Human ortholog of Drosophila Melted impedes SMAD2 PNAS PLUS release from TGF-β receptor I to inhibit TGF-β signaling Premalatha Shathasivama,b,c, Alexandra Kollaraa,c, Maurice J. Ringuetted, Carl Virtanene, Jeffrey L. Wranaa,f, and Theodore J. Browna,b,c,1 aLunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital, Toronto, ON, Canada M5T 3H7; Departments of bPhysiology, cObstetrics and Gynaecology, dCell and Systems Biology, and fMolecular Genetics, University of Toronto, Toronto, ON, Canada M5S 3G5; and ePrincess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada M5G 1L7 Edited by Igor B. Dawid, The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, and approved May 5, 2015 (received for review March 11, 2015) Drosophila melted encodes a pleckstrin homology (PH) domain- The gene locus encompassing human VEPH1, 3q24-25, lies containing protein that enables normal tissue growth, metabo- within a region frequently amplified in ovarian cancer (6, 7). Tan lism, and photoreceptor differentiation by modulating Forkhead et al. (8) found that this locus was also amplified in 7 of 12 ep- box O (FOXO), target of rapamycin, and Hippo signaling pathways. ithelial ovarian cancer cell lines. A gene copy number analysis of Ventricular zone expressed PH domain-containing 1 (VEPH1) is the 68 primary tumors by Ramakrishna et al. (9) identified frequent mammalian ortholog of melted, and although it exhibits tissue- (>40%) VEPH1 gene amplification that correlated with tran- restricted expression during mouse development and is poten- script levels. We determined the impact of VEPH1 on gene ex- tially amplified in several human cancers, little is known of its pression in an ovarian cancer cell line using a whole-genome function. Here we explore the impact of VEPH1 expression in ovar- expression array. The results indicate a gene-expression profile ian cancer cells by gene-expression profiling. In cells with elevated that is partially consistent with that reported for Melted and raises VEPH1 expression, transcriptional programs associated with me- the possibility that VEPH1 may modulate TGF-β signaling. tabolism and FOXO and Hippo signaling were affected, analogous TGF-β is a pleiotropic cytokine that regulates tissue develop- to what has been reported for Melted. We also observed altered ment, repair, remodeling, and homeostasis by affecting cell pro- β β CELL BIOLOGY regulation of multiple transforming growth factor- (TGF- ) target liferation, differentiation, survival, and migration. TGF-β signals genes. Global profiling revealed that elevated VEPH1 expression by inducing the formation of a heterotetrameric complex of type II suppressed TGF-β–induced transcriptional responses. This inhibitory β β β (T RII) and type I (T RI; ALK5) serine/threonine kinase trans- effect was verified on selected TGF- target genes and by reporter membrane receptors (10). Ligand-bound, constitutively active gene assays in multiple cell lines. We further demonstrated that TβRII phosphorylates TβRI, resulting in TβRI association with VEPH1 interacts with TGF-β receptor I (TβRI) and inhibits nuclear and C-terminal phosphorylation of Sma- and Mad-related protein accumulation of activated Sma- and Mad-related protein 2 (SMAD2). β 2 (SMAD2) and/or SMAD3 (SMAD2/3) (11). In the canonical We identified two T RI-interacting regions (TIRs) with opposing β effects on TGF-β signaling. TIR1, located at the N terminus, inhibits TGF- signaling pathway, phosphorylated SMAD2/3 rapidly β β dissociates from TβRI and oligomerizes with SMAD4. The canonical TGF- signaling and promotes SMAD2 retention at T RI, – similar to full-length VEPH1. In contrast, TIR2, located at the C-ter- SMAD2/3 SMAD4 complex then accumulates in the nucleus minal region encompassing the PH domain, decreases SMAD2 re- tention at TβRI and enhances TGF-β signaling. Our studies indicate Significance that VEPH1 inhibits TGF-β signaling by impeding the release of ac- tivated SMAD2 from TβRI and may modulate TGF-β signaling during Ventricular zone expressed pleckstrin homology domain-con- development and cancer initiation or progression. taining 1 (VEPH1) is among genes on chromosome 3q24-26, a region amplified in several cancers. Although little is known of VEPH1 | TGF-β | SMAD2/3 | Melted | ALK5 mammalian VEPH1, its Drosophila ortholog, Melted, is involved in neural and eye development, metabolism, and size deter- entricular zone expressed pleckstrin homology domain-con- mination through effects on Forkhead box O, target of Vtaining 1 (Veph1) was initially identified as a novel gene rapamycin, and Hippo signaling. We show that VEPH1 expression encoding an 833-amino-acid protein expressed in the developing affects similar gene categories as Melted and potently inhibits β β murine central nervous system (1). In the adult mouse, Veph1 transforming growth factor- (TGF- ) signaling. VEPH1 interacts with TGF-β type I receptor (TβRI) and inhibits dissociation of acti- expression is restricted to the eye and kidney. Although the β function of Veph1 in mammals is unknown, the Drosophila vated Sma- and Mad-related protein 2 from T RI, resulting in impaired TGF-β signaling. TGF-β acts initially as a tumor suppres- ortholog melted impacts tissue growth and metabolism (2). Dis- sor through its cytostatic activity, but subsequently promotes tu- ruption of melted in Drosophila results in a 10% reduction in adult melted mor progression. These findings suggest that VEPH1 could affect size, which is rescued by transgenic expression of or human TGF-β activity during cancer development/progression. VEPH1. Mutant flies have reduced fat (triglyceride) content at- tributable to decreased Melted, specifically within the fat body. Author contributions: P.S., A.K., M.J.R., J.L.W., and T.J.B. designed research; P.S. and A.K. Comparison of gene expression profiles of fat bodies from wild- performed research; P.S., J.L.W., and T.J.B. contributed new reagents/analytic tools; P.S., type (WT) and Melted-deficient flies identified altered expression A.K., C.V., and T.J.B. analyzed data; and P.S., A.K., M.J.R., J.L.W., and T.J.B. wrote the paper. of genes involved in metabolism, protein degradation, and im- mune response. Melted inhibits Forkhead box O (FOXO) and The authors declare no conflict of interest. promotes target of rapamycin (TOR) signaling—pathways in- This article is a PNAS Direct Submission. volved in regulating metabolism and tissue growth—by localizing Data deposition: The microarray data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. FOXO and Tsc1/Tsc2 complexes to the plasma cell membrane. GSE67765). Melted has also been shown to regulate R8 color photoreceptor 1To whom correspondence should be addressed. Email: [email protected]. warts differentiation by opposing the expression of , a tumor sup- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pressor and key component of the Hippo signaling pathway (2–5). 1073/pnas.1504671112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1504671112 PNAS Early Edition | 1of10 Downloaded by guest on September 28, 2021 A 30 Dataset: B C ^ Cancer Cell, 2010 Amplificaon ‡ Cancer Research, 2012 25 Deleon ≠ Cell, 2012 3.5 & Cell, 2013 c HOC7 OVCAR3 SKOV3 OVCA429 ES2 HEY HepG2 COS7 3.0 20 ø JCO, 2013 VEPH1 ¶ Nature, 2011 * Nature, 2012 2.5 Acn 15 # Nature, 2013 2.0 % Hepatology, 2014 mRNA levels 10 = Nature, 2014 1.5 b ¥ Nature Genecs, 2010 b TBP D EGFP-VEPH1 DAPI † Provisional / 1.0 5 § In revision 0.5 Alteraon frequency (%) frequency Alteraon a a a 0 VEPH1 0.0 ES2 HEY HOC7 † ACC SKOV3 * CCLE † DLBC † GBM † Liver % Liver OVCAR3 & GBM † ccRCC # ccRCC = Breast‡ NCI-60 † Breast * Breast OVCA429 † Uterine # Uterine † Glioma † Ovarian† Cervical * Prostate¶ Ovarian † Bladder= Bladder† Prostate ø Bladder ^ Prostate † Lung* Lung squ squ = Stomach† Stomach ¥ Sarcoma & Prostate † Sarcoma† Pancreas Melanoma † Esophagus † Uterine CS † † Lung adeno = Lung adeno ≠ Lung adeno † Head§ Head & neck & neck Fig. 1. VEPH1 is differentially expressed in ovarian cancer. (A) Histogram showing frequency of VEPH1 gene amplification or deletion in large-scale DNA copy-number datasets accessed through the cBioPortal for Cancer Genomics. Individual datasets are identified by the tissue. ACC, adrenocortical carcinoma; adeno, adenocarcinoma; CCLE, Cancer Cell Line Encyclopedia; ccRCC, kidney renal clear cell carcinoma; CS, carcinosarcoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; GBM, glioblastoma; NCI-60, NCI-60 cell lines; Squ, squamous. (B) RT-qPCR analysis of VEPH1 mRNA expression in six ovarian cancer cell lines, normalized to TATA-binding protein (TBP) mRNA expression. Bars represent mean ± SEM, n = 3. Bars with different letters are statistically different from one another as determined by ANOVA followed by the Student–Neuman–Keuls (SNK) post hoc test (P < 0.05). (C) Western blot of VEPH1 and actin levels in total lysates from ovarian cancer, HepG2, and COS7 cell lines. (D) GFP immunofluorescence staining showing localization of VEPH1 (green) in HepG2 cells transfected with pEGFP-VEPH1. Chromatin is visualized by DAPI staining (blue). (Magnification, 40×; scale bar, 9 μm.) to modulate gene transcription in association with additional pression in subpopulations of ovarian cancer tumors (8, 9). VEPH1 transcriptional coregulators (10, 11). protein was predominantly localized to the cell