Increased androgen receptor levels and signaling in ovarian cancer cells by VEPH1 associated with suppression of SMAD3 and AKT activation.

Studies indicate androgens contribute to initiation or progression of epithelial ovarian cancer through poorly understood mechanisms. We provide evidence that the androgen receptor (AR) interacts in a ligand-independent manner with the putative armadillo repeat domain of ventricular zone expressed PH domain-containing 1 (VEPH1). This interaction was increased by mutation of the two nuclear receptor-interacting LxxLL motifs present within the VEPH1 armadillo repeat domain. Androgen treatment did not result in nuclear co-localization of VEPH1 with AR, suggesting that VEPH1 does not function as a nuclear co-regulatory protein. VEPH1 expression decreased SMAD3 and activated AKT levels in ovarian cancer cell lines and increased AR activity and protein levels, consistent with an impact on receptor stability. Treatment of cells with dihydrotestosterone (DHT) increased AR protein levels measured 24 h after treatment, an effect augmented in VEPH1-transfected cells, and inhibited by knock-down of endogenous VEPH1. SMAD3 overexpression decreased AR protein levels and prevented the VEPH1-dependent increase in AR; however, silencing of SMAD3 paradoxically also decreased AR levels. DHT treatment led to a rapid and sustained decrease in phosphorylated AKT (pAKT) levels that was enhanced by VEPH1 expression. Inhibition of PI3K resulted in increased AR protein levels. These studies indicate that VEPH1 acts to enhance AR activity in ovarian cancer cells by decreasing SMAD3 and pAKT levels, resulting in increased levels of AR protein.

Ventricular Zone Expressed PH Domain Containing 1 (VEPH1): an adaptor protein capable of modulating multiple signaling transduction pathways during normal and pathological development.

Ventricular Zone Expressed PH Domain-Containing 1 (VEPH1) is an 833-amino acid protein encoded by an evolutionarily conserved single-copy gene that emerged with pseudocoelomates. This gene has no paralog in any species identified to date and few studies have investigated the function of its encoded protein. Loss of expression of its ortholog, melted, in Drosophila results in a severe neural phenotype and impacts TOR, FoxO, and Hippo signaling. Studies in mammals indicate a role for VEPH1 in modulating TGFß signaling and AKT activation, while numerous studies indicate VEPH1 expression is altered in several pathological conditions, including cancer. Although often referred to as an uncharacterized protein, available evidence supports VEPH1 as an adaptor protein capable of modulating multiple signal transduction networks. Further studies are required to define these adaptor functions and the role of VEPH1 in development and disease progression.

VEPH1 expression decreases vascularisation in ovarian cancer xenografts and inhibits VEGFA and IL8 expression through inhibition of AKT activation.

BACKGROUND: VEPH1 is amplified in several cancers including ovarian but its impact on tumour progression is unknown. Previous work has shown that VEPH1 inhibits TGFß signalling while its Drosophila ortholog increases tissue growth, raising the possibility that VEPH1 could impact tumour growth or progression. METHODS: A CRISPR approach was used to disrupt VEPH1 expression in ovarian cancer ES-2 cells, while VEPH1-negative SKOV3 cells were stably transfected with VEPH1 cDNA. The impact of altered VEPH1 expression was assessed using in vitro and in vivo assays and mechanistic studies were performed in vitro. RESULTS: VEPH1 expression in SKOV3 cells resulted in a reduced tumour growth rate associated with increased necrotic area, and decreased microvessel density and VEGF-A levels relative to tumours formed by mock-transfected cells. VEPH1 expression also decreased VEGFA and IL8 expression in SKOV3 cells and was associated with decreased activated AKT levels. These effects were not observed in ES-2 cells, which bear a BRAFV600E activating mutation that leads to constitutively increased IL8 and VEGFA expression. CONCLUSIONS: VEPH1 expression in SKOV3 ovarian cancer cells inhibits AKT activation to decrease VEGFA and IL8 expression, which leads to decreased tumour vascularisation and progression.

Human ortholog of Drosophila Melted impedes SMAD2 release from TGF-ß receptor I to inhibit TGF-ß signaling.

Drosophila melted encodes a pleckstrin homology (PH) domain-containing protein that enables normal tissue growth, metabolism, and photoreceptor differentiation by modulating Forkhead box O (FOXO), target of rapamycin, and Hippo signaling pathways. Ventricular zone expressed PH domain-containing 1 (VEPH1) is the mammalian ortholog of melted, and although it exhibits tissue-restricted expression during mouse development and is potentially amplified in several human cancers, little is known of its function. Here we explore the impact of VEPH1 expression in ovarian cancer cells by gene-expression profiling. In cells with elevated VEPH1 expression, transcriptional programs associated with metabolism and FOXO and Hippo signaling were affected, analogous to what has been reported for Melted. We also observed altered regulation of multiple transforming growth factor-ß (TGF-ß) target genes. Global profiling revealed that elevated VEPH1 expression suppressed TGF-ß-induced transcriptional responses. This inhibitory effect was verified on selected TGF-ß target genes and by reporter gene assays in multiple cell lines. We further demonstrated that VEPH1 interacts with TGF-ß receptor I (TßRI) and inhibits nuclear accumulation of activated Sma- and Mad-related protein 2 (SMAD2). We identified two TßRI-interacting regions (TIRs) with opposing effects on TGF-ß signaling. TIR1, located at the N terminus, inhibits canonical TGF-ß signaling and promotes SMAD2 retention at TßRI, similar to full-length VEPH1. In contrast, TIR2, located at the C-terminal region encompassing the PH domain, decreases SMAD2 retention at TßRI and enhances TGF-ß signaling. Our studies indicate that VEPH1 inhibits TGF-ß signaling by impeding the release of activated SMAD2 from TßRI and may modulate TGF-ß signaling during development and cancer initiation or progression.