Loss of PRP4K drives anoikis resistance in part by dysregulation of epidermal growth factor receptor endosomal trafficking

Abstract

Anoikis acts as a critical barrier to metastasis by inducing cell death upon cancer cell detachment from the extracellular matrix (ECM), thereby preventing tumor cell dissemination to secondary sites. The induction of anoikis requires the lysosomal-mediated downregulation of epidermal growth factor receptors (EGFRs) leading to termination of pro-survival signaling. In this study, we demonstrate that depletion of pre-mRNA splicing factor 4 kinase (PRP4K; also known as PRPF4B) causes dysregulation of EGFR trafficking and anoikis resistance. We also report a novel cytoplasmic localization of PRP4K at the late endosome, and demonstrate both nuclear and cytoplasmic localization in breast, lung and ovarian cancer tissue. Mechanistically, depletion of PRP4K leads to reduced EGFR degradation following cell detachment from the ECM and correlates with increased TrkB, vimentin and Zeb1 expression. As a result, PRP4K loss promotes sustained growth factor signaling and increased cellular resistance to anoikis in vitro and in a novel zebrafish xenotransplantation model of anoikis sensitivity, as well as increased metastasis in a mouse model of ovarian cancer. Thus, PRP4K may serve as a potential biomarker of anoikis sensitivity in ovarian and other epithelial cancers.

Haploinsufficient tumour suppressor PRP4K is negatively regulated during epithelial-to-mesenchymal transition

“…Similarly, when PRP4K is depleted in the mesenchymallike TNBC cell line MDA-MB-231, mesenchymal markers such as fibronectin, Zeb1, and Slug/SNAI2 were upregulated, as was epithelial marker Zo-1 by 2-fold (Fig 1). In previous work, we also observed the upregulation of Zeb1 following depletion of PRP4K in the epithelial-like breast cancer cell line MCF7 but this occurred without loss of E-cadherin [21]. Thus, depletion of PRP4K seems to drive partial EMT in normal mammary epithelial cell lines ( Fig 10A).…”

“…However, recent studies have linked aggressive cancer phenotypes to both mesenchymal-to-epithelial transition (MET) and developmental states caught "in-between" EMT and MET, otherwise known as partial EMT [1,5,34]. In this study, we examined the inter-relationship between EMT and PRP4K, a protein that when depleted can induce aggressive cancer phenotypes including anoikis and chemotherapy resistance [21,23].…”

“…Recently we demonstrated that depletion of PRP4K in transformed mouse ID8 ovarian and human MCF7 breast cancer cells leads to dysregulation of EGFR signalling and upregulation of mesenchymal gene expression, including vimentin and fibronectin [21]. From these data we hypothesized that reduced PRP4K expression might lead to cancer progression by and claudin-1 (in MCF10A); with the overall effect being a mix of both epithelial and mesenchymal gene expression consistent with a "partial" EMT phenotype [5], particularly in the non-transformed mammary epithelial cell lines.…”

The Zebrafish Xenograft Platform—A Novel Tool for Modeling KSHV-Associated Diseases

“…A recent study conducted by our group suggested that the yolk sac likely does not provide the same extracellular matrix context present in a solid tumor [33]. As a result of this microenvironment feature, resistance to anoikis, a mode of programmed cell death initiated after loss of contact with the extracellular matrix, is required to support increased XT proliferation in this compartment.…”

“…Even though we could detect KSHV lytic gene products in the xenograft, it remains unclear whether this is sufficient to support production of infectious virions in situ, which will require further development of sensitive detection methods. It has been reported that the normal maintainence temperature of zebrafish (28 • C) can hinder replication of human viruses that have evolved to replicate at higher temperatures [15,33]. We have addressed this issue by housing xenotransplanted zebrafish larvae at 35 • C to enable KSHV replication.…”