The adaptor protein VEPH1 interacts with the kinase domain of ERBB2 and impacts EGF signaling in ovarian cancer cells.

Upregulation of ERBB2 and activating mutations in downstream KRAS/BRAF and PIK3CA are found in several ovarian cancer histotypes. ERBB2 enhances signaling by the ERBB family of EGF receptors, and contains docking positions for proteins that transduce signaling through multiple pathways. We identified the adaptor protein ventricular zone-expressed pleckstrin homology domain-containing protein 1 (VEPH1) as a potential interacting partner of ERBB2 in a screen of proteins co-immunoprecipitated with VEPH1. In this study, we confirm a VEPH1 - ERBB2 interaction by co-immunoprecipitation and biotin proximity labelling and show that VEPH1 interacts with the juxtamembrane-kinase domain of ERBB2. In SKOV3 ovarian cancer cells, which bear a PIK3CA mutation and ERBB2 overexpression, ectopic VEPH1 expression enhanced EGF activation of ERK1/2, and mTORC2 activation of AKT. In contrast, in ES2 ovarian cancer cells, which bear a BRAFV600E mutation with VEPH1 amplification but low ERBB2 expression, loss of VEPH1 expression enabled further activation of ERK1/2 by EGF and enhanced EGF activation of AKT. VEPH1 expression in SKOV3 cells enhanced EGF-induced cell migration consistent with increased Snail2 and decreased E-cadherin levels. In comparison, loss of VEPH1 expression in ES2 cells led to decreased cell motility independent of EGF treatment despite higher levels of N-cadherin and Snail2. Importantly, we found that loss of VEPH1 expression rendered ES2 cells less sensitive to BRAF and MEK inhibition. This study extends the range of adaptor function of VEPH1 to ERBB2, and indicates VEPH1 has differential effects on EGF signaling in ovarian cancer cells that may be influenced by driver gene mutations.

The impact of interval between primary cytoreductive surgery with bowel resection and initiation of adjuvant chemotherapy on survival of women with advanced ovarian cancer: a multicenter cohort study.

OBJECTIVE: Our aim was to determine if the time interval between bowel resection and initiation of adjuvant chemotherapy impacts survival in advanced ovarian cancers. METHODS: This was a retrospective cohort study using data from two cancer centers, Princess Margaret Cancer Centre in Toronto, Ontario, Canada and Samsung Comprehensive Cancer Center in Seoul, South Korea. Patients with International Federation of Gynecology and Obstetrics (FIGO) stage III or IV ovarian cancer that underwent large bowel resection during primary cytoreductive surgery (PCS) were included. RESULTS: Ninety-one women were eligible of which the majority (90.1%) were diagnosed with high-grade serous cancer. The median interval from PCS to chemotherapy for all patients was 21 days (7-86 days). Patients were stratified into 3 groups: 1) Interval ≤14 days, 32 (35.2%) patients; 2) Interval between 15-28 days, 27 (29.6%) patients; and 3) Interval between 29-90 days, 32 (35.2%) patients. Surgical procedures and postoperative outcomes were similar between groups. Multivariate analysis indicated that PCS to chemotherapy interval of 2-4 weeks, younger age, and completion of 4 or more adjuvant chemotherapy cycles were independent prognostic factors of favorable overall survival. CONCLUSION: Initiation of adjuvant chemotherapy between 2 to 4 weeks after PCS with bowel resection may improve survival outcomes in women with advanced ovarian cancer by maximizing the benefit of PCS plus adjuvant chemotherapy.

A mouse model of neoadjuvant chemotherapy followed by interval cytoreductive surgery indicates impaired efficacy of perioperative cisplatin.

BACKGROUND: Investigate the impact of interval cytoreductive surgery (ICS) on progression in an orthotopic mouse model of ovarian cancer and the impact of chemotherapy delivered at various timelines following surgery. METHODS: Luciferase-expressing ID8 murine ovarian cancer cells were implanted intra-bursally and IP to C57BL/7 mice. Once disease was established by bioluminescence, 2 cycles of neoadjuvant cisplatin were administered, and animals received either ICS (removal of the injected bursa/primary tumor) or anesthesia alone. Postsurgical chemotherapy was administered on the same day as the intervention (ICS/anesthesia), or on day 7 or day 28 following the intervention. Progression was quantified serially with in vivo bioluminescence imaging. Volume of ascitic fluid volume collected at necropsy was measured. RESULTS: Animals were matched for tumor burden at stratification. There was no accelerated growth of residual tumor after interval cytoreduction compared to controls. Animals who received chemotherapy on postoperative day (POD) 7 had better disease control compared to standard-of-care POD 28. Animals who underwent surgery had less ascites at necropsy compared to those who had anesthesia alone. CONCLUSIONS: In this animal model, surgical wounding with suboptimal cytoreduction after neoadjuvant chemotherapy did not cause accelerated expansion of residual disease. Surgical wounding appears to impair cisplatin activity when given at time of surgery.

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.

Banking Mesenchymal Stromal Cells from Umbilical Cord Tissue: Large Sample Size Analysis Reveals Consistency Between Donors.

Mesenchymal stromal cells (MSCs) have emerged as candidate cells with therapeutic potential to treat different pathologies. The underlying mechanism is paracrine signaling. The cells secrete proteins that can impact inflammation, apoptosis, angiogenesis, and cell proliferation. All are important in wound healing and tissue regeneration. Although the bone marrow has been the most widely used source of MSCs, umbilical cord tissue (CT) presents a source that is just starting to be used in the clinic, yet can be obtained with more ease and easily stored. Here, we characterize CT-MSCs obtained from multiple donors by analyzing cell surface proteins, differentiation capacity, and proteome profile. Analysis of low, medium, and high passage cells indicates that the morphology and proliferation rate stay constant and with the exception of cluster of differentiation (CD) 105 at late passage, there are no changes in the cell surface protein characteristics, indicating the population does not change with passage. TNF-stimulated gene 6 protein was measured in a subset of samples and variable expression was observed, but this did not impact the ability of the cells to enhance skin regeneration. In conclusion, CT-MSC represents a consistent, easily accessible source of cells for cell therapy. Stem Cells Translational Medicine 2019;8:1041-1054.

Impact of interval from primary cytoreductive surgery to initiation of adjuvant chemotherapy in advanced epithelial ovarian cancer.

OBJECTIVE: To determine the optimal timing of adjuvant chemotherapy after primary cytoreductive surgery for advanced epithelial ovarian cancer. METHODS: In a retrospective cohort analysis, data were assessed from women with advanced epithelial ovarian carcinoma treated at Princess Margaret Cancer Centre, Toronto, Canada between 2002 and 2012, and at Samsung Medical Centre, Seoul, Korea, between 2002 and 2015. The treatment interval was defined as the time period between primary cytoreductive surgery and the first cycle of adjuvant chemotherapy. RESULTS: Overall, 711 women met the inclusion criteria. Among them, 247 (34.7%) had optimal cytoreduction (residual 1-9 mm), 229 (32.2%) had microscopic residual disease (0 mm), and 235 (33.1%) had suboptimal cytoreduction (≥10 mm). The median time of treatment interval was 10 days (range 3-86 days). In the optimal (1-9 mm) group, a longer treatment interval was significantly associated with poor overall survival (hazard ratio 1.02, 95% confidence interval 1.01-1.03; P=0.001) in multivariate analysis. Treatment interval was not associated with a significant difference in overall survival in the microscopic or suboptimal residual disease groups. CONCLUSION: Overall survival might be negatively affected by longer treatment intervals among women with advanced epithelial ovarian carcinoma.

Wounding promotes ovarian cancer progression and decreases efficacy of cisplatin in a syngeneic mouse model.

BACKGROUND: Primary cytoreductive surgery followed by adjuvant chemotherapy is the standard treatment for advanced epithelial ovarian cancer. The average interval between surgery and chemotherapy initiation is approximately 4-weeks at most centers; however, since surgery may accelerate residual tumor growth, a shorter interval may be more beneficial. METHODS: The murine ID8 cell model of ovarian cancer was used to examine the efficacy of cisplatin treatment administered perioperatively or 7 days after surgical wounding. Luciferase-expressing cells ID8 cells were injected intraperitoneally (i.p.) into female C57/Bl6 mice. Fourteen days post-injection, animals received an abdominal incision or anesthesia alone and received i.p. cisplatin either on the surgical day or 7 days later, or received no chemotherapy. Additional animals received cisplatin 28 days after wounding for comparison. RESULTS: Abdominal tumor mass increased 2.5-fold in wounded vs. unwounded animals as determined by bioluminescent in vivo tumor imaging. Cisplatin administered on the day of wounding decreased tumor burden by 50%, as compared to 90% in unwounded animals. Cisplatin on day 7 or day 28 decreased tumor burden by 80 and 37% respectively. CONCLUSIONS: Surgical wounding increases ovarian tumor mass and decreases perioperative cisplatin efficacy in this animal model. Administration of cisplatin 1 week after surgery was more effective than cisplatin administered perioperatively or 4 weeks after surgery.

BRCA1 Mutation Status and Follicular Fluid Exposure Alters NFκB Signaling and ISGylation in Human Fallopian Tube Epithelial Cells.

Germline BRCA1 or BRCA2 mutations (mtBRCA1 and mtBRCA2) increase risk for high-grade serous ovarian cancer (HGSOC), the most commonly diagnosed epithelial ovarian cancer histotype. Other identified risk factors for this cancer, which originates primarily in the distal fallopian tube epithelium (FTE), implicate ovulation, during which the FTE cells become transiently exposed to follicular fluid (FF). To test whether mtBRCA1 or mtBRCA2 nonmalignant FTE cells respond differently to periovulatory FF exposure than control patient FTE cells, gene expression profiles from primary FTE cultures derived from BRCA1 or BRCA2 mutation carriers or control patients were compared at baseline, 24 hours after FF exposure, and 24 hours after FF replacement with culture medium. Hierarchical clustering revealed both FF exposure and BRCA mutation status affect gene expression, with BRCA1 mutation having the greatest impact. Gene set enrichment analysis revealed increased NFκB and EGFR signaling at baseline in mtBRCA1 samples, with increased interferon target gene expression, including members of the ISGylation pathway, observed after recovery from FF exposure. Gene set enrichment analysis did not identify altered pathway signaling in mtBRCA2 samples. An inverse relationship between EGFR signaling and ISGylation with BRCA1 protein levels was verified in an immortalized FTE cell line, OE-E6/E7, stably transfected with BRCA1 cDNA. Suppression of ISG15 and ISGylated protein levels by increased BRCA1 expression was found to be mediated by decreased NFκB signaling. These studies indicate that increased NFκB signaling associated with decreased BRCA1 expression results in increased ISG15 and protein ISGylation following FF exposure, which may be involved in predisposition to HGSOC.

The Two Faces of Adjuvant Glucocorticoid Treatment in Ovarian Cancer.

Adjuvant glucocorticoid treatment is routinely used in the treatment of ovarian cancer to mitigate the undesirable side effects of chemotherapy, thereby enhancing tolerability to higher cytotoxic drug doses and frequency of treatment cycles. However, in vitro and preclinical in vivo and ex vivo studies indicate that glucocorticoids may spare tumor cells from undergoing cell death through enhanced cell adhesion, promotion of anti-inflammatory signaling, and/or inhibition of apoptotic pathways. The implications of laboratory studies showing potential negative impact on the efficacy of chemotherapy have been long overlooked since clinical investigations have found no apparent survival detriment attributable to adjuvant glucocorticoid use. Importantly, these clinical studies were not randomized and most did not consider glucocorticoid receptor status, a vital determinant of tumor response to glucocorticoid administration. Additionally, the clinically beneficial elements of increased chemotherapy treatment adherence and dosing afforded by adjuvant glucocorticoids may offset and therefore mask their anti-chemotherapy activities. This review summarizes the current evidence on the impact of glucocorticoids in ovarian cancer and discusses the need for further research and development of alternative strategies to ameliorate untoward side effects of chemotherapy.

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.

Slow binding kinetics of secreted protein, acidic, rich in cysteine-VEGF interaction limit VEGF activation of VEGF receptor 2 and attenuate angiogenesis.

VEGF-A (VEGF) drives angiogenesis through activation of downstream effectors to promote endothelial cell proliferation and migration. Although VEGF binds both VEGF receptor 1 (R1) and receptor 2 (R2), its proangiogenic effects are attributed to R2. Secreted protein, acidic, rich in cysteine (SPARC) is a matricellular glycoprotein thought to inhibit angiogenesis by preventing VEGF from activating R1, but not R2. Because R2 rather than R1 mediates proangiogenic activities of VEGF, the role of human SPARC in angiogenesis was reevaluated. We confirm that association of SPARC with VEGF inhibits VEGF-induced HUVEC adherence, motility, and proliferation in vitro and blocks VEGF-induced blood vessel formation ex vivo. SPARC decreases VEGF-induced phosphorylation of R2 and downstream effectors ERK, Akt, and p38 MAPK as shown by Western blot and/or phosphoflow analysis. Surface plasmon resonance indicates that SPARC binds slowly to VEGF (0.865 ± 0.02 × 10(4) M(-1) s(-1)) with a Kd of 150 nM, forming a stable complex that dissociates slowly (1.26 ± 0.003 × 10(-3) s(-1)). Only domain III of SPARC binds VEGF, exhibiting a 15-fold higher affinity than full-length SPARC. These findings support a model whereby SPARC regulates angiogenesis by sequestering VEGF, thus restricting the activation of R2 and the subsequent activation of downstream targets critical for endothelial cell functions.

Molecular profiling and clinical outcome of high-grade serous ovarian cancer presenting with low- versus high-volume ascites.

Epithelial ovarian cancer consists of multiple histotypes differing in etiology and clinical course. The most prevalent histotype is high-grade serous ovarian cancer (HGSOC), which often presents at an advanced stage frequently accompanied with high-volume ascites. While some studies suggest that ascites is associated with poor clinical outcome, most reports have not differentiated between histological subtypes or tumor grade. We compared genome-wide gene expression profiles from a discovery cohort of ten patients diagnosed with stages III-IV HGSOC with high-volume ascites and nine patients with low-volume ascites. An upregulation of immune response genes was detected in tumors from patients presenting with low-volume ascites relative to those with high-volume ascites. Immunohistochemical studies performed on tissue microarrays confirmed higher expression of proteins encoded by immune response genes and increased tumorinfiltrating cells in tumors associated with low-volume ascites. Comparison of 149 advanced-stage HGSOC cases with differential ascites volume at time of primary surgery indicated low-volume ascites correlated with better surgical outcome and longer overall survival. These findings suggest that advanced stage HGSOC presenting with low-volume ascites reflects a unique subgroup of HGSOC, which is associated with upregulation of immune related genes, more abundant tumor infiltrating cells and better clinical outcomes.

Glucocorticoid-induced reversal of interleukin-1ß-stimulated inflammatory gene expression in human oviductal cells.

Studies indicate that high-grade serous ovarian carcinoma (HGSOC), the most common epithelial ovarian carcinoma histotype, originates from the fallopian tube epithelium (FTE). Risk factors for this cancer include reproductive parameters associated with lifetime ovulatory events. Ovulation is an acute inflammatory process during which the FTE is exposed to follicular fluid containing both pro- and anti-inflammatory molecules, such as interleukin-1 (IL1), tumor necrosis factor (TNF), and cortisol. Repeated exposure to inflammatory cytokines may contribute to transforming events in the FTE, with glucocorticoids exerting a protective effect. The global response of FTE cells to inflammatory cytokines or glucocorticoids has not been investigated. To examine the response of FTE cells and the ability of glucocorticoids to oppose this response, an immortalized human FTE cell line, OE-E6/E7, was treated with IL1ß, dexamethasone (DEX), IL1ß and DEX, or vehicle and genome-wide gene expression profiling was performed. IL1ß altered the expression of 47 genes of which 17 were reversed by DEX. DEX treatment alone altered the expression of 590 genes, whereas combined DEX and IL1ß treatment altered the expression of 784 genes. Network and pathway enrichment analysis indicated that many genes altered by DEX are involved in cytokine, chemokine, and cell cycle signaling, including NFκΒ target genes and interacting proteins. Quantitative real time RT-PCR studies validated the gene array data for IL8, IL23A, PI3 and TACC2 in OE-E6/E7 cells. Consistent with the array data, Western blot analysis showed increased levels of PTGS2 protein induced by IL1ß that was blocked by DEX. A parallel experiment using primary cultured human FTE cells indicated similar effects on PTGS2, IL8, IL23A, PI3 and TACC2 transcripts. These findings support the hypothesis that pro-inflammatory signaling is induced in FTE cells by inflammatory mediators and raises the possibility that dysregulation of glucocorticoid signaling could contribute to increased risk for HGSOC.

Altered expression of inflammation-associated genes in oviductal cells following follicular fluid exposure: implications for ovarian carcinogenesis.

Evidence indicates that high-grade serous ovarian carcinoma (HGSOC) may originate from lesions within the distal fallopian tube epithelium (FTE). Our previous studies indicate that fallopian tube epithelial cells from carriers of germline mutations in breast cancer susceptibility genes exhibit a pro-inflammatory gene expression signature during the luteal phase, suggesting that delayed resolution of postovulatory inflammatory signaling may contribute to predisposition to this ovarian cancer histotype. To determine whether exposure of tubal epithelial cells to periovulatory follicular fluid alters expression of inflammation-associated genes, we used an ex vivo culture system of bovine oviductal epithelial cells. Oviductal cells grown on collagen IV-coated transwell membranes assumed a cobblestone appearance and immunocytochemistry for FoxJ1 and Pax8 indicated that both ciliated and secretory epithelial cells were maintained in the cultures. Oviductal cells were exposed to human follicular fluid or culture medium for 24 h following which total cellular RNA was extracted at various time points. Expression of genes associated with inflammation was determined by quantitative real-time RT-PCR. Exposure to follicular fluid transiently increased the transcript levels of interleukin 8 (IL8) and cyclooxygenase 2 (PTGS2), and decreased the expression of mitochondrial superoxide dismutase (SOD2), glutathione peroxidase 3 (GPX3), disabled homolog 2 (DAB2), and glucocorticoid receptor (NR3C1). Tumor necrosis factor (TNF) and IL6 levels were also decreased while those of nicotinomide phosphoribosyltransferase (NAMPT) were unaffected. This study demonstrates that periovulatory follicular fluid can act directly upon oviductal epithelial cells to alter gene expression that might contribute to early carcinogenic events. Furthermore, these findings illustrate the potential use of bovine oviductal cells to study signaling events implicated in ovarian carcinogenesis.

C-terminal region of teneurin-1 co-localizes with the dystroglycan complex in adult mouse testes and regulates testicular size and testosterone production.

Testicular size is directly proportional to fertility potential and is dependent on the integration of developmental proteins, trophic factors, and sex steroids. The teneurins are transmembrane glycoproteins that function as signaling and cell adhesion molecules in the establishment and maintenance of the somatic gonad, gametogenesis, and basement membrane. Moreover, teneurins are thought to function redundantly to the extracellular matrix protein, dystroglycan. Encoded on the last exon of the teneurin genes is a family of bioactive peptides termed the teneurin C-terminal-associated peptides (TCAPs). One of these peptides, TCAP-1, functionally interacts with ß-dystroglycan to act as a neuromodulatory peptide with trophic characteristics independent from the teneurins. However, little is known about the localization and relationship between the teneurin-TCAP-1 system and the dystroglycans in the gonad. In the adult mouse testis, immunoreactive TCAP-1 was localized to spermatogonia and spermatocytes and co-localized with ß-dystroglycan. However, teneurin-1 was localized to the peritubular myoid cell layer of seminiferous tubules and tubules within the epididymis, and co-localized with α-dystroglycan and α-smooth muscle actin. TCAP-1-binding sites were identified in the germ cell layers and adluminal compartment of the seminiferous tubules, and epithelial cells of the epididymis. In vivo, TCAP-1 administration to adult mice for 9 days increased testicular size, seminiferous and epididymal tubule short-diameter and elevated testosterone levels. TCAP-1-treated mice also showed increased TCAP-1 immunoreactivity in the caput and corpa epididymis. Our data provide novel evidence of TCAP-1 localization in the testes that is distinct from teneurin-1, but is integrated through an association with the dystroglycan complex.

C-terminal processing of the teneurin proteins: independent actions of a teneurin C-terminal associated peptide in hippocampal cells.

Many neuropsychiatric conditions have a common set of neurological substrates associated with the integration of sensorimotor processing. The teneurins are a recently described family of proteins that play a significant role in visual and auditory development. Encoded on the terminal exon of the teneurin genes is a family of bioactive peptides, termed teneurin C-terminal associated peptides (TCAP), which regulate mood-disorder associated behaviors. Thus, the teneurin-TCAP system could represent a novel neurological system underlying the origins of a number of complex neuropsychiatric conditions. However, it is not known if TCAP-1 exerts its effects as part of a direct teneurin function, whereby TCAP represents a functional region of the larger teneurin protein, or if it has an independent role, either as a splice variant or post-translational proteolytic cleavage product of teneurin. In this study, we show that TCAP-1 can be transcribed as a smaller mRNA transcript. After translation, further processing yields a smaller 15 kDa protein containing the TCAP-1 region. In the mouse hippocampus, immunoreactive (ir) TCAP-1 is exclusively localized to the pyramidal layers of the CA1, CA2 and CA3 regions. Although the localization of TCAP and teneurin in hippocampal regions is similar, they are distinct within the cell as most ir-teneurin is found at the plasma membrane, whereas ir-TCAP-1 is predominantly found in the cytosol. Moreover, in mouse embryonic hippocampal cell culture, FITC-labeled TCAP-1 binds to the plasma membrane and is taken up into the cytosol via dynamin-dependent caveolae-mediated endocytosis. Our data provides novel evidence that TCAP-1 is structurally and functionally distinct from the larger teneurins.

Expression and function of nuclear receptor co-activator 4: evidence of a potential role independent of co-activator activity.

Nuclear receptor coactivator 4 (NcoA4), also known as androgen receptor-associated protein 70 (ARA70), was initially discovered as a component of Ret-Fused Gene expressed in a subset of papillary thyroid carcinomas. Subsequent studies have established NcoA4 as a coactivator for a variety of nuclear receptors, including peroxisome proliferator activated receptors α and γ, and receptors for steroid hormones, vitamins D and A, thyroid hormone, and aryl hydrocarbons. While human NcoA4 has both LXXLL and FXXLF motifs that mediate p160 coactivator nuclear receptor interactions, this ubiquitously expressed protein lacks clearly defined functional domains. Several studies indicate that NcoA4 localizes predominantly to the cytoplasm and affects ligand-binding specificity of the androgen receptor, which has important implications for androgen-independent prostate cancer. Two NcoA4 variants, which may exert differential activities, have been identified in humans. Recent studies suggest that NcoA4 may play a role in development, carcinogenesis, inflammation, erythrogenesis, and cell cycle progression that may be independent of its role as a receptor coactivator. This review summarizes what is currently known of the structure, expression, regulation, and potential functions of this unique protein in cancerous and non-cancerous pathologies.

Dynamic distribution of nuclear coactivator 4 during mitosis: association with mitotic apparatus and midbodies.

The cytoplasmic localization of Nuclear Receptor Coactivator 4 (NcoA4), also referred to as androgen receptor associated protein 70 (ARA70), indicates it may possess activities in addition to its role within the nucleus as a transcriptional enhancer. Towards identifying novel functions of NcoA4, we performed an in silico analysis of its amino acid sequence to identify potential functional domains and related proteins, and examined its subcellular distribution throughout the cell cycle. NcoA4 has no known or predicted functional or structural domains with the exception of an LxxLL and FxxLF nuclear receptor interaction motif and an N-terminal putative coiled-coil domain. Phylogenetic analysis indicated that NcoA4 has no paralogs and that a region referred to as ARA70-I family domain, located within the N-terminus and overlapping with the coiled-coil domain, is evolutionarily conserved in metazoans ranging from cnidarians to mammals. An adjacent conserved region, designated ARA70-II family domain, with no significant sequence similarity to the ARA70-I domain, is restricted to vertebrates. We demonstrate NcoA4 co-localizes with microtubules and microtubule organizing centers during prophase. Strong NcoA4 accumulation at the centrosomes was detected during interphase and telophase, with decreased levels at metaphase and anaphase. NcoA4 co-localized with tubulin and acetylated tubulin to the mitotic spindles during metaphase and anaphase, and to midbodies during telophase. Consistent with these observations, we demonstrated an interaction between NcoA4 and α-tubulin. Co-localization was not observed with microfilaments. These findings indicate a dynamic distribution of NcoA4 with components of the mitotic apparatus that is consistent with a potential non-transcriptional regulatory function(s) during cell division, which may be evolutionarily conserved.