but its zinc-ribbon domain is required for exosome-mediated mRNA decay. These results suggest that specific exosome domains contribute to specific functions, and that different RNAs probably interact with the exosome differently. The combination of an endoRNase and an exoRNase activity seems to be a widespread feature of RNA-degrading machines.

Determining in vivo activity of the yeast cytoplasmic exosome.

A 3'-exoribonuclease complex, termed the exosome, has important functions in the cytoplasm, as well as in the nucleus, and is involved in 3'-processing and/or decay of many RNAs. This chapter will discuss methods to study cytoplasmic exosome function in yeast with in vivo approaches. The first section will describe mutants that are available to study the processing or decay of a specific RNA by the nuclear or cytoplasmic exosome. The second section will discuss methods to determine whether the cytoplasmic exosome is functional under a specific condition(s) with reporter mRNAs that are known substrates of this complex.